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+{"text": "Intravascular fasciitis (IVF) is a rare benign condition characterised by reactive myofibroblastic proliferation arising from the superficial or deep fascia and involving arteries and/or veins. It is a distinct variant of the more common condition of nodular fasciitis, which possesses similar clinical and histological features to IVF, but lacks vascular invasion. A thorough review of the literature revealed 26 reported cases of IVF.We report a case of IVF in a 16-week pregnant lady affecting the hypothenar eminence of the hand associated with the ulnar artery.The characteristic involvement of muscular arteries and veins by reactive myofibroblastic proliferation in IVF suggests a malignant component and often leads to an inappropriate diagnosis for this benign condition. We propose that hormone-related changes associated with pregnancy may play an important role in the aetiopathogenesis of this myofibroblastic lesion. Intravascular fasciitis (IVF) is a term originally described by Patchefsky and Enzinger to describe this distinctive variant of nodular fasciitis . It is ahousewife. There was no history of trauma, insect bites or drainage from the area. There was no significant past medical or surgical history. She was not on any prescribed medication. There was no family history of tumours. She denied smoking, alcohol and drug abuse. On examination, a non-tender, firm to palpation, well-demarcated and tethered to the subcutaneous tissues mass, surrounded by mild erythema, and measuring approximately 2.5 cm \u00d7 3.0 cm was revealed, in the absence of palpable lymph nodes at the ipsilateral elbow or axilla. There were no other abnormal signs.In August 2003, a 20-year-old 16-week pregnant mother of one, presented to the orthopaedic outpatient clinic with a 2-month history of a slowly growing painless mass located in the right hypothenar eminence. She was right-hand dominant and a Haematological and biochemical investigations were within the normal range. A plain X-ray of the right hand and forearm was unremarkable. Magnetic resonance imaging showed the mass to have infiltrated all skin layers and to have encircled the ulnar artery without involvement of the underlying bones Figure . The absThree weeks after initial presentation, the lesion was excised preserving the ulnar nerve and artery. Histology showed a multi-nodular, well-circumscribed uniform spindle cell lesion with varying cellularity and fibrosis. In one section, central cyst formation was observed. Occasional mitoses of normal morphology were seen. Necrosis was not a feature. In several areas, the lesion appeared to be intravascular, the vessel was lined by an apparent endothelium Figure . ScatterThe patient had an uneventful recovery and required no further therapy. She continues to be evaluated for local recurrence every 6 months, with no sign of recurrence at the 2-year follow-up.IVF was first described as a subset of cases closely resembling the clinical and histological picture of nodular fasciitis (NF) . RecogniPre-existing trauma, viral infection, and venous thrombosis resulting in myofibroblastic transformation of the vessel wall have all been implicated in the aetiopathogenesis of IVF, but there was no evidence for either of these in our case affecting the hypothenar eminence. Pregnancy was the only significant co-existing medical condition.We hypothesised that hormone-related changes seen during pregnancy may have contributed to the development of IVF in our patient. Oestrogen in particular, has for long been known to stimulate fibroblast and smooth muscle cell types and has been implicated in fibroproliferative diseases such as carpal tunnel syndrome, Dupuytren's contracture, and dermoid tumours. A weak presence of oestrogen receptors has also previously been demonstrated in nodular fasciitis . OestrogIVF is a benign myofibroblastic proliferative lesion that involves arteries and/or veins, a feature distinguishing it from other pseudosarcomatous proliferative lesions. Nevertheless, it may be easily mistaken for a malignant sarcomatous invasion of the vasculature. Based on our case presentation, we propose that pregnancy and oestrogen-related changes may be another risk factor for IVF and play an important role in the aetiopathogenesis by influencing proliferative changes in fascia myofibroblasts, however, more research and further cases need to be studied prior to establishing such hypothesis.The author(s) declare that they have no competing interests.1 reviewed the literature and prepared the manuscript. SRC5 provided the case and revised the manuscript. AN3 obtained patient consent and helped in preparation of the manuscript. ET6 and EMT2 conception and identification of case hypothesis, critical revision of the manuscript and coordination of the study. AA4 provided the histology images and histology information. All authors read and approved the final manuscript.AA"}
+{"text": "Subsequent clinical follow-up and CT examination of the patient has not revealed recurrent disease at 14 months.Carcinoma within a long-standing fistula-in-ano is rare and may be defined by specific neoplastic involvement of the fistulous track in the absence of rectal mucosal carcinoma. The presence of a carcinoma of mucinous histology occurring synchronously in the perianal region and the colon is exceptionally rare. We present a case with a review of the literature concerning its aetiopathogenesis and treatment. A 72-year-old man with a 2 months history of dark red rectal bleeding and mucus per rectum with alternating constipation and diarrhoea, was observed. Clinical examination and a barium enema showed a perianal fistula and an annular stenosing lesion of the rectosigmoid. Preoperative CT scan confirmed the colonic lesion. Colonic resection and wide fistula excision were performed. Histology showed an adenocarcinoma with a clear resection margins. The fistula also showed a similar histology. Chemoradiation (5-Fluorouracil (425 mg/m Carcinoma within a long-standing fistula-in-ano is rare and may A 72-year-old Afro-Caribbean male presented to our University Hospital with a 2 months history of dark red rectal bleeding and mucus per rectum with alternating constipation and diarrhoea as well as 20 lb. weight loss. There was no family history of colorectal neoplasia. There was an attendant history of perianal discharge for the previous 2 years and the patient volunteered that he had been seen in the United Kingdom and told that he had a perianal fistula in 1999. Clinical examination was unremarkable apart from the perianal area where a perianal fistula was evident which appeared to be low trans-sphincteric in nature. It was firm and indurated. A barium enema which had been performed outside the hospital showed an annular stenosing lesion of the rectosigmoid. Preoperative CT scan confirmed the colonic lesion but was otherwise unremarkable. In view of the presentation he was taken to the operating theatre after full bowel preparation for elective sigmoid colectomy and wide fistula excision.2) and leucovorin (20 mg/m2) before and after 4500 cGy external beam radiotherapy (EBRT) to the perineum and inguinal portals. Clinical and CT examination of the patient has not revealed any signs of recurrent disease at 14 months.Histology of the colon showed a moderate to well differentiated adenocarcinoma with extensive mucinous components and none out of 10 lymph nodes resected was involved with tumour. (Stage IIA American Joint Committee Cancer) The resection margins of the tumour were clear. The fistula also showed a similar histology Figure  The postSynchronous adenocarcinoma of the rectosigmoid and the perianum is exceptionally rare and we could only find 6 previously reported cases -10. VeryThe presence of perianal malignancy of mucinous histology may have several aetiologies. One possibility is malignant degeneration within a long-standing anal fistula and this, although uncommon, has been well reported. The histology of such cases tends to be either squamous or mucinous, with Rosser first describing in 1931 the 3 basic criteria for definitional purposes which determine that a fistula has undergone malignant transformation. For thiet al.[Viable tumour cells which represent clones capable of transplantation and proliferation have been retrieved from the lumen of the large intestine distal to established rectal tumours. In thiset al. where tuet al. where thThe management of this patient is unclear since too few cases have been reported to provide any evidence base for therapy. In our case, we performed a sphincter saving procedure as opposed to that of Hyman and Kida who recommended an extended abdominoperineal resection The r\u00f4leNS: Assisted in the photography of the case and in the literature searchPP: Assessed the histology and the photomicrographsAC: Assisted in the format and design of the paperAPZ: Assisted in the literature search and the writing of the articleAll authors read and approved the final manuscript."}
+{"text": "This article reports measurements of household levels of gamma and cosmic rays at the addresses of children with cancer at the time of diagnosis and six months before, and of similar data at the addresses of control children. There is no indication of increased risk with increasing dose rates either in matched or unmatched analyses, with or without adjustment for deprivation. Sub-division by diagnostic group did not reveal any association with any specific types of malignancy. Studies of the relationship between household gamma rays and radon concentration show no evidence of any interactions.British Journal of Cancer (2002) 86, 1727\u20131731. doi:10.1038/sj.bjc.6600277www.bjcancer.com\u00a9 2002 Cancer Research UK To address this question separate household measurements were made of radon gas concentration and of penetrating external radiation . Whilst there was special interest in the biological effects of high linear energy transfer (LET) irradiation from radon and its short term decay products  variation in dose rate . The UK The UKCCS was designed as a population-based matched case\u2013control study covering the whole of Great Britain. Ten regional centres  Family Health Services Authority  or Scottish Health Board of residence. Children were eligible if they were born in Great Britain, had no prior malignancy and were not in local authority care. Subjects were ineligible if they themselves or their parents had lived outside Great Britain for the three months leading up to diagnosis. Eligible controls who declined to participate were replaced until two controls were enrolled .A face-to-face interview was conducted with each child's parents, covering social, occupational and medical histories of the child and parents. A full residential history for the child was also collected and all addresses lived in by the child for 6 months or more were targeted for measurement. A written request to participate was then made to each residence. Following agreement, two radon and two thermoluminescent dosimeters (TLD) or \u2018gamma\u2019 detectors were sent to each household with instructions to place one of each in the main bedroom and in the main living area. After 6 months had elapsed a letter was sent recalling the detectors, which were returned to the NRPB for processing and measurement of the cumulative exposure. This study reports on the results of the address of the case at diagnosis, that is, those who had lived at that address for at least the previous 6 months.The system used for passive environmental photon monitoring consisted of a modification of a standard body TLD used for personal photon monitoring and the assessment of dose is based on two 30% LiF;Mg;Ti detector elements. These detectors respond to all types of penetrating ionising radiation including terrestrial gamma rays and secondary particles resulting from cosmic rays.The results are expressed as absorbed dose to air. Annual estimates are in micro gray (\u03bcGy) per year and hourly dose rates in nano gray (nGy) per hour. The results given in this study include the dose from cosmic rays; this varies with height above sea level, but differences within the UK are small. The mean has been estimated to be 32 nGy per hour  or 280\u2009\u03bcThe main problems encountered with the detectors were occasional damaged detectors, faulty readouts, and differences between the recorded doses on the two detectors of greater than 20%. In all these cases the result was excluded from the analysis. It is estimated that this occurred for between 5 and 10% of measurements. Where the readings of the two detectors differed by less than 20%, the mean value was used. Doses accumulated away from the household of measurement were allowed for by subtracting 2.3\u2009\u03bcGy for each day in storage, and 0.9\u2009\u03bcGy for each day in transit.Detectors were intended to be in place for 6 months but the period over which measurements were actually made varied. The analyses were restricted to the measurements of the doses that had accumulated with the detectors in place within a household for both living-room and bedroom for between 5 and 7 months. Over 99% of all detectors met this requirement.An area-based index of deprivation for the child's residence at the time of diagnosis, and 6 months before, based on an index used in a previous study of childhood leukaemia . The indAll statistical analyses were performed using Stata version 6 . All estThe parents of 3838 children with cancer and 7629 children without cancer were interviewed, representing 87% of eligible cases and 64% of eligible controls. Following interview, measurements were obtained from the home at diagnosis of 2165 cases and 5086 controls. Nearly all (97%) estimates were based on readings obtained from both the bedroom and the living room.\u22121) to a maximum of 2027\u2009\u03bcGy\u2009y\u22121.The case household participation for TLD \u2018gamma\u2019 detection was virtually identical to that for radon measurements. Thus all the issues that arose from the differential response rates of the case and control families and from the overall response rates are similar to those discussed in the accompanying radon paper . Table 1Table 1Table 2land See . This isP=0.15)Studies of dose rate by measurement year and months by region showed little variation over the period of the study (results not shown). Table 3\u22123 is shown in Table 5The relationships between TLD-derived doses divided into thirds  and radon concentrations split into the five predefined levels  of 0\u201324, 25\u201349, 50\u201399, 100\u2013199 and 200+ BqmTo our knowledge this is the first case\u2013control study directly measuring domestic gamma ray and cosmic ray levels and relating this to the risk of all childhood cancers. The results relate to the households of the affected children at the time of diagnosis, and are limited to children who had lived at the address for a minimum period of 6 months, the control households having the same limitations. The study is limited by accuracy of the TLD measurements, including inherent detector variability, storage, transport, householder compliance and the assumption of constant cosmic ray dose rate irrespective of altitude of the household. Some indication of combined uncertainties can be obtained from Cosmic ray dose rates increase systematically with increasing altitude. However, most of the population live at relatively low altitudes (see below), so it is reasonable to assume the variation in dose rates seen in the analysis is mostly due to terrestrial gamma rays.The geographical trends in gamma-ray dose rate results by Region that might be expected from knowledge of geological variations are obscured by variation in population density. For example: studies of Terrestrial gamma rays have been estimated to contribute about 30% of the total natural annual low-LET dose to bone marrow, most of the remainder being due to cosmic rays (\u223c40%) and natural internal radio nuclides with the body (\u223c30%) , then ab\u22121 is very similar to that found in the NRPB material  positive trend. That one such trend should be observed when six groups are examined is hardly surprising. When the estimated mean cosmic ray dose rate is taken into account, the mean household terrestrial gamma-ray dose rate of 62.1 nGy hThe increase in measured household exposure with increase in the deprivation index necessitated adjustment for socio-economic factors, but this makes no significant difference to the results. With the exception of the highest measured gamma-ray dose rate and radon concentration there was no evidence of any association between the gamma-ray and the results of the radon analysis. The number of households for which the highest levels of both were recorded (8) was, however, very small.In conclusion, in line with standard risk estimates, the findings from this study are broadly reassuring.RA Cartwright, G Law, E Roman, E Gilman, OB Eden, M Mott , K Muir, D Goodhead, G Kendall.KK Cheng, Central Region; N Day, East Anglia Region; RA Cartwright, A Craft, North East Region; JM Birch, OB Eden, North West Region; PA McKinney, Scotland; J Peto, South East Region; V Beral, E Roman, South Midlands Region; P Elwood, South Wales Region; FE Alexander, South West Region; CED Chilvers, Trent Region; R Doll, Epidemiological Studies Unit, University of Oxford; GM Taylor, Immunogenetics Laboratory, University of Manchester, Manchester; M Greaves, Leukaemia Research Fund Centre, Institute of Cancer Research; DT Goodhead, Medical Research Council, Radiation and Genome Stability Unit, Harwell; FA Fry, National Radiological Protection Board; G Adams, UK Co-ordinating Committee for Cancer Research.KK Cheng, E Gilman, Central Region; N Day, J Skinner, D Williams, East Anglia Region; RA Cartwright, A Craft, North East Region; JM Birch, OB Eden, North West Region; PA McKinney, Scotland; J Deacon, J Peto, South East Region; V Beral, E Roman, South Midlands Region; P Elwood, South Wales Region; FE Alexander, M Mott, South West Region; CED Chilvers, K Muir, Trent Region.RA Cartwright, G Law, J Simpson, E Roman.Br J Cancer (2000) 82: 1073\u20131102A complete list of investigators is given in: The United Kingdom Childhood Cancer Study: objectives, materials, and methods."}
+{"text": "Biomedical research in the post-genome era is intensely data-driven and increasingly more integrative as new technologies are introduced, such as next- or third-generation sequencing, mass spectrometry, and imaging to identify novel biological insights. The volume and complexity of biomedical data is increasing exponentially as faster high-throughput machines are introduced. As a result, many research institutes, biotech companies, pharmaceutical companies, and computational labs are considering cloud computing as a cost-effective alternative to process and store this vast amount of data. Efforts in next-generation sequencing (NGS) There are multiple cloud providers, both commercial and open source, including Amazon Web Services (AWS), Rackspace, GoGrid, Nimbus, and Eucalyptus, each contributing to the popularity and globalization of cloud computing. For the purposes of this guide, we focus on the use of AWS as the cloud computing platform and adopt the definition of Vaquero, who states that the cloud is \u201ca large pool of easily usable and accessible virtualized resources . These resources can be dynamically re-configured to adjust to variable load , allowing for optimum resource utilization\u201d http://docs.amazonwebservices.com/AWSEC2/2009-11-30/GettingStartedGuide/): Elastic Compute Cloud (EC2), Elastic Block Storage (EBS), and Simple Storage Service (S3). For additional AWS products beyond the scope of this overview, we refer the reader to the AWS Web site (http://aws.amazon.com/). EC2 contains a variety of user selectable instance types that range in computing power and cost (http://aws.amazon.com/publicdatasets/). Access to all of AWS's services can be done using either a Web-based console, for beginners, or through the command line using an AWS-specific application programming interface (API), for advanced users. AWS costs are generally based either on an hourly rate or amount of data transferred or stored or other services used , networking, and operating system, and is an example of \u201cinfrastructure as a service\u201d (IaaS). IaaS is popular with computational biologists because it offers more flexibility for designing projects ad hoc. The majority of computational projects will make use of three AWS products (see \u201cGet Started with EC2\u201d at and cost . An instces used .http://media.amazonwebservices.com/pdf/AWS_Security_Whitepaper.pdf).Before beginning to use the cloud, it is important to understand the basic best practices for cost control and data security  or Secure Copy (scp). Both the AWS Web console and command line tools provide a simple interface to generate key pairs when launching an instance. The public key is automatically installed onto an instance, and the private key can then be used on a computer that will ssh into that instance.Recently, AWS introduced Identity and Access Management (IAM) to offer greater control and management of multiple users. Each user has their own set of security credentials to access cloud resources, eliminating the need to share login information and keys for the master AWS account owner. This is important because the master account contains the personal billing information, which, for obvious reasons, should not be accessible to all users. IAM can restrict services based on specific users or group policies. For example, it is possible to restrict a user to a specific S3 bucket between 9 A.M. and 5 P.M. from a specific IP address.A security group defines a set of rules that govern how traffic (data or communication) reaches the AWS instance. By default, the security group restricts all inbound traffic, allows all outbound traffic, and allows other instances within the group to communicate. These rules can be completely customized; for example, it is possible to restrict access to a specific IP address on a specific port address and not allow that instance to communicate with other instances within the AWS account.http://aws.amazon.com/vpc/), encrypted file systems, and encrypted data volumes that may be used by those who have security needs beyond these basic best practices.All security keys should be replaced with new ones every 30\u201390 days. Installing regular software updates is essential to protect the operating system and third-party software from vulnerabilities. There are many additional security features such as private clouds  and also per gigabyte for persistent storage . It is ehttp://web.mit.edu/stardev/cluster/), Boto (http://code.google.com/p/boto/), Condor (http://www.cs.wisc.edu/condor/), and Hadoop (http://hadoop.apache.org/mapreduce/) are making cluster creation, termination, and job queuing more automated and accessible to bioinformatics specialists with perhaps only a limited understanding of systems administration and architecture. Within AWS, there is also the option to use Elastic Map Reduce (AWS's implementation of Hadoop) or high performance computing instances (http://aws.amazon.com/ec2/hpc-applications/) for an additional cost per instance. Note that an important consideration for a large cluster is to shut down instances when the number of CPUs is greater than the number of jobs. This will reduce the amount of money spent on idle CPU time, which can be substantial for hundreds or thousands of CPUs A large-scale computing environment that scales up or down in response to computational demand is the most commonly perceived use of cloud computing because it takes full advantage of rapid replication and linear scaling of cheap commodity compute cycles. However, it is important to remember that the cloud does not \u201cmagically\u201d enable programs to run more efficiently or in parallel . Instead, it requires an understanding of how to connect multiple instances together to form a cluster and knowledge of how to divide a computational task into sub-components that can run simultaneously. Until recently, cluster creation was onerous, requiring substantial amounts of customized solutions handled best by an expert in systems administration and computer science. Fortunately, new advances in open source cluster management software such as StarCluster  and a parallel computing technique called MapReduce To put the previous concepts into practice, we will walk through the analysis of a large amount of NGS data. Specifically, we detail the creation of a pipeline to process an entire human genome's worth of NGS reads using a short read mapping algorithm. We use the \u223c4 billion paired 35-base reads sequenced from a Yoruba African male http://www.gnu.org/software/wget/). Next, we format and mount the EBS volume and create three directories for testing the mapping data\u2014small , medium , and all (entire genome). Then, we upload the African genome and the smaller testing files into the appropriate directories. Following the MAQ instructions (http://maq.sourceforge.net/maq-man.shtml) and executing the mapping and assembly commands, we learn that it takes 2 hours to analyze one pair of read files from the \u201csmall\u201d directory on the EBS volume. However, only one of the possible eight CPUs on the extra-large instance is in use because we only issued one MAQ map command. While we could manually launch eight MAQ commands, a better approach would be to use cluster management software to automatically take advantage of all eight CPUs and include additional instances.The NGS mapping example begins by prototyping and testing the whole-genome mapping pipeline . At thishttp://gridscheduler.sourceforge.net/index.html) to manage batch queuing across distributed systems, along with OpenMPI to manage job distribution and instance communication. The cluster is composed of a master instance, which is responsible for managing a larger set of worker instances. In this example, each worker instance is able to process eight jobs concurrently and will contain the necessary software to run the analysis. In order to get StarCluster running, we need to work through a few steps that involve configuring the StarCluster instance type, setting the proper security, and installing StarCluster on your local computer to remotely create and terminate a cluster.Next, we will introduce the use of StarCluster to create and manage a small test cluster of two instances. StarCluster is customized for use on AWS and uses the open source version of Sun Grid Engine (http://docs.amazonwebservices.com/AWSEC2/2011-02-28/UserGuide/), we record the AMI ID\u2014we will use this later in the StarCluster configuration file. We take a snapshot of the EBS volume to back it up in S3. We will use the snapshot ID later in the StarCluster configuration file to allow each instance access to the data.First, we will configure the StarCluster base instance type or Amazon Machine Image (AMI) with our required software. An AMI packages the operating system, installed programs, and user settings into a binary file that can be launched to exactly replicate an environment. Amazon creates a unique private ID (default) or public ID for each AMI to launch identical instances. We locate the StarCluster AMI through the AWS console under Community AMIs , launch it, and attach the previously created EBS volume containing the NGS data to the running instance. Then we install MAQ and any additional processing scripts as before. Next, we need to bundle the instance into an AMI in order to allow StarCluster to launch multiple identical instances. After bundling the AMI . The installation package includes the necessary scripts and configuration files to manage a cluster. The configuration file contains the various parameters to specify the cluster creation such as AMI ID, number of instances to launch, AWS account credentials, instance type, key pair, EBS snapshot ID containing the NGS data, and security group.Third, we install StarCluster on our local computer following their documentation . When the jobs are finished, we can save the results on the EBS volume and shut down the cluster.At this point in our case study, we are interested in testing the scalability of the NGS mapping pipeline by creating a small cluster and confirming that the environment is functioning as expected . Using SWe now expand our case study to the next level of usage, one that best exemplifies the most common conception of cloud computing: a virtually unlimited computational environment, which an analysis task will harness for rapid completion. However, getting to this point requires successful prototyping of an application, namely the prior two stages outlined above, on the cloud and ensuring that your application and pipeline can run on two or more inter-communicating instances.Returning to our case study, we want to create the environment to process the entire human genome . The prehttp://aws.amazon.com/importexport/). More information about cloud computing, detailed cost analysis, and security can be found in references In this overview to biomedical computing in the cloud, we discussed two primary ways to use the cloud (a single instance or cluster), provided a detailed example using NGS mapping, and highlighted the associated costs. While many users new to the cloud may assume that entry is as straightforward as uploading an application and selecting an instance type and storage options, we illustrated that there is substantial up-front effort required before an application can make full use of the cloud's vast resources. Our intention was to provide a set of best practices and to illustrate how those apply to a typical application pipeline for biomedical informatics, but also general enough for extrapolation to other types of computational problems. Our mapping example was intended to illustrate how to develop a scalable project and not to compare and contrast alignment algorithms for read mapping and genome assembly. Indeed, with a newer aligner such as Bowtie"}
+{"text": "BCAM promoter and repressed transcription. Thus, these data indicate that BCAM is a suppressive oncoprotein, and that FBI1/Akirin2 is involved in tumorigenicity and metastasis of hepatoma through the downregulation of suppressive oncogenes.Basal cell adhesion molecule (BCAM), known to be a splicing variant of Lutheran glycoprotein (LU), is an immunoglobulin superfamily membrane protein that acts as a laminin \u03b15 receptor. The high affinity of BCAM/LU for laminin \u03b15 is thought to contribute to the pathogenesis of sickle red blood cells and to various developmental processes. However, the function of BCAM in carcinogenesis is poorly understood. Based on microarray expression analysis, we found that BCAM was one of the target genes of the oncogenic 14-3-3\u03b2-FBI1/Akirin2 complex, which acts as a transcriptional repressor and suppresses MAPK phosphatase-1 gene expression. To elucidate the detailed function of BCAM in malignant tumors, we established BCAM-expressing hepatoma K2 cells. These cells lost the malignant characteristics of parental cells, such as anchorage-independent growth, migration, invasion, and tumorigenicity. Moreover, luciferase reporter assays and chromatin immunoprecipitation analysis revealed that the 14-3-3\u03b2-FBI1/Akirin2 complex bound to the  Furthermore, overexpression of 14-3-3\u03b2 in NIH3T3 cells confers tumorigenicity in nude mice via activation of the mitogen-activated protein kinase (MAPK) cascade. Mutations in ras family oncogenes and suppressive oncogenes such as p53 and Rb are not detected in K2 cells 14-3-3 proteins regulate many cellular processes, including the cell cycle, metabolism, signal transduction, malignant transformation, and apoptosis. We previously reported that 14-3-3\u03b2 is implicated in the positive regulation of cell cycle progression and tumorigenesis mitogen-activated protein kinase phosphatase 1 (MKP-1) transcription, resulting in the promotion of tumorigenicity and metastasis basal cell adhesion molecule (BCAM) gene as one of the genes downregulated by FBI1/Akirin2.To further analyze the oncogenic function of 14-3-3\u03b2, we screened for 14-3-3\u03b2 binding partners by the yeast two-hybrid system using 14-3-3\u03b2 as a bait BCAM, known to be a splicing variant of Lutheran glycoprotein (LU), is an immunoglobin superfamily membrane protein and acts as a laminin \u03b15 receptor 1-induced rat hepatoma were cultivated with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS). Cells were maintained at 37\u00b0C in a humidified atmosphere of 5% CO2 in air (2).K2 cells established from AFB32P-labelled probes as previously described NotI-BamHI fragment of full-length BCAM, and the 0.7-kb NotI-XhoI fragment of FBI1/Akirin2.Total RNAs were isolated from various cell lines by the acidic guanidine thiocyanate/phenol/chloroform method Total RNAs were prepared from various cell lines as described above. Reverse transcription (RT) reactions were performed using total RNAs and Moloney murine leukemia virus reverse transcriptase, according to the manufacturer's instructions (Invitrogen). The RT reaction mixtures were subjected to PCR amplification using specific primers as follows: mmp9-forward, 5\u2032-TGG CTC TAG GCT ACA GCT TTG CTG C-3\u2032, mmp9-reverse, 5\u2032-CGA AGG AGT CAT CGA TCA CGT GTC G-3\u2032; cyclinD1-forward, 5\u2032-CTC CAT GTT CCA AAA CCA TTC C-3\u2032, cyclinD1-reverse, 5\u2032-GGG CAA CCT TCC CAA TAA ATA C-3\u2032; FBI1-forward, 5\u2032-TGG ATT TCG ACC CAC TGC TTA GC-3\u2032, FBI1-reverse, 5\u2032-GAT CAT CCC AAC CTG CCT TAG AG-3\u2032; mkp1-forward, 5\u2032-CCA TGG TGA TGG AGG TGG GCA TCC T-3\u2032, mkp1-reverse, 5\u2032-CCT TCA GCA GCT CGG AGA GGT TGT G-3\u2032.BCAM expression vector was constructed by the insertion of a BCAM open reading fragment into the pcDNA3 expression vector. K2 cells were transfected with BCAM expression vector or empty vector using Lipofectamine regent (Invitrogen) according to the manufacturer's instructions. After 2 weeks of selection with 1 mg/ml G418, resistant clones were expanded and analyzed for the expression level of BCAM by western blotting.Western blotting was performed as described previously 4) were plated in 24-well plates containing DMEM supplemented with 5% FCS and cultivated for various times. The cell number was counted using a hemocytometer. For soft agar assays, cells (1\u00d7103) were suspended in 0.3% agar medium containing 5% FCS and layered on a 0.5% agar-coated 35-mm dish and cultivated for 2 weeks. The colonies formed were stained with 0.25% 1-p-iodophenyl-p-nitrophenyl-5-phenyltetrazolium chloride  for 12 h, and the number of colonies (>0.2 mm in diameter) was counted Cells . Cells were allowed to migrate for 12 and 24 h at 37\u00b0C, and the same fields were photographed again. Scratched areas were measured with ImageJ software , and recovered surface areas over 6, 12, and 24 h were calculated compared to control cells. Photographs of the wounded areas were taken at different times after wounding the monolayer.4 cells were plated on the top chamber with a Matrigel-coated membrane   in medium with 0.1% FCS, and medium supplemented with 10% FCS was used as a chemoattractant in the lower chamber. After 48 h, cells on the lower surface of the membrane were stained with crystal violet and counted.For the invasion assay, 6\u00d7104/200 \u00b5l of phosphate-buffered saline/flank) were inoculated subcutaneously into 6-week-old SCID mice . Tumor volume was calculated according to the formula V\u200a=\u200aa\u00d7b2\u00d70.52, where a is the largest diameter and b the smallest diameter of the tumor. The average volumes of the tumors were represented by the mean tumor value \u00b1 SE (n\u200a=\u200a5).Cells .5\u2032-CGT CCT AAA ACT CAA CAA TAG CCA AAG-3\u2032, and 3\u2032-primer for -1942Luc, 5\u2032-ATT CCC TGC AGT GGC GGC AG-3\u2032. For each transfection, 1\u00d7104 cells/96-well plate were transfected with 30 ng of each pGL4.10 luciferase reporter vector, 3 ng of Renilla luciferase expression vector pGL4.74, and a total of 10 ng of pcDNA3 empty vector and pcDNA3-FBI1 in various combinations. Twenty-four hours after transfections, cells were lysed with the lysis buffer of the Dual-Luciferase Reporter Assay System (Promega), and luciferase activities were determined according to the manufacturer's instructions using an ARVO Light (Perkin Elmer). Reporter gene activities were normalized using Renilla luciferase activity as an internal control.Luciferase reporter gene plasmids driven by the rat BCAM promoter were constructed as follows. Rat BCAM promoters were amplified by PCR using K2 cell genomic DNA and introduced into pGL4.10 vector (Promega) and designated as -1942Luc. The plasmids -1281Luc, -705Luc, -208Luc, -61Luc, and -4Luc were generated by cloning the region of -1942Luc extending from \u22121281, \u2212705, \u2212208, \u221261, or \u22124 to +10. The primer sequences used were as follows: 5\u2032-primer for -1942Luc,  GCC AGC AGG ACT GCG AGC AAC AG -3\u2032).Cells were cross-linked with 1% formaldehyde for 10 min. The nuclear fraction was isolated and sonicated to shear genomic chromatin. Chromatin extracts were precleaned with protein G-Sepharose/salmon sperm DNA beads at 4\u00b0C for 3 h and then incubated with anti-FBI1 and anti-14-3-3\u03b2 antibodies at 4\u00b0C overnight. Protein G-Sepharose/salmon sperm DNA beads were then added to the mixture for 3 h, and immunoprecipitated DNA-protein complexes were isolated from beads after several washing steps. Reversal of the cross-linking of chromatin was performed at 65\u00b0C for 6 h, and proteinase K digestion was allowed to proceed for 1 h at 55\u00b0C. DNA was extracted by the phenol/chloroform method. PCR was carried out on purified DNA using primers corresponding to -309 to +49 of the BCAM promoter region  degrade extracellular matrix, and the expression level of MMPs is correlated with the metastatic ability of cancer cells observed . Also, cobserved .3, whereas BCAMS1 and BCAMS2 cells formed smaller tumors with mean volumes of 2.41 and 0.87 cm3, respectively , casein kinase II (CKII), and PKA MKP-1 transcription via binding with the GC box in the promoter region BCAM gene expression, K2 cells were transfected with various amounts of Flag-FBI1 expression vector, and after 24 h expression levels of BCAM protein were analyzed. BCAM protein expression was dose-dependently downregulated by the ectopic expression of FBI1, up to a maximum of 0.2 \u00b5g of FBI1, at which point an approximately 50% reduction was observed Click here for additional data file.Figure S2Expression levels of FBI1/Akirin2 mRNA in various cell lines. Total RNAs were extracted from various cell lines, including rat hepatocarcinoma cells , rat glioblastoma cells (C6), embryonic carcinoma cells (P19), human hepatocarcinoma cells (HepG2), cervical carcinoma cells (HeLa), and normal prostate tissue analyzed by northern blotting using 32P-labeled BCAM cDNA as a probe.(TIF)Click here for additional data file."}
+{"text": "The temperature dependence of ionic conductivity from 303\u2009K to 353\u2009K exhibits Arrhenius plot behaviour. The thermal stability of the polymer electrolyte system is studied by using thermogravimetric analysis (TGA) while the structural and morphological properties of the polymer electrolyte is studied by using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction analysis (XRD), respectively.Gel polymer electrolytes (GPEs) are developed using poly(1-vinylpyrrolidone-co-vinyl acetate) [P(VP-co-VAc)] as the host polymer, lithium bis(trifluoromethane) sulfonimide [LiTFSI] as the lithium salt and ionic liquid, and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide [EMImTFSI] by using solution casting technique. The effect of ionic liquid on ionic conductivity is studied and the optimum ionic conductivity at room temperature is found to be 2.14\u2009\u00d7\u200910 Polymer electrolytes are gaining great interest due to their high ionic conductivity and wide electrochemical windows. They also possessed interesting properties such as thin-film forming ability, flexibility, and transparency \u20133. There3SO3\u2212) and BF4\u2212. The obtained films are freestanding and flexible with ionic conductivities ranging from 1.1 to 5.8\u2009mS\u2009cm\u22121 at room temperature /lithium bis(trifluoromethane) sulfonimide [LiTFSI] polymer electrolytes system is investigated using ac impedance spectroscopy at different temperatures. In addition, the thermal stability, structural and morphological properties of the polymer electrolyte system is analysed using thermogravimetric analysis (TGA), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction analysis (XRD), respectively.4\u2009g\u2009mol\u22121 (Sigma-Aldrich), lithium salt LiTFSI with 97% purity (Sigma-Aldrich), and ionic liquid EMImTFSI with 98% purity (Basionics) which were used without further purification in this study. The gel polymer electrolytes were prepared by using solution casting technique using distilled water as a solvent.The materials used in this research work are P(VP-co-VAc) with a molecular weight of ~5.0 \u00d7 10The casted thin films were then characterized on its electrical, thermal, structural, and morphological properties.Electrical Properties: Ac Impedance Spectroscopy. HIOKI 3532-50 LCR HiTESTER was used to measure the impedance of the samples. The thickness of the samples was measured using a micrometer screw gauge. The impedance measurement for each sample was done in the frequency range from 50\u2009Hz to 5\u2009MHz at room temperature to 80\u00b0C. The samples were placed on the sample holder in between two stainless steel electrodes with diameter of 4.9087\u2009cm2. Ionic conductivity, \u03c3, was determined from the following equation:L is the sample thickness (cm), A is the cross-sectional area of electrode and sample contact (cm2), and Rb is the bulk resistance (\u03a9) of the sample.Thermal Properties: Thermogravimetric Analysis (TGA). TA TGA Q500 was used to study the thermal stability of the samples. The samples were cut and weighed ~2.0\u2009mg. Then, the samples were put into the sample holder and heated under nitrogen atmosphere from 25\u00b0C to 505\u00b0C at a heating rate of 10\u00b0C min\u22121.Structural Properties: Fourier Transform Infrared (FTIR) Spectroscopy. The FTIR studies were carried out by using Thermo Scientific Nicolet iS10 spectrophotometer at room temperature in the wave region between 4000\u2009cm\u22121 and 600\u2009cm\u22121 with a resolution of 4\u2009cm\u22121. Morphological Properties: X-Ray Diffraction Analysis (XRD). The XRD study was conducted by using D5000 diffractometer to determine the polymer electrolytes films which are either crystalline or amorphous in nature based on the characteristic pattern obtained from X-ray diffractogram. Coherent length study is done to justify that the crystallinity of P(VP-co-PVAc)\u2014LiTFSI\u2014EMImTFSI system is reduced. The coherent length, C, is calculated from the Scherrer equation:\u03bb is the X-ray wavelength; \u03b8b is the glancing angle; and \u03942\u03b8b is the full width at half maximum (FWHM).\u221210\u2009S\u2009cm\u22121. The maximum ionic conductivity 2.14 \u00d7 10\u22126\u2009S\u2009cm\u22121 is achieved with addition of 25\u2009wt% of EMImTFSI to the polymer electrolyte system.\u03c3) against 1000/T (in Kelvin) are shown in Ea, for each system can be determined from the slope of its Arrhenius plot. Ea for the polymer electrolytes.Plots of linear variation of log ionic conductivity . The addition of LiTFSI into the polymer system shows that the diffraction peaks corresponding to the salt are disappearing, suggesting that LiTFSI is fully complexed with P(VP-co-VAc).The diffractograms of P(VP-co-VAc)-LiTFSI with different wt% of EMImTFSI are shown in Figures + and TFSI\u2212 ions in the ionic liquid are mobile, thus increasing the number of charge carriers available for conduction. Besides, with the low viscosity of EMImTFSI, the crystallinity of the polymer matrix can be reduced, hence increasing the mobility of the charge carriers [Incorporation of ionic liquid, EMImTFSI, to the polymer electrolyte gives plasticizing effect which increases the flexibility of the polymer backbone and enhances the amorphous phase of the system. In addition, both Imcarriers . The inccarriers .The increase in ionic conductivity as a function of temperature is due to the decrease in viscosity of the polymer systems that increases the chain flexibility . This ph\u03c3o is the preexponential factor; Ea is the activation energy; k is the Boltzmann constant; and T is the absolute temperature.From Ea, can be defined as the energy needed to overcome the reorganization and reformation of Li+ to relocate to neighbouring sites. From + to move faster inside the polymer network. Upon addition of EMImTFSI, the interaction between the polymer system and lithium ions is disturbed causing less energy which is needed for the Li+ hopping process [Activation energy,  process .There are three degradation stages which can be observed from Figures For the second degradation stage, it is shown that the pure polymer is starting to degrade at 295\u00b0C with 24% weight loss. In EMIm 0, the second degradation stage occurred at 275\u00b0C with 30% weight loss while in EMIm 10 and EMIm 25 it occurred at temperature 265\u00b0C with weight loss of 30% and 275\u00b0C with 25% weight loss, respectively. This stage corresponds to the degradation of poly(vinyl acetate) (PVAc) due to the total loss of acetic acid as shown in 3 [ cis-trans isomerisation, aromatization, and cross-linking [The third stage of degradation happened due to the degradation of vinylpyrrolidone  and viny3 . The pol-linking . These r-linking . For theAs EMImTFSI is nonflammable and nonvolatile, these properties contribute to the increase in heat resistivity of the samples resulting in EMIm 25 to have less weight loss compared to pure P(VP-co-VAc) with residual mass of 12.44% and 9.66%, respectively. Therefore, it can be concluded that samples with addition of EMImTFSI are more thermally stable than pure P(VP-co-VAc).\u22121 and 2954\u2009cm\u22121 which is assigned to O\u2013H stretching and C\u2013H stretching of P(VP-co-VAc), respectively, is observed to be shifted to 3405\u2009cm\u22121 and 2958\u2009cm\u22121, respectively, in EMIm 0. It is also observed that the peaks of C=O stretching of both PVAc and PVP at 1731\u2009cm\u22121 and 1655\u2009cm\u22121 have shifted to 1732\u2009cm\u22121 and 1651\u2009cm\u22121, respectively, in the PE system. These shifting might be due to the interaction between free Li+ with the ester C=O in PVAc and amide C=O in PVP. Besides, the peak shift of C=O in PVP (\u0394f = 4\u2009cm\u22121) is more intensive as compared with PVAc (\u0394f = 1\u2009cm\u22121); therefore it is claimed that interaction of Li+ with C=O of PVP is more favourable [vourable .\u22121 ), 781\u2009cm\u22121  + vibrational (S\u2013N)), 1193\u2009cm\u22121 s), and 1356\u2009cm\u22121 a) are shifted to 739\u2009cm\u22121, 788\u2009cm\u22121, 1182\u2009cm\u22121, and 1350\u2009cm\u22121 in the EMIm 0, respectively. These shifting can be attributed by the presence of free TFSI\u2212 in the EMIm 0. Two characteristic peaks of LiTFSI \u03b4CH3 at 2979\u2009cm\u22121 and 2876\u2009cm\u22121 have disappeared after interacting with P(VP-co-VAc) and C\u2013H stretching peaks of P(VP-co-VAc) at 2954\u2009cm\u22121 shifted to 2958\u2009cm\u22121 in EMIm 0. Besides, the characteristic peak at 1423\u2009cm\u22121 in P(VP-co-VAc) has shifted to 1441\u2009cm\u22121 in EMIm 0. Characteristic peak at 1021\u2009cm\u22121 in P(VP-co-VAc) has also shifted to wavenumber 1022\u2009cm\u22121 and becomes the shoulder peak to LiTFSI characteristic peak that shifted from wavenumber 1065\u2009cm\u22121 in LiTFSI to 1055\u2009cm\u22121 in EMIm 0.From the figure, we can also observe the shifting in the LiTFSI characteristic peaks. The peaks of LiTFSI at 740\u2009cm\u22121 in the EMIm 0 is observed to be shifted to 3390\u2009cm\u22121 and 3403\u2009cm\u22121 in EMIm 10 and EMIm 25 systems, respectively. The C=O stretching of PVAc and PVP in the EMIm 0 system is also shifted from 1732\u2009cm\u22121 and 1651\u2009cm\u22121, respectively, to 1731\u2009cm\u22121 and 1652\u2009cm\u22121 in EMIm 10 and to 1731\u2009cm\u22121 and 1654\u2009cm\u22121 in EMIm 25.\u22121 which shifted to a shoulder peak in EMIm 10 and disappears in EMIm 25.Upon adding EMImTFSI into the polymer electrolyte system, there are no significant changes occurring to LiTFSI characteristic peaks except for the peak of wavenumber 1022\u2009cm\u22121 and 1573 cm\u22121 have disappeared after being added to EMIm 0 system as shown in Figures \u22121 and 1168\u2009cm\u22121 in \u22121 in \u22121 in It can also be observed from As shown in Based on Figures In general, coherent length defines the crystallite size. As the diffraction peak width is longer, the crystallite size is shorter and thus brings higher amorphous phase and lower crystallinity of the polymer system . Based o\u22126\u2009S\u2009cm\u22121, which suggested that addition of ionic liquid in the polymer electrolytes systems increases the ionic conductivity values. The temperature dependence ionic conductivity study shows that all samples exhibit Arrhenius type which is favourable for Li+ ion hopping mechanism at higher temperature as proved by the increasing in ionic conductivity values with temperature. In addition, the addition of EMImTFSI also enhanced the thermal stability of the polymer electrolytes systems. The FTIR and XRD studies showed the complexation of the materials inside the polymer electrolytes systems which revealed that with the incorporation of EMImTFSI the amorphous fraction is increased, resulting in the increasing of the ionic conductivity values.In this work, GPEs are developed by incorporating different amount of EMImTFSI into the polymer electrolytes system. Among all the polymer electrolytes systems, EMIm 25 has the highest ionic conductivity, 2.14 \u00d7 10"}
+{"text": "Multiple independent genomic profiling efforts have recently identified clinically and molecularly distinct subgroups of ependymoma arising from all three anatomic compartments of the central nervous system . These advances motivated a consensus meeting to discuss: (1) the utility of current histologic grading criteria, (2) the integration of molecular-based stratification schemes in future clinical trials for patients with ependymoma and (3) current therapy in the context of molecular subgroups. Discussion at the meeting generated a series of consensus statements and recommendations from the attendees, which comment on the prognostic evaluation and treatment decisions of patients with intracranial ependymoma (WHO Grade II/III) based on the knowledge of its molecular subgroups. The major consensus among attendees was reached that treatment decisions for ependymoma  should not be based on grading (II vs III). Supratentorial and posterior fossa ependymomas are distinct diseases, although the impact on therapy is still evolving. Molecular subgrouping should be part of all clinical trials henceforth. Ependymoma is a histologically defined intrinsic tumor that involves the three major anatomic compartments  of the central nervous system and affects both children and adults. The current standard of care therapy for patients with intracranial ependymoma remains surgical resection combined with radiotherapy. The survival benefit of chemotherapy for ependymoma and the prognostic ability of histopathological grading criteria to risk-stratify patients are still both inconclusive and contentious. No molecular or tumor-specific immunohistochemical markers are in routine current clinical use for ependymoma. Recent advances in the biological characterization of ependymal tumors have demonstrated the existence of nine clinically, demographically, and molecularly distinct entities, with three occurring in each anatomic compartment. These findings offer new opportunities to create a precise, reliable, and objective platform for stratification of ependymoma patients, and the potential for altering therapeutic decisions based on molecular features. Herein, we discuss the current consensus on the molecular subgroups of intracranial ependymoma (WHO Grade II/III) in children and adults, as well as recommendations for integration into future clinical trial designs. These discussions and recommendations were made by a collection of neuro-oncologists, neurosurgeons, neuro-pathologists, radiation oncologists, and basic scientists, meeting at the global ependymoma consensus conference   would dramatically benefit from a matched control to correct for aberrations inherent to the germline. As such, an agreement among most attendees was established that submission of blood samples should also be mandatory for enrollment in a clinical trial. It should be recognized that arguments were made against the mandate of fresh-frozen tissue, owing to the logistical issues of collection, storage, and submission, particularly in small community centers. Additionally, there were ethical concerns regarding the mandated submission of blood. Attendees recognized that efforts would need to be established to create standard operating procedures in smaller centers to enable reliable collection and submission of frozen tissue. Many of those agreeing on a mandate of frozen tissue and blood argued that given the rapid developments in the field of molecular genetics, with the emergence of increasingly powerful analytical devices and computational tools, the time is now to collect tissue specimens in combination with high-quality clinical data. This would enable the use of such advances to improve the care of future ependymoma patients.Clinical management of intracranial ependymomas (WHO Grade II/III) is challenging and the optimal treatment strategy is contentious. Intracranial ependymoma, particularly before administration of any therapy, demonstrates predominantly locally invasive growth patterns and has only very low metastatic potential. Surgery plays a primary role for local tumor control and the extent of neurosurgical resection has been the most consistent independent prognostic factor reported in the last decades , 6, 34. In addition to surgery, post-operative field radiotherapy dosed at 54\u201359.4\u00a0Gy is considered the standard of care for patients with non-disseminated ependymoma to lower the risk of local recurrence . Radiatin\u00a0=\u00a0820 cases) found that patients with either PF-EPN-A or PF-EPN-B tumors benefit from gross total resection, with the survival rates being particularly poor for sub-totally resected PF-EPN-A, even in the setting of radiation therapy [It should be emphasized that all prior studies that evaluated the therapeutic value of neurosurgical interventions and external beam radiation in posterior fossa ependymoma have not accounted for molecular subgroup affiliation and might therefore be confounded by clinical differences in response to therapy between these subgroups. Data from a current retrospective study on four independent non-overlapping cohorts of posterior fossa ependymomas  and were associated with a poor prognosis with 5-year progression-free and overall survival of 29 and 75%, respectively. Interestingly, the level of resection did not significantly affect the outcome within the ST-EPN-RELA-positive subgroup in this retrospective analysis in patient samples collected over a long period of time (>20\u00a0years). The two remaining supratentorial subgroups, ST-SE and ST-EPN-YAP1, were restricted only to adults  and predominantly to children , respectively, with both of these variants showing an excellent prognosis. As the cited studies and other available collections of single cases markedly differ regarding age distribution, therapy modalities and availability of molecular data, variations in outcome cannot be reliably linked to specific treatment approaches or molecular subgroups. It was, therefore, concluded that there was not enough evidence yet to recommend distinct treatment approaches for ST-EPN-RELA ependymoma. Molecular analyses of supratentorial ependymomas from clinically well-annotated international trial cohorts as well as from large retrospective cohorts with long-term follow-up have now been initiated. The authors expect that this approach will help to clarify questions about the clinical outcome of the molecular variants of supratentorial ependymoma and result in explicit therapy recommendations.Observation for gross totally resected supratentorial ependymomas has also been advocated based on retrospective series that were not molecularly characterized. For example, a retrospective, multicenter study comprising 92 patients  with gross totally resected and non-anaplastic supratentorial ependymal tumors did not find evidence of decreased progression-free or overall survival with the omission of external beam radiation . The 5\u20131In contrast to surgery and radiotherapy, the role of chemotherapy in the management of ependymoma remains unproven despite extensive investigation. Cohorts of pediatric or adult patients in which the role of chemotherapy was retrospectively analyzed either failed to demonstrate a survival advantage or showed substantial variation between individual patients , 13, 28.EPHB2)-driven ST ependymoma models\u2014also highly expressed in ST-EPN-RELA tumors\u2014have pinpointed 5-fluorouracil treatment as a potential cytotoxic therapy with efficacy in murine models and is currently being evaluated in early phase ependymoma clinical trials [Because of the recognition that ependymal tumors comprise molecularly distinct subtypes, with potentially distinct clinical management, the generation of subgroup-specific pre-clinical models for the development and assessment of novel therapies is required. The identification of candidate cells of origin for ependymoma has permitted the generation of novel mouse models that can be leveraged for novel therapeutic discovery and evaluation , 27, 30.l trials , 16, 38.l trials , 39. In We now recognize that ependymal tumors from different compartments of the central nervous system are biologically distinct and there are phenotypically divergent subgroups within each anatomic compartment. Future clinical trials, the development of pre-clinical model systems, and the identification and testing of subtype-specific therapeutics must accompany molecular classification to be useful to ependymoma patients and to the neuro-oncology community. The differentiation between histologically defined grade II versus grade III/anaplastic ependymomas is problematic and of limited utility for clinical decision-making, and therefore should be used with great caution outside the setting of a clinical trial. For patients with PF-EPN-A ependymoma over the age of 12\u00a0months of age, the recommended standard of care is maximal safe micro-neurosurgical removal followed by local radiotherapy, but probably does not include the routine use of chemotherapy outside the setting of a clinical trial. A subset of PF-EPN-B ependymoma patients who undergo gross total micro-neurosurgical resection are likely cured in the absence of radiotherapy, and a clinical trial to test the possibility to avoid radiotherapy in the context of complete resection for PF-EPN-B patients is indicated. The characteristics and heterogeneity between molecular subgroups of supratentorial ependymoma require additional study before specific treatment recommendations can be made. The division of an already uncommon entity (\u201cependymoma\u201d) into nine new entities will necessitate great co-operation and international collaboration with the pediatric and adult neuro-oncology community if clinical trials are to be properly and expeditiously completed."}
+{"text": "The ability to preoperatively predict postoperative complication risks is valuable for individual counseling and (post)operative planning, e.g. to select low-risk patients eligible for short stay surgery or those with higher risks requiring special attention. These risks however, are not well established in pituitary surgery.We conducted a systematic review of associations between preoperative characteristics and postoperative complications of endoscopic transsphenoidal surgery according to the PRISMA guidelines. Risk of bias was assessed through the QUIPS tool.In total 23 articles were included, containing 5491 patients (96% pituitary adenoma). There was a wide variety regarding the nature and number of risk factors, definitions, measurement and statistics employed, and overall quality of mainly retrospective studies was low. Consistent significant associations were older age for complications in general, and intraventricular extension for cerebrospinal fluid (CSF) leaks. Associations identified in some but not all studies were younger age, increased BMI, female gender, and learning curve for CSF leaks; increased tumor size for complications in general; and Rathke\u2019s cleft cysts for diabetes insipidus. Mortality (incidence rate 1%) was not addressed as a risk factor.Based on current literature, of low to medium quality, it is not possible to comprehensively quantify risk factors for complications. Nevertheless, older age and intraventricular extension were associated with increased postoperative complications. Future research should aim at prospective data collection, reporting of outcomes, and uniformity of definitions. Only then a proper risk analysis can be performed for endoscopic pituitary surgery.The online version of this article (doi:10.1007/s11102-017-0839-1) contains supplementary material, which is available to authorized users. Over the past two and a half decades, pituitary surgery has undergone major technical developments, the introduction of the endoscope perhaps being the most important one. Several systematic reviews show relatively better results in terms of gross total resection, with reduced complication rates for endoscopic surgery compared to microscopic surgery \u201310. ThesA systematic review was conducted according to a predefined protocol, which was based on the PRISMA criteria for systematic reviews  and regiA literature search was conducted on May 15 2017, with the guidance of a trained clinical librarian (J.S.). The following databases were searched: PubMed, Embase, Web of Science, Cochrane, CINAHL, Academic Search Premier and ScienceDirect. Terms included were \u2018pituitary adenoma\u2019, \u2018non-functioning adenoma\u2019, \u2018acromegaly\u2019, \u2018Cushing\u2019s disease\u2019, \u2018prolactinoma\u2019, \u2018Craniopharyngioma\u2019, \u2018Rathke\u2019s cleft cyst\u2019, \u2018complications\u2019, \u2018risk factors\u2019 and \u2018prognosis\u2019, and derivatives or synonyms of these words. The complete search strategy can be found in online supplement 1. Reference checking of included studies was performed to screen for additional studies.Inclusion criteria were: (1) articles reporting on outcomes of ETS for pituitary tumors; (2) describing an association between \u22651 preoperative characteristics and \u22651 postoperative complications; (3) published in English; (4) peer-reviewed; (5) containing original clinical data; and (6) including >10 adult patients (>18\u00a0years). Excluded studies were: (a) microscopic, endoscopic-assisted surgery, or combined microscopic and endoscopic approaches without a separate description of endoscopic results, (b) articles without a described association, and (c) articles including >10% other pathologies than pituitary adenoma.A meta-analysis appeared to be infeasible because of heterogeneity in (the definition of) risk factors and outcomes. In addition, the number of studies assessing the same association for a complication was too small. This review focuses on complications that directly intricate the postoperative course. Perioperative CSF leaks can be managed adequately during surgery and were therefore not included. Other reviews have addressed specific complications occurring during surgery; e.g. internal carotid artery (ICA) injuries  or laterThe selection consisted of two phases: (1) title and abstract screening for potentially eligible articles, and (2) full text screening of these articles. During both phases, the same inclusion and exclusion criteria were used. During phase 1, in case of doubt, the full text paper was retrieved. Since a variety of risk factors can be investigated within the same cohort, a decision was made not to omit overlapping cohorts.Extracted study characteristics included: institution, study period, study design, number of patients, number of procedures, percentage females, tumor type, approach, length of stay, and duration of follow-up. Preoperative factors were categorized into groups  and all potential associations were categorized into complications in general, neurosurgical and endocrine complications. Risk factors were considered consistent when they were reported as significant in \u22652 independent studies. Inconsistent when \u22652 positive or negative and \u22651 neutral (non-significant) associations were reported and conflicting when \u22651 positive and \u22651 negative associations were reported.Assessment of risk of bias was done by means of the Quality in Prognostic Studies (QUIPS) tool . The QUIThe search resulted in 2596 unique titles and abstracts. The screening of titles and abstracts resulted in the selection of 472 full-text articles retrieved for the second phase of the selection process. Finally, 23 articles were included in the present systematic review Fig.\u00a0.The study characteristics are summarized in Table\u00a0The results of the scoring of the methodological quality of the studies are shown in Table\u00a0Overall, the methodological quality was low: only one study had a low risk of bias (4.3%), two a moderate risk (8.7%) and the remaining twenty studies had a high risk of bias (87.0%). A high risk of bias was found twelve times for study confounding .The incidence rates of complications described in the included studies are described in Table\u00a0Eight studies investigated the potential risk factors for complications in general , 31, 35.Age was assessed in two studies. Both studies found an increased risk for older age . Age was defined as a categorical parameter: (1) age \u226570 versus <60\u00a0years   and (2) 3 versus <10\u00a0cm3 OR 6.3 (1.6\u201325.0) [Tumor size and volume were significantly associated with increased complications in general in two out of three studies. Definitions used were (1) macroadenoma versus microadenoma OR 3.98  , (2) tum.6\u201325.0) , (3) tum.6\u201325.0) , and (4).6\u201325.0) .Four out of six investigated risk factors showed increased risks for complications in general. Tumor extension was defined in five different ways: (1) intraventricular extension, (2) Knosp grade, (3) supra-/parasellar extension, (4) extension into the anterior cranial fossa (ACF), and (5) cavernous sinus invasion. Intraventricular extension OR 7.85 (2.88\u201321.43) , supra-/Tumor type was investigated in one study; however, no significant effect was detected .Previous radiation was associated with an increased risk in one study  . The surFourteen studies investigated the potential risk factors for postoperative CSF leaks , 36, 37.Younger age was inconsistently associated with a higher risk of CSF leaks. Three studies, including two with overlapping cohorts , 34, fouIn two of five studies, female gender was associated , 33, 34 As expected, three out of four studies found a significant increase in postoperative CSF leaks in patients with a higher BMI , 25, 34.One study evaluated various comorbidities in relation to CSF leaks Table\u00a0 and founOnly one out of five studies looking at tumor size or volume found a significant association with CSF leaks , 33, 36.Tumor extension was analyzed in seven studies , 36, 37,Four studies looked at the relationship between various forms of pathology and CSF leaks , 25, 34.Previous surgery was not reported as a risk factor for CSF leaks , 33, 34.Even though the frequency of surgical resection after radiotherapy is low, one study found an increased risk for patients with prior radiotherapy; 4/14 patients had a postoperative CSF leak . The othOne out of four studies considering the surgeon\u2019s learning curve found a significant increase in postoperative CSF leaks , 23, 32.Two out of three studies reporting associations found a significant association for intracranial infections , 27, 38.Only two studies looked at risk factors for bleedings: (1) ICA injury , and (2)Eight studies looked at risk factors for DI , 32, 33;Age (2 studies) , 33, gen3 (no significant effect) [One study reported an association with permanent or overall DI , 29, 33. effect) , 33, and effect) .Knosp grade was not associated with a significant increase of DI .In overlapping cohorts, RCC was significantly associated with an increased risk of DI compared to other tumor types: (1) 47.6 versus 20.2% (p\u2009<\u20090.05) , and (2)Previous surgery was defined as (1) prior non-endoscopic surgery , (2) priLearning curve was assessed in three studies, but not associated with an increased risk of DI , 23, 32.One out of three studies looking at adrenal insufficiency addressed potential associations . IncidenThree complications were only analyzed once: cranial nerve injury, vision loss and sinusitis. One study found a significant relationship between patients with a history of an extrasellar tumor and cranial nerve injury  . One stuThis systematic review on preoperative risk factors for postoperative complications in ETS identified only two consistent risk factors: older age for complications in general and intraventricular extension for CSF leakage. Clear and uniform definitions of postoperative complications were mostly missing and almost all studies were retrospective. This resulted in a lack of standard reporting of complications, causing a large variation between studies regarding reported risk factors and incidence rates of complications.The most frequently studied complication, CSF leaks, was consistently associated with intraventricular extension. Other risk factors were not consistent, but did not report conflicting results. At this stage, we conclude that intraventricular extension increases the risk of CSF leaks and lower age, female gender, and high BMI potentially increase the risk Table . The secA distinction can be made between amendable and non-amendable risk factors. Even though often difficult to change, these should be taken into consideration in cases with increased risks. The learning curve is perhaps one of the easiest to amend. Experience (learning curve), for instance, appears to be an important risk factor for a lower risk of CSF leaks. This was confirmed in some but not all studies, while learning curve was not associated with any other complication. Obviously, learning curve statistics can be biased since a more experienced surgeon will operate on a more complex case mix with an innate higher risk of CSF leaks, which cannot be extracted from the currently available series. In a national survey, Ciric found that for most complications, surgical learning curve is an important factor . This waIncreased BMI and younger age were risk factors for postoperative CSF leaks. This might be explained by the increased intra-abdominal pressure . PerhapsSince generally only large tumors have suprasellar, intraventricular extension or extension into the ACF, these risk factors can be considered correlated and classified under tumor size. When taking this into account, large or giant pituitary tumors are associated with complications in general and postoperative CSF leaks. Literature on endoscopic resection of giant adenoma is scarce, however increased risk of complications can be found . In partPathology, also a non-amendable risk factor, might also be an important risk factor for postoperative complications. In particular, several associations were described for RCCs and craniopharyngiomas; however, only those for DI have been reported more than once (in overlapping cohorts). These two tumors have an increased risk of DI, possibly also for postoperative CSF leaks. The relationship with pathology can likely be explained by the tumor etiology. Whereas RCCs are typically located between the anterior and posterior lobe, compression/manipulation of the posterior lobe is likely to occur prior to or during surgery. Craniopharyngiomas commonly arise in the pituitary stalk, which is vulnerable to surgical manipulation; therefore, surgery is more likely to cause DI. Another risk factor found for RCCs was postoperative CSF leaks.Despite being addressed only in one study, previous radiation showed an association between complications in general and carotid artery injury. Even though radiotherapy prior to surgery is not common, some adenomas are very therapy resistant and need additional surgery. Boling et al. presented data from nine patients who had received radiotherapy prior to surgery, showing a complication rate of 33% . This miOne of the most reviewed subjects in pituitary surgery literature is the comparison between endoscopic and microscopic surgery. We found several reviews assessing the topic. Because the influence of surgical technique was the primary interest of comparison, patient-related risk factors were not investigated in these reviews. Typically, gross-total resection and complications have been compared between microscopy and endoscopy, most showing equal or superior results in favor of endoscopy; however, patient-related risk factors have not been further determined \u201310. In tThe main purpose of the study was to improve preoperative patient counseling and to identify high- and low-risk patients. The low quality of the studies precludes firm conclusions based on this review. Overall, most studies were retrospective and too small to allow multivariable analyses. Also, no meta-analysis could be performed because of the heterogeneity and low number of associations. Complications were often defined differently and mostly gave limited descriptions. This complicates generalizability, and future researchers should aim at clearly defining (presented) complications in an effort to improve the clinical impact of future research on daily practice. Furthermore, reporting of outcomes, not only by centers of excellence, and prospective registration will lead to further evolvement in the field. Many examples, like the Value Based Healthcare concept , have shWe realize that only a subset of the total number of studies reporting on complication rates in ETS could be included in this study since the vast majority did not perform risk factor analysis. Furthermore, studies that presented only pooled data between microscopic and endoscopic surgery did not give a utilizable overview of potential risk factors for complications specific for patients treated through an endoscopic transsphenoidal approach, as in many daily practices nowadays.Although many studies assessed individual risk factors of different types of postoperative complications, there were no prognostic models found in the current literature. Prognostic models in other fields have shown added value in individualized decision-making and patient counseling. Such a model could have different types of outcomes, based on the aim of the model: complications in general, prediction of potential candidates for short stay. Before implementation of such a model, it should be thoroughly internally and externally validated.Although on average the reported mortality rate is around 0.6%, unfortunately no associations were found in the current literature. Even though incidence rates are low, they are not negligible. Suggested improvements for definitions and registration of complications might give us a better understanding of the etiology of these complications.We present an overview of preoperative risk factors for postoperative complications. Only two risk factors were consistently associated with increased risks: older age for complications in general and intraventricular extension for CSF leakage. This does not mean that there are no other important risk factors, and further emphasizes the need for uniform definitions, reporting of outcomes and prospective registration. The low methodological quality of included studies, inconsistent results, and lack of uniform definitions make firm conclusions difficult. Nevertheless, we believe that awareness of presented risks may benefit patient counseling and surgical case selection.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 15 KB)Supplementary material 2 (DOCX 19 KB)Supplementary material 3 (DOCX 33 KB)"}
+{"text": "Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-\u03b1. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3+ cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1\u03b1 levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes.LEN, at the concentration range reached In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity. The development of these drugs represented a paradigm shift in the treatment of multiple myeloma (MM) [The Immunomodulatory drugs (IMiDsoma (MM) , 2. LEN-oma (MM) \u20135. Moreooma (MM) .\u00ae have been recently elucidated highlighting the role of Cereblon (CRBN) and its target factors [Several mechanisms of action have been described, , 8 inclu factors , 11. LEN factors . Through factors . Recentl factors  and in M factors .Currently, few data are reported on the possible effects of LEN on dendritic cells (DCs) populations. , 16\u201318 IDifferent studies reported an increased incidence of acute Graft versus host disease (aGvHD), with a possible enhancement of the graft versus MM effect, in patients treated with LEN after allo-transplantation \u201323. SincBased on these evidences, in this study we investigated whether LEN may affect maturation, phenotype and functionality of DCs as antigen presenting cells (APCs), either directly or through the modulation of human mesenchymal stromal cell (hMSC) effect on DCs.in vivo in MM patients, [vs LEN 0.1 \u03bcM, 45.82 \u00b1 4.55 vs 59.45 \u00b1 8.21, p = 0.029; DMSO vs LEN 1 \u03bcM, 45.82 \u00b1 4.55 vs 73.52 \u00b1 7.71, p = 0.001), CD86  and CD209    differentiated from BM aspirates and PB of MM patients. Despite a reduction of both number and % of mature DCs, LEN, at the concentration range reached atients,  signific) Figure , comparevs LEN 0.1 \u03bcM, 147.49 \u00b1 45.08 vs 200.44 \u00b1 44.22, p = 0.002; DMSO vs LEN 1 \u03bcM, 147.49 \u00b1 45.08 vs 249.61 \u00b1 42.10, p = 0.016) and CD209   expression was found in DCs differentiated from PB CD14+ cells  (Friedman test)  and the percentage of DCs obtained in vitro    at 10vs LEN 0.1 \u03bcM vs LEN 1 \u03bcM: 1076 vs 1755 vs 2193 pg/ml, p < 0.05), CC chemokine ligand (CCL)2 , CCL5  and TNF-\u03b1  and slightly decreased the production of IL-6  , by mo-DCs differentiated from MM patients, compared to vehicle  assay in the presence of LEN or vehicle.+ cell proliferation was significantly higher in co-culture with LEN-treated DCs, compared to DMSO-treated DCs      Figure . However) Figure .+ cells of MM patients were differentiated into DCs in the presence of LEN or DMSO treated human telomerase reverse transcriptase transduced hMSC (hTERT-hMSC) conditioned medium (CM).To investigate a possible MSC-mediated indirect effect of LEN on DCs, BM CD14in vitro system  and CD86    Figure .PTGS2) gene expression levels at all tested concentrations  , IL6, CCL5 and transforming growth factor beta 1 (TGFB1).Thereafter, we examined whether LEN treatment affected the expression of immunosuppressive factors in hTERT-hMSCs, by Real-time PCR (RT-PCR). Interestingly, we found that LEN significantly down-regulated prostaglandine 2  Figure  but not To investigate the molecular mechanism involved in the effect of LEN on MSCs, firstly we checked the expression profile of Cereblon and its target proteins in hTERT-hMSCs showing that they expressed Cereblon Figure  but not in vitro DC differentiation from BM CD14+ cells of 2 MM patients. Interestingly, we found that the effect of hTERT-hMSCs on DC maturation markers were reverted by the down-regulation of CK1-\u03b1   .in vivo LEN treatment on DC maturation markers, we compared the expression profile of DCs differentiated from PB CD14+ cells of 9 MM relapsed patients, purified at the baseline (DAY 0) and after 7 days of LEN treatment, just before the start of the weekly treatment with Dex. All the patients were responsive to LEN treatment.Finally, to evaluate the effect of in vivo LEN treatment significantly increased the expression of HLA-DR  and CD209 , and CD86 without reaching statistical significance   . Flow-cytometry histograms from one representative MM patient were reported in Interestingly, we found that DCs of MM patients are known to be functionally defective, with a decreased expression of maturation markers and antigen presentation ability . The proin vivo in MM patients treated with this drug 5\u201325 mg daily, [\u22128 M, consistent with several studies that reported Dex inhibitory effects on DC maturation and functions [vs LEN alone [Studies performed on murine models, showed that both LEN and POM treatment increased the expression of DC maturation markers, enhanced DC endocytotic activity, increased the production of TNF-\u03b1 and CCL2, and the DC-dependent T-cell expansion , 17. Howg daily, , 34 signg daily, \u201337 both unctions , 39. Howunctions  on high-EN alone . ConversEN alone , 42.in vitro DC differentiation was associated with an increased DC functional activity to stimulate T cell proliferation that was abrogated by the combination with Dex.Consistent with the effect of LEN on DC maturation markers, we found that LEN treatment increased DC production of IL-8, CCL2, CCL5, and TNF-\u03b1, in line with data observed on murine models , 17. TheSubsequently, we investigated the molecular mechanism beyond LEN effects on DCs. It is widely demonstrated that LEN exerts its anti-MM activity through the modulation of Ikaros and Aiolos , 13. MorPTGS2, known to inhibit the transitional processes of differentiation of monocytes into DCs [Along with a direct effect of LEN on DC maturation, our data suggested a potential indirect effect, through the modulation of MSC immunomodulatory properties such as the production of cytokines and chemokines. \u201326, 45 Winto DCs , 47. Theinto DCs , 48 receinto DCs , 48. Intinto DCs .in vitro evidences were expanded and confirmed by ex vivo DC cultures in relapsed MM patients treated with LEN 25 mg/day, as mono-therapy for one week, just before the start of the weekly treatment with Dex. After 7 days of treatment we found an increased PB DC differentiation. Of note, all analyzed patients were responsive to LEN treatment. This early effect was in agreement with recent data reporting the in vivo increase of T and NK cells, with a rapid decline of Ikaros, after 7 days of POM treatment without Dex in MM patients [Lastly, our patients .in vitro and in ex vivo cultures, enhancing DC ability to stimulate T cell proliferation and to release chemokines involved in the immune response. LEN treatment also reduces the immunosuppressive properties of hMSCs, suggesting new possible effects of IMiDs\u00ae on the allo-reactivity against MM cells.In conclusion, our data indicate that LEN increases the expression of mature DC markers both BM and/or PB were obtained from 30 consecutive patients with active MM , including both newly diagnosed and relapsed MM, admitted to our hematological Unit. Patient samples were obtained after informed consent, according to the Declaration of Helsinki. The study was approved by the Institutional Ethical Review Board of our Hospital.Moreover, PB were obtained from 9 patients with relapsed MM  I: 4, International Staging System (ISS) II: 3, International Staging System (ISS) III: 2), at the baseline and after 7 days of treatment with LEN 25 mg/day (days 1\u201321), just before the start of the weekly treatment with Dex.in vitro studies.Mononuclear cells (MNCs) were isolated from BM and PB samples after Ficoll gradient separation and used for further \u22126M). All cell lines were authenticated and tested for mycoplasma contamination.The human myeloma cell line (HMCL) JJN3, purchased by DSMZ  and the human monocytic cell line THP-1, obtained from the American Type Culture Collection , were maintained in culture in RPMI 1640 medium with 10% FBS; hTERT-hMSCs were kindly gifted from Dr Giuseppe Gaipa  and maintained in culture with RPMI 10% FBS with hydrocortisone . CD3+ cells were isolated following the same protocol, using anti-CD3 mAb from PB of MM patients. The presence of potential contaminating cells in each fraction was evaluated by flow cytometry analysis, using the fluorescence-activated flow cytometer BD FACS Canto II with Diva software ; Franklin Lakes, NJ). Purity of cell samples was > 92%.BM and PB CD14+ cells, cultured in vitro at 1 \u00d7 106 cells/ml in RPMI 10% FBS, with recombinant human (rh) granulocyte macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and IL-4 (50 ng/ml) , for 8 days , in the presence of LEN  or vehicle (DMSO), at concentration 0.1 and 1 \u03bcM. TNF-\u03b1 at 10 ng/ml  was added to the culture medium for the last 24 h, in order to induce DC terminal maturation. At the end of culture period, both cells and CM were collected for further analysis. In some experiment, the combination of LEN and Dex  was tested on DC differentiation. Briefly, DCs were differentiated from BM CD14+ cells of MM patients, in the presence of LEN (0.1 and 1 \u03bcM) or vehicle, as reported above. At the end of culture period, cells were collected and reseeded (5 \u00d7 104/ml) in fresh medium with Dex (10\u22128M) or vehicle (EtOH) for 48 h. After Dex treatment, cells were collected and analyzed for DC maturation markers. For the ex vivo studies, PB CD14+ cells were isolated from MM patients at day 0 and after one week (day 7) of LEN (25 mg/day) treatment, just before the start of the weekly treatment with Dex. Cells were then differentiated into DCs, following the above protocol, without LEN in vitro treatment.DCs were differentiated from purified CD14DCs were also differentiated from THP-1 cell line, by adding rhIL-4 (200 ng/ml), rhGM-CSF (100 ng/ml), ionomycin (200 ng/ml)  and rhTNF-\u03b1 (20 ng/ml) for 72 h to the culture medium ; then LEN or DMSO were added for the last 24 h of culture period. THP-1-derived DCs (THP1-DCs) were detached with EDTA 2 mM on ice for 2 h and cell pellets collected for further analysis.+ cells of MM patients, in the presence or absence of the CM  of hTERT-hMSCs treated with LEN or DMSO. Briefly, 1 \u00d7 104 hTERT-hMSCs were seeded in T75 flasks and cultured in RPMI 10% FBS, in presence of LEN (0.1 and 1 \u03bcM) or DMSO, for 5 days. At the end of culture period, the medium was replaced with RPMI 10% FBS in order to discard LEN, and after 48 h, the CM was collected and used during DC differentiation, as previously reported. In some experiments, after 5 days of LEN treatment, hTERT-hMSC pellets were collected for immunoblotting and RT-PCR analysis.In some experiments DCs were differentiated from BM CD143 cells/w) in round-bottomed 96well-plates, in RPMI 15% AB human serum. DCs were co-cultured with autologous PB CD3+ cells (1 \u00d7 104) for 6 days. At the end of culture period, an MTT assay  was performed in order to measure T cell proliferation.DCs were differentiated, as previously reported, in presence of LEN or vehicle , from BM of 6 MM patients, for 8 days. Then, treated cells were collected, analyzed by flow-cytometry and partly re-seeded : 1) anti-CD14-FITC/anti-CD83-PE/isotype control-PE-Cy5/isotype control-APC, 2) anti-CD14-FITC/anti-CD83-PE/anti-CD86-PE-Cy5/anti-HLA-DR-APC, 3) anti-CD14-FITC/anti-CD83-PE/anti-CD80-PE-Cy5/anti-CD209-APC. Four color, six-parameter acquisition and analysis were perfomed on a two-laser FACSCalibur instrument (BD Biosciences) using CellQuest software (BD Biosciences). Mature DCs were identified as CD14\u2212CD83+ cells and the MFI of the maturation markers was compared between cells treated with LEN and/or Dex vs the relative control, for each experiment.After in vitro DC differentiation, by using multiplex bead-based sandwich immunoassay kits , following the manufacturer's instructions. Measurement were performed by a reader . For TNF-\u03b1 level evaluation, the obtained results were normalized for TNF-\u03b1 concentration measured in the control medium .The concentration of Interferon (IFN)-\u03b3, IL-6, IL-8, IL-10, IL-12, IFN-\u03b3 induced protein (IP)-10, CCL2, CCL5 and TNF-\u03b1 was evaluated on DC CM, collected after Nuclear and cytosolic extracts were obtained using a commercial kit  following the manufacturer's protocol from THP1-DCs and hTERT-hMSCs, treated with LEN or DMSO. Immunoblotting was performed as previously reported  using th4 cells were infected with a multiplicity of infection (MOI) of 4, in the presence of 8 \u03bcg/ml polybrene . 24 h later, the infected medium was replaced with fresh growing medium. Puromycin selection (0.5 \u03bcg/ml) was initiated 2 days after transduction. Once a cellular clone was established, to induce CK1\u03b1 silencing, cells were incubated with 500 \u03bcM IPTG  every 2\u20133 days for a total of one week. Then, fresh medium without IPTG and puromycin was added for further 48 h. At the end of culture period, cell pellets were collected and analyzed by western blotting to check CK1-\u03b1 down-regulation and select the more efficient clone. CM was also collected and used for in vitro DC differentiation.RNAi was performed through the generation of inducible shRNA stable cell lines. hTERT-hMSC were transduced with the IPTG inducible lentiviral particles carrying CSNK1A1-specific shRNA . Two independent shRNAs  sequences were chosen. 3 \u00d7 10IDO1 (Assay ID:103804), IL6 (Assay ID: 144013), CCL5 (Assay ID: 113395), PTGS2 (Assay ID: 102471), TGB1 (Assay ID: 101210), and GAPDH (Assay ID: 102052). The expression of selected genes was checked by Real Time PCR by Light Cycler 480 . To normalize the differences in RNA quality and reverse transcription efficiency, we applied the comparative Ct method using the endogenous reference gene GAPDH.Total RNA was extracted from hTERT-hMSCs, after all different experimental conditions, using the RNeasy total RNA isolation kit . RNA (1 \u03bcg) was reverse-transcribed with 400 U Moloney murine leukemia reverse transcriptase  in accordance with the manufacturer's protocol. Real Time PCR was performed by adding complementary DNA to a universal Light Cycler 480 Probes Master and RealTime ready Catalog Assay  for the following genes: t-test was used to analyze flow cytometry data of in vitro DC differentiation from BM and PB of MM patients and for the ex vivo studies. Non-parametric Friedman test, Wilcoxon test and Mann-Whitney test were used for the other experiments with a lower number of samples. Results were considered significant at p < 0.05. GraphPad Prism 6.1\u2122  was used for all the statistical analyses.Data were expressed as mean \u00b1 SEM or median values. Paired Student's"}
+{"text": "This Special Issue relates to the 18th biannual International Gap Junction Conference (IGJC2017), held at the Crowne Plaza Hotel, Glasgow, U.K., from the 29 July\u20132 August 2017. The special issue, entitled: Interplay of Connexins and Pannexins in Tissue Function and Disease focused on six key state of the art reviews written by chairs of the sessions highlighting the assembly and functional interactions of connexins and pannexins in diverse tissues and disease states, with translational outputs emerging. A further 14 articles detail specific contributions that were presented as oral communications . The meeConnexins and pannexins are tetramembrane spanning channel proteins with a shared topology and related functional properties. They oligomerise to form channels in the plasma membrane with two extracellular loops that project into the extracellular space and an intracellular carboxyl tail is subject to post-translational modification. Within the connexin family (21 members in man), these extracellular loops interdigitate and dock with loops from neighbouring cells to form dodecameric intercellular gap junction channels (GJCs) that link the cytoplasm\u2019s of neighbouring cells. These GJCs enable the regulated exchange of over 300,000 metabolites of less than 1000 Da in size, in so doing co-ordinating specific cell and tissue homeostasis. Connexin compatibility is highlighted by phylogenetic classification into three\u2013four specific subgroups, with only specific heteromeric channel combinations possible and in so doing facilitating the segregation of tissue compartments.Over the last 15 years, it has emerged that connexin hemichannels can be triggered to open under conditions of cell stress releasing signalling molecules such as ATP, glutamate and Nicotinamide adenine dinucleotide (NAD) into the extracellular environment and subsequently elicit localised extracellular signalling cascades via purinergic receptors. Pannexins, proteins forming hemichannel-like structures, identified about 15 years ago, share a common topology but no sequence homology with connexins, are thought to be evolutionary related to the innexins, gap junction forming proteins in invertebrates. Unlike connexins (and innexins) pannexins are highly glycosylated proteins and act to release ATP, engaging with downstream signalling pathways. In particular, pannexin signalling has been closely linked with inflammatory mediated events, where caspase1 cleavage of the carboxyl tail renders these channels constitutively open and triggers cell death \u2018find me\u2019 signals.Due to the diversity of connexin and pannexin expression in tissue networks, their importance cannot be underestimated and they are now firmly established as key proteins in diverse tissue networks including the cardiovascular system, the skin, the nervous system, the liver, the ocular, respiratory and immune system. Further, changes in connexin expression and function occur in disease states associated with all of these tissues: Tumorigenesis, diabetic retinopathy, cardiac arrhythmia, atherosclerosis, stroke, Alzheimer\u2019s disease, chronic non\u2013healing skin wounds, and inflammation of epithelial tissue, to name a few. A range of mutations in connexins are associated with clinical disease, where mutations in Cx26 are among the most common in recessively inherited hearing impairment, Cx32 with the demyelinating disorder Charcot Marie Tooth-Linked disease, Cx43 with oculodentodigital dysplasia, and Cx46 and Cx50 with familial cataract formation. All these diseases impact on the quality of life, healthcare resources, ageing populations and many with conditions that can be managed with current therapies, but not cured.Thus, connexins and pannexins have emerged as prime therapeutic targets for a diverse range of disease states. These channels are amenable to targeting therapeutically and a range of antisense and peptidomimetic strategies have emerged as key regulators of channel function. Such regulators were first identified over 25 years ago when Evans and Warner synthesised the first mimetic peptides and antibodies that mimicked amino acid sequences on the extracellular loops. These tools are now widely used by the research community to define the role of connexins and pannexins in tissue function and are proving successful in translational research where the success of clinical trials of a connexin targeted therapy, from bench to bedside, for improving wound healing events is reported in this special issue.The Editors thank all contributors to this special issue, delegates and in the conference support team for the successful running of IGJC2017. The follow-up meeting will be held in Victoria, Vancouver Island, July 2019 (#IGJC2019).ReviewsInt. J. Mol. Sci.2018, 19(5). [Aasen, T.; Johnstone, S.; Vidal-Brime, L.; Lynn, K.S.; Koval, M. Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease. Int. J. Mol. Sci.2018, 19(5). [Sorgen, P.L.; Trease, A.J.; Spagnol, G.; Delmar, M.; Nielsen, M.S. Protein\u2013Protein Interactions with Connexin 43: Regulation and Function. Participant ContributionsInt. J. Mol. Sci.2018, 19(6). [Ek-Vitorin, J.F.; Pontifex, T.K.; Burt, J.M. Cx43 Channel Gating and Permeation: Multiple Phosphorylation-Dependent Roles of the Carboxyl Terminus. Int. J. Mol. Sci.2018, 19(6). [Spagnol, G.; Trease, A.J.; Zheng, L.; Gutierrez, M.; Bazu, I.; Sarmiento, C.; Moore, G.; Cervantes, M.; Sorgen, P.L. Connexin43 Carboxyl-Terminal Domain Directly Interacts with \u03b2-Catenin. Int. J. Mol. Sci.2018, 19(7). [Sanchez-Pupo, R.E.; Johnston, D.; Penuela, S. N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1. , 19(7). Int. J. Mol. Sci.2018, 19(9). [Batissoco, A.C.; Salazar-Silva, R.; Oiticica, J.; Bento, R.F.; Mingroni-Netto, R.C.; Haddad, L.A. A Cell Junctional Protein Network Associated with Connexin-26. 2018, 19(9). [Schadzek, P.; Helmes, D.; Stahl, Y.; Dilger, N.; Ngezahayo, A. Concatenation of human connexin26 (hCx26) and human connexin46 (hCx46) for the analysis of heteromeric gap junction hemichannels and heterotypic gap junction channels. , 19(9). ReviewInt. J. Mol. Sci.2018, 19(6). [Molica, F.; Figueroa, X.F.; Kwak, B.R.; Isakson, B.E.; Gibbins, J.M. Connexins and Pannexins in Vascular Function and Disease. Participant ContributionsInt. J. Mol. Sci.2018, 19(1). [Carballo, S.; Pfenniger, A.; Carballo, D.; Garin, N.; James, R.W.; Mach, F.; Shah, D.; Kwak, B.R. Differential Association of Cx37 and Cx40 Genetic Variants in Atrial Fibrillation with and without Underlying Structural Heart Disease. Int. J. Mol. Sci.2018, 19(4). [Noureldin, M.; Chen H, Bai D. Functional Characterization of Novel Atrial Fibrillation-Linked GJA5 (Cx40) Mutants. , 19(4). Int. J. Mol. Sci.2018, 19(4). [Viczenczova, C.; Kura, B.; Egan Benova, T.; Yin, C.; Kukreja, R.C.; Slezak, J.; Tribulova, N.; Szeiffova Bacova, B. Irradiation-Induced Cardiac Connexin-43 and miR-21 Responses Are Hampered by Treatment with Atorvastatin and Aspirin. , 19(4). Int. J. Mol. Sci.2018, 19(6) [Boucher, J.; Simonneau, C.; Denet, G.; Clarhaut, J.; Balandre, A.C.; Mesnil, M.; Cronier, L; Monvoisin, A. Pannexin-1 in Human Lymphatic Endothelial Cells Regulates Lymphangiogenesis. 8, 19(6) Int. J. Mol. Sci.2018, 19(7). [Htet M, Nally, J.E.; Shaw, A.; Foote, B.E.; Martin, P.E.; Dempsie, Y. Connexin 43 Plays a Role in Pulmonary Vascular Reactivity in Mice. , 19(7). ReviewInt. J. Mol. Sci.2018, 19(6). [Graham, S.V.; Jiang, J.X.; Mesnil, M. Connexins and Pannexins: Important Players in Tumorigenesis, Metastasis and Potential Therapeutics. , 19(6). Participant ContributionsInt. J. Mol. Sci.2018, 19(3). [Busby, M.; Hallett, M.T.; Plante, I. The Complex Subtype-Dependent Role of Connexin 43 (GJA1) in Breast Cancer. , 19(3). Int. J. Mol. Sci.2018, 19(7). [Iikawa, N.; Yamamoto, Y.; Kawasaki, Y.; Nishijima-Matsunobu, A.; Suzuki, M.; Yamada, T.; Omori, Y. Intrinsic Oncogenic Function of Intracellular Connexin26 Protein in Head and Neck Squamous Cell Carcinoma Cells. , 19(7). ReviewsInt. J. Mol. Sci.2018, 19(5). [Chanson, M.; Watanabe, M.; O\u2019Shaughnessy, E.M.; Zoso, A.; Martin, P.E. Connexin Communication Compartments and Wound Repair in Epithelial Tissue. , 19(5). Participant ContributionsInt. J. Mol. Sci.2018, 19(2). [Faniku, C.; O\u2019Shaughnessy, E.; Lorraine, C.; Johnstone, S.R.; Graham, A.; Greenhough, S.; Martin, P.E. The Connexin Mimetic Peptide Gap27 and Cx43-Knockdown Reveal Differential Roles for Connexin43 in Wound Closure Events in Skin Model Systems. Int. J. Mol. Sci.2018, 19(5). [Chen, J.; Liang, C.; Zong, L.; Zhu, Y.; Zhao, H.B. Knockout of Pannexin-1 Induces Hearing Loss. ReviewInt. J. Mol. Sci.2018, 19(6). [Montgomery, J.; Ghatnekar, G.S.; Grek, C.L.; Moyer, K.E.; Gourdie, R.G. Connexin 43-Based Therapeutics for Dermal Wound Healing."}
+{"text": "The clinical significance of hematogenous and lymphatic metastasis in ovarian cancer has been increasingly addressed, as it plays an imperative role in the formation of both intraperitoneal and distant metastases. Our objective is to identify the key molecules and biological processes potentially related to this relatively novel metastatic route in serous ovarian cancer.Since lymphovascular space invasion (LVSI) is considered as the first step of hematogenous and lymphatic dissemination, we developed a gene signature mainly based on the transcriptome profiles with available information on LVSI status in the Cancer Genome Atlas (TCGA) dataset. We then explored the underlying biological rationale and prognostic value of the identified gene signature using multiple public databases.We observe that primary tumors with increased risk of hematogenous and lymphatic metastasis highly express a panel of genes, namely POSTN, LUM, THBS2, COL3A1, COL5A1, COL5A2, FAP1 and FBN1. The identified geneset is characterized by enhanced deposition of extracellular matrix and extensive stromal activation. Mechanistically, both the recruitment and the activation of stromal cells, especially fibroblasts, are closely associated with lymphovascular metastasis. Survival analysis further reveals that the elevated expression of the identified genes correlates to cancer progression and poor prognosis in patients with serous ovarian cancer.Our findings indicate that tumor stroma supports the hematogenous and lymphatic spread of ovarian cancer, increasing tumor invasiveness and ultimately resulting in worse survival. Thus stroma-targeted therapies may improve the clinical outcomes in combination with cytoreductive surgery and chemotherapy. Ovarian cancer is the most lethal gynecological malignancy, and a fair number of patients are diagnosed in advanced stage with extensive intraperitoneal spread and distant metastases . ExploraIt has long been assumed that direct shedding of ovarian cancer cells from the primary site into the intraperitoneal cavity is the most predominant route for the formation of metastatic diseases . HematogLymphovascular space invasion (LVSI), describing the presence of tumor cells within the lumen of lymphatic or vascular capillaries of the primary tumor, is demonstrated to associate with worse clinical outcomes in patients with ovarian cancer , 7. As thttp://www.ncbi.nlm.nih.gov/geo) and preprocessed by the robust multi-array average algorithm (RMA). Clinical and pathological characteristics of the cohorts of patients analyzed in this study was listed in Additional\u00a0file\u00a0Microarray transcriptome profiles from TCGA, GSE26712, GSE9891, GSE49997 and the corresponding clinical metadata were downloaded from the curatedOvarianData database ) in patients with late-stage diseases undergoing optimal cytoreduction  and possess multiple tumor-promoting functions, facilitating tumor invasion and metastasis through direct and indirect crosstalk with tumor cells as well as with other non-tumor components like immune cells and endothelial cells .Firstly, the co-evolution of cancer cells and CAFs facilitate tumor growth and spread via multiple interactions. CAFs produce autocrine and paracrine cytokines, chemokines and growth factors like CXCL1, CCL5, HB-EGF and TGF-\u03b1, subsequently promoting cancer cell invasiveness through upregulation of matrix metalloproteinases (MMPs) and induction of EMT. CAFs can additionally reprogram ovarian cancer cell metabolism via producing metabolites or altering the key enzymatic activities . In turnBesides, angiogenesis and inflammatory response are involved in the tumor-stroma crosstalk, which was also revealed in the above enrichment analysis. Pathological angiogenesis, supplying adequate nutrients and oxygen to tumor cells, often indicates the metastatic potential in tumors. CAFs secretes key pro-angiogenic factor VEGF-A as a result of HOXA9 upregulation from ovarian cancer cells, promoting proliferation and invasiveness of endothelial cells so as to trigger angiogenesis . Many ofReferred to as \u201cwounds that never heal\u201d, cancer and chronic inflammation are closely correlated. CAFs overexpress pro-inflammatory chemokines and cytokines such as IL-6, COX-2, and CXCL1, which mediate tumor-related inflammation and induce carcinogenesis . These CConsidering the essential role of tumor stroma in promoting cancer progression, strategies targeting stromal cells exert great potential in improving clinical outcomes of ovarian cancer patients. Notably, low resistance rate is a distinct advantage of anti-stromal therapies, largely relying on the genetic stability of stromal cells. Among the genes identified in the present study, the safety of FAP-antibody sibrotuzumab has been validated in phase I trials in patients with tumors highly expressing FAP . AlthougIn addition, TGF-\u03b2-targeted therapies are currently investigated as a promising approach to impede cancer progression, since TGF-\u03b2 has been demonstrated as the key driver of fibroblast activation and CAF formation in different cancer types. In line with the available researches , 40, somCurrently, there are over 50 clinical trials evaluating TGF-\u03b2-targeted therapies in cancers . For patOf note, some genes we explored overlap with previously identified gene signatures associated with cancer progression and poor survival in various solid tumors including ovarian , 43, breIn this manuscript, we identified a panel of genes closely correlated with hematogenous and lymphatic metastasis of serous ovarian cancer. The upregulation of this gene signature, predominantly expressed by the increased infiltration of reactive stroma, is further confirmed to associate with tumor invasiveness and poor survival. Utilizing multiple transcriptome profiles available in the public database, we explored the potential biological rationale underlying this relatively novel metastatic route of ovarian cancer, highlighting the imperative role of tumor stroma in the development of ovarian cancer metastasis.Additional file 1: Table S1. Clinical and pathological characteristics of the cohorts of patients analyzed in the manuscript. Table S2. Flowchart of this study. Table S3. Eight genes common to both LVSI- and metastasis-related DEGs were listed. Fold changes and adjusted P values were generated by limma package. Table S4. Results of purity-corrected correction analysis from TIMER database showed a significant but weak correlation between the expression levels of the identified genes and the infiltration of immune cells in ovarian cancer samples. Table S5. The expression of the LMGS in the ovarian cancer CCLE cell lines were ranked, with the corresponding EMT phenotypes annotated based on two public sourcesAdditional file 2: Figure. S1. (a) Paired t-test revealed that all eight genes were significantly elevated in omental metastases compared with the corresponding primary ovarian tumors in the dataset GSE30587. (b) Four genes  of the LMGS were remarkably elevated, while COL5A1 was significantly down-regulated in SKOV3-OM3 , compared to SKOV3ip1 intraperitoneal injected to the host mice (representing the primary tumors) in the dataset GSE52999. (c) Genes of the LMGS were likely to form a biologically functional network based on PPI analysis. Primary OV: primary ovarian cancer samples, Metastatic OM: omental metastases of ovarian cancer. * P\u2009<\u20090.05, ** P\u2009<\u20090.01, *** P\u2009<\u20090.001Additional file 3: Figure. S2. All eight genes of the LMGS were highly enriched in the C1 molecular subtype of (a) the Tothill dataset and (b) in the TCGA mesenchymal subtype. (c) The expression levels of individual gene of the LMGS were significantly elevated in tumor stroma compared with the epithelial components in dataset GSE38666. A similar tendency was observed in paired samples in (d) dataset GSE115635, as well as in stromal components compared with normal ovarian stroma in (e) dataset GSE40595.Additional file 4: Figure. S3. The significant and negative correlation between the activation of the LMGS and tumor purity was validated in (a) GSE9891 and (b) GSE26712. (c-e) The positive correlation between the expression of the LMGS and immune cell infiltration was significant but relatively weak. The activation of the LMGS was positively correlated with mesenchymal infiltration in serous ovarian cancer samples from (f) GSE9891 and (g) GSE26712. The infiltration of immunocytes was similar between the primary ovarian cancer samples with LVSI-positive status versus LVSI-negative ones. A similar trend was observed in omental metastases compared with primary lesions in (h) dataset GSE2109 and was validated in (i) paired samples from dataset GSE30587Additional file 5: Figure. S4. Genes of the LMGS were remarkably elevated in (a-c) patients undergoing suboptimal cytoreduction and (d-f) those with late-stage serous ovarian cancer"}
+{"text": "The opioid crisis can be seen as a double crisis or a dual epidemic: the crisis of nonmedical use and the crisis of uncontrolled pain.It is important to ensure both crisis are adequately addressed.The evidence base of clinical guidelines to ensure safe and appropriate opioid prescribing as well as compliance with\u00a0 these \u00a0guidelines\u00a0 can\u00a0 be\u00a0 further\u00a0 improved.There is a striking absence on research related to the lack of equal access to medically justified opioids. Additionally, there is a lack of evidence on unintended consequences of policy and regulatory actions.Strategies to battle nonmedical use of opioids should not go at the expense of access for patients in legitimate medical need. Close monitoring is needed to minimize unintended consequences.1Pharmacoepidemiology and Drug Safety, several research papers address the safe and appropriate use of opioids. This is important, given the critical situation on opioid use in the United States (US). According to preliminary data from the Centers for Disease Control and Prevention (CDC), more than 72\u00a0000 people in the United States died because of drug overdose in 2017 with over two\u2010thirds involving opioids.In this issue of 2We acknowledge that abuse and diversion of opioids constitute a serious threat to public health. But it is also important to recognize that there is another side of the coin: opioids are an indispensable pharmaceutical treatment option for patients in pain, and many patients in medical need have inadequate access. Data from the International Narcotics Control Board (INCB) show that 95.7% of the global consumption of opioid analgesics in 2011 to 2013 took place in regions representing only 15% of the global population.3In recent years, there has been a large increase in the number of scientific studies reporting on the nonmedical use of opioids. Despite this scientific focus, there is still a lack of evidence\u2010based guidance on the safe prescribing of opioid medicines and on the pathways from opioid dependence to opioid overdosing. Several studies have identified medication\u2010related and patient\u2010related factors associated with the risk of opioid overdose. Medication\u2010related factors include the use of long\u2010acting or extended release formulations , combined use with benzodiazepines, high daily doses, and long\u2010term opioid use., Ranapurwala et al revealed several internal and external validity concerns in the opioid safety studies.Prescribing guidelines are important to guide clinicians in the safe and appropriate prescribing of opioids. However, the CDC guidelines are also criticized for making some recommendations that are not supported by current scientific evidence, which is also acknowledged by the guidelines themselves.4Pharmacoepidemiology and Drug Safety, two papers have assessed compliance with CDC recommendations.Aside from the level of evidence, it is useful to know to what extent these guidelines are followed in practice. In the current issue of Compliance with clinical guidelines is an essential prerequisite for the functioning of health systems. But clinical guidelines are typically based on the average patient; there may be certain patients with individual circumstances that justify deviating from guidelines. For example, there may be patients that require treatment with an extended release formulation, or with a higher dose than 90 MME at onset of their treatment. This also applies to patients with a history or high susceptibility of opioid dependence; this group represents a particularly disadvantaged and challenging population. Providing these patients with opioids in a balanced fashion remains critical. Since this is a high\u2010risk population, close clinical monitoring, management of abuse risk, and adequate access to opioid dependence treatment are crucial when opioid analgesics are justifiably used for pain management.5Although the focus on appropriate prescribing and dispensing is understandable given the current opioid abuse and misuse crisis, there is a striking absence on research related to the other crisis, ie, the lack of equal access to medically justified opioids. A review of 46 articles published between 2007 and 2013 showed that 31.8% of the patients with cancer did not receive adequate pain relief.Societal attitudes regarding the medical use of opioid analgesics may have changed because of the opioid epidemic. In discussions addressing this crisis, people may not always distinguish between overdose, misuse or illegal diversion of prescribed opioids, and use of illicit opioids. This confusion may result in a disproportionate generalized fear of opioids, limiting access for patients in medical need. Some experts believe that most patients with opioid dependence are recreational drug users who become dependent, rather than patients with pain becoming patients with opioid dependence.Although it is beyond any doubt that nonmedical use and diversion of opioids should be battled, this should not go at the expense of balanced strategies to ensure access to medicines that are legitimately on the market for patients in need of essential pain relief. The issue is how to monitor and minimize potential unintended consequences for these patients. The Access to Opioid Medication in Europe (ATOME) project\u2014aimed at the increase of access to opioid medicines in 12 countries with statistical evidence of low opioid consumption\u2010signaled clearly the importance of sustained investments in public health and education, and improved legal and regulatory systems to ensure safe and appropriate treatment of pain.www.tipharma.nl), is accepted under the condition that no company\u2010specific product or company related study is conducted. The Centre has received unrestricted research funding from public sources, eg, Netherlands Organization for Health Research and Development (ZonMW), the Dutch Healthcare Insurance Board (ZIN), EU 7th Framework Program (FP7), the Dutch Medicines Evaluation Board (MEB), and the Dutch Ministry of Health, Welfare and Sport.The Division of Pharmacoepidemiology and Clinical Pharmacology of Utrecht University is designated as a WHO Collaborating Centre for Pharmaceutical and Regulation. The WHO Collaborating Centre for Pharmaceutical Policy and Regulation receives no direct funding or donations from private parties, including pharma industry. Research funding from public\u2010private partnerships, eg, IMI, TI Pharma ("}
+{"text": "As a result, our model performed well in the global and local cross-validations, which indicated that IDLDA had a great performance in predicting novel associations. Case studies of colon cancer, breast cancer, and gastric cancer were also implemented, all lncRNAs which ranked top 10 in both databases were verified by databases and related literature. The results showed that IDLDA might play a key role in biomedical research.It has been demonstrated that long non-coding RNAs (lncRNAs) play important roles in a variety of biological processes associated with human diseases. However, the identification of lncRNA\u2013disease associations by experimental methods is time-consuming and labor-intensive. Computational methods provide an effective strategy to predict more potential lncRNA\u2013disease associations to some degree. Based on the hypothesis that phenotypically similar diseases are often associated with functionally similar lncRNAs and  Non-coding RNA (ncRNA) is a kind of RNA molecule that is not translated into protein . In decaRecently, exploiting potential lncRNA\u2013disease associations have become a growing significant research area. Many associations between lncRNA and human diseases have been identified by medical experiments, but which is costly and time-consuming. Predicting potential associations by the mathematical method and computational inference for experimental verification is a quite certain well-selected alternative .vice versa. IDLDA achieved reliable predictions with global and local cross-validations and it obtained higher AUROC than some previously proposed methods. Our results showed that the predicted top 10 lncRNAs in both databases were confirmed by databases and literature, and there were only 2, 2, and 1 lncRNAs which ranked top 50 by IDLDA in both databases that were not confirmed. All these results demonstrated the effectiveness and value of IDLDA in identifying potential lncRNA\u2013disease associations. Data and code are freely available for research purposes only, you can email the author for it.In this paper, we developed an improved diffusion model for predicting lncRNA\u2013disease associations (IDLDA) based on the hypothesis that phenotypically similar diseases are often associated with functionally similar lncRNAs and LncRNADisease  and Lnc2G as follows. V=L\u222a\u200bD is the vertex set, where L is the lncRNA set { l1,l2,\u2026,lNl }, D is the disease set { d1,d2,\u2026,dNd }, and denote the edge set E={ eij:di\u2208D,lj\u2208L }. Nd and Nl represent the number of diseases and the number of lncRNAs, respectively. Here, the lncRNA\u2013disease association can be represented by an adjacency matrix A={aij}Nd\u00d7Nl, where aij=1 if disease di and lncRNA lj have experimentally validated relation in the databases, while the unknown associations are set to 0 indicating that they will be ranked.We constructed lncRNA\u2013disease associations as a bipartite graph dj in the MeSH database, we constructed a directed acyclic graph DAG(dj) based on the MeSH descriptors of Category C downloaded from the National Library of Medicine. For example, V(DAG(dj)) indicated the vertex set including vertex dj and its ancestor vertices, and E(DAG(dj)) was the edge set of corresponding direct links from a parent vertex to a child vertex, which represented the relationship between different diseases.For every disease term dj, in the DAG(dj), the contribution of each disease semantic term Cdj(di) of disease di was defined as follows  \u2229\u200b DAG(dj) should have a higher semantic similarity. Thus, the semantic score of disease dj was acquired by adding up all the contributions from ancestor diseases and disease dj itself. Define the semantic score (C) of disease dj as follows:According to this way to measure disease semantic similarity, we thought that two diseases SS) between disease di and disease dj can be written as  between disease di and disease dj.Based on the basic assumption that two lncRNAs with more functional similarity prefer to be more related to similar diseases and ce versa , we coulce versa . Then weIP(di) was the i-th column of matrix A. The parameter \u03b3d was a parameter for adjusting the bandwidth of the kernel, which should be updated by using a new bandwidth parameter where us study , \u03b3d\u2019wa\u03b3d could be defined as follows:Thus, DS) between disease di and disease dj as follows:Define the disease ensemble similarity  and D(lj) be the set of diseases related to lncRNA li and lncRNA lj, respectively. Define similarity score S between D(li) and D(lj) as follows:Let vice versa  | and | D(lj) | were the numbers of diseases associated with lncRNA li and lncRNA lj, respectively.where | li and lncRNA lj was defined as follows  between lncRNA li and lncRNA lj as follows:Define the lncRNA ensemble similarity  of the dj vertex was shown as follows:First of all, we selected one disease dj vertex returned back to L by LA and DA. Then the final comprehensive index (resources) of the li vertex as shown below:Each disease scattered the received resources to its associated lncRNAs, the resources located on the LA and DA. Therefore, for a given disease Du, we could obtain the comprehensive index IDLDA-score of every lncRNA. Accordingly, we got the predicted ranks of all lncRNAs for every disease. This predicted result can be represented by a rank matrix R={rij}Nd\u00d7Nl, where rij indicated the relevance score between disease di and lncRNA lj. The larger the value of rij, the more likely disease di and lncRNA lj are to be related. Thus, IDLDA can predict not only new disease-related lncRNAs but new lncRNA-related diseases. The flow chart of IDLDA is shown in Here the parameters \u03b1, \u03b2 were used to balance the contribution between In this section, we first analyzed some properties of the lncRNA\u2013disease association network. Next, we used global and local cross-validations and performed enrichment analysis to evaluate the performance of IDLDA. Then, we conducted case studies to verify the efficiency of IDLDA in discovering some potential disease-related lncRNAs.We analyzed the lncRNA\u2013disease association network\u2019s characteristics to obtain a whole view of it Table 1.A receiver operating characteristic (ROC) curve is a graphical plot that shows the diagnostic ability of the binary classifier system because its recognition thresholds are different . AUROC /(MN) , where mCase studies were implemented to examine the capability of IDLDA in discovering potential lncRNA\u2013disease associations. For some special diseases, we ranked those candidate lncRNAs based on their corresponding IDLDA-scores. Case studies included three common human diseases . Prediction results were verified based on not only the recent updates in the Lnc2Cancer and LncRNADisease but recently published experimental literature. Then we observed the number of the verified lncRNAs in the top 10 and 50 predictions in both databases, all the ranking results have been listed in Colon cancer is one of the most common malignant tumors in the world , killingBreast cancer is the second leading cause of cancer deaths in women, accounting for 22% of all cancer deaths in women . Some reGastric cancer is the second major reason for cancer-related death in the world . A myriaAccording to previous literature, lncRNAs are associated with a mass of diseases. With the emergence of many biological data about lncRNA, it is urgent to design a powerful and effective computing method to predict the underlying disease-related lncRNAs. In this paper, disease semantic similarity, lncRNA functional similarity, disease/lncRNA Gaussian kernel similarity, and lncRNA\u2013disease associations were integrated on a large scale. We developed a computational model named IDLDA, which based on the diffusion model to predict potential lncRNA\u2013disease associations. IDLDA achieved higher AUROC than other methods in the combined dataset. Meanwhile, local cross-validation, enrichment analysis could also show the reliability of the model. Moreover, case studies of colon cancer, breast cancer, and gastric cancer were also implemented, all lncRNAs which ranked top 10 in both databases were verified, only 2, 2, and 1 lncRNAs which ranked top 50 in both databases were not confirmed by databases and related literature. What is more, the results of local cross-validation showed IDLDA can predict not only new disease-related lncRNAs but new lncRNA-related diseases.Here are the reasons why IDLDA performs better than some aforementioned methods. Firstly, the lncRNA ensemble similarity and disease ensemble similarity can make full use of the information about known lncRNA\u2013disease associations by integrating lncRNA functional similarity, disease semantic similarity, and the Gaussian kernel similarity. Secondly, both disease ensemble similarity and lncRNA ensemble similarity are used in the diffusion process, IDLDA could predict not only new lncRNAs but also new diseases, overcoming some limitations of previous methods. Thirdly, IDLDA as a semi-supervised method is superior to the supervised methods when the data is incomplete. In particular, semi-supervised method could be implemented without any negative lncRNA\u2013disease associations, which are closer to reality. In short, IDLDA will be an important and powerful bioinformatics tool in biomedical research of the lncRNA\u2013disease association prediction, and even disease treatment.Although IDLDA is effective, this work has several limitations. Firstly, IDLDA contains two parameters, and finding suitable parameters for different datasets is a challenging task. Additionally, some specific lncRNAs are not associated with certain diseases. If this kind of data can be added to the model in the future, it will certainly be helpful to improve the predictive ability. Successfully established models in the other computational fields would inspire the development of lncRNA\u2013disease association prediction. Perhaps we can improve the predictive performance of IDLDA by integrating more information, such as lncRNA\u2013miRNA information  and disehttp://www.cuilab.cn/lncrnadisease , http://www.bio-bigdata.net/lnc2cancer. Publicly available datasets were analyzed in this study. This data can be found here: QW conceived the project, developed the prediction method, designed the experiments, implemented the experiments, analyzed the result, and wrote the paper. GY analyzed the result and revised the paper.GY was supported by the National Natural Science Foundation of China under Grant No. 11631014.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Long non-coding RNAs (lncRNAs) play a crucial role in the pathogenesis and development of complex diseases. Predicting potential lncRNA\u2013disease associations can improve our understanding of the molecular mechanisms of human diseases and help identify biomarkers for disease diagnosis, treatment, and prevention. Previous research methods have mostly integrated the similarity and association information of lncRNAs and diseases, without considering the topological structure information among these nodes, which is important for predicting lncRNA\u2013disease associations. We propose a method based on information flow propagation and convolutional neural networks, called LDAPred, to predict disease-related lncRNAs. LDAPred not only integrates the similarities, associations, and interactions among lncRNAs, diseases, and miRNAs, but also exploits the topological structures formed by them. In this study, we construct a dual convolutional neural network-based framework that comprises the left and right sides. The embedding layer on the left side is established by utilizing lncRNA, miRNA, and disease-related biological premises. On the right side of the frame, multiple types of similarity, association, and interaction relationships among lncRNAs, diseases, and miRNAs are calculated based on information flow propagation on the bi-layer networks, such as the lncRNA\u2013disease network. They contain the network topological structure and they are learned by the right side of the framework. The experimental results based on five-fold cross-validation indicate that LDAPred performs better than several state-of-the-art methods. Case studies on breast cancer, colon cancer, and osteosarcoma further demonstrate LDAPred\u2019s ability to discover potential lncRNA\u2013disease associations. Many studies have indicated that protein-coding genes only account for ~2% of the human genome, whereas non-coding protein sequences account for ~98% ,2,3,4,5.The calculation methods employed for predicting potential lncRNA\u2013disease associations can be broadly divided into three categories. The first method uses the biological information of lncRNA to identify lncRNA\u2013disease associations, such as the expression profile, tissue specificity, and genome location. Li et al.  predicteThe second method uses machine learning models to predict the potential associations. Chen et al.  proposedThe third method establishes heterogeneous networks, based on which lncRNA\u2013disease associations can be predicted. Zhang et al.  construcIn this study, we propose a method, called LDAPred, based on information flow propagation and a convolutional neural network, to predict potential lncRNA\u2013disease associations. LDAPred utilises the similarities, associations, and interactions among lncRNAs, miRNAs, and diseases. On the left side of the network, the original feature matrix of lncRNA\u2013disease node pairs was constructed from the biological perspective. On the right side, according to the information flow propagation of the bi-layer network formed by lncRNA, miRNA, and disease, the possibility of interconnections between them was calculated, and the characteristic matrix was formed. Dual convolution was used to learn deeper features and make association predictions. Combined with five-fold cross-validation experiments, the results indicate that LDAPred is better than several existing methods for the prediction of candidate lncRNAs. Moreover, the results of the case study on breast cancer, colon cancer, and osteosarcoma also indicate that LDAPred has a strong ability to identify potential disease lncRNAs.To achieve the best prediction result, we repeatedly verified the results by conducting experiments. Finally, the filter used in the convolutional layer and the pooling layer in the dual channel system was set to the dimension of To evaluate the performance of the prediction model, we used five-fold cross-validation. First, the known 2687 lncRNA\u2013disease associations were divided into five groups, four of which were used as the training set and one as the test set. Second, we deleted the association in the test set when calculating the similarity of lncRNAs. We regarded those with lncRNA-related diseases in the test set as positive cases and those without any association as negative cases.TP is the number of positive samples that are considered positive, and TN is the number of counterexamples that are considered counterexamples. FN is the number of positive examples that are considered counterexamples, and FP is the number of counterexamples that are considered positive examples. Finally, the average of all disease AUCs was taken to represent the performance of the predictive model. The higher the value, the higher the global performance of the model.After using our prediction model to evaluate the associated scores of the test samples, the scores of the samples were ranked in descending order. The higher the ranking of the positive examples, the better the prediction performance of the model. We measured the global performance of our prediction model by drawing the receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). The true positive rate (TPR) and false positive rate (FPR) can be defined as follows:Because the lncRNA\u2013disease sample has a number of associated positive examples that are smaller than the unrelated or unrecognized counterexamples, there is a serious imbalance ratio. Therefore, we also used the precision\u2013recall (PR) curve to measure the overall performance of the model. The larger the area under the PR curve (AUPR), the better the prediction performance. The precision and recall can be calculated as follows:k  samples, i.e., the ratio of the positive samples in the first k samples to all the predicted positive samples, as another performance index.Biological experiments are costly and time-consuming and limited by equipment precision and human error; thus, biologists choose to predict the top lncRNA to verify the disease associated with it. Therefore, we also calculated the recall rate of the first To reveal the advantages of considering network topology information in lncRNA\u2013disease association prediction modeling and demonstrate the strong performance of our model, we selected four latest lncRNA\u2013disease association prediction methods, namely SIMCLDA , Ping\u2019s As shown in As shown in p-value of 0.05. In addition, to assess whether the AUC performance of LDAPred for all 405 diseases is better than those of the other four methods, we performed a paired Wilcoxon test. The statistical results are shown in k lncRNAs, the greater the number of correctly identified lncRNAs that are related to the disease. k samples of all 405 diseases. LDAPred is superior to the other methods at different k values, accounting for 86.4% in the top 30, 92.8% in the top 60, 95.1% in the top 90, and 96.3% in the top 120. The recall rate of Ping\u2019s method is very close to that of LDAP. The former accounts for 68.9%, 81.2%, 87.5%, and 92.7% among the top 30, 60, 90, and 120, whereas the latter accounts for 68.5%, 81.7%, 88.0%, and 93.3%, respectively. SIMCLDA accounts for 49.3% in the top 30, 63.0% in the top 60, 74.1% in the top 90, and 80.3% in the top 120, exhibiting lower values than Ping\u2019s method and LDAP. Compared to the four methods, MFLDA always shows the worst performance, accounting for 42.0%, 53.9%, 60.9%, and 65.5%, respectively.The higher the recall rate of the top To further demonstrate the LDAPred\u2019s ability to detect disease-related lncRNAs, we used two separate databases (Lnc2Cancer and lncRNADisease) and related literature to validate candidate genes for breast cancer, colon cancer, and osteosarcoma. The top 15 candidate lncRNAs associated with these cancers were analysed separately .Lnc2Cancer is an experimentally supported lncRNA manual management database for various human cancers . It contThe remaining three candidates reported in previous studies are marked as the \u201cliterature\u201d in To predict the relationships between lncRNAs and diseases, we needed to integrate the attributes and characteristics of each node of the lncRNAs, miRNAs, and diseases. Therefore, we downloaded 2687 lncRNA\u2013disease associations from the LncRNADisease  and Lnc2A disease can be expressed as a directed acyclic graph (DAG), which can be obtained from Medical Subject Headings (MeSH), and it includes all relevant annotated items of the disease. Studies have shown that the more common the DAG of two diseases, the more similar the two diseases. Wang et al.  measuredThe more similar the functions of two lncRNAs, the more similar the related diseases. Therefore, we calculated the similarity of two lncRNAs by calculating the similarity of the two lncRNA-associated diseases. For example, lncRNA n et al. , the simSimilar to the lncRNA similarity calculation, the miRNA similarity was calculated based on the associated diseases. We used the matrix A, B, C, In this study, heterogeneous data resources were synthesized and the interaction matrix was established: the lncRNA\u2013disease association matrix We constructed a dual convolutional neural network (CNN) predictive model to predict the lncRNA\u2013disease associations. The left side uses the original information of the lncRNA L, and the association vector A, and combined them together. Second, if C, vector C, and We utilized lncRNA d+nl+nm) .Inspired by Chen et al. , we consL represents the original information between lncRNA nodes; i.e., the one-hop similarity information. In a network comprising lncRNAs, Similarly, In a network comprising lncRNAs and diseases, A represents the one-hop information between lncRNA and disease node pairs, and Similarly, the association information between the disease and miRNA is expressed by In the network composed of lncRNA and miRNA, B represents the original interaction information between lncRNA and miRNA node pairs, i.e., the one-hop information, and Finally, we took the second row of matrix g matrix .S. In addition, to fully learn the edge information, we applied wide convolution by padding zeros before convolution. The definitions of ith row and jth column element of the embedded layer S, and kth filter is slid to position Because the left and right convolution processes are similar, we will only describe the left convolution process in detail herein. In the pooling layer, After two convolutions and pooling were completed, we obtained the final representation H is the weight matrix between the fully connected layer and the output layer, and Finally, we flattened K is the weight matrix between the fully connected layer and the output layer, and Similarly, we employed To fully utilize the dual prediction score matrix, we designed a dual combination strategy to train the model and obtain the final prediction score. We used M represents the number of training samples, and a and b represent the probabilities obtained by the Softmax function. The dual convolution and combining processes are displayed in The loss functions of the left and right CNNs can be defined asLDAPred, which is a new method based on a dual convolutional neural network, was developed to predict the potential associations between lncRNAs and diseases. According to the biological premise that lncRNAs are likely to possess associations with diseases, the embedding layer was established from a biological perspective. The left and right embedding layers capture the original similarities, associations, and interactions among lncRNAs, miRNAs, and diseases, as well as the topological structures of bi-layer networks. The original representation of lncRNA\u2013disease pairs and their network representations were learned by the new framework based on dual convolutional neural networks and information flow propagation. Cross-validation results for 405 diseases and case studies on three diseases indicated that LDAPred has a strong ability to predict potential associations between lncRNAs and diseases."}
+{"text": "Long non-coding RNAs play an important role in human complex diseases. Identification of lncRNA-disease associations will gain insight into disease-related lncRNAs and benefit disease diagnoses and treatment. However, using experiments to explore the lncRNA-disease associations is expensive and time consuming.Integrating Diverse Heterogeneous Information sources with positive pointwise Mutual Information and Random Walk with restart algorithm (namely IDHI-MIRW). IDHI-MIRW first constructs multiple lncRNA similarity networks and disease similarity networks from diverse lncRNA-related and disease-related datasets, then implements the random walk with restart algorithm on these similarity networks for extracting the topological similarities which are fused with positive pointwise mutual information to build a large-scale lncRNA-disease heterogeneous network. Finally, IDHI-MIRW implemented random walk with restart algorithm on the lncRNA-disease heterogeneous network to infer potential lncRNA-disease associations.In this study, we developed a novel method to identify potential lncRNA-disease associations by https://github.com/NWPU-903PR/IDHI-MIRW.Compared with other state-of-the-art methods, IDHI-MIRW achieves the best prediction performance. In case studies of breast cancer, stomach cancer, and colorectal cancer, 36/45 (80%) novel lncRNA-disease associations predicted by IDHI-MIRW are supported by recent literatures. Furthermore, we found lncRNA LINC01816 is associated with the survival of colorectal cancer patients. IDHI-MIRW is freely available at The online version of this article (10.1186/s12859-019-2675-y) contains supplementary material, which is available to authorized users. Long non-coding RNAs (lncRNAs) are the biggest part of non-coding RNAs with at least 200 nucleotides and no observed potential to encode proteins , 2. To dIn recent years, the number of experimentally verified lncRNA-disease associations is gradually increasing. Several databases for lncRNA functions and disease associations have been published, such as LncRNAdb , LncRNADThe network-based methods, such as RWRlncD , RWRHLD To address the aforementioned issues (or limitations) and further improve the prediction accuracy, we proposed a novel network-based method, namely IDHI-MIRW, to predict the potential lncRNA-disease associations by constructing a large-scale lncRNA-disease heterogeneous network with Random Walk with Restart (RWR) algorithm and the positive pointwise mutual information (PPMI). Instead of constraining lncRNA and disease on those with at least one known lncRNA-disease association, IDHI-MIRW calculates the lncRNA similarities for all the lncRNAs involved in lncRNA expression profiles, lncRNA-miRNA interactions, and lncRNA-protein interactions, and also calculates the diseases similarities for all the diseases involved in disease ontology, disease-miRNA associations, and disease-gene associations. Then, IDHI-MIRW uses the RWR algorithm on each similarity network to capture network topological structural features for measuring the lncRNA/disease topological similarity through the PPMI. By integrating the lncRNA/disease topological similarity, and introducing the known lncRNA-disease association information, a large-scale lncRNA-disease heterogeneous network is built. Finally, the random walk with restart on heterogeneous network (RWRH) algorithm  is appliIn this section, we first introduced the evaluation method and metrices for evaluating the performance of the IDHI-MIRW method. Then, we compared our IDHI-MIRW method with other existing state-of-the art methods on a small-scale lncRNA-disease heterogeneous network, explored the predictive power of IDHI-MIRW on a large-scale lncRNA-disease heterogeneous network, and discussed the effect of different parameters. In the end, we analyzed several predicted potential lncRNA-disease associations with our IDHI-MIRW.di, each known lncRNA associated with di is left out in turn as a test sample, and corresponding association edge between test lncRNA and di is removed, and the remaining lncRNAs associated with di are considered as training samples.The leave-one-out cross validation (LOOCV) test method was used to evaluate the performance of the IDHI-MIRW method. In LOOCV test method, each known lncRNA-disease association in the dataset is singled out in turn as a test sample, and the remaining lncRNA-disease associations are used as training samples. That is, for a given disease The area under the receiver operating characteristic (ROC) curve (AUC) and the area under the precision-recall (PR) curve (AUPR) were used as evaluation metrices in our experiments. The ROC curve is the plot of the true-positive rate  versus the false-positive rate (FPR) at different rank cutoffs. The PR curve is the plot of the ratio of true positives among all positive predictions for each given recall rate.S) which contains 362 lncRNAs, 370 diseases, and 2169 known lncRNA-disease associations. Most existing methods often built this small-scale lncRNA-disease heterogeneous network in which each lncRNA (or disease) has at least an associated disease (or lncRNA) to predict the potential lncRNA-disease associations. LRLSLDA [We compared our IDHI-MIRW method with other six state-of-the-art methods of LRLSLDA , LNCSIM  LRLSLDA  and LNCS LRLSLDA  adopt th LRLSLDA , IRWRLDA LRLSLDA , KATZLDA LRLSLDA  and GrwL LRLSLDA  are the To further evaluate the performance of IDHI-MIRW for predicting the associated lncRNAs for new diseases without any known lncRNA association information, we removed all the known lncRNA associations for the query disease in the small-scale lncRNA-disease heterogeneous network. Due to RWRlncD implemented the RWR algorithm on an lncRNA similarity network, we just compared our IDHI-MIRW method with other five methods of LRLSLDA, LNCSIM, IRWRLDA, KATZLDA and GrwLDA for predicting the associated lncRNAs of the query diseases. The comparison results are shown in Fig.\u00a0L) by introducing 2169 known lncRNA-disease associations, then implemented our IDHI-MIRW method on HNetL. Additional\u00a0files\u00a0S and HNetL heterogeneous networks in LOOCV test are listed in Table\u00a0In order to illustrate the effectiveness of introducing multiple information sources, we collected 7637 lncRNAs and 6453 diseases from EMBL-EBI E-MTAB-5214), starBase v2.0 , NPInter4, starBaS) and large-scale ncRNA-disease heterogeneous network (HNetL) in LOOCV test are shown in Table\u00a0In order to evaluate the effectiveness of using the topological similarity network to construct the lncRNA-disease heterogeneous network for improving the predictive performance, we designed another method of IDHI-AVG by adopting the strategy of averaging three lncRNA similarity matrices of LncNet1, LncNet2 and LncNet3 to form the lncRNA integration network , averaging of three disease similarity matrices of DisNet1, DisNet2, and DisNet3 to form the disease integration network . IDHI-AVG combines these two integration similarity networks of LncINet and DisINet with known lncRNA-disease bipartite network to construct the lncRNA-disease heterogeneous network on which RWRH algorithm is implemented to predict the potential lncRNA-disease associations. The compared results of IDHI-AVG and IDHI-MIRW on the small-scale lncRNA-disease heterogeneous network . Additional\u00a0file\u00a0S heterogeneous network in LOOCV test. In this work, we selected \u03b1\u2009=\u20090.9, \u03b3\u2009=\u20090.9, \u03b7\u2009=\u20090.2, and \u03b2\u2009=\u20090.6.There are four main parameters in our method, which are the restart probability We used breast cancer, stomach cancer, and colorectal cancer as the cases to predict their potential associated lncRNAs with our IDHI-MIRW. For a given disease, all known lncRNAs associated with this given disease were considered as the seed nodes, and other remaining lncRNAs  were considered as the candidates associated with the given disease. By implementing our IDHI-MIRW algorithm on the large-scale lncRNA-disease heterogeneous network, and according to the lncRNA-disease associations ranking scores from large to small, we extract top 15 potential association lncRNAs for each cancer. These top potential association lncRNAs are listed in Additional\u00a0files\u00a0For breast cancer which is one of most common cancers and the second leading cause of cancer death , 13 out For stomach cancer (or gastric cancer) which is the third leading cause of cancer mortality in the world , 54, 11 For colorectal cancer which is the third most commonly diagnosed cancer in males and the second in females , 12 out To further discover the evidences for the predicted lncRNAs associated with cancers, we analyzed the RNAseq and clinical data from TCGA for breast cancer, stomach cancer and colorectal cancer. For colorectal cancer, the RNASeq data including 19,676 protein coding genes, 15,513 lncRNA genes in 41 normal samples and 474 tumor samples were downloaded from TCGA. Using DESeq2  algorithAdditional\u00a0files\u00a0In summary, 36  out of 45 potential association lncRNAs have been supported by recent literatures. By analyzing the nine unvalidated potential association lncRNAs, we found that six lncRNAs are differentially expressed in corresponding cancers, and lncRNA LINC01816 is associated with the survival of patients with colorectal cancer. Results of these three case studies show that IDHI-MIRW can effectively predict the new association lncRNAs for a disease.LncRNAs play important roles in the development of human complex diseases. More and more attentions have been paid to discover the lncRNA functions related with human complex disease. Most previous computational methods only focus on the small-scale lncRNA-disease heterogeneous network  to predict the lncRNA-disease associations. To address this issue, IDHI-MIRW was developed to predict the potential lncRNA-disease associations based on a large-scale lncRNA-disease heterogeneous network (containing 7637 lncRNAs and 6453 diseases). Instead of calculating similarities of lncRNAs and diseases only involving in known lncRNA-disease associations, IDHI-MIRW used three lncRNA-related information  to form three lncRNA similarity networks, and three disease-related information  to form three disease similarity networks. Furthermore, instead of directly fusing those similarity networks, IDHI-MIRW applied the RWR algorithm on each lncRNA/disease similarity network to capture the topological similarity, and the PPMI to generate lncRNA/disease topological similarity network. The large-scale lncRNA-disease heterogeneous network was constructed by combing the lncRNA topological similarity network, disease topological similarity network, and the known lncRNA-disease bipartite graph. Then, the RWRH algorithm was used to prioritize candidate lncRNAs for each query disease. Our experiment results show that IDHI-MIRW achieves a better performance than other existing methods. We evaluated the effectiveness of introducing multiple information sources and capturing topological similarities, Tables\u00a0Although IDHI-MIRW can effectively predict potential lncRNA-disease associations, there are still several issues need to be further addressed in the future. First, IDHI-MIRW used three lncRNA-related and three disease-related information to generate similarity matrices, we still expect to integrate more information  to better predict lncRNA-disease association. Second, the averaging strategy was used to integrate the lncRNA/disease topological similarity matrices, we expect to design better integration approaches in future work to measure the different contributions of multiple lncRNA/disease similarities.In this study, we proposed a novel network-based method (namely IDHI-MIRW) for identifying potential lncRNA-disease associations. We built a large-scale lncRNA-disease heterogeneous network by integrating multiple lncRNA-related information , multiple disease-related information , and known lncRNA-disease association information using RWR and PPMI. Our experimental results show that IDHI-MIRW can achieve higher performance than other state-of-the-art methods, and we found lncRNA LINC01816 is associated with the survival of colorectal cancer patients. These results indicate that IDHI-MIRW will contribute to the identification of potential lncRNA-disease associations.We collected lncRNA expression profile, lncRNA-miRNA interaction, and lncRNA-protein interaction data for constructing the lncRNA similarity networks, and Diseases Ontology (DO) information, disease-miRNA association, and disease-protein association data for constructing the disease similarity networks. All lncRNAs are annotated by ensembl gene ID, and all diseases are annotated by Disease Ontology ID.LncRNA expression profiles were downloaded from EMBL-EBI (E-MTAB-5214), which includes the expression profiles in 53 human tissue samples. LncRNA-miRNA interactions and lncRNA-protein interactions were collected from starBase v2.0 , NPInterOur IDHI-MIRW algorithm consists of the following four steps. Step 1, build three lncRNA similarity networks  based on lncRNA expression profiles, lncRNA-miRNA interactions, and lncRNA-protein interactions, and also build three disease similarity networks  based on disease ontology, disease-miRNA associations, and disease-gene associations. Step 2, form the lncRNA topological similarity network (LncTSNet) and disease topological similarity network (DisTSNet) by fusing lncRNA and disease multiple topological similarities obtained through implementing RWR on lncRNA similarity network  and disease similarity network , respectively. Step 3, construct a large-scale lncRNA-disease heterogeneous network by integrating lncRNA topological similarity network (LncTSNet), disease topological similarity network (DisTSNet), and known lncRNA-disease associations. Step 4, implement RWRH on the lncRNA-disease heterogeneous network for predicting the potential lncRNA-disease associations. The flowchart of IDHI-MIRW is shown in Fig.\u00a0P-value threshold (<\u20090.01), we built the LncNet1 lncRNA similarity weighted network. Based on Gaussian interaction profile kernel similarity [li and lncRNA lj, then built the LncNet2 and LncNet3 lncRNA similarity weighted networks, respectively. Gaussian interaction profile kernel similarity between lncRNA li and lncRNA lj is calculated.IP(li) is the binary vector of lncRNA-miRNA (or lncRNA-protein) interactions encoding the presence or absence of interactions between lncRNA li and miRNA (or protein) in the lncRNA-miRNA (or lncRNA-protein) interaction dataset, \u03bal controls the kernel bandwidth, and Nl is the total number of lncRNAs.By calculating the Pearson correlation coefficient of any lncRNA pair with expression profiles and fixing the milarity , 63 of ldi and dj, then built the DisNet2 and DisNet3 disease similarity weighted networks, respectively.IP(di) is the binary vector of disease-miRNA (or disease-gene) associations encoding the presence or absence of associations between di and miRNA (or gene) in the disease-miRNA (or disease-gene) association dataset. \u03bad controls the kernel bandwidth, and Nd is the total number of diseases.Based on the structure of a directed acyclic graph (DAG) in Disease Ontology, we used the function \u201cdoSim\u201d form R package \u201cDOSE\u201d  to obtaiSt is the distribution matrix in which the -th element denotes the distribution probability of node j being visited from node i after t iterations in the random walk process and S0 is the initial distribution matrix in which S0\u2009=\u20091, S0\u2009=\u20090, \u2200j\u2009\u2260\u2009i.\u00a0\u03b1 is restart probability controlling the relative influence of local and global topological information. B is the weighted adjacency matrix of lncRNA (or disease).Instead of directly fusing six similarity networks , we captured the network topological structural features by implementing the RWR algorithm on each similarity network. The RWR algorithm is a network diffusion algorithm, which has been extensively applied to analyze the complex biological network \u201369. By cS\u2009=\u2009St\u2009+\u20091\u2009\u2212\u2009Stis less than a small positive \u03b5 (we set \u03b5\u2009=\u200910\u221210), we can obtain a stationary distribution matrix S, which was referred as the diffusion state of each node [S in diffusion state matrix S represents the probability of RWR starting node i and ending up at node j in equilibrium. When the diffusion states of two nodes are close, which suggests that they may have similar positions with respect to other nodes in the network and they probably share similar functions.When the L1 norm of \u0394ach node . The eleMotivated by Gligorijevic et.al. , we thenMI is a non-symmetric matrix, thus we use the average of MI and MI to represent the topological similarity of node i and node j. After obtaining three lncRNA topological similarity matrices The matrix AL and AD represent the weighted adjacency matrices of LncTSNet and DisTSNet, respectively; ALD is the adjacency matrix of the lncRNA-disease bipartite graph; ADL represents the transpose of ALD. If there is association between lncRNA i and disease j in known lncRNA-disease associations, ALD\u2009=\u20091, otherwise, ALD\u2009=\u20090.By integrating the LncTSNet and DisTSNet networks with known lncRNA-disease bipartite network, we can construct the lncRNA-disease heterogeneous network whose adjacency matrix can be defined as:pt is a probability vector in which the i-th element holds the probability of finding the random walker at node i at step t; \u03b2\u2009\u2208\u2009 is restart probability; p0 is the initial probability vector for lncRNA-disease heterogeneous network which is defined as u0 and v0 represent the initial probability of LncTSNet and DisTSNet, respectively. The initial probability u0 of LncTSNet network is set such that all the seed nodes are assigned to the equal probabilities with the sum of probabilities equal to 1. Similarity, the initial probability v0 of DisTSNet network is given. The parameter \u03b7\u2009\u2208\u2009 is used to weight the importance of each subnetwork.To predict the association between lncRNA and disease, we adopted the RWRH  algorithm  to priorML and MD are the intra-subnetwork transition matrices, MLD and MDL are the inter-subnetwork transition matrices. Let \u03b3 be the jumping probability, that is, the probability of random walker jumping from lncRNA network to disease network or vice versa. Thus, the transition probability ML from lncRNA li to lncRNA lj and the transition probability MD\u2009 from disease di to disease dj are defined asli to disease dj and the transition probability from disease di to lncRNA lj are described as:The transition probability from lncRNA p\u2217\u2009=\u2009p\u221e can be obtained by performing the iteration until the difference between pt and pt\u2009+\u20091 (measured by the L1 norm) fall below 10\u221210. p\u2217 gives the ranking score of every lncRNA for a query disease. The lncRNAs with maximum in p\u2217 are considered as the most probable associated lncRNAs of the query disease.After some steps, the steady state probability vector Additional file 1:LncRNA data processing procedure. (TIF 1447 kb)Additional file 2:Disease data processing procedure. (TIF 1340 kb)Additional file 3:\u03b1. (B) AUC values with different \u03b3. (C) AUC values with different \u03b7. (D) AUC values with different \u03b2. (E) AUPR values with different \u03b1. (F) AUPR values with different \u03b3. (G) AUPR values with different \u03b7. (H) AUPR values with different \u03b2. (TIF 3520 kb)AUPR values of IDHI-MIRW on the large-scale lncRNA-disease heterogeneous with different parameters in LOOCV test. (A) AUC values with different Additional file 4:\u03b1. (B) AUC values with different \u03b3. (C) AUC values with different \u03b7. (D) AUC values with different \u03b2. (E) AUPR values with different \u03b1. (F) AUPR values with different \u03b3. (G) AUPR values with different \u03b7. (H) AUPR values with different \u03b2. (TIF 3705 kb)AUC and AUPR values of IDHI-MIRW on the small-scale lncRNA-disease heterogeneous with different parameters in LOOCV test. (A) AUC values with different Additional file 5:The top 15 predicted associated lncRNAs for breast cancer. (XLSX 9 kb)Additional file 6:The top 15 predicted associated lncRNAs for stomach cancer. (XLSX 9 kb)Additional file 7:The top 15 predicted associated lncRNAs for colorectal cancer. (XLSX 9 kb)Additional file 8:The results of RNASeq data analysis for breast cancer. (A) heatmap of top 200 most significantly dysregulated lncRNA expression values. (B) heatmap of lncRNA AL157395.1 expression values. (C) boxplot of lncRNA AL157395.1 expression in normal and tumor samples. (D) heatmap of lncRNA AP001528.1 expression values. (E) boxplot of lncRNA AP001528.1 expression in normal and tumor samples. (TIF 9850 kb)Additional file 9The results of RNASeq data analysis for stomach cancer. (A) heatmap of top 200 most significantly dysregulated lncRNA expression values. (B) heatmap of lncRNA KCNQ1OT1 expression values. (C) boxplot of lncRNA KCNQ1OT1 expression in normal and tumor samples. (D) heatmap of lncRNA DLEU2 expression values. (E) boxplot of lncRNA DLEU2 expression in normal and tumor samples. (F) heatmap of lncRNA LINC00299 expression values. (G) boxplot of lncRNA LINC00299 expression in normal and tumor samples. (TIF 9211 kb)Additional file 10:The predicted lncRNA-disease associations. (TXT 180 kb)Additional file 11:Details and statistics of collected data. (DOCX 34 kb)"}
+{"text": "Current studies have shown that long non-coding RNAs (lncRNAs) play a crucial role in a variety of fundamental biological processes related to complex human diseases. The prediction of latent disease-lncRNA associations can help to understand the pathogenesis of complex human diseases at the level of lncRNA, which also contributes to the detection of disease biomarkers, and the diagnosis, treatment, prognosis and prevention of disease. Nevertheless, it is still a challenging and urgent task to accurately identify latent disease-lncRNA association. Discovering latent links on the basis of biological experiments is time-consuming and wasteful, necessitating the development of computational prediction models. In this study, a computational prediction model has been remodeled as a matrix completion framework of the recommendation system by completing the unknown items in the rating matrix. A novel method named faster randomized matrix completion for latent disease-lncRNA association prediction (FRMCLDA) has been proposed by virtue of improved randomized partial SVD (rSVD-BKI) on a heterogeneous bilayer network. First, the correlated data source and experimentally validated information of diseases and lncRNAs are integrated to construct a heterogeneous bilayer network. Next, the integrated heterogeneous bilayer network can be formalized as a comprehensive adjacency matrix which includes lncRNA similarity matrix, disease similarity matrix, and disease-lncRNA association matrix where the uncertain disease-lncRNA associations are referred to as blank items. Then, a matrix approximate to the original adjacency matrix has been designed with predicted scores to retrieve the blank items. The construction of the approximate matrix could be equivalently resolved by the nuclear norm minimization. Finally, a faster singular value thresholding algorithm with a randomized partial SVD combing a new sub-space reuse technique has been utilized to complete the adjacency matrix. The results of leave-one-out cross-validation (LOOCV) experiments and 5-fold cross-validation (5-fold CV) experiments on three different benchmark databases have confirmed the availability and adaptability of FRMCLDA in inferring latent relationships of disease-lncRNA pairs, and in inferring lncRNAs correlated with novel diseases without any prior interaction information. Additionally, case studies have shown that FRMCLDA is able to effectively predict latent lncRNAs correlated with three widespread malignancies: prostate cancer, colon cancer, and gastric cancer. Long non-coding RNAs are RNA molecules whose transcripts are not less than 200 nucleotides, including intronic/exonic lncRNAs, antisense lncRNAs, overlapping lncRNA and long intergenic ncRNAs (lincRNAs). LncRNAs have long been considered as transcriptional noise, because of their absence in encoding proteins. Recently, it has been found that some lncRNAs regulate the expression of target genes after transcription, whose malfunction may lead to a number of diseases. For example, abnormal lncRNA expression may be involved in certain stages of cancer progression, which can serve as a potential biomarker for early tumor diagnosis . In addiIn the first major category, models for single-interaction data sources are based on diseases-lncRNAs interaction (association/link) data, which is unique known interaction information. According to its method, the model can be divided into two minor branches. The first minor branch is composed of machine-learning based models, in which the prediction of latent disease-lncRNA association takes experimentally validated disease-lncRNA associations as labeled data (training set) and unknown associations as unlabeled samples . For example, a method named Laplacian Regularized Least Squares (LRLSLDA) was first proposed by Chen et al. to infer disease-lncRNA associations with a semi-supervised learning model . It is aAdditionally, the second minor branch is composed of network-based models, random walk and a variety of propagation algorithms implemented on a heterogeneous network to infer latent disease-lncRNA associations. The heterogeneous network is constructed by integrating lncRNA-disease interaction network, disease similarity network and lncRNA similarity network. For instance, based on the hypothesis that functional lncRNAs are associated with diseases with similar phenotypes, a lncRNA functional similarity network (LFSN) is constructed and a novel computational framework RWRlncD is proposed for predicting latent disease-lncRNA associations through random walk with restart . HoweverDue to the fact that known experimentally validated disease-lncRNA interactions are still rare, the second major category of computational models is models based on multi-interaction data sources proposed for association prediction. Multi-interaction data sources, such as lncRNA-gene interaction, lncRNAs-miRNAs interaction, disease-gene interaction and miRNA-disease interaction, are also included to infer latent disease-lncRNA associations. For example, a TPGLDA method is proposed to identify the underlying relationships by a tripartite graph of disease-lncRNA-gene and to develop an efficient resource allocation algorithm in the graph . TPGLDA Inferring latent disease-lncRNA association can also be modeled as a recommendation system which recommends top-ranked lncRNAs for given diseases. Based on matrix completion, the establishment of the disease-lncRNA recommendation system aims to complete unknown terms in the association matrix according to its ranked scores. Similar to the hypothesis in the user-item recommendation system that users with similar behaviors prefer similar items, the prediction of disease-lncRNA association assumes that phenotypically similar diseases tend to interact with functionally similar lncRNAs . In our Our work main contributions are threefold: first, the integrated similarities for diseases and lncRNAs were properly calculated by different methods dealing with different types of data sources. The ratio of cosine similarity in integrated similarity was better determined by learning, which extracted similarity information based on known disease-lncRNA interaction. Therefore, FRMCLDA was able to offset biased predictions by similarity integration which was not entirely dependent on known interaction. Second, diseases and lncRNAs were mapped into the same network by constructing a heterogeneous bilayer network. FRMCLDA completed the disease-lncRNA interaction matrix by completing the adjacency matrix of the large-scale heterogeneous network. Thus, the similar information was included in the association prediction. Third, we took advantage of an effective fSVT algorithm which adopted rSVD-BKI and a novel subspace reuse technique to expeditiously approximate the dominant singular values and homogeneous singular vectors in an adaptive manner. Hence, the recommendation system could be extended for comprehensive adjacency matrices of heterogeneous bilayer networks.For evaluating the performance of our method, cross validation experiments were performed on three benchmark databases, Dataset 1, Dataset 2 and Dataset 3. FRMCLDA obtained reliable AUCs of 0.92068, 0.91224 in global LOOCV and local LOOCV respectively in Dataset1, at least 5% higher than other comparison models. In Dataset 2 and Dataset 3, FRMCLDA achieved an AUC of 0.9182 and 0.8999 by global 5-fold CV, higher than other comparison methods. In addition, a case study on inferring latent lncRNAs associated with prostate cancer, colon cancer and gastric cancer in Dataset 3 were performed. In terms of the results, 16, 15, 16 out of the top 20 predicted lncRNAs associated with prostate cancer, colon cancer and gastric cancer respectively were confirmed by recent literature and public databases. The results show that FRMCLDA is able to effectively infer the associations between diseases and lncRNAs with higher accuracy than the other existing models.id is associated with lncRNA jl, the value of DL in the association matrix is 1. And if the link between id and jl is unknown or uncertain, DL is 0. It is noted that the unlinked evidence between id and jl is difficult to obtain. The known experimentally validated disease-lncRNA links can be retrieved from the public association database, based on which disease-lncRNA interaction matrix DL is established. If the number of nonzero elements is far smaller than that of zero elements, and the distribution of nonzero elements in the matrix is irregular, the matrix will be called sparse matrix. Generally, matrix DL is a sparse matrix, because, due to the insufficient number of studies, there are much more unknown associations  in matrix DL than known ones   see Table 1.In our work, a new method called FRMCLDA is proposed to infer latent disease-lncRNA associations on the basis of fast matrix completion. The mechanism of FRMCLDA is shown in All the known diseases-lncRNA interactions were obtained from three gold standard databases in three benchmark datasets respectively: MNDR database, Lnc2Cancer database and LncRNADisease database .The known associations between lncRNA and disease in Dataset 1 were retrieved from the MNDR database in 2015. After removing all the duplicate records of lncRNAs and diseases, and what do not belong to human beings, and correcting the names of the lncRNAs  and diseases , we finalized 352 disease-lncRNA associations, including 156 lncRNAs and 190 diseases.In Dataset 2, the known associations between lncRNA and disease were obtained from the Lnc2Cancer database in 2016. After eliminating the duplicate associations on account of different evidences, we obtained 540 distinct known disease-lncRNA associations, including 115 lncRNAs and 178 diseases.http://cmbi.bjmu.edu.cn/lncrnadisease) in 2015. In the same way as data preprocessing, we downloaded 621 known disease-lncRNA associations, including 248 lncRNAs and 226 diseases. The details of the three datasets are shown in In Dataset 3, the known disease-lncRNA associations were downloaded from the manually curated LncRNADisease database  is utilized to label a disease, which includes overall relevant annotation labels acquired from the U.S. National Library of Medicine (MeSH)  e (MeSH) . It is aontology .Disease functional similarity: Disease functional similarity was calculated using the Jaccard similarity coefficient on account of gene-gene ontology relationships and disease-gene relationship, as reported in previous studies , and can be calculated by formula (1):b)  studies . DiseaseGOdi represents the gene ontology terms related to disease di, and the symbol |\u00b7| represents the number of items in a set. where Disease cosine similarity: Widely used in information retrieval and data mining, the cosine similarity is a popular method for calculating the similarity as the cosine of the angle between vectors. Here we used cosine similarity to extract disease feature information from the known interaction matrix DL. The disease cosine similarity denoted as DS\u2013cosinecan be calculated by formula (2):c) IP(id) is the interaction profile of disease id, the i-th row vector of the interaction matrix DL. If disease id is associated with lncRNA kl, the k-th element in IP(id) is 1, otherwise the value is 0. The value 0 does not mean that association does not exist but means it is uncertain. ||IP(id)|| is the 2-norm of IP(id). where Integrated disease similarity DS: To illustrate the adaptability of our model to different similarity data, we adopted two different integrated disease similarities in three benchmark datasets. In Dataset 1 and Dataset 2, DS was calculated by: DS was calculated by: d) To calculate lncRNA integrated similarity, we adopted four different sources of similarity data: lncRNA sequence similarity, lncRNA expression similarity, lncRNA functional similarity and lncRNA cosine similarity.LncRNA sequence similarity: Most of the RNA sequences of lncRNAs were downloaded mainly from the database LncRNADisease (http://www.cuilab.cn/lncrnadisease). The sequences not available in LncRNADisease were retrieved from the databases UCSC and LNCipedia. The sequence similarity between two lncRNAs were calculated with Needleman-Wunsch global alignment algorithm (Emboss-Needle tool) (a) le tool) . We set SW is the alignment score calculated by Emboss-Needle, which is equal to the sum of the matches taken from the scoring matrix, minus penalties arising from opening and extending gaps in the aligned sequences. where LncRNA expression similarity: The expression profiles of lncRNA can be obtained from the dataset E-MTAB-513 in ArrayExpress (LS_exp with the absolute Spearman correlation coefficient (b) yExpress . Based officient .LncRNA functional similarity: Based on an accepted assumption that lncRNAs with similar functions have similar interaction patterns to those of diseases, the functional similarity of lncRNA can be obtained via computation of disease semantic similarity from a previous study by il is correlated with a set of diseases iD = {id1, id2,\u2026, dim}, and lncRNA jl is correlated with a set of diseases Dj = {jd1, jd2,\u2026jnd}. Semantic similarity between ild and Dj is calculated as formula (4):c)  And then the functional similarity of lncRNAs can be computed as formula (5):LS_func denotes the functional similarity between lncRNA il and lncRNA jl. where LncRNA cosine similarity: In the same way, we used cosine similarity to extract lncRNA feature information from the known interaction matrix DL. lncRNA cosine similarity can be calculated as formula (6):d) IP(jl) is resulted from the j-th column of the interaction matrix DL. IP(jl) is a vector which denotes the feature vector for lncRNA jl. where Integrated lncRNA similarity LS: In three benchmark datasets, we adopted three different similarity computation methods to fully demonstrate the robustness of FRMCLDA. In Dataset 1, integrated lncRNA similarity LS was calculated as: LS was calculated as: LS was calculated as: e) DS and LS calculated above, disease similarity network and lncRNA similarity network can be constructed. Let D = {d1, d2,\u2026, nd} represent the set of n diseases in the disease similarity network. The edge between disease id and jd is weighted by integrated disease similarity DS. Let L = {l1, l2,\u2026, ml} represent the set of m lncRNAs in the lncRNA similarity network. The edge between lncRNA il and jl is weighted by integrated lncRNA similarity LS. Besides, the disease-lncRNA interaction network can be modeled as G, where V(G) = {D,L}, E(G) \u2286 D \u00d7 L, E(G) = {ije, edge between disease id and lncRNA jl}. The edge ije is initialized to 1, if there exists a known link between disease id and lncRNA jl, otherwise, ije is initialized to 0. DL is the adjacency matrix for the disease-lncRNA interaction network.Based on the integrated disease and lncRNA similarity matrices via disease-lncRNA association network, as shown in Finally, a heterogeneous bilayer network is constructed by connecting disease similarity network and lncRNA similarity network DS and LS are the adjacency matrix of the disease similarity network and the lncRNA similarity network. The off-diagonal sub-matrix DL is the adjacency matrix for the disease-lncRNA interaction network, TDL is the transpose of DL. Usually, the interaction between lncRNAs and diseases is mutual, and values of the matrix DL are nonnegative, therefore the adjacent matrix A is meristic and positive semi-definite. The singular values of the adjacent matrix A are nonnegative real numbers and equivalent to the eigenvalues. In conclusion, the prediction of disease-lncRNA association can be remodeled as the matrix completion of the adjacency matrix A. If matrix A is only comprised of matrix DL, rather than the large-scale matrix of the heterogeneous network, then the completion based on rank minimization will not generate significant results. That is because all known disease-lncRNA associations are positive in matrix. Only restoring the matrix DL will result in an optimized solution to rank minimization problem, i.e., all-one matrix with rank 1.where diagonal sub-matrices A* with the same size (m + n) \u00d7 (m + n). It is assumed that A have rank r(r \u226a (m + n)). \u03a6 is denoted as an index set for all the known entries of matrix A. The problem of matrix completion can be converted to solving the rank minimization problem by formula (8):Our goal is to restore the unknown entries of the adjacent matrix A by constructing a proximate matrix P\u03a6(A) is denoted as an orthogonal projector onto the span of matrix A. Its value is 0 when the element  is not in the set \u03a6. Matrix A is the adjacency matrix of the heterogeneous network constructed in Datasets and Data Processing. However, the problem of rank minimization is generally considered as a NP-hard problem (*) of the matrix, which is known to be solved by standard singular value threshold (SVT) algorithm (in which  problem . An apprlgorithm . Therefolgorithm . MinimizEquation (9) can be solved by the iterative processes in formula (10) and (11):shrink(Y(i), \u03c4) is a soft thresholding operator that computes the singular value of the matrix Y at level \u03c4 (Yat the ith iteration. Y(i) is usually a relatively large matrix with high sparsity, and usually can be stored with a sparse matrix. Starting computation from X(i) and Y(i) can be generated through the linearized Bregman iteration.where the function  level \u03c4 . \u03b4 is thflops) with the runtime benefit. Faster matrix completion even incorporates a block Krylov sub-space iteration rSVD-BKIr scheme and a novel sub-space reuse mechanism  (LD. Our faster randomized matrix completion method is illustrated in Algorithm 1 in the rSVD-BKI(\u00b7) performs singular value decomposition and the details of realization can be found in an earlier study  . The faseration) . FRMCLDAer study We first put forward the evaluation metrics for the methods of association prediction. Second, we tested the effects of cosine similarity on diseases and lncRNAs and fine-tuned the weights of cosine similarity. Third, we implemented permutation test to assess the influence of different data sources on optimization procedure. Fourth, we recorded the time usage of FRMCLDA for different sizes of heterogeneous network. Fifth, we compared FRMCLDA with other existing methods by global LOOCV experiments, local LOOCV experiments and global 5-fold cross-validation experiments. Finally, we implemented case studies to validate the practicability of FRMCLDA.In order to assess the performance of FRMCLDA in inferring latent disease-correlated lncRNAs, global LOOCV experiments, local LOOCV experiments and global 5-fold cross-validation experiments are implemented on three benchmark datasets. Under the framework of LOOCV, each known experimentally validated association is picked in turns as a test sample, and all the other known associations are considered as training samples. The test sample is sorted together with the candidate samples without known association evidence. The test sample whose rank exceed the given threshold would be considered as a successful prediction. The main difference between global and local LOOCV is whether to investigate all diseases simultaneously or only query one disease at a time to select candidate samples. That is to say, global LOOCV considered all the unknown associations as candidate samples, whereas local LOOCV only focused on one disease in the test sample and selected the corresponding unknown associations as candidate samples. In global 5-fold cross-validation, all of the known experimentally validated associations are divided into five uncrossed sets, whose size must be strictly equal. Each set of the five is taken in turns as the test sample, but the other 4 sets are served as training samples. After matrix completion is performed, the test samples are ranked together with candidate samples and then are sorted in the descending order of their predicted scores.Furthermore, false negative (FN), false positive (FP), true negative (TN) and true positive (TP) are summarized based on the ranked results for each specific threshold. The receiver operating characteristic curves are made by plotting the true positive rate  against false positive rate (FPR) based on varying thresholds. The precision-recall (PR) curve is also plotted to fully evaluate the performance of the prediction. The area under the ROC curve (AUC) and the area under the PR curve (AUPR) are finally calculated to evaluate the overall performance of the prediction. An AUC value of 0.5 implies a random prediction and an AUC value of 1 implies a perfect prediction performance. Therefore, AUC and AUPR are used as primary evaluating measures.lw and dw in integrated similarity calculations can be fine-tuned by cross validation in three benchmark datasets separately. Let dw and lw vary from 0.1 to 1 at the increment of 0.1. According to AUC values of LOOCV based on Dataset1, FRMCLDA performed best when dw1 = 0.9 and lw1 = 0.7. Likewise, on Dataset2, we chose dw2 = 0.7 and lw2 = 0.5. On Dataset3, we chose dw3 = 0.5 and lw3 = 0.5. All can be seen in Both integrated disease similarity and integrated lncRNA similarity in three benchmark datasets are calculated with cosine similarity combined, which can extract feature information from the known interaction matrix. The weights of In Dataset2, we implemented 5-fold cross validation 20 times to test the effects of the cosine similarity on model performance. The four test settings were: 1) using cosine similarity both for integrated similarity of neither lncRNAs nor diseases; 2) only the lncRNA similarity integrating the cosine similarity; 3) only the disease similarity integrating the cosine similarity; 4) both lncRNA similarity and disease similarity integrating cosine similarity. The results can be seen in To evaluate the influence of different data sources on the optimization procedure of matrix completion, we have implemented a permutation test on disease-lncRNA interaction matrix DL, lncRNA similarity matrix LS, and disease similarity matrix DS separately. Based on the LOOCV framework, we randomized each of the three matrices in turns, while keeping the other two matrices unchanged. We carried it out 20 times and recorded the average AUC value for each type of data source. Usually, if a matrix contributes more to the optimization procedure, the result of the permutation test based on it will be closer to the stochastic value. As shown in In FRMCLDA model, we implement matrix completion through fSVT as proposed by a previous study . Algoritp = 2 and \u03b5 = 0.4. We executed 20 times FRMCLDA in three benchmark datasets. Average time usage of FRMCLDA and standard deviations are shown in Here, we set On Dataset 1, the performance of FRMCLDA is compared with four popular methods: LRLSLDA , KATZLDAOne advantage of FRMCLDA is that it is able to infer latent correlated lncRNAs with queried diseases, even novel diseases. To show the performance of FRMCLDA in predicting novel disease-correlated lncRNAs, we implemented local LOOCV on Dataset 1. The results of FRMCLDA with local LOOCV on Dataset 1 were recorded in The robustness of FRMCLDA was further validated by inferring latent associations on Dataset 2 and Dataset 3. We conducted 20 times global 5-fold cross-validation experiments to validate the precision of prediction by FRMCLDA on Dataset 2 and Dataset 3. The results of ROC curve, PR curve, precision-rank bars and recall-rank bars using different methods are shown in After performing cross validation to confirm the ability of FRMCLDA, we conducted a global prediction of potential related disease-lncRNA pairs. All known lncRNAs-diseases links were considered as training samples, while other unknown associations constituted the candidate samples. FRMCLDA can infer the latent correlated lncRNAs for all diseases simultaneously by faster random matrix completion. All candidate lncRNAs correlated with a queried disease were ranked according to predicted scores generated by FRMCLDA. The predicted and ranked lncRNAs (excluding known correlated lncRNAs) correlated with 226 diseases on Dataset 3 can be seen in via genistein, and the expression of HOTAIR in castration-resistant PCa cell line is higher than that of standard prostate cell lines  and the Natural Science Foundation of Hunan, China (Grant No. 2018JJ2461).JY and GT were employed by company Geneis Beijing Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest."}
+{"text": "Over the past decades, a large number of long non-coding RNAs (lncRNAs) have been identified. Growing evidence has indicated that the mutation and dysregulation of lncRNAs play a critical role in the development of many complex human diseases. Consequently, identifying potential disease-related lncRNAs is an effective means to improve the quality of disease diagnostics and treatment, which is the motivation of this work. Here, we propose a computational model (LncDisAP) for potential disease-related lncRNA identification based on multiple biological datasets. First, the associations between lncRNA and different data sources are collected from different databases. With these data sources as dimensions, we calculate the functional associations between lncRNAs by the recommendation strategy of collaborative filtering. Subsequently, a disease-associated lncRNA functional network is built with functional similarities between lncRNAs as the weight. Ultimately, potential disease-related lncRNAs can be identified based on ranked scores derived by random walking with restart (RWR). Then, training sets and testing sets are extracted from two different versions of a disease-lncRNA dataset to assess the performance of LncDisAP on 54 diseases.A lncRNA functional network is built based on the proposed computational model, and it contains 66,060 associations among 364 lncRNAs associated with 182 diseases in total. We extract 218 known disease-lncRNA pairs associated with 54 diseases to assess the network. As a result, the average AUC (area under the receiver operating characteristic curve) of LncDisAP is 78.08%.In this article, a computational model integrating multiple lncRNA-related biological datasets is proposed for identifying potential disease-related lncRNAs. The result shows that LncDisAP is successful in predicting novel disease-related lncRNA signatures. In addition, with several common cancers taken as case studies, we found some unknown lncRNAs that could be associated with these diseases through our network. These results suggest that this method can be helpful in improving the quality for disease diagnostics and treatment. Long non-coding RNAs (lncRNAs), which compose the largest portion of the mammalian non-coding transcriptome , are emeLncRNAs are the key to explaining disease mechanisms. As analysing lncRNAs is very appealing to researchers, many researchers have devoted their work to lncRNAs for exploring complex human diseases at the molecular level. For example, BCYRN1 has been demonstrated to induce the proliferation and migration of non-small cell lung cancer (NSCLC) cells and play an important role in NSCLC progression . LncRNA Although a large number of lncRNAs have been recorded in public databases, such as GENCODE , NONCODEThe workflow of LncDisAP is shown in Fig.\u00a0DO  databaseRNAcentral  is a datLncRNADisease  is a datSTRING  is a datstarBase v2.0  systematThe differences in different data sets bring some difficulties to the integration of lncRNA data. Two problems must be solved before constructing the lncRNA functional association network. One is the mapping of disease terms. MEDIC and DO are both comprehensive disease corpuses and contain abundant disease terms, so we can annotate DO entries with the vocabulary from MEDIC and create a combined vocabulary of disease terms. Referring to this vocabulary, we build mappings between the DO terms and the disease terms of LncRNADisease. The other problem that must be addressed is the unification of lncRNA identifications. As mentioned above, the lncRNA naming rules of different lncRNA databases are different. Therefore, we employ the RNAcentral id as the unified identification system of lncRNAs considering that the RNAcentral database provides mapping data among various public lncRNA databases.da and db is defined as follows:Ga\u2009=\u2009{ga1, ga2, \u2026} and Gb\u2009=\u2009{gb1, gb2, \u2026} are related to disease da and db, respectively; num(G) represents the numbers of genes related to one disease; and RG(g) represents the degree of association between a gene g and a gene set G . ConsidSTRING provides human protein-protein interactions, and in the above section, the functional similarities between lncRNA-related diseases have been calculated. Therefore, we can obtain the relational degrees between one lncRNA and a certain disease or protein based on the similarities of lncRNA-related diseases or proteins. Then, these degrees can be used to make a multi-dimensional vector for each lncRNA. Hence, we can calculate lncRNA functional similarity by cosine similarity in a multi-dimensional space, which is defined by lncRNA-related diseases and proteins. The workflow of calculating lncRNA functional similarity based on the recommendation strategy of collaborative filtering is shown in Fig.\u00a0L as the set of lncRNAs, D as the set of lncRNA-related diseases and P as the set of lncRNA-related proteins. DRl is defined as the set of diseases directly related to lncRNA l. The predicted association score between disease d and lncRNA l is defined as follows:l\u2009\u2208\u2009L, d\u2009\u2208\u2009D, DRl\u2009\u2286\u2009D and 1\u2009\u2264\u2009i\u2009\u2264\u2009|DRl|; here, |DRl| represents the number of diseases in the set of DRl. Similarly, for lncRNA-related proteins, PRl is defined as the set of proteins directly related to lncRNA l. The predicted association score between protein p and lncRNA l is defined as follows:l\u2009\u2208\u2009L, p\u2009\u2208\u2009P, PRl\u2009\u2286\u2009P and 1\u2009\u2264\u2009i\u2009\u2264\u2009|PRl|; here, |PRl| represents the number of proteins in the set of PRl and SPscore  represents the relevance score between protein p and pi from STRING.In this multi-dimensional space, neither all diseases nor all proteins are directly related to one lncRNA. To predict the score of a disease that is not directly related to one lncRNA, we define D|\u2009+\u2009|P| dimensions. |D| and |P| represent the size of the disease set D and the protein set P, respectively. For each lncRNA, we can define its vector l in this multi-dimensional space. AS and AS are the scores of disease dk and protein pj, respectively, for lncRNA l. Now, we can obtain |L| vectors of lncRNAs.Subsequently, we define a vector of each lncRNA with |l1 and lncRNA l2 is defined as follows:ASk,i represents the association score in the i-th dimension of the vector lk. The range of CR is 0 to 1 because these values of ASk,i are positive numbers.In this multi-dimensional space, each lncRNA can be depicted by a multi-dimensional vector. Therefore, we can measure the similarity between any two vectors of lncRNAs based on cosine similarity. The similarity between lncRNA mRNALinkSet. First, the relevance between an mRNA k and an mRNA set M is defined as follows:links represents the number of links between mRNA k and members in the mRNA set M, and these links have to be included in mRNALinkSet. Let a pair of mRNA sets M1\u2009=\u2009{m11, m12, \u2026} and M2\u2009=\u2009{m21, m22, \u2026} be related to lncRNA l1 and l2, respectively. The similarity between lncRNA l1 and l2 based on mRNA is defined as follows:M1| and |M2| represent the numbers of mRNAs related to lncRNA l1 and l2, respectively. Finally, we complete the calculation of lncRNA similarities based on different lncRNA-related knowledge.In addition, mRNA can also be seen as a factor to calculate lncRNA functional similarity because of the existing links between lncRNAs and mRNAs. In view of the relationships between mRNAs, we can extract links between them from HPRD denoted as l1 and l2 is defined as follows:LncFunNet is 0 to 1, as in CR and MR. Utilizing this lncRNA network, we can identify novel candidate disease-related lncRNAs.We can take lncRNA similarities as weight to construct a lncRNA functional association network. In this network, the weight between lncRNA To identify novel candidate disease-related lncRNAs, we employ RWR to fully exploit the global functional associations between lncRNAs in this network. RWR, as a global optimization method, can reveal more information between one lncRNA and all the others in the network. The random walker in the network starts from the root node and moves to adjacent nodes with the probabilities from that node to the others. After enough iterations, the probabilities from the root node to all the other nodes will become stable, which can be used as scores for predicting novel disease-related lncRNAs . There were 5,600,133 relationships between 13,716 mRNA and 1034 lncRNAs extracted from starBase v2.0. We found 15,622 associations between 33 proteins and 2750 lncRNAs from starBase v2.0 and STRING.We calculated similarity among 374 lncRNAs and removed lncRNA pairs that had a similarity of 0. Finally, we built a lncRNA functional network, which contains 66,060 associations among 364 lncRNAs associated with 182 diseases.To assess the performance of the lncRNA functional network, we compared two different versions of LncRNADisease and extracted 218 known disease-lncRNA pairs associated with 54 diseases from the newer version of LncRNADisease (released in June 2018). The detailed statistics for evaluating disease-related lncRNA networks are given in Additional\u00a0file\u00a0As a result, the disease-related lncRNA functional network has a good performance in predicting disease-lncRNA pairs for the 54 diseases with an average AUC value of 78.08%. The performance in predicting lncRNAs associated with cholangiocarcinoma is shown in Fig.\u00a0Many studies have indicated that lncRNAs play critical roles in the development of various cancers . To furtThe relationship between diseases and lncRNAs in the data source was extracted from LncRNADisease released in July 2017. Hence, we evaluated the impact of different data sources and different test sets on the performance of predicting disease-lncRNA pairs in the disease-related lncRNA functional network. First, with the LncRNADisease data set released in June 2018 as the test set, we compared the two lncRNA functional networks that were built based on data sources from the 2015 and 2017 versions. After the abovementioned validation strategy was carried out, the lncRNA functional network based on the data source from LncRNADisease released in 2015 had an AUC value of 72.6%, while the AUC of the network based on the 2017 version reached 78.08%. Simultaneously, we assessed the performance of the lncRNA functional network based on the data source of the 2015 version with a test set extracted from the 2017 version, whose AUC reached 72.8%, as shown in Fig.\u00a0l1 and l2, denoted as ER. The similarity between lncRNA l1 and l2 is defined as follows:The introduction of lncRNA expression similarity has been considered before. However, the results are not ideal. We obtained lncRNA expression profiles from NONCODE . This daSubsequently, we built two lncRNA functional networks based on data sources from the 2015 and 2017 versions with the lncRNA expression similarity operator introduced. LncRNADisease released in June 2018 was taken as the test set. The lncRNA functional network based on LncRNADisease released in 2015 had an AUC value of 68.3%, while the AUC of the network based on the 2017 version achieved 75.46%. As shown in Fig.\u00a0In this article, a computational model for potential disease-related lncRNA identification was proposed based on multiple biological datasets. The results showed that LncDisAP was proven to be successful in predicting novel disease-related lncRNA signatures with an average AUC value of 78.08% and can be an effective solution to improve the quality of disease diagnostics and treatments. To further evaluate the performance of our computational model, we used several common cancers as case studies. We found some unknown lncRNAs that could be associated with these diseases through our network. In addition, we discussed the impact of different data sources and different test sets on the performance of the disease-related lncRNA functional network in predicting disease-lncRNA pairs.Additional file 1: Statistics of 54 diseases for evaluating disease-related lncRNA functional network."}
+{"text": "Accumulating studies have shown that long non-coding RNAs (lncRNAs) are involved in many biological processes and play important roles in a variety of complex human diseases. Developing effective computational models to identify potential relationships between lncRNAs and diseases can not only help us understand disease mechanisms at the lncRNA molecular level, but also promote the diagnosis, treatment, prognosis, and prevention of human diseases. For this paper, a network-based model called NBLDA was proposed to discover potential lncRNA\u2013disease associations, in which two novel lncRNA\u2013disease weighted networks were constructed. They were first based on known lncRNA\u2013disease associations and topological similarity of the lncRNA\u2013disease association network, and then an lncRNA\u2013lncRNA weighted matrix and a disease\u2013disease weighted matrix were obtained based on a resource allocation strategy of unequal allocation and unbiased consistence. Finally, a label propagation algorithm was applied to predict associated lncRNAs for the investigated diseases. Moreover, in order to estimate the prediction performance of NBLDA, the framework of leave-one-out cross validation (LOOCV) was implemented on NBLDA, and simulation results showed that NBLDA can achieve reliable areas under the ROC curve (AUCs) of 0.8846, 0.8273, and 0.8075 in three known lncRNA\u2013disease association datasets downloaded from the lncRNADisease database, respectively. Furthermore, in case studies of lung cancer, leukemia, and colorectal cancer, simulation results demonstrated that NBLDA can be a powerful tool for identifying potential lncRNA\u2013disease associations as well. In recent years, accumulating evidence studies have shown that non-coding RNAs (ncRNAs) are involved in various biological processes in the human body ,2,3, andCurrently, with the rapid development of bioinformatics, some lncRNA\u2013disease association databases such as LncRNADisease  and Lnc2\u03b2k to each node in the network, where k is the degree of the node and \u03b2 is a freely adjustable parameter. Moreover, considering that traditional mass diffusion-based algorithms focused on unidirectional mass diffusion only, we further applied a consistence-based mass diffusion algorithm via bidirectional diffusion on NBLDA to predict potential lncRNA\u2013disease associations by adopting a label propagation algorithm. Finally, in order to estimate the prediction performance of NBLDA, the framework of leave-one-out cross validation (LOOCV) was implemented, and simulation results show that NBLDA can achieve reliable AUCs of 0.8846, 0.8273, and 0.8075 in LOOCV based on three versions of known lncRNA\u2013disease association datasets downloaded from the lncRNADisease database, respectively, which demonstrates the excellent prediction performance of NBLDA. In addition, in case studies of lung cancer, leukemia, and colorectal cancer, simulation results show that there are 9, 10, and 7 out of the top 10 predicted disease-related lncRNAs of these three kinds of diseases having been validated by evidence from studies in the PubMed literature and Lnc2Cancer database, respectively, which further indicates NBLDA has a satisfactory prediction performance in discovering potential lncRNA\u2013disease associations as well.Inspired by the above-mentioned state-of-the-art methods, a network-based computational model NBLDA was proposed for this paper to predict potential lncRNA\u2013disease associations based on the assumption that functionally similar lncRNAs show similar interaction patterns with similar diseases. In NBLDA, two new networks were constructed first based on known lncRNA\u2013disease associations and Gaussian interaction profile kernel similarity for lncRNAs and diseases, and then we assigned an attraction that is proportional to In order to estimate the prediction performance of NBLDA, and described in this section, we implemented LOOCV on NBLDA based on known lncRNA\u2013disease associations downloaded from the LncRNADisease database. While implementing LOOCV, each known lncRNA\u2013disease association was left out in turn as a test sample and the other remaining known lncRNA\u2013disease associations were taken as training samples. Moreover, all lncRNA\u2013disease pairs without known relevance evidences were considered as candidate samples. Thereafter, we obtained the ranking of each test sample within all candidate samples according to their scores predicted by NBLDA, and then, the test sample was regarded as successfully predicted if its ranking exceeded a given threshold. Furthermore, the receiver operating characteristic (ROC) curves were drawn based on true positive rate  and false positive rate  obtained at different thresholds. Here, the sensitivity represents the proportion of test samples with a ranking higher than the given threshold to all positive samples, whereas 1-specifcity indicates the ratio between candidate samples with a ranking above a given threshold and all candidate samples. Then, the areas under the ROC curve (AUCs) were further calculated to evaluate the predictive performance of our model NBLDA, and it is obvious that the larger the value of AUC, the better the prediction performance of NBLDA will be.DS1, respectively  is one of the most common types of cancer in the United States and the second leading cause of cancer death . The aveAccumulating evidence studies have shown that lncRNAs are closely related to a variety of biological processes. Identifying potential lncRNA\u2013disease association not only helps us understand the pathogenesis of disease at the molecular level of lncRNA, but also contributes to the diagnosis, treatment, prognosis, and prevention of diseases. In this paper, we presented a computational model NBLDA to reveal potential lncRNA\u2013disease associations based on known lncRNA\u2013disease associations and Gaussian interaction profile kernel similarity for lncRNAs and diseases. We improved the baseline algorithm of bipartite network recommendation based on the network topological similarity of the lncRNA\u2013disease association network and resource allocation strategy of unequal allocation and unbiased consistence. A label propagation algorithm was then used to predict potential lncRNA\u2013disease associations. NBLDA achieved AUCs of 0.8846, 0.8273, and 0.8075 in the validation framework of LOOCV based on three versions of known lncRNA\u2013disease association datasets, which significantly improved the previous classic models. Furthermore, we conducted case studies of lung cancer, leukemia, and colorectal cancer, and simulation results show that there are 9, 10, and 7 out of the top 10 predicted candidate lncRNAs having been confirmed by previous studies in the literature respectively. As a result, both cross validation and case studies have shown that NBLDA has a good performance in potential lncRNA\u2013disease association prediction.D (or a given lncRNA L) may also contribute resources to D (or L), we then constructed novel networks based on known lncRNA\u2013disease associations and the Gaussian interaction profile kernel similarity for diseases and lncRNAs. Third, we adopted a resource allocation strategy of unequal allocation and unbiased consistence. Certainly, there are still some limitations in NBLDA which must be improved in the future. First of all, the similarity measures for diseases and lncRNAs are relatively simple, and more effective similarity measures such as disease semantic similarity, disease phenotypic similarity, and lncRNA functional similarity can improve the performance of our model. Moreover, although the numbers of lncRNA\u2013disease associations data have increased compared to before, the known lncRNA\u2013disease associations in our dataset are still too sparse, and the performance of NBLDA can be further improved when more lncRNA\u2013disease associations datasets are available and more reliable types of biological datasets are integrated. Last but not least, increasing lncRNA\u2013disease association data can be used as training samples for model learning with the development of biological experimental techniques.The novel and reliable performance of NBLDA is mainly attributed to the following aspects. First, the method proposed by us is based on a classical approach that has already achieved excellent performance in predicting associations in other biological networks. Second, considering that the lncRNAs (or diseases) which are not associated with a given disease http://www.cuilab.cn/lncrnadisease), respectively (see DS1) from the LncRNADisease database, and after removing duplicated records and associations that do not belong to human beings, we finally obtained 1695 known lncRNA\u2013disease associations involving 314 diseases and 828 lncRNAs. Next, we downloaded the 2015 version of the dataset (denoted as DS2) from the LncRNADisease database, and after removing duplicated data, we finally obtained 621 known lncRNA\u2013disease associations including 226 diseases and 285 lncRNAs. Finally, we downloaded the 2012 version of the dataset (denoted as DS3) from the LncRNADisease database, and after removing duplicated data, we finally obtained 293 known lncRNA\u2013disease associations including 167 diseases and 118 lncRNAs. Thereafter, we adopted an adjacency matrix Y to indicate known associations between lncRNAs and diseases. In the adjacency matrix Y, if there is a known association between lncRNA il and disease jd, then there is Y = 1; otherwise, there is Y = 0. Moreover, for convenience, we further introduced DN and LN to denote the number of diseases and lncRNAs collected above, respectively.Three versions of the datasets were downloaded from the LncRNADisease database  is the ith row of the adjacency matrix Y and represents the interaction profile of lncRNA il with all diseases. The parameter lS based on these lncRNAs collected above.Based on the hypothesis that functionally similar lncRNAs are always associated with similar diseases , for anyous work . Obviousid and jd, we can obtain the Gaussian interaction profile kernel similarity between id and jd according to Equation (3) as follows:IP(id) is the ith column of the adjacency matrix Y and represents the interaction profile of disease id with all lncRNAs. The parameter dS based on these diseases collected above.In a similar way, for any given diseases set to 1 . ObviouslS, dS, and Y as follows:As illustrated in SL, we can construct a bipartite network first, and then, for a randomly given node \u03c8 in the newly constructed bipartite network, supposing that \u03c8 has been assigned an attraction such as \u03b2k(\u03c8), where k(\u03c8) represents the degree of node \u03c8 in the bipartite network and \u03b2 is a freely adjustable parameter, it is obvious that \u03b2 = 0 means the average allocation of resources, \u03b2 < 0 means that nodes with lower degrees are more attractive and will obtain more resources, and \u03b2 > 0 indicates that nodes with higher degrees have greater attraction and will be allocated more resources  and jDN].From the above descriptions, it is easy to see that"}
+{"text": "A lot of studies indicated that aberrant expression of long non-coding RNA genes (lncRNAs) is closely related to human diseases. Identifying disease-related lncRNAs (disease lncRNAs) is critical for understanding the pathogenesis and etiology of diseases. Most of the previous methods focus on prioritizing the potential disease lncRNAs based on shallow learning methods. The methods fail to extract the deep and complex feature representations of lncRNA-disease associations. Furthermore, nearly all the methods ignore the discriminative contributions of the similarity, association, and interaction relationships among lncRNAs, disease, and miRNAs for the association prediction. A dual convolutional neural networks with attention mechanisms based method is presented for predicting the candidate disease lncRNAs, and it is referred to as CNNLDA. CNNLDA deeply integrates the multiple source data like the lncRNA similarities, the disease similarities, the lncRNA-disease associations, the lncRNA-miRNA interactions, and the miRNA-disease associations. The diverse biological premises about lncRNAs, miRNAs, and diseases are combined to construct the feature matrix from the biological perspectives. A novel framework based on the dual convolutional neural networks is developed to learn the global and attention representations of the lncRNA-disease associations. The left part of the framework exploits the various information contained by the feature matrix to learn the global representation of lncRNA-disease associations. The different connection relationships among the lncRNA, miRNA, and disease nodes and the different features of these nodes have the discriminative contributions for the association prediction. Hence we present the attention mechanisms from the relationship level and the feature level respectively, and the right part of the framework learns the attention representation of associations. The experimental results based on the cross validation indicate that CNNLDA yields superior performance than several state-of-the-art methods. Case studies on stomach cancer, lung cancer, and colon cancer further demonstrate CNNLDA's ability to discover the potential disease lncRNAs. Long non-coding RNA genes (lncRNAs) are transcripts longer than 200 nucleotides which are not translated into proteins Reik, . AccumulPredicting disease-related lncRNAs (disease lncRNAs) can screen the potential candidates for the biologists to discover the real lncRNA-disease associations with the wet-lab experiments  that includes all the disease terms related to the disease. If two diseases have more common disease terms, they are more similar, which is the basic idea for semantic similarity between Gene Ontology terms . LS is defined as,As the lncRNAs associated with the similar diseases are generally possible to have more similar functions, Chen et al. measured the similarity of two lncRNAs based on their associated diseases  is the semantic similarity of disease of dai and dbj which belong to DTa and DTb respectively. m and n are the numbers of diseases that are included by DTa and DTb. The lncRNA similarities are denoted by matrix Lij is the similarity of two lncRNAs li and lj   .l1 and d2, P, is input to the convolutional module on the left to learn a global deep representation for l1 and d2. The convolutional module includes two convolutional layers and two pooling layers  is the element at the ith row and the jth column of P\u2032, and P\u2032k,i,j represents a region within the filter when the kth filter slides to the position P\u2032. The formal definitions of P\u2032k,i,j and Zconv1,k are as follows,For the first convolutional layer, the length of a filter is set as bconv1 is the bias vector, f is a relu function  is the element at the ith row and jth column of the kth feature map Zconv1,k.where Zconv1. ng and np are the length and width of a filter of pooling layer, respectively. The pooling outputs of all the feature maps are Zconvpool1,We apply the max pooling to extract the robust features from the feature maps Zconvpool1,k is the kth feature map, and Zconvpool1,k is the element at its' ith row and jth column.where l1 and disease d2. Thus, the module consists of the attention mechanism at the feature level and the one at relationship level , and it contains the probability that a lncRNA and a disease is determined to have an association relationship and the probability that they have no association.p is defined as In our model, the cross-entropy loss between the ground truth distribution of lncRNA-disease association and the prediction probability z \u2208 \u211d2 is the classification label vector and T is a set of training samples. If l1 is associated with d2, the second dimension of the vector z is 1 and the first one is 0. On the contrary, if l1 is not associated with d2, the first dimension of z is 1 and the second one is 0.where We denote all neural network parameters by \u03b8. The objective function in our learning process is defined as follows,where \u03bb is a trade-off parameter between the training loss and regularization term. We use Adam optimization algorithm to optimize the objective function  and the false positive rates (FPRs) to get a receiver operating characteristic (ROC) curve by changing threshold \u03b8. TPR and FPR are defined as follows,The samples are ranked by their association scores after the association probabilities of the testing samples are estimated. The higher the node pairs of the positive samples are ranked, the better CNNLDA performs. If an observed association exists in lncRNA-disease node pair samples, and its association score is greater than a threshold \u03b8, it is a successfully determined positive sample. If the prediction score of a negative sample is smaller than \u03b8, it is a determined correctly negative sample. We calculate the true positive rates   and the unobserved ones (the negative samples) form the serious imbalance. In such case, we also use the precision-recall (PR) curve and its area (AUPR) to assess the performance of a prediction method  for each of the 5 folds when used as a test set, and the 5 performances are averaged to give the final performance.k among the total positive samples.In addition, the biologists usually select lncRNA candidates from the top part of the ranking list, and then further validate their associations with diseases. Therefore, the recall values of top 30, 60, \u2026, 240, are calculated, and they represent the fraction of the successfully recovered positive samples in the top list To evaluate the performance of CNNLDA, we compare it with several state-of-the-art methods including SIMCLDA (Lu et al., Wilcoxon-test to evaluate whether CNNLDA's AUCs and AUPRs across all of the tested diseases are significantly higher than those of another method. CNNLDA achieves significantly higher performance than the other methods in terms of both AUCs and AUPRs as the corresponding P-values are smaller than 0.05 (We perform a paired han 0.05 .k ranked lncRNA-disease associations is, the more genuine associations are determined correctly. Under different k cutoffs, the performance of CNNLDA consistently outperforms other methods (The higher the recall rate on the top  methods , and ranIn addition, to validate the effectiveness of exploiting the information related to the miRNAs, we construct another instance of CNNLDA that is trained without this kind of information, and the instance is referred to as CNNLDA-nM. The instance of CNNLDA that is trained by using the miRNA-related information is still named as CNNLDA. CNNLDA's AUC and AUPR are 0.2%and 0.94% greater than CNNLDA-nM, which confirms the importance of integrating the information for improving CNNLDA's prediction performance.stomach cancer, lung cancer, and colon cancer and analyze the top 15 candidates respectively related to these cancers (To demonstrate CNNLDA's ability to discover potential candidate disease lncRNAs, we execute the case studies on  cancers .First, a database named Lnc2Cancer curates the lncRNAs that have different expression in the disease tissues compared to the normal ones. Lnc2Cancer contains lncRNAs related to cancers that have been identified by analyzing the results of northern blot experiments, microarray experiments, and quantitative real-time polymerase chain reaction experiments (Gao et al., 1\u201d are supported by several published literature. These lncRNAs are confirmed to have dysregulations in the cancers when compared with the normal tissues (Bahari et al., Next, 2 candidates of stomach cancer, 1 candidate of lung cancer and 2 candidates of colon cancer labeled with \u201cliterature2,\u201d and \u201cmiRCancer, StarBase\u201d are related to the important factors affecting the development of the corresponding cancers. In the metabolic network, lncRNA HCP5 is regulated by three miRNAs, and the miRNAs are downregulated in stomach cancer. It indicates that the expression of HCP5 is more likely to associate with stomach cancer (Mo et al., Finally, 5 candidates labeled with \u201cliteratureAfter evaluating its prediction performance through the cross-validation process and case studies, CNNLDA is applied to all 402 diseases. All the positive samples and the negative ones are used to train CNNLDA to predict the novel disease-associated lncRNAs. The potential candidate lncRNAs for these diseases are listed in A novel method based on dual convolutional neural networks, CNNLDA, is developed for predicting the potential disease-related lncRNAs. We respectively construct the attention mechanism at feature and relationship levels to discriminate the different contributions of features and learn the more informative representation of lncRNA-disease associations. The new framework based on dual convolutional neural networks is developed for learning the global representation and the attention of lncRNA-disease associations. The experimental results indicate that CNNLDA is superior to the compared other methods in terms of both AUCs and AUPRs. The case studies on 3 diseases demonstrate CNNLDA's ability for discovering potential disease-associated lncRNAs.All datasets analyzed for this study are cited in the manuscript and the PX and YC conceived the prediction method, and they wrote the paper. YC and ZZ developed computer programs. TZ and RK analyzed the results and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Evidences have increasingly indicated that lncRNAs (long non-coding RNAs) are deeply involved in important biological regulation processes leading to various human complex diseases. Experimental investigations of these disease associated lncRNAs are slow with high costs. Computational methods to infer potential associations between lncRNAs and diseases have become an effective prior-pinpointing approach to the experimental verification.In this study, we develop a novel method for the prediction of lncRNA-disease associations using bi-random walks on a network merging the similarities of lncRNAs and diseases. Particularly, this method applies a Laplacian technique to normalize the lncRNA similarity matrix and the disease similarity matrix before the construction of the lncRNA similarity network and disease similarity network. The two networks are then connected via existing lncRNA-disease associations. After that, bi-random walks are applied on the heterogeneous network to predict the potential associations between the lncRNAs and the diseases. Experimental results demonstrate that the performance of our method is highly comparable to or better than the state-of-the-art methods for predicting lncRNA-disease associations. Our analyses on three cancer data sets  also indicate the usefulness of our method in practical applications.Our proposed method, including the construction of the lncRNA similarity network and disease similarity network and the bi-random walks algorithm on the heterogeneous network, could be used for prediction of potential associations between the lncRNAs and the diseases. Long non-coding RNAs (lncRNAs) form a new class of important ncRNAs, with length longer than 200nt \u20133. AccumComputational models have been developed to predict potential associations between lncRNAs and diseases. Chen et al.  had an aBased on the fact that non-coding genes are often cooperated in human diseases to predict potential lncRNA-disease association, Peng et al.  proposedSome models predict novel associations without referring to known associations between lncRNAs and diseases. Chen  proposedSome computational models have been applied to predict lncRNA-disease associations based on random walk on networks. Chen et al.  considerIn this study, we propose a novel computational model of Laplacian normalization and bi-random walks on heterogeneous networks for predicting lncRNA-disease associations (Lap-BiRWRHLDA). Firstly, the method calculates the Gaussian interaction profile kernel similarity of lncRNAs and diseases by known lncRNA-disease associations. Next, we integrate the two sources of similarity to construct an lncRNA-lncRNA similarity network. The disease-disease similarity network can be constructed by the profile kernel similarity of diseases. Subsequently we perform Laplacian normalization on the similarity matrices of lncRNAs and diseases as the transpose matrices. Furthermore, we apply random walks on the lncRNA similarity network and the disease similarity network, respectively. Finally, we use a weighted average of random walks on both networks as a predictor of lncRNA disease associations. We believe that the higher scores of lncRNA-disease associations will have greater possibility for further verification. To evaluate our proposed method, we utilize leave-one-out cross-validation experiments to demonstrate its superior performance compared with existing approaches. Furthermore, the analyses of three cancers  effectively support the practical application of our method. We then use Lap-BiRWRHLDA to infer potential lncRNA-disease associations. Some high-score results are successfully verified by the LncRNADisease and Lnc2Cancer databases.To assess the performance of our proposed method, we use the leave-one-out cross-validation to perform the assessment. We leave out each known lncRNA-disease association in turn as test sample, while other known relationships are used as training samples and all unknown relationships are taken as candidate samples. Since disease similarity and lncRNA similarity depend on the Gaussian interaction profile kernel similarity of the known lncRNA-disease association, the disease similarity and lncRNA similarity will change when we delete a known lncRNA-disease association, so we will get different similarities.A receiver-operating characteristics (ROC) curve is applied to determine the predictive performance, which plots the correlation between true-positive rate (TPR) indicating sensitivity and false-positive rate (FPR) indicating specificity at different thresholds. Sensitivity represents the percentage of the left-out associations achieving the ranking higher than a given threshold; specificity means the percentage of candidate associations achieving the ranking lower than this given threshold. When we vary thresholds, we will obtain the corresponding different TPRs and FPRs. In this way, ROC is drawn and AUC is calculated. As a result, Lap-BiRWRHLDA achieved the AUC of 0.8409, 0.8527 and 0.8429 for three datasets used, respectively.\u03b1 controls the probability of the random walk restart. To optimize the parameter \u03b1, we increased \u03b1 from 0.1 to 1 with step size 0.1, and then calculated the corresponding AUC value by LOOCV. After experimental verification, we chose \u03b1=0.9, and we achieved the AUC values of 0.8409. The experimental results indicate that Lap-BiRWRHLDA offers better performance on the LncRNADisease dataset on October 2012, when \u03b1=0.9 is selected. Similarly we achieved the AUC values of 0.8527 (\u03b1=0.2) and 0.8429 (\u03b1=0.8) based on Lnc2Cancer dataset on July 2016, and the LncRNADisease dataset on April 2016.Parameter We compared Lap-BiRWRHLDA with previous published methods in LOOCV based on the LncRNADisease dataset on October 2012. (1) LRLSLDA  computesWe also compared the LncRNADisease dataset on April 2018 with the LncRNADisease dataset on October 2012, then selected 50 lncRNA-disease associations which were unverified in the LncRNADisease dataset on October 2012 but were verified in the LncRNADisease dataset on April 2018. We compared our method with LRLSLDA, GrwLDA by independently testing the ranking of the 50 relationships. Through experimental tests, our method has 30 rankings higher than the LRLSLDA method, and 41 rankings higher than the GrwLDA method.To further highlight the performance of Lap-BiRWRHLDA, we studied the predictive performance of three cancers: breast cancer, lung cancer, and liver cancer. For each type of cancer, we take the top 10 most probable lncRNAs as candidates associated with this cancer. Next, we manually checked these lncRNAs by mining biomedical literature from the LncRNADisease dataset and the Lnc2Cancer dataset.Breast cancer is the second leading cause of female cancer deaths, comprising 22% of all cancers in women , 17. LapAccumulated experimental evidences have shown that lncRNAs play an important role in the human complex disease mechanism, and mutations or disorders of lncRNAs are associated with various complex diseases. More and more evidences show that it is crucial to propose an effective computational model to infer potential lncRNA-disease associations. In this article, we proposed a novel computational model of Laplacian normalization and bi-random walks on heterogeneous networks for predicting lncRNA-disease associations. Our method shows better performance in LOOCV experiments by comparison with previous methods. In 50 unverified lncRNA-disease associations experiments, We compared our method with LRLSLDA, GrwLDA. The results indicated that our method has the higher ranking. Furthermore, the study of the cases of breast cancer, lung cancer, and liver cancer shows that our method improves the performance of predicting potential relationships.Although our method can improve the prediction accuracy, it still has some limitations. For example, construction of the disease-disease similarity matrix relies on the Gaussian interaction profile kernel similarity matrix for diseases from the known disease-lncRNA associations. In further work, we will improve our method in the following aspects: Firstly, Lap-BiRWRHLDA relies on the calculation of similarity matrix when constructing an lncRNA similarity network, and so the incompleteness of data may affect the final performance. Therefore, the integration of gene disease correlation data or the addition of more bioinformatics data may improve the performance of our method. These aspects have been considered in previous methods such as TPGLDA  and BRWLIn this study, we proposed a method called Lap-BiRWRHLDA to predict the relationship between lncRNA and diseases. This model utilizes the Laplacian normalization of the lncRNA similarity matrix and the disease similarity matrix. Then constructs a heterogeneous network based on lncRNA similarity network, disease similarity network and available lncRNA-disease associations. Next, it applies bi-random walks on the heterogeneous network to predict potential associations between lncRNAs and diseases. Our method can be used to better identify potential associations between lncRNAs and diseases.The reason why our method has good results is mainly due to two factors. On the one hand, we exploit the similarity of lncRNAs by integrating Gaussian interaction profile kernel similarity of lncRNA and lncRNA expression similarity, and then apply Laplacian normalization. We also rely on lncRNA similarity matrices to construct an lncRNA similarity network. On the other hand, the bi-random walk algorithm simulates random walk restarts on the lncRNA similarity network and disease similarity network; we then infer the relationship between lncRNAs and diseases by weighted averaging. We believe that the higher the score of potential lncRNA-disease relationship is, the higher the probability of association is.A, where the value A of row i and column j is 1 if disease d(i) is related to lncRNA l(j), otherwise it is 0. Let L={l(1),l(2),\u22ef,l(nl)} denote the set of lncRNAs, and D={d(1),d(2),\u22ef,d(nd)} denote the set of diseases.We downloaded three data sets of lncRNA-disease associations from the supplementary files of published articles , 8, whicL1, where L1 is composed of lncRNAs with lncRNA expression profiles (L1\u2286L). According to the previous approaches [l(i), l(j)\u2208L1, we calculated the Spearman correlation coefficient of l(i) and l(j) as the lncRNA expression similarity. The lncRNA expression similarity matrix is represented by matrix SPC, where SPC(l(i),l(j)) is the expression similarity between l(i) and l(j) if they belongs to L1, otherwise 0.We also downloaded lncRNA expressions and the gene expression levels from the supplementary files of the published articles , 8, whicproaches , if l(i)M=M,i,j=1,2,\u22ef,N, is a symmetric matrix, D is a diagonal matrix of which D is the sum of row i of M and D=0 for i\u2260j. M is normalized by Suppose that M. It is often used to normalize a weighted matrix of a network [This process is called Laplacian normalization of  network , 20, 21.IP(l(i)) is a binary vector which is 1 if lncRNA l(i) is related to the disease, 0 otherwise, defined as the i-th column of the adjacency matrix A of the known lncRNA-disease association network constructed above. Then we can calculate the Gaussian interaction profile kernel similarity of lncRNA l(i) and lncRNA l(j) from their interaction profiles as Based on the assumption that similar diseases tend to show a similar interaction or non-interaction with the lncRNAs, the Gaussian interaction profile kernel similarity of lncRNAs can be calculated from known lncRNA-disease associations . The lnc\u03b3l controls the kernel bandwith, which is calculated based on the new kernel bandwidth parameter where the parameter nl denotes the number of lncRNAs. For simplicity we set where us works , 22.SL the lncRNA similarity matrix, where the element SL defines the similarity between lncRNA l(i) and lncRNA l(j) as Following previous approaches , we consSPC(l(i),l(j)) represents the expression profile similarity of lncRNA l(i) and lncRNA l(j), and its value is the Spearman correlation coefficient of lncRNA l(i) and lncRNA l(j), so the matrix SPC is a symmetric matrix. KL(l(i),l(j)) represents the Gaussian interaction profile kernel similarity of lncRNA l(i) and lncRNA l(j), so the matrix KL is also a symmetric matrix. Therefore the lncRNA similarity matrix SL is a symmetric matrix. In Eq.  is calculated through two steps: Next, using Laplacian normalization, the element Similar to lncRNAs, the Gaussian interaction profile kernel similarity of diseases can be constructed as IP(d(i)) is defined as the i-th row of the adjacency matrix A of the known lncRNA-disease association. It is a binary vector representing the relationship between disease d(i) and each gene. The Gaussian interaction profile kernel similarity matrix KD is a symmetric matrix. The parameter \u03b3d is calculated as Here nd denotes the number of diseases; for simplicity we set where us works , 22.From relevant research , 19, to c and d are two parameters, for which we adopt the same parameter selection as in the previous studies [c=\u221215,d= log(9999). The disease similarity matrix SD is a symmetric matrix. Next, using Laplacian normalization, the element SD is calculated through two steps: where  studies , 19, i.eLD, LL to construct two networks, namely a disease similarity network, and an lncRNA similarity network. In the lncRNA similarity network, the edge between l(i) and l(j) is weighted by the similarity value of these two lncRNAs.We first use the two matrices d(i) and d(j) is weighted by the similarity value of these two diseases.Likewise, in the disease similarity network, the edge between d(i) and lncRNA l(j), the weight of the edge is 1; otherwise it is 0. We divide the nodes of the heterogeneous network into two types. Those nodes connecting the lncRNA similarity network with the disease similarity network are called bridging nodes, and the other nodes are named internal nodes [Besides, the lncRNA-disease association network can be modeled as a bipartite graph. In this graph, the heterogeneous nodes correspond to either lncRNA or disease, and edges denote the presence or absence of the associations between them. If there is a known association between disease al nodes .The heterogeneous network can be constructed by connecting the lncRNA similarity network and the disease similarity network via the known lncRNA-disease associations. A simple example of a heterogeneous network is illustrated in Fig.\u00a0In this study, we develop a novel computational method called Lap-BiRWHLDA to predict human lncRNA-disease associations. Figure\u00a0d(1) to l(1) and then to l(2). We can take d(1) as the starting node for the random walk. To simulate this process, we apply a random walk on the lncRNA similarity network. The iterative process can be described as Suppose a random walker can jump from Similarly, we can also apply a random walk on the disease similarity network as follows: \u03b1 is a parameter to control the restart probability for the random walker, l and disease d in the t-th iteration, d and lncRNA l in the t-th iteration, with where t-th step, Lap-BiRWHLDA further combines RTt as follows: After the bi -random walks in the disease similarity network and in the lncRNA similarity network in the RTt+1 and RTt is less than 10\u221210, we obtain the steady prediction score matrix RT, where RT is the probability of potential association disease d(i) and lncRNA l(j).After several steps, when the change between"}
+{"text": "Ecological carrying capacity is an important factor of sustainable development for cities, and a critical part of achieving the coordinated development of the social economic and ecological environment for urban agglomerations. In order to evaluate the regional ecological carrying capacity and provide a basis for decision-making for regional sustainable development, this paper constructs an ecological carrying capacity model for the urban agglomeration from two dimensions: ecological carrying elastic force and ecological carrying pressure. The analytic hierarchy process is utilized to determine the weights of nine indices in these two dimensions. For the Yangtze River urban agglomeration, the comprehensive index of its ecological carrying capacity is investigated quantitatively, and the spatial distribution map of its comprehensive index measuring ecological carrying capacity is computed. The results show that Nanjing, Yangzhou, Taizhou, and Changzhou are in the stage of high load carrying; Suzhou, Wuxi, Nantong, and Zhenjiang are in the stage of low load carrying. In addition, the environmental protection investment has the greatest impact on ecological carrying elastic force, followed by the proportion of the tertiary industry; wastewater discharge has the greatest impact on ecological carrying pressure. The level of ecological carrying capacity varies within the region. It is necessary to take measures to increase the ecological carrying elastic force and reduce the ecological carrying pressure according to the actual conditions in each region. Meanwhile, exchanges and cooperation between different regions should be strengthened to stimulate the coordinated and sustainable development. In recent years, China\u2019s economy has rapidly developed, and the level of urbanization has continuously upgraded. By the end of 2017, the urbanization rate of China\u2019s permanent residents was 58.52%, increasing 40.6 percentage points from 1978, with an annual average increase of one percent  , 11, 20]  cIn summary, domestic and foreign scholars pay more attention to the study of ecological carrying capacity, but there are two shortcomings: (1) The systematic nature of the evaluation index system for regional ecological carrying capacity has not been well studied in depth. The selection of evaluation indicators and the methods for determining the weight of indicators vary also. In particular, since the selection of indicators needs to be based on the actual socio-economic development of urban agglomerations, the scientific nature of the indicator system for urban agglomerations needs to be explored. (2) In terms of the evaluation method of ecological carrying capacity, most evaluation methods are not comprehensive, and there is a lack of integrated evaluation from the perspectives of the ecology, resources, environment, society, and economy.Therefore, this paper focuses on constructing a comprehensive evaluation index system for the ecological carrying capacity for the Yangtze River urban agglomeration from two dimensions: ecological carrying elastic force and ecological carrying pressure, and utilizes the analytic hierarchy process to determine the index weight according to the connection between the indicators. Moreover, the spatial distribution of the ecological carrying capacity for Yangtze River urban agglomeration is analyzed. The research aims at exploring the comprehensive evaluation method of regional ecological carrying capacity, so as to provide effective suggestions for realizing regional sustainable development. In the remainder of the paper, the content is structured as follows. With the issuance of \u201cOpinions on accelerating the construction of Yangtze river urban agglomeration\u201d by the Jiangsu provincial party committee and provincial government and the implementation of the \u201cDevelopment plan of Yangtze river urban agglomeration\u201d, the Yangtze River urban agglomeration was born.The Yangtze River urban agglomeration is located along the Yangtze River in Jiangsu Province, China, covering Nanjing, Zhenjiang, Changzhou, Wuxi, Suzhou, Yangzhou, Taizhou, and Nantong . With anHowever, its traditional industry occupies a dominant position in industrial structure, and its economic development and environmental protection are not coordinated due to its large population density, large resource occupation and irregular agricultural development. Assessing the ecological carrying capacity of this region has become an important issue that cannot be ignored for sustainable development.We selected some relevant indicators from two aspects: the ecological carrying elastic force and ecological carrying pressure, which was motivated by the literature research and the findings of Li and Ma  (2013), This index system covers several aspects of the development of the Yangtze River urban agglomeration, including the social economy, population, industrial structure, environmental pollution, resource consumption, etc., which reflects the concept of sustainable development and the comprehensive evaluation of the whole index system.The specific evaluation index system is shown in Constructing a reasonable evaluation model of ecological carrying capacity is the premise and basis for the evaluation of ecological carrying capacity. In this paper, the ecological carrying capacity system is divided into the ecological carrying elastic system and the ecological carrying pressure system, so as to construct a comprehensive index model for the ecological carrying elastic force and ecological carrying pressure. Among them, we define ecological carrying capacity as the comprehensive reflection of ecosystem service capacity and social and economic development pressure; ecological carrying elastic force as the self-repairing ability of the ecological environment system; ecological carrying pressure as the direct negative impact of human activities on the natural ecological environment.The urban ecological carrying elastic index is : (1)S=\u2211jThe urban ecological carrying pressure index is :(2)P=\u2211j=The comprehensive index of urban ecological carrying capacity is :(3)D=P/SThe combination of the ecological carrying pressure index with the ecological carrying elastic index can reflect the ecological carrying status of a city. Among them, the value of the ecological carrying elastic index reflects the level of urban ecological carrying capacity; the value of the ecological carrying pressure index reflects the level of urban ecological environment carrying pressure. The higher the pressure, the higher the comprehensive index of ecological carrying capacity. If the comprehensive index of ecological carrying capacity of a city is high, indicating that the urban ecosystem is in a stage of high load, the lower the ecological carrying capacity. The comprehensive index of urban ecological carrying capacity reflects the overall carrying status of a city.According to the research of Li et al.  (2017) aAt present, various methods are used to determine factor weights, including the analytic hierarchy process, principal component analysis, and the entropy method. Considering the fuzzy connection between the selected indicators and the complexity of the selected indicators in our study, we used the analytic hierarchy process to determine the index weights. The main steps are as follows :CI is the consistency index, RI the random consistency index, n the number of characteristic roots of the judgment matrix. when CR < 1.0, the judgment matrix is considered to be consistent, and the smaller the value of CR, the better the consistency of the judgment matrix. When CR \u2265 1.0, it is considered that the judgment matrix does not conform to the consistency and needs to be revised again. Firstly, the hierarchical model is established; secondly, a judgment matrix is established, in which the scale method of 1\u20139 is utilized, and the value is assigned according to its importance; thirdly, we calculate the weight vector, perform the consistency test, and calculate the weight value. In order to avoid the interference of other factors on the judgment matrix, the judgment matrix is required to meet the general consistency in practice, and the consistency test should be conducted according to Formula (4).The data in this paper were selected from the 2017 statistical yearbook of Jiangsu province, the Environmental Statistical Bulletin of Jiangsu province, and the Statistical Yearbook and Environmental Statistical Bulletin of all cities in the Yangtze River urban agglomeration in 2017. Due to the differences in the measurement units, the nature and order of magnitude among the indicators in the indicator system, a unified dimensionless treatment was needed to eliminate the differences among the indicators. The specific method is as follows:Standardized treatment formula of the benefit index:Standardized treatment formula of the cost index:Through the judgment matrix of the analytic hierarchy process, Ten experts from government agencies, universities, and enterprises engaged in ecological and environmental research were invited to score the relative importance of the indices in the criterion Layer B and the indicator Layer C to calculate the index weights of the indicator layer. Then, the weighted average was utilized to summarize the scores of ten experts, so as to get the judgment matrix. The final results are shown in It can be seen from In the ecological carrying pressure system, C6 accounted for the largest proportion, indicating that industrial wastewater discharge had the greatest impact on the ecological carrying pressure, followed by C9, industrial tailpipe emission. With the development of the city, the population has been expanding, and the discharge of wastewater and waste gas has also been increasing, which has caused tremendous pressure on the ecological environment. Based on the ecological carrying elastic index model, the ecological carrying elastic indices by city were calculated. Then, the histogram of the ecological carrying elastic indices  can be oIt follows from (1) Suzhou has the highest ecological carrying elastic index, which belongs to the medium carrying level. Due to the high level of urbanization in Suzhou, the proportion of the tertiary industry is relatively high, and the industrial structure is relatively perfect. At the same time, the environmental protection investment in Suzhou is higher, and the environmental protection is also stronger.(2) Wuxi\u2019s ecological carrying elastic index ranks the second, which belongs to a low carrying level. It has a relatively developed economy and a complete industrial structure, but its environmental investment is relatively lower when compared with Suzhou.(3) The ecological carrying elastic indices for Nanjing, Nantong, and Changzhou are similar, at a weak carrying level. However, the reasons are slightly different. Nanjing and Changzhou have higher economic development levels and better industrial structures, so their ecological carrying elastic indices are relatively higher. Nantong has more investment in environmental protection, which partly compensates for the impact of the low economic development level and industrial structure imbalance on the ecological carrying elastic index. The ecological carrying capacities of Zhenjiang, Yangzhou, and Taizhou are also weaker and lag behind. Zhenjiang city, due to its low investment in environmental protection, has a lower ecological carrying elastic force; The economic development levels of Yangzhou and Taizhou are lower, and their industrial structures are not balanced, resulting in low levels of ecological carrying elastic force. In general, the ecological carrying elastic force of most cities in Yangtze River urban agglomeration is not high, at a weak carrying level, implying that the ecological carrying capacities of most cities are not high, and the environmental conditions are not optimistic. It is necessary to improve their ecological carrying capacities according to the specific conditions of each city.Through the ecological carrying pressure index model, the ecological carrying pressure indices of each city were calculated. Combined with the urban ecological carrying capacity grading evaluation criteria , a histoFrom (1) Suzhou has the highest ecological carrying pressure index, which belongs to the medium pressure level. Suzhou\u2019s industry has developed. Meanwhile, the population density is high, and hence, the exhaust emissions are large, while the occupancy of resources is large, and hence, the pressure on the ecological environment is relatively large.(2) Nanjing, Changzhou, Yangzhou, and Wuxi are at low pressure levels, but the pressures are still high in Yangtze River urban agglomeration. The reason lies in their high population density and wastewater discharge. Among them, Yangzhou and Changzhou have increased their ecological carrying capacities to some extent due to the large amount of chemical fertilizers and pesticides applied.(3) Taizhou, Zhenjiang, and Nantong are under weak pressure. Primarily due to the low discharge of wastewater and the small population pressure, the ecological carrying pressure is relatively small.On the whole, the higher the level of economic development, the higher the corresponding ecological carrying pressure. At the same time, most of the cities in the Yangtze River urban agglomeration have low ecological carrying pressure, and most of them are in a stage of low carrying pressure or weak carrying pressure. However, with the further development of the Yangtze River urban agglomeration, the ecological carrying pressure will increase, and measures still need to be taken to decrease the ecological carrying pressure of each city. Especially in Suzhou, the economic development and population density lead to the large amount of resources and the maximum ecological carrying pressure.Through the comprehensive index model of urban ecological carrying capacity, with the ecological carrying elastic indices  and ecolFrom (1) Nanjing, Changzhou, Taizhou, and Yangzhou are under high load. The economic development levels of Nanjing, Changzhou, Suzhou, and Wuxi are higher and the population density higher, and so are the waste and pollutant emissions of production and living. Therefore, the ecological carrying elastic indices and ecological carrying pressure indices of these four cities are higher than other cities. Among them, the ecological carrying elastic indices of Nanjing and Changzhou are lower than those of their ecological carrying pressure indices because the environmental protection investment cannot maintain their huge population and economic development. Taizhou and Yangzhou have a relatively low level of economic development, an underdeveloped industrial structure, a high use of chemical fertilizers and pesticides in agricultural development, and a low investment in environmental protection, resulting in huge ecological carrying pressure. Therefore, the comprehensive indices of their ecological carrying capacities are high, and they are in a stage of high load.(2) Zhenjiang, Wuxi, Suzhou, and Nantong are under low load. Among them, Zhenjiang has a low comprehensive index of ecological carrying capacity due to low population density and low production and living emissions. Nantong has more environmental protection investment, and the ecological carrying elastic index is higher than the ecological carrying pressure index, so the comprehensive index of ecological carrying capacity is low. Although Suzhou and Wuxi have large emissions from production and living, they have a large investment in environmental protection. Therefore, they are in a low-load stage. However, the ecological carrying pressure indices of Suzhou and Wuxi are slightly lower than the ecological carrying elastic indices, and the comprehensive indices of ecological carrying capacity are close to one, which means close to ecological balance. In short, the comprehensive indices of the ecological carrying capacity of Yangzhou, Taizhou, Changzhou, and Nanjing are higher in the Yangtze River urban agglomeration and are in the stage of high load carrying, indicating that the development of these cities has exceeded the capacity of the regional ecological environment and is in an unsustainable development state. These cities should pay more attention to environmental inputs while developing their economies. For Wuxi, Suzhou, Nantong, and Zhenjiang, although they are in a low-load stage, they still need to pay attention to environmental protection and reduce the comprehensive indices of ecological carrying capacity, especially Wuxi and Suzhou: their comprehensive indices of ecological carrying capacities were close to one.According to the ecological carrying elastic indices , the ecoThrough further analysis, we found that environmental protection investment and industrial structure had the greatest impact on ecological carrying elastic force. The proportion of tertiary industry and the investment in environmental protection in Nanjing, Suzhou, Nantong, and Wuxi were higher than other cities, making their ecological carrying elastic indices higher than other cities. It can be seen that a reasonable industrial structure can enhance the ecological carrying capacity of the region; the environmental protection investment can strengthen the cycle mechanism of the ecosystem, thereby improving the ecological carrying elastic force. Suzhou\u2019s environmental protection investment had a larger proportion, which improved its ecological carrying elastic force to a certain extent, therefore improving its comprehensive carrying capacity.Secondly, the wastewater discharge had the greatest impact on ecological carrying pressure, higher for Suzhou, Wuxi, Nanjing, and Yangzhou than other cities, making their ecological carrying pressure indices higher. The use of chemical fertilizers and pesticides in agricultural production also exerted a certain pressure on the ecological environment, most significant for Changzhou and Yangzhou. The use of large amounts of chemical fertilizers and pesticides aggravated the regional ecological carrying pressure and had a large negative impact on the ecological environment.By utilizing the comprehensive indices and ranking table of the ecological carrying capacity for the Yangtze River urban agglomeration  and the (1) Nanjing, Yangzhou, Taizhou, and Changzhou are under high load of carrying conditions. First of all, these cities are farther away from the estuary of the Yangtze River, and their own ecosystem circulation is weaker. Among them, Nanjing\u2019s industrial structure is relatively complete, but the environmental protection investment is relatively low; meanwhile, as a political and cultural center of Jiangsu province, Nanjing is densely populated, resulting in high wastewater and gas emissions. For the economically-developed cities like Nanjing, we should pay more attention to environmental protection investments, optimize the population structure, and reduce the environmental pollution and resource waste. Yangzhou and Taizhou have lower economic development levels, imperfect industrial structure, and insufficient investment in environmental protection, and their comprehensive indices of ecological carrying capacity are higher due to the higher discharge of wastewater and waste gas and the heavier use of chemical fertilizers and pesticides. For such cities, local resources should be fully utilized to develop the economy and improve the industrial structure; at the same time, increasing investment in environmental protection, raising public awareness of environmental protection, and improving resource utilization. Changzhou\u2019s industrial structure is improper; the agricultural production behavior is not standardized; and environmental protection investment is insufficient. For such cities, we should first focus on the transformation and upgrading of the industrial structure while developing the economy, and at the same time increase investment in environmental protection; second, increasing investment in science and technology, stimulating the transformation of the agricultural production mode, and reducing the use of fertilizers and pesticides.(2) Suzhou, Wuxi, Nantong, and Zhenjiang are under a lower load of carrying conditions. Except Zhenjiang, other cities are close to the estuary of the Yangtze River, and their ecosystems have stronger circulation capacities. Like Suzhou, it is densely populated and has a large amount of resources, but the industrial structure is good and the environmental protection investment higher. Therefore, although having a higher load of carrying and higher pressure state, it has only a lower load. For such cities, it is still necessary to optimize the industrial structure and strengthen the formulation of relevant environmental policies. On the contrary, Zhenjiang has a better environmental condition; at the same time, wastewater discharge is lower, and it is a lowlier loaded, lower pressure state. For such cities, we should vigorously encourage the tertiary industry such as the service industry and tourism on the basis of making full use of the advantages of local natural resources and continuously increase environmental protection investments in the later stage of development. The economic development level of Nantong is a little lower; the proportion of tertiary industry is low; but its environmental investment is high, and population density is low. For such cities, it is necessary to develop the tertiary industry on the basis of geographical advantages and accelerate the transformation and upgrading of the industrial structure. Wuxi has a relatively high level of economic development and a relatively complete industrial structure. At the same time, due to the higher investment in environmental protection and less pollution in agricultural development, its ecological environment is good. For such cities, the industrial structure should be continuously optimized during the development process, and investment in environmental protection should be increased. On the other hand, we should also increase the investment in science and technology, improve resource utilization, and formulate relevant policies such as sewage tax to reduce pollutant emissions, thereby maintaining the low-load carrying stage.The comprehensive evaluation index system of urban ecological carrying capacity is the basis for urban carrying capacity assessment and the focus of sustainable development. The assessment of the ecological carrying capacity for urban agglomerations is conducive to guiding the industrial structure and development direction for each city and has positive significance for promoting urbanization. We have established such a system for the Yangtze River urban agglomeration from two dimensions, ecological carrying elastic force and ecological carrying pressure, and analyzed the spatial difference of its ecological carrying capacity. The results revealed that:(1) The scientific evaluation of ecological carrying capacity requires comprehensive evaluation from two dimensions: ecological carrying elastic force and ecological carrying pressure. The more developed areas in the region tended to have larger resource occupations and thus higher ecological carrying pressure; moreover, due to the relatively complete industrial structure and high technical level, the ecological carrying elastic force of these areas was also higher. In areas where the ecological carrying elastic index was lower than the ecological carrying pressure index, the ecosystem was in a higher load stage because it could not maintain the ecological pressure brought about by its production and life; the areas where the ecological carrying elastic index was higher than the ecological carrying pressure index had lower ecological pressure and were in the stage of lower load.(2) Environmental protection investment had the greatest impact on ecological carrying elastic force, followed by the proportion of the tertiary industry; wastewater discharge had the greatest impact on ecological carrying pressure. For example, the proportion of tertiary industry and environmental protection investment in Nanjing, Suzhou, Nantong, and Wuxi were higher than other cities, and so were their ecological carrying elastic indices. Such densely-populated and economically-developed cities as Suzhou and Nanjing had larger relatively complete systems treating wastewater and waste gas, and therefore, the pressure on the ecological environment was naturally heavier.(3) The ecological conditions of cities in the Yangtze River urban agglomeration were different. It is urgent to build an exchange and cooperation mechanism for urban agglomerations to improve the quality of the overall ecological environment. First of all, municipal governments should take measures to improve the ecological carrying elastic force and reduce the ecological carrying pressure according to the actual conditions of each city. For example, for cities with large problems in industrial structures and an insufficient proportion of tertiary industry like Yangzhou, Taizhou, Zhenjiang, and Nantong, the key is to stimulate the transformation and upgrading of their industrial structures. Secondly is strengthening communication and cooperation among cities in the development process and highlighting the leading role of such central cities as Suzhou, sharing green development experiences with cities for decision-making. Thirdly is setting up special supervision departments to strengthen the supervision and inspection of the ecological environment in urban agglomerations, such as strict control of wastewater and waste gas emissions, strengthening the supervision and guidance of agricultural practices. Finally is coordinating regional development and establishing a complete urban ecological economic system. The mutually beneficial urban ecological network is used to facilitate the coordinated development of ecology and economy among cities and between urban and rural areas.The innovations of this paper are as follows: (i) most of the existing research is on typical provinces, cities, or regions, but there is a lack of research from the perspective of the urban agglomeration. The construction of the index system and the evaluation of ecological carrying capacity for the urban agglomeration in this paper enrich the research of ecological carrying capacity for urban agglomerations. (ii) Different from previous studies evaluating ecological carrying capacity from a single perspective, we established a comprehensive evaluation index system for the Yangtze River urban agglomeration from the two dimensions of ecological carrying elastic force and ecological carrying pressure, which is a combination of ecological and social economic perspectives. Furthermore, the advantage of this paper is that it is not limited to specific research conclusions. Through the comprehensive evaluation of ecological carrying capacity for the Yangtze River urban agglomeration, it points out the general standard for evaluating regional ecological carrying capacity, which is more universal. Meanwhile, the indicator system established in this study lays a foundation for the establishment of a more scientific indicator system in the future and also provides a decision-making basis for the sustainable development of urban agglomerations. However, due to the large amount of data required for the evaluation of the ecological carrying capacity of the ecological composite system in this study, the complicated process of obtaining the data, the limitation of creating indicators, and the subjectivity in determining the weight of the index, subsequent research for other areas with significant conditions needs to be conducted to further optimize the indicator system, the method of determining the index weight can also choose a more scientific method combining subjective and objective weighting, especially for the creation of indicators; for example, tailpipe emissions are derived from the conversion of various types of exhaust gases into standard conditions. However, some air pollutants may not be suitable for conversion to a standard state, or simply adding them together has little impact on research problems; follow-up research will carefully consider these issues, and the creation of indicators will be more rigorous. Then, the improved ecological carrying capacity evaluation model will have better scientific and universal value."}
+{"text": "Listeria monocytogenes through a microbial challenge test. Sliced cured pork loins can be stored at 6 \u00b1 1 \u00b0C for 105 days while maintaining a consumer acceptance of more than 75%. The freshness loss was associated mainly with a decrease in aromatic notes (particularly the smoke and cured aroma), and with the appearance of spoiled characteristics, specifically a sour/vinegar aroma and acidic taste that were detected by a reduced proportion of participants. The freshness evaluation was positively influenced by the typical characteristics of cured products, such as color and a garlic and wine aroma. Sour/vinegar aroma and acidic taste were the attributes most associated with higher freshness penalization. During the period of the test, Listeria monocytogenes inoculated onto the cured loin slices did not grow.Cured pork loins are sausages with a production tradition in several regions worldwide. They are made from one of the noblest cuts of pork, and for this reason cured loins are one of the most expensive pork meat products. Establishing the correct shelf life allows products to be accepted by the consumer, and to avoid the costs associated with shorter shelf lives. The aim of this study is: (1) to establish proper shelf life by evaluating the willingness of participants to consume and the sensory modifications that occur during prolonged storage via Check All That Apply (CATA) questions; and (2) to study the behavior of  Cured pork loins are different according to the production region. Variations in seasoning, size, cut  and smoking determine the differences. All of these products are cured using salt and/or nitrate, and are dried to achieve a sliceable texture and an activity of water (aw) reduced enough to contribute to the safety and preservation capacity of the product ,2,3,4,5.Clostridium botulinum and Listeria monocytogenes, among others [Salmonella or enteropathogenic Escherichia coli is commonly overcome by the use of high microbial quality raw materials and application of mild heat treatments at the beginning of the drying [Salmonella population resulting from the processing. The hurdles used to achieve that reduction might include chemical preservatives, mild heat treatments, and drying [Cured loins are made from one of the noblest cuts of pork and are, therefore, one of the most expensive pork meat products . They usg others ,9. The pe drying ,11. Whene drying ,13,14. Rd drying . Increasd drying  has beend drying . Chemicad drying . The stad drying . Anotherd drying ,21. In td drying ,23.L. monocytogenes or other psychotropic pathogens and if the product has intrinsic parameters, namely aw and pH, that allows their multiplication [Despite the very high hygiene measures used in slicing, any accidental contamination with foodborne pathogens might result in a lack of food safety. It is of particular concern if the contamination occurs with lication ,25. Takilication . When salication . There alication ,29. For lication .L. monocytogenes through a microbial challenge test.The aim of the present study was: (1) to establish proper shelf life by evaluating the willingness of consumers to consume and the sensory modifications the occur during prolonged storage via a Check All That Apply (CATA) test; and (2) to study the behavior of 2, water vapor transmission < 3 g/m2.d, and oxygen permeability < 25 cm3/m2.d.bar; lower part\u20140.12 mm, 118.00 g/m2, water vapor transmission < 3 g/m2\u00b7d, and oxygen permeability < 25 cm3/m2.d.bar. The upper film was 0.17 mm, 167.50 g/m2, water vapor transmission < 2 g/m2\u00b7d, and oxygen permeability \u2264 30 cm3/m2.d.bar.Cured pork loins, locally named salpi\u00e7\u00e3o, were obtained from an industrial unit on the week of their production. The cured loins were produced using the normal industrial manufacturing process. White commercial crossbreed pork carcasses (80 to 90 kg) of both sexes were obtained two days after slaughter. The loins were excised during the carcass dressing. Cured pork loins were prepared by cutting the loin transversely in single pieces about 5-cm thick and weighing about 400 g, then seasoning with red wine, salt, dextrose, garlic, and spice extracts. Non-meat ingredients were also included, specifically soya protein concentrate, milk powder, pork hemoglobin, and the food additives sodium nitrite, trisodium citrate, and pentasodium triphosphate, according to the limits of addition previewed in the Regulation UE 1129 . The mixThe sampling procedure used in the present experiment is presented in For the consumer test, 90 packages were transported to the laboratory under refrigeration, then divided into seven groups and stored at 6 \u00b1 1 \u00b0C for a total of 126 days, corresponding to seven storage periods with expirations spaced 21 days apart . Upon expiration of the stipulated period, the samples were frozen to \u221218 \u00b0C until sensory evaluation, to allow a reverse storage approach. This approach enabled us to analyze samples with different storage durations simultaneously . Before The challenge test was made using samples from the two batches. Once they arrived at the laboratory with one week of difference, we kept the samples from the first batch at 2 \u00b0C for one week. The packages were opened in aseptic conditions, and the slices were distributed for new packages, with five slices per package .Focus groups (FGs) were used to evaluate the effect of freezing on the sensory characteristics of the cured loins and validate the usefulness of the reverse storage strategy used, as well as to identify vocabulary with the potential to describe the freshness and spoilage of cured pork loins . Three fConsumer test was performed with total of 81 consumers. They were recruited from the professional and personal relationships of the researchers. Less than 20% of the participants were students. The group was composed of 58% women, and the mean age was 39 \u00b1 15 years (21\u201365). Of these participants, 90% were regular consumers of dry-cured meat products, while the reaming 10% were sporadic consumers. Those not liking cured sliced loins did not accept the invitation. It was not possible to conduct this work with a randomly selected group of consumers that might represent a more appropriate approach. Nonetheless, the group of consumers represented several socio-demographic groups and were consumers of the product. On the other hand, none of the participants had any relationship with the manufacturer or the meat products industry, and most of the respondents were not connected to food science, aspects that are usually pointed out as being potential biases of a group ,36.The test was divided into two sessions with the same participants to avoid the overload of analyzing seven samples in the same session. Storage durations were distributed for the two sessions using alternate times . The preFor each sample, participants were asked to rate its freshness using a five-point scale (where 1 corresponded to \u201cnot fresh\u201d and 5 corresponded to \u201cvery fresh\u201d) and to indicate their consuming and purchasing intentions using a binary yes/no question. They were also asked to fill in the CATA part of the form, which consisted of a list of 24 attributes . Water activity was measured in a Rotronic Hygroscope DT apparatus with a WA40 probe . Color was measured directly in the slices 30 min after opening the packages via a tristimulus color analyzer Minolta CR 310  with a standard illuminant DEnterobacteriaceae and Pseudomonas spp. LAB counts were determined on De Man, Rogosa and Sharpe Agar and incubated under at 30 \u00b0C for 2\u20133 days; mold and yeasts were determined on Rose-Bengal Chloramphenicol Agar Base and incubated at 25 \u00b0C for 5 days; Enterobacteriaceae were determined on Violet Red Bile Glucose Agar and incubated at 37 \u00b0C for 24 h; and Pseudomonas spp. were determined on Pseudomonads Agar Base with Cetrimide, Fucidin and Cephalosporin supplements and incubated at 25 \u00b0C for 48 h. All culture media were from Biokar . Results are expressed as log cfu/g. For statistical purposes, when the microorganism count was below the detection limit, it was considered to be zero. Estimated counts were considered for data analysis when countable colonies were present but below the countable range.Microbiological analysis was performed at days 0, 42, 63, 84, and 126 of storage. After opening each of the three packages considered for each storage duration, 10 g was randomly cut from at least three slices then weighed and suspended in 90 mL of peptone water, as described in the reference , and anaL. monocytogenes during the storage of sliced cured pork loins, as described in the literature [L. monocytogenes was performed immediately after packaging, then again at 42, 63, 84, and 126 days. For each storage duration, three packages were reviewed to perform the L. monocytogenes count. For each sample, slices were unpacked, weighed, and homogenized with the necessary volume of NaCl 0.85% to achieve an initial dilution of 0.1 g/mL. Serial decimal dilutions in NaCl 0.85% were prepared, and 0.1 mL of appropriate dilutions was spread onto selective Oxford Agar  followed by incubation at 30 \u00b0C for 24 to 48 h. When low counts were expected, 0.5 mL of the initial dilution was inoculated on two Petri dishes (0.25 mL each), dried in the laminar flow (to avoid biofilm formation), and counted as the total colonies in both dishes. Results are expressed in log cfu/g.A challenge test was performed to study the behavior of terature . FreshlyL. monocytogenes counts\u2014were made using ANOVA for the three packages analyzed for each storage duration (p < 0.05). An evaluation of freshness was compared between the consumption and purchasing intentions (yes/no using a t-test). The Pearson\u2019s correlation between the LAB counts (expressed in log cfu/g) and pH was computed. The CATA results were recorded as 1 when a participant checked an attribute and 0 when they did not. The data were analyzed by factor analysis. Each attribute was compared between formulations using a Cochran test. The mean impact of each CATA attribute in the freshness evaluation was computed. The impact of each CATA attribute on the freshness evaluation was calculated only for the longest storage duration, when the spoilage notes were more easily detected. All the statistics were calculated using XLStat . Raw data might be accessed at http://www.mdpi.com/2304-8158/9/5/621/s1  than those not accepted by the participants (2.51 \u00b1 1.73). A similar trend was observed for purchasing intention . Still, in the period of high acceptance, the freshness evaluation was statistically different (p < 0.05) only between the first day of storage and all the other storage durations. After the initial more accentuated slope, the freshness assessment was similar from the 42nd day of storage until the end. The median values . Accordn values  also refTo understand what modifications in the product were associated with a reduced consumption intention/freshness evaluation, participants were asked to identify those attributes of the cured loins (from a list provided) that they considered relevant at different storage durations. The results are illustrated in From the initial list of 24 attributes, 11  were withdrawn from the analysis if they were used by less than 20% of participants for all the storage durations . That stp < 0.05) between storage durations (p = 0.097).Even though the attributes \u201csour/vinegar\u201d and \u201cacid\u201d had a frequency of use lower than 20% for most of the storage durations, they had frequencies or 20% or more at the longer durations, and so were kept. The first factor (F1) in urations . Still, p > 0.05) between any storage durations. The cured color, which seems to be discriminative in the factor analysis (p = 0.097) to be noted by fewer participants, from 79% at the beginning of the experiment to 61% at 126 days of storage. In the same sense, the color parameters a* and b* did not have differences among the tested storage durations , which is a common occurrence since the production of lactic acid, and eventually acetic acid, is the main factor responsible for pH reductions in meat products that support the growth of LAB and incorporate carbohydrates into their formulation [Brochothrix thermosphacta, which can dominate the microbiota of packaged thermal processed meat products [Enterobacteriaceae and Pseudomonas are frequently associated with the occurrence of pungent spoilage odors due to strong amino acid catabolic activity [The formation of aromatic ketones due to lipid oxidation is further associated with the characteristic aroma of cured meat products , and in mulation . Cured lmulation ). Duringmulation ,65. In cproducts . In the activity . In the activity ,68,69,70activity . In the activity ,73)\u2014but activity , and itsCured pork loins usually have a low amount of fat. Combining that reduced fat with vacuum packaging and the presence of food additives that are not classified as antioxidants but have a role in preventing lipid oxidation , it was not assumed that the sliced cured loins experienced lipid oxidation. However, this attribute was maintained for the CATA test, as the initiation of oxidation might occur in some products before packaging, and the further modification can occur, albeit slowly, during long storage periods ,75. As oThe CATA analysis allowed us to understand the loss of freshness of sliced cured loins during storage. The impact each attribute had on the freshness evaluation was assessed only using the data of the longest storage duration (126 days) .In L. monocytogenes during the storage of sliced cured loins. The main inference from this test is that the pathogen did not grow during storage. L. monocytogenes has a reputation for high resistance under adverse environmental conditions [L. monocytogenes did not grow, probably due to the presence of nitrite. For the longest storage durations, the LAB could also contribute to pathogen inhibition Additionally, L. monocytogenes couts showed a slight decrease for the longest storage duration, and determined significant differences (p < 0.05) between 0 days (3.21 \u00b1 0.09 log cfu/g) and all other storage durations. At 126 days, the count of the pathogen was 1.61 \u00b1 0.25 log cfu/g, the lowest of the experiment. On average, the count of the pathogen decreased by around 1.5 log cfu/g. This behavior was probably associated with the start of the LAB growth. The first shift with a more pronounced slope\u2014between zero and 42 days\u2014matched the major reduction in pH observed  (L. monocytogenes has been observed previously in cured products [nditions . In the 6 units) . The secproducts .L. monocytogenes. Testing should be performed to guarantee that, if the product leaves the industry with L. monocytogenes, it does not grow [L. monocytogenes. According to that legal framework, only meat products with a pH \u2264 4.4 or an aw \u2264 0.92, or products with a pH \u2264 5.0 or aw \u2264 0.94, are exempted from the need to perform challenge tests with L. monocytogenes if the manufacturer chooses to allow the presence of L. monocytogenes in the finished product. The results of the present work allow the industry to be unconcerned with this seriously concerning biological hazard. The sliced cured loins used in the present work meet all the conditions necessary to be L. monocytogenes-free. Besides the high level of hygiene in the industry and the qualification of suppliers, , the hot-smoking process and presence of nitrite contribute to the elimination of potential contaminations [L. monocytogenes, knowing how pathogens behave in cold chains allows the industry to ponder the risks associated with sliced meat products if one accidental or unpredicted contamination occurs.The sliced cured loins had an average aw of 0.93 and a pH of 5.8, which, according to Regulation EC 2073 , might snot grow . The indinations . The mosinations . The indinations . DespiteSliced cured loins can be stored at 6 \u00b1 1 \u00b0C for 105 days and maintain a consumer acceptance of more than 75%. Freshness loss is due to two concomitant mechanisms. On one hand, sliced cured loins lose intrinsic aromatic notes, particularly the smoke and cured aromas, probably due to scalping as a result of the packaging material. On the other hand, the product develops spoiled notes putatively related to the fermentative activity of LAB. A sour/vinegar aroma and acidic taste are the main sensory characteristics associated with spoilage. Any sensory trait related to rancidity or non-protein-nitrogen catabolic activity of the microbiota was detected in this study. Although lightness (L*) decreased during the storage, participants did not detect any difference in the \u201cbright\u201d or \u201cdull: aspects, nor in the \u201cmoisten\u201d or \u201cdry\u201d aspect.The freshness evaluation was influenced mainly by the typical characteristics of cured products with a positive impact. The spoilage notes of a sour/vinegar aroma and acidic taste were responsible for the major penalizations in the mean evaluation of freshness.L. monocytogenes inoculated onto the surface of the cured loin slices did not grow. A slight reduction of about 1.5 log cfu/g was observed, and thus shelf life extension of the vacuum packaged sliced cured loins did not compromise the safety of the product.During the period of the test, This study had the limitation of using a convenience consumer group. As pointed out in the materials and methods section, the group was considered acceptable due to its demographic profile, consumption habits, lack of connections with the manufacturer, and small proportion of food science-related professionals."}
+{"text": "Precise control and maintenance of population size is fundamental for organismal development and homeostasis. The three cell types of the mammalian blastocyst are generated in precise proportions over a short time, suggesting a mechanism to ensure a reproducible outcome. We developed a minimal mathematical model demonstrating growth factor signaling is sufficient to guarantee this robustness and which anticipates an embryo's response to perturbations in lineage composition. Addition of lineage-restricted cells both in vivo and in silico, causes a shift of the fate of progenitors away from the supernumerary cell type, while eliminating cells using laser ablation biases the specification of progenitors toward the targeted cell type. Finally, FGF4 couples fate decisions to lineage composition through changes in local growth factor concentration, providing a basis for the regulative abilities of the early mammalian embryo whereby fate decisions are coordinated at the population level to robustly generate tissues in the right proportions. Across metazoa, coordination between cell fate specification and population size ensures robust developmental outcomes. Integration of cell behavior at the population level allows a coordinated response to injury in both embryos and adults . The preThe blastocyst-stage embryo is a hallmark of mammalian preimplantation development. It comprises three cell types \u2013 the pluripotent epiblast, which gives rise to the fetus, and the extra-embryonic trophectoderm (TE) and primitive endoderm , which predominantly form supporting tissues . In the Nanog, Gata6, Fgf4 or Fgfr1 alter these proportions and cause peri-implantation lethality . In the mouse embryo, uncommitted ICM progenitors, which co-express NANOG and GATA6, adopt epiblast or PrE identity asynchronously and irreversibly over the course of blastocyst development . In wildethality . The ratethality . HoweverIn this study, we combine manipulations of ICM composition with predictions from in silico simulations to address the question of regulation of the number of cells allocated to each ICM lineage. We develop a minimal mathematical model in which cell fate decisions in the ICM are mediated solely by intercellular signaling. In this model, ICM cells spontaneously and robustly segregate into two lineages, which scale with embryo size as they do in vivo. The model has only two free parameters, which are adjusted to recapitulate the observed wild-type behavior. The robustness of this in silico decision is evidenced by the response of the system to perturbations that alter lineage composition. Specifically, the model predicts  that reducing or increasing the number of cells in one lineage, would change the pattern of progenitor differentiation to restore lineage composition. This effect is also observed experimentally by using two-photon laser excitation for ablation of specific cells in embryos, and by adding exogenous, lineage-restricted cells to embryos to generate chimeras. The ability to recover from these perturbations is reduced over time, as the number of uncommitted progenitor cells is depleted. Finally, we alter the size of the PrE by experimentally tuning the size of the epiblast compartment. Using this system, we show that FGF4 is the growth factor providing the feedback necessary to couple lineage size with cell fate decisions. Our results provide a mechanistic basis for the regulative and scaling abilities of the early mouse embryo and illustrate how a self-organizing system can develop robustly and reproducibly without the need for external inputs.Epiblast and PrE cells originate from a population of bipotent progenitor cells that co-express the lineage-associated transcription factors NANOG (epiblast) and GATA6 (PrE) , which wEpiblast and PrE size (with respect to cell number) scale with embryo size to maintain a consistent ICM composition . To dete\u2212/\u2212Gata6 cells, instead of wt cells , wt cells contribute almost exclusively to the PrE, whereas in chimeras with fewer mutant cells (>60% wt GFP+ cells), wt cells contribute to both the epiblast and PrE, and generate ICMs with a normal lineage composition (We first mixed labeled and unlabeled wild type-(wt) cells, which have equivalent developmental potential, in different proportions . The prowt cells , and monepiblast , left  of epiblast:PrE cells , charactBox 1.A minimal model of growth factor-mediated cell-fate decisions.We use the following model to represent the dynamics of a population of cells in which the transcription factors NANOG and GATA6 mutually repress each other through extracellular growth factor signaling:i, and x over the immediate neighborhood of cell i, including Here, This model can be derived from a specific molecular-level circuit explicitly involving NANOG, GATA6 and the growth factor FGF4, and implicitly ERK signaling downstream of FGF receptors, as detailed in Appendix 1. However, due to its minimal character, the model is not unique to the molecular interactions assumed to be involved in this cell fate decision; other molecular circuits can likely be reduced to it to achieve the same result.In the model, the level of NANOG is inhibited by that of its neighbors, in a manner that resembles lateral inhibition-mediated signaling. We ask whether such a model can sustain solutions in which cells cluster into two distinct cell types, expressing NANOG at two different levels (which we could interpret as epiblast and PrE cells). Epiblast and PrE cells display a salt-and-pepper distribution in the ICM at early blastocyst stages . Therefoa and b of the two clusters are interchangeable.The dynamics of this potential two-cluster solution can be examined via the phase-plane portrait shown in The existence of the two symmetric stable equilibria ensures that the two-cluster state is a solution of the system, and that the population splits spontaneously into two distinct fates, as we show by means of agent-based simulations (described in Appendix 1) throughout the text.ix per cell, which in our case can be considered to represent the amount of NANOG in that cell. FGF4 is assumed to be activated by NANOG and feeds back onto NANOG and GATA6 via ERK  and are rearranged as the embryo develops and grows. To that end, we implemented an agent-based model to simulate the growth of the ICM (see Appendix 1) in three dimensions, in which cells divide and interact with one another via a soft-sphere potential . The bioThe fate decisions ascribed by the model are also biologically robust, since altering the absolute cell number within the in silico ICM leads to scaling of lineage size to maintain ICM composition , in agreThe attractor solution found in the model described suggests that the cell fate decisions reached by the embryo are robust to perturbations, in particular to those affecting the size of each population. To probe the capacity of the system to perceive and adjust to changes in cell numbers, we expanded the epiblast by introducing increasing amounts of mouse embryonic stem cells (ESCs) into embryos . ESCs ar+/\u2212Gata6 or +/\u2212Fgf4 blastocysts, which exhibit a reduction in PrE cell numbers relative to epiblast  , althougWe next asked whether this experimental perturbation would be recapitulated by our mathematical model in silico. To do so, increasing amounts of epiblast-equivalent cells (ESCs) were added to the system before activation of the molecular circuit  and a uWe used laser ablation  to test Pdgfra expression (Pdgfra and mKate2), as well the overrepresentation of PrE cells within the ICM. Progenitor cells in embryos where the PrE was targeted showed a comparable trend to control embryos, generally upregulating Pdgfra expression and acquiring a PrE identity  to recovery of ICM composition after cell ablation. In addition to changes in progenitor specification , we obseOverall, these results indicate that changes in ICM composition are primarily compensated for by a shift in the differentiation pattern of progenitor cells. Although the small sample size precludes any definitive conclusion regarding the roles of cell death and proliferation, both our cell ablation and chimera data suggest that cell death and division play only accessory roles in this process, and that uncommitted progenitors are the primary substrate for regulation.\u2212/\u2212Fgf4 embryos, which cannot make PrE . Embryos were obtained from natural matings of 4-\u00a0to\u00a012 week-old females to stud males. Alleles used and their original source are summarized in Embryonic days (E) were determined by considering noon of the day of detection of the copulation vaginal plug as E0.5. Higher-resolution staging based on total cell number was used throughout as previously . To deteEmbryos were flushed from dissected oviducts (8\u201316 cell stage) or uterine horns (blastocyst stages) using forcing Flushing and Holding Media , as previously described . Live emEmbryos were lysed for genotyping in 10 \u00b5l of lysis buffer  for 50 min at 50\u00b0C, followed by 10 min at 90\u00b0C to inactivate Proteinase K . 2 \u00b5l of2 atmosphere. Prior to culture, embryos were rinsed 3x in drops of KSOM-AA. For live imaging, embryos were cultured on 35 mm glass-bottom dishes (MatTek) within 5\u201310 \u00b5l drops of KSOM-AA under mineral oil.Embryos were cultured on 35 mm Petri dishes  within microdrops (10\u201315 \u00b5l) of Potassium Simplex Optimized Media with amino acids  under mineral oil (Sigma), at 37\u00b0C, in a humidified 5% COImages of live embryos were acquired using Zeiss LSM880 laser-scanning confocal microscopes. In cell ablation experiments, images were acquired using a Zeiss C-Apochromat 40x/NA1.1/WD0.21mm objective. For all other experiments, images were acquired using a Zeiss EC Plan-Neofluar 40x/NA1.3/WD0.17mm. GFP was excited using a 488 nm Argon laser at 20\u00b5W. mKate2 was excited using a 543 nm HeNe laser or a 561 nm DPSS 561\u201310 laser at 90\u00b5W. Laser power was measured through a Zeiss Plan-Neofluar 10x/NA0.3 objective prior to each imaging session with a light meter (Coherent) and the laser output adjusted to match laser power across experiments. 80 \u00b5m stacks were acquired through embryos, at 2 \u00b5m intervals, every 15 min. Although these stacks do not capture an entire blastocyst, they encompass the ICM while limiting laser exposure to about 30\u201340 s per time point and embryo. Time lapse movies were 16\u201320 hr long.Tg/+CAG:H2B-EGFP R1 ESCs  used were  R1 ESCs  and HexVE14 ESCs . ESCs weTg/+CAG:H2B-EGFP (Tg/+CAG:H2B-EGFP) were used in the generation of both wt \u2194 GFP+ and \u2212/\u2212Gata6 \u2194 GFP+ chimeras  and those with four or fewer cells were discarded. Morulae (8\u201316 cells) from CD1 x EGFPTg/+  crosses chimeras , whereasGdf9iCre  and a fle  attached to an Eppendorf CellTram microinjectiong system was used to assist with injections. Blastocyst-stage embryos were collected in the morning of the 4th day of development and monitored for development to the mid-blastocyst stage (chimeric embryos were retrospectively estimated to have had ~60\u201380 cells at the time of injection). Individual embryos were held with a holding pipette  by the ICM end, while a Piezo Drill flat tip needle (Eppendorf) was used to introduce ESCs into the blastocyst cavity . After iPdgfra expression    , in whicteTg/Tg) . For ablud males . For ablTg males .H2B-GFPPdgfra allele was markedly higher than in their neighbors on each optical plane  to cover the period of PrE and epiblast specification  in which a number of ICM cells equivalent to the total number of PrE or epiblast cells found in GFP+ littermates  was randomly selected and ablated using the same settings.Cells were eliminated by repeatedly focusing the beams of an 800 nm Ti:Sapphire femtosecond laser at 10\u201312% output (Coherent), onto the central region of each nucleus to be ablated (approximately 30\u201350% of the nuclear area in the section). The number of pulses was determined empirically on a test embryo for each experiment, so that a clear wound was observed on the fluorescent nucleus, but no obvious damage was inflicted to the neighboring cells  . AlthougAfter cell ablation, live intact and targeted embryos, were imaged for 16\u201320 hr, as described above. Laser ablation generated a visible wound in nuclei expressing H2B-GFP, which allowed tracking of targeted and intact cells and assessing nuclear fragmentation as a mark of cell death ; Video 4Whole-mount embryo immunofluorescence was performed as described previously . PrimaryImmunolabeled embryos were mounted on 35 mm glass-bottom dishes (MatTek), within micro drops of a 5 \u00b5g/ml solution of Hoechst 33342 in PBS and imaged using a Zeiss LSM880 laser-scanning confocal microscope, equipped with an oil-immersion Zeiss EC Plan-Neofluar 40x/NA1.3/WD0.17mm. Z-stacks were acquired through whole embryos with 1 \u00b5m step between optical slices. Laser power was measured for each laser line prior to each imaging session and parameters adjusted so as to keep laser power consistent for each primary-secondary antibody combination across experiments over time \u2013 except for the 405 channel, which was used to excite the nuclear label (Hoechst 33342), and was solely used for image segmentation.https://github.com/therealkatlab/MINS (requires MATLAB license). Missing nuclei, or multiple nuclei segmented as one (under-segmentation) were measured manually using ImageJ  and the measured values for each channel, as well as XYZ coordinates, added to the data table.Nuclear image segmentation of still images and manual image correction was performed using the MINS software  as previPdgfra expression and allow the classification of cell types over time (Time lapse images were processed using Imaris (Oxford Instruments). Individual ICM cells were identified based on the presence of H2B-GFP and/or nuclear mKate2 and tracked manually over the course of the movie using the spots function. Cell death or mitotic events were labeled as such for each individual cell . GFP levver time . Cell idver time  and verihttps://github.com/nestorsaiz/saiz-et-al_2020/blob/master/notebooks/_data-processing-wf.pdf and in https://github.com/nestorsaiz/saiz-et-al_2020/blob/master/notebooks/Z-correction.ipynb\u00a0, PrE (GATA6+), double positive  and NANOG-low or NANOG- epiblast (NANOG-lo) . A fifthon.ipynb\u00a0.Pdgfra expression) for ICM cells were used to determine changes in cell identity over time in time-lapse movies. To reduce noise in the data, a moving average was calculated for each cell, with a window of 4 timeframes (1 hr). A simple classifier was thus devised to assign cell identity automatically to individual cells based on these GFP levels. Thresholds for fluorescence were manually determined for each litter analyzed, both at the start and the end of the movie, which were used to determine a threshold slope for PrE and epiblast identities. The classifier followed two rules: (1) cells classified as PrE or epiblast maintain that identity for the remainder of the movie \u2013 based on Corrected fluorescence values of H2B-GFP .Thank you for submitting your article \"Growth factor-mediated coupling between lineage size and cell fate choice underlies robustness of mammalian development\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:In this study Saiz et al. explore the mechanism responsible for the balanced generation of epiplast and primitive endoderm cells from the inner cell mass of the mouse embryo. The authors use a combination of molecular and embryological assays coupled with mathematical modelling to conclude that non-autonomous regulative feedback, mediated by FGF4, is responsible for robustly maintaining Epi and PrE ratios.Essential revisions:The reviewers think this is a very well done study, which can be published without any further experiments. However they all commented on the extend of the modelling presented and suggest that this could be strengthened in 3 specific areas with regard to:\u2013 FGF spread;\u2013 How geometry affects the behaviour;\u2013 How asynchrony in decisions is implemented in the model and relates to the biology. Essential revisions:The reviewers think this is a very well done study, which can be published without any further experiments. However they all commented on the extend of the modelling presented and suggest that this could be strengthened in 3 specific areas with regard to:\u2013 FGF spread;\u2013 How geometry affects the behaviour;\u2013 How asynchrony in decisions is implemented in the model and relates to the biology.Our mathematical model was designed to capture the basic interactions posited to underlie the Epi-PrE decision taking place in the early mouse embryo. In spite of its simplicity, the model allows us to examine the influence of a variety of biological factors, in particular those highlighted by the reviewers: (i) the signaling range of FGF, (ii) the geometry of the population, and (iii) the role of cell division variability on the asynchronicity of cellular decisions.We have systematically studied the influence of these factors in the behavior of our model, by performing a sensitivity analysis of the final cell fate ratio exhibited by the model, when all model parameters are varied independently by increasing or decreasing their values 20%, 30% and 50% beyond the nominal values given in Appendix 1\u2014table 1. The results are shown in the new Figure 2\u2014figure supplement 1F. We describe these results in what follows.i) FGF signaling range (spread)An increase in the range of signaling  does not noticeably change the decision pattern exhibited by the model, which reproduces qualitatively the experimental observations. Only when the signaling range is substantially reduced  is the decision prevented. This is to be expected, since the latter case a cell only detects the FGF molecules that it itself secretes. In that limit the decision cannot occur, since in our model the decision is fully mediated by intercellular FGF signaling.We have also systematically varied the range of FGF signaling, from the autocrine limit , in which cells only sense the FGF they produce, to the all-to-all coupling limit , in which the FGF secreted by a cell is sensed by all cells in the embryo. The results show that as soon as cells are coupled with their nearest neighbors , the decision is reached with appropriate cell-fate ratios, which are maintained even when signaling extends over the entire population. Local nearest-neighbor coupling is thus not necessary for the correct decision to arise.ii) GeometryThe decision is also robust to most perturbations in the mechanical parameters of the model, which partly define, and are defined by, the geometry of the system. This can be seen again in Figure 2\u2014figure supplement 1F, which shows that changes in the effective friction and adhesion between cells, and in the adhesion range, do not for the most part affect the cell fate decision ratios produced. Only when the effective friction decreases substantially  is the decision lost. In this latter case, cell mobility becomes too high and intercellular signaling stops taking place efficiently, which mimics the autocrine limit discussed in the previous point.A stronger geometrical constraint is the dimensionality of the system. The main results reported in this manuscript were obtained in a three-dimensional configuration, which corresponds to the experimental situation studied here. However, we have also examined how our model behaves in a more constrained two-dimensional topology. The response of the system when cells are restricted to move  on a 2D plane are displayed in Figure 2\u2014figure supplement 1E. Simulations show that the cell fate decision process is maintained in these conditions, which manifests the robustness of the mechanism to the dimensionality of the system.iii) Variability and asynchrony of the decisionGiven the key role of cell-cell signaling set forth in this study, it is important for our model to describe cell proliferation adequately: when cells divide the population is reorganized, and thereby the signals sensed by cells from their neighbors change. Experimental observations from ourselves and others show that, as the early embryo develops, cells divide in an asynchronous manner.Following previous modeling approaches ), we implemented this asynchrony in our model, by considering that the cell division time of each cell varies randomly (according to a uniform distribution) with respect to a baseline in which cells would divide at times proportional to an average division time Both In the revised manuscript we discuss the results described above, which we have included in new panels (E-H) that have been added to Figure 2\u2014figure supplement 1. We have also taken the opportunity to revise the text in order to discuss the similarities and differences between our model and previous pattern formation frameworks, such as the Turing and lateral inhibition mechanisms. While our model has conceptual similarities to both scenarios, its specific form, and the way in which it derives from a minimal set of assumptions regarding the regulation of the Epi-PrE cell fate choice, is to our knowledge, new. We have also attempted to clarify further the way in which FGF signaling affects NANOG and GATA6 in our model."}
+{"text": "Scientific Reports 10.1038/s41598-020-60654-7, published online 27 February 2020Correction to: The original version of this Article contained an error in Figure 1 where the two maps were overlapping. This has now been corrected in the PDF and HTML versions of the paper."}
+{"text": "Scientific Reports 10.1038/s41598-020-65327-z, published online 26 May 2020Correction to: The version of this Article previously published contained a corrupted version of Figure 5. This has now been corrected in the PDF and HTML versions of the paper."}
+{"text": "First reported in China, the coronavirus responsible for coronavirus disease 2019 (COVID-19)\u00a0has spread to 213 countries and territories around the world as of April 26, 2020. This study was designed to explore COVID-19 trends in the Eastern Mediterranean Region (EMR), with a particular focus on Pakistan. Daily reports and updates from the Ministry of National Health Services Regulations and Coordination COVID-19 Pakistan\u00a0and the European Centre for Disease Prevention and Control\u00a0were collected and study-specific data were extracted and analyzed. Our analysis revealed that, as of April 26, 2020, a total of 22 countries and territories in the EMR have reported COVID-19 cases.\u00a0Iran had the highest number of cases  followed by Saudi Arabia , Pakistan , and the United Arab Emirates . Egypt (7.1%), Iran (6.3%), and Iraq (4.9%) had high case fatality rates; Lebanon (3.4%) and Pakistan (2.1%) had moderate case fatality rates; Saudi Arabia and the United Arab Emirates had low case fatality rates of 0.8% and 0.7%, respectively. Iran (76.3%) and Iraq (69.4 %) had the highest recovery rate followed by Pakistan (22.5%), the United Arab Emirates (19.2%), and Saudi Arabia (13.6%). If the current trend continues, based on the susceptible, infected, recovered (SIR) epidemiological model, we predict that EMR countries might\u00a0experience a surge in the number of COVID-19 cases, resulting in as many as 2.12 million cases in Iran, 0.58 million in Saudi Arabia, and 0.51 million in Pakistan by June 20, 2020. Pakistan is the most populated country in the EMR and was the third most-affected country in terms of the number of cases with moderate case fatality and recovery rates. We predict that Pakistan\u2019s weak healthcare system would not be able to sustain care if there is an explosive increase in the number of cases due to insufficient\u00a0and inconsistent disease prevention and control policies. The best strategy for mitigating the COVID-19 pandemic is to strictly follow recommendations based on epidemiological principles. Coronaviruses have previously been known for causing Severe Acute Respiratory Syndrome (SARS-CoV) in China in 2003 and Middle East Respiratory Syndrome (MERS-CoV) in Saudi Arabia in 2012. The novel virus responsible for coronavirus disease 2019 (COVID-19), termed SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2), is more contagious than SARS and MERS-CoV [Scientific reports suggest that 81% of COVID-19 patients have a mild or asymptomatic disease, 14% have severe disease\u00a0manifesting respiratory distress\u00a0or pneumonia and require medical care, and 5% of hospitalized patients require transfer\u00a0to intensive care units\u00a0.After originating from China, the novel coronavirus has\u00a0spread to 213 countries and territories throughout the world as of April 26, 2020. A World Health Organization (WHO) report confirmed the first 100,000 cases in two months , however, the number of cases increased to 200,000\u00a0in the next 12 days\u00a0 .The available global data on April 26, 2020 reflects 2,804,796 COVID-19 confirmed cases with 193,710\u00a0deaths. In the Americas region, the United States of America has the highest number of confirmed cases  with 46,204 deaths, followed by Brazil with 52,995 confirmed cases and\u00a03,670 deaths. In the Eastern Mediterranean region, Iran has reported 89,328 confirmed cases and 5,650 deaths followed by the Kingdom of Saudi Arabia with 16,299 confirmed cases and 136 deaths. Spain in the European region has reported 219,764 confirmed cases and\u00a022,524 deaths, followed by Italy with 195,351 confirmed cases and 26,384 deaths. In the Western Pacific region, China has the highest number of confirmed cases  with 4,642 deaths, followed by Japan with 13,182 confirmed cases and 353 deaths. Reports from Southeast Asia showed 26,496 confirmed cases in India with 824 deaths, while Indonesia has reported 8,607 confirmed cases\u00a0with\u00a0720 deaths. In the African region, South Africa has reported 4,361 confirmed cases with 88\u00a0deaths, and Algeria has reported 3,256 confirmed cases with 419\u00a0deaths\u00a0[The Eastern Mediterranean Region (EMR) comprises\u00a022 countries and territories and is home to over 679\u00a0million people\u00a0.In mid-February 2020, the first nine confirmed cases of COVID-19 were reported in the Eastern Mediterranean region. Eight cases from the United Arab Emirates tested positive between January 29 and February 9, 2020. One case from Egypt was confirmed on\u00a0February 14, 2020. The Ministry of Health and Prevention of the United Arab Emirates announced that three of the reported cases recovered and were discharged from the hospital during the second week of February 2020. The confirmed case in Egypt was asymptomatic and had a history of travel to China\u00a0.On January 30, 2020, the WHO declared COVID-19 a Public Health Emergency of International Concern (PHEIC)\u00a0. Since tData were collected daily from the Pakistan Ministry of National Health Services Regulations and Coordination COVID-19 dashboard and situation updates from the European Centre for Disease Prevention and Control\u00a0-12. The The true numbers of cases in the EMR countries were calculated based on the\u00a0estimation that a substantial number of COVID-19 cases do not get reported and only 14% of the cases have been reported\u00a0. The numa) Case fatality rate =  x 100b) Recovery rate =  x 100The number of cases\u00a0that EMR countries might expect by June 20, 2020, was estimated by using a calculator based on the susceptible, infected, recovered (SIR) model, a standard epidemiological model that is used to estimate disease dynamics\u00a0. These ca)\u00a0Every infected symptomatic person interacts with seven people a dayb)\u00a0Every infected asymptomatic person interacts with 15 people a day\u00a0As of April 26, 2020, Iran had the highest number of total of COVID-19 confirmed cases  in the EMR,\u00a0with 15,485 active cases, 3,096 critical cases, 5,630 deaths, and 68,193 recoveries. The number of confirmed cases per million population was 1,064 , the number of confirmed COVID-19 cases was 9,813, with 7,457 active cases, 71 deaths, one critical case, and 1,887 recoveries. The number of confirmed cases per million population was 938 Table\u00a0. The numIn Qatar, the number of confirmed COVID-19 cases was 9,358, with 8,419 active cases, 72 critical cases, 10 deaths, and 929 recoveries. The number of confirmed cases per million population was 3,248  followed by Bahrain , Iran , UAE (938), Kuwait (677), Saudi Arabia (468), Lebanon (103), and Pakistan (55) , Bahrain (229), Pakistan (187), Egypt (166), Saudi\u00a0Arabia (133), Kuwait (130), Iraq (124), Lebanon (109), and UAE (98).Pakistan, along with many countries around the world, is facing a historical public health challenge. As of April 26, 2020, Pakistan had\u00a012,723 COVID-19 cases. In Pakistan, the COVID-19 outbreak initially presented as sporadic. Two cases were reported on February 26, 2020, and the volume of cases remained quite low (53 cases) until March 15, 2020\u00a0. On MarcThe number of confirmed cases was doubled in Pakistan in\u00a011 days Table\u00a0, which wThe number of deaths doubled in Pakistan in 10 days. However, Iraq 27 days) and Iran (26 days) had a longer deaths doubling time , Iran (6.3%), and Iraq (4.9%) ,\u00a0which describes the average number of people who could be infected by a sick person. Currently, Pakistan has a local transmission rate of 80.6%\u00a0. As showAll countries and territories in the EMR\u00a0have reported cases of COVID-19, however, the number of cases and CFR varies among the EMR countries. Pakistan has the third-highest confirmed cases with moderate case fatality and recovery rates. If unswerving preventative and control measures are not adopted to prevent the widespread transmission of SARS-CoV-2, Pakistan could experience an explosive surge in the number of COVID-19 cases amounting to an estimated\u00a0half a million cases by June 20, 2020, as determined by the SIR epidemiological model.\u00a0This study highlights the need for strategic development and the implementation of policies and programs\u00a0focused on enhanced testing, contact tracing, quarantine, and social distancing\u00a0in Pakistan."}
+{"text": "Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified  Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens.Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host. Viruses are the largest known reservoir of genetic diversity. As such, they have given rise to an incredible assortment of genetic tools and potential therapeutics used as sustainable substitutes for antibiotics , 2, targPhaeobacter inhibens is a marine bacterium and member of the Roseobacteraceae family of Alphaproteobacteria. Organisms in this clade are found worldwide, especially near coastal waters , which protects its natural algal symbiont from marine pathogens  and whole-genome sequencing. Based on the morphology and genomic comparison to other related phages, we found that this isolate represents a novel species of siphophage, which we name Phaeobacter virus MD18. Finally, we used a barcoded transposon insertion mutant library  to characterize the relationship between Phaeobacter phage MD18 and P. inhibens. We identified the type IV pilus system, the ChvI/ChvG two-component system, and regulators of cell division as key determinants of infection. This work characterizes the genetic basis of phage infection in an underexplored nonmodel host system.In this study, we isolated a siphophage capable of infecting P. inhibens. We enriched for phages infecting P. inhibens by incubating filtered environmental samples together with exponentially growing liquid cultures of P. inhibens overnight. Then, we filtered the enrichment cultures and spotted each enrichment supernatant onto a lawn of P. inhibens grown on agar plates and monitored for the formation of plaques. Of the 20 environmental samples, only 1 sourced from a seashore environment produced a clear plaque, indicating the presence of a lytic phage. This enriched phage sample contained a high titer of PFU (\u223c1010 PFU/ml [Bdellovibrio spp.) were not responsible for the observed inhibition. Propagation of clear plaques indicated essentially complete bacterial host lysis and thus was likely the result of a lytic phage, henceforth referred to as Phaeobacter phage MD18.We obtained samples from 20 aquatic environments around Woods Hole, MA, to isolate a wild bacteriophage capable of infecting 0 PFU/ml ) and rep0 PFU/ml ). GrowthCaulobacter crescentus. MD18 was incapable of forming plaques on C. crescentus under conditions in which \u03a6CbK readily formed plaques  and C. crescentus (bottom). Phages demonstrated host-specific lytic potential. Download FIG\u00a0S1, PDF file, 1.4 MB.Phaeobacter phage MD18 exhibits lytic specificity to Copyright \u00a9 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Siphoviridae morphotype . Highlighted regions indicate sections exhibiting over 50% sequence similarity with Roseobacter phage DSS3P8. Despite sequence similarity, gene order appears to differ between these two phages  , eight (D4), and five (D6) species clusters. At the genus level, two (D0), five (D4), and two (D6) clusters resulted. The number of clusters determined at the family level were one (D0), five (D4), and one (D6). Download FIG\u00a0S3, PDF file, 0.2 MB.Phylogenetic analysis of Copyright \u00a9 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Phaeobacter virus MD18 corresponds to a unique species that has not previously been identified.Finally, we assessed whether MD18 had been previously identified in metagenomes. We used BLAST to search the MD18 genome against 27,346 marine virome contigs assembled from 78 marine viromes . This seN-acetylmuramoyl-l-alanine amidase, which have been demonstrated to facilitate bacterial cell wall degradation and cell lysis  . Phage genomes frequently encode tRNA genes, which facilitate the translation of phage transcripts in host bacteria  and less significantly correlated with a distantly related nonhost bacterium, Escherichia coli   . These tRNA gene   .10.1128/mSphere.00898-20.6TABLE\u00a0S2Table\u00a0S2, XLSX file, 0.02 MB.tRNA genes identified in Phaeobacter phage MD18 genome. Download Copyright \u00a9 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. inhibens by identifying host gene products that confer susceptibility to infection. Recent work has used transposon insertion sequencing to rapidly perform reverse-genetic screens in hosts and identify genes that contribute to phage susceptibility (\u2013P. inhibens transposon insertion mutants with decreased susceptibility to phage by exposing a previously constructed barcoded transposon mutant library (P. inhibens genes annotated using RAST (8 PFU) for 8 h, conditions which allowed for significantly delayed growth in wild-type P. inhibens  was originally isolated in Galicia, Spain, and is available through the Leibniz Institute DSMZ. P. inhibens DSM 17395 was grown in Difco marine broth 2216 liquid (MB), on plates supplemented with 1.5% agar, or in 0.5 to 0.7% top agar overlays at 30\u00b0C. The P. inhibens-infecting bacteriophage was isolated from Woods Hole Waterfront Park in Woods Hole, MA. A sterile 15-ml tube was loaded with a sample of seagrass and filled to 15\u2009ml with seawater. After vigorous vortexing for 5\u2009s, 5\u2009ml of this sample was filtered using a 0.22-\u03bcm polyethersulfone (PES) syringe filter and divided into 1-ml aliquots. A single aliquot was incubated overnight at room temperature with 5\u2009ml of log-phase  P. inhibens in MB, centrifuged for 2 min at 2,000\u2009\u00d7\u2009g, and filtered using a 0.22-\u03bcm PES syringe filter to obtain the enriched bacteriophage population. This enriched sample was used for all downstream experiments with no further purification. The presence of the isolated phage was confirmed by spotting onto 0.7% top agar inoculated with mid-exponential-phase P. inhibens. The isolated phage was named MD18 because it was isolated as part of the microbial diversity course at the Marine Biological Laboratory in Woods Hole, MA, during the summer of 2018.The P. inhibens, and this mixture was spread evenly across a petri dish containing MB agar. Once solidified, 5\u2009\u03bcl of each dilution was spotted and incubated for 16\u2009h at 30\u00b0C. As a control to exclude the presence of a cellular predator, 10\u2009\u03bcl of the undiluted phage stock was incubated with 1\u2009\u03bcl of 2.0% chloroform for 1\u2009h with shaking at 4\u00b0C before spotting on the same plate. Medium control was prepared by incubating 10\u2009\u03bcl of MB with 1\u2009\u03bcl of 2.0% chloroform for 1\u2009h with shaking at 4\u00b0C. To perform the liquid growth assays in the presence of bacteriophage, a log-phase culture of P. inhibens was diluted 1:100 in MB and 190\u2009\u03bcl was aliquoted into a 96-well Corning clear-bottom plate. To each well, 10\u2009\u03bcl of each bacteriophage dilution or sterile MB was added  in triplicate. The plate was grown for 24\u2009h at 30\u00b0C with shaking at 150\u2009rpm, and the OD600 of each culture was monitored using a Promega GloMax Explorer multimode microplate reader.Top agar spotting and liquid growth assays were used to measure the concentration of PFU in the lysate and to characterize the plaque phenotype of the enriched phage MD18 isolate. Eight 10-fold dilutions of the enrichment were prepared by serial dilution in MB. To perform the spotting assay, 4\u2009ml of MB with 0.5% molten agarose was cooled to \u223c50\u00b0C before mixing with 1\u2009ml of log-phase 10 PFU/ml) was incubated with a glow-discharged Formvar-coated 200-mesh copper grid for 3 min before being washed three times with sterile 0.22-\u03bcm-filtered water. The sample was negatively stained by incubation in a 2% uranyl acetate solution under darkness for 1 min before undergoing another series of washes. For imaging host cells and phage MD18 together, 5\u2009\u03bcl of the same phage preparation was preincubated with 5\u2009\u03bcl of exponential-phase P. inhibens culture for 10\u2009min and adhered to the grid and stained as described above. Samples were imaged using a Zeiss 10CA transmission electron microscope with help from the Marine Biological Laboratory Central Microscopy Core.To prepare the bacteriophage for transmission electron microscopy, 10\u2009\u03bcl of the enriched bacteriophage isolate lysate (\u223c1010 PFU/ml) in 100\u2009ml of mid-log-phase P. inhibens in MB. This culture was grown for 24\u2009h with shaking before centrifugation and 0.22-\u03bcm filtration of the supernatant. This phage fraction was concentrated to \u223c1\u2009ml with Corning 30,000-molecular-weight-cutoff (MWCO) Spin-X UF 20 concentrators .Phage MD18 was prepared by inoculating 100\u2009\u03bcl of the phage enrichment  purification kit . Briefly, DNase I was added to 300\u2009\u03bcl of phage sample at a final concentration of 1\u2009\u03bcg/ml and incubated at room temperature for 1\u2009h. To the virus sample, 480\u2009\u03bcl of 50\u2009mM EDTA and 900\u2009\u03bcl of cell lysis solution were added, vortexed, and incubated for 30\u2009min at 30\u00b0C. To this mixture, 600\u2009\u03bcl of nucleus lysis solution was added, vortexed, and incubated for 5\u2009min at 80\u00b0C. To this mixture, 3\u2009\u03bcl of RNase A solution was added, vortexed, and incubated for 30\u2009min at 37\u00b0C. To this mixture, 200\u2009\u03bcl of protein precipitation solution was added, vortexed for 20 s, and incubated for 5\u2009min on ice. Cellular debris was pelleted by centrifugation at 13,000\u2009\u00d7\u2009https://rast.nmpdr.org/rast.cgi) to identify putative genes and the tRNAscan-SE 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/). Genome feature tables were generated by converting GenBank (.gbk) files using the GB2sequin online server . The DNA was sequenced on an Illumina NovaSeq 6000 with a NovaSeq SP reagent kit, yielding 647,478 250-nucleotide (nt) read pairs. Reads were trimmed using Trimmomatic version 0.38, using the parameters SLIDINGWINDOW:4:15 LEADING:2 TRAILING:2 MINLEN:35, resulting in 623,304 reads. These reads were assembled using SPAdes (version 3.11.1) using default parameters. The primary assembled contig was submitted to the RAST , 21 onlin-SE 2.0 , 25 onlie server . The cire server .Phaeobacter virus MD18 and potentially related species was performed using the Virus Classification and Tree Building Online Resource web server (VICTOR). C. crescentus phages were selected from reference JX100813; CcrMagneto, JX100812; CcrSwift, JX100809; CcrKarma, JX100811; CcrRogue, JX100814; and CcrColossus, JX100810. These accession numbers, along with those for Phaeobacter phage MD18 and Roseobacter phage DSS3\u04248 (accession no. KT870145), were submitted to the Virus Classification and Tree Building Online Resource web server .Phylogenetic analysis of VICTOR processed these genomes in the following manner. All pairwise comparisons of the nucleotide sequences were conducted using the genome BLAST distance phylogeny (GBDP) method  under sehttps://genoplotr.r-forge.r-project.org/). Genomes were aligned using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using discontiguous megablast, which is optimized for more dissimilar sequences. The hit table (text) file from the alignment along with the genome GenBank records (.gb) were used as input for GenoPlotR.Genome maps between Phaeobacter phage MD18 and Roseobacter phage DSS3\u04248 were generated in R (version 4.0.2) using the GenoPlotR package (version 0.8.9) .To determine whether MD18 had been previously identified in metagenome libraries, the MD18 genome was searched against a data set of 27,346 marine virome contigs assembled from 78 marine viromes . BLAST , 60 commP. inhibens library was originally produced by Wetmore et al. , and 1.5\u2009ml of 100-fold-diluted plaque-purified bacteriophage lysate (\u223c1010 PFU/ml) was added to three of these cultures. These six cultures were grown for 8 h, then 1\u2009ml of each culture was centrifuged at 2,000\u2009\u00d7\u2009g for 2 min, and the pellets were frozen for later DNA preparation.The e et al.  and geneIn total, nine samples were processed for BarSeq. Three of these samples represented the original library, three represented the library grown without phage, and three more represented the library grown in the presence of phage. Genomic DNA from all samples was harvested using a Promega Maxwell RSC PureFood GMO and authentication kit. To prepare these samples for Illumina sequencing, sequencing primer sites, Illumina flow cell adapters, and sample indices were added using two successive PCRs. Primers used are described in 10.1128/mSphere.00898-20.8TABLE\u00a0S4Table\u00a0S4, XLSX file, 0.01 MB.Primers used for BarSeq libraries (PCR 1 and PCR 2). PCR 1 added sequencing primers, whereas PCR 2 added indices and flow cell adapter sequences. Download Copyright \u00a9 2020 Urtecho et al.2020Urtecho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. inhibens DSM 17395 genome (GenBank no. CP002976.1) was annotated using the RAST (https://rast.nmpdr.org/), and barcodes were grouped by which RAST-identified gene they overlapped. Barcode transposon insertion sites were previously described by Wetmore et al. . The the RAST , 21 onlie et al. . All insFirst, the gene enrichment scores with and without phage were calculated. This was determined by dividing the RPM-normalized counts of all barcode insertions overlapping each gene after treatment by the RPM-normalized counts before treatment.RPM-normalized counts were calculated from the mean of three biological replicates. Then, the fitness score of each gene disruption was calculated by dividing the sum of all enrichment scores within the phage treatment by the sum of all respective enrichment scores without phage added (see equations).https://string-db.org/) and searched against Phaeobacter inhibens DSM 17395. Default settings were utilized and online STRING tools were used to identify three gene clusters using K-means clustering.Fitness scores were considered significant if they were greater or less than 3 standard deviations of the bootstrapped mean fitness score. All gene annotations were generated using RAST with default parameters and viewed using SEED to acquire Gene Ontology terms. To generate the gene network, amino acid sequences of the genes whose knockouts exhibited significantly greater fitness scores were uploaded as multiple sequences to the STRING online tool (MT270409). P. inhibens transposon mutant fitness data are available on NCBI Gene Expression Omnibus (accession no. GSE148502). Code to analyze BarSeq data and generate figures is available on Github (https://github.com/gurtecho/PhaeobacterphageMD18).The Phaeobacter phage MD18 genome is available in GenBank (accession no."}
+{"text": "Acute restraint stress (ARS) is an unavoidable stress situation and may be encountered in different clinical situations. The aim of the current study was to investigate the effects of ARS on the hippocampus and cerebellum, assess the impact of these effects on the behavior and cognitive function, and determine whether pretreatment with ceftriaxone would attenuate the damages produced by ARS on the hippocampus and cerebellum. Four groups of male mice were included in this study: The control group, ARS group, ceftriaxone group, and ARS + ceftriaxone group. Tail suspension test, Y-maze task, and open field tests were used to assess depression, working spatial memory, and anxiety. The biochemical analyses included measurements of serum cortisol, tumor necrotic factor (TNF), interleukin-6, hippocampal expression of bone morphogenetic protein 9 (BMP9), lysosomal-associated membrane protein 1 (LAMP1), glutamate transporter 1 (GLT1), heat shock protein 90, cerebellar expression of S100 protein, glutamic acid decarboxylase (GAD), and carbon anhydrase. Histopathological examination of the brain sections was conducted on the hippocampus and cerebellum by hematoxylin and eosin stains in addition to ultrastructure evaluation using electron microscopy. Our results suggested that ceftriaxone had neuroprotective properties by attenuating the effects of ARS on the hippocampus and cerebellum in mice. This effect was demonstrated by the improvement in the cognitive and behavioral tests as well as by the preservation of the hippocampal and cerebellar architecture. Acute restraint strain (ARS) is an inevitable stress situation that might cause autonomic and behavioral alternation . The hipThe cerebellum is the area of the hind brain that plays an important role in cognitive and emotional processes . The dysCeftriaxone is an antibiotic that has shown neuroprotective effects in animal models of stroke , traumatThe aim of the current study was to investigate the effects of ARS on the hippocampus and cerebellum, assess the impact of these effects on the behavior and cognitive function, and evaluate whether pretreatment with ceftriaxone would attenuate the damages produced by ARS on the hippocampus and cerebellum.In total, 24 male albino mice aged 10 weeks (25\u201330 g) constituted the animal model of the current study. The guidelines of the Ethical and Scientific Committee of the Department of Medical Physiology at the Faculty of Medicine, Cairo University were followed. Rats were divided into 4 groups with 6 mice per group:Vehicle control group: Where mice received normal saline (0.9% NaCL solution) intraperitoneally (i.p), with a 24-h interval for 3 consecutive days.-Acute restraint stress (ARS) group: Where each mouse experienced a single stress session following 18 h of fasting (food deprivation). Each stress session consisted of 2.5 h of immobilization by firmly securing all four limbs of each mouse to a grid using a quartz tape [-rtz tape .Ceftriaxone group: Received ceftriaxone , i.p., as a single dosage of 200 mg/kg/day dissolved in normal saline [-l saline  for 3 coARS + ceftriaxone group: Where each mouse experienced ARS (in a similar way to that in the ARS group) and received 3 doses of ceftriaxone .-The following tests were performed before drug administration and grouping of mice and after acclimatization to the laboratory environment to avoid previously reported problems in the literature. The tests were then repeated 24 h after exposure to ARS.Open field test: Open field testing measures locomotion, exploration, and anxiety. A wooden box (1 m \u00d7 1 m \u00d7 0.5 m) was divided by a marker pen into equally spaced squares. A mouse was positioned in the center of the open field and was examined in a quiet room illuminated by controlled light for 5 min [or 5 min . The behTail suspension test (TST): It tests depression-like behavior in mice. Mice were held 50 cm above the floor by adhesive tape positioned about 1 cm from the tip of the tail. Immobility time was recorded for a period of 6 min. Mice were considered immobile only when they hung passively and became completely immobile [immobile .Y-maze test: Mice were placed in the middle of the maze and were allowed to explore the three arms for 8 min to check the working memory of the space. The three successive decisions taken by the mouse with three different arms were counted as a correct choice. The alternation score was calculated by dividing the total number of alternations by the total number of choices minus 2 multiplied by 100 [d by 100 . After 24 h, serum samples were received from the retro-orbital sinuses. The mice were euthanized by cervical decapitation. The cerebella and hippocampi were dissected, and their tissues were used for biochemical analyses and histopathological examination.Serum was analyzed for cortisol, tumor necrotic factor-alpha (TNF-\u03b1), and interleukin-6 (IL-6) and cortisol levels by ELISA (Quantikine R&D system USA) as instructed by the manufacturer. Hippocampus tissue was assessed for bone morphogenetic protein 9 (BMP9), lysosomal-associated membrane protein 1 (LAMP1), glutamate transporter 1 (GLT1) and heat shock protein 90 and cerebellar tissue was evaluated for S100 protein levels, GLT1, glutamic acid decarboxylase (GAD) and carbonic anhydrase by RT-PCR .Total RNA extraction: Total RNA was extracted from the homogenized tissue using the SV Total RNA Isolation System  according to the manufacturer\u2019s instructions. RNA concentrations and purity were calculated using an ultraviolet spectrophotometer .2, 2 \u03bcL of 0.1 M DTT, and 1 \u03bcL of SuperScript\u00ae III RT (200 U/\u03bcL) was applied to the total mixture and incubated at 25 \u00b0C for 10 min followed by another 50 min at 50 \u00b0C.The cDNA was synthesized from 1 \u03bcg of RNA using the SuperScript III First-Strand Synthesis System according to the manufacturer\u2019s protocol . To summarize, 1 \u03bcg of total RNA was combined with 50 \u03bcM oligo (dT) 20, 50 ng/\u03bcL random primers, and 10 mM dNTP. The total mixture volume was 10 \u03bcL. The mixture was incubated at 56 \u00b0C for 5 min, then put in ice for another 3 min. The total mixture comprising 2 \u03bcL of 10\u00d7 RT solution, 4 \u03bcL of 25 mM MgClReal-time PCR amplification and analysis were performed using the Applied Biosystem software version 3.1 StepOne . The reaction included SYBR Green Master Mix , a gene-specific primer pair , which wTo summarize, proteins were extracted from tissue homogenates using ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors , then centrifuged at 12,000 rpm for 20 min. The protein concentration for each sample was determined using Bradford assay. Equal amounts of protein  were separated by SDS/polyacrylamide gel electrophoresis (10% acrylamide gel) using a Bio-Rad Mini-Protein II system. The protein was transferred to polyvinylidene difluoride membranes  with a Bio-Rad Trans-Blot system. After transfer, the membranes were washed with PBS and were blocked for 1 h at room temperature with 5% (w/v) skimmed milk powder in PBS. The manufacturer\u2019s instructions were followed for the primary antibody reactions. Following blocking, the blots were developed using antibodies for GLT1and beta actin supplied by Thermoscientific  and incubated overnight at pH 7.6 at 4 \u00b0C with gentle shaking. After washing, peroxidase-labeled secondary antibodies were added, and the membranes were incubated at 37 \u00b0C for 1 h. Band intensity was analyzed by a ChemiDocTM imaging system with Image LabTM software version 5.1 . The results were expressed as arbitrary units after normalization for \u03b2-actin protein expression.The dissected mice brains were fixed in a 10% formalin solution and enclosed in paraffin. Sections of 5 \u00b5m thickness were obtained at the level of the hippocampus and cerebellum, stained with hematoxylin and eosin, and examined by a light microscope for histological examination .Morphometric measurements were performed in Cairo University-Research Park (CURP), Faculty of Agriculture, Cairo University using a Leica Qwin 500 image analyzer computer system . The image analyzer consisted of a colored video camera, Panasonic wv. GP 210, colored monitor, and a hard disk of a Leica IBM personal computer connected to a BX41 Olympus microscope  and controlled by Lecia Qwin 500 software. Each parameter was measured by two observers blinded to the experimental group by using 10 non-overlapping readings from each animal in different groups.The following structures were measured :(1) Measurements of the molecular cell layer (M) and granular cell layer (G) thickening; at the mid sagittal section of the cerebellum; at 3 different areas; cortical thickness of the folium surface (1), cortical thickness facing the fissure (2), and cortical thickness at the fissure base (3) . They we(2) Morphometric analysis of the Purkinje cell count at higher magnification .(3) Morphometric analysis of pyramidal neurons counts in the CA3 region of the hippocampus .Small blocks (1\u20132 mm thick) of the brain at the level of the hippocampus and cerebellum were fixed in 2.5% glutaraldehyde at 0.1 M sodium cacodylate buffer at 4 \u00b0C for 6 h. All procedures for the preparation and analysis of the samples were conducted according to Grimaud and Borojevic .p \u2264 0.05 [Using SPSS 21 , data presented as mean \u00b1 standard deviation (Mean \u00b1 SD), a comparison of quantitative variables between groups was made using variance analysis (ANOVA) with Bonferroni post-hoc test. Results were considered statistically significant for p \u2264 0.05 . p \u2264 0.05) in the number of line crossings, the duration of central square entry, and the time spent in the central square. The ARS group also experienced increased anxiety compared to the control group as indicated by a significant increase in rearing, grooming, freezing, and stretching frequencies (p \u2264 0.05) compared to the control group. Ceftriaxone pretreatment significantly (p \u2264 0.05) improved exploratory activity and reduced anxiety relative to the ARS group  in the ARS group compared to the control group  compared to the control group and the same test was significantly decreased (p \u2264 0.05) in the ARS + ceftriaxone group compared to the ARS group (The length of TST immobility suggesting depression in the ARS group was significantly increased (RS group .p \u2264 0.05) in the ARS group compared to the control group. Serum cortisol was decreased and serum IL-6 and TNF-\u03b1 were significantly decreased in the ARS + ceftriaxone group compared to the ARS group. The same markers were significantly increased (p \u2264 0.05) in the ARS + ceftriaxone group compared to the control group.The biochemical analyses in blood, hippocampal, and cerebellar tissues are presented in p \u2264 0.05) in the ARS group and the level of hippocampal HSP90a was significantly increased (p \u2264 0.05) in the same group compared to the control group. The levels of BMP9, LAMP1, and GLT1 were significantly (p \u2264 0.05) increased in the ARS + ceftriaxone group and the level of HSP90 was significantly decreased in the same group to levels that were similar to those of the control group  increased in the ARS group compared to the control group. The levels of cerebellar S100B and GAD were significantly (p \u2264 0.05) decreased in the ARS + ceftriaxone group compared to the ARS group to levels similar to those of the control group. Cerebellar S100 B, carbonic anhydrase, and GAD were significantly (p \u2264 0.05) decreased in ARS + ceftriaxone group compared to the ARS group, although it was still significantly increased (p \u2264 0.05) compared to the control group. Carbonic anhydrase was significantly (p \u2264 0.05) decreased in the ARS group and significantly (p \u2264 0.05) increased in the ceftriaxone group compared to the ARS group  were uniform in size and evenly arranged. Each neuron had a rounded central vesicular nucleus with a prominent nucleolus. The cytoplasm contained prominent basophilic cytoplasmic Nissel\u2019s granules and was surrounded by thin neuropil. The molecular layer (M) contained many glial cells (G) among the neuronal processes A. The H&E-stained sections of the mouse hippocampus in the ARS group demonstrated pathologic changes in most of the nuclei of the pyramidal neurons as well as pathologic cytoplasmic changes in the form of vacuolated cytoplasm (V). Some pyramidal cells had vesicular nuclei with clogged marginated chromatin and prominent nucleoli (P) while others had pyknotic nuclei (white arrow). Few neurons had homogenous nuclei with eosinophilic cytoplasm (arrows), and others had ghost changes (G) B. The H&E-stained sections of the mouse hippocampus in the ARS group demonstrated other specimens with markedly affected pyramidal cells of the CA3 region. Most of the neurons were shrunken, with hyperchromatic nuclei (thick arrows) and vacuolated cytoplasm; others had pyknotic nuclei (thin arrows) or karyolysis (K). Areas devoid of pyramidal neurons (asterisk) were detected C. The H&E-stained sections of the mouse hippocampus in the ARS + ceftriaxone group demonstrated marked improvement of the pyramidal neurons similar to the normal architecture of the control group. They were heavily crowded with thin neuropil in between and had basophilic cytoplasm, well-formed Nissel\u2019s granules, and vesicular nuclei (P). Few pyknotic nuclei with vacuolated cytoplasm were detected (arrows) compared to other groups D.The electron micrographs of the control group demonstrated a normal ultra-structure of the pyramidal cell as the neuron had an euchromatic, central, and rounded nucleus (N) with a smooth bilaminar nuclear envelope and finely dispersed chromatin. Well-formed rough endoplasmic reticulum (R) and intact mitochondrial (M) were seen A. The ARS group demonstrated a distorted dense neuron with an irregular outline. The nucleus (N) had marginated chromatin, chromatin clumps, and was surrounded by perinuclear space (thick black arrow). The cytoplasm was shrunken and showed deposition of electron-dense bodies (e). Some mitochondria were distended with disrupted cristae (black arrow) while others were comparable to the control (white arrow). The rough endoplasmic reticulum was markedly dilated (r) B. The ARS group demonstrated markedly affected pyramidal cells. The nucleus was irregular in shape (N) and the cytoplasm contained multiple ballooned mitochondria (arrows) with disrupted cristae C. The ARS + ceftriaxone group demonstrated a marked improvement of the pyramidal cell architecture; the nucleus (N) was regular in shape and was surrounded by the bilaminar nuclear membrane. The cytoplasm revealed multiple mitochondria comparable to the control group (black arrow) while others were ruptured (white arrow) D.The H&E-stained sections in the mouse cerebellar tissues of the control group demonstrated the characteristic pattern of the cerebellar cortex formed of three layers: The molecular cell layer (M), Purkinje cell layer (P), and granular cell layer (G). The medulla formed of white matter fibers (W) was also seen A. The ARThe H&E-stained sections in the mouse cerebellar tissues of the control group demonstrated the molecular cell layer (M) and showed small scattered basket cells and stellate cells. The Purkinje cell layer (P) showed large pyriform-shaped cells having vesicular open-face nuclei, eosinophilic cytoplasm, and prominent Nissl\u2019s granules. The granular cell layer (G) showed crowded small deeply stained cells A. The ARS group demonstrated few Purkinje cells (P) with deeply stained nuclei and eosinophilic cytoplasm, surrounded by vacuolated neuropil. Other Purkinje cells were shrunken with pyknotic nuclei and vacuolated cytoplasm (arrows) B.The ARS group demonstrated other specimens with marked degeneration of the Purkinje cells, exhibiting vacuolated cytoplasm (V) and pyknotic nuclei (black arrows). Basket cells and stellate cells were surrounded by perineuronal spaces (white arrows) C. The ARS + ceftriaxone group demonstrated a remarkable improvement and restoration of the normal architecture of the three cortical layers of the cerebellum; Purkinje cells were increased in number, and most of them were comparable to the control group with open face nuclei (P) while few cells had darkly stained nuclei and eosinophilic cytoplasm (arrow) D.The electron micrographs of the control group demonstrated Purkinje cells with an euchromatic nucleus (N) with finely dispersed chromatin and well-formed bi-laminar nuclear envelope. The cytoplasm contained scattered cell organelles. A primary dendrite (D) was also seen projecting from the cell membrane A. The ARS group demonstrated Purkinje cells with a dark electron-dense nucleus (N) and corrugated nuclear membrane. The cytoplasm contained multiple swollen mitochondria with rarified matrix and fragmented or lost cristae (M) B.The ARS group demonstrated markedly affected Purkinje cells with signs of degeneration. The nucleus was shrunken, darkly electron dense, and irregular in shape with indentation of its nuclear membrane (I). Many dispersed irregularly shaped chromatin aggregates were seen throughout the nucleus. The cytoplasm contained multiple ballooned mitochondria (arrows) with disrupted cristae C. The ARS + ceftriaxone group demonstrated an improvement of the Purkinje cell architecture, with an euchromatic nucleus (N) and finely dispersed chromatin. The cytoplasm revealed multiple mitochondria comparable to the control group (M) while others were disrupted with separated outer and inner mitochondrial membranes (arrows) D. p < 0.05)  .p < 0.05)  were significantly restored in the ARS + ceftriaxone group ( < 0.05) .p < 0.05) (There was a significant decrease in the number of cerebellar Purkinje cells of the ARS group compared to the control and ceftriaxone groups. The number of Purkinje cells was significantly restored in the ARS + ceftriaxone group ( < 0.05) .Stress leads to the production of mental, emotional, and cognitive alterations . Acute rIn the current study, ceftriaxone (CTX)  demonstrated a substantial neuroprotective impact on plasma stress markers and tissue markers in the hippocampus and cerebellum. In accordance with our results, Wei et al.  demonstrThe pathological changes in the hippocampal and cerebellar cells in the untreated ARS group can be attributed to the high level of serum cortisol, which is involved in stress reactions. High levels of serum cortisol have previously been found to lead to multiple structural and physiological changes in the nervous system .Cortisol has also been shown to influence cognition and decrease activity in the cerebellum. For example, cerebellar volume has been shown to be similarly decreased in individuals with Cushing\u2019s disease . The conDysmetria is thought to be related to the role of the cerebellum in sensory, cognitive, and emotional regulation . Stress Cerebellar activation in response to acute stress involves the expression of the c-fos gene , which was associated the action potential of the neurons . Stress Stress impairs long term potentiation (LTP) in the hippocampus. High doses of cortisol in vivo or in vitro have also been shown to impair LTP , indicatThe effect of ceftriaxone on improving behavior, cognition, and depression in the current study can be explained by the fact that ceftriaxone and corticosterone modulate specific frequency bands in opposite directions, and reveals the potential role of ceftriaxone in counteracting the effects of corticosterone . CeftriaThe number of crossed lines of the mice in the ceftriaxone group was significantly decreased compared to mice in the control group and those in the ARS + ceftriaxone group. This could be attributed to the upregulation of GLT1, and this is in agreement with Matos-Ocasio et al. .The results of the current study showed that mice in the ARS group showed reduced hippocampal BMP9 and LAMP1 and increased hippocampal HSP90 compared to the control group. Mice in the ceftriaxone group showed increased BMP9 and LAMP1 and reduced HSP90 to levels similar to those in the control group.Basal forebrain cholinergic neurons (BFCNs) project into the hippocampus and cerebral cortex where the activation of their neurotransmitter, acetylcholine (ACh), is essential for attention, learning, and memory processes . Bone moThe lysosomal membrane is implicated in the cell death pathway. Lysosomal-associated membrane protein-1 (LAMP-1), an abundant lysosomal membrane protein, produces a sugar coat or glycocalyx on the inner side of the lysosomal membrane and protects the membrane from hydrolytic enzymes and degradation . LAMP1 iCells react to environmental stress through the synthesis of several molecular chaperones. One of the amplest molecular chaperones is the heat shock protein 90 (Hsp90). The expression of HSPs is a sensitive marker for metabolic activation or oxidative stress . RegardiIn the current study, mice of the ARS group showed increased cerebellar S100 B and carbonic anhydrase relative to those in the control group. Mice in the ceftriaxone group showed normalized cerebellar S100B and decreased carbonic anhydrase compared to those in the ARS group; however, it was still upregulated compared to those in the control group. These findings may be explained by the affinity of S100 proteins to bind with calcium as a calcium-binding protein The conformational change caused by calcium binding results in the display of two adjacent hydrophobic-binding surfaces. S100 proteins also interact with calcium-dependent partner proteins, such as . S100B aCarbon anhydrase in the nervous tissue is considered a marker for glial cells. pH has a strong modulatory effect on the function and excitability of the central nervous system. Calcium, even if catalytically inactive, may function as \u2018proton-collecting antennas\u2019, thus increasing cerebellar S100 B and carbonic anhydrase in the net transmembrane proton flux and suppressing the development of HC microdomains . CA III \u03b3-aminobutyric acid (GABA) from glutamate. GABAergic neuron dysfunction impairs the cerebellar output [Glutamic acid decarboxylase (GAD) catalyzes the formation of glutamate to r output . The neuIn conclusion, the harmful effects of ARS on the hippocampus and cerebellum  could be ameliorated by ceftriaxone, which appears to have neuroprotective properties. This deduction is supported by the improvement in the cognition and behavior of the mice, and was confirmed structurally by the preservation of the hippocampal and cerebellar architecture, as revealed by tissue markers\u2019 expression, and microscopic and ultrastructural evaluations."}
+{"text": "The coronavirus disease 2019 (COVID-19) is an epidemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Populations at risk as well as those who can develop serious complications are people with chronic diseases such as diabetes, hypertension, and the elderly. Severe symptoms of SARS-CoV-2 infection are associated with immune failure and dysfunction. The approach of strengthening immunity may be the right choice in order to save lives. This review aimed to provide an overview of current information revealing the importance of bee products in strengthening the immune system against COVID-19. We highlighted the immunomodulatory and the antiviral effects of zinc and polyphenols, which may actively contribute to improving symptoms and preventing complications caused by COVID-19 and can counteract viral infections. Thus, this review will pave the way for conducting advanced experimental research to evaluate zinc and polyphenols-rich bee products to prevent and reduce the severity of COVID-19 symptoms. On 11 March 2020, the World Health Organization recognized the new coronavirus disease 2019 (COVID-19) as a pandemic. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected for the first time in Wuhan, China, at the end of 2019; the virus has not reported before in humans . Most in\u00ae combined with standard care  were used for hospitalized COVID-19 patients in a trial registered under the number NCT04480593 ,1918,19]+ cytotoxic T lymphocytes and then induce their restriction by the release of the effectors\u2019 molecules such as perforin and granzymes , which are subsequently recognized by antigen-specific CD8ranzymes C 19]. L. L+ cyto+ helper T lymphocytes with the transition into a T helper (Th) which has a double function: cytokines produce and promote B Cells to generate antibodies. Being stimulated by intracellular pathogens, T helper lymphocytes 1 (Th1) phenotype promotes the cytotoxic T lymphocyte activity by releasing IL-2 and enhances the differentiation of B lymphocytes to plasma cells which produce specific antiviral antibodies by liberating IFN-\u03b3  via MHC I (MHC I); [Human Leukocyte Antigen (HLA) in humans] and MHC II (HLA-Cw\u221708) . Furtherng IFN-\u03b3 B.T helper lymphocytes (Th2) activate and promote the degranulation and the release of chemokines, proteases, and histamines by innate immune system cells including mast cells and basophils, which increased vascular permeability and enhanced the recruitment of macrophages and other inflammatory cells . Besides+ and CD4+ memory T cells in SARS-CoV-2 patients were effective in prompting a specific immune response from 3 months to 6 years without the presence of antigens ..194].Meanwhile, there is evidence that viral infections are associated with an increase in reactive oxygen species (ROS) levels and a decrease in antioxidant defenses . ExposurPolyphenols are natural compounds well known for their antioxidant and anti-inflammatory effects. The antioxidant activity of polyphenols is related to their capacity to react with ROS using different reaction pathways such as the inhibition of the enzymes responsible for ROS production, the up-regulation of endogenous antioxidant defenses, or scavenging reactive oxygen species . 2 pathways. Resveratrol exerted an anti-inflammatory effect by inhibiting NF-\u03baB and TPA-induced COX-2 expression. Similarly, caffeic acid phenethyl ester (CAPE) inhibits inflammatory reaction via the reduction of NF-\u03baB [It seems that the main pathways, by which polyphenols-rich bee products can attenuate SARS-CoV-2 infection, are the modulation of the immune system, the prevention of inflammatory responses, and the reduction in oxidative stress . It was of NF-\u03baB .pro with efficacy and binding energies equivalent to an already claimed N3 protease inhibitor [Recently, numerous published reports revealed that phenolic compounds found in bee products can react with SARS-CoV-2 and blocks its entry into human cells . It was nhibitor .In addition, the molecular docking of ten flavonoid compounds: apigenin, chrysin, fisetin, galangin, hesperitin, luteolin, morin, naringin, quercetin, revealed that all of the flavonoids studied have a high binding affinity with the active site of the spike protein of SARS-CoV-2 .pro and replication of the virus [The antiviral effect of 6 compounds present in honey and propolis  was studied in silico. The results showed that four compounds  had a strong binding affinity with a good glide score and may inhibit the COVID-19 Mhe virus .pro) and Spike (S) protein) revealed that cyanidin may suppress rdrp by binding at asp761 catalytic residue and halting the viral replication process. In addition, the following flavonoids can interact on the spike proteins\u2019 key rbd and inhibit the spread to receptors and thus limit viral spread: daidzein, eriodictyol, fisetin, genistein, kaempferol, myricetin, quercetin, arbutin, chalconaringenin, phloretin, and liquiritin. In a study conducted by Vijayakumar et al. , the molO-glucoside can be considered lead compounds in the inhibition of SARS-CoV-2 papain-like protease (PLpro) [A total of 220 phenolic compounds were tested in a study published by Pitsillou et al. The results of in silico and in vitro using an enzymatic inhibition assay indicated that hypericin, rutin, and cyanidin-3- (PLpro) .p-cumaric acid, and benzoic acid revealed that rutin and caffeic acid phenethyl ester have the highest affinity to COVID-19 3CL-protease and S1 Spike [In the same vein, docking studies of rutin, caffeic acid phenethyl ester, quercetin, kaempferol, pinocembrin, pinobanksin, galangin, chrysin, S1 Spike  . Antioxidant compounds and other food micronutrients with valuable additional functionalities are important in resolving other COVID-19-related issues. For instance, human trial studies documented that ferulic acid and resveratrol have strong antidiabetic, cardioprotective, and renoprotective effects ,206. Thuhttps://clinicaltrials.gov/ct2/home, accessed on 10 February 2022). For instance, the NCT04578158 trial, a randomized, open-labeled, and controlled study, investigates the adjuvant benefits of quercetin Phytosome  in community-based subjects with confirmed SARS-CoV-2 infection. In addition to quercetin, resveratrol, a stilbenoid that highly presents in bee products, has been the object of 4 clinical trials . The NCT04400890 trial aims to evaluate the safety and explore the effectiveness of resveratrol treatment  against COVID-19 infection. Published data support that resveratrol inhibits coronavirus replication through the down-regulation of mRNA and nucleocapsid protein expressions of SARS-CoV-2 [Furthermore, as of 10 February 2022, quercetin has been approved in 14 clinical trials for profylaxis and managing COVID-19 symptoms (available online at RS-CoV-2 ,208.In the absence of specific antiviral drugs against SARS-CoV-2, natural remedies such as apitherapy could alleviate the complications associated with COVID-19. Considering the crucial role of the immune system in fighting SARS-CoV-2 infection, boosting immunity was highly recommended. In this direction, the present review highlights and provides an overview of zinc and phenolic compounds found in bee products and their direct and indirect actions on the immune system in fighting against this emergent public health crisis. The problems which arise are that the nature, quality, and quantity of these bioactive molecules in bee products differ from one country to another. To overcome this major drawback, an international norm, ISO/TC 34/SC 19, is currently in progress to standardize the whole process and circulation of bee products."}
+{"text": "In the two studies presented here, we explore the significance of word class for concreteness and imageability in human and computationally obtained ratings. The observed correlations in the CPD indicate correspondences between psycholinguistic measures expected from the literature. Word classes exhibit differences in subjective frequency, age of acquisition, concreteness and imageability, with significant differences between nouns, verbs, adjectives and adverbs. In the computational study which focused on concreteness and imageability, concreteness obtained higher correlations with human ratings than imageability, and the system underpredicted the concreteness of nouns, and overpredicted the concreteness of adjectives and adverbs. Overall, this suggests that word class contains schematic conceptual and distributional information. Schematic conceptual content seems to be more significant in human ratings of concreteness and less significant in computationally obtained ratings, where distributional information seems to play a more significant role. This suggests that word class differences should be theoretically explored.Psycholinguistic databases containing ratings of concreteness, imageability, age of acquisition, and subjective frequency are used in psycholinguistic and neurolinguistic studies which require words as stimuli. Linguistic characteristics  are frequently coded, but word class is seldom systematically treated, although there are indications of its significance for imageability and concreteness. This paper presents the Croatian Psycholinguistic Database , containing 6000 Croatian nouns, verbs, adjectives and adverbs, rated for concreteness, imageability, age of acquisition, and subjective frequency. Moreover, we present computationally obtained extrapolations of concreteness and imageability to the remainder of the Croatian lexicon (available at:  Psycholinguistic databases containing human ratings of characteristics such as concreteness, imageability, age of acquisition, and subjective frequency are routinely used in a variety of psycholinguistic and neurolinguistic studies which require words as stimuli  are often based on subjective ratings which have been found to correlate with objective measures of AoA , will thus have similar vector values and appear as close in the spatial representation of that particular corpus. What is even more interesting for our work, the N values of the resulting n-dimensional vectors were proven to encode various linguistic properties of words.In machine learning studies, word embeddings are often used. Word embeddings are numerical representations of words, usually in the form of n-dimensional vectors. Considering that vectors can mathematically be represented as positions in an n-dimensional Cartesian space, every word-related vector assigns a unique \u201caddress\u201d in space to every word. Word vectors are calculated using the positions of words within large language corpora, based on their neighboring words and in line with observations first made by Harris , and latThere have been warnings that extrapolation may create semantic artifacts absent from the human ratings for AoA, concreteness and affective variables, even if correlations are high  entities defined in time and being diffuse in space  from the object (table). Adverbs are more varied than adjectives, and may refer to the spatial or temporal domain, as well as various other circumstances .This last view hinges on treating word class and other grammatical information as meaningful, which is in line with cognitive linguistic treatments . Finally, adjectives and adverbs have the grammatical valence of exactly 1, because the way they describe qualities and circumstances requires a noun/verb, respectively, whose quality/circumstance is realized. In this sense, adverbs are further removed from adjectives, because they work with verbs which have their separate grammatical valence of at least 1.This information is also related to grammatical valence as conceptually defined by Croft . GrammatThese explanations shed new light on the three leading models that have been developed to account for the processing differences of concrete and high-imageable words on the one hand, and abstract and low-imageable words on the other: the context availability theory  and the University of Rijeka . No monetary compensation was offered, but some students were awarded class credit for their participation. The mean age of the participants was 21.18 (SD = 2.61), and their age range was 18\u201350. There were 78.45% females and 21.55% of males. Most participants (69.32%) reported speaking two or more foreign languages . Most participants (79.67%) reported that they spent between 1 and 4 hours reading per day on average. Every word was rated by an average of 30 participants (see Table hrast \u201coak\u201d) and groups of people (e.g. razred \u201cclass\u201d) were marked as inanimate, whereas supernatural, anthropomorphic and dead entities as animate , based on the differences in morphological marking of masculine nouns. Preliminary results of the first round of data collection, focusing on the dimensions of concreteness and imageability and testing the dual-coding theory, were published in Croatian  to control for the method (order) effect. The participants rated two variables on the same sheet (concreteness and subjective frequency or imageability and AoA).tuljan \u201cseal\u201d, plivati \u201cto swim\u201d, slan \u201csalty\u201d; abstractness: pravda \u201cjustice\u201d, morati \u201cto have to\u201d, poetski \u201cpoetic\u201d; high imageability: ku\u0107a \u201chouse\u201d, grmjeti \u201cto thunder\u201d; low imageability: nedosljedan \u201cinconsistent\u201d, smjeti \u201cbe allowed to\u201d, aspekt \u201caspect\u201d).The instructions see  were forSubjective frequency was operationalized using a Likert scale, where the participants were asked to report how frequently they encounter a word: almost never (1), once a year (2), once a month (3), once a week or (4) once/several times a day (5). AoA was defined as \u201cunderstanding the meaning of a word at a certain age\u201d, so the participants were asked to estimate the age at which they could say they knew the meaning of a word. Such a continuous measure of AoA correlates highly (above .80) with more frequently used Likert-like rating-scale measures, where the participants have to mark a number indicating an age range (defined in advance by researchers) in which they acquired a word  for the psycholinguistic variables . Moreover, linguistic characteristics of each word are also coded, i.e. word length in number of characters, word class , animacy and gender for nouns and raw frequency in the hrWaC corpus. Currently, the rated words appear only in Croatian, but English translations will be provided by the end of the project.M = .88) for concreteness, from .81 to .88 (M = .85) for imageability, from .89 to .93 (M = .91) for AoA, and from .87 to .90 (M = .89) for subjective frequency. Moreover, there were no significant correlations between the place on the list and ratings for any of the variables, except for imageability ratings on list A and AoA ratings on list B which were both very small at .04 (p < .01). This corroborates the reliability and validity of the collected ratings.The reliability of the ratings for concreteness, imageability, AoA and subjective frequency was calculated by randomly dividing the participants into two subgroups of equal size and computing the correlation between averaged estimation by item. The reliability indexes were calculated on 5000 different randomizations of the participants. The obtained split-half correlations ranged from .86 to .90 . The correlation for concreteness with \u0106oso et al. .Results of ANOVAs revealed significant differences between word classes in all the psycholinguistic word features, as well as in word length (p < .001) in all pairs of word classes in mean concreteness ratings, with nouns being rated as the most concrete, followed by verbs (nouns \u2013 verbs Cohen\u2019s d = 0.51), adjectives (verbs \u2013 adjectives Cohen\u2019s d = 0.42), and finally, adverbs as the most abstract words (adjectives \u2013 adverbs Cohen\u2019s d = 0.38). The same pattern was observed for imageability, with nouns rated as the most imageable, followed by verbs (nouns \u2013 verbs Cohen\u2019s d = 0.36), adjectives (verbs \u2013 adjectives Cohen\u2019s d = 0.31), and finally, adverbs as the least imageable words (adjectives \u2013 adverbs Cohen\u2019s d = 0.41).Post hoc Bonferroni tests revealed a statistically significant difference (p < .001) in subjective frequency except for the difference between nouns and adjectives (p = .052). Adverbs were rated as the most frequent, followed by verbs (adverbs \u2013 verbs Cohen\u2019s d = 0.65), and nouns and adjectives as the least frequent (verbs \u2013 nouns Cohen\u2019s d = 0.29). The same pattern was observed for the AoA. The Bonferroni test indicated a statistically significant difference (p < .001) in all pairs of word classes, except for the difference between adverbs and verbs (p = .205). Adverbs and verbs were rated as the earliest acquired words, followed by nouns (verbs \u2013 nouns Cohen\u2019s d = 0.23), and adjectives as acquired the latest (nouns \u2013 adjectives Cohen\u2019s d = 0.20) (see Table p < .001). The shortest word class\u2014adverbs\u2014were shorter than nouns (Cohen\u2019s d = 0.27), nouns were shorter than adjectives (Cohen\u2019s d = 0.41), and finally, adjectives were shorter than the longest word class\u2014verbs (Cohen\u2019s d = 0.23). We report effect sizes for the smallest differences obtained in the post hoc tests .As for subjective frequency and AoA, a Bonferroni test indicated that all pairs of word classes differed statistically significantly , nov\u010di\u0107 \u201ccoin\u201d , normalizacija \u201cnormalization\u201d  and neshvatljiv \u201cincomprehensible\u201d . However, given the .819 correlation, the CPD also contains words that diverge from this pattern exhibiting high concreteness and low imageability, e.g. primatelj \u201crecipient\u201d , kositar \u201ctin\u201d , and low concreteness and high imageability, e.g. mi\u0161ljenje \u201copinion\u201d , obe\u0107anje \u201cpromise\u201d . Reflecting the high association between imageability and concreteness, the pattern of relations of the two variables with other word features was similar. Thus, words rated as more concrete and more imageable tended to be estimated as acquired at a younger age, as being subjectively and objectively more frequent, and as being shorter. AoA had the highest correlation with subjective frequency, suggesting that words reported to be acquired at a younger age also tended to be rated as more subjectively frequent. Although a lower correlation was obtained between AoA and objective frequency, it reveals that words estimated as being acquired at a younger age are objectively more frequent than those reported to be acquired at an older age. Furthermore, words rated as being acquired at a younger age tended to be shorter than those rated as being acquired at an older age. In addition to the mentioned correlations between subjective frequency and the psycholinguistic variables, subjective frequency had a high positive correlation with objective frequency, and a low negative correlation with word length. Finally, the two linguistic word features correlated moderately negatively: words which are objectively more frequent tend to be shorter. Pearson correlations calculated separately in the subsamples of the four word classes showed that the overall pattern and the magnitude of associations between the word features were very similar across the four word classes.Pearson correlations between all word features included in the CPD, calculated for all 6000 words, are presented in Fig. The values of all word features are comparable to values available in databases for other languages cited earlier. AoA means in our study may seem somewhat higher than those obtained in other studies using a continuous AoA measure for French , with all post hoc differences significant (p < .001), except for the difference between nouns and adjectives. This indicates that an explanation based on word class-related factors may be worth exploring, although the overall effect size of word class on AoA is small.All the correlations obtained in the data follow the expected pattern found in previous studies and databases  to which extent we can extrapolate concreteness and imageability ratings in our lexicon of 6000 entries to the remainder of the Croatian lexicon and (2) to extend the existing body of research on extrapolation of psycholinguistic variables by focusing on (a) variation between word classes and (b) the interaction between concreteness and imageability.Extrapolations in this work were performed by using word embeddings as explanatory variables and one of the two variables, concreteness or imageability, as response variable. In line with Ljube\u0161i\u0107 et al. , we usedDifferent to our previous work , rs =.87 (MRC) and rs = .89 (BWK), whereas they were rs = .68 (MEGAHR) and rs = .80 (MRC) for imageability. A larger dataset seems to facilitate machine learning, which is why English extrapolations are better than Croatian. The correlation for concreteness for the MRC database for our model is slightly better than the one published by Hollis et al. , and in the case of MRC . Differences in predicting concreteness and imageability between MEGAHR and MRC are also significant .Concreteness predictions correlate with human predictions better than predictions for imageability, and the difference between them is significant, both in the case of MEGAHR  with a clear cline between them. The results for imageability show the strongest correlation for nouns (r = .68), and weaker correlations for the other word classes .We performed a separate evaluation of the models trained and evaluated on the MEGAHR dataset, by separating the predictions by word class. The results show that the overall correlations for concreteness for nouns are stronger than those for verbs, adjectives and adverbs (r = .93) than they are in human ratings (r = .82). We then compared the direction of the predictions for a set of 5301 nouns, verbs, adjectives and adverbs. The sample was obtained by removing the training set and manually discarding any obvious errors in the predicted data . We subtracted the predicted concreteness or imageability mean ratings from the human-rated concreteness or imageability mean ratings separately for each variable, obtaining a score that shows the direction of the prediction. Negative scores indicate that the mean of the predicted value is greater than the mean of the human rating; i.e. in the case of negative scores, extrapolated values are overpredicted in relation to human ratings. Positive scores represent underprediction by machine learning. We performed an ANOVA on the means to test for word class differences.Finally, we compared concreteness and imageability extrapolations with human ratings for all the word classes. The results show that the overall correlations between concreteness and imageability are higher in machine predictions . Post hoc Bonferroni tests revealed that all pairs of word classes except for nouns and verbs differed statistically significantly (p < .001), with nouns being consistently underpredicted, and adjectives and adverbs consistently overpredicted . The results for imageability also showed a significant difference between word classes, but the effect size was minimal .The results for concreteness showed a significant difference between word classes, with a small effect size (imagine comes to mind).Concreteness was easier to predict than imageability, which may mean that concreteness\u2014i.e. availability to the senses\u2014is captured more easily using distributional data. Notionally, concreteness is verifiable by our senses; it refers to entities that are present in our environment and is textually related to a number of lexical items . In contrast, imageability is an \u201cinternal\u201d (\u201cone\u2019s minds\u2019 eye\u201d) capacity of a human being to invoke a mental image. The mental image is based on the knowledge of an external stimulus; however, invoking a mental image makes sense only if the stimulus is not present (you do not need to imagine a desk or a foul smell when you can see it or smell it). Imageability is not as clearly related to a number of basic and readily available lexical items . In the remaining cases, distributional evidence may not be sufficient. This is evident from a comparison of predictions of concreteness and imageability for words that participants rated as abstract and highly imageable. A total of 98 such words, with concreteness rated below 2.5 and imageability above 3.5, were found in our set (some of them mentioned in the first study). Among them, imageability was predicted within .5 of the mean of the human rating in four cases, and in 94 cases going beyond this. In contrast, in the same set, concreteness was predicted within .5 of the mean of the human rating in 59 cases, with the remaining 39 cases going beyond the arbitrary .5 limit. This suggests that distributional evidence works better for concreteness than for imageability. This is in line with Crossley et al.\u2019s  study whThe results of the human rating study showed expected correlations between psycholinguistic variables, as well as an effect of word class on the psycholinguistic variables of AoA, subjective frequency, concreteness and imageability. For concreteness and imageability, there was a cline nouns > verbs > adjectives > adverbs. In the machine learning study, we focused on concreteness and imageability and obtained results which, in the whole sample, correlated with human predictions. However, the size of the correlations across word classes followed the same cline. Finally, a similar cline was obtained for the direction of the predictions for concreteness. The system underpredicted the concreteness of nouns, and overpredicted the concreteness of adjectives and adverbs. Imageability predictions followed suit, but the effect size was minimal.These results are in line with the conceptual definition of word classes given in the introduction, which suggests that word class should be taken as a shorthand for semantic and distributional characteristics, at least within a single language. Simply put, nouns typically denote objects, verbs denote relations between objects, and adjectives and adverbs denote qualities and external characteristics. There is a clear conceptual decrease in concreteness and imageability: nouns are most highly concrete and imageable because they are conceptually related to entities that are less diffuse and have a spatial basis, and the decrease in the spatial basis of verbs, adjectives and adverbs, and their increasingly relational nature leads to lower concreteness and imageability. In other words, word class adds a schematic conceptual frame to the lexical meaning of words.This is also visible from the differences in the direction of the prediction for concreteness in the machine learning experiment, where this \u201cconceptual frame\u201d seems to be more significant for humans than when using machine learning based on collocational evidence. It seems that, at least for some nouns, humans give more weight to the conceptual factor of \u201cnouniness\u201d (i.e. thing-like representation) rather than to distributional evidence. By the same token, people see some adjectives and adverbs as more diffuse and hence less concrete, perhaps because adjectives and adverbs require conceptual separation of the quality from the object it describes. In contrast, relying on collocational evidence (where no such separation occurs), the system predicts them as more concrete than humans.Regarding the theoretical significance of word class for the context availability theory, the dual-coding theory and grounding theories, it seems that all of them should incorporate grammatical information as part of their program. It seems that various flavors of grounding theories  convincingly, so that the artifacts are not a result of using only one type of evidence (and the wrong kind of evidence at that). Characteristics other than imageability that require more than just textual evidence seem to include emotional valence, arousal and dominance . Given the role of word class in this work, and the relation between affective variables and concreteness and imageability (Kousta et al.,"}
+{"text": "An 11-year-old female presents to the sleep clinic for evaluation for possible sleep-disordered breathing (SDB). She has a history of frequently intractable seizures for which she was tried on multiple antiepileptic medications. She had vagus nerve stimulation (VNS) implantation two years ago to treat her focal seizures. Nine months later, her seizures were controlled, but the family raised concerns about louder snoring and more frequently witnessed apneas. She had polysomnography (PSG) that showed severe obstructive sleep apnea (OSA) related to her VNS. The patient was diagnosed with SDB secondary to electrical activations of the implanted VNS. We describe an epilepsy patient whose case illustrates the possible respiratory complications (primarily OSA) associated with VNS. We will discuss the possible mechanisms of VNS related SDB and the importance of screening for SDB and advocate for a PSG both before and after VNS implantation. Vagus nerve stimulation (VNS) can worsen sleep-disordered breathing (SDB) after implantation. Patients with VNS can have central apneas, obstructive hypopneas, and/or obstructive apneas. Patients with VNS often have an increase in apneic events after implantation . This caAn 11-year-old female presented to the sleep clinic for evaluation of snoring and witnessed apneas. She has had a history of focal epilepsy since age four years and was tried on multiple antiseizure medications, including levetiracetam, clobazam, zonisamide, and valproate. Her most recent antiepileptic medications include clonazepam, lacosamide, and\u00a0topiramate for the last three years. She continued to have up to 10 seizures every week, and so two years ago, the VNS was implanted to manage her intractable epilepsy. Nine months following her VNS implantation her seizures were reduced dramatically, but her mother raised concerns about more frequent nocturnal respiratory pauses during her sleep as well as louder snoring. This was reported by the family for the first time-only after her VNS was implanted. Thus, the patient was referred to our Pediatric Sleep Medicine clinic for an initial evaluation. Epworth Sleepiness Scale (ESS) assessment was six of 24. She had a consistently regular sleep/wake cycle with 10 hours of sleep with a bedtime of 9:30 PM. She usually would fall asleep within 15 minutes and would wake up between 7:30 AM and 8:00 AM. She had no daily naps. The patient had no other significant medical history. Family history included paternal sleep apnea and maternal generalized seizures.2\u00a0. She had a Mallampati score of 1; tonsil size of 0. The remainder of the physical examination was normal.Examination revealed patient weight at 34.8 kg; height, 127.5 cm; Z-scores of -0.89 and -3.05 were based on the Centers for Disease Control (CDC) two to 20 years\u2019 weight-for-age and stature-for-age data, respectively. Body mass index was 21.43 kg/m2 (EtCO2) was 33 mmHg. Periodic limb movement index was 1.0 events/hour. The patient\u2019s polysomnography (PSG) demonstrates the respiratory events  was 30.7 events/hour; central apnea index, 0.1 events/hour. Sleep latency was five minutes, lowest oxygen saturation was 90%, and mean end-tidal CODifferential diagnosisThe patient has evidence of severe obstructive sleep apnea (OSA) on the PSG. Respiratory events were unusually rhythmic and occurred with the accuracy of a metronome . Each reThese findings on the polysomnographic signals would raise questions about their possible etiologies that can include possibilities like an underlying arrhythmia or an artifact not related to the VNS\u00a0versus a VNS related phenomena. Recognizing the exact etiology and accurately answering these questions is pivotal to decide what will be the best next step for management.Questions and answersThe\u00a0findings on the polysomnographic signals would raise questions about their possible etiologies that can include possibilities like an underlying arrhythmia or an artifact not related to the VNS\u00a0versus a VNS related phenomena. Recognizing the exact etiology and accurate\u00a0interpretation of these findings is pivotal to decide what will be the best next step for management.\u00a01.\u00a0What is the most possible etiology\u00a0of the electromyogram (EMG) and the electrocardiogram (EKG) artifacts in the figures above?(A)\u00a0Myocardial fiber stretch during episodes of apnea; (B)\u00a0electrical activations of the implanted VNS; (C)\u00a0undiagnosed supraventricular arrhythmia; (D)\u00a0ungrounded recording equipment affected by a nearby air-conditioner .The answer is B. Electrical activations of the implanted VNS. 2.\u00a0What will be the most appropriate next step to address this finding?These respiratory events can be reduced with changes in the VNS operational parameters or with the use of\u00a0continuous positive airway pressure (CPAP). (This will be discussed further below in detail.)Treatment20 during which the (O-AHI) was 1.3. The patient was started on PAP therapy. The VNS could have also worsened a preexisting OSA and thus the patient was also referred to ear, nose, and throat (ENT) for evaluation of possible surgical options like Tonsillectomy and Adenoidectomy (T&A). The ENT evaluation did not identify any additional surgical options.The patient had a repeat PSG for positive airway pressure (PAP) therapy. Her repeat PSG one month later with the VNS \u201con\u201d showed an optimal pressure of 7 cm HOutcome and follow-upOn the three-month follow-up after PAP therapy was started the patient tolerated the PAP therapy well with 99.3% of days usage-an average usage of seven hours and 51 minutes per night and 90% of days with usage >=4 hours. There were no reported side effects to PAP therapy. The patient remained seizure-free for the last year and is currently on clonazepam\u00a0and lacosamide.There is a paucity of cases demonstrating this finding in children. A robust\u00a0PubMed database search for the search terms VNS & sleep apnea yielded 69 articles\u00a0on this topic, most of these articles were discussing either\u00a0the VNS role in epilepsy and/ or the respiratory complications associated with vagus nerve stimulators, however, only one article matched\u00a0citation for VNS and obstructive sleep apnea in adults. Also, a systematic review on the topic has come to light by \u00d3scar Romero-Osorio et al.\u00a0that describes only four case reports in adults between the ages of 20 years and 57 years at the time of the evaluation in the United States .\u00a0A similFew case reports in the literature have reported a possible association between the VNS and the respiratory events. Shaman reported a 50-year-old male who presented with snoring, witnessed apneas, unrefreshing sleep, and excessive daytime sleepiness (ESS 17) despite adequate sleep duration . This pa2) of 41. Bilevel positive airway pressure (BiPAP) was initiated and titrated to 11/7 cm H20 with a backup rate of 12. BiPAP with the VNS \u201con\u201d resulted in an O-AHI of 0.0 and oxygen nadir of 94%. This case, however, did not diagrammatically illustrate the VNS-related respiratory events similar to our case and did not emphasize the importance of screening before and after the VNS implantation. The AHI was also much higher in our case (13.5 events/hour versus 30.7 events/hour in this case presented). Our case adds to the existing literature and raises awareness about the importance of screening for OSA and SDB even before the VNS is implanted.\u00a0Recently, an abstract by Ochoa et al.\u00a0described a 12-year-old female with medically intractable epilepsy who had a VNS implantation as an additional therapy . She wasIntermittent VNS can reduce the frequency of seizures in patients with refractory epilepsy. Although the exact mechanism of seizure reduction remains unclear, Mithani, et al. reported that the vagus afferent network (VagAN) is thought to be the neural substrate of VNS efficacy. This recent study provides the first multi-institutional, multimodal, connectomic prediction algorithm to reliably identify VNS responders based on the reported group differences in their white matter microstructure that was demonstrated on the diffusion tensor imaging used in this study. This is important in order to mitigate surgical risks for those children predicted not to benefit from the VNS implantation and to ensure\u00a0cost-effective allocation of health care resources . Stimula2O resulted in the effective treatment of his severe OSA. This study also suggests PSG before VNS implantation should be considered in order to identify pa\u00adtients with OSA\u00a0[Manipulating VNS settings such as stimulus intensity, frequency, and cycle times may decrease the respiratory disturbance ,9. Howevwith OSA\u00a0. A similwith OSA\u00a0.There are complex relationships between epilepsy and OSA. Patients with refractory epilepsy need assessment for undiagnosed and untreated SDB before implantation of VNS devices. Patients with VNS often have an increase in their apneic events after implantation, and these patients need screening for sleep apnea before and after implantation\u00a0.This case demonstrates the importance of screening for SDB, preferably before, but also after the VNS implantation.\u00a0This case illustrates these concepts, especially in children, where scarce cases are describing these findings.\u00a0We also elaborate on the possible mechanisms of VNS related SDB and discuss the variable management options that the providers can choose to pursue like CPAP utilization\u00a0or possible changes in the VNS operational application to address the medical condition.Additional research including a larger group of patients is needed in order to conduct a subgroup analysis\u00a0to determine the concrete clinical practice guidelines of which subgroup of patients for which routine screening for SDB and recommending the PSG either before or after the VNS implantation or both will be most beneficial. This case raises awareness about this phenomenon and serves as a valuable proponent in the literature for providers to consider screening for SDB as well as a PSG both before and after the VNS implantation."}
+{"text": "The microstructure and phase composition of the composite material were analyzed, and the influence of laser energy density on the microstructure and mechanical properties of composite materials was discussed. The results showed that the needle-like ZrB2 ceramic reinforcement was successfully synthesized via an in-situ synthesis reaction. The composites were mainly composed of needle-like ZrB2, Ni dendrites and a Cu matrix. The morphological changes of Ni dendrites could be observed at the interface inside the composite material: cellular crystals \u2192 large-sized columnar dendrites \u2192 small-sized dendrites . The continuous Ni dendritic network connected the ZrB2 reinforcements together, which significantly improved the mechanical properties of the composite material. At a laser energy density of 0.20 kJ/mm2, the average microhardness of the composite material reached 294 HV0.2 and the highest tensile strength was 535 MPa. With the laser energy density increased to 0.27 kJ/mm2, the hardness and tensile strength decreased and the elongation of the Cu composites increased due to an increase in the size of the ZrB2 and a decrease in the continuity of the Ni dendritic.Laser additive manufacturing is an advanced material preparation technology, which has been widely used to prepare various materials, such as polymers, metals, ceramics and composites. Zirconium diboride (ZrB Laser additive manufacturing (LAM) is an advanced material preparation technology, which adopts laser as an energy source and powders or wires as raw material to construct a 3D specimen under the control of a computer ,2. LAM h2/Cu5Zr reinforced Cu composites have exhibited a good combination of electrical conductivity and mechanical properties [2 ceramic whisker is a single crystal with a very small diameter [Copper is a common and important metal material due to its excellent thermal conductivity, corrosion resistance and ductility ,17, and operties . ZrB2 cediameter . Its ato4C, and they acted as the heterogeneous nucleation sites for the Ti matrix, which promoted the formation of fine equiaxed \u03b1-Ti grains [3Fe, CrB, M23C6 and Cr2.8Fe1.2B4) were synthesized in the molten pool, which significantly improved the high temperature wear resistance of the coating [2 and CuZr were synthesized in the Cu matrix, which improved the high temperature hardness of Cu composites [With regard to ceramic reinforced metal matrix composites (CMC), the manufacture of defect-free CMCs with excellent mechanical properties has always presented a challenge and has been the focus of research due to large differences in the physical and chemical properties of ceramic reinforcements and metal matrices ,25. Accoi grains . In the  coating . During mposites .2 ceramic whisker reinforced copper matrix composites. The ZrB2 reinforcement was in-situ synthesized via the designed chemical reaction in the molten pool, and the crystal structure of its growth tip was analyzed. The microstructure, phase composition and interface characteristics of the composites were determined. The influence of energy input density on the microstructure of composites was discussed. In addition, the microhardness distribution and tensile properties of the samples prepared under different energy input densities were studied.In this study, the exploration goal was to use LDED technology to prepare ZrB2, Al, Ni-B4C , Ni and Cu were used as the raw material for laser direct energy deposition. The sizes and weight percentages of the composite powders were listed in 2, Al and B4C were the reactants of the in-situ synthesis reaction, and the molar ratio between them was set from the designed reaction:The composite powders ZrO2 ceramic reinforcements using this ratio of reactants [The Gibbs free energy  of 180 mm/min, a spot diameter (D) of 3 mm and a laser power (P) of 1.8 or 2.4 kW. The laser energy density (E = P/VD) represented the laser energy applied per unit area during the deposition process, which was used to comprehensively analyze the influence of process parameters for each sample. Two samples were prepared under different laser energy densities; the E of samples 1 and 2 were 0.20 and 0.27 kJ/mm2, respectively.Pure copper with a size of 50 \u00d7 50 \u00d7 15 mm3 (10 g), HCl  and H2O (100 mL). The phase composition of the composite material was analyzed using an X-ray diffractometer  at a speed of 0.5\u00b0/min. A tungsten wire scanning microscope  equipped with an energy spectrometer (EDS) was used to observe the microstructure of the composites and the morphology of the reinforcements. The growth direction and interface characteristics of the reinforcement were characterized using transmission electron microscopy . A disc with a diameter of 3 mm was cut on the SD-LD surface of the composites, and then a TEM sample was obtained via mechanical thinning and ion beam thinning. The microhardness distribution of the samples on the BD-LD was tested using a Vicker\u2019s hardness tester (HVS-1000) under a load of 0.2 kg and the dwell time of 10 s; the distance between the test points was 100 \u03bcm. The tensile samples of the composites were cut on the SD-LD plane . According to the build direction, the left side of the interface was N + 1 track deposition, in which small-sized dendrites and large-sized columnar dendrites could be observed. Compared with the area in sample 1  was [112 crystals grew into regular geometric shapes with multiple symmetry planes. The interface structure and surface energy played key roles in the growth of the crystal plane. The interface structure could be divided into a smooth interface and a rough interface according to the crystal structure and bonding bonds between atoms. The Jackson factor (\u03b1) was used to determine the interface type  [k is Boltzmann\u2019s constant; mT is the melting point; \u03b7 is the coordination number between atoms in the layer and the atoms in the same layer (only the surface layer); v is the coordination number between the surface layer atoms and the next layer of solid atoms; R is the gas constant and 2 with a C32 hexagonal close-packed structure was P6/mmm (191). \u03b7/v could be calculated for each crystal plane from the positions of atoms in the hexagonal close-packed structure. The \u03b7/v of {0001}, {10R = 8.31 J/mol\u00b7K, and so the \u03b1 of the ZrB2 low-index were \u03b1{0001} = 5.84, \u03b1{10-10} = 1.95 and \u03b1{11-20} = 1.95. The \u03b1 of {10uvtw) of a crystal plane (uvtw) was inversely proportional to the atomic layer distance (duvtw) of the crystal plane. According to the interplanar spacing of the common low-index crystal plane families of ZrB2  :(2)\u03b1=\u0394Hf J/mol\u00b7K , R = 8.3H) model ,39, the  of ZrB2 , the ord2. The regular crystal planes at the tip of ZrB2, which are a typical morphology of the single crystal, could be observed. Furthermore, the angles between the crystal planes were approximately 120\u00b0, which is similar to the ideal hexagonal morphology. 2 as marked by the dashed box. Two symmetrical facets could be clearly observed, and the normal angle \u03b8 between the two facets was about 62\u00b0. The space group of ZrB2 with a C32 close-packed hexagonal structure was P6/mmm (191), and the 4-axis coordinate system of its crystal structure is shown in 2 crystal was mainly composed of {0001} and {102 crystal nucleus grew into a whisker reinforcement.0.2. The average microhardness, 294.26 HV0.2, was about five times higher than that of pure copper. The fine ZrB2 needle-like phase and Ni dendritic network structure improved the microhardness of sample 1. However, there were fewer needle-like phases and the large-size columnar Ni dendrites at the remelting interface , the tensile strength reduced, but the elongation increased.2 reinforced Cu composites were successfully fabricated using laser direct energy deposition. The microstructure of the composites was mainly a composite structure composed of needle-like ZrB2, Ni dendrites, and a Cu matrix.The needle-like ZrB2 was shown to be a high-strength whisker reinforcement. In sample 1, the diameter of ZrB2 was less than 2 \u03bcm, and the aspect ratio was about 30:1. The long axis direction with the fastest growth rate of ZrB2 ceramic whiskers was {11-20}.In-situ synthesized ZrB2. The morphological changes of Ni dendrites could be observed at the interface inside the composite material: cellular crystals \u2192 large-sized columnar dendrites \u2192 small-sized dendrites .In the Cu matrix, a network of Ni dendrites connected the needle-like ZrB2 ceramic reinforcement and Ni dendrites significantly improved the mechanical properties of the composite material. The microhardness and ultimate tensile strength of the composite material reached 322 HV0.2 and 535 MPa, respectively, and the tensile fracture of sample 1 was a mixed mechanism of ductile and brittle fracturing. As the laser energy density increased, the size of the in-situ synthesized ZrB2 increased, and the number and continuity of Ni dendrites decreased, which decreased the hardness and strength of the composite material, and the fracture mechanism of sample 2 was changed to ductile fracturing.ZrB"}
+{"text": "Chronic bronchitis in patients with chronic obstructive pulmonary disease (COPD) is associated with poor respiratory health outcomes. However, controversy exists around whether non-obstructive chronic bronchitis (NOCB) is associated with airflow obstruction, lung function decline, and all-cause mortality in ever smoker or never smoker.This systematic review and meta-analysis aimed to clarify the relationship between NOCB and incident COPD, lung function decline, and all-cause mortality, and to quantify the magnitude of these associations.We searched PubMed, Embase, and Web of Science for studies published up to October 1, 2021. Eligibility screening, data extraction, and quality assessment of the retrieved articles were conducted independently by two reviewers. Studies were included if they were original articles comparing incident COPD, lung function decline, and all-cause mortality in normal spirometry with and without chronic bronchitis. The primary outcomes were incident COPD and all-cause mortality. The secondary outcomes were respiratory disease-related mortality and lung function decline. Pooled effect sizes and 95% confidence intervals (CIs) were calculated using the random-effects model.I2 = 76.3% and relative risk: 1.44, 95%CI: 1.13\u20131.85, I2 = 56.1%), all-cause mortality , and respiratory disease-related mortality . Data on the decline in lung function could not be quantitatively synthesized, but the five articles that assessed the rate of decline in lung function showed that lung function declines faster in individuals with NOCB. The mean difference in the additional decline in forced expiratory volume in 1 s ranged from 3.6 to 23.2 mL/year.We identified 17,323 related references and included 14 articles. Compared with individuals without NOCB, individuals with NOCB had an increased risk of incident COPD (odds ratio: 1.98, 95% CI: 1.21\u20133.22, Individuals with NOCB are at a higher risk of incident COPD and all-cause mortality than individuals without NOCB, highlighting the crucial need for strategies to screen for and reduce NOCB risk.https://www.crd.york.ac.uk/PROSPERO/ PROSPERO, identifier CRD42020202837 Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable disease that is characterized by persistent respiratory symptoms and airflow obstruction. It is currently a leading cause of death and disability worldwide , 2. IdenChronic bronchitis is defined epidemiologically as cough and sputum production for \u22653 months each year for \u22652 consecutive years . The maiUnderstanding the association between NOCB and incident COPD and respiratory health outcomes has important implications for disease management, such as in terms of targeted treatments and cautions about drug usage. Independent studies have shown a significantly increased risk of airflow obstruction and all-cause mortality in individuals with NOCB, and this has been summarized in a recent narrative review , 15\u201317. The study protocol was registered with the International Prospective Register of Systematic Reviews (registration number: CRD42020202837). This systematic review and meta-analysis was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .In this systematic review and meta-analysis, two reviewers (FH and JL) independently searched Embase, Web of Science, and PubMed to identify studies published from database inception to October 1, 2021. Keywords and subject terms were customized for each database. The search terms combined the respiratory symptoms related to chronic bronchitis  and prognostic terms . The references of related studies were also consulted to identify potentially relevant articles.The eligibility of identified studies was independently verified, and disagreements were resolved by discussion or, where necessary, by consulting a third researcher (FW). For primary inspection, the titles and abstracts were reviewed, and articles were excluded mainly for COPD patients with chronic bronchitis and non-extractable data . For secondary inspection, full-text review was performed, and articles were excluded based on the inclusion and exclusion criteria. Studies that satisfied the following criteria were included: (1) prospective cohort study or retrospective cohort study; (2) inclusion of odds ratios (ORs), relative risks (RRs), or hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) to estimate COPD risk; the risk of mortality  in individuals with NOCB; or enough data to calculate these risks; (3) comparison of individuals without NOCB; (4) independent studies. Studies that were the same as the published dataset were not considered independent. We deemed studies as eligible if they were longitudinal cohort studies that enrolled adults and reported the association between NOCB and the study outcomes.1]/forced vital capacity [FVC] ratio of <0.7) was the preferred definition of COPD . The secondary outcomes were respiratory disease-related mortality and lung function decline.NOCB was defined as chronic cough and sputum production for \u22653 months for \u22652 consecutive years in subjects without airflow obstruction. Without NOCB was defined as normal spirometry and no chronic bronchitis. Without NOCB serves as a healthy control of NOCB, which does not limit smoking status. The primary study outcomes were incident COPD and all-cause mortality. The GOLD criterion (a post-bronchodilator forced expiratory volume in 1 s [FEVTwo reviewers (FH and JL) independently extracted the study information and independently verified the quality of each study. The extracted information included first author, year of publication, location, study design, year of enrolment, study outcome, follow-up time, and the characteristics of the study participants . Two reviewers (HF and JL) independently conducted quality assessments of the included studies. The quality of each study was based on the Newcastle\u2013Ottawa Scale (NOS) for cohort studies . StudiesI2 statistic was used to evaluate study heterogeneity. An I2 value of 0\u201324% was considered as no heterogeneity. Greater values represented greater heterogeneity, with values of 25\u201349% representing low heterogeneity, 50\u201374% representing moderate heterogeneity, and >75% indicating high heterogeneity  software was used for the meta-analysis.The outcomes of the synthesis included the OR and the RR of incident COPD, the HR of all-cause mortality and respiratory disease-related mortality, and the mean difference in the rate of lung function decline. For eligible studies that reported the HR and 95% CI of incident COPD, the HR and 95% CI were pooled with the RR. We could use the calculation formula to convert the OR into the RR, but some studies did not report the proportion of the control group that developed COPD, and the OR of most studies was adjusted for confounding factors . Therefoogeneity . Publicaogeneity . All sta1/FVC ratio of \u22650.7 as the main definition of normal spirometry, and one study used a pre-bronchodilator FEV1/FVC ratio \u2265 the LLN as the main definition of normal spirometry. Coronary Artery Risk Development in Young Adults Lung Study data were published in two articles  with significant inter-study heterogeneity   . The numP < 0.001) with low inter-study heterogeneity  (P < 0.001) with no inter-study heterogeneity   . Two stu= 0.722) .1 declined significantly faster in individuals with NOCB than normal spirometry in men without chronic bronchitis, but there was no significant difference in women   and FVC  among ever smokers  . In the oduction . In the  in FEV1 . The mos smokers .P < 0.001) and respiratory disease-related mortality  than in individuals with normal lung spirometry without NOCB (P = 0.206) or respiratory disease-related mortality  compared with individuals with normal spirometry and without NOCB . Finally, almost all studies included in our meta-analysis used pre-bronchodilator lung function data to diagnose COPD. A previous study has demonstrated that pre-bronchodilator lung function overestimates the prevalence of COPD compared with post-bronchodilator lung function . This maTo conclude, this systematic review and meta-analysis demonstrated that NOCB is associated with an increased risk of incident COPD, all-cause mortality, and respiratory disease-related mortality, as well as faster lung function decline. Individuals with NOCB should be identified early through screening, and strategies aimed at controlling NOCB should be implemented.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.PR, YZ, FW, HF, and JL contributed to study conception, protocol of the review, and interpretation of data. FW, HF, JL, SZ, and WZ performed the systematic review. FW and HF performed the statistical analysis. YZ, PR, FW, HF, and JL drafted the paper and all authors provided critical revisions and contributed to the editing of the paper. All authors are guarantors of this work.This study was supported by the National Key Research and Development Program (2016YFC1304101), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01S155), the National Natural Science Foundation of China (81970045), and Zhongnanshan Medical Foundation of Guangdong Province .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Insects tend to feed on related hosts. Coevolution tends to be dominated by interactions resulting from plant chemistry in defense strategies, and evolution of secondary metabolisms being in response to insect herbivory remains a classic explanation of coevolution. The present study examines whether evolutionary constraints existing in host associations of economically important fruit flies in the species\u2010rich tribe Dacini (Diptera: Tephritidae) and to what extent these species have evolved specialized dietary patterns. We found a strong effect of host phylogeny on associations on the 37 fruit flies tested, although the fruit fly species feeding on ripe commercially grown fruits that lost the toxic compounds after long\u2010term domestication are mostly polyphagous. We assessed the phylogenetic signal of host breadth across the fruit fly species, showing that the results were substantially different depending on partition levels. Further, we mapped main host family associations onto the fruit fly phylogeny and Cucurbitaceae has been inferred as the most likely ancestral host family for Dacini based on ancestral state reconstruction. The present study examines whether evolutionary constraints existing in host associations of economically important fruit flies in the species\u2010rich tribe Dacini (Diptera: Tephritidae) and to what extent these species have evolved specialized dietary patterns. We found a strong effect of host phylogeny on associations on the 37 fruit flies tested, although the fruit fly species feeding on ripe commercially grown fruits that lost the toxic compounds after long domestication are mostly polyphagous. Further, we discussed the role of secondary metabolisms in feeding preference, and other suites of traits in the specialization of fruit fly dietary patterns. To obtain the interacted hosts of the tephritid species, we extracted records from the pest\u2010oriented database of the Centre for Agricultural and Biosciences  which reports fruit flies occurring and feeding on plants. Host associations were cross\u2010checked for completeness with the COFFHI database . Synonyms on the host list were eliminated and only accepted names adopted for each plant species. In general, our database of interactions between fruit flies and host plants contains 1,841 associations, including 37 Dacini fruit flies and 706 host species belonging to 87 families.The tribe Dacini, primarily comprises species of three genera  model of evolution, values of K\u00a0>\u00a01 imply strong phylogenetic signals, and K\u00a0=\u00a00 implies the absence of phylogenetic conservatism. While there is no clearly defined K value cutoff in which to apply phylogenetic comparative methods, nonsignificant value of <1, or more conservative <0.5, are typical for characters that are phylogenetic independent. Pagel's \u03bb examines the effect of ancestral relatedness on character evolution, with a value close to 1 indicates phylogenetic signal and a value approaching 0 indicates phylogenetic independence.To test whether host breadth and namely states of polyphagy would be phylogenetically distributed across the tephritid species tree, we used Blomberg's \u03bb Pagel,\u00a0 to asses10 (phylodistance\u00a0+\u00a01) according to empirical experiments from Gilbert et al. (\u03b20) and slope coefficient (\u03b21) and the 95% confidence interval were obtained from the mean coefficients across all 1,000 sets from these regressions for all fruit flies.We took a logistic regression approach to calculate the probability of a host plant being infected by, or found in association with, a particular tephritid species , and its relationship with phylogenetic distances. We compiled associations at the genus level, and the associations were modeled as a binary trait . In the regression analysis, we only considered fruit fly species with three or more plant associations to make it more reasonable, and host phylogenetic distances were calculated in million years. We integrated the associations between fruit flies and host plants with the matrix of pairwise phylogenetic distances among host genera to investigate the role of host phylogeny. To calculate the possibility of fruit fly host affiliation, we applied the phylogenetic distance as predictor variable , 2.4We applied the DEC  model  \u201ctwo families\u201d and \u201cfour families\u201d were not apparent using the K estimate, but signals were detected under the \u03bb estimate . The character (feeding on) \u201cthree families\u201d showed strong phylogenetic signals using both the te Table\u00a0. SignifiThe probability that two host genera sharing one tephritid species declined significantly with phylogenetic distance, supporting that Dacini fruit flies prefer to feed on closely related plants Figure\u00a0. The logZeugodacus and Dacus.There were 87 families being used as hosts; however, only a handful of families are \u201cimportant\u201d host families which might play a decisive role in the evolutionary history of Dacini fruit flies. It contained nine main host families exclusively in the ancestral host state reconstruction analysis for studying purpose. Families of Cucurbitaceae and Myrtaceae were the most frequently used two families in terms of number of associated host plants belong to these families; families of Cucurbitaceae and Rutaceae were the most commonly used two families in terms of number of fruit fly species feeding upon these plant species. In addition, host use family Cucurbitaceae was distributed widely in the diet on genera of Bactrocera, and two Zeugodacus species Zeugodacus cucurbitae and Zeugodacus tau were not included in the reconstruction, because that these fruit flies feeding on more than 20 host families seemed to be out of limitations due to shifts in geographic distribution and could do little to estimate ancestral hosts. There were 25 tephritid species that had been expected to be contributed to estimate the ancestral host state in the DEC model. We mapped main host families onto simplified phylogeny of tephritid species, most likely ancestral host states were shown on each node  by host shifts, that fruit odors as key traits help distinguish among respective host plants  that can evolve as adaptive syndromes may be relevant to plant\u2013insect interactions. Although ripe, fleshy fruits function primarily to attract seed dispersers, they must also defend against diverse communities of frugivores. In particular, fruits can employ a variety of strategies such as ripening in seasons in which herbivores population densities are low or, reducing nutrient content and evolving a thick protective exocarp . Alternatively, inducible volatile organic compounds involved in host recognition may function as indirect defenses  is an evolutionarily dynamic phenomenon and host breadth should be treated as a continuum (expand or shrink over evolutionary time), its evolution is thus particularly difficult to test due to the inherent uncertainties. It requires systems approaches that could be capable of handling the practical situation of preference and performance on a case\u2010by\u2010case basis especially for highly polyphagous species which exhibit a great degree of variability in their host use patterns. However, combining host utilization with information of bioinformatics and phylogenetic analyses is still a proven approach to investigate insect species dietary pattern and feeding strategy. Furthermore, although the association data included were cross\u2010checked, the identification of these fruit flies on hosts might actually be incorrect  or based upon accidentally incidence on putative hosts . The existence of complexes could also confuse fruit fly host affiliations. We assigned the host records at the level of host genus or family that could be more reliable and reasonable for the analyses, and dubious host records are omitted. Nonetheless, an extremely polyphagous fruit fly might consist of a complex of cryptic species, leading to overestimation of host breadth in the fruit flies. However, it means that the conservatism should be more evolutionarily constrained than we investigated.5We found that Dacini fruit flies feeding on economically important host plants which normally lost secondary metabolisms after long\u2010term domestication still show specialized host associations, implying a decrease in the possibility of host sharing with increasing phylogenetic distance of the host plant taxa. Earlier study proposed that polyphagous fruit flies should be able to infest a broad range of phylogenetically distant host plants without absolute limitations and overcome plant defenses with little effort. Our study disaccords with this view and suggests that host specialization cannot be explained solely by secondary metabolites. Cucurbitaceae was recognized as the most probably ancestral host family in ancestral host reconstruction analysis. Diverse associations outside ancestral hosts for extremely polyphagous fruit flies are potentially attributable to capabilities of long\u2010distance dispersal and being generalist opportunists.The authors declared no conflicts of interest.Jiayao He: Conceptualization ; Data curation (supporting); Funding acquisition ; Methodology (supporting); Visualization (supporting); Writing\u2010original draft (supporting); Writing\u2010review & editing (lead). Ke Chen: Conceptualization ; Funding acquisition (supporting); Project administration ; Supervision ; Validation ; Visualization . Fan Jiang: Conceptualization ; Funding acquisition (supporting); Investigation ; Validation (lead); Writing\u2010review & editing . Xubin Pan: Conceptualization ; Investigation ; Project administration ; Supervision (lead); Writing\u2010review & editing .Data S1Click here for additional data file."}
+{"text": "Vitamin D deficiency is common, but no data have been reported on vitamin D levels in light chain (AL) amyloidosis.2D] and vitamin D binding protein (DBP). Measurements were made by liquid chromatography-tandem mass spectrometry. Kidney survival and overall survival (OS) were assessed in association to vitamin D status.In this exploratory study, stored serum samples from 173 patients with newly diagnosed AL amyloidosis were analyzed for vitamin studies which included 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D deficiency was noted in 37.6% of patients and was independently associated with low eGFR and hypoalbuminemia. Progression to ESRD occurred in 23.7% of evaluable patients. Patients who progressed to ESRD had lower serum 25(OH)D and 1,25(OH)2D levels compared to those who did not progress to ESRD. On a multivariate analysis, severe 25(OH)D deficiency was an independent predictor of progression to ESRD as was renal stage, while 1,25(OH)2D deficiency was not.Cardiac and kidney involvement occurred in 69% and 63% of patients, respectively. 25(OH)D deficiency (<20 ng/mL) was seen in 56.6% of the patients and was notably found among patients with heavy proteinuria (96%), hypoalbuminemia (84.3%) and morbidly obese patients (68.3%). Heavy proteinuria (>5 gr/24-h) and vitamin D supplementation were independent predictors of 25(OH)D level on nominal multivariate regression analysis. 1,25(0H)Hypovitaminosis D is common in AL amyloidosis, particularly among patients with heavy proteinuria. Severe 25(OH)D deficiency at time of diagnosis predicts progression to ESRD. Light chain (AL) amyloidosis is a rare B-cell secreting clonal disorder characterized by circulating immunoglobulin light chains with amyloidogenic properties . The clo2D]. 1,25(OH)2D reflects 1-alpha hydroxylation of 25(OH)D, a process that occurs primarily within the kidney. Circulating 1,25(OH)2D levels are approximately 1000-fold lower than those of 25(OH)D.Vitamin D deficiency is assessed by measurement of the storage form of vitamin D, namely 25-hydroxyvitamin D [25(OH)D]. The prevalence of vitamin D deficiency, defined as a 25(OH)D level below the optimal range of <20 ng/ml, has been reported to range from 15-50% within the general United States (US) population \u20138. IdentAside from its roles in maintaining serum calcium levels and skeletal homeostasis, vitamin D has been shown to play an important role in regulation of differentiation, proliferation, apoptosis, metastatic potential and angiogenesis in a variety of malignancies \u201311. Low No studies have explored vitamin D levels in light chain (AL) amyloidosis. We therefore performed a study to examine vitamin D levels among patients with AL and to explore the impact vitamin D has in this rare disease.2D] and vitamin D binding protein (DBP). All vitamin D measurements were made by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total 25(OH)D and 1,25(OH)2D was assessed by the additive sums of the 25(OH)D2 + 25(OH)D3 and 1,25(OH)2D2 + 1,25(OH)2D3 components, respectively. Samples obtained for vitamin D studies were collected within 90 days of diagnosis (median 20 days) and were stored at -20\u00b0C.In this study patients with biopsy-proven AL amyloidosis diagnosed between January 1, 2000 and September 30, 2013 were included if they had stored serum samples. The stored serum samples were obtained for vitamin D studies, which included 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D  was ascertained for 162 patients (94% of the study population). Ten patients presented or progressed to ESRD within 3 months of AL diagnosis and were excluded from this analysis. Of the remaining patients, 36 patients developed ESRD, representing 23.7% of patients assessed for progression to ESRD. Patients who developed ESRD had lower baseline 25(OH)D levels compared to patients who did not progress to ESRD  are provided in vs 49.2 months; P=0.03; 2D \u226518 pg/mL and those with lower levels D levels >20 ng/mL had shorter overall survival (OS) compared to patients with lower 25(OH)D values D level <20 ng/mL showed a trend towards longer OS , whereas a 1,25(OH) did not Table\u00a05.In this study we explored, for the first time, the prevalence and risk factors for vitamin D deficiency in patients with AL amyloidosis. We found that more than half of our study population had vitamin D deficiency (defined as 25(OH)D <20 ng/mL), while over a fifth of the study cohort had severe vitamin D deficiency [25(OH)D <10 ng/mL]. Vitamin D deficiency was particularly common among patients with significant proteinuria, an established risk factor for vitamin D deficiency. We have also found that severe vitamin D deficiency was associated with increased risk for progression to end-stage renal disease. Overall, the findings in this study support measurement of baseline vitamin 25(OH)D in patients with AL amyloidosis, particularly among those with significant proteinuria.The proportion of patients with vitamin D deficiency in this study (56.6%) is higher compared to the general US population. In the National Health and Nutrition Examination Survey (NHANES), conducted in a similar time period to our study (2001 to 2010), the prevalence of vitamin D deficiency among US adults age 18 or above ranged between 27.1% and 30.8% (depending on age group)  and popuWe identified several risk factors for hypovitaminosis D in AL amyloidosis. The most significant risk factor for low 25(OH)D levels was significant proteinuria. Patients with nephrotic syndrome are known to be at risk for vitamin D deficiency . Kidney via modulation of several pathways including the renin-angiotensin, NF-kB and Wnt/\u03b2-catenin signaling pathways D deficiency is an independent predictor of progression to ESRD. This finding has been reported in previous studies with patients with chronic kidney disease \u201334. The pathways . In addipathways . HoweverIrrespective of the mechanisms by which vitamin D deficiency increases the risk of ESRD, correction of vitamin D deficiency in patients with CKD , should be assessed in prospective studies given the potential benefit of such treatment. A large, randomized trial among type II diabetes mellitus participants did not demonstrate a renoprotective effect for vitamin D supplementation versus placebo . However2D deficiency, was associated with improved survival compared to patients with normal vitamin D levels. This observation stands in contrast to observations among cancer patients (25(OH)D deficiency, but not 1,25(OH)patients , 18\u201322. patients . As survpatients , 40 and patients , vitamin2D levels to be normal in subjects with t(11:14) as compared to those with non-t. t is the most common genetic abnormality in patients with AL amyloidosis  (In this study there was a trend for 25(OH)D and 1,25(OH)poptosis \u201343. As a (BCL-2) , an antivia the gold standard method of liquid chromatography-tandem mass spectrometry. We were also powered for several observations including the positive effect of concurrent vitamin D supplementation on serum 25(OH)D and the expected seasonal variation on serum 25(OH)D levels. The study follow-up period was long (median >16 years), allowing sufficient time of observation for events . The primary limitation to our study was its retrospective design. We did not preselect number of samples based on predefined hypothesis, which may limit the study power. These limitations negatively affect the generalizability of the above findings and require further studies.Our study has several strengths. These include that this is the first report on the incidence of vitamin D deficiency in patients with AL amyloidosis, and the ability to measure vitamin D metabolites and vitamin D binding protein In conclusion, we have shown that patients with renal AL amyloidosis have a high incidence of vitamin D deficiency. We anticipate our findings will serve as the basis for future prospective clinical efforts to improve vitamin D levels in these patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Mayo Clinic Institutional review Board. The patients/participants provided their written informed consent to participate in this study.EM, MTD, and MG designed the study, analyzed the data, wrote the first draft and approved the final version of the manuscript; NL, AD, ML, FB, DD, SH, PK, YH, AF, MH, WG, TK, RW, SR, RG, MB, SR, and SK provided care for patients, revised the manuscript critically, and approved the final version of the manuscript. RK performed patients\u2019 follow-up, revised the manuscript critically, and approved final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by CA90628-21 Pal Calabresi K12 Career Development Award.EM: honorarium from Janssen and consultation fee from Protego (fee paid to institution). NL served on advisory board for Takeda Pharmaceuticals. AD: research funding from Celgene, Millennium Pharmaceuticals, Pfizer, and Janssen and received a travel grant from Pfizer. ML: Research Funding from Celgene. PK is a principal investigator of research studies for which Mayo Clinic has received funding from AbbVie, Takeda, Sanofi, Janssen, Karyopharm, Glaxo SmithKline, Regeneron Pharmaceuticals, Ichnos Sciences and Amgen. He has served on the Medical advisory board meetings of Sanofi, Pharmacyclics, BeiGene, Cellectar, GSK, X4 and Karyopharm. SK received research funding for clinical trials to the institution from Abbvie, Amgen, BMS, Carsgen, Janssen, Astra-Zeneca, Novartis, Roche-Genentech, Takeda, Tenebio, Molecular Templates, received consulting/Advisory Board participation  from Abbvie, Amgen, BMS, Janssen, Roche-Genentech, Takeda, Astra-Zeneca, Bluebird Bio, Epizyme, Secure Biotherapeutics and  Oncopeptides, Beigene, Antengene. MG served as a consultant for Millennium Pharmaceuticals and received honoraria from Celgene, Millennium Pharmaceuticals, Onyx Pharmaceuticals, Novartis, GlaxoSmithKline, Prothena, Ionis Pharmaceuticals, and Amgen.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "We analyzed the usefulness of vitamin D biomarkers for the evaluation of MBD in patients with CKD. We analyzed blood and urine samples from 208 outpatients with CKD stage G2\u2013G5. 25(OH)D showed a poor correlation with the estimated glomerular filtration rate (eGFR). Conversely, the 24,25(OH)2D level and VMR were significantly correlated with eGFR and the intact parathyroid hormone level. In conclusion, 24,25(OH)2D and VMR have the potential to be vitamin D biomarkers for the detection of MBD in CKD patients.The appropriate management of vitamin D deficiency and hyperparathyroidism is essential to prevent metabolic bone disorder (MBD) and cardiovascular diseases in chronic kidney disease (CKD). Recently, the 24,25-dihydroxyvitamin D [24,25(OH) In case of vitamin D toxicity, negative feedback turns on the catabolism mechanism to reduce blood vitamin D levels. The activated 24(OH)D hydroxylase enzyme (CYP24A1) converts 25(OH)D to 24,25(OH)2D by the stimulation of 1,25(OH)2D [2D or VMR could be a physiologically better biomarker than the currently used 25(OH)D, in that they represent the result of a feedback mechanism. In addition, previous studies have suggested that VMR may have a stronger association with bone health than that of 25(OH)D [2D level or VMR might be biomarkers of vitamin D status, particularly in CKD patients [Recently, serum 24,25(OH) D level ,19. 24,225(OH)2D ,21. Ther 25(OH)D . The kid2D, and VMR in patients with CKD. In this present study, we compared various vitamin D biomarkers according to renal function in CKD patients, as well as the association of the biomarkers with intact PTH (iPTH). Since there are several limitations to the current method for evaluating vitamin D status with 25(OH)D, we aim to evaluate whether 24,25(OH)2D or VMR can be a biomarker alternative to 25(OH)D in CKD.There are rare studies comparing vitamin D biomarkers, including 25(OH)D, 24,25(OH)This prospective study enrolled outpatients with CKD aged >18 years who visited the Department of Nephrology, Gyeongsang National University Hospital, between June and December 2021. We analyzed a subset of blood samples from a total of 208 patients who provided informed consent. Patients taking calcimimetics, phosphate binders, any medications and supplements containing calcium, or phosphorus, as well as vitamin D, were excluded. This study was approved by the Institutional Review Board of Gyeongsang National University Hospital .2D was measured by the method mentioned in a previous study with slight modifications [2D can currently be measured only by the LC-MS/MS method. While 25(OH)D can also be measured by a electrochemiluminescence binding assay, it was measured by the LC-MS/MS method for consistency in the measurement method. Following the addition of the internal standard, stable isotope-labeled d6-24,25(OH)2D and d6-25(OH)D were added to 200 \u03bcL of serum sample. After this, methanol was mixed with the solution. To precipitate protein, the solution was mixed by vortexing and kept at 4 \u00b0C for 10 min. After centrifugation at 4 \u00b0C and 12,000 g for 10 min, phosphate-buffered saline was added into the supernatant and applied onto an SPE cartridge. After performing SPE, the vacuum system was applied to the eluted fraction for evaporation. The dried remnant was reconstituted in 75% methanol. In total, 5 \u03bcL of the reconstituted solution was analyzed by the LC-MS/MS system. The LC-MS/MS system in our study was an Agilent 1260 HPLC system  with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies) and an electrospray ionization source. For the separation of 24,25(OH)2D and 25(OH)D by HPLC, a Kinetex\u00ae Biphenyl column  and Poroshell\u00ae 120 EC-C 18 column  were utilized, respectively. The mobile phase in HPLC used 0.1% aqueous formic acid and methanol, and was processed by a gradient program at a flow rate of 0.4 mL/min. The monitored transitions were 417 \u2192 381 m/z for 24,25(OH)2D, 423 \u2192 387 m/z for d6-24,25(OH)2D, 401 \u2192 383 m/z for 25(OH)D, and 407 \u2192 389 m/z for d6-25(OH)D. In the LC-MS/MS system for the present study, the limits of quantitation of 24,25(OH)2D and 25(OH)D were 0.2 and 2 ng/mL, respectively.The serum concentration of 25(OH)D and 24,25(OH)2D concentration by 25(OH)D concentration and then multiplying it by 100.The VMR was calculated according to a previous study . It was 2/dL2), and iPTH were recorded. Medical records were screened to identify patients with a history of diabetes mellitus and hypertension. The estimated glomerular filtration rate (eGFR) was calculated using the 2012 CKD-EPI creatinine equation. The CKD stage was classified as G2-G5, according to the Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guidelines [The following data were collected through the Electrical Medical Record (EMR) to analyze the correlation between the CKD stage and vitamin D biomarkers in the patients with CKD. Age, sex, height, serum creatinine (SCr), serum albumin, urine protein to creatinine ratio , serum calcium (mg/dL), serum phosphate (mg/dL), calcium\u2013phosphate product , the Akaike information criterion, and the Bayesian information criterion. All the tests were two tailed, and p < 0.05 was considered statistically significant. We used R software  and SPSS software  for statistical analyses and the construction of graphics.To assess the correlation between two variables, Pearson\u2019s correlation test was used. For variables with a normal distribution, differences between the groups were evaluated using an analysis of variance. For multivariate analysis, multivariate linear regression and logistic regression analyses were used. We confirmed the goodness-of-fit of the regression models using an adjusted R-squared , 5 (10.2%), 18 (29.0%), 19 (35.2%), and 8 (57.1%), respectively.2D, and VMR levels were 22.81 \u00b1 11.24 ng/mL, 0.83 \u00b1 0.63 ng/mL, and 3.55 \u00b1 1.91, respectively. In CKD stage G3a, the mean iPTH level was 42.10 \u00b1 20.43 pg/mL, which was lower than that of G2. However, in general, the mean iPTH level increased as the CKD stage increased from G2 to G5 (p < 0.001). The 25(OH)D level did not differ according to the CKD stage (p = 0.353), whereas the 24,25(OH)2D level and VMR showed a significant difference according to the CKD stage .2 = 0.0874, p < 0.001)  . The eGFR and VMR were also positively correlated  (A negative correlation was found between iPTH level and eGFR (R< 0.001) a. The 25= 0.632) b. There < 0.001) c,d.2 = 0.0277, p = 0.016)   (The iPTH level was negatively correlated with the 25(OH)D (R= 0.016) a, 24,25(< 0.001) b, and VM< 0.001) c levels.< 0.001) . 2D, and VMR levels, according to the CKD stage. The mean iPTH level was the lowest in CKD stage G3a and gradually increased with the increasing stage to CKD stage G5, except in CKD stage G2. However, the 24,25(OH)2D level and the VMR decreased with the increasing CKD stage, from CKD stage G2; this trend was more prominent for the VMR. The 25(OH)D level did not change with the 24,25(OH)2D level or the VMR between different CKD stages.2, the Akaike information criterion, and the Bayesian information criterion. In models using the eGFR as a dependent variable (models 4\u20136), model 6, which explained the eGFR using the VMR, had the best fit. Of the three vitamin D biomarkers, the VMR was the most strongly associated with the eGFR. In the models using iPTH as a dependent variable (models 7\u20139), model 8, which explained iPTH using 24,25(OH)2D, had the best fit. Of the three vitamin D biomarkers, 24,25(OH)2D was most strongly associated with the iPTH level D, 24,25(OH)2D and VMR as the alternative biomarkers of 25(OH)D. In the present study, we compared biomarkers of the vitamin D level in patients with CKD, particularly to determine the usefulness of 24,25(OH)2D level and VMR tend to decrease consistently from stage G2 to G5. Although various biochemical indicators related to CKD-MBD are altered in CKD stage 3, clinical features, such as degree of severity and rate of change, vary among patients. The KDIGO guideline recommends that serum calcium, phosphorus, and PTH levels should be monitored from CKD stage 3 in adults and from CKD stage 2 in children [As shown in children .2; additionally, the proportion of patients with SHPT increased rapidly with a decreasing eGFR [2D level and VMR were more strongly correlated with eGFR than iPTH or the 25(OH)D level, particularly in the early stages of CKD.In children with slowly progressive kidney disease, the PTH level may be elevated from CKD stage 2 ,30. In aing eGFR . Therefo2D level and the VMR, but not the 25(OH)D level, were significantly correlated with the KDIGO CKD stage and the eGFR; this suggests that the 24,25(OH)2D level and the VMR are better indicators of vitamin D level than 25(OH)D, the currently used vitamin D biomarker in CKD patients. In several previous studies, the 24,25(OH)2D, VMR and 25(OH)D have been controversial as vitamin D biomarkers. A study found that VMR may be used as a complementary indicator of vitamin D level for the evaluation of endogenous metabolism in premenopausal women [Levin et al.  found loal women . In a stal women . On the al women . Therefo2D, and VMR, were significantly correlated with the iPTH level, the 24,25(OH)2D level showed the strongest correlation with the iPTH level in the model fitness analysis. A previous study showed that the 24,25(OH)2D level had a stronger association with iPTH than the 25(OH)D level [2D and the VMR are better predictors of the effects of vitamin D supplementation on MBD than 25(OH)D, particularly in CKD patients. Our study suggests that 24,25(OH)2D and the VMR may be more sensitive vitamin D biomarkers than 25(OH)D in CKD patients, because they started to decrease at an earlier stage than 25(OH)D in Although all the evaluated vitamin D biomarkers, including the 25(OH)D, 24,25(OH))D level . However2D or the VMR is a better biomarker than 25(OH)D, especially for bone health. Ginsberg et al. showed that the VMR had stronger correlation with both BMD and fracture risk compared to 25(OH)D [2D or the VMR differs from 25(OH)D in bone health are unclear. In Some studies suggest that 24,25(OH) 25(OH)D . Another 25(OH)D . This su2D is decreased in CKD due to a reduced 1\u03b1-hydroxylase function. The vitamin D level is regulated by a precise feedback mechanism that maintains homeostasis between its production and catabolism [2D and FGF-23, and is inhibited by PTH [2D [2D reflects the vitamin D level more accurately than 25(OH)D, particularly because it is an indicator of catabolism that maintains the concentration of active vitamin D in vivo through feedback. Additionally, 24,25(OH)2D is an appropriate vitamin D biomarker. First, the circulating half-life of 24,25(OH)2D in blood is relatively long . Second, it has a relatively high level, which is why it is measured in ng/mL [In general, the synthesis of 1,25(OH)tabolism ,35. The d by PTH ,37,38. D PTH [2D . Therefoin ng/mL . For comThis study has several strengths. First, 24,25(OH)2D and the VMR were compared with 25(OH)D as vitamin D biomarkers in the clinical setting with CKD patients. Second, our study included patients of various CKD stages from G2 to G5, suggesting the possibility of 24,25(OH)2D and the VMR as a potential vitamin D biomarker in a wide range of CKD situations. Third, the measurement of 25(OH)D in a clinical laboratory commonly proceeds with an electrochemiluminescence assay, a type of immunoassay. However, in our study, the measurement of 25(OH)D and 24,25(OH)2D was performed with the same test platform, LC-MS/MS, to minimize errors due to differences in the analyzers and to increase the accuracy. Our study had two main limitations. First, this was a cross-sectional study, and follow-ups of patients or blood investigations were not performed. Second, several factors that could affect the serum vitamin D level, including seasonal variation, food, outdoor activity duration, and use of sunscreen, were not analyzed. However, to minimize seasonal variation, we conducted the study over two seasons, from summer to autumn, in South Korea. According to the website, which provides year-round weather statistics for Seoul, the capital of South Korea, there is an average of 2428 h of sunlight per year (of a possible 4383) with an average of 6:38 of sunlight per day . Because2D and the VMR were significantly correlated with the eGFR and iPTH levels in patients with CKD stage G2\u2013G5. Therefore, we suggest that 24,25(OH)2D and the VMR have the potential to be alternative vitamin D biomarkers to 25(OH)D for the diagnosis of MBD in CKD patients.Although 25(OH)D is the most commonly performed test to assess vitamin D levels in CKD patients, it was poorly correlated with the eGFR. On the other hand, 24,25(OH)"}
+{"text": "Saturating concentrations of 1,25(OH)2D3, 25(OH)D3 and 25(OH)D2 resulted after 24 h stimulation in a comparable number and identity of target genes, but below 250 nM 25(OH)D3 and 25(OH)D2 were largely insufficient to affect the transcriptome. The average EC50 values of 206 common target genes were 322 nM for 25(OH)D3 and 295 nM for 25(OH)D2 being some 600-fold higher than 0.48 nM for 1,25(OH)2D3. The type of target gene, such as primary/secondary, direct/indirect or up-/down-regulated, had no significant effect on vitamin D metabolite sensitivity, but individual genes could be classified into high, mid and lower responders. Since the 1\u03b1-hydroxylase CYP27B1 is very low expressed in PBMCs and early (4 and 8 h) transcriptome responses to 25(OH)D3 and 25(OH)D2 were as prominent as to 1,25(OH)2D3, both vitamin D metabolites may directly control gene expression. In conclusion, at supra-physiological concentrations 25(OH)D3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs possibly by directly activating the vitamin D receptor.Human peripheral blood mononuclear cells (PBMCs) represent a highly responsive primary tissue that is composed of innate and adaptive immune cells. In this study, we compared modulation of the transcriptome of PBMCs by the vitamin D metabolites 25-hydroxyvitamin D The vitia cells , as wellia cells , 31. Prihe donor .3 is 100- to 1,000-fold lower than for 1,25(OH)2D3 2D3 (0.05\u20130.15 nM) D5(OH)2D3 , 34, whi0.15 nM) . This re the VDR . The molg pocket , 38. In g pocket .3 and 25(OH)D2 in comparison to that of 1,25(OH)2D3 in freshly isolated human PBMCs. We will demonstrate that 25(OH)D3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs and cannot exclude the possibility that both vitamin D metabolites directly activate the VDR.In this study, we analyzed the transcriptome-wide effects of 25(OH)DPeripheral blood was collected from a single healthy individual  the vitamin D responsiveness of whom had already been characterized in the VitDbol trial (NCT02063334) , 40. The2 incubator and treated for 4, 8 or 24 h either with 1,25(OH)2D3  25(OH)D3 , 25(OH)D2  or solvent (0.1% EtOH). All experiments were performed in three repeats. Deconvolution of RNA-seq data using the algorithm CIBERSORTx (+ T cells (32%), CD4+ T cells (20%), natural killer cells (21%) and monocytes/macrophages (20%) within the PBMC pool.PBMCs were isolated immediately after collecting 100 ml peripheral blood using Vacutainer CPT Cell Preparation Tubes with sodium citrate (Becton Dickinson) according to manufacturer's instructions. After washing with phosphate-buffered saline, the cells were grown at a density of 0.5 million/ml in 5 ml RPMI1640 medium supplemented with 10% charcoal-depleted fetal calf serum, 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin at 37 \u00b0C in a humidified 95% air/5% COBERSORTx  and its www.ncbi.nlm.nih.gov/geo) with accession number GSE199273.Total RNA was isolated using the High Pure RNA Isolation Kit (Roche) according to manufacturer's instructions. RNA quality was assessed on an Agilent 2100 Bioanalyzer system (RNA integrity number \u2265 8). rRNA depletion and cDNA library preparation were performed using the New England Biolabs kits NEBNext rRNA Depletion, NEBNext Ultra II Directional RNA Library Prep for Illumina and NEBNext Multiplex Oligos for Illumina (Index Primers Sets 1 and 2) according to manufacturer's protocols. RNA-seq libraries went through quality control on an Agilent 2100 Bioanalyzer and were sequenced on a NextSeq 500 system (Illumina) at 75 bp read length using standard protocols at the Gene Core facility of the EMBL . The libraries were sequenced as four batches. Fastq files of the 66 libraries can be found at Gene Expression Omnibus  (via the R package BiomaRt (version 2.46.0)  with Ensembl annotation (version 103) by using default settings of the nf-core/rnaseq STAR-Salmon pipeline (version 3.0) 4323183) . The num23183 . TEdgeR (version 3.36.0)   between the samples. This distance was calculated as the root mean square deviation (Euclidean distance) of the largest 500 log2FCs between a given pair of samples, i.e., for each pair a different set of top genes was selected. The inspection of the MDS plot showed that (i) the time-dependent divergence from the native transcriptome state and (ii) the concentration-dependent modulation by treatment with 1,25(OH)2D3, 25(OH)D3 or 25(OH)D2 are the two principal factors driving differences in the gene expression profiles of PBMCs. The gene-wise statistical test for differential expression was computed using the generalized linear model quasi-likelihood pipeline  and FC > 2 at 24 h. Taking all treatments together, 553 genes with a Benjamini-Hochberg corrected p-value  < 0.05 and a total trimmed mean of M-value normalized CPM count > 10 2D3 and EtOH-treated samples at each time point) were considered as vitamin D targets .Differential gene expression analysis was computed in R (version 4.1.2) in the Rocky Linux 8.5 operating system using the tool  3.36.0) . The anang (MDS) . MDS waspipeline . The emppipeline . The glm targets . Mean-Digets 3A;  for the 2FC) was modeled with the three-parameter log-logistic function LL.3 from the R package drc  and family-wise error rate (FWER)-adjusted p-values retrieved. Comparisons with a FWER < 0.05 were considered as significant. The code of the analysis can be found at https://github.com/andreahanel/2022_Doseresponse.The effect of vitamin D metabolites on the change in mRNA levels D3, 1,000 nM 25(OH)D2 or solvent (0.1% EtOH). In three repeats RNA-seq was performed on the basis of total RNA. As in comparable studies D3  and 385 genes modulated by 25(OH)D2  , FN1 (fibronectin 1), LOXHD1 (lipoxygenase homology PLAT domains 1) and SLC24A4 (solute carrier family 24 member 4) were the top up-regulated and the genes CXCL10 (C-X-C motif chemokine ligand 10), STEAP4 , CXCL9 and OLFM1 (olfactomedin 1) the most down-regulated.In order to obtain maximal responses of the transcriptome, PBMCs from an healthy individual were treated immediately after isolation for 24 h with either 10 nM 1,25(OH) studies , 47, 48,gulated) . For all2D3, with 100, 250, 500, and 750 nM 25(OH)D3 as well as with 100, 250, 500, and 750 nM 25(OH)D2. This resulted in 7 and 179 target genes for 0.1 and 1 nM 1,25(OH)2D3, no targets for 100 and 250 nM 25(OH)D3, 322 and 392 responding genes for 500 and 750 nM 25(OH)D3, no targets for 100 nM 25(OH)D2 as well as 30, 342, and 397 genes that reacted on a stimulation with 250, 500, and 750 nM 25(OH)D2 D3 as well as 25, 278, 307, and 299 to 250, 500, 750, and 1,000 nM 25(OH)D2 D2 25(OH)D2 . These i)D2 (2D3 . This le25(OH)D2 . Venn di25(OH)D2 . From bo3 and 25(OH)D2 were insufficient for general gene regulation, while 500 nM of both vitamin D metabolites was clearly above this threshold.The 206 common targets represent a stable set of genes responding to variant concentrations of all three tested vitamin D metabolites . Combine2D3, 25(OH)D3 and 25(OH)D2 are very comparable. At concentrations of 250 nM and below, 25(OH)D3 and 25(OH)D2 are largely insufficient to significantly modulate the expression of vitamin D target genes.In summary, both by number as well as by most responsive target genes the transcriptome-wide responses to saturating concentrations of 1,25(OH)50 values 0.48 nM for 1,25(OH)2D3, 322 nM for 25(OH)D3 and 295 nM for 25(OH)D2  between the potencies of 25(OH)D3 and 25(OH)D2, but their average EC50 is some 640-fold higher than that of 1,25(OH)2D3.Plotting the FC of the 206 common vitamin D target genes over vitamin D metabolite concentration and using the three-parameter log-logistic model, determined the EC25(OH)D2 . There i2D3 the EC50-values of the eight different categories varied from 0.29 to 0.73 nM but did not differ significantly  between each other (3 the range was 318 to 389 nM and for 25(OH)D2 192 to 313 nM, but the difference was not statistically significant .Based on the reference dataset , the 206ch other . Similar2D3 the EC50-values ranged from 0.10 nM [ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2)] to 2.39 nM [LMNA (lamin A/C)], for 25(OH)D3 from 121 nM [NXPH4 (neurexophilin 4)] to 461 nM [ENTPD7 (ectonucleoside triphosphate diphosphohydrolase 7)] and for 25(OH)D2 from 132 nM (SLC11A1) to 421 nM [STAB1 (stabilin 1)] , 59 mid responders (0.41 to 1.06 nM) and 12 low responders (1.20 to 2.39 nM). Representative genes for the three categories are HBEGF (heparin binding EGF like growth factor) as high responding gene (PPARGC1B (PPARG coactivator 1 beta) as mid responding gene  as low responding gene D3 and 25(OH)D2 did not allow a categorization of the vitamin D target genes by their response to these metabolites.Since neither gene categories nor up- or down-regulation allowed a distinction of the sensitivity of vitamin D target genes to vitamin D metabolites, the dose responses of the 206 genes were investigated on an individual gene level. Manual inspection of the dose response curves provided for 130 genes convincing fits for all three vitamin D metabolites. Interestingly, for 1,25(OH)ilin 1)] . The widing gene , PPARGC1ing gene  and MYCLing gene . Importa50 values of the response of vitamin D target genes to 25(OH)D3 and 25(OH)D2 are not significantly different and are in the order of 300 nM, i.e., some 600-fold higher as those for 1,25(OH)2D3. Categorization of the target genes into primary/secondary, direct/indirect or up-regulated/down-regulated does not allow any significant distinction in their response to the three vitamin D metabolites. However, individual target genes can be classified by their response to 1,25(OH)2D3 (but not to 25(OH)D3 and 25(OH)D2) as high, mid and low responding genes.Taken together, the average EC3 and 25(OH)D2 may activate vitamin D signaling without enzymatic conversion by CYP27B1 to 1,25(OH)2D3 and 1,25(OH)2D2, respectively, the transcriptome-wide response to saturating concentrations of all three vitamin D metabolites was assessed by RNA-seq at earlier time points. After 4 h stimulation with 10 nM 1,25(OH)2D3, 16 genes (13 up- and 3 down-regulated) passed the statistical threshold , with 1,000 nM 25(OH)D3 32 genes (27 up- and 5 down-regulated) and with 1,000 nM 25(OH)D2 20 genes (19 up- and 1 down-regulated) (HBEGF and G0S2 (G0/G1 switch 2). After filtering with the reference dataset of vitamin D target genes in PBMCs D3 or 1,000 nM 25(OH)D2 at 4, 8 and 24 h (In order to investigate whether 25(OH)Dgulated) . Common in PBMCs  31), Ve, Ve3 andin PBMCs . As obsein PBMCs , 31, at and 24 h .DHCR7, CYP2R1 and CYP27A1 show comparable mid-range expression, while the average expression of CYP27B1 is 10- to 32-fold lower. In this way, CYP27B1 belongs to the 5% lowest expressed genes in PBMCs. Since the CYP24A1 gene is a well-known vitamin D target gene 2D on the basis of 25(OH)D is far less prominently covered by enzyme expression as the degradation of 25(OH)D and 1,25(OH)2D to 24,25(OH)D and 1,24,25(OH)3D, respectively.The enzymes DHCR7 (7-dehydrocholesterol reductase), CYP2R1, CYP27A1, CYP27B1 and CYP24A1 are critical nodes in the vitamin D metabolism pathway , left. Tget gene , its expiD study  are dispiD study . The ind3 and 25(OH)D2 as prominently as to 1,25(OH)2D3. The expression of CYP27B1 protein in PBMCs is very likely too low for an efficient conversion of 25(OH)D3 and 25(OH)D2 into 1,25(OH)2D3 and 1,25(OH)2D2, respectively, in particular in the presence of highly expressed CYP24A1 protein. However, it cannot be excluded that metabolic formation of 1,25(OH)2D is partly contributing to the results obtained.In summary, already at early time points (4 and 8 h) the PBMC transcriptome responds to a stimulation with saturating concentrations of 25(OH)D3 and 25(OH)D2 to each other and in reference to 1,25(OH)2D3. At supra-physiological concentrations of 10 nM 1,25(OH)2D3  and 1,000 nM 25(OH)D (10-times the recommended serum level) the response of the PBMC transcriptome is very comparable, i.e., the expression of some 300 genes is significantly modulated showing after 24 h stimulation some 3-times more down-regulation than up-regulation. Moreover, the target gene lists of 25(OH)D3, 25(OH)D2 and 1,25(OH)2D3 are to 85% identical, i.e., our experimental series had relatively low transcriptional noise. These observations suggest that all three vitamin D metabolites use identical mechanisms in the modulation of vitamin D target gene expression. Form a structural point of view this is obvious, since 25(OH)D will bind in the same agonistic VDR conformation as 1,25(OH)2D D25(OH)2D .2D3 time course study in PBMCs D3 and 295 nM for 25(OH)D2 compared to 0.48 nM for 1,25(OH)2D3 is the first report on the sensitivity of the transcriptome to vitamin D metabolites. These results provide an additional argument that there is no difference in the response of the transcriptome to 25(OH)D3 and 25(OH)D2. Moreover, our findings indicate with the factor of ~600 a good estimation of the relative gene regulatory potential of 25(OH)D compared to 1,25(OH)2D. Since serum levels of 25(OH)D are even 1,000-times higher than that of 1,25(OH)2D, this supports the option that 25(OH)D can directly modulate the expression of vitamin D target genes. However, 25(OH)D concentrations in the order of 300 nM are far higher than the recommended 100 nM. Therefore, irrespective of the mechanism of action, for persons with a normal vitamin D status the transcriptome as a whole may not be regulated by 25(OH)D. However, there are a few very sensitive genes, such as NXPH4, SLC11A1, ADGR3 (adhesion G protein-coupled receptor G3), G0S2, HBEGF, and PMEPA1 , which showed EC50 values for 25(OH)D below 150 nM. Thus, in healthy persons with a very high vitamin D status, a few genes may be directly affected by elevated 25(OH)D serum levels.The 206 common vitamin D target genes, on which we focus in this study, represent the majority of the vitamin D sensitive part of the PBMC transcriptome, although  they had been filtered by a reference dataset from a recent 1,25(OH)in PBMCs . The est3 or D2 supplementation, there are clinical settings, where supplementation with higher doses of 25(OH)D3 or 25(OH)D2 are recommended D supplementation.In addition to the control of the vitamin D status of healthy individuals through careful sun exposure and vitamin Dommended . These p chicken . Also inHBEGF D2, it does not matter, if the observed effects on the PBMC transcriptome are explained either (i) by the enzymatic conversion of 25(OH)D into 1,25(OH)2D during the 4\u201324 h duration of stimulation phase, which then activates the VDR, (ii) by a direct binding of 25(OH)D to the VDR or (iii) a combination of both. The very low expression of the CYP27B1 gene, in particular in relation to CYP24A1 expression, in PBMCs of one person used in this study is representative for other individuals. This calls into question, whether there was enough 1\u03b1-hydroxylase activity to convert within 4 h a sufficient amount of 25(OH)D into 1,25(OH)2D, which then stimulated primary vitamin D target genes. For example, in order to reach a 1,25(OH)2D level of 10 nM, 1% of the 1,000 nM 25(OH)D pool need to be handled within 4 h. However, as it is typical for in vitro cell culture stimulation experiments, supra-physiological concentrations of 1,25(OH)2D3 and 25(OH)D are compared. In fact, the tight regulation of the 1,25(OH)2D3 level in vivo via the molecule's rapid degradation by the enzyme CYP24A1 D CYP24A1 , indicat3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs and regulate the same set of vitamin D target genes as the most potent VDR ligand, 1,25(OH)2D3. However, in order to observe consequences of the gene regulatory potential of 25(OH)D, concentrations of 300 nM or higher need to be available. This is three times the recommended serum level, i.e., it does not apply to healthy individuals with a regular vitamin D status.In conclusion, 25(OH)DFastq files of the raw data can be found at GEO with accession number GSE199273.The studies involving human participants were reviewed and approved by the Ethics Committee of the Northern Savo Hospital District had approved the study protocol (#9/2014). The patients/participants provided their written informed consent to participate in this study.AH performed RNA-seq data analysis. CC did RNA isolation and RNA-seq library preparation. CV synthesized the tested compounds. AH and CC wrote the manuscript, which was reviewed by CV. All authors contributed to the article and approved the submitted version.Parts of this study were sponsored by Carbogen Amics, Ltd. CC was supported by the WELCOME2\u2014ERA Chair European Union's Horizon2020 research and innovation program under Grant Agreement No. 952601.This study received funding from Carbogen Amics, Ltd. The funder had the following involvement with the study: Providing vitamin D metabolites. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Osteosarcoma is a malignant tumor derived from primitive osteogenic mesenchymal cells, which is extremely harmful to the human body and has a high mortality rate. Early diagnosis and treatment of this disease is necessary to improve the survival rate of patients, and MRI is an effective tool for detecting osteosarcoma. However, due to the complex structure and variable location of osteosarcoma, cancer cells are highly heterogeneous and prone to aggregation and overlap, making it easy for doctors to inaccurately predict the area of the lesion. In addition, in developing countries lacking professional medical systems, doctors need to examine mass of osteosarcoma MRI images of patients, which is time-consuming and inefficient, and may result in misjudgment and omission. For the sake of reducing labor cost and improve detection efficiency, this paper proposes an Attention Condenser-based MRI image segmentation system for osteosarcoma (OMSAS), which can help physicians quickly locate the lesion area and achieve accurate segmentation of the osteosarcoma tumor region. Using the idea of AttendSeg, we constructed an Attention Condenser-based residual structure network (ACRNet), which greatly reduces the complexity of the structure and enables smaller hardware requirements while ensuring the accuracy of image segmentation. The model was tested on more than 4000 samples from two hospitals in China. The experimental results demonstrate that our model has higher efficiency, higher accuracy and lighter structure for osteosarcoma MRI image segmentation compared to other existing models. Osteosarcoma is one of the most common bone malignancies, which develops from mesenchymal cell line . AlthougThe diversity of osteosarcoma leads to scattered information about it in the medical literature, particularly in imaging results. The images of diverse osteosarcoma generated in the identical environment and process are various as well, and it is hard to tell the difference between healthy tissue and lesion areas manually in some cases ,15. GeneThe mortality of osteosarcoma is very high, but early detection and timely treatment can greatly improve the survival rate . HoweverIn recent years, with the increasing attention to AI, some medical image detection technologies have also been applied to the diagnosis of osteosarcoma ,30. AlthAccording to the above contents, we propose an MRI image segmentation and AI-assisted diagnosis system based on Attention Condenser for osteosarcoma (OMSAS) to assist doctors. This intelligent medical system is designed to assist doctors in identifying MRI images of patients with osteosarcoma and to automatically segment osteosarcoma, thus providing doctors with more powerful and intuitive tips and aids. The analysis results provided by the system can be used as an auxiliary basis for doctors to diagnose patients, which can effectively improve the efficiency and accuracy of diagnosis and reduce the cost of diagnosis and treatment for patients. Firstly, we carry out data augmentation in the dataset and use a variety of methods to expand, standardize and classify the original dataset. Data preprocessing reduces the degree of over-fitting and makes the model more generalized. In terms of model design, we improve AttendSeg , replace(1)In this article, the region in the image can be focused more accurately through the Attention Condenser. The multilayer condenser structure can further locate the boundary of the tumor, and reduce a large number of unnecessary calculations in the later stage, to improve the efficiency and accuracy of training.(2)When using the model to predict, OMSAS uses a compound decision module that turns a single input into multiple copies of the input. Then, the model makes predictions by multiple copies simultaneously, and then combines the multiple outputs to get the final result. Multiple decisions can reduce the wrong prediction caused by some unknown situations and make the output more accurate. Meanwhile, it can improve the stability of the model.(3)More than 4000 images provided by the First People\u2019s Hospital of Huaihua and the Second People\u2019s Hospital of Huaihua were used for testing. The result indicates that our ACRNet in OMSAS is better than other existing segmentation models. The model has high training efficiency and prediction accuracy as well as small resource consumption, which is important in assisting doctors to diagnose osteosarcoma in patients.The specific contributions of the essay are divided into the following points:The paper is arranged as follows: Through investigation and research, we find that many technologies use artificial intelligence knowledge for medical decision-making and image processing. In modern medical systems, more and more artificial intelligence (AI) algorithms are used for image segmentation, health prediction, and other functions. In the diagnosis of osteosarcoma, how to process MRI images and accurately mark the tumor area has become a research hotspot. This section will introduce some mainstream algorithms in related directions.Nasor et al.  proposedArunachalam et al.  used supDionisio f.c.f et al.  took HauIn the field of image segmentation, there are many good models in recent years, which can be divided into threshold-based, cluster-based, edge-based, region-based, etc . U-Net pThrough the above research, we found that with the continuous development of computer technology, the research scope of artificial intelligence technology in the field of paramedicine, especially in the direction of image recognition, is expanding. However, due to the variability of the morphology and structure of osteosarcoma, existing medical image recognition techniques are difficult to achieve the expected results in MRI image segmentation of osteosarcoma. To improve the segmentation accuracy and better adapt to some medical devices with relatively poor performance, we design a new strategy for MRI image segmentation for osteosarcoma based on Attention Condenser. The approach enhances the efficiency and accuracy of osteosarcoma detection by reducing the device requirements via tactics such as data preprocessing, residual network, LayerNorm and attention condenser.Considering that we have submitted two similar papers ,60, the We have conducted a comparative analysis of the innovations in the three papers. First, all three papers address the fact that osteosarcoma is extremely dangerous for human beings and that the diagnosis and treatment of osteosarcoma in developing countries is extremely difficult due to the shortage of medical resources. Moreover, there are few specialized physicians due to the large volume and complexity of patient data. Therefore, we designed an osteosarcoma artificial intelligence method to assist physicians in picture analysis and clinical diagnosis.However, the three studies have different emphases, and there are differences in the problems they each address.For literature , it usesFor literature , this meCompared with the above two papers, our paper uses a more novel MRI image segmentation model for osteosarcoma, ACRNet. The ACRNet model is more novel, and the model training is more efficient and achieves better segmentation results. This is reflected in the following points.In terms of data preprocessing, we use a simpler and more efficient processing method that maintains a good segmentation performance. We binarize and regularize the images to filter out the valid regions in the images and eliminate the effect of different brightness levels between images on the training. Additionally, we enhance the dataset to improve the generalization of the model.In terms of model design, we refactored the main component of AttendSeg, \u201cAttention Condenser\u201d. We use the attention mechanism and combine it with the residual structure to further enhance the attention of the model to the osteosarcoma region, so that the model can shift more attention to refine the details between regions in the osteosarcoma MRI image, and understand the global view of the image, correct the results in the reconstruction, and effectively improve the segmentation effect. In addition, we transformed the original general convolutional layer combination into a reserved block and a shrinkage block, which greatly reduces the number of parameters of the general convolutional operation and allows the model to better extract various features of the image and run more efficiently.In the output part of the model, we use the composite decision to integrate the output of multiple different angles of the same image and unify the results of the same source, which effectively enhances the accuracy and robustness of the output results. We plot the output results as a black-and-white image to assist the doctor\u2019s diagnosis, thus greatly reducing the burden of the doctor\u2019s film reading.In terms of the practicality of the segmentation system, the network has a lightweight structure with only 6.91 M parameters and a SETT value of 174, which makes the model simpler and more efficient in training and more adaptable to low-configuration medical equipment, which helps the implementation of the medical-assisted segmentation system on the ground. As a result, ACRNet ensures high accuracy in segmenting osteosarcoma MRI images while improving efficiency, which will save more human and financial resources for developing countries and improve the efficiency of osteosarcoma diagnosis in hospitals.This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.At this stage, in most developing countries, the distribution of medical resources is very unbalanced, and many advanced medical resources are concentrated in the region where very few people are located. Additionally, generally speaking, a particularly good analytical instrument can greatly reduce the working pressure of doctors. However, the high manufacturing and maintenance costs make it very difficult to promote the medical equipment of image-assisted diagnosis.Considering the osteosarcoma patients living in developing countries, if the early diagnosis is not timely, the lack of technology or the very high cost of later medical treatment will often make them give up their last chance of survival. Therefore, what we need to do is to improve the early diagnosis rate. For hospitals, the prolonged manual diagnosis and the shortage of talents result in the difficulty of diagnosis. It is necessary to introduce an osteosarcoma MRI image segmentation medical system to assist doctor. Moreover, to promote this system more widely, we expect that the built-in process should be simple and efficient enough, the model should have high accuracy, and the requirements for equipment should be as low as possible. Thus, based on these cognitions, this paper proposes a segmentation method for osteosarcoma MRI image based on Attention Condenser (OMSAS), which can accurately depict the tumor area in the image, relieving the burden of reading films for doctors. The overall design of the system is shown in According to Some symbols involved in the chapter are explained, as shown in (1)The osteosarcoma MRI images in the dataset have different brightness and darkness due to different instruments, contrast agent dose, and other external environmental reasons;(2)The amount of data is still insufficient for training a model with high accuracy, and the training is prone to instability.We find that the data in the original dataset is not suitable for model training directly, and there are the following problems:To address the above two problems, we have carried out a series of data preprocessing to optimize the effect of the model by improving the quality of data and eliminating interference factors. The specific operation is shown in First, we select the region of all the osteosarcoma MRI images. After the image is binarized by (1), it will be filled to obtain a picture with only pure black and pure white areas. Then, we select the smallest rectangular area in the picture that can just contain the pure white area, which is the selected effective area. Selecting the effective region can simplify the training and recognition of the model, and make the model pay more attention to the feature extraction of the effective region.In addition, we regularize all the osteosarcoma MRI images to avoid the impact of some excessively bright and dim images on the training. The brightness gap and different density distribution between images make it easy for the model to take these unnecessary factors into account, which not only reduces the speed of model training, but also may affect the effect of the model. Therefore, we use a unified regularization process to transform all MRI images with (2).The regularized image addresses the first problem. At this time, the image has met the minimum standard of training. However, to enhance the robustness of our model and the generalization of it, we also need data augmentation. For data augmentation, we rotate each picture by 90\u00b0, 180\u00b0, and 270\u00b0 at first, and then flip each picture horizontally and vertically. After that, the amount of data is 8 times that of the original dataset. We then randomly select some images and add noise to them to reduce the overfitting phenomenon of the model when learning high-frequency features.After data preprocessing, the quality of the MRI images of osteosarcoma in the dataset was improved, and the high-quality dataset can be used as a reference basis for clinical diagnosis, which is beneficial for doctors to make more effective film reading. At the same time, the enhanced dataset can provide a good fit for the model training.3.2. Brief Introduction of Attention.The model of this paper involves the mechanism of attention, so attention will be briefly introduced in this part.M. and the size of M is . Moreover, w means width, h represents its height, and c is its number of channels. Then, the compression process can be expressed as (3).We know that the inspiration of the attention mechanism comes from our physiological perception of the environment. Our visual system will actively select the information we need to pay attention to in the impression and ignore the irrelevant information in the field of vision. Similarly, in the neural network, when we segment an osteosarcoma MRI image or recognize the boundary, we also hope the network to pay more attention to the region we expect, and ignore the irrelevant information. The attention mechanism can be divided into three steps: compression, activation, and attention. For example, we now have an osteosarcoma MRI image input In order to evaluate the importance of each channel, we need to activate the previous operation results as a whole. The weight calculation formula on each channel is shown in (4), where After the activation, we assign In our model design, attention can be understood as the process of (1)The osteosarcoma MRI images will be rotated and flipped to obtain a group of pictures before each prediction of it.(2)The set of pictures from the previous step will be used as input for the residual network with multiple attention condensers, and a set of results will be output.(3)This group of results is inversely changed according to the original change direction to obtain a group of results consistent with the original image direction. This group of results is calculated and processed to obtain the final prediction tumor region.On the osteosarcoma image segmentation model, we designed a residual network based on Attention Condenser (ACRNet), and its overall structure is shown in In recent years, a large number of scholars have emerged to conduct research on attention networks. The attention condenser proposed by Alexander Wong et al.  uses theAttention Condenser consists of four parts: a condensed layer that reduces the dimension of spatial channels, an embedded structure that represents the activation relationship of joint spatial channels, an extended layer that increases the dimension, and an attention mechanism that applies selective attention. In ACRNet, we use two linear structures and the middle embedding layer to perform the above scaling operation. In addition, we introduce a variable called ratio, which can manually adjust the expansion and contraction times of the condenser, to meet the needs in different situations better.The residual structure is used many times in the whole network. The residual unit is realized in the form of layer skip connection. Some layers are regarded as a unit. The initial input of the unit and the final output will be added before activation. The residual structure addresses the degenerate problem of the deep neural network. Under the same number of layers, the training speed of the residual network is faster, which can make the operation efficiency of the model higher.In order to better extract various features of the osteosarcoma MRI image, we use the idea of the block to combine and transform the original general convolution layer into the reservation block and the retracting block. The reservation block includes a 1 \u00d7 1 convolution layer, a Layer Normalization, and a ReLU activation function, which only changes the number of channels of input without affecting the size of the image. Adding LayerNorm can normalize the data before entering the activation function to avoid the problem of vanishing gradient in the saturated region of the activation function. Meanwhile, the use of LayerNorm accelerates the convergence speed of the model again.The retracting block consists of a 3 \u00d7 3 and a 1 \u00d7 1 convolution layer. Its purpose is to simply extract image features. A retracting block instead of single 3 \u00d7 3 convolution layer makes a convolution kernel responsible for only one channel. A channel will be convoluted by only one convolution kernel, and the change of channel is handed over to 1\u00d71 convolution layer. This can significantly reduce the parameters number of the ordinary convolution operation.In the output, we use compound decision-making to integrate multiple outputs from different angles of the same image, since that for neural networks, every rotated or flipped image is a new image, and the prediction results are not necessarily the same. Unified processing of homologous output can enhance the accuracy and robustness of results. The specific formula for output calculation is as follows.Medical image segmentation models often encounter the phenomenon of data imbalance. Training unbalanced data is easy to cause the problem of high precision but low recall. Moreover, in the diagnosis of osteosarcoma, missed diagnosis of tumor area is a more intolerable serious error than the wrong diagnosis of the normal area. Therefore, when designing the model, we choose Tversky Loss as the loss function. Tversky loss is a special dice loss, which is a combination of dice loss and Jaccard coefficient. Generally, the weight of dice loss for FP and FN is equal, so the model will not focus on the improvement of recall Sufficiently during training. Therefore, we use Tversky Loss with parameters \u03b2 as the loss function in model training. The specific definition is as shown in (7).\u03b1 and \u03b2 control the penalty strength of the loss function for FP and FN, respectively. The higher the \u03b2, the greater the penalty of the loss function on FN, and the easier it is to improve the recall. After testing, we adopt \u03b1 = 0.25, \u03b2 = 0.75.By using our model, we can easily perform region detection on osteosarcoma MRI images and the annotation regions are comparable to the \u201cGold Standard\u201d of doctor annotation. Our model ensures accuracy while greatly reducing the burden on doctors when reading MRI images. For hospitals, the time and money spent on processing related cases are greatly reduced.The data in the article are collected from the First People\u2019s Hospital of Huaihua and the Second People\u2019s Hospital of Huaihua. During the experiment, we collected more than 4000 MRI images and other related index data of 210 patients with different degrees of osteosarcoma. During the experiments, all segmentation models are built on the same dataset, so the performance tests of the models are comparable. To ensure that the sample is closer to the standard of developing countries, we selected data mostly from patients with not very high income in the lower social class. Additionally, the data are mostly from early imaging and a small percentage from mid to late. We selected three different cross-sectional slices to allow the model to better fit multiple input cases and to achieve stable segmentation results. All MRI images are selected and manually labeled by doctors. We divided the dataset into the training set and the test set in the ratio of 7:3. Of the 210 real cases of patients we owned, 147 are put into training set and 63 are in test set. We used a 10-fold cross-validation to ensure the validity of experimental results.The training, test environment, and some important parameters settings during the training of the model in this paper are shown in In the process of training and testing, better, we will use some common comparative index and single epoch training time (SETT) as metrics to evaluate the performance of our ACRNet model and other segmentation models. Moreover, we will also make a comparison of the number of parameters of the different models.We used the confusion matrix to analyze and explain the model. TP indicates that both the predicted results and the actual results are osteosarcoma areas, and TN indicates that both the predicted results and the actual results are normal areas. FP represents the normal area misjudged as the disease area, and FN represents the lesion area misjudged as the normal area. The specific definition and calculation formula of the above indicators are as follows.Definition\u00a01.Accuracy. It represents the proportion of all samples with correct prediction. Its definition is as shown below.Definition\u00a02.Precision. It represents the proportion of all samples judged as tumor areas that are actual tumor areas. The formula for Precision is:Definition\u00a03.Recall. It represents the percentage of all samples judged as normal areas that are really normal areas. Compared with precision, we expect the emergence of high recall in medical experiments, and false negative is more intolerable than false positive. Its definition is as follows.Definition\u00a04.F1-Score. It is an index based on precision and recall, which can represent the robustness of a model. The higher the F1-Score, the greater the robustness. Its definition is as shown below.Definition\u00a05.IOU. It represents the intersection and union ratio between the tumor region in the prediction result and the tumor region in the real result, reflecting the coincidence between the prediction result and the real region. Its definition formula is as follows, where represents the tumor region in the predicted result and  represents the tumor region in the real result.Definition\u00a06.DSC. It describes the similarity between the prediction result and the truth and is an index with a value of 0-1. The closer DSC is to 1, the closer the result is to the real situation. Its definition formula is as follows.Definition\u00a07.SETT. It represents the average time required for an epoch of model training under the same amount of data, and the unit is seconds (s).For the purpose of further evaluating the segmentation effectiveness as well as the structural complexity of our ACRNet, we used U-Net , MSFCN , MSRN 5, FPN 5757 and FCFirstly, we trained the model without preprocessing. The training and test results are shown in We trained the model and finally got a trained ACRNet model. Then, we put the prepared test data into it for testing and test it synchronously with other prepared image segmentation models. The tumor region prediction images are shown in According to To compare each model more accurately and digitally, we counted the segmentation results of each model and calculated and sorted out its indicators. We used several indicators selected and defined in section C to compare our model with other models. To achieve a better prediction effect, we often need to train more epochs to make the model fit the dataset better. We often expect a good model to converge to a higher accuracy as soon as possible, and the higher the final convergence value, the better. Based on this, we tested different models as shown in In addition to comparing accuracy, we also made statistics on recall and F1-Score. Similarly, we took the first 50 epochs for research. During the experiment, we also conducted additional research on the attention part. We visualized the attention mechanism and superimposed the binary mask output by the middle layer with the input picture to obtain a more intuitive information range of attention focus, namely the attention map. The brighter the area, the more focused the attention. We selected 18 maps of different categories for comparison and display, and the results are shown in In the article, the ACRNet was trained and tested by more than 4000 osteosarcoma MRI images. Through comparison and statistical analysis, we find that OMSAS has good prediction results in many indicators such as accuracy and recall. OMSAS not only improves the efficiency and accuracy of the model but also saves the cost with fewer parameters and shorter training time. The experimental results show that the segmentation model ACRNet in OMSAS has a better effect and more obvious advantages in osteosarcoma MRI image segmentation, and is suitable for application in developing countries with unbalanced medical resources distribution and poor medical care.In the future, with further expansion and improvement of the dataset, we can continuously improve the prediction ability of the model, so as to detect the region of more complex osteosarcoma MRI images or even achieve similar good results on MRI of other cancers. At the same time, with the improvement of computing power and the need for later medical treatment, we can expand more modules in the model, such as outputting the size of the tumor area and predicting the future diffusion of tumor area, to provide more professional assistance for medical diagnosis."}
+{"text": "Osteosarcoma is one of the most common primary malignancies of bone in the pediatric and adolescent populations. The morphology and size of osteosarcoma MRI images often show great variability and randomness with different patients. In developing countries, with large populations and lack of medical resources, it is difficult to effectively address the difficulties of early diagnosis of osteosarcoma with limited physician manpower alone. In addition, with the proposal of precision medicine, existing MRI image segmentation models for osteosarcoma face the challenges of insufficient segmentation accuracy and high resource consumption. Inspired by transformer's self-attention mechanism, this paper proposes a lightweight osteosarcoma image segmentation architecture, UATransNet, by adding a multilevel guided self-aware attention module (MGAM) to the encoder-decoder architecture of U-Net. We successively perform dataset classification optimization and remove MRI image irrelevant background. Then, UATransNet is designed with transformer self-attention component (TSAC) and global context aggregation component (GCAC) at the bottom of the encoder-decoder architecture to perform integration of local features and global dependencies and aggregation of contexts to learned features. In addition, we apply dense residual learning to the convolution module and combined with multiscale jump connections, to improve the feature extraction capability. In this paper, we experimentally evaluate more than 80,000 osteosarcoma MRI images and show that our UATransNet yields more accurate segmentation performance. The IOU and DSC values of osteosarcoma are 0.922\u2009\u00b1\u20090.03 and 0.921\u2009\u00b1\u20090.04, respectively, and provide intuitive and accurate efficient decision information support for physicians. Osteosarcoma is the most common malignant primary bone tumor, with the highest incidence among adolescents . Among aFor patients with osteosarcoma, if not diagnosed and treated early, there is a tendency to develop extensive metastases in other soft tissues . In addiMost developing countries face a huge dilemma in the diagnosis and treatment of osteosarcoma. Take China as an example. China is a vast country with unbalanced medical development between regions, and the awareness of osteosarcoma is still unfamiliar in primary hospitals and even some provincial and municipal hospitals. And the occurrence, development, transformation, or deterioration of osteosarcoma are a dynamic process of change. With the different age, gender, physique, and even living conditions of patients, the morphology and size of osteosarcoma often show great variability and randomness. This places a high demand on the expertise of physicians. However, the poor and backward regions of China have poor medical equipment and shortage of professional physicians . On the Modern medical imaging technology is developing rapidly, and computer-aided diagnosis (CAD) system is also constantly innovating. Sathiyamoorthi et al.  used adaAs the most popular encoder-decoder network in medical image segmentation , U-Net  segmenteInspired by the self-attention mechanism of transformer , this paIn this paper, we optimize the classification of dataset by mean-teacher model to alleviate the influence of noisy labels on model training; remove irrelevant backgrounds in osteosarcoma MRI images through dataset preprocessing to reduce the burden of input images on segmentation network; and then improve the accuracy and segmentation efficiency of osteosarcoma segmentation.The incorporation of multilevel guided self-aware attention module (MGAM) into the encoder-decoder architecture of U-Net, which combines deep semantic information and spatial information through jump connections, provides the necessary fine-grained and high-resolution features for deconvolution, and facilitates the recovery of spatial information of osteosarcoma. In addition, the application of dense residual learning to the convolution module, combined with multiscale jump connections and aggregation of different semantic amplification sampling functions, facilitates the reduction of interference from osteosarcoma MRI image noise, and better preserves detailed information such as osteosarcoma edge features.Two components, transformer self-attention component (TSAC) and global context aggregation component (GCAC), are designed in UATransNet to enhance the ability to construct remote feature dependencies and capture global contextual information. Among them, TSAC adaptively integrates local features with their global dependencies and GCAC optimizes the feature representation effect of osteosarcoma by aggregating contexts to learned features. Meanwhile, the UATransNet method has few parameters, simple computation, and low hardware equipment requirements, which alleviates the difficulties of backward medical equipment in developing countries and achieves a balance of speed and accuracy in osteosarcoma detection and segmentation, greatly improving the diagnostic efficiency of physicians.Experimental analysis was performed using over 80,000 osteosarcoma MRI images acquired from Xiangya II Hospital of Central South University. Our proposed osteosarcoma image segmentation architecture exhibits better segmentation performance compared with some state-of-the-art baselines. Furthermore, it can enable the diagnostic accuracy of osteosarcoma images to approach the level of expert physicians, providing information support for decision-making in regions and countries lacking high-level physicians.The detailed contributions of this study are as follows:Osteosarcoma is an aggressive malignant bone tumor that occurs mostly in the extremity bones of children and adolescents, with an extremely poor natural prognosis and a tendency to develop blood metastases at an early stage. Imaging is an important tool for the diagnosis and clinical evaluation of osteosarcoma. In recent years, computer-aided decision-making systems have become a popular research direction in the medical field to analyze the health status of patients mainly through healthcare data and diagnostic images.Due to the cellular heterogeneity in the dataset, pathologists are often faced with high complexity processing and disagreement in classifying osteosarcoma tumors. Similarly, segmentation and classification of tissues in tumor images remain extremely challenging due to interclass similarity and intraclass variability, despite H&E staining. Chen and Zhao  proposedVarious osteosarcoma detection methods are used for early detection of osteosarcoma, but evaluation of slides under the microscope to detect the extent of tumor necrosis and tumor outcome remains a major challenge in the medical field. Therefore, Badashah et al.  developeDiffusion-weighted imaging (DWI) can capture cellular changes in tumor tissue without contrast injection in the early stages of patient treatment, facilitating early diagnosis and prognosis of malignant disease . OsteosaIn recent years, automatic segmentation based on deep learning has been widely used. Among them, U-Net is one of the most important semantic segmentation frameworks for convolutional neural networks (CNNs) and is widely used for lesion segmentation and classification in the field of medical image analysis. U-Net  is succeIn order to better extract edge features and texture features, etc. using the shallow edge output module, we extract semantic features using the deep output module, calculate the loss value between the predicted map and the actual tumor image by the side output module, and then backpropagate the loss information. Zhang et al.  proposedBased on the above analysis, in order to better assist clinical diagnosis and treatment, the research on osteosarcoma image preprocessing, image segmentation, and image analysis by model construction has been developed significantly in recent years. However, the MRI images of osteosarcoma are susceptible to noise and have complex edge contours, making the segmentation effect poor. To optimize the segmentation effect of MRI images of osteosarcoma, we combine the depth-separable U-shaped network with transformer and propose a new perspective to improve the performance of semantic segmentation, which achieves more effective feature fusion by multilevel guided attention, i.e., collaborating with transformer's self-attention and global spatial attention, and multiscale exploration of connected contextual semantic information to ensure the semantic embedding consistency.With the development of intelligent medicine, image processing plays an indispensable role in the diagnosis, treatment, and prognosis of diseases. Osteosarcoma MRI images have complex features and contain a lot of redundant data, which makes manual screening and detection by physicians alone time-consuming and laborious. U-Net is widely used for MRI image segmentation of osteosarcoma because of its better segmentation of small targets and scalable structure. However, as the performance requirement of medical image segmentation increases, U-Net shows the limitation of information decline due to the lack of ability to effectively construct remote feature dependencies and capture global contextual information. On the other hand, osteosarcoma MRI images are susceptible to noise and prone to overfitting resulting in loss of edge features. Based on this, we propose a lightweight UATransNet osteosarcoma image segmentation model by combining the encoder-decoder architecture of U-Net with transformer. We successively perform dataset classification optimization and remove irrelevant backgrounds from MRI images by averaging teacher models and normalizing preprocessing. In order to break the limitation of information degradation exhibited by U-Net due to the lack of ability to effectively construct remote feature dependencies and capture global contextual information, we designed two components: transformer self-attentive component (TSAC) and global contextual aggregation component (GCAC). TSAC adaptively integrates local features with their global dependencies to better preserve osteosarcoma edge features and other detailed information, and GCAC optimizes the osteosarcoma feature representation by aggregating context to the learned features.In addition, this paper applies dense residual learning to the convolution module, combined with multiscale jump junction, to improve the feature extraction capability. Using over 80,000 osteosarcoma MRI images acquired at Xiangya II Hospital of Central South University for experimental analysis, our proposed osteosarcoma image segmentation architecture exhibits better segmentation performance compared to some state-of-the-art baselines. In addition, the diagnostic accuracy of osteosarcoma images is close to that of expert physicians, providing intuitive and accurate information support for decision-making in regions and countries that lack high-level physicians.In this section, the proposed UATransNet model is introduced in terms of dataset optimization, normalized preprocessing, multilevel guided attention mechanism, and multiscale jump connection. The overall design of the proposed UATransNet osteosarcoma image segmentation model in this paper is shown in We selected over 80,000 osteosarcoma MRI images as the dataset for our experiments, but among them, some of the images had too small or blurred osteosarcoma regions, which led to significant degradation of the experimental model performance. To improve the accuracy of the test, we combined the mean-teacher network , ResNet To cope with the possible gradient disappearance problem as the number of network layers increases, we use residual connections and three 3\u2009\u00d7\u20093 convolutional blocks to capture the high-dimensional feature information of the image and use group normalization instead of batch normalization in the Res-Block to cope with the possible performance degradation caused by small batches, as shown in D1 and D2. Among them, the D1 dataset contains Label1 and the D2 dataset does not contain the label. The overall design of the mean-teacher semisupervised algorithm is shown in First, we randomly divide the original dataset into two parts, D1 and D2 datasets are input to the student model, and the predicted likelihood of Pstu\u2009\u2009de\u2009\u2009nt1 and Pstu\u2009\u2009de\u2009\u2009nt2 is output; the D2 dataset is input to the teacher model, and the predicted likelihood Pteacher2 is output. Second, the loss value V1 is calculated based on the loss function Loss1 and Pstu\u2009\u2009de\u2009\u2009nt1 and Pteacher1 can calculate the loss value V1, which is calculated as follows [Mean-teacher semisupervised algorithm: first, the  follows :(1)Loss1V2 is calculated based on the loss function Loss2 and Pstu\u2009\u2009de\u2009\u2009nt2 and Pteacher2. The network parameters of the teacher model of UATransNet \u2009\u03b8t\u2032 are obtained by updating the network parameters of the student model \u2009\u03b8t by moving average.Similarly, the loss value \u03b8t of student model are obtained by updating the parameters through loss gradient descent. Among them, the loss function contains two parts: the first part is the supervised loss function, as shown in equation  scattering relative entropy loss function [The network parameters equation . Loss1 ifunction ; the KL Loss2 is calculated as follows:However, there is a problem of asymmetry in the KL function. In order to make the prediction distribution of the teacher model and the student model consistent, we adopted the Jensen\u2013Shannon (JS) algorithm  to compeFinally, we divided the osteosarcoma MRI image dataset into effective part and difficult part with the proportion of 39.4% and 60.6%, respectively, and input them into the UATransNet network model sequentially to achieve better training effect.To remove irrelevant backgrounds from the osteosarcoma MRI images and reduce the burden of the input images on the network, we segmented the input osteosarcoma MRI images into osteosarcoma-suspected regions and the processing steps are shown in First, a multiscale deep belief network (m-DBN) and Gaussian mixture classifier (GMC)  were useIn the image matrix, the minimum weight of histogram is 5%. The upper bound and the next bound of the adjusted weight are less than 5%. The image is then median filtered to remove noise using a window of a certain size, which is 5% of the maximum value of the length height of the region of interest. However, since the use of median filtering  leads toThe Otsu method  was used\u03bc is the average intensity of the image pixels in the segmented region, Ix\u2032,y\u2032 is the intensity of the candidate pixel points located at , and \u03b8 is the determined threshold. Then, the threshold value obtained in the third step of osteosarcoma size estimation is used as the initial threshold value, and when no remaining pixels satisfy the seed expansion conditions specified by the above two equations, the size of the segmented region obtained in the current evolutionary stage is checked in relation to the predicted osteosarcoma size; if the currently obtained region size is smaller than the estimated value, the threshold value is lowered to continue expanding the region; if the currently obtained region is larger than the estimated value, the segmentation is stopped.Based on a priori knowledge, in osteosarcoma MRI images, the tumor region is generally brighter than the surrounding healthy tissue. Therefore, the brighter multiple pixel points in the center of the region of interest are selected as the initial seed points. The following two principles are followed when selecting the pixel points adjacent to the seed points to be added to the seed points:The threshold is adjusted adaptively according to the size of the osteosarcoma, and the input osteosarcoma MRI image is segmented into the suspected region of the osteosarcoma. The grayscale characteristics of the image can be directly used, the calculation is simple, and the calculation efficiency is high, but for a few grayscale differences that are not obvious, images with a large disparity in the size and proportion of the target and the background, you need to combine manual experience selection to have a better effect. Therefore, this paper mainly focuses on most of the MRI images of osteosarcoma with obvious gray difference.The U-Net network model for segmenting osteosarcoma MRI images is able to fuse deep semantic information (such as the color and shape of the osteosarcoma) with the information contained in high-precision features but is unable to model the contextual relationships of features that are far away, resulting in insufficient accuracy for osteosarcoma segmentation. Like the opposite, the transformer network model employs a self-attentive mechanism, which facilitates the extraction of global information but lacks information at local details. Based on this, we propose a lightweight UATransNet osteosarcoma image segmentation model with a multilevel guided self-aware attention module (MGAM) between the bottom encoder and decoder of the U-shaped architecture, which helps to capture a broader and richer contextual representation and better fuses the features of semantic inconsistency between transformer and U-Net network models. It is beneficial to reduce the interference of osteosarcoma MRI image noise, so that detailed information such as osteosarcoma edge features is better preserved and excellent segmentation results can be obtained. In addition, represented by MGAM, UATransNet has a small number of parameters and simple computation, which reduces the consumption of resources and achieves a balance of accuracy and efficiency of osteosarcoma MRI image segmentation.The core component of the UATransNet proposed in this paper: the multilevel guided self-aware attention module (MGAM) consists of the transformer self-attention component (TSAC) and the global context aggregation component (GCAC).Referring to transformer's multihead self-attention mechanism, UATransNet focuses on semantic information from the global contextual representation subspace and first adds the learned position encoding of the osteosarcoma MRI image to the input of the encoder features and shares it among all attention layers for a given query/key value sequence, enabling the capture of contextual information about absolute and relative positions , which iRc\u00d7h\u00d7w as input is first reconstructed as a query matrix Q\u2009\u2208Rc\u00d7(h \u00d7 w) and key matrices K\u2009\u2208Rc\u00d7h\u00d7w and V\u2009\u2208Rc\u00d7h\u00d7w [Q, K, and V matrices contain the texture features of the osteosarcoma MRI image.Wq, Wk, and Wv are embedding matrices for different linear projections. Then, a scaled dot product operation with softmax normalization between the transposed versions of the query and key matrices is used to generate a matrix of contextual attention maps CAM\u2009\u2208Rc\u00d7c and captures the necessary fine-grained and high-resolution features in the osteosarcoma MRI images. The matrix exhibits similarity to the global elements of the key matrix compared to the given elements from the query matrix. To further implement the aggregation of values weighted by attention weights, we multiplied the contextual attention map CAM by V and recovered the detailed information in the osteosarcoma MRI images. Finally, in the TSAC, we can represent the multiheaded attention as follows:The concrete implementation of the TSAC is shown in \u2009\u2208Rc\u00d7h\u00d7w , and theIt should be noted that equation . FinallyUATransNet encodes broader contextual location information into local features via GCAC and selectively aggregates global context into the learned features. The specific implementation is shown in Featurepc0 \u2208 Rc0\u00d7h\u00d7w and Featurepc1 \u2208 Rc1\u00d7h\u00d7w, where c1=c0/8, respectively, using two convolution operations via the encoder feature Featureenco\u2009\u2009de\u2009\u2009r. Then, Featurepc0 is reshaped into Upc0 \u2208 Rc0\u00d7(h \u00d7 w) and Featurepc1 is reshaped into Vpc1 \u2208 Rc1\u00d7(h \u00d7 w) and Wpc1 \u2208 Rc1\u00d7(h \u00d7 w). Next, the matrix multiplication operation with softmax normalization is executed on the reshaped V and W to obtain the position attention map L \u2208 Rh \u00d7 w)\u00d7(h \u00d7 w).1 and \u221d2 are scale parameters initialized to 0. In this way, we can further optimize the feature representation of osteosarcoma MRI images with semantic consistency to obtain more accurate segmentation of osteosarcoma MRI images and provide intuitive, accurate, and efficient decision information support for physicians.In order to fully utilize the obtained contextual information and spatial relationships, the multilevel guided self-aware attention function module takes a dynamic planning approach to optimally combine the original features and attention feature embeddings of osteosarcoma through attention embedding fusion, which is the important reason why UATransNet can acquire spatial information of osteosarcoma and accurately segment the MRI images of osteosarcoma.\u03c5 (\u00b7), \u2295, and f (\u00b7) are the upsampling, concatenation, and mixed convolution operations, respectively.In this paper, feature maps from all scales are directly connected to form a unified tensor through cascade connection, and then, the features of the target are extracted, as shown in The higher-level network has a larger perceptual field with strong semantic information representation capability; however, this structure results in low resolution of feature and lacks spatial geometric feature details; the lower-level network has a smaller perceptual field with strong geometric detailed information representation capability and weak semantic information representation capability though high resolution. In order to fuse multiscale features to be equally effective in encoding global and local contexts, UATransNet guides the upsampling process in the decoder subnetwork through a new multiscale jump connection scheme, i.e., residual connection and dense connection, and the network structure is shown in As an input module, the model improves the structure of MRI images of osteosarcoma and solves the problem of unbalanced classification of MRI images of osteosarcoma .(12)F=fnTo cope with the medical dilemma of outdated hardware equipment in developing countries, UATransNet employs a dense connection module in the encoder-decoder architecture. Dense concatenation takes the upsampled feature set of the previous encoder block as the input to the current block and the output feature map as the input to all subsequent blocks, as expressed in the upcoming equation. One advantage of dense connectivity is that it has fewer parameters than traditional convolutional networks because it does not need to relearn redundant feature maps, reducing the requirement for hardware equipment and alleviating the difficulties of poor medical equipment in poor areas.UATransNet aggregates multiple decoder features at different semantic scales by bilinear interpolation, which better preserves detailed information such as osteosarcoma edge features. In addition, the dense concatenation mechanism generates the most discriminative feature representation in contrast to using one-time cascade concatenation. In these ways, the UATransNet osteosarcoma image segmentation model can not only mitigate the loss of details due to excessive upsampling but also alleviate the problems of gradient disappearance and overfitting.UATransNet successively performs dataset classification optimization and removes MRI image irrelevant background by averaging the teacher model and normalized preprocessing, which reduces the waste of computational resources, reduces the requirement for hardware devices, and greatly improves the efficiency of osteosarcoma segmentation. Then, UATransNet integrates local features and global dependencies in TSAC and GCAC at the bottom of the encoder-decoder architecture, which effectively reduces the interference of osteosarcoma MRI image noise. In addition, UATransNet applies dense residual learning to the convolution module and combines multiscale jump connections to better retain detailed information such as osteosarcoma edge features, which can provide doctors with fast and accurate decision suggestions, effectively simplify the diagnosis process, save time, and reduce the burden of tedious medical work for doctors.The dataset used in the experiment is over 80,000 osteosarcoma MRI images and related indexes of 286 osteosarcoma patients in Xiangya II Hospital of Central South University in recent years provided by the Ministry of Education Mobile Health Information-China Mobile Joint Laboratory and Xiangya II Hospital of Central South University . In ordeU-Net: the U-structure U-Net combines deep semantic information and spatial information through skip connections of the encoder and decoder, providing necessary high-resolution features for deconvolution, which is beneficial to recover valuable spatial informationThe core of pyramid scene parsing network (PSPNet): PSPNet aggregates contextual information from different regions to obtain global information through the pyramid pooling moduleMSFCN: based on a fully convolutional network, multiscale feature learning is performed with three supervised output layers, and both local and global features are capturedMultiscale residual network (MSRN): convolutional kernels of different sizes are introduced for adaptive detection of image features at different scalesFully convolutional network: FCN classifies images at the pixel level and uses a jump structure to achieve fine segmentationFeature pyramid network (FPN): both high-resolution features in the lower layers and high semantic information features in the higher layers are fused and predicted separately for each fused feature layerIn our experiments, we applied two different approaches to implement UATransNet, UATransNet Residual, and UATransNet Dense osteosarcoma segmentation models based on residual connections and dense connections, respectively. In addition, we also used a wide range of algorithms such as U-Net, PSPNet , MSFCN , MSRN 5, FCN, an1=1, \u221d2=0, \u221d1=0.75, \u221d2=0.25, \u221d1=0, \u221d2=1, \u221d1=0.5, \u221d2=0.5, \u221d1=0.25, \u2009and\u2009\u221d2=0.75 by setting different \u221d1 and \u221d2 values and observe PRE, IOU, DSC, and other changes in the evaluation indicators to find the parameter combination with the best performance of the model.UATransNet was implemented using PyTorch, Cuda 11.3, and all experiments were run on 1 RTX A4000 GPU with 32\u2009G of memory. Before training the UATransNet model, we extended the dataset by scaling up  images, rotating images, and flipping images to enhance the robustness of the model. During training, our proposed UATransNet framework was trained for 100 epochs with an initial learning rate set to 0.001, which changed to 0.0001 when training reached 50 epochs and was dynamically adjusted using CosineAnnealingLR. Through experiments, it can be seen that each epoch in the training process takes about 1\u20135\u2009minutes. Finally, 100 epochs of verification are performed using the checkpoints that store the trained models, and the changes in the evaluation metrics for each epoch are recorded. The verification time of each epoch is about 6\u201310\u2009s. In the process of parameter adjustment, we mainly set different parameter combinations such as \u221dTo measure the similarity between the predicted mask and ground truth, we used accuracy (ACC), precision (PRE), recall (REC), dice similarity coefficient (DSC), intersection over union (IOU), and F1-score (F1) as measures, which were calculated in pixels and used to quantitatively evaluate the performance of the UATransNet model for osteosarcoma image segmentation. The specific definitions of each indicator are as follows:In the UATransNet osteosarcoma image segmentation model, we first perform classification optimization of the dataset, as shown in In order to evaluate the performance of different osteosarcoma segmentation models more clearly and accurately, we performed a quantitative representation of the segmentation effect. We tested by training with 100 epochs, using accuracy (ACC), precision (PRE), recall (REC), dice similarity coefficient (DSC), intersection over union (IOU), and F1-score (F1) as measures for comparative analysis. The comparative results of the evaluation metrics are shown in As shown in However, our proposed UATransNet Residual and UATransNet Dense showed good performance in the osteosarcoma image segmentation task, and both models outperformed other baseline models in accuracy (ACC), precision (PRE), recall (REC), intersection over union (IOU), dice similarity coefficient (DSC), and F1-score(F1). UATransNet Residual and UATransNet Dense not only greatly optimize the segmentation accuracy, but also their FLOPs were only 161.01G and 163.20G, respectively, as shown in As shown in For medical disease detection, if the recall rate is low, a bad situation such as a missed diagnosis can easily occur. Therefore, we pay particular attention to the analysis of the recall rate when we evaluate the performance of the osteosarcoma image segmentation model. In this experiment, we compared the recall rate of the UATransNet model with other baseline models, as shown in In addition, we carefully analyzed how the accuracy of UATransNet and the individual baseline models changed with the progression of 100 epochs, as shown in The F1-score is a statistical measure of the accuracy of a binary classification model. It takes into account both the accuracy and recall of the classification model. As can be seen from Osteosarcoma MRI image segmentation helps physicians in clinical diagnosis by providing precise contours of osteosarcoma and assisting them in the clinical process. We designed a novel network, UATransNet, based on a modified U-Net, by introducing a self-attentive mechanism, incorporating a self-aware attention module, and combining it with a mean-teacher model, which produced good segmentation performance with IOU and DSC of 0.922\u2009\u00b1\u20090.03 and 0.921\u2009\u00b1\u20090.04, respectively. To further explore the effectiveness of MRI image segmentation in supporting the neuro-radiosurgery treatment planning stage, Rundo et al.  and MiliThis paper proposes a novel lightweight osteosarcoma image segmentation model, UATransNet, based on over 80,000 osteosarcoma image datasets collected from Xiangya II Hospital of Central South University for extensive experiments. UATransNet significantly improves the accuracy of osteosarcoma segmentation based on the original computational power through dataset classification optimization and preprocessing, and achieves the trade-off between accuracy and speed; utilizes TSAC and GCAC components are used to optimize the feature representation effect of osteosarcoma; applying dense residual learning to the convolution module, combined with multiscale jump junction, better preserves the edge features of osteosarcoma, provides effective decision information support for clinicians, and provides a new option for early diagnosis and treatment of osteosarcoma in developing countries.However, this paper is not clear enough for edge segmentation of osteosarcoma MRI images with little grayscale difference. With the development of medical image segmentation technology, in future research work, we will comprehensively utilize and optimize the edge features and texture features of osteosarcoma MRI images to further optimize the segmentation effect of osteosarcoma MRI images."}
+{"text": "Porphyromonas gingivalis , activates the host immune response to release numerous proinflammatory cytokines. Here, we aimed to clarify Leuconostoc mesenterica (L. mesenteroides) LVBH107 probiotic effects based on the inhibition of P.gingivalis activities while also evaluating the effectiveness of an in vitro P.gingivalis lipopolysaccharide-stimulated RAW 264.7 cell-based inflammation mode. L. mesenteroides LVBH107 survived at acid, bile salts, lysozyme, and hydrogen peroxide conditions, auto-aggregated and co-aggregated with P. gingivalis, exhibited strong hydrophobicity and electrostatic action, and strongly adhered to gingival epithelial and HT-29 cells . Moreover, L.mesenteroides LVBH107 exhibited sensitivity to antibiotics erythromycin, doxycycline, minocycline, ampicillin, and others (thus indicating it lacked antibiotic resistance plasmids), effectively inhibited P.gingivalis biofilm formation and inflammation (in vitro inflammation model), reduced the secretion of pro-inflammatory cytokines  and inflammatory mediators (NO and PGE2), and decreased the expression levels of inflammation related genes. Thus, L.mesenterica LVBH107 holds promise as a probiotic that can inhibit P.gingivalis biofilm formation and exert anti-inflammatory activity to maintain oral health.Probiotics, active microorganisms benefiting human health, currently serve as nutritional supplements and clinical treatments. Periodontitis, a chronic infectious oral disease caused by  Probiotics are defined by the FAO/WHO as \u201clive microorganisms which when administered in adequate amounts confer a health benefit on the host\u201d . The effLactobacillus and Lactococcus species as prospective probiotic treatments for oral diseases [Lactobacillus reuteri (L. reuteri), Tweetman et al. [Lactobacillus brevis to patients with periodontal disease led to decreased gingivitis severity and dental calculus deposition [L. reuteri [Periodontitis is a common chronic inflammatory oral disease that is caused by oral pathogenic microbes , which idiseases . In an en et al.  found maposition , while r reuteri . Further reuteri  demonstrPorphyromonas gingivalis  is currently recognized as an important pathogen involved in mature dental plaque formation from biofilm generated through activities of a variety of associated pathogenic or nonpathogenic bacteria that adhere to and copolymerize with P. gingivalis cells. In addition, a variety of virulence factors  that participate in biofilm formation are expressed during interactions of these bacteria with host cells [P. gingivalis by the use of probiotic organisms holds promise as an important approach for preventing and slowing the progression of destructive periodontal disease.st cells , wherebyst cells ,21. TherLeuconostoc mesenteroides (L. mesenteroides), a Gram-positive, spherical or short-chain facultative anaerobic lactobacillus, is widely used for the production of fermented foods and bacteriocins. Several studies have shown that L. mesenteroides treatments can exert antioxidant activities, improve immunity, reduce cholesterol levels, and alleviate hyperlipidemia [L. mesenteroides administration could effectively reduce levels of the inflammatory factor IL-6, prompting researchers to speculate that L. mesenteroides may serve as a safe immunomodulatory treatment [L. mesenteroides LVBH107 were isolated from a traditional fermented food (spicy cabbage) and shown to exert an obvious inhibitory effect on P. gingivalis activities as based on Oxford cup assay results -stimulated inflammation, prompting the current study. Here, potential probiotic properties of L. mesenteroides LVBH107 were investigated in vitro, including resistance, surface characteristics, antibiotic resistance, and anti-P. gingivalis activity, assessed inflammatory responses of L. mesenteroides LVBH107 using a model of inflammation based on P. gingivalis LPS-stimulated RAW 264.7 macrophage. Thereby, our results provide experimental and theoretical basis for further using LVBH107 as an auxiliary drug to inhibit periodontal pathogens and treat periodontitis.ipidemia ,23. For reatment . In our  results . Those rLeuconostoc mesenteroides (subsp. mesenteroides) LVBH107 (CGMCC No. 21362) that obtained from a fermented food product (spicy cabbage) were deposited in the China General Microbiological Culture Collection Center . L. mesenteroides LVBH107 that was inoculated  into de Man, Rogosa, and Sharpe (MRS) broth  was cultured in an anaerobic incubator  at 37 \u00b0C for 16 h.L. mesenteroides LVBH107 was collected by centrifugation ; then, the pellet was washed three times with phosphate-buffered saline  to obtain the bacteria-free supernatant, which was filtered through a 0.22 \u03bcM filter membrane to remove bacterial cells. In addition, a heat-killed L. mesenteroides LVBH107 preparation was prepared by heating the bacteria at 90 \u00b0C for 30 min.After culture, P. gingivalis (ATCC 3327) was obtained from the China General Microbiological Culture Collection Center and was propagated on Columbia blood agar  at 37 \u00b0C in an anaerobic incubator  for 48 h. Next, brain heart infusion (BHI) broth (Hopebio Company) was inoculated with a P. gingivalis culture  followed by incubation at 37 \u00b0C for 48 h.v/v) fetal bovine serum  and 100 U/mL Penicillin-Streptomycin  at 37 \u00b0C . Cells were subcultured once every 2 days by splitting cultures 1:3 in fresh medium after harvesting adherent cells by detaching them from flask surfaces using 0.25% (w/v) trypsin-EDTA solution (Sigma-Aldrich).Human colorectal adenocarcinoma HT-29, human gingival epithelial (HGE), and RAW 264.7 (mouse mononuclear macrophage-derived) cell lines were obtained from Otwo Biotech  and cultured in Dulbecco\u2019s Modified Eagle Medium  containing 10% (L. mesenteroides LVBH107 was inoculated at 3% (v/v) into MRS broth containing 0.3% (w/v) pepsin , bovine bile , lysozyme , and hydrogen peroxide ; then, cultures were incubated at 37 \u00b0C for 20 h under aerobic conditions. During the culture period, optical density (OD) values at 600 nm were measured using a UV-VIS spectrophotometer  every 2 h.An overnight culture of L. mesenteroides LVBH107 cells were collected by centrifugation  and resuspended in PBS. The OD600 of the suspension was adjusted to 1.0 using a UV-VIS spectrophotometer; then, the suspension was incubated at 37 \u00b0C for 8 h, during which OD600 values were measured at 2 h, 4 h, 6 h, and 8 h [T is the OD600 value at 2 h, 4 h, 6 h, and 8 h, and A0 is the OD600 value at 0 h. After overnight culture,  and 8 h . Auto-agL. mesenteroides LVBH107 and P. gingivalis strains were harvested at 16 h or 48 h by centrifugation , cells of each strain were resuspended in sterile PBS; then, the OD600 values of the two strains were adjusted to 0.50 and 0.60, respectively. Next, equal volumes (2 mL) of suspensions of both strains were shaken , and each suspension was separately incubated without shaking for 8 h at 37 \u00b0C. During the 8 h incubation, OD600 readings of each suspension were taken at 2 h, 4 h, 6 h, and 8 h [X and AY are absorbance values of L. mesenteroides LVBH107 and P. gingivalis at 0 h and Amix is the absorbance value of the mixture containing both organisms after incubation for 2 h, 4 h, 6 h, and 8 h.After  and 8 h . Co-aggrL. mesenteroides LVBH107 was determined using the microbial adhesion to hydrocarbons (MATH) method with toluene and xylene serving as hydrophobic organic solvents. Chloroform was selected as Lewis acid and ethyl acetate as Lewis base in order to determine surface charge characteristics of L. mesenteroides LVBH107 [L. mesenteroides LVBH107 preparation was the same method as described above except that the bacterial pellet was resuspended in 0.1 M KNO3 buffer (pH 6.2) and adjusted to an OD600 value of 0.60. Next, 1 mL of organic solvent  to 3 mL of the L. mesenteroides LVBH107 suspension were combined to form a two-phase solution that was preincubated at room temperature for 10 min then vortexed for 2 min followed by incubation at room temperature for 30 min. Thereafter, the OD600 value of the aqueous phase was determined; then, the adherence of L. mesenteroides LVBH107 to hydrocarbons was calculated as follows:0 is the OD600 value before treatment with the organic solvent and A1 is the OD600 value of the aqueous phase after treatment with the organic solvent. The surface hydrophobicity of  LVBH107 . The met5 cells/well); then, the plates were incubated until cells adhered completely to well surfaces and reached 70\u201380% confluence. Next, medium in the wells was replaced with high glucose DMEM medium (without penicillin-streptomycin); then, the plates were incubated at 37 \u00b0C for 24 h. Thereafter, monolayers of cells were washed three times with PBS; then, 500 \u03bcL of L. mesenteroides LVBH107 (8 Log CFU/mL) was added per well, and then the plates were incubated for 2 h at 37 \u00b0C. Next, non-adherent bacteria were removed from each well then 2 mL of 1% (v/v) Triton X-100  was added to each well to detach adherent cells [L. mesenteroides LVBH107 was calculated as follows:1 is the total count of adhered L. mesenteroides LVBH107, and V0 is the number of total added L. mesenteroides LVBH107.HT-29 and HGE cells were transferred to 12-well tissue culture plate wells  culture was evenly spread onto the surface of each newly prepared MRS agar plate after the agar solidified at room temperature. Next, commercial antibiotic disks were placed onto plate surfaces, and then the plates were incubated at 37 \u00b0C for 48 h. After incubation, the diameter of each inhibition zone around each disk was measured and expressed as susceptible (S), intermediate (I), or resistant (R), as specified within the instructions provided by the manufacturer . All analyses were performed in triplicate.The antibiotic susceptibility of P. gingivalis cultures that had been incubated for 48 h at 37 \u00b0C were adjusted to 6 Log CFU/mL by dilution in BHI broth. Next, L. mesenteroides LVBH107 culture medium or supernatant and P. gingivalis were added to upper and lower chambers of 24-well transwell plates , respectively, at a ratio of 1:1 (v/v). After the incubation of transwell plates for 48 h at 37 \u00b0C, the fluid culture medium within the lower chamber was gently removed and discarded; then, the P. gingivalis biofilm at the bottom of each well was gently washed three times with PBS to remove planktonic P. gingivalis. Thereafter, P. gingivalis biofilm in each lower chamber was fixed in 2.5% (v/v) glutaraldehyde solution at 4 \u00b0C overnight and dried at room temperature. Next, 100 \u03bcL of 0.1% (w/v) crystal violet was added to each lower transwell chamber; then, staining was allowed to proceed at room temperature for 15 min, after which wells that had received stain were rinsed gently with 75% ethanol to remove unbound crystal violet and dried at room temperature [P. gingivalis biofilm structure was observed under a microscope  and photographed then biofilm absorbance values were measured at 540 nm using a microplate reader. The rate of L. mesenteroides LVBH107 inhibition of P. gingivalis biofilm formation was calculated as follows:540 value of P. gingivalis biofilm in the absence of L. mesenteroides LVBH107 culture medium or supernatant, and AC is the OD540 value of the P. gingivalis biofilm treated with L. mesenteroides LVBH107 culture medium or supernatant.perature . ThereafL. mesenteroides LVBH107 culture medium or supernatant and P. gingivalis were prepared in transwell plates as described above. After incubation of transwell plates for 72 h at 37 \u00b0C, coverslips coated with P. gingivalis biofilm were stained for 15-20 min in the dark using a LIVE/DEAD\u00ae BacLight\u2122 Bacterial Viability Kit  [P. gingivalis biofilm structure on each coverslip was assessed and recorded using a confocal laser scanning microscope .After sterilized coverslips (10 mm \u00d7 10 mm) were placed into lower chambers of transwell plates, co-cultures containing MA, USA) . Next, PL. mesenteroides LVBH107 for RAW 264.7 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay . After RAW 264.7 cells were inoculated into wells of 96-well plates and allowed to adherently grow on well surfaces to 70\u201380% confluence (1 \u00d7 106 cells/well), different concentrations of live or heat-killed L. mesenteroides LVBH107 were added to the wells  followed by co-incubation of plates for 6 h at 37 \u00b0C. Thereafter, the culture medium was removed from the wells; then, 300 \u03bcL of CCK-8 working reagent was added to each well, followed by the incubation of plates at 37 \u00b0C for 4 h. Next, the optical density of each well at 450 nm was measured using a multi-function microplate reader . Cell viability was calculated as follows:450 value of RAW 264.7 cells co-incubated with L. mesenteroides LVBH107, and Ac is the OD450 value of RAW 264.7 cells in the absence of L. mesenteroides LVBH107.The cytotoxicity of P. gingivalis anti-inflammatory activity, RAW 264.7 cells were treated with live or heat-killed L. mesenteroides LVBH107 suspensions for 3 h or 6 h and then 1 \u03bcg/mL of P. gingivalis LPS  was added to cells followed by incubation of stimulated cells for 24 h . Next, the production of nitric oxide (NO) was determined in P. gingivalis LPS-stimulated RAW 264.7 cells using assay kits to measure levels of each compound . In addition, the effects of L. mesenteroides LVBH107 on the production of TNF-\u03b1, interleukin-6 (IL-6), interleukin-1\u03b2 (IL-1\u03b2), and prostaglandin E2 (PGE2) were assessed in P. gingivalis LPS-induced RAW 264.7 cells using enzyme linked immunosorbent assay (ELISA) kits  [To assess , China) .TNF-\u03b1, IL-6, IL-1\u03b2, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). The 2\u2212\u0394\u0394Ct method was used to calculate qRT-PCR results. Melting curve analysis was used to evaluate the specificity of the reaction. \u03b2-actin served as the internal control. Primer sequences are listed in RAW 264.7 cells were transferred to centrifuge tubes; then, total RNA was extracted according to instructions provided with the TaKaRa MiniBEST Universal RNA Extraction Kit . Next, the Primescript\u2122 RT reagent Kit (TaKaRa) was used to reverse transcribe approximately 1.0 \u03bcg of total RNA into cDNA . The cDNp < 0.05 were considered statistically significant.Data are provided as mean \u00b1 standard deviation. All experiments were repeated three times. All statistical analyses were performed using SPSS 22.0  software. Significant differences between groups were assessed using one-way ANOVA and LSD post hoc analysis . Values L. mesenteroides LVBH107 survived and exhibited high growth activity after culture in 0.3% pepsin (pH 5.0) and bovine bile salts (0.3% and 0.5%) for 20 h, while in 0.3% pepsin at pH 2.0 and 3.0, the organisms grew slowly and survived only for as long as 10 h. In addition, L. mesenteroides LVBH107 .In order to function within the oral environment of the human host, probiotic organisms must possess strong tolerance to harsh conditions found there in order to remain viable and carry out activities that benefit human health.  LVBH107 B exhibitL. mesenteroides LVBH107 auto-aggregation (5.80%) was observed  revealed a highly organized and uneven biofilm structure containing a large number of P. gingivalis organisms  and decreased numbers of cells exhibiting green fluorescence (live bacteria), as shown in P. gingivalis biofilm generally stained red, indicating that most cells had died  and a portion exhibiting green fluorescence , as shown in L. mesenteroides LVBH107 could effectively inhibit P. gingivalis biofilm formation, with the L. mesenteroides LVBH107 culture medium inhibitory effect found to be greater than of the bacteria-free culture supernatant.Adhesion and aggregation of L. mesenteroides LVBH107 when the strain was added to RAW 264.7 macrophages in vitro, we evaluated the viability of RAW 264.7 macrophage-like cells when exposed to different L. mesenteroides LVBH107 concentrations. As shown in L. mesenteroides LVBH107 to RAW 264.7 cells were associated with obvious greater RAW 264.7 cell viability (to varying degrees) as compared to the control group without observed cytotoxic effects, with cell viability reaching 103.76\u2013126.45% after 6 h co-culture with L. mesenteroides LVBH107. Importantly, the addition of live L. mesenteroides LVBH107 to RAW 264.7 cells achieved a significantly greater effect than was achieved by the addition of heat-killed bacteria. By contrast, under the same conditions as mentioned above, the addition of 9 Log CFU/mL bacteria to RAW 264.7 cells led to decreases in RAW 264.7 cell viability and proliferation. These results suggest that the cytotoxic effect induced by the addition of 9 Log CFU/mL L. mesenteroides LVBH107 resulted in a partial cell death that ultimately led to reduced viable cell numbers. Therefore, 7 Log CFU/mL and 8 Log CFU/mL concentrations of both live and heat-killed L. mesenteroides LVBH107 were used in subsequent experiments.In order to assess potential cytotoxic effects of P. gingivalis LPS for 24 h clearly stimulated the cells and led to increased levels of proinflammatory cytokines . However, the treatment of RAW 264.7 cells with L. mesenteroides LVBH107 suppressed levels of secreted TNF-\u03b1, IL-6, and IL-1\u03b2 to varying degrees after P. gingivalis LPS stimulated  of L. mesenteroides LVBH107 were significantly reduced after P. gingivalis LPS stimulation, while the exposure of these RAW 264.7 cells to heat-killed L. mesenteroides LVBH107 led to a reduced inhibition of proinflammatory cytokine secretion than was observed after addition of live L. mesenteroides LVBH107 to RAW 264.7 cells.As expected, the exposure of RAW 264.7 cells to imulated A\u2013C, thusP. gingivalis LPS stimulation also significantly increased the secretion levels of inflammatory mediators PGE2 and NO in macrophage RAW264.7  were up-regulated by LPS stimulation, the treatment of RAW 264.7 cells with L. mesenteroides LVBH107 led to relative down-regulated expression of these cytokines  and Lactobacillus fermentans (L. fermentans) could survive under conditions with low pH, bile salts, and lysozyme.As a probiotic that must exert its beneficial activity within the oral cavity, lactobacillus must be able to survive within the harsh oral environment and adhere to oral mucosal cells for a certain period of time. Indeed, our results demonstrated that olerance A in addilysozyme B while ml et al. ,35, indiL. mesenteroides LVBH107 was confirmed here to possess an auto-aggregation ability  [L. mesenteroides LVBH107, through auto-aggregation and co-aggregation activities, may form a barrier that protects the host epithelium by blocking binding of potential pathogens to host cell receptors.Importantly, a previous study demonstrated that lactobacillus strains with strong auto-aggregation ability could colonize mucosal surfaces and consequently carry out their probiotic functions better than strains without this ability . Notably ability C that gral cells ,37. In ttococcus  and resutococcus . In addiG (6.1%) . MeanwhiG (6.1%) , with stG (6.1%) . TherefoL. mesenteroides LVBH107 possessed good biosafety characteristics (lacked antibiotic resistance plasmids), as reflected by its sensitivity to erythromycin, doxycycline, minocycline, ampicillin, chloramphenicol, and cefuroxime. However, here, we must note that tolerance to antibiotics can differ depending on species of lactobacillus under study and even among isolates of a given strain obtained from different sources. For example, Tulumoglu et al. [L. plantarum isolated from traditional cured meat in Tunisia revealed that most of them were resistant to rifampicin [L. plantarum isolates obtained from traditional Marseille dairy products were sensitive to penicillin [Due to the fact that antibiotic genes encoded by plasmids can be transferred among bacterial strains , this acu et al.  reportedfampicin . Moreovenicillin , a resulP. gingivalis, the main pathogenic bacterial species that has been reported to be associated with chronic periodontitis, has been detected at a high rate (60.9%) within subgingival tissues of patients with periodontitis [P. gingivalis and subsequent dental plaque formation are main pathogenic factors associated with periodontitis. Dental plaque, a type of bacterial biofilm, is a soft, nonmineralized bacterially derived material that sticks to teeth and covers tooth surfaces. Importantly, dental plaque provides ideal attachment sites that support the colonization and growth of oral pathogens that play key roles in the pathogenesis of periodontitis. Therefore, inhibiting dental plaque biofilm formation is an important way to prevent periodontitis. In the present study, L. mesenterica LVBH107 significantly inhibited formation of P. gingivalis biofilm . Notably, biofilm damage increased after the addition of cell-free L. mesenterica LVBH107 culture supernatant to P. gingivalis cultures, with some P. gingivalis organisms found to be dead (as detected based on red fluorescence) and some found to be alive (as detected based on green fluorescence). Previously reported observations indicate the protection of microorganisms within biofilm due to the encapsulation of organisms by LPS, peptidoglycan, gingival protease, and flagellin that were produced via plaque biofilm metabolic activities [L. mesenterica LVBH107 may inhibit growth of P. gingivalis and biofilm formation by inhibiting P. gingivalis activities within the biofilm, which ultimately reduces the production of extracellular products that support P. gingivalis survival within the oral microenvironment.Biofilm activities can be observed quickly and intuitively using confocal laser scanning microscopy (CLSM) and fluorescent staining. Two commonly used fluorescent stains include SYTO9, which can enter all bacterial cells to produce green fluorescence, and propidium iodide (PI), which can only enter bacterial cells with damaged cell membranes to produce red fluorescence. Here, CLSM results revealed that tivities . Taken t2), reactive oxygen species, proinflammatory cytokines , and cell chemokines [2, iNOS, and COX-2 is regulated by upstream enzymes, with nitric oxide synthase (iNOS) acting as the rate-limiting enzyme that controls the NO synthesis pathway. In fact, the triggering of iNOS synthesis by inflammatory processes induces high-level expression and the release of large amounts of NO that, in turn, can combine with superoxide anion to form nitroso anions that play crucial roles in killing pathogenic microorganisms and tumors [2) that participate in the inflammatory response [2 dilates blood vessels and lowers blood pressure, leading to up-regulated expression of other pro-inflammatory mediators (including cytokines) that may cause severe cell damage resulting from inflammatory disease processes [2 levels in P. gingivalis LPS-stimulated RAW 264.7 cells that were both decreased after treated with L. mesenterica LVBH107 due to L. mesenterica LVBH107 inhibition of mRNA-level expression of iNOS and COX-2. This result aligned with results of similar studies showing that heat-inactivated Weissella cibaria JW15 and L. brevis K65 bacteria could inhibit production of NO and PGE2 by down-regulating iNOS and COX-2 mRNA-level expression [Macrophages are important immune system cells that play key roles in various inflammatory processes . During emokines  that supemokines . Importad tumors . Meanwhiresponse . PGE2 dirocesses . Here, apression ,59. HoweP. gingivalis LPS activated the macrophage inflammatory response and significantly increased mRNA-level expression of pro-inflammatory cytokines TNF-\u03b1, IL-6, and IL-1\u03b2. By contrast, treated with L. mesenterica LVBH107 bacteria attenuated the RAW 264.7 cell inflammatory state by inhibiting mRNA-level expression of TNF-\u03b1, IL-6, and IL-1\u03b2 to reduce the production of these pro-inflammatory factors. Therefore, these findings collectively suggest that L. mesenterica LVBH107 may function as an anti-inflammatory probiotic, as consistent with results of numerous other studies demonstrating anti-inflammatory effects of other probiotics, such as Bifidobacterium, Lactobacillus paracei, and Lactobacillus swiss, that inhibited the expression and/or secretion of pro-inflammatory cytokines TNF-\u03b1, IL-6, and IL-1\u03b2 [L. mesenterica LVBH107 produced an extracellular metabolite that exerted an anti-inflammatory effect that inhibited TNF-\u03b1, IL-1\u03b2, and IL-8 production by specifically targeting an anti-inflammatory molecular pathway. Notably, a similar mechanism was reported previously for L. mesenteroides exopolysaccharides S81 and BioE-LMD18 [Pro-inflammatory cytokines mainly include TNF-\u03b1, IL-6, and IL-1\u03b2, which are produced by macrophages and endothelial cells. IL-1\u03b2 and TNF-\u03b1 promote inflammatory cell aggregation and activation, stimulate the release of inflammatory mediators, stimulate fever production, and exacerbate inflammatory responses ,61. IL-6nd IL-1\u03b2 ,64,65. WoE-LMD18 ,67, whicL. mesenterica LVBH107, a probiotic isolated from a traditional fermented food (spicy cabbage). This biosafe strain can survive in extreme environments, such as low-pH, bile salts, hydrogen peroxide, and lysozyme; at the same time, it has auto-aggregation, co-aggregation, and adhesion capacity abilities that encourage L. mesenterica LVBH107 adhesion and colonization of the oral environment. In vitro studies have shown that L. mesenterica LVBH107 effectively inhibited P. gingivalis biofilm formation, as well as bacterial activities within established biofilm. In addition, L. mesenterica LVBH107 attenuated the inflammatory response of P. gingivalis LPS-stimulated RAW 264.7 cells and reduced both expression and secretion of inflammatory mediators and pro-inflammatory factors, thus exhibiting anti-inflammatory potential (L. mesenterica LVBH107 could serve as a highly promising probiotic with beneficial antibacterial and immunomodulatory activities, which could be one candidate for doctors in the treatment of periodontal inflammation and protecting teeth far from periodontal pathogen infection.In summary, results of this work demonstrated antibacterial and anti-inflammatory effects of otential . Taken t"}
+{"text": "COX2 were analyzed by real-time quantitative PCR (RT-qPCR); cell viability was measured by cell counting kit-8 (CCK-8) assay; and apoptosis was detected by flow cytometry. In addition, the release of cytokines was determined by Enzyme-linked immunosorbent assay (ELISA), and the binding sites between miR-367-5p and circ_0000080/COX2 were predicted by bioinformatics analysis and confirmed by dual-luciferase reporter and RNA pull-down assays. circ_0000080 was upregulated in patients with MI and in H9c2 cells treated with H2O2 and hypoxia/reoxygenation (H/R). Silencing circ_0000080 reduced the H/R-mediated apoptosis of cardiomyocytes and secretion of pro-inflammatory cytokines. Moreover, circ_0000080 functioned as an miR-367-5p sponge to regulate the expression of COX2. Downregulated miR-367-5p or overexpressed COX2 degraded cellular functions of cardiomyocytes. circ_0000080 knockdown alleviated myocardial hypoxia injury through the miR-367-5p/COX2 axis.This study aimed to investigate the potential role of circRNA circ_0000080 in myocardial hypoxia injury and the underlying mechanisms. Patients with myocardial hypoxia injury who were admitted to Xi\u2019an No. 1 Hospital, China, were included in this study. The expression levels of circ_0000080, miR-367-5p, and   MyocaCompared to traditional linear RNA, circRNA, with a closed circular structure, is more stable ,7. CircuMicroRNAs are a class of non-coding single-stranded RNA, approximately 21\u201323 nucleotides long ,14. MiR-COX2 axis on myocardial viability, apoptosis, and inflammation. Overall, this study provides an important theoretical basis for the development of therapeutic targets for treating MI.Here, we aimed to reveal the protective effect of circ_0000080 on oxidative stress injury in myocardial cells induced by hypoxia/reoxygenation. We try to elucidate the effect of circ_0000080/miR-367-5p/A total of 38 healthy heart tissues and 45 heart tissues from patients with MI were obtained. The human heart tissue samples were obtained from patients with their consent and their use for research was approved by the ethical review board of Xi\u2019an No. 1 Hospital (ethics number: 2021JM-587).2.Rat embryonic cardiomyocyte H9c2 was purchased from Shanghai Cell Bank of Chinese Academy of Sciences . The cells were cultured in Dulbecco\u2019s modified Eagle medium (DMEM) in an incubator at 37\u00b0C, with saturated humidity and 5% CO2 cells exceeded 90%, the supernatant of the culture medium was replaced by DMEM without fetal bovine serum. Thereafter, H9c2 cells were placed in an incubator (5% CO2 and 95% N2) for 4 h, and then cultured in DMEM containing 10% glycerol (5% CO2 and 95% O2) for 3\u00a0h, followed by 24\u00a0h of oxygen.When the growth density of H9CCardiomyocytes were seeded in a 6-well plate. According to the operating instructions, transfection was performed when the degree of cell fusion exceeded 60%. Briefly, Lipofectamine 2000 and plasmid were diluted in Opti-MEM, respectively. After 5\u00a0min, the diluted Lipofectamine 2000 and plasmid were mixed evenly. The transfection complex was then added to the 6-well plate and replaced with fresh medium after 4\u20136\u00a0h of transfection. The transfection plasmid used in this study was constructed by Tsingke Biotechnology . SiRNA was transfected with Lipofectamine 2000. The sequences of siRNAs were as follows: si-NC: 5\u2019-AGTGGGTCTACGGCGATA-3\u2019, si-circ_0000080 1#: 5\u2019-GTTTGGAGGAACTCAACCCTA-3\u2019, and si-circ_0000080 2#: 5\u2019-TTTGGAGGAACTCAACCCTAT-3\u2019.GAPDH, as a reference, the relative content of the target sample gene was calculated. Commercial TaqMan primers for qPCR were purchased from Life Technologies, USA. The primers were: circ_0000080\u00a0F: 5\u2019-GAGGAACACTCCATATAATTGGTGA-3\u2019; R: 5\u2019-TCTGTTTTTCTTTGAAGGGCTACCT-3\u2019, miR-367-5p F: 5\u2019-TGCGGACTGTTGCTAATATG-3\u2019; R: 5\u2019-CCAGTGCAGGGTCCGAGGT-3\u2019, COX2 F: 5\u2019-TTCAAATGAGATTGTGGGAAAAT-3\u2019; R: 5\u2019-AGATCATCTCTGCCTGAGTATCTT-3\u2019.For fluorescent probe PCR, the following components were added to a 20\u00a0\u03bcL PCR mixture : 10\u00a0\u03bcL TaqMan Gene Expression PCR Master Mix, 1\u00a0\u03bcL template DNA, 1\u00a0\u03bcL 20\u00d7\u00a0Prime Probe mixture, 8\u00a0\u03bcL DNase-free water, and 9.4\u00a0\u03bcL DNase-free water. The following cycling conditions were used for PCR: 95\u00b0C 10\u00a0min; 95\u00b0C 15s, 60\u00b0C 30s, 68\u00b0C 30s, 40 cycles. Using the internal reference gene, After H9c2 cells were transfected as indicated and treated with hypoxia/reoxygenation (H/R), 10\u00a0\u03bcL CCK-8  was added to each well. After 2 h, the absorbance of each sample was measured at 450\u00a0nm according to the kit instruction.Flow cytometry was performed according to a previous study . After HCells were lysed using RIPA buffer. After detecting protein concentration using BCA kit , equal protein was added and run on 10% SDS-PAGE. Then the protein was transferring to PVDF membranes, followed by blocked with 5% skim milk for 1\u00a0h. The membranes were incubated with primary antibodies  overnight at 4\u00b0C, and incubated with secondary antibody  at room temperature for 1\u00a0h. The bands were visualized using ECL plus . The original bands were provided in supplemental material 1.After H9c2 cells were transfected as indicated and treated with H/R, the cell culture supernatant was collected and the concentrations of IL-6, TNF-\u03b1, and IL-1\u03b2 were detected according to the instructions of commercial kits . ELISA was performed as previous described .COX2-wt or pMIR-COX2-mut was inserted by COX2 cDNA or mutant COX2. When the growth density of H9C2 cells exceeded 90%, cells were cultured and co-transfected with pMIR-circ_0000080-wt or pMIR-COX2-wt, pMIR-circ_0000080-mut or pMIR-COX2-mut, miR-367-5p mimics, and miR-NC, respectively. After 48\u00a0h, luciferase was detected with the dual-luciferase reporter gene assay system  as instructed.Target genes were predicted by Starbase v3.0. PMIR-circ_0000080-wt was formed by cloning circ_0000080 cDNA into the pMIR-Vector; pMIR-circ_0000080-mut was inserted by the mutant circ_0000080. The cloned sequences were provided in supplemental material 2. Similarly, pMIR-COX2 in cells was determined by an RNA pull-down assay [The interaction between circ_0000080 and miR-367-5p or the interaction between miR-367-5p and wn assay . Biotin SPSS 25.0 statistical software was used for data analysis, and the data are expressed as mean \u00b1 SEM. One-way ANOVA was used for comparison among multiple groups, and the LSD test was used for pairwise comparison. P <\u00a00.05 was considered statistically significant.In this study, we sought to explore the expression of circ_0000080 in MI and its role in H/R-treated H9c2 cells. We analyzed the effects of circ_0000080 on cell viability, apoptosis, and inflammation. The goal of this study is to provides an important theoretical basis for the development of therapeutic targets for treating MI.This experiment was conducted to detect the expression level of circ_0000080 between MI patients and healthy individuals (controls) to understand the role of circ_0000080 in the heart. The qRT-PCR results revealed that the expression level of circ_0000080 was significantly elevated in MI patients than in healthy individuals ). The coThe expression level of circ_0000080 was markedly reduced by si-circ_0000080 1# compared to si-circ_0000080 2#, while si-circ_0000080 1# did not affect the linear GAPDH expression level ). Such fInflammation after MI is necessary for heart repair; however, inflammation is also involved in the pathophysiological processes, such as heart remodeling and heart failure after MI . As showBased on increasing evidence, circRNA acts as a miRNA sponge ,21. The A rescue experiment was conducted to explore the relationship between circ_0000080 and miR-367-5p in H/R treated H9c2 cells. Based on qPCR, the miR-367-5p inhibitor significantly reduced the expression level of miR-367-5p, while the miR-367-5p mimic markedly increased the expression level of miR-367-5p ). The CCTo determine the effects of miR-367-5p in H/R-treated H9c2 cells, an experiment was conducted to further assess the effects of miR-367-5p on the secretion of inflammatory factors in H9c2 cells in vitro. Knockdown of circ_0000080 reduced the release of inflammatory cytokines including IL-1\u03b2, IL-6, and TNF-\u03b1, and the miR-367-5p inhibitor was found to increase the secretion of IL-1\u03b2, IL-6, and TNF-\u03b1 ).FigureCOX2 are shown in COX2-wt. Further, the RNA pull-down assay suggested that biotin-miR-367-5p could notably pull down more circ_0000080 . The mRcontrols ), and thcontrols ). Besidec2 cells ) but wasc2 cells ).FigureCOX2 in H/R-treated H9c2 cells. The transfection efficiency of COX2 is shown in COX2 overexpression (COX2 overexpression promoted apoptosis in H9c2 cells (A rescue experiment was performed to confirm the relationship between miR-367-5p and pression ). Moreovc2 cells ).FigureCOX2 significantly increased the release of inflammatory cytokines, such as IL-1\u03b2, IL-6, and TNF-\u03b1, as shown in The overexpression of COX2. MiR-367-5p knockdown or COX2 overexpression inhibited the role of circ_0000080 in MI.The cardiovascular system is the engine of life. Severe cardiovascular disease is a potentially fatal disease ,23. AlthApoptosis is a form of programmed cell death; it affects cardiomyocytes during MI . The incWe proceeded to explore the mechanism of circ_0000080 in MI. Most circRNAs play regulatory roles in MI by targeting downstream genes ,31. Liu COX2 knockdown may increase the risk of MI [COX2 in H9c2 cells, and the overexpression of COX2 reversed the effect of miR-367-5p in cells. Therefore, circ_0000080 may weaken the targeted inhibitory effect of miR-367-5p on COX2 by binding to miR-367-5p, thereby inhibiting the progression of MI. The circ_0000080/miR-367-5p/COX2 axis may provide a new therapeutic option for MI.To further study the mechanism of miR-367-5p\u2019s biological function in MI, we investigated the downstream target gene of miR-367-5p. Zhang et al. found that sk of MI . Herein,COX2 axis in MI needs to be confirmed using several clinical studies, which will be the focus and a challenge of future research. Additionally, only the roles of miR-367-5p and COX2 were explored in this study. whether other miRNAs and genes influence circ_0000080-mediated regulation in MI still needs to be elucidated.The main limitation of this study is that the role of the circ_0000080/miR-367-5p/COX2 signal axis was found to promote proliferation and impair the apoptosis of H/R treated cells. This study not only revealed the novel roles of the circ_0000080/miR-367-5p/COX2 axis in MI, but also provided a new target for the management of MI.In summary, the circ_0000080/miR-367-5p/Click here for additional data file."}
+{"text": "NAC genes in poplar, the functional characterization of drought-related NAC genes have been scarcely reported in Populus species. Here, we identified a total of 170 NAC domain-containing genes in the P. trichocarpa genome, 169 of which were unevenly distributed on its nineteen chromosomes. These NAC genes were phylogenetically divided into twenty subgroups, some of which exhibited a similar pattern of exon\u2013intron architecture. The synteny and Ka/Ks analysis indicated that the expansion of NAC genes in poplar was mainly due to gene duplication events occurring before and after the divergence of Populus and Salix. Ten PdNAC (P. deltoids \u00d7 P. euramericana cv.\u2019Nanlin895\u2019) genes were randomly selected and cloned. Their drought-responsive expression profiles showed a tissue-specific pattern. The transcription factor PdNAC013 was verified to be localized in the nucleus. Our research results provide genomic information for the expansion of NAC genes in the poplar genome, and for further characterizing putative poplar NAC genes associated with water-deficit.The NAC  is a large gene family of plant-specific transcription factors that play a pivotal role in various physiological processes and abiotic stresses. Due to the lack of genome-wide characterization, intraspecific and interspecific synteny, and drought-responsive expression pattern of  Petunia NAM [Arabidopsis ATAF1/2 and CUC2 [cis-element at their promoters [NAC  gene, which encodes an important regulator in ABA-dependent signaling pathway [Arabidopsis VND (vascular related NAC-domain) genes, including VND6 and VND7, are involved in xylem differentiation and cell fiber differentiation [Physcomitrella patens are related to water transport in cellular tissues [CarNAC3 gene from Cicer arietinum, can lead to an increase in proline content and antioxidant enzyme activities, and a decrease in MDA  concentration of transgenic poplar NL895 (Populus deltoides \u00d7 P. euramericana cv. \u2032Nanlin895\u2019) [PeNAC034, PeNAC045, PeNAC036 and PeNAC122 genes of P. euphratica, have been functionally characterized to date [Identification of NAC TFs at the genome-wide level has been documented in the genomic sequences of angiosperm species, such as 75 in a sativa , 105 in thaliana , 74 in Vvinifera , 189 in persicum . Meanwhiin yield . ANAC016 pathway . Two Arantiation . The NAC tissues . Overexp to date ,18.Populus species, as a settled and woody perennial species, may have evolved a sophisticated regulatory mechanism by which genes are regulated in response to water deficit occurring over the poplar lifetime. However, the regulating role of NAC TFs in poplar in response to drought stress remains largely unknown. This is, in part, due to the lack of the basic information of poplar NAC TFs, such as their coding sequence, physical and chemical properties, subcellular localization, drought stress-responsive expression profiling, and divergent evolutionary pattern.The frequency of extreme meteorological events and the change of rainfall pattern aggravate the degree of drought at a regional scale . Severe P. trichocarpa genome were identified, in which comprehensive bioinformatics analyses were performed, including the physicochemical attribution, chromosomal distribution, phylogenetic classification, and intraspecific and interspecific collinearity detection. Ten poplar NAC genes were randomly selected and cloned from the genome of the poplar clone NL895. Their drought-responsive expression patterns were analyzed in three tissues  of poplar NL895 using quantitative real-time PCR (qRT-PCR). Finally, the subcellular localization of PdNAC013 protein, which was encoded by one of the selected ten NAC genes, was verified in poplar NL895 protoplasts.In this research, NAC transcription factors in the whole P. trichocarpa genome. These transcripts were transcribed from 170 gene loci of the P. trichocarpa genome. The 170 NAC genes were reassigned as PtrNAC001-PtrNAC170 according to their corresponding chromosome number and physical location. Only one (PtrNAC170) of all PtrNAC genes was not located on any of the nineteen P. trichocarpa chromosomes.Here, we identified a total of 289 putative NAC (Pfam No.PF02365) domain-containing transcripts located in the entire PtrNAC genes had an average GC content of 43.89%, with a relatively wide range of from 37.84% (PtrNAC120) to 52.44% (PtrNAC055). Detailed information on these data was summarized in The PtrNAC proteins ranged from 122 amino acid (aa) (PtrNAC125) to 1524 aa (PtrNAC170) in size, with an average length of 350.71 aa. Their corresponding molecular weights were between 14.84 kDa (PtrNAC125) and 174.42 kDa (PtrNAC170). Their predicted pI (isoelectric point) varied from 4.28 (PtrNAC138) to 10.73 (PtrNAC021). The coding sequences of these PtrNAC genes, 169 were located on all nineteen chromosomes of P. trichocarpa. The 169 PtrNAC genes shared an uneven chromosomal distribution  to sixteen (Chr01). The average number of PtrNACs per 10 Mb (megabase pair) also differed among nineteen chromosomes in P. trichocarpa, ranging from 2.36 (Chr18) to 6.87 (Chr14). It seemed obvious that PtrNAC genes were not evenly distributed over a few chromosomes. Three NAC gene-rich clusters were found in Chr02 (PtrNAC025-PtrNAC029), Chr06 (PtrNAC060-PtrNAC067) and Chr14 (PtrNAC129-PtrNAC134). In addition, there were eight tandem duplicated gene pairs distributed on eight chromosomes, including Chr01 (PtrNAC011 and PtrNAC012), Chr02 (PtrNAC022 and PtrNAC023), Chr06 (PtrNAC063 and PtrNAC064), Chr09 (PtrNAC089 and PtrNAC090), Chr12 (PtrNAC112 PtrNAC113), Chr14 (PtrNAC132 and PtrNAC133), Chr16 (PtrNAC146 and PtrNAC147) andand Chr19 (PtrNAC168 and PtrNAC169).Of all 170 ribution . The totPtrNAC genes during evolutionary innovation. A total of 117 PtrNAC genes were identified to be derived from WGD events, suggesting that WGD contributed mainly to the expansion of the NAC gene family in poplar.Gene duplication events, such as tandem duplication and whole-genome duplication (WGD), were considered as a common process during plant evolutionary process, and could lead to the expansion of multigene families . In ordePtrNAC gene pairs were less than 1, demonstrating that PtrNAC members had experienced purifying selective pressure and their functions might be conserved after the expansion of NAC genes in poplar [PtrNAC pairs could be estimated based on Ks-based distribution. Their divergence times ranged from 9.5 to 208.09 million years ago (Mya). The divergence times for 55 out of all 90 collinear gene pairs were less than 31 Mya. This showed that the expansion of PtrNACs occurred after and before the split between Populus and Salix (45 Mya) [The chromosomal synteny analysis discovered a total of 90 collinear gene pairs . The synn poplar ,24. The (45 Mya) .P. trichocarpa and four other plants, including Salix purpurea, Eucalyptus grandis, Vitis vinifera and Arabidopsis thaliana  tree. These subfamilies were designated alphabetically as A through T. The number of PtrNAC genes in these subfamilies were between two (subfamily D) and twenty-two (subfamily A) (To explore the evolutionary relationships of amily A) . MoreovePtrNAC genes, we investigated the exon\u2013intron patterns of PtrNAC genes (PtrNAC genes were transcribed at the forward (+) and reverse (\u2212) strand of all P. trchocarpa chromosomes. Exons or introns were unequally distributed over PtrNAC genes, but most related members of the same subfamily had similar intron/exon structures. For example, all PtrNAC genes contained introns except for all members belonging to the subfamily G, five genes  belonging to the subfamily H, and PtrNAC136 in the subfamily R. In contrast, each of the six subfamilies  shared three exons pattern. The exons in the PtrNAC genes had an average number of 3.34. Two PtrNAC genes, including PtrNAC083 and PtrNAC107, possessed the maximum number of eight exons.To further understand the characteristics of the AC genes . A totalcis-elements in the upstream region of 289 PtrNAC transcripts  and G-box) [cis-elements belonging to stress-responsive element were consisted of STRE (stress response element), TC-rich repeat, MYB and MYB-like sequence. A large proportion of PtrNACs contained MYB binding sites in their promoter region, suggesting that MYB might be a major kind of transcription factors binding to PtrNACs [PtrNAC genes were implicated in abiotic stress response , and plant growth and development.Transcription factors have a close association with the regulation of gene expression via binding the promotor region of their target genes . Here wenscripts . Cis-actd G-box) , ethylend G-box) . ABA andd G-box) . The cis PtrNACs . This rePtrNAC genes under drought stress, ten PtrNAC genes were randomly selected for cloning these genes from the NL895 poplar. They were named according to the homology with their corresponding genes in P. trichocarpa. The NAC gene sequences of the NL895 poplar were compared with that of P. trichocarpa and I-69 poplar (Populus deltoides bartr. cv. \u2018Lux\u2019). Each of these genes shared a pairwise sequence identity of nearly 99% between the three Populus sepcies.Based on the RNA-seq profiling of PtrNAC genes in different tissues of P. trichocarpa. The majority of NAC genes were induced by drought stress in poplar. Among them, ten genes were selected for cloning genes from the NL895 poplar, and their drought-responsive expression pattern in P. trichocarpa had been shown in PtrNAC genes could be a marked difference among three tissues, including roots, stems and leaves.The external environment stimulus had a major influence on plant growth and development, and the plant could respond to environmental stress by regulating crucial molecular processes. The RNA-seq profiling data provided a drought-responsive expression pattern of PdNAC genes were implicated in poplar responses to drought, we adopted qRT-PCR to analyze their tissue-specific patterns under drought stress. As shown in PdNACs changed after 3% (w/v) PEG6000 treatment. The results shown that the drought-responsive expression patterns of a few PdNACs were the difference among the leaf, root and stem tissues. In the leaf tissues, drought-induced expression of all but PdNAC027 genes shared a single-pulse pattern. The expression abundance of PdNAC013, PdNAC86 and PdNAC105 genes reached the peak at 72 h after PEG6000 treatment. The expression levels of PdNAC013 and PdNAC105 genes increased by nearly 85-fold and 20-fold at 72 h compared to control group (0 h), respectively. The highest expression level of PdNAC049, PdNAC55 and PdNAC78 genes occurred at 24 h after PEG6000 treatment. In the root tissues, drought-induced expression of all but PdNAC055 genes could be clustered into three patterns, including single-peak , double-peak  and triple-peak (PdNAC078) pattern. In the stem tissues, only three genes  exhibited a single-pulse pattern in response to drought stress, and genes  showed a double-peak pattern. In short, transcriptional time series of some PdNAC genes under PEG6000 treatment might have an impulse pattern [Thus, to investigate whether the ten  pattern .PdNAC013 constructed in protoplasts of NL895 poplar, with expressing a red nuclear marker D53-mCherry protein [PdNAC013 fusion was observed microscopically only to localize in the nucleus with the red nuclear marker D53-mCherry protein  [A. thaliana (105) [V. vinifera (74) [S. lycopersicum (93) [P. trichocarpa. The intraspecific and interspecific collinearity analysis suggested that the expansion of NAC genes in poplar was caused mainly by WGD events. Recent studies also suggest that WGD event is a major driving force for expansion of multigene family in plant genome [Pyrus pyrifolia [Zea mays [PtrNACs showed diverse expression, indicating that modifications, such as point mutations, might occur in regulatory regions of duplicated genes, which affected the function and expression patterns [In this study, we identified a total of 170 iva (75) , A. thalna (105) , V. viniera (74)  and S. lcum (93) . It was t genome ,35,36. Tyrifolia  and Zea Zea mays . In addipatterns ,40.Populus and Salix are two sister lineages which shared common WGD events  happening in approximately 58 Mya [P. trichocarpa and S. purpurea were more than twice those of between P. trichocarpa and the remaining three species . Likewise, the analysis of Ks-based distribution showed that the divergence times for more than half of homologous PtrNAC pairs were less than 31 Mya. These results suggested that the expansion of PtrNACs occurred after and before the separation time of Populus and Salix (45 Mya) [PtrNACs arisen after the divergence of Populus and Salix play a role in the adaptation of Populus species to their local environment  [y 58 Mya ,42. This(45 Mya) . Additiond heat) ,44.PtrNACs under drought treatment, ten NAC (PdNAC) genes were randomly selected and cloned from P. deltoids \u00d7 P. euramericana cv.\u2019NL895\u2019. The promoter region of these NAC genes contained many cis-acting elements related to drought stress, such as ABRE, DRE and G-box binding sites [cis-acting elements in the downstream promoter region of drought-related genes, thereby activating the expression of this gene and improving drought resistance in plants [cis-acting ABA-responsive element (ABRE elements) of TaSNAC8-6A and TaNAC48 promoter and activated their expression in wheat, thus enhancing drought tolerance of plants [ONAC066 could bind the JBS-like (JBSL) cis-element in the promoter region of OsDREB2A gene to positively regulate drought and oxidative stress response [Based on the RNA-seq profile of ng sites ,46,47. Pn plants ,49,50. Ff plants ,52. ONACresponse .PdNAC genes in three tissues of poplar NL895. Six PdNAC genes had sequence similarity to ATAF1 (ANAC002) and RD26 (ANAC072) genes, which were found to be drought-responsive NAC genes in A. thaliana [ATAF1 is an Arabidopsis NAC transcription factor that regulates ABA synthesis [OsNAC6, a member of ATAF family, can mediate the adaptation of plant root structure and up-regulate the expression of drought-resistant genes, which enhances the drought tolerance of rice plants [PdNAC055 (Potri.005G180200), PdNAC021 (Potri.002G081000), PdNAC049 (Potri.005G069500) and PdNAC078 (Potri.007G099400), shared high sequence similarity to ATAF1 from A. thaliana. It seemed obvious that the expression levels of the four PdNAC genes were induced by drought stress. PagNAC045 (Potri.007G099400), which was isolated from P. alba \u00d7 P. glandulosa cv.84K, might be implicated with NaCl and ABA-mediated stresses [PdNAC genes were implicated with ABA remains unknown.Therefore, we analyzed the drought-responsive expression patterns of ten thaliana . ATAF1 iynthesis ,56. OsNAe plants ,58,59. Fstresses . HoweverANAC072 /RD26 is a positive regulator of ABA and drought stress [ANAC072 /RD26 can up-regulated under drought and salt stress, and the plants showed obvious drought resistance [PdNAC105|PtrNAC105 (Potri.011G123300) and PdNAC013|PtrNAC013 (Potri.001G404100) were two highly homologous sequences of ANAC072 /RD26. PtrNAC013 gene is overexpressed in poplar 84k , and the overexpressed plants had better salt tolerance than the wild type [PdNAC013 and PdNAC105 had significant changes in gene expression levels in leaves, roots and stems under PEG-stress treatment. PdNAC013 shared a drought-responsive single-impulse pattern in the three tissues, which reached its peak at 72 h after PEG6000 treatment. PdNAC013 protein was localized in the nucleus in the protoplasts of NL895 poplar, which was consistent with the subcellular localization of PtrNAC013 gene in tobacco [PdNAC013 and PdNAC105 may play an important role in drought stress.t stress . The gensistance ,62. PdNAild type . PdNAC01 tobacco . In concPdNAC genes were induced by drought, and might be implicated in poplar response to drought. However, the regulatory role of NAC genes in drought response in Populus species remains complex and elusive. For example, almost all PtrNAC genes had MYB binding sites (MBS) in their upstream regions, suggesting that MBS elements of a few PtrNAC genes might be bound by PtrMYB proteins. In wheat, TaMYBL1 protein can bind to two MBS cis-acting elements inserted in the promoter region of a drought-related gene TaNAC071-A, causing an increase in its expression level and drought tolerance in plant [cis-acting elements and trans-acting factors, transcriptional activation domain and DNA-binding domain of poplar NAC genes associated with drought stress.The study indicated that a few in plant . Thus, wP. trichocarpa (v3.0), Salix purpurea (v5.1), Eucalyptus grandis (v2.0) and Vitis vinifera L (v2.1) were downloaded from the Phytozome database (https://phytozome.jgi.doe.gov/ (accessed on 2 March 2022)). The GFF/GTF, transcript and protein files for Arabidopsis thaliana were retrieved from the EnsemblPlant release 53 (https://plants.ensembl.org/ (accessed on 2 March 2022)). The A. thaliana NAC (AthNAC) sequences were searched and acquired from the Arabidopsis information resource ).Genomic sequence (transcript and protein sequences) and GFF/GTF annotation files of P. trichocarpa NAC (PtrNAC) proteins, the HMM (hidden Markov model) profile of NAM domain (PF02365.15) was searched against all P. trichocarpa proteins by using HMMER (v3.3.2) with an E-value threshold of 1 \u00d7 10\u22123 [\u22125 [https://www.ebi.ac.uk/interpro/about/interproscan/ (accessed on 8 July 2022)) and Pfam (http://pfam.xfam.org/ (accessed on 8 July 2022)) service. The length, GC-content, isoelectric point (pI), and molecular weight (MW) of all PtrNACs were calculated with Biopython (v1.79).To identify the ain PF023.15 was sain PF023.15 was shttp://www.clustal.org/clustal2/ (accessed on 2 March 2022)) with default parameter. The best-fitted substitution model \u2018JTT+F+R7\u2032 was inferred from the PtrNAC MSA file by ModelFinder implemented in IQ-TREE (v1.6.12) based on minimum BIC value [Multiple sequence alignment (MSA) of PtrNAC protein sequences was performed via ClustalW ). Conserved motifs on the PtrNAC proteins were analyzed using MEME ). The MEME parameters were specified as follows: the optimum motif width was between 6 and 50 in length, and the maximum number of motifs was 15.The PtrNACs genome database and gff3 annotation file were used to investigate the exon and intron distributions of the http://cello.life.nctu.edu.tw/ (accessed on 5 May 2022)), WoLFPSORT (https://wolfpsort.hgc.jp/ (accessed on 5 May 2022)) and BUSCA (http://busca.biocomp.unibo.it/ (accessed on 5 May 2022)).The subcellular localization of PtrNAC proteins were predicted using three web tools, including Cello (P. deltoids \u00d7 P. euramericana cv. \u2032Nanlin895\u2032), we did the following experiments. First, the newly expanded leaves were harvested from 8-week-old plantlets, and used for the poplar protoplast isolation with Cellulase RS  and Pectolyase Y23  according to the method of Yoo et al. [PdNAC013::GFP (green fluorescence protein) was constructed according to homologous recombination method. D53-mCherry is a nuclear marker plasmid fused with rice protein D53 [2+ (PEG-4000) mediated transformation [In order to verify the subcellular localization of PdNAC013 (Potri.001G404100.1) in poplar NL895  [P. trichocarpa, S. purpurea, E. grandis, V. vinifera and A. thaliana, was carried out by BLASTp in the BLAST+ (v2.9.0) package. The resulting BLASTp outputs were used to analyze the collinearity of NAC proteins between P. trichocarpa and the other four species, by using Multiple Collinear Scan Toolkit ). The collinearity relationship of between P. trichocarpa and each of the other four species was plotted with JCVI ). To further evaluate the characteristics of the influence of evolutionary on PtrNACs family, a simple Ka/Ks calculator was used to calculate the synonymous (Ks) and non-synonymous (Ka) of PtrNAC gene pairs using ParaAT (v2.0) and KaKs-Calculator (v2.0) [\u22129 [The chromosomal locations of v0.4.15) . Pairwisr (v2.0) . The div2.0) [\u22129 ,73.P. trichocarpa genomic sequence with SeqKit (v0.8.0) [cis-regulatory elements using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 March 2022)).The 2 kb sequence in the front of the PtrNACs start codon (ATG) was obtained from (v0.8.0) . The 2 kP. trichocarpa under drought treatment were derived from NCBI SRA database (BioProject no. PRJEB19784). These RNA-seq data were pre-processed and trimmed with FastQC (v0.11.8) and Trimmomatic (v0.38). The spliced mapping of the trimmed RNA-seq reads against the P. trichocarpa genome (v3.0) was performed using STAR (v2.7.3a) [p-value cutoff of 0.01 [The RNA-seq data for the three tissues  of v2.7.3a) . Differe of 0.01 . The hea(P. deltoids \u00d7 P. euramericana cv. \u2018Nanlin895\u2032) were grown on the 1/2 Murashige and Skoog (MS) medium (pH = 5.8). The poplar NL895 seedlings were cultivated in a greenhouse at a room temperature (23 \u00b0C) and a relative humidity of 74%. They were cultured in 1/2 MS medium for two months and then transferred to 1/2 MS medium with 3% (w/v) PEG6000 for drought treatment. Samples were harvested at 12 h, 24 h, 36 h, 72 h and 168 h (7 days) after 3% PEG6000 treatment, respectively. These samples were immediately frozen in liquid nitrogen and stored at \u221280 \u00b0C for subsequent RNA isolation. The total RNA was extracted from the leaf, stem and root tissues of the poplar NL895 with plant RNA extraction kit , respectively.Plantlets of hybrid poplar NL895 The total RNA was isolated from three vegetative organs  of the poplar NL895 using a plant RNA extraction kit . The concentration and integrity of the extracted RNA was determined using a combination of an ultraviolet-visible (UV-Vis) spectrophotometer  and 1% agarose gel electrophoresis. The extracted RNA was reverse-transcribed into cDNA by a reverse transcription kit . Then the cDNA was diluted 10 times for qRT-PCR experiment.TM 5\u03b1 chemoreceptor cells . Finally, a single colony was screened to verify the size of bands in gel-electrophoresis, and sequenced on an ABI 3730xl DNA analyzer.Prior to qRT-PCR (quantitative reverse transcription PCR), the specific primers were designed with Oligo (v7.37) using poplar CDSs (coding sequences) as reference. The primer sequences were listed in PdNAC genes were designed by Primer Premier (v5.0) to analyze their expression patterns  genes were identified at the entire genome level. The physicochemical property calculating, chromosomal locating, phylogenetic classifying, intraspecific/interspecific collinearity inferencing, and dehydration-responsive expression profiling of these PtrNAC genes were performed. Analysis of cis-acting elements and drought-responsive expression pattern of PtrNACs indicated that these genes might be involved in drought stress response. In addition, ten NAC genes from poplar NL895 were cloned. The PdNAC013 exhibited a tissue-specific and pulsing expression pattern under drought treatment. The subcellular localization analysis suggested that PdNAC013 protein localized in the nucleus. In summary, these findings will provide a valuable clue for further screening and characterization of NAC genes in relation to poplar drought response.In the research, a total of 170"}
+{"text": "RXFP2 and TWIST1. This study identified these changes for the first time and summarized them. The results suggested that the Pc mutant locus may inhibit neural crest cell EMT generation and keratin expression, leading to failures in neural crest cell migration and keratinization of the horn bud tissue, regulating the production of the polled phenotype.The Polled Celtic (Pc) mutation locus is a genetically simple single mutation that is the best choice for breeding polled cattle using gene editing. However, the mechanism of the Pc locus for regulating horn development is unclear, so we used gene editing, somatic cell nuclear transfer and embryo transfer to obtain polled Holstein fetal bovine (gestation time 90 days) with a homozygous Pc insertion  and the wild-type 90 days Holstein fetal bovine (WH) as controls. The hematoxylin-eosin (HE) staining results showed that, compared to the WH, the EH horn buds had no white keratinized projections or vacuolated keratinocytes and no thick nerve bundles under the dermal tissue. Furthermore, DNA sequencing results showed that the Pc locus was homozygously inserted into the fetal bovine genome. A total of 791 differentially expressed genes were identified by transcriptome sequencing analysis. Enrichment analysis and protein interaction analysis results of differentially expressed genes showed that abundant gene changes after Pc insertion were associated with the adhesion molecule regulation, actin expression, cytoskeletal deformation and keratin expression and keratinization. It was also noted that the results contained several genes that had been reported to be associated with the development of horn traits, such as  Growth of horns have been a common vertebrate development, especially for bovines, where they are diverse in form and have medical and application value. However, with the rapid development of farming, horns will cause injury to other cattle and farm staff on intensive farms. The sharpness of the horns can increase the risk of physical damage to vital areas such as the udder, which can reduce the lifetime economic benefit of the cow ,2. In toMedugorac et al. 2012) first identified a single mutation associated with the polled trait in several European beef breeds, such as polled Angus and Galloway cattle [2 first iCarlson et al. in 2016, successfully produced polled calves by editing the Pc locus with Transcription Activator-like (TAL) Effector Nucleases (TALEN). Subsequently, Young et al. selected one of polled calves and crossed it with a horned cow (pp) to obtain six heterozygous (Pcp) polled calves. The Pc allele segregated in the offspring of this bull, and inheritance of either allele produced polled calves ,6. SchusUntil recently, the exact molecular mechanism through which the Pc mutation locus causes the polled trait in cattle was not known . In a prThis laboratory successfully obtained a monoclonal cell line with a Pc locus homozygous insertion in the ear margins of Holstein bull by Tild-CRISPR/Cas9 technology. Both PCR detection and DNA sequencing identified that the Pc locus was homozygous and inserted into the genome of the cell line. The homozygous mutant cell was subsequently used as a donor cell and the donor was transplanted into a stage MII enucleated oocyte by somatic cell nuclear transfer technique to prepare reconstituted embryos and perform electrofusion to blastocysts in In this experiment, the horn buds of EH fetal bovines and WH Holstein fetal bovines obtained by slaughter were identified morphologically and histologically. A distinct white keratinized projection could be observed in the WH horn bud tissue at 90 days by morphological observation, while none was observed in the EH group, seen in To investigate the regulatory mechanisms of bovine horn development, cDNA libraries of WH and EH horn bud tissues were constructed, and raw data subsequently obtained by Illumina high-throughput sequencing, with raw reads ranging from 49,208,676 to 56,395,082 for each sample. After eliminating many low-quality bases, splice-containing, and N-containing reads from the raw data, 56,135,334 (99.54%) to 48,996,840 (99.57%) clean reads were obtained in the WH and EH horn bud tissues and the Q30 was greater than 92.5% in all groups.Bos taurus, UMD 3.1) with more than 46,947,510 (91.5%) clean reads and more than 95.5% of the above reads were uniquely mapped to the bovine reference genome. Based on the expression information, the Pearson correlation coefficient between any two samples was higher than 0.987. The heat map results showed that the inter-group sample reproducibility was good in the WH group as shown in Sequence comparison showed that all groups were localized to the bovine reference genome  were screened in WH and EH as shown in The differential gene screening and analysis were performed by DE-Seq. The differential gene screening criteria were |log2FC| \u2265 1 and The GO enrichment, KEGG pathway and Reactome enrichment analyses were performed to explore the molecular mechanisms of the Pc locus regulating the polled trait in Holstein, as shown in Similarly, KEGG pathway analysis results showed that Focal adhesion (ko04510), ECM-receptor interaction (ko04512) and Calcium signaling pathway (ko04020) were also enriched significantly in http://string-db.org, accessed on 30 August 2022) for differentially expressed genes in In this experiment, protein interaction networks were analyzed using the STRING protein interaction database , laminin subunit gamma 2 (LAMC2), and laminin subunit alpha 5 (LAMA5)  . The promotility . The genigration ,25.L1CAM, LAMC2, and LAMA5, was significantly lower in EH horn buds compared to WH , actinin alpha 2 (ACTN2), actin alpha cardiac muscle 1 (ACTC1), and actin alpha 2, smooth muscle (ACTA2). The ACTA1, ACTC1, and ACTA2 genes encode one of six different actin proteins and Hu et al. demonstrated that overexpression of ACTA1 inhibited cell proliferation and migration [ACTC1 significantly inhibited cell migration [ACTA2 inhibited migration of neural stem cells by increasing Rho A expression and decreasing Rac1 expression to hinder actin filament polymerization [ACTA1, ACTN2, ACTC1, and ACTA2, was down-regulated in EH horn buds . This is a regulator of the interaction between E-cadherin and the cytoskeleton, regulating cytoskeletal remodeling and cell polarization by participating in the linkage between the two and promoting cell motility . The expThe above results suggested that the Pc locus may inhibit the development of EMT in neural crest cells by regulating the expression of cell adhesion molecules, preventing neural crest cells from initiating migration. At the same time, the insertion of the Pc locus may also inhibit the expression of actin and cytoskeletal deformation in neural crest cells, limiting the migration of neural crest cells. The neural crest cells are unable to migrate to the site of the horn bud to proliferate and differentiate into neuronal and mesenchymal cells required for horn development and due to the lack of a material basis for horn development, mutations at the Pc locus led to the creation of the polled trait.Studies of horn development found that keratin may play a key role in it. A brief white keratinized projection appears on the horn bud tissue during development in horned fetal calves and disappears later in development, whereas no keratinized projection appears on the horn bud tissue throughout the development of hornless fetal calves . It suggBased on the above experimental results, it seems that the Pc locus completed the regulation of key events in the formation of the polled trait early in fetal bovine development. The possible regulatory mechanisms include two points. First, the Pc locus may initiate the EMT event by regulating the expression of cell adhesion molecules, actin, and cytoskeletal deformation in neural crest cell migration, resulting in a decrease in the ability of neural crest cells to migrate. As a result, large numbers of neural crest cells are unable to migrate successfully to their designated sites at the horn buds for value-added differentiation. Secondly, the Pc locus may inhibit the expression of keratin at the horn bud and the process of keratinization by keratinocytes, failing to provide the material basis for later horn development. Based on the above process, we have drawn a hypothetical model of Pc regulation, see The donor cells for somatic cell nuclear transplantation were mutant cell lines with homozygous insertion of the Pc locus mediated by Tild-CRISPR/Cas9 technology in this laboratory. In previous experiments, we obtained the above cell lines carrying the Pc variant by the following steps: (1) First, we obtained the ear marginal tissue of a Holstein bull and established cell lines. (2) Second, the sgRNA was designed for the Pc locus controlling the polled in cattle, and its mutation efficiency was detected by T7El digestion and sequencing. (3) Third, the sgRNA was designed for the Pc locus controlling the polled form in cattle, and its mutation efficiency was detected by T7El digestion and sequencing. The PUC57 vector was used as the skeleton to produce homologous recombinant fragment through connecting the Pc fragment and homology length of 1600 bp (800 bp homology on each arm), then the homologous recombinant fragment was obtained by double enzyme digestion and sgRNA were co-transfected into the fibroblasts of the auricular margin in Holstein bulls. Finally, the Holstein ear margin fibroblast cell line with the homozygous Pc insertion was successfully obtained . OvariesThe results of a study by Aldersey et al. in 2020 showed that fetal bovines exhibit significant differences in horn traits at 90 days of gestation . In hornHomozygous mutant cell lines were cultured for two to three generations in 15% Dulbecco\u2019s modified eagle medium .2 saturation humidity.Slaughterhouse ovaries were washed repeatedly with saline supplemented with penicillin and streptomycin at 27 \u00b0C. In sterile conditions, cumulus-oocyte complexes (COCs) were aspirated from 3 to 6 mm follicles using a vacuum peristaltic pump with an 18-gauge needle. Selection of COCs with homogeneous cytoplasm and dense oocytes was made using a home-made oral pipette under the microscope with a heated stage. After washing the selected COCs twice with HEPES-buffered TCM199  medium and three times with culture media, the COCs were transferred into the culture media and incubated for 22 h in an incubator at 38.5 \u00b0C and 5% CO\u22121 and pulses of 10 \u00b5s and two pulses. The reconstituted embryos were transferred into the embryo culture medium and incubated in the incubator for 30 min. The reconstituted embryos were transferred to the A23187 solution for 5 min, then transferred to the 6-DMAP solution and incubated in the incubator for 6 h. The reconstituted embryos were then transferred to the embryo culture solution and the blastocyst formation was counted after 7 days in the incubator. The reconstructed embryos were finally transferred into the embryo culture medium, and the blastocyst formation was counted after 7 days of culture in the incubator.The COCs cultured to maturity were digested with 1% hyaluronidase and oocytes with first polar bodies, intact morphology and homogeneous cytoplasm were selected for transfer into the manipulation solution. The microscopic manipulation aspirated the first polar body and a little of the surrounding cytoplasm. The donor cells were injected into the enucleated oocytes at the denuded pinhole and subsequently transferred into the electrical fusion medium for 1 min. The oocytes were arranged in batches in the cell electrofusion bath in a uniform manner while ensuring that the electric field was perpendicular to the contact surface of both cells. The parameters of the CF150B Cell fusion instrument  were adjusted to a field strength of 2.4 kV\u00b7cmYoung heifers of 14 to 15 months of age were selected for uniform body structure, good body condition, good genetic performance, and the presence of the corpus luteum on the ovaries on rectal examination. Recipient heifers were treated with 6 mL intramuscular cloprostenol for estrus synchronization, followed by observation and recording of estrus over 2 to 3 days.At day 7 of embryo culture, embryos were graded according to embryo quality and quality blastocysts were stored in separate 0.25 mL plastic tubes in units of one embryo for transport. The remaining blastocysts were preserved by cryopreservation by vitrification. Referring to the report of Roper et al. (2018), the best recipient cattle were selected for embryo transfer in this experiment . Ultraso2O). The PCR reaction conditions included pre-denaturation at 93 \u00b0C for 30 s, denaturation at 93 \u00b0C for 20 s, annealing at 58 \u00b0C for 30 s and extension at 72 \u00b0C for 70 s, for 33 cycles; followed by an extension at 72 \u00b0C for 2 min and storage at 4 \u00b0C. The PCR products were identified by agarose gel electrophoresis (AGE) at a concentration of 1.2% and the amplified products were sequenced by Sanger at Huada Genetics .The experiment randomly selected eight embryos labelled a to h to extract the whole genome of single cell clones using the cell tissue blood whole genome DNA extraction kit . The whole genome was used as the template and btHP-1963-F/btHP-1963-R shown in Horn bud tissue was obtained from the WH and EH Holstein male fetal bovines by vertical cutting. Tissue samples were fixed using 10% formalin and dehydrated in a graded ethanol series. They were washed in xylene and embedded in paraffin and 4 \u03bcm microtome sections were stained with hematoxylin and eosin (HE). Digital image acquisition was with a Prog Res C5 camera .The whole genome of the skin tissue was extracted separately using the cell tissue blood whole genome DNA extraction kit  and was used as a template and NEW-F/NEW-R as primers as shown in \u00ae Reagent kit  was used to extract tissue RNA. Quality-qualified Total RNA was selected as the starting sample for mRNA sequencing. The quality required was RIN \u2265 7, the ratio of 28S to 18S RNA was greater than or equal to 1.5:1 by Agilent 2100 BioAnalyzer  and the starting volume required was in the range of 2 to 3 \u03bcg. The Qubit RNA assay kit  provided accurate quantification of starting Total RNA. The Ribo-Zero\u2122 Magnetic Kit  was used for the isolation and removal of rRNA from Total RNA. Quality control of rRNA removed from total RNA was performed using agarose gel electrophoresis, Nanodrop microspectrophotometer , and Agilent 2100 .The TRIzolThe raw reads from the downstream machine were quality controlled using fastp  to filtep-value \u2264 0.05. Hierarchical clustering analysis was also done on the screened differentially expressed genes using R 4.1.2 software  [The clean reads were compared to the reference genomic sequence using HISAT2 2.1.0 software   with an Austria)  and genep-value < 0.05 were significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using Kobas 3.0  software to obtain the enrichment of differentially expressed genes in the pathway. Differentially expressed genes were mapped to each term of the Reactome database  and the number of differential genes per term was calculated to obtain a list of differential genes with Reactome function and a count of the number of differential genes. The experiment applied a hypergeometric test to identify Reactome terms that were significantly enriched in differential genes compared to the whole background. A STRING diagram  was constructed for interaction networks of differentially expressed proteins. Visual protein interaction network maps and Hub protein interaction analysis were produced using Cytoscape software , Bethesda, MD, USA).In this experiment, Gene Ontology (GO) analysis of the differentially expressed genes were performed using R 4.1.2 software  . The GO GAPDH) expression using the 2\u2212\u0394\u0394CT method. The sequences of the primers used are shown in Total RNA extracted from tissues was synthesized using the Revertra ace qPCR kit  according to the manufacturing instructions. The complementary DNA (cDNA) was synthesized using a QuantStudio5 real-time fluorescent quantitative PCR system  in a total volume of 15 \u03bcL. The reaction system consisted of 10 \u03bcL 2 \u00d7 SYBR green PCR buffer , 1 \u03bcL of 10 \u03bcM PCR forward primer, 1 \u03bcL of 10 \u03bcM PCR reverse primer, 1 \u03bcL of template, and 6 \u03bcL of de-RNAase water. The reaction procedure was one cycle, denaturation at 50 \u00b0C for 2 min, denaturation at 95 \u00b0C for 2 min, followed by 40 cycles of amplification, denaturation at 95 \u00b0C for 15 s, and annealing extension at 60 \u00b0C for 1 min, followed by melt curve analysis in triplicate. The expression data were normalized to glyceraldehyde-3-phosphate dehydrogenase . All experiments were repeated at least in three independent experiments.We used SAS software version 9.2  for statistical analysis. Groups were compared by one-way analysis of variance (ANOVA) followed by the Duncan post-hoc test for multiple comparisons. The present research will facilitate further studies on horn development and provide further insight into the molecular mechanisms of Pc locus regulation of the polled trait. The present research will provide ideas and a theoretical basis for exploring the mechanism of horn development and Pc locus regulation in Holstein cattle and provide methods for breeding polled Holstein bulls to improve the safety and economic efficiency of cattle farm breeding."}
+{"text": "Population size has increasingly been taken as the driver of past human environmental impact worldwide, and particularly in the Arctic. However, sedimentary ancient DNA (sedaDNA), pollen and archaeological data show that over the last 12,000 years, paleoeconomy and culture determined human impacts on the terrestrial ecology of Arctic Norway. The large Mortensnes site complex  has yielded the most comprehensive multiproxy record in the Arctic to date. The site saw occupation from the Pioneer period  with more intensive use from c. 4,200 to 2,000\u00a0cal. years BP and after 1,600\u00a0cal. years BP. Here, we combine on-site environmental archaeology with a near-site lake record of plant and animal sedaDNA. The rich animal sedaDNA data (42 taxa) and on-site faunal analyses reveal switches in human dietary composition from early-Holocene fish\u00a0+\u00a0marine mammals, to mixed marine\u00a0+\u00a0reindeer, then finally to marine\u00a0+\u00a0reindeer\u00a0+\u00a0domesticates , with highest reindeer concentrations in the last millennium. Archaeological evidence suggests these changes are not directly driven by climate or variation in population densities at the site or in the region, but rather are the result of changing socio-economic activities and culture, probably reflecting settlers\u2019 origins. This large settlement only had discernable effects on its hinterland in the last 3,600 years (grazing) and more markedly in the last 1,000 years through reindeer keeping/herding and, possibly domestic stock. Near-site sedaDNA can be linked to and validate the faunal record from archaeological excavations, demonstrating that environmental impacts can be assessed at a landscape scale. Significance StatementHuman impacts in the Arctic (70\u00b0N) are assessed over 12,000 years from one of the largest site complexes known using plant and animal sedaDNA, pollen, nonpollen palynomorphs , and archaeological data. From these data, it is shown that human activities rather than population levels are most important for driving vegetation changes. These activities are connected to the changing marine environment in the Holocene and a switch from marine to terrestrial resources, which, in turn, impacted the terrestrial environment. We show that combining plant and animal sedaDNA, pollen, and archaeology can provide detailed ecological reconstructions and describe shifting socio-ecological baselines, which in this case were determined by culture (including grazing/herding) associated with waves of immigration.The Arctic is warming up to three times more rapidly than any other biome, and it is critical to understand how human populations have affected and will continue to impact Arctic ecosystem biodiversity . EstimatRangifer tarandus) and moose , as well as being related to marine resources. The latter reflect water temperatures and by the earliest Holocene, the Polar Front had returned to close to Svalbard, with the 5\u00b0 isotherm being close to Varanger (Gadus morhua) and haddock (Melanogrammus aeglefinus), with Arctic cold-water fish such as Polar cod (Boreogadus saida)  .\u22122, which is one of the lowest in Europe, although the coastal density population at over 10 persons km\u22122 is comparable with the Troms\u2013Finnmark county as a whole (15 persons km\u22122); thus, it is not atypically low and likely a reflection of waves of inward migration . The remarkable concentration of archaeological sites in Varanger (over 100) has attracted research for over a 100 years. The fjord represents a \u201cMaritime Core Area,\u201d where social complexity arose in hunter\u2013gatherer\u2013fisher communities , and the lack of taxonomic precision . Althoug2 of raised beach, bedrock, scree, and coastal sand-flats. The archaeology covers almost all the archaeological periods and technological traditions of northern Norway/Fennoscandinavia and comprises tent-rings, semi-subterranean pit houses , middens, hearths, graves, a standing stone, and a labyrinth , and whale (Cetacea) but no reindeer (G. morhua) and some pollock (Pollachius virens) (Rissa tridactyla), guillemots (Uria), and great auk (Pinguinus impennis), whilst the more recently analyzed shellfish are dominated by black clam (Artica islandica) and periwinkle (Littorina littorea)  (Ovis/Capra), reindeer, cattle (Bos taurus), and pig (Sus scrofa). There are some seal but no whale. The fish are dominated by very large quantities of cod and haddock (Melanogrammus aeglefinus) and the birds are dominated by rock ptarmigan (Lagopus mutus), kittiwake, and guillemots. This assemblage implies both summer grazing and the collection of hay and fodder for the overwintering of the domesticated stock. This pastoral farming is combined with reindeer, boat fishing, and the use of bird-cliffs, the nearest of which is located only 1,200\u00a0m to the east of archaeological area D  dated to c. 7,600 to 5,500\u00a0cal. years BP  revealed evidence of diet, local resources, and hot-stone technology , 43. Thereindeer . A problreindeer . The fis virens) , 43. Thewere cod , revealea molva) , 44. Fewa molva) . It reveArtemisia), ribwort plantain (Plantago lanceolata), and microcharcoal from 6,900\u00a0cal. years BP. The record of cultivars fits with the archaeology with rye  first appearing c. 1,320\u00a0cal. years BP (630 CE) and then again at 880\u00a0cal. years BP, whilst barley (Hordeum) appears at 660\u00a0cal. years BP.Pollen and spore data are also available from a mire within the archaeological site, Fig.\u00a0 40) and and40) aAnalysis of a 3.2\u00a0m sediment core from Lake Nordvivatnet  along with Arctic tundra shrubs  also appears in this zone in both sedaDNA (and pollen) at levels that imply it was in the lake catchment  and Ribes (currant), respectively.Zone N1 covers the Younger Dryas and is not considered further here due to it predating human occupation at this site, which at the time was a marine embayment. Zone N2 , willow-herb (Epilobium), peavine (Lathyrus), field forget-me-not (Myosotis arvensis), common buttercup (Ranunculus acris), globeflower (Trollius europaeus), and bird vetch (Viccia cf. cracca). Several of these taxa, including willow-herb, are also found in the earliest Holocene as they are pioneer, r-selected strategists suited to open, and disturbed ground, which is common under both periglacial conditions and human disturbance (In the absence of cultivars (apochores) from the lake, human impact is assessed using a combined sum of facultatively synanthropic  due to the similarity to values found in the Late Holocene when it is known that the lake was surrounded by birch-willow forest and other sites in northern Norway  and heather , which also increase at this time . An increasing proportion of pine pollen is observed and remains high until the uppermost sample at c. 500\u00a0cal. years BP. Birch, pine, and the dwarf shrubs remain dominant throughout Zone Npo3. There is a low number of facultatively synanthropic species, and they show no coherent increase over time. However, there are distinct peaks in coprophilous fungal spores with a rise in the early Holocene  during the period of birch-forest establishment with fluctuations in the middle Holocene (Npo4) and a reduction in the late Holocene (Npo5 to Npo6) except for a spike at c. 3,400\u00a0cal. years BP, which is dominated by Sporormiella sp. which suggest phases of more intense grazing around the lake  in the relatively open environment prior to human arrival due to natural fires. The microcharcoal which is supported by a peak in Neurospora (HdV 55\u2014a genus of ascomycete carbonicolous fungus) is common in the Younger Dryas and early Holocene and its reduction in the remainder of the Holocene could be related to a reduction in natural fires due to lower summer temperatures and increase in precipitation , suggesting that Nordvivatnet was a lagoon or beach-flat at this time prior to the development of the beach barrier that today confines the lake. This would explain the rapid deposition of sediments with low organic matter and glacially scoured stones (Rangifer tarandus). Several marine taxa were detected with sporadic occurrences thereafter, which we explore in the Rana temporaria), moose , and red fox (Vulpes vulpes) from c. 9,000 to 3,500\u00a0cal. years ago, are consistent with a warmer than present climate and forest cover. Intriguingly, between c. 6,500 and 4,500, cold-adapted taxa of open habitats, such as reindeer and Norwegian lemming (Lemmus lemmus), drop out of the record and at c. 5,700\u00a0cal. years BP, we observe the only occurrence of Eurasian beaver (Castor fiber), a taxon that is absent from northern Norway today, suggesting warmth and forest cover were at their maxima over this interval. This occurrence of beaver also falls within the age range covered by the Old Stone Age house R12 from Mortensnes, which contained beaver bones  was applied to the Nordvivatnet plant sedaDNA, pollen data, and summed probability distribution (SPD) of 14C dates from archaeological sites in northern Norway  Fig.\u00a03b3b. The pure Fig.\u00a0. The plaure Fig.\u00a0. The beture Fig.\u00a0.Ribes), and strong evidence of reindeer. In this period, the on-site evidence is dominated by reindeer with domesticates, principally sheep, and cereal cultivation. Mortensnes records the second-most northerly, and the most easterly, occurrence of cereal pollen in Fennoscandia; the only more northerly occurrence is from Risfjorddalen, Melham (71\u00b0N), which is dated to 2,450 and 2,800\u00a0cal. years BP  superimposed on the five traditional archaeological phases, that show different ecological impacts around Lake Nordvivatnet and the Mortensnes archaeological site Fig.\u00a0. In the years BP . At MortCombining the data, it is clear that the economic character of Mortensnes changed over time, from one dominated by the use of the sea and coast in the Older Stone Age to a more mixed economy using more parts of the landscape in the Younger Stone Age. This may reflect a change in hunting pattern, with early hunting occurring further inland, but also it signals a trend towards sedentism, which can also be argued from the archaeology . FinallyIn trying to interpret this temporal pattern, it is pertinent to return to the location and marine environment of Mortensnes. The importance of the Atlantic Boreal marine species increases with warmer sea temperatures and thus they became dominant in the Older Stone Age; this is happening again today with the warming of the Barents Sea . VarangeUsing the most comprehensive multiproxy paleoecological record in the Arctic to date , we show that a site with a nearly complete Holocene record of human occupation had little or no discernable effects on its hinterland until the last 3,600 years, when grazing and probably wood-cutting is evidenced but was still low. It is only in the last 2,000 years that these effects increased and in the last millennium that we get continuous and sedaDNA reads of reindeer, suggesting herding. The plant record is shown here to diverge from climatic control in the last 2,000 years, and this is ascribed to human impact. Comparison with the archaeological data suggests that it is the changing economy associated with in-migration, from marine-orientated pioneers (from west and/or east), to mixed fishing  and reindeer hunting, and finally terrestrially dominated pastoral agriculture in the Sami Iron Age and historical period, that has driven this record, rather than population per se. This implies that it is what people do, and probably where they came from, that drives environmental impact in this Arctic region, both in the past and probably into the future.Only a brief summary of the methods is given here and for more detail see the The study is part of the ECOGEN project \u201cEcosystem change and species persistence over time: A genome-based approach,\u201d financed by Research Council of Norway grant 250963/F20. The publication charges for this article have been partially funded by a grant from the publication fund of UiT The Arctic University of Norway.pgac209_Supplemental_FilesClick here for additional data file."}
+{"text": "An estimated 19 million people are infected with rifampicin-resistant/multidrug-resistant strains of tuberculosis worldwide. There is little done to prevent these individuals from becoming sick with RR/MDR-TB, a disease that is associated with high rates of morbidity, mortality, and suffering. There are multiple phase III trials currently being conducted to assess the effectiveness of treatment of infection  for RR/MDR-TB, but their results are likely years away. In the meantime, there is sufficient evidence to support a more comprehensive management of people who have been exposed to RR/MDR-TB so that they can maintain their health. We present a patient scenario and share our experience in implementing a systematic post-exposure management program in South Africa with the goal of inspiring similar programs in other high-burden RR/MDR-TB settings. Each year, more than a half million people become newly sick with rifampicin-resistant/multidrug-resistant forms of tuberculosis (RR/MDR-TB) . The pasRR/MDR-TB can have a major impact on a family. In our work in a peri-urban township located outside of Cape Town, we encountered many households struggling with the disease. As an example, the Makhanda  family struggled to meet the growing household\u2019s most basic needs. With only one working household member, money was tight, and there was often not enough to eat. When the oldest adult brother died in a motor vehicle accident, the family matriarch, Busisiwe, had to make room for his three children\u2014all under the age of ten years\u2014to come and live in the already crowded shack where she was living with her daughter, her son, and four of her other grandchildren. Over the coming months, Busisiwe attributed her weight loss to the meagre food supply and the stress, but it was harder to explain away her cough. When she finally presented to the health center and was told she had RR/MDR-TB, she was devastated at the loss of her income that the daily visits to the health center for observed therapy would cause and she was worried about the family members living with her. Her son and two of the grandchildren were coughing as well, and although the nurse told her to bring all the household members to clinic for evaluation, they only had taxi fare to bring the sickest one. When she used that money to bring her grandson to clinic, she was grief-stricken to learn he was also sick and wept as they took him away to the hospital. She worried endlessly about the other children but was told there was nothing else they could do. \u201cJust bring them to the clinic if they are sick\u201d the nurse instructed her, as Busisiwe left the health center once again with a heavy heart, worried that she had brought this misfortune on them all. Mycobacterium tuberculosis means that a person has the bacilli living in the lungs but is not yet sick with TB disease. In this paper, we will refer to this state as RR/MDR M.tb infection. The precise data on the number of people who are infected with RR/MDR-TB are not possible to calculate since current tests for TB infection cannot differentiate between drug-susceptible and drug-resistant strains. This is because drug susceptibility testing can only be done when M. tuberculosis or its DNA has been isolated, a condition that usually defines TB disease. Modelling studies suggest that there are more than 19 million people worldwide who harbor mycobacterial strains that are resistant to rifampicin and isoniazid [M.tb infection far outstrips treatment capacity. That fact alone is reason for concerted action, but until recently, the only measure recommended to prevent RR/MDR-TB among those who were exposed was frequent clinical monitoring [Infection with soniazid  . Some of this also stems from the historical debate about the transmissibility of RR/MDR M.tb strains, with some studies suggesting that the fitness costs associated with some mutations leading to resistance could reduce the transmissibility of these forms of TB [Much of this stems from the fact that there are not yet randomized controlled trials supporting the use of a specific medication for the treatment of RR/MDR ms of TB . Severalms of TB ,9.Rapid diagnosis and prompt initiation of effective therapy are among the most effective means for preventing RR/MDR-TB since primary transmission is driving most of the burden of RR/MDR-TB in many settings in the world ,11. ThusWhen someone is newly diagnosed with RR/MDR-TB, working with them to compassionately disclose this diagnosis to other household members is key. If done in a supportive manner\u2014as is the case with HIV and other infectious diseases \u2014and by aBox 1Empathize with the person who was just diagnosed;Assess any physical or mental risks that might result if disclosure happens;Identify any individuals who might be able to support disclosure with other household members and share the news with that individual first;Practice the disclosure and review what is the best way to say things for the newly diagnosed person;Emphasize the concepts of \u201cshared air\u201d rather than contagion and blame;Offer to assist the newly diagnosed person with the disclosure if s/he would like;Stress that there are many actions that can be done to keep household members healthy;Schedule a time to follow up and review how the disclosure went.Most RR/MDR-TB transmission occurs prior to a diagnosis of RR/MDR-TB being made . Many trThe principles of what has traditionally been called preventive therapy but what should more accurately be referred to as the treatment of infection for drug-susceptible TB and RR/MDR-TB are essentially the same . The goaM.tb infection treatment, the first step is ruling out active TB disease. This is usually conducted through a symptom assessment, weight monitoring, physical examination, bacteriologic testing, and chest radiography [M.tb infection perform poorly as they require a functioning immune system and are, thus, more likely to yield false-negative results in the populations most at risk of becoming sick with TB, such as in young children or persons living with HIV [M.tb infection can be presumed if the index individual is sick with a strain of RR/MDR-TB (either confirmed or possible).For both drug-susceptible and RR/MDR iography . Tests fwith HIV . In addiwith HIV . RR/MDR M.tb infection [M.tb infection treatment were conducted several decades ago, access to drug susceptibility testing for fluoroquinolones was limited and took weeks to months to obtain. This led to the use of multidrug regimens for RR/MDR M.tb infection treatment, a strategy that increased both the pill burden and the likelihood of adverse events [M.tb infection was used in a larger cohort of persons exposed to RR/MDR-TB in the household in Karachi, Pakistan and found the treatment of infection to be effective but that the use of ethionamide resulted in poor adherence, largely mitigated by adverse events [M.tb infection, isoniaizid-based preventive therapy regimens lasting six months should be effective.What differs between the treatment of infection for drug-susceptible and RR/MDR-TB is the medications that can be used for therapy. By definition, RR/MDR-TB strains are resistant to the medications used to prevent drug-susceptible TB, including rifamycins and (for persons with MDR-TB) isoniazid. For this reason, fluoroquinolones have been the mainstay in the treatment of RR/MDR nfection . When the events . In spite events . A largee events . A combie events ,35. Of ne events  or bedaqe events . Of noteM.tb infection treatment globally have access to it. Currently, RR/MDR M.tb infection treatment is usually only implemented in low-burden TB settings. Some of this may be due to a lack of randomized controlled trials leading to a rather lukewarm policy recommendation from the WHO on RR/MDR-TB preventive therapy. However, given the notable morbidity and mortality of RR/MDR-TB disease worldwide and existing programmatic data the balance of risk/benefit ratio would likely favor wider-scale implementation of RR/MDR M.tb treatment of infection even while randomized trial data are being collected. There are three ongoing trials of RR/MDR M.tb infection treatment, two of which are assessing the role of fluoroquinolones and one of which is testing the novel nitroimidazole medication delamanid [M. tb infection, including inhaled therapies, vitamin supplementation, and nutritional support.Very few of the persons who could benefit from RR/MDR elamanid . The staMuch of what is driving the global RR/MDR-TB crisis is poverty, hunger, lack of housing, and co-morbid conditions, such as HIV, substance use disorder, malnutrition, and diabetes . EffectiAll too often, programs and providers report feeling too overwhelmed to take on TB prevention, and this may be even truer for the prevention of RR/MDR-TB. At the same time, RR/MDR-TB prevention activities are critical, since even with improved diagnostic and treatment strategies, outcomes for people living with RR/MDR-TB are far below those achieved for people with drug-susceptible form of TB. While many high-risk TB settings face unique issues and challenges, there are often cross-cutting themes of poverty, injustice, and inequity driving the transmission of RR/MDR-TB in communities. We were able to successfully implement a program aimed at RR/MDR-TB prevention in a peri-urban township outside of Cape Town, South Africa beginning in 2020. With the aim of sharing lessons learnt with policy makers and TB care providers, below is a description of the program as well as the key factors supporting its success .The Khayelitsha post-exposure management program  was implemented as a collaborative effort between M\u00e9decins Sans Fronti\u00e8res, the City of Cape Town, and the Western Cape Department of Health. Household contacts (particularly children and adolescents) of persons newly diagnosed with RR/MDR-TB, were assessed for signs or symptoms of TB, and provided TB counselling, either in their home or at their primary health care clinic. This was conducted by a PEP-dedicated TB nurse who worked across all 10 Khayelitsha clinics. Contacts with any suggestive signs or symptoms of TB were referred for further evaluation by a doctor and, where indicated, for chest radiograph and/or bacteriological testing. Following best practices in the South African setting, tests of infection were not routinely performed given that all these individuals were close contacts of persons newly diagnosed with TB. Contacts without clinical evidence of active TB disease would be offered 6 months of treatment of infection most commonly with a fluoroquinolone (high-dose isoniazid or delamanid was used where there was suspected or confirmed fluoroquinolone resistance in the index case).Patient-centered and differentiated care was a key aspect of this program , with persons given the option to follow up for treatment of infection in their home, at the clinic, or telephonically. Follow-up consults focused on ongoing treatment literacy/counseling support, and eliciting if there were any emerging TB symptoms or adverse events. To minimize disruptions to people\u2019s lives, follow-up visits were conducted every two to three months, with multi-month medication refills provided. Young children were offered child friendly formulations of medication to support treatment adherence, including dispersible tablets of levofloxacin provided by the Global Drug Facility. Where socio-economic concerns were noted in the household, persons could be referred to a TB counsellor or social worker and could be provided with food parcels. Transport allowance was provided where needed for travel to attend any clinic visits.The outcomes of the initial phase of this program (112 children and adolescents on treatment of infection) are described in more detail in a recent publication. Notably, this program resulted in a high level of case detection (10% of those screened were started on treatment for TB disease), as well as high retention in care for those who received treatment for infection (80% completed treatment); therapy was well tolerated with minimal adverse events. None of those who started treatment for infection developed active TB disease (median 200-day follow-up). After the initial pilot success of this program, the work was integrated into the routine TB services offered at the clinic.It is imperative that more work is conducted to prevent RR/MDR-TB in the coming years. RR/MDR-TB is one of the most serious aspects of the global crisis in antimicrobial resistance, but efforts to prevent RR/MDR-TB have narrowly focused on resistance amplification, while ignoring other important interventions. RR/MDR-TB preventative efforts require a fundamental shift in the thinking and actions of the TB community, to a focus on providing person and family-centered care along the whole continuum. Rapid diagnosis of all forms of TB and initiation of the shortest most effective regimen is key to RR/MDR-TB prevention. So too is compassionate post-exposure care for all close contacts and members of the family coupled with supportive counselling. In this review, we have outlined key preventative strategies along the spectrum of care and shared our experiences of a family centered RR/MDR-TB preventive care model. Implementation of these strategies by committed TB programs and care providers promises to dramatically improve RR/MDR-TB prevention efforts and could, with time, reduce the implementation burdens of treating RR/MDR-TB for countries and programs. Doing so will dramatically reduce the suffering of persons, such as Busisiwe and her family."}
+{"text": "In 2020, almost half a million individuals developed rifampicin-resistant tuberculosis (RR-TB). We estimated the global burden of RR-TB over the lifetime of affected individuals. We synthesized data on incidence, case detection, and treatment outcomes in 192 countries . Using a mathematical model, we projected disability-adjusted life years (DALYs) over the lifetime for individuals developing tuberculosis in 2020 stratified by country, age, sex, HIV, and rifampicin resistance. Here we show that incident RR-TB in 2020 was responsible for an estimated 6.9  million DALYs, 44%  of which accrued among TB survivors. We estimated an average of 17  DALYs per person developing RR-TB, 34%  greater than for rifampicin-susceptible tuberculosis. RR-TB burden per 100,000 was highest in former Soviet Union countries and southern African countries. While RR-TB causes substantial short-term morbidity and mortality, nearly half of the overall disease burden of RR-TB accrues among tuberculosis survivors. The substantial long-term health impacts among those surviving RR-TB disease suggest the need for improved post-treatment care and further justify increased health expenditures to prevent RR-TB transmission. Rifampicin-resistant tuberculosis (RR-TB) requires longer, more toxic therapy than rifampicin-sensitive disease and is associated with a higher occurrence of long-term sequelae. In this mathematical modeling study, the authors estimate that incident RR-TB in 2020 will be responsible for ~6.9 million disability-adjusted life years; 44% due to post-tuberculosis sequelae. Rifampicin is one of the key drugs in the WHO-recommended first-line tuberculosis treatment regimen, and rifampicin resistance represents a major challenge for effective tuberculosis treatment. For individuals diagnosed with resistant disease, the most commonly used treatment regimens have required an extended duration of therapy, using drugs that are both more toxic and less effective compared to the first-line regimen . As a result, individuals treated for RR-TB experience lower cure rates and higher mortality rates compared to individuals with drug-susceptible tuberculosis treated with the first-line regimen3. Moreover, many individuals diagnosed with tuberculosis are not tested for drug resistance, such that many of those with a drug-resistant strain will receive an inappropriate treatment regimen, leading to high rates of treatment failure and recurrence5. Due to these factors, many individuals who develop RR-TB will die during the disease episode or experience a prolonged period of ill health before achieving a cure.In 2021, it is estimated that 450 thousand individuals developed rifampicin-resistant tuberculosis (RR-TB)7, elevated mortality rates8, income losses9, and persistent social and psychological sequalae10. These ongoing effects contribute a substantial fraction of the overall disease burden caused by tuberculosis12. Evidence suggests that these post-TB sequelae are more common and severe for individuals surviving RR-TB14. Due to delays in initiating appropriate treatment15 and the lower efficacy of treatment regimens, individuals treated for RR-TB are likely to accumulate greater lung damage before the disease is effectively controlled by treatment. Moreover, individuals with RR-TB may receive several treatment courses before achieving a cure due to receipt of inappropriate first-line treatment or lower success rates even when appropriate treatment is available, with additional lung damage occurring during this extended disease period. The greater toxicity of drugs included in RR-TB regimens can also cause long-term sequelae. A meta-analysis of data on tuberculosis survivors found greater lung impairment among individuals surviving multi-drug resistant (MDR) tuberculosis compared to individuals with drug-susceptible tuberculosis14, and high levels of impairment among MDR-TB survivors have been reported across a range of functional domains16.Accumulating evidence indicates that tuberculosis survivors frequently experience ongoing disability1, these trends\u2014as well as the absolute fraction of incident tuberculosis cases with rifampicin resistance\u2014vary substantially across world regions. Similarly, national tuberculosis programs vary greatly in their capacity to identify RR-TB and provide effective treatment. The development of better diagnostics and RR-TB regimens promises to improve patient care in the future, yet the adoption of these new tools has been uneven. These differences between settings will produce differences in the magnitude of disease burden attributable to RR-TB. In this study, we synthesized evidence on the incidence, detection, and outcomes of RR-TB treatment across 192 countries and used mathematical modeling to simulate the lifetime burden of disease due to incident RR-TB in 2020, including health losses incurred during the disease episode as well as among individuals surviving tuberculosis. Based on this analysis, we report the number of disability-adjusted life years (DALYs) attributable to incident RR-TB in 2020, stratified by country, age, sex, HIV status, and disease stage.While global RR-TB incidence is estimated to be decliningIncident RR-TB occurring in 2020 was estimated to result in 6.9  million DALYs over the lifetime of affected individuals, representing 5.4%  of lifetime DALYs attributed to all incident tuberculosis in this year. The majority ) of RR-TB DALYs were estimated to result from premature mortality  million DALYs), as compared to reductions in quality of life  million DALYs). Just over half ) of all RR-TB DALYs were estimated to be incurred during the disease episode  million DALYs), with the remainder resulting from post-TB sequelae among tuberculosis survivors  million DALYs). Figure\u00a0 million Table\u00a0Table\u00a0Supplementary Table\u00a017 for estimating tuberculosis-attributable mortality among tuberculosis survivors and a scenario that assumed that post-TB disability weights for RR-TB were the same as for RS-TB. For both scenarios, total RR-TB DALYs were estimated to be 15\u201316% lower than in the main analysis.Supplementary Table\u00a0We assessed the global disease burden due to incident RR-TB in 2020, including mortality and morbidity during the disease episode as well as the lifetime impact of post-TB sequelae among those surviving the disease. We found that RR-TB occurring in 2020 was responsible for 6.9  million DALYs, or 5.4% of the total DALYs due to all TB in 2020. An average of 17.3  DALYs were associated with each individual developing RR-TB, with nearly half of the total DALYs estimated for RR-TB accruing among tuberculosis survivors. While the South-East Asia Region was estimated to have the greatest absolute burden of RR-TB, former Soviet Union countries and southern African countries had the highest burden of RR-TB per population.18, with a large fraction of current tuberculosis cases experiencing resistance to rifampicin and, in some cases, many other tuberculosis drugs1. While approaches to tuberculosis diagnosis and treatment in the region have been formulated to address this challenge19, it still represents a major barrier to effective tuberculosis care, and the poor outcomes experienced by individuals with drug-resistant tuberculosis (as compared to drug-susceptible disease) are reflected in the high RR-TB burden estimates.For former Soviet Union countries, this high RR-TB burden results from historical approaches to tuberculosis treatment that produced high resistance levelsFor southern African countries, the high RR-TB burden estimates are likely related to the dual burden of tuberculosis and HIV in these countries. The relationship with HIV is notable, with HIV-infected individuals estimated to experience 40 times greater DALYs from RR-TB compared to HIV-uninfected individuals, a consequence of both elevated tuberculosis incidence for individuals with HIV and rapid disease progression and death in the absence of prompt, effective treatment. For southern African countries, the close overlap of tuberculosis and HIV epidemics may be exacerbated by poverty and health system weaknesses that make it difficult to achieve successful treatment outcomes for individuals with both HIV and RR-TB.20. While for some countries we needed to impute data gaps using other evidence, the major sources of uncertainty in this study derive from the challenge of extrapolating future health outcomes for individuals with RR-TB. This additional uncertainty is reflected in the wider uncertainty intervals reported for post-TB DALYs, as compared to DALYs estimated for the disease period. While much of the additional DALYs per RR-TB case (as compared to RS-TB cases) resulted from higher mortality during the disease episode, post-TB DALYs were also estimated to be higher for RR-TB, reflecting more prevalent and more severe sequelae following RR-TB. A substantial body of evidence now describes the elevated mortality and health burden experienced by tuberculosis survivors, motivating efforts to establish clinical standards21 and systems of care for this large cohort of individuals22. However, some part of this excess morbidity and mortality will relate to pre-existing health conditions and behaviors that co-occur with tuberculosis. Moreover, given the concentration of tuberculosis in poor and marginalized communities, the lower health outcomes for tuberculosis survivors will also partly reflect the impact of socioeconomic determinants of health that would still be present in the absence of tuberculosis. In this study, we extended a published approach to separating the causal impact of tuberculosis from these other factors12, which requires several assumptions. For this reason, additional empirical research on the causal contribution of tuberculosis and RR-TB to future health outcomes is needed.This study relies on accurate epidemiological data on drug resistance patterns across countries and population groups. This highlights the utility of efforts that have been made to develop and extend systems for monitoring drug resistance trends across many countries23. Uncertainty in pediatric estimates is compounded by limited data on post-TB outcomes for pediatric tuberculosis survivors, though the data that exist suggest persistent disease sequelae24. Second, our analysis makes assumptions about healthcare interventions and access for RR-TB survivors that may not hold in the future. In particular, the development and scale-up of effective rehabilitation interventions for post-TB lung disease could improve outcomes for RR-TB survivors25 and reduce the burden relative to the estimates in this study. Similarly, the development and deployment of better diagnostics26 and treatments27 for RR-TB may reduce the excess disease burden experienced by individuals with RR-TB relative to drug-susceptible disease. In particular, the adoption of new all-oral 6-month regimens for RR-TB could produce major improvements in RR-TB treatment outcomes in the future. An MDR/RR-TB regimen composed of bedaquiline, pretomanid, linezolid and moxifloxacin (BPaLM) was included in a 2022 update to WHO treatment guidelines2, and many countries have subsequently moved to introduce this regimen. Other short-course regimens are under development28. Given the better end-of-treatment outcomes achieved by the BPaLM regimen compared to older long-course regimens27 and the fact that cure is achieved more rapidly, it is possible that the introduction of new short-course RR-TB regimens will improve treatment outcomes and reduce post-treatment morbidity for RR-TB cohorts in the future.This research has several additional limitations. First, there is additional uncertainty around RR-TB outcomes in pediatric populations due to greater uncertainties in incidence estimates M. tuberculosis strain, clinical presentation, and treatment regimen. A second priority is interventional studies that examine approaches to protect and restore lung function during TB disease treatment and identify best practices for providing long-term rehabilitation for TB survivors. Finally, policy analyses are needed to identify the most impactful and cost-effective approaches along the care\u00a0continuum  for RR-TB disease.This study suggests several directions for future research. First, empirical studies are needed that adopt quasi-experimental designs to separate the long-term effects of TB on post-TB health outcomes from the impact of comorbid conditions, health behaviors, and living conditions. These studies will be more valuable if they are able to describe how the causal effects of TB vary by individual-level factors such as RR-TB represents a major disease burden globally and a fundamental, lifelong health challenge for affected individuals. While the burden estimates reported by this study highlight the regions, countries, and population groups most impacted by RR-TB, they provide little guidance on the appropriate response to this burden. New interventions to facilitate the rapid detection and effective care for RR-TB are urgently needed, as well as concerted efforts to expand access to the best current diagnostics and treatments and policy analyses that compare actions to prevent, treat, and rehabilitate RR-TB in order to devise the most impactful approaches for reducing RR-TB burden in the future.12. This model stratifies the cohort developing tuberculosis in a single year by country, age, sex, and HIV status and tracks this cohort over the remaining lifetime of the affected individuals. Over this projection period, we recorded excess deaths and reductions in health-related quality of life causally attributable to TB or post-TB sequelae. For this analysis, we additionally stratified the model by the presence/absence of rifampicin resistance. For individuals without rifampicin resistance, we stratified the cohort according to whether the individual received or did not receive tuberculosis treatment. For individuals with rifampicin resistance, we stratified the cohort according to whether the individual (1) did not receive tuberculosis treatment, (2) received tuberculosis treatment with an inappropriate regimen (defined as the standard first-line regimen recommended for rifampicin-susceptible tuberculosis), or (3) received treatment with an appropriate regimen (defined as a WHO-recommended RR-TB regimen). Analytic inputs and methods are described below and summarized in Fig.\u00a0We adapted a previously published mathematical model of future health outcomes among the global cohort of individuals with tuberculosis disease29. The fraction of cases receiving treatment was based on the number of notified tuberculosis cases within each country, sex, and age group for 2020, divided by the estimated incidence for each stratum. For countries with missing notification data, we used WHO-estimated tuberculosis treatment coverage. We removed countries with less than 10 estimated cases for 2020 .Within each stratum, we assumed that the fraction receiving any tuberculosis treatment was independent of rifampicin resistance. We estimated the fraction receiving a diagnosis of rifampicin resistance (among the population with rifampicin resistance diagnosed with tuberculosis) by dividing country-reported data on RR-TB diagnoses by WHO estimates of RR-TB incidence scaled by the fraction detected. We estimated the fraction of RR-TB initiated on second-line treatment by comparing data on RR-TB diagnoses and second-line treatment initiations for each country. Several countries had missing values for the data used to construct the study cohort. Supplementary Fig. We based assumptions around the duration of disability during the tuberculosis disease episode on values estimated by the WHO, stratified by treatment and HIV status . Mortality odds ratios for HIV and for receipt vs. non-receipt of tuberculosis treatment were based on mortality risks estimated by the WHO Global Tuberculosis Programme. We allowed for differences in survival between individuals with RS-TB receiving a first-line regimen , individuals with RR-TB receiving a second-line regimen, and individuals with RR-TB inappropriately receiving a first-line regimen, using country-reported data (details in Supplementary Information).There is little evidence of survival among individuals with RR-TB inappropriately receiving a first-line regimen, and we calculated this value as a weighted average of survival probabilities for individuals with RR-TB receiving a second-line regimen and untreated individuals, with a wide prior distribution specified for this weighting parameter  was based on a systematic review of observational cohort studies  mortality among individuals surviving tuberculosis disease, following a published approach (details in Supplementary Information)12. These methods were also used to estimate post-TB disability weights that were applied to tuberculosis survivors. We assumed elevated mortality rates and disability weights among individuals surviving RR-TB vs. RS-TB based on studies reporting a higher prevalence and severity of tuberculosis sequelae among individuals surviving RR-TB as compared to RS-TB13  YLLtb, representing DALYs lost to mortality during the tuberculosis disease episode; (3) YLDptb, representing DALYs lost to post-TB disability among TB survivors; and (4) YLLptb, representing DALYs lost due to post-TB mortality.Health outcomes were quantified as the years of life lost due to premature mortality (YLLs) and the years of healthy life lost due to non-fatal disability (YLDs). For each modeled stratum, we calculated total tuberculosis-attributable DALYs as the sum of: (1) YLD30. To do so, we re-estimated the model for each of the 1000 parameter sets created from a Latin hypercube sample from the parameter probability distributions and calculated intervals as the 2.5th and 97.5th percentiles of the resulting distribution. We also calculated partial-rank correlation coefficients (PRCCs), describing the sensitivity of the estimate for total global DALYs due to RR-TB to uncertainty in individual parameters31. For parameters that varied at the country level  post-TB mortality rates and disability weights for RR-TB assumed to be the same as for RS-TB. For each alternative specification, we estimated total global RR-TB DALYs, average DALYs per incident RR-TB case, and the fraction of global tuberculosis DALYs attributable to RR-TB.We recalculated results for several alternative analytic assumptions: (1) the duration of RR-TB disease assumed to be 50% greater than for RS-TB, (2) the case fatality for RR-TB inappropriately treated with a first-line regimen assumed to be the same as for untreated individuals, (3) case fatality for RR-TB inappropriately treated with a first-line regimen assumed to be the same as for individuals treated with a second-line regimen, (4) case fatality for untreated RR-TB assumed to be greater than for RS-TB (OR\u2009=\u20091.5), (5) post-TB mortality rates based on estimates reported by Lee Rodriguez et al.Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"}
+{"text": "IL4R, IL12RB1, IL12RB2, IL6, TMPRSS2, and ACE2. Furthermore, a generalized linear mixed model identified statistically significant associations between prostate cancer risk and SNPs in IL12RB2, IL13, IL17A, IL4R, MAPT, and TFNRS1B. Finally, a statistically significant association was observed between IL2RA and TNFRSF1B and Gleason scores, and between SLC11A1, TNFRSF1B and PSA values. We identified SNPs in inflammation and two prostate cancer-associated genes. Our results provide new insights into the immunogenetic landscape of prostate cancer and the impact that SNPs on immune genes may have on affecting the susceptibility to prostate cancer.The immune system plays a critical role in modulating cancer development and progression. Polymorphisms in key genes involved in immune responses are known to affect susceptibility to cancer. Here, we analyzed 35 genes to evaluate the association between variants of genes involved in immune responses and prostate cancer risk. Thirty-five genes were analyzed in 47 patients with prostate cancer and 43 healthy controls using next-generation sequencing. Allelic and genotype frequencies were calculated in both cohorts, and a generalized linear mixed model was applied to test the relationship between prostate cancer risk and nucleotide substitution. Odds ratios were calculated to describe the association between each single nucleotide polymorphism (SNP) and prostate cancer risk. Significant changes in allelic and genotypic distributions were observed for  Prostate cancer (PCa) is one of the most widespread noncutaneous cancers in men. The incidence of PCa appears to be affected by several risk factors, among which inflammation plays a prominent role ,2. InflaACE, ACE2, ATG7, CD28, IFNG, IFNGR1, IFNGR2, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL17A, IL18, IL18R1, IL2, IL2RA, IL4, IL4R, IL5, IL6, IL7R, IRF5, LAG3, MAPT, NR1I2, PACGR, PARK2, PTPN22, SCLC11A1, TMPRSS2, TNFRS, TNFSF1B, TRAF6, and VDR). We then evaluated the possible association between the identified significant genetic polymorphisms and the clinical parameters of patients with PCa.Advances in next-generation sequencing (NGS) technologies have enabled the simultaneous sequencing of millions of nucleotides and investigation of unique sets of genes for diagnostic, prognostic, therapeutic, and research purposes. Many studies have investigated the association between polymorphisms in immune system-related genes and cancer risk ,7,8,9,10IL4R, IL12RB1, IL12RB2, IL6, TMPRSS2, and ACE2 were found associated with PCa risk. Both the C allele and the CC genotype of IL4R rs2301807 were inversely associated with PCa risk . The AA genotype in the 3\u2032 UTR of IL12RB1 rs3746190, along with the C allele of IL12RB2 rs2307145, were found associated with a lower risk of PCa . The G allele of the intronic variant IL6 rs2069832 was positively associated with a higher risk of PCa . In addition, both the A allele and CA heterozygous genotype of the intronic variant TMPRSS2 rs140141551 were both associated with a higher risk of PCa . Conversely, the G allele and the AG genotype of TMPRSS2 rs11701576 were found to be associated with a decreased risk of PCa . Finally, a synonymous variant was identified within the ACE2 gene, and the T allele of rs35803318 was found to be associated with a higher risk of PCa .The present study analyzed a total of 47 patients with PCa and 43 HCs using NGS, and comprehensive gene profiling was conducted on a panel of 35 genes. p < 0.05, with a few exceptions for borderline values, which were retained to avoid premature selection in intermediate models  was implemented. The first GLMM runs were applied individually to each gene to identify loci statistically associated with the occurrence of PCa. Statistical significance was set at e models . Odds rae models . StatistIL12RB2 rs2228420  = 0.000347), IL13 rs20541  = 0.000138), IL17A rs7747909  = 0.015979), MAPT rs2258689  = 0.012260), TNFRSF1B rs2275416  = 0.027425). A negative association with PCa risk was instead observed for IL12RB2 rs2229546  = 0.001876), IL4R rs2301807  = 1.87 \u00d7 10\u22126), and TNFRSF1B rs636964  = 0.034034). Additionally, no statistically significant association between PCa risk and the nucleotide substitutions found within ACE, ATG7, CD28, IFNG, IFNGR1, IFNGR2, IL12A, IL12B, IL18, IL18R1, IL2, IL2RA, IL4R, IL5, IL7R, IRF5, LAG3, R1I2, PACGR, PTPN22, TNFRS, TRAF6, and VDR.Using the predictors identified in Finally, GLMM analysis was performed to explore the relationship between statistically relevant variants identified as useful predictors in the initial model  and the IL2RA rs7093069 and TNFRSF1B rs2275416) were significantly associated with GS. Accordingly, the variables rs7093069 and rs2275416  represent good predictors for the PCa severity. Indeed, with GS with values of around 6, rs7093069 and rs2275416 do not appear as informative variables, while with values of around 9, they appear strongly associated with the severity of the pathology. In addition, SLC11A1 rs17221959 and both TNFRSF1B rs636964 and rs2275416 were found to be significantly associated with serum PSA levels. Overall, only GS and PSA have been positively selected by the model, while other variables  were removed during the backward selection. Indeed, in the dataset here, analyzed age, cTNM, and BMI demonstrated to have no association with PCa, nor alone or taken together.Two polymorphisms (ACE2 and TMPRSS2) in a cohort of patients with PCa and a cohort of HCs. We identified several novel polymorphisms that contributed to PCa susceptibility. In particular, we found that six SNPs in several key genes, namely IL4R, IL12RB1, IL12RB2, TMPRSS2, and TNFRSF1B, were associated with a lower risk of PCa. In contrast, the eight SNPs identified in IL6, TMPRSS2, ACE2, IL12RB2, IL13, IL17A, MAPT, and TNFRSF1B were associated with a higher risk of PCa. PCa patients with specific polymorphisms in T-helper cytokine genes (IL13 rs20541 and IL4R rs2301807) may have an altered T-helper response and consequently, a potential imbalance in their immune system. Altered expression and increased circulating levels of both pro- and anti-inflammatory cytokines have been reported in several types of cancer [IL6 rs2069832 was positively associated with a higher risk of PCa is consistent with that of Slattery et al. [IL12RB1 rs3746190, IL12RB2 rs2307145, and rs2229546 were associated with decreased PCa risk. Interleukin 12 (IL-12) plays a pivotal role in stimulating natural killer (NK) cell cytotoxicity, inflammation, and boosting T-helper type I cells [IL12RB2 rs2229546, our results are in line with another study that found an association between rs2229546 and a reduced risk of cervical squamous cell carcinoma and HPV-18-positive cervical cancer [IL17A rs7747909 belongs to the 3\u2032-UTR region of interleukin 17A (IL-17A) and was found associated with a higher risk of PCa. Interestingly, Bedoui et al. reported a strong association between IL17A polymorphisms, including rs7747909, and the development of tolerance to colon rectal cancer therapy [IL17 has been reported more frequently in CRPC and neuroendocrine prostate cancer than in hormone-na\u00efve PCa [IL13 rs20541 polymorphism, located in exon 4, appears to be strongly associated with increased serum IgE levels in children [IL13 rs20541 was positively associated with the risk of PCa, whereas it was previously reported to be associated with a decreased risk of glioma [TMPRSS2 rs140141551 and ACE2 rs35803318 were associated with a higher risk of PCa than TMPRSS2 rs11701576, which was negatively associated with PCa risk. TMPRSS2 encodes a transmembrane serine protease 2 (TMPRSS2), which is expressed by prostate epithelial cells. To date, TMPRSS2 physiological function remains unclear, albeit its expression appears to be regulated by androgens in PCa cells [ACE2 encodes the angiotensin-converting enzyme (ACE2), which is aberrantly expressed in several cancers [The importance of the immune system in cancer onset and progression has been well established in cancer research. In the last decade, the notion of engaging the immune system as a patient-personalized weapon against cancer cells has undoubtedly revolutionized cancer therapy. A growing body of evidence has investigated the presence of variants in genes implicated in anti-tumor immune responses and cancer risk. Therefore, we investigated the presence of polymorphisms in a panel of 35 genes involved in pro- and anti-inflammatory responses, autophagy-lysosome pathway and two PCa-associated genes  whose expression is not restricted to neurons. In truth, MAPT overexpression was previously associated with lower GS, suggesting its potential as marker of PCa prognosis [IL2RA rs7093069 and TNFRSF1B rs2275416, implying the strong impact of genetic variability within immune genes on cancer development, progression, and staging. Alterations in IL2RA relative expression have been reported in groups of patients with different GS [TNFRSF1B rs2275416 was associated with serum PSA levels, GS, and PCa risk. TNFRSF1B encodes the receptor for tumor necrosis factor-\u03b1 (TNFR2), which plays a crucial role in promoting tumor growth, invasion, and metastasis. TNFR2 is highly expressed in T regulatory cells (Tregs) and its presence is crucial in the TME [TNFRSF1B rs2275416 may also affect PCa prognosis, given its association with both GS and PSA levels. SLC11A1 rs17221959 was also associated with serum PSA levels. This finding is also consistent with the results recently reported by Zhu et al. in which SLC11A1 rs7573065 C > T was associated with increased risk of PCa and low overall survival; meanwhile, higher mRNA expression was found in PCa tissues compared to normal tissues [tability ,42,43,44rognosis  and resprognosis . In our erent GS . On the  the TME . Hence,  the TME ,50,51. A the TME , it is p tissues . Despite+-EDTA test tubes. PCa assessment was based on the clinical examination and histopathological analysis of isolated biopsies performed in all patients. The ethical committee of the AOU, Sassari, approved this study. Two cohorts, comprising 47 patients with PCa and 43 HCs, were recruited. The demographic and clinical characteristics of the patients with PCa and HCs are summarized in PBMCs were isolated by Ficoll-Histopaque gradient centrifugation . PBMCs were washed twice using phosphate-buffered saline (PBS 1X) and stored at \u221280 \u00b0C in fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) . Genomic DNA extraction from PCa and HCs samples was performed using the DNeasy Blood and Tissue Kit . After thawing, cells were washed twice in PBS 1X to remove DMSO and FBS traces and then resuspended in 200 \u03bcL PBS 1X before proceeding according to the manufacturer\u2019s instructions. The final DNA concentration of each sample was measured using Nanodrop One , and DNA quality was assessed by calculating the ratios of absorbance at A260/A280 and A260/A230.Library preparation and DNA sequencing were performed at the CRS4 Research Center . Thirty-five genes involved in both innate and adaptive immune responses as well as PCa-associated genes were sequenced. Two AmplySeq primer pools were designed and purchased from Illumina for the gene panel analysis. The PCR protocol and reagents were adapted from QIAGEN Application Note (Qiagen 1104745 10/2016) to amplify the entire length of each gene. The PCR products were quantified using Qubit, and equimolar amounts were pooled. Gene-panel PCR pools were grouped to achieve the appropriate sequencing coverage. Libraries were obtained using Nextera DNA Flex with 100 ng of DNA and indexed with IDT for the Illumina Nextera DNA UD Indexes Primer Set . PCR products were purified with 1X AMPure XP beads , and the libraries were quantified using Qubit. A loading pool consisting of 90 samples (47 PCa and 43 HCs) was diluted to 9 pM before sequencing using the MiSeq Reagent Kit v3 600-cycle (Illumina). The BaseSpace Sequence Hub (Illumina) was used to conduct demultiplexing and fastq file generation, and two distinct analyses were performed for each sample. Gene panel sequences were analyzed using an in-house pipeline. http://ihg2.helmholtzmuenchen.de/cgi-bin/hw/hwa1.pl, accessed on 1 January 2022). Fisher exact test, chi-square test, and both allelic and genotypic odds ratios (ORs) were used to compare genetic variants between PCa and HC cohorts. To test the relationship between cancer onset and nucleotide substitution for a given locus, multivariate regression analysis was carried out by applying backward selection, in which non-significant covariates were removed step by step until the final model was obtained. A GLMM implemented in the R package lme4 [https://www.r-project.org/, accessed on 1 January 2022), was used to perform the analysis. Odds ratios with 97.5% confidence interval were calculated using the R-package \u201coddsratio\u201d .The allele and genotype frequency estimates were calculated using (age lme4  in the R"}
+{"text": "Our results show that a small amount of Rb+ was enough to induce the crystallization of the \u03b1-FAPbI3 phase and suppress the formation of the yellow non-photoactive phase; the grain size increased, and the product of the carrier mobility and the lifetime (\u03bc\u03c4) improved. As a result, the fabricated photodetector exhibited a broad photo-response region, from ultraviolet to near-infrared, with maximum responsivity (R) up to 11.8 mA W\u22121 and excellent detectivity (D*) values up to 5.33 \u00d7 1011 Jones. This work provides a feasible strategy to improve photodetectors\u2019 performance via additive engineering.Photodetectors are widely employed as fundamental devices in optical communication, automatic control, image sensors, night vision, missile guidance, and many other industrial or military fields. Mixed-cation perovskites have emerged as promising optoelectronic materials for application in photodetectors due to their superior compositional flexibility and photovoltaic performance. However, their application involves obstacles such as phase segregation and poor-quality crystallization, which introduce defects in perovskite films and adversely affect devices\u2019 optoelectronic performance. The application prospects of mixed-cation perovskite technology are significantly constrained by these challenges. Therefore, it is necessary to investigate strategies that combine crystallinity control and defect passivation to obtain high-quality thin films. In this study, we incorporated different Rb The family of all-inorganic perovskites has fewer members owing to the limitations of the tolerance factor and octahedral factor [6]4\u2212. Numerous chemically versatile multidimensional OIHP structures can be obtained by modifying the A-site organic cation component due to the wide variety of organic cation groups. OIHPs can adapt to various optoelectronic applications such as solar cells, photodetectors, and light-emitting diodes [3) has been extensively investigated and exhibits outstanding photoelectric conversion efficiency as a potential absorber in solar cells [3 crystal structure [3 has a tetragonal rather than cubic structure at room temperature, with a band gap of 1.51 eV due to the small size of the MA ion [3, formamidinium lead iodide (FAPbI3) possesses higher symmetry and is closer to a cubic structure [3 exhibits superior thermal stability and a favorable band gap closer to the optimal band (1.4 eV) [3 one of the most promising absorbing layers for high-performance photodetectors [3 perovskite has only two phases at room temperature: one is a photoactive cubic phase (\u03b1-phase), and the other is a non-photoactive hexagonal phase (\u03b4-phase) [3 without the \u03b4-phase and stabilizing the \u03b1-phase have become urgent problems. In particular, mixed-cation engineering has been demonstrated as an effective strategy for improving perovskites\u2019 phase stability. Jeon et al. proved that a small amount of MA+ is enough to induce improved crystallization of black \u03b1-FAPbI3 [+ can effectively suppress the formation of yellow-phase impurities in MA/FA perovskite compounds; a device prepared based on a Cs/MA/FA triple-cation yielded stabilized power-conversion efficiency exceeding 21% [\u2212 and Br\u2212) to form different halide compositions, which incorporate various impurities and defects into the film [+ or Rb+ in triple-cation perovskites has been proven to alleviate lattice strain, which is beneficial for suppressing the formation of FA+ and I\u2212 vacancies [+ does not dope the lattice of Cs/MA/FA perovskite, avoiding the introduction of extra lattice strains. Instead, Rb+ may form various Rb-containing compounds with excess PbI2 and can act as a potential passivation layer, which is beneficial for decreasing non-radiative recombination and improving carrier transport [In the past decade, solution-processed organic\u2013inorganic hybrid perovskites (OIHPs), which can be prepared by a simple and low-cost spin coating method, have garnered widespread attention in the field of optoelectronics due to their superior properties, such as high absorption coefficient, high carrier mobility, low exciton binding energy, and adjustable band gap ,2,3. In l factor . Compareg diodes ,6,7. OIHg diodes ,9. In paar cells ,11,12. Htructure . Moreovee MA ion . Comparetructure ,16. Furt(1.4 eV) . The redetectors ,19,20. G\u03b4-phase) . Unfortu\u03b1-FAPbI3 . Furtherding 21% . Neverthding 21% ,24,25. Pthe film . It is wthe film . To addrthe film . For exathe film . F4TCNQ the film ,31. Receacancies ,33. Moreransport . These f+ into the triple-cation perovskite precursor solution Cs0.05(MA0.17FA0.83)0.95Pb(I0.83Br0.17)3 (Cs0.05(MA0.17FA0.83)0.95 is abbreviated as CsMAFA in this paper). We demonstrated via X-ray diffraction and scanning electron microscopy that the optimal ratio of Rb+ can improve crystallinity and suppress the formation of non-photoactive phases. Incorporating Rb+ increased the grain size and reduced grain boundaries, which contributed to the inhibition of non-radiative complexation and improved carrier mobility. Therefore, the fabricated photodetector exhibited a broad photo-response region from ultraviolet (UV) to near-infrared (NIR) with a maximum responsivity of up to 11.8 mA W\u22121 and an excellent D* value of up to 5.33 \u00d7 1011 Jones. The maximum switching ratio was 7252, and the rise and decay times were 208.3 \u03bcs and 288.4 \u03bcs, respectively. During measurement under ambient light, no significant photocurrent decays were found during multiple cycles.In this study, we incorporated different ratios of Rb2, 99.0%, Acros, Geel, Belgium) were purchased from Beijing Innochem Science & Technology Co., Ltd., Beijing, China. Lead (II) iodide  was purchased from Xi\u2019an Polymer Light Technology Corp., Xi\u2019an, China. Super-dehydrated dimethylformamide  was purchased from Beijing J&K Scientific Technology Corp., Beijing, China. Dimethyl sulfoxide  was purchased from Alfa Aesar Chemical Corp., Taiwan, China. All chemicals were purchased from commercial suppliers and used without further processing.Formamidinium iodide , cesium iodide , methylammonium bromide , and lead (II) bromide (PbBr2 layer was deposited from a precursor of titanium diisopropoxide (0.6 mL) and bis(acetylacetonate) (0.4 mL) in anhydrous propan-2-ol (7 mL). The precursor solution was sprayed onto the glass substrate, which was subsequently annealed on a hot plate at 460 \u00b0C for an hour and then left to cool to room temperature. The Cs/MA/FA perovskite precursor solution was prepared by dissolving 1 M FAI, 1.1 M PbI2, 0.2 M MABr, 0.063 M CsI, and 0.22 M PbBr2 in anhydrous DMF/DMSO 4:1 (v/v). Next, the quadruple cation was prepared by adding 0%, 5%, and 10% of the 1.0 M RbI solution to the Cs/MA/FA perovskite precursor solution. The quadruple-cation precursor solution was spin-coated onto the glass/c-TiO2 substrate at 1500 rpm for 5 s and then at 4000 rpm for 30 s; the glass/c-TiO2/perovskite substrate was subsequently annealed on a hot plate at 100 \u00b0C for 60 min. A 60 nm gold interdigitated electrode (distance between electrodes: L = 50 \u00b5m) was deposited via thermal evaporation under a pressure of 4 \u00d7 10\u22124 Pa. A black mask was pasted on to guarantee a standard illumination area of 5 mm2.The glass substrates were cleaned twice with deionized water, and then with ethanol. After each cleaning, the glass substrates were sonicated for 20 min. After sonication, the glass substrates were blown dry with nitrogen, and then a UV\u2013ozone treatment was performed for 30 min. A compact TiO\u22121). The morphologies of thin films were measured using a SU8010 SEM . The photodetector\u2019s performance was measured using an LED light source with different wavelengths. All current data were recorded using an electrochemical workstation with a CIMPS system .UV\u2013vis spectra were measured using the UV\u20132450  from 400 nm to 900 nm. XRD patterns were obtained with an X-ray diffractometer  using Cu\u2013K\u03b1 radiation (1.5418)  perovskites have achieved impressive high-efficiency solar cells (25.7%) and become a popular material in the optoelectronic devices field. However, FAPbI3\u2019s octahedral skeleton is unstable owing to the lattice distortion caused by FA ions, which may form vacancies in the perovskite lattice. According to the principle of mechanical force, internal stresses occur and resist this effect when large cations stretch the octahedral skeleton, leading to an unstable state in the whole system [+ and Cs+, at the A-site [+, MA+, and Br\u2212 into the FA-based perovskite\u2019s lattice, yielding a higher symmetry.Possessing a broad optical absorption spectrum and high absorption coefficient, black-phase FAPbIe system . Figure e system . One stre A-site . Figure + incorporation on the growth of CsMAFA perovskite films, X-ray diffraction measurements were taken. As shown in +) film, a typical characteristic peak appeared at 14.06\u00b0. The main reflection peak of \u03b1-FAPbI3 (14\u00b0) shifted to a higher angle, indicating that the doping of Cs+ caused the contraction of the MAFA-based perovskite lattice, which facilitated the release of lattice strain. Moreover, we noted small side peaks at 11.56\u00b0 and 12.96\u00b0, corresponding to non-photoactive (\u03b4-FAPbI3) and excess PbI2, respectively. This was a typical observation of the incomplete conversion of mixed-cation perovskites to the photoactive phase. Subsequently, when we introduced 5% of Rb+, the main reflection peak further shifted to 14.10\u00b0 and the peak intensity increased, demonstrating that the introduction of Rb+ could further alleviate lattice distortion in the CsMAFA-based perovskite. In particular, the intensity of the \u03b4-phase diminished when we introduced Rb+, and 5% of Rb+ suppressed the formation of the \u03b4-phase most effectively, which indicated that a small amount of Rb+ was able to promote the CsMAFA-based perovskite crystallizing to the \u03b1-phase. Notably, the decrease in the small side peak at 12.96\u00b0 may suggest that Rb+ consumed the residual PbI2 reaction to form the Rb-containing mixture [+ may compete with the incorporation of Cs+ into the perovskite lattice by forming a stable mixed Cs/Rb hexagonal phase.To further investigate the effect of Rb mixture . Moreove+ concentration was increased to 10%. Therefore, excessive Rb+ may be detrimental to the crystallization of CsMAFA-based perovskite. In terms of crystal quality, the optimal ration of Rb+ was 5%.The main reflection peak shifted slightly to 14.08\u00b0 and the diffraction peak\u2019s intensity decreased when the Rb0.05(CsMAFA)0.95 were investigated using scanning electron microscopy (SEM) (0.05(CsMAFA)0.95 film improved from 211 nm to 226 nm, which indicates that Rb+ promoted crystallization and matched the XRD result. Energy Dispersive Spectroscopy (EDS) on the surface of the Rb 0.05(CsMAFA)0.95 film also confirmed the presence of Rb+, as shown in + to the perovskite precursor solution could effectively control the crystallization process of the film, which was beneficial for yielding highly crystalline and crack-free perovskite films to improve photodetectors\u2019 performance.It is well known that films with a large grain size and fewer grain boundaries are essential for reducing carrier recombination and enhancing carrier mobility, as pinholes or cracks tend to form non-radiative recombination centers that adversely affect devices\u2019 performance. The surface morphologies of CsMAFA and Rbpy (SEM) a,b. As s+ ratios. The CsMAFA film has a strong absorbance in the region of wavelengths less than 450 nm, and the absorption band could extend beyond 800 nm. The Rb0.05(CsMAFA)0.95 film\u2019s absorbance significantly improved in the region of wavelengths less than 752 nm, with a band gap of 1.65 eV calculated using the Tauc formula  product. To quantitatively characterize the effect of different Rb+ ratios on the detector\u2019s charge collection efficiency, we fabricated three devices separately and performed photoconductivity measurements. In this paper, we used a gold interdigitated electrode to collect carriers, as shown in \u00b5\u03c4 values. The Hecht equation is expressed as follows [I0 is the saturated photocurrent and d is the distance between electrodes [\u00b5\u03c4 values of CsMAFA, Rb0.05(CsMAFA)0.95, and Rb0.1(CsMAFA)0.9 were 8.37 \u00d7 10\u22126 cm2 V\u22121, 1.03 \u00d7 10\u22125 cm2 V\u22121, and 9.01 \u00d7 10\u22126 cm2 V\u22121, respectively. The Rb0.05(CsMAFA)0.95 device yielded the highest \u00b5\u03c4 value, further demonstrating the beneficial effect of small amounts of Rb+ on the crystallization of films. \u22122 and a bias of 4 V. The calculated on\u2013off values for CsMAFA, Rb0.05(CsMAFA)0.95, and Rb0.1(CsMAFA)0.9 were 64, 1519, and 37, respectively. Notably, the photocurrent of the Rb0.1(CsMAFA)0.9 device decreased instead, which could probably be attributed to cesium\u2019s tendency to form a stable Cs0.5Rb0.5PbI3 phase in the presence of excess rubidium [+ could yield the highest switching ratio. Based on the above analysis, we determined that the optimal Rb+ ratio was 5% and measured the current\u2013voltage (I\u2013V) characteristics (R) is an important parameter characterizing the ability of a photodetector to convert photons into a current signal, and it can be calculated using the following equation [pI is the photocurrent, dI is the dark current, P is the light intensity, and S is the effective area of the device. R versus light intensity. The photocurrent increased linearly with increasing light intensity under 454 nm illumination, whereas the responsivity decreased with increasing light intensity due to the dominance of bimolecular recombination under strong light [R was 11.8 mA W\u22121 at a light intensity of 0.5 mW cm\u22122. Linear dynamic range (LDR) is a figure-of-merit that represents the range of light intensity over which a detector can output photocurrent linearly. LDR can be calculated using the following formula [maxP is the saturation light intensity under which the photocurrent begins to deviate from linearity and minP is the initial light intensity. \u22122 to above 20 mW cm\u22122, yielding an LDR of 32 dB. We further evaluated the specific detectivity, which is a parameter characterizing a detector\u2019s ability to detect weak light and is calculated using the following expression:R is the responsivity, e is the unit charge, dI is the dark current, and S is the active area. D* with light intensity. The maximum value of D* was 5.33 \u00d7 1011 Jones under light irradiation at 454 nm, 0.5 mW cm\u22122, which proves its potential application in weak near-ultraviolet light sensing and imaging. External quantum efficiency (EQE) is also a key photodetector parameter and is defined as the ratio of the number of electron\u2013hole pairs collected by the external circuit to the number of incident photons. It is calculated as follows:R is the responsivity, h is Planck\u2019s constant, c is the speed of light, e is the elementary charge, and \u03bb is the wavelength of the incident light. The EQE versus wavelength is shown in EQE reached a peak of 1.5% at a wavelength of 454 nm, with a D* up to 2.49 \u00d7 1011 Jones due to the high absorption coefficient in the near-UV band. The EQE gradually decreased in the region of wavelengths above 632 nm; the lowest D* was 9.38 \u00d7 108 Jones at 868 nm.Detectors\u2019 charge collection efficiencies can be evaluated using the carrier mobility\u2013lifetime ( follows :(1)I=I0\u03bcectrodes . The calrubidium . As a reeristics a of the equation :(2)R=Ip\u2212ng light . The max formula :(3)LDR=2I0 is the amplitude of the current and \u03c4 is the time constant. The calculated rise time (r\u03c4) and fall time (f\u03c4) were 208.3 \u03bcs and 288.4 \u03bcs, respectively, which demonstrated the effective extraction of carriers at the interface between the perovskite and Au electrodes and the perovskite thin film\u2019s high charge mobility. The quadruple-cation perovskite films\u2019 ultra-fast response time and broad absorption spectrum from the ultraviolet to the near-infrared demonstrated their promising application as broadband photodetectors. A summary of the state-of-the-art research regarding perovskite photodetectors\u2019 performance is presented in Moreover, response time is a key parameter that represents the speed of detecting transient light signals. Rise time is the time duration from 10% to 90% of the saturation current. As shown in \u22122 light illumination; this demonstrated good photosensitive stability under ambient conditions.The repeatability of photodetectors in detecting light is an important factor limiting their practical application. It is well known that detectors based on mixed-cation perovskite thin films suffer from severe baseline drift during continuous on\u2013off due to ion migration and phase segregation. As shown in + proportion into the precursor solution helped obtain highly crystalline perovskite films with large grain sizes and few grain boundaries. A reduction in nonradiative or undesired recombination improved the carrier mobility\u2013lifetime product (\u03bc\u03c4). Hence, the photodetector based on mixed-cation perovskite (Rb0.05(CsMAFA)0.95) thin film exhibited a good linear response over a light intensity range of 0.5 mW cm\u22122 to 20 mW cm\u22122 with a maximum responsivity of 11.8 mA W\u22121 and an excellent on\u2013off ratio of up to 7252. Simultaneously, the photodetector showed a broad absorption spectrum between 400 and 765 nm, with a maximum D* of 5.33 \u00d7 1011 Jones. The effective extraction of carriers at the interface between the perovskite and Au electrodes facilitated the device\u2019s ultra-fast response time; the r\u03c4 and the f\u03c4 were 208.3 \u03bcs and 288.4 \u03bcs, respectively, with no significant decrease in photocurrent during multiple cycle measurements. These results verify perovskite\u2019s potential as a next-generation high-performance photodetector.In conclusion, we developed a photodetector based on a mixed-cation perovskite thin film. The incorporation of an optimal Rb"}
+{"text": "Effects of hot pixels on pixel performance in light and dark environments have been investigated in pinned photodiode 0.18 \u03bcm backside illuminated CMOS image sensors irradiated by 10 MeV protons. After exposure to protons, hot pixels and normal pixels are selected from the whole pixel array, and their influences on key parameters are analyzed. Experimental results show that radiation-induced hot pixels have a significant impact on pixel performance in dark environments, such as dark signal nonuniformity, long integration time, and random telegraph signal. Hot pixels are caused by defects with complex structures, i.e., cluster defects. Furthermore, the dark current activation energy result confirms that the defects causing the hot pixels have defect energy levels close to mid-gap. Backside illuminated CMOS image sensors (BSI CIS) feature a higher sensitivity compared to the frontside illuminated CIS, and have been extensively used for space imaging missions ,2,3. TheMuch effort has been made to study displacement damage-induced hot pixels ,8,9,10, t damage ; (c) thet damage . Although these mechanisms have been proposed in previous work, it can be known that the generation of hot pixels depends on many factors, such as the electric field in the pixel, the defect types and density, and the defect energy level within the Si bandgap. Hence, displacement damage-induced hot pixel is still a complicated issue, and the dominant mechanism needs to be identified. Moreover, the previously published papers mainly focus on the behaviors of hot pixels. Additional work is needed to investigate the effects of hot pixels on pixel performance in light and dark environments. The purpose of this paper is to investigate the effects of hot pixels on pixel performance in light and dark environments and to provide a new perspective for understanding hot pixels. We randomly select the hot pixels and normal pixels from the whole array, and study their influences on key parameters, including full well capacity, quantum efficiency (QE), conversion gain (CVG), dark signal nonuniformity, and random telegraph signal (RTS). From the perspective of the defects, the correlation between the hot pixels and RTS pixels is discussed in detail. In addition, the physical mechanism of radiation-induced hot pixels is identified through dark current activation energy measurement.\u2212, and the full well capacity is 90 ke\u2212. The devices under study are the BSI CIS manufactured in a 0.18 \u03bcm CMOS process dedicated to imaging. The image sensors are constituted of 2048 \u00d7 2048-11 \u03bcm-pitch-4T pixels using the pinned photodiode (PPD). The image sensor supports two modes: STD mode at 48 fps and HDR mode, which is designed to achieve a high dynamic range (93 dB) at 24 fps. The HDR mode is selected to acquire raw images in this work. The shutter type of the image sensor is an electronic rolling shutter. The temporal dark current is 1.6 e9 p\u00b7cm\u22122, and the irradiation duration is 100 s. The cumulative displacement damage dose is 39.4 TeV/g, and the total ionizing dose is 2.8 krad(Si). The proton flux is 5 \u00d7 107 p\u00b7cm\u22122\u00b7s\u22121, and the proton beam homogeneity is within \u00b15%. The image sensors were irradiated at room temperature with all the pins grounded.BSI CIS was irradiated by 10 MeV protons at the Institute of Heavy Oon Physics, Peking University, CHN. The proton fluence is 5 \u00d7 10Key parameters test and characterization were performed at 24 \u00b0C one week after exposure to protons according to the standard for characterization of image sensors and cameras (EMVA1288). RTS pixel test was carried out with the successive acquisition of the raw images. The duration of raw image acquisition is 3 h, and the integration time is set to 1 s. RTS pixels can be detected from the subarray of the interest by an automated RTS test software, which is implemented using MATLAB software and is based on the sharp edge detection technique described in ref. . The RTSdark), which is independent of the particle types and energy [d energy ,17. Thus\u2212/s, and the one is 415.4 e\u2212/s for normal pixels). In order to obtain the response of the selected pixels in light and dark environments, the locations of the selected hot pixels and normal pixels are recorded to track these pixels.Hot pixels are located at the exponential tail, and normal pixels are in the Gaussian peak. According to the definition of the hot pixels, we select 10 hot pixels with the maximum dark current from the exponential tail and 10 normal pixels from the Gaussian peak. The normal pixel is randomly selected from the whole array and meets statistical requirements. 5 e\u2212/s (corresponding to the photo-generated current). When the integration time is 0.2 s, the pixel output reaches full well capacity. Full well capacity is defined as the maximum amount of electrons stored in the pixel and is related to the light flux, VLOTG, and temperature [perature . The stu\u03b7\u03bb) and can be converted into an electron (e\u2212) through the CVG. CVG is defined as how much digital number variations are produced by an electron. \u2212 in the studied BSI CIS, and there is no difference in CVG for the hot pixels and normal pixels. Therefore, it can be concluded that radiation-induced hot pixels have no influence on the optoelectric performance in BSI CIS.QE represents the pixel\u2019s ability to convert the incident photons to electrons at distinct wavelengths and is expressed as follows:\u2212. Dark signal nonuniformity refers to dark signal variations from pixel to pixel, and its unit is eExcept for the hot pixel, the RTS pixel is another anomalous pixel in solid-state imaging devices and refers to dark current fluctuation between two or more discrete levels ,22. RTS dcI is the dark current, A is the preexponential factor, aE is the activation energy, k is the Boltzmann constant, and T is the temperature. Considering the temperature dependence of each parameter [tE is the defect energy level, iE is the mid-gap energy level, and gE is the Si bandgap. Considering the temperature dependence of the Si bandgap, we obtain the expression of activation energy as a function of the energy level into the silicon bandgap as:To harvest the energy level of the defects, dark current was tested at distinct temperatures, and the corresponding activation energy was calculated according to the Arrhenius law ,26,27. that the defect energy level is located at the mid-gap when the dark current activation energy is 0.649 eV, and the generation ability of the defect is maximized. In this paper, the effects of hot pixels on the pixel performance are investigated in pinned photodiode 0.18 \u03bcm backside illuminated CMOS image sensors irradiated by 10 MeV protons up to 39.4 TeV/g. After proton irradiation, hot pixels are produced by displacement damage through the elastic and inelastic nuclear scattering. Hot pixels are selected from the exponential tail of the dark current distribution, and normal pixels are randomly selected from the whole array. The method of selecting two kinds of pixels meets statistical requirements.Hot pixel has no influence on optoelectric performance since the photo-generated current is much larger than the dark current, and the impact of the dark current can be neglected. However, it is worth pointing out that the effects of hot pixels on pixel performance in dark environments should be a concern. Hot pixel behaves as a \u201cbright spot\u201d in the captured dark images and gives rise to the increase of dark signal nonuniformity, which has an impact on the dim target detection mission. Dark signal nonuniformity degradation is strongly dependent on the number of hot pixels. A hot pixel is able to reach the situation of full well capacity at the long integration time without light illumination. Above the specific integration time, the hot pixel makes no response and is not an effective pixel anymore. This case has an impact on the astronomical observation mission, in which the observation at the long integration time is required for the image sensors. It is confirmed that there is a correlation between hot pixels and RTS pixels, which is the hot pixels tend to exhibit RTS characteristics. The possible explanation is that the defects responsible for the hot pixel have a complex structure. The complex defects not only spontaneously transition from one configuration to another configuration due to the instability of the defects, but also have a big probability of introducing energy levels close to mid-gap. According to the characteristics of displacement damage-induced defects, cluster defects, which have complex defect structures, are more likely to cause the production of hot pixels. Furthermore, the result of dark current activation energy suggests that radiation-induced hot pixel is caused by defects with energy levels close to mid-gap."}
+{"text": "Numerous studies have demonstrated that hepatic fibrosis, a progressive condition as an endpoint of multiple chronic hepatic diseases, is largely characterized with the extensive activation of hepatic stellate cells (HSCs). The precise effect of miR-488-5p in HSCs during hepatic fibrosis has not been elucidated.4), high-fat diet (HFD) and bile duct ligation (BDL). In vitro, the effects of miR-488-5p in HSCs were examined through cell proliferation assay and apoptosis. Luciferase reporter assay was applied to identify the underlying target of miR-488-5p. In vivo, the effects of miR-488-5p were explored through mouse liver fibrosis models.In our study, qRT\u2010PCR was applied to assess the level of miR-488-5p in activated HSCs stimulated by TGF-\u03b21. We built murine liver fibrosis models with carbon tetrachloride  expression via straightly binding onto the 3\u2032 UTR of its mRNA, which sequentially restrained the TGF-\u03b2/Smad2/3 pathway. TET3 inhibition induced by the overexpression of miR-488-5p reduced extracellular matrix deposition, which contributed to mitigating mouse liver fibrosis.We highlight that miR-488-5p restrains the activation of HSCs and hepatic fibrosis via targeting TET3 which is involved in the TGF-\u03b2/Smad2/3 signaling pathway. Collectively, miR-488-5p is identified as a potential therapeutic target for hepatic fibrosis.The online version contains supplementary material available at 10.1007/s12072-022-10404-w. However, under the effect of the longtime harmful factors, liver fibrosis will sequentially transform into liver cirrhosis, hypohepatia and even hepatocellular carcinoma [Hepatic fibrosis, as a self-repair mechanism, is quite common when stimuli such as alcohol, hepatitis B virus (HBV), carbon tetrachloride , belonging to a class of small non\u2010coding RNAs, serve as post-transcriptional regulators by negatively regulating downstream target gene expression , 10. Inc4, high-fat diet (HFD) and bile duct ligation (BDL) would be mitigatory once the mice were treated with ago-miR-488-5p. To sum up, our research findings demonstrate that miR-488-5p acts as a vital role in the hepatic fibrosis and might be a possible therapeutic target.The purpose of our research was intended to detect the role of miR-488-5p in hepatic fibrosis. First, the data presented that miR-488-5p were remarkably downregulated in the activated HSCs stimulated by TGF-\u03b21 and mouse liver fibrosis models. Degree of activation of HSCs was restrained by miR-488-5p overexpression, as evidenced by the inhibited proliferation, promoted apoptosis and the reduction of profibrotic marker expression. We identified that tet methylcytosine dioxygenase 3 (TET3), serving as a target gene of miR-488-5p, played a pivotal role in hepatic fibrosis. More importantly, the degree of hepatic fibrosis attributed to CClAll fibrotic specimens from patients with advanced liver disease and normal tissues from patients with hepatic hemangioma were collected in the first Affiliated Hospital of Nanjing Medical University. All samples were straightly frozen in liquid nitrogen once the surgical resection was completed.We purchased the LX-2 cells, as a kind of cell line of hepatic stellate cells (HSCs), from the Cell Center of Shanghai Institutes for Biological Science. LX-2 cells were cultured with the Dulbecco\u2019s Modified Eagle Medium  which contained 10% fetal bovine serum (FBS) and 1% antibiotics . Addition of TGF-\u03b21 (10\u00a0ng/mL) into DMEM with no serum was intended to activate the LX-2 cells for 0, 3, 6, 12, and 24\u00a0h.4  through intraperitoneally injection twice a week for 8\u00a0weeks was intended to establish the hepatic fibrosis model. Meanwhile, olive oil (2\u00a0mL/kg) was used by the same way as the control group. The high-fat diet  was used to feed the mice for 24\u00a0weeks to establish the HFD model. Meanwhile, normal diet was used in the control group. For the control and bile duct ligation (BDL) group, sham or BDL surgery was performed in the mice, respectively.Male C57BL/6 mice, 8 weeks old, were obtained and fed based on standard conditions at the animal house of Nanjing Medical University. Treatment with CClThe RIPA buffer embracing 1% protease inhibitor and 1% PMSF was employed to extract the total protein based the standard protocol from the manufacturer (Beyotime). The extracted samples were separated via 10% SDS-PAGE and sequentially transferred to PVDF membranes. All antibodies were used in this study: TET3, TIMP-1 (Abcam), TGF-\u03b2, collagen-I, \u03b1-SMA, Smad2, p-Smad2, p-Smad3, Smad3, and GAPDH rabbit antibodies and HRP-conjugated anti-rabbit IgG antibodies .The TRIzol reagent was employed to extract total RNA on the basis of the standard instructions. Subsequently, RNA reversion and cDNA synthesis were carried out through using Reverse Transcription Kit (Vazyme). mRNA amplification and cDNA quantification were carried out through using SYBR green (Vazyme) on the basis of standard protocols. The levels of miR-488-5p and U6 were assessed via using TaqMan miRNA assay system (Life Technologies Corporation). The normalization of genetic level rests with U6 or \u03b2-actin level, respectively. The primers used are presented in Supplementary material.4 and HFD liver fibrosis models for 2\u00a0weeks was finished, we administrated ago-miR control and ago-miR-488-5p into the mice twice a week. After the treatment with ago-miR control and ago-miR-488-5p twice a week for 2\u00a0weeks, the liver tissues from BDL model mice were obtained. Eventually, we collected the liver tissues for further detection.miR-488-5p mimics, scrambled miRNA (SCR-miRNA), TET3 lentivirus (LV-TET3), and control lentivirus (LV-NC), ago-miR-488-5p and ago-miR control were obtained from GenePharma. Transfection process was auxiliary with the existence of lipofectamine 2000 (Invitrogen) for 48\u00a0h for further researches. Mice were subjected to the treatment with ago-miR control and ago-miR-488-5p (20\u00a0nmol/200\u00a0mL) through tail injection. Once establishing the CClWe planted LX-2 cells into 6-well plates at a density of 1000 cells per well and cultured for 15\u00a0days. The supernatant was discarded, and the clone was fixed with 4% paraformaldehyde. Staining was performed using a solution of crystal violet (staining time: 30\u00a0min). Eventually, the number of clone formations was counted.We planted LX-2 cells into 96-well plates at a density of 2000 cells per well and cultured them with DMEM supplemented with CCK-8 reagent (10\u00a0\u03bcL/well) for 5\u00a0days to evaluate the cell viability. Once incubation for 2\u00a0h was completed, the absorbance (450\u00a0nm) of each well was observed.The apoptotic level of LX-2 cells was evaluated through the Apoptosis Detection Kit . After incubation with 5 \u03bcL Annexin V-FITC and 5 \u03bcL PI for 20\u00a0min, each cell sample was mixed with 400 \u03bcL binding buffer. All steps were followed by manufacturer\u2019s protocol.After liver tissues were totally fixed with 4% paraformaldehyde, they were embedded in paraffin. Subsequently, the paraffin block was made into slice and dyed with hematoxylin and eosin (HE). The Masson\u2019s-stained or Sirius red\u2019s-stained sections were used to assess the degree of liver fibrosis using the METAVIR scoring method. Eventually, the positive area was evaluated by pathologist to present the collagen deposition.After we obtained the tissue samples, the paraffin embedding was completed followed by standard steps. The paraffin block was made into slices (4\u00a0\u03bcm) which were incubated with anti-TET3 and anti-\u03b1-SMA for 12\u00a0h at 4\u00a0\u00b0C. Afterwards, the secondary antibody conjugated with HRP was utilized to incubate the slices for 1\u00a0h and eventually were dyed with 3,3\u2032-diaminobenzidine and hematoxylin.All steps obeyed to the standard protocols of IF staining. In short, LX-2 cells were incubated with primary antibody \u03b1-SMA, and followed by secondary goat anti-rabbit Texas Red-conjugated IgG. At the same time, DAPI was used to present the nuclei.The luciferase reporter vector embracing the wild-type sequences of 3\u2032\u2010UTR of TET3, and the mutated vector was constructed based on the binding sites. The luciferase reporter vectors were then co-transfected with miR\u2010488\u20105p mimic or miR-SCR. At 48\u00a0h after transfection, cells were harvested and tested with the Dual-Luciferase reporter assay system  followed with manufacturer\u2019s protocol. The relative luciferase activity was assessed as the ratio between firefly and Renilla luciferase activities detected by a dual-luciferase system .p value. Once p value\u2009<\u20090.05, was considered statistically significant.Statistical analysis results were shown as mean\u2009\u00b1\u2009SEM. The SPSS software and GraphPad Prism were employed to manage the data and calculate the First, the LX-2 cells were cultured with medium containing TGF-\u03b21 (10\u00a0ng/mL) for 0, 3, 6, 12 and 24\u00a0h. As shown in the Fig.\u00a04, BDL and HFD were administrated to the mice to, respectively, develop liver fibrosis. As shown in the Fig.\u00a04, BDL and HFD. Compared with the respective control (CTL) group, the histological structure was seriously damaged in CCl4 and BDL group, meanwhile the lipid vacuoles were abundant in the HFD group. At the same time the massive deposition of collagen was presented in CCl4, HFD and BDL group. Compared to their respective CTL group, the mRNA level of fibrosis-related markers containing \u03b1-SMA, tissue inhibitor of metalloproteinases 1 (TIMP-1) and Collagen-I were significantly upregulated  was a probable target gene of miR-488-5p. After consulting plentiful literatures, we found that tet methylcytosine dioxygenase 3 (TET3) was closely related with fibrosis , 15. ForFew studies have reported that TET3 played an essential role in activating TGF-\u03b2 pathway, which is closely involved in the liver fibrosis , 16. For4, HFD and BDL group were, respectively, subjected to the transfection with ago-miR control (CTL) and ago-miR-488-5p. As presented in the Fig.\u00a04 and BDL group; meanwhile, the number of lipid vacuoles was reduced in the HFD group   Fig.\u00a0d. EventuHepatic fibrosis, as a common pathological condition in clinic, is usually caused by the long-term exposure to viral hepatitis, excessive drinking, fat-rich diet, drug-induced liver injury and biliary obstruction, and so on \u201319. One The role of miRNAs in HSCs activation and hepatic fibrosis has attracted plenty of attention for the past few years , 23. For4, BDL and HFD models and human fibrotic liver samples. Therefore, our study indicated that miR-488-5p might play a vital role in inhibiting hepatic fibrosis, which was worthy of further study.Through referring many literatures concerning miRNA and liver fibrosis, we noticed that the microarray analysis for miRNA expression from a previous study revealed miR-488-5p was significantly downregulated in activated LX-2 cells which hinted that miR-488-5p may be involved with the progression of liver fibrosis . Our datBioinformatics analyses were executed to explore the underlying target of miR-488-5p through miRWalk and TargetScan, the further experiments indicated that tet methylcytosine dioxygenase 3 (TET3) was the direct target of miR-488-5p in LX-2 cells. TET3 belongs to the member of ten\u2013eleven translocation (TET) family proteins that participate in oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) . It had As we know, TGF-\u03b2 participates in the plenty of biological processes, containing cellular migration, development, proliferation and differentiation, and so on . MoreoveIn conclusion, we identified that miR-488-5p, as a fibrosis-inhibitory factor, was downregulated in the activated HSCs and fibrotic liver tissues. miR-488-5p overexpression restrained HSCs activation and hepatic fibrosis via restraining TET3/TGF-\u03b2/SMAD2/3 signaling pathway.Supplementary file1 (DOCX 16 KB)Below is the link to the electronic supplementary material."}