The current COVID-19 pandemic highlights the importance of novel molecular technologies and methods for rapid pathogen control . In justThis Research Topic collects contributions at the edge of the transition era to new sequencing technologies and their application to integrative omics in molecular surveillance . The selRíos-Castro et al. use DNA metabarcoding to analyze environmental samples from sediment and marine water samples of a marine ecosystem. They identified pathogens with high relevance for the aquacultural sector and which are of medical and veterinary interest. This study demonstrates the potential of NGS technology for environmental monitoring. Although 16S high-throughput sequencing is widely used worldwide, most protocols do not take into account the variable biomass of microbes, which affects the relative bacterial abundances per sample. Here, Cruz et al. describe a novel equivolumetric protocol that generates library sizes proportional to the total microbial load in 16S rRNA amplicon sequencing. Therefore, the authors introduced 16S amplicon sequencing with short reads in combination with Bayesian cumulative probability models to address this issue and to ultimately achieve higher proportionality between library sizes and total microbial load.Our journey into the era of new sequencing technologies begins with the monitoring of microbial communities in environmental samples. Studies focus on the detection of unknown microbes, the survey of the global distribution of known taxa, and the causes and implications of the composition of certain microbial communities. As an example, via bioinformatic approaches comprising pangenome comparisons, Multilocus Sequence Typing (MLST), Genomewide Association Studies (GWAS), and core-genome based phylogenies. All four studies involve at least short-read based Whole-Genome Sequencing (WGS) but also highlight the advantages and challenges of integrating data derived from technologies such as Oxford Nanopore long-read sequences (ONT). In the first study, Karthik et al. performed comparative genomics and WGS short-read sequencing of Indian isolates of Brucella melitensis, a zoonotic pathogen prevalent worldwide. Via pangenome analysis and MLST, the authors were able to predict several virulence genes and prophage regions and they were able to phylogenetically resolve different lineages of this species within India, where many isolates have been previously reported. The decreasing costs of NGS, open-source bioinformatics tools, and more easily operated laboratory technology make this approach more globally accessible for monitoring  proteins of Cronobacter sakazakii strains by adding MALDI-TOF-MS data to WGS data. The strains were isolated from powdered and dairy formulas and showed diverse virulence factors as well as resistance to beta-lactam antibiotics both in silico and in vitro. Their results reinforce a governmental decision to recall all involved powdered and dairy formulas in Chile in 2017 and further highlight the importance of continued molecular surveillance to detect product contamination by this pathogen. A third study deals with a foodborne pathogen, Listeria monocytogenes. Here, Chiaverini et al. performed phylogenetic analysis and GWAS on WGS short-read data to describe an outbreak due to this pathogen in Central Italy. Besides they were not able to identify any genomic differences between the outbreak and a reemergent strain, they found a possible differentiation by transcriptional factors and phage genes. Phages also play an important role in the dissemination of AMR genes of an unusual chimeric methicillin-resistant Staphylococcus aureus (MRSA) strain, as presented by Burgold-Voigt et al. In this study, the authors combined short-read WGS with long-read ONT sequencing to perform hybrid-assembly of an MRSA strain and its bacteriophages. By combining different sequencing technologies, they were able to shed light on several interesting features and the evolution of S. aureus.Current NGS technology allows for in-depth characterization and molecular surveillance of certain bacterial pathogens. The authors of four studies in the Research Topic utilized different technologies to characterize outbreaks of these pathogens nitoring . In anotBrandt et al. present an option to tackle this problem with an easy-to-use, fast, and robust workflow for the reconstruction of SARS-CoV-2 genomes, namely poreCov. The study highlights the importance of integrating bioinformatics software into reproducible pipelines using state-of-the-art workflow management systems and containers to finally bring new technologies into production and to allow the usage by non-experts (Due to the novelty and rapid development of molecular technologies, there are still many challenges in data acquisition, standardization, and reproducibility; especially concerning downstream bioinformatics analysis . This re-experts .Within this Research Topic, we have assembled a diverse portfolio of studies on the molecular surveillance of bacteria, eukaryotic microbes, and viruses; analyzed using a variety of technologies and computational approaches. The results of these studies could impact important areas such as the development of new vaccines, government decisions on molecular surveillance of pathogens in food, control of pathogens in livestock, and methods for tracking outbreaks. In addition, we are particularly pleased to have attracted studies from researchers around the world: India, Chile, Italy, Spain, Germany, and Brazil; and we are grateful to all authors and reviewers for their contribution to this Research Topic.


Psychological Science. Taken together, our results suggest diversity of geographic regions and periods in research activities in psychology. More importantly, our findings indicate that universality holds neither for human behavior nor research activities on human mental processes.Recent cultural studies have discussed universality and diversity in human behavior using numerous samples investigated worldwide. We aimed to quantitatively extend this discussion to various research activities in psychology in terms of geographic regions and time trends. Most psychology departments have specialists in various fields of psychology. Further, research institutions in all regions typically aim to provide systematic and balanced research education. Nevertheless, most researchers recognize universal features and patterns of diversity in research activities in psychology in terms of regional differences and time trends. However, these arguments remain intuitive and vague, and no studies have conducted quantitative analyses. To this end, we conducted topic modeling for the abstracts of psychological articles with the regions of author affiliations and publication periods as covariates. The results showed that the topic proportions related to basic research were high in North-Central America, whereas those related to clinical research were high in Europe. Interestingly, the regional differences shown by topic modeling were not observed in the frequency analysis of keywords, indicating that topic modeling revealed implicit characteristics. Moreover, we observed an increasing trend of neuroscience topics across publication periods. However, this trend was not valid for the psychology journal  Homo sapiens worldwide . However, no studies have quantitatively examined the tacit knowledge described above. To facilitate discussion about appropriate research and education activities in psychology worldwide, quantitative evidence should be provided based on an objective data analysis of whether there is diversity in research activities in psychology at a higher level compared with cultural differences in human behavior and the WEIRD problem.Proceedings of the National Academy of Sciences from 1991 to 2001 and showed that the topics extracted with topic modeling were consistent with the article classifications that the authors had selected. Recently, To this end, we conducted topic modeling, a text mining approach based on probabilistic modeling. Topic modeling with latent Dirichlet allocation LDA;  is used Psychological Science).Moreover, we examined whether specific proportions of topics related to these psychological themes differ across geographic regions worldwide. This study is the first quantitative test of diversity of geographic regions and time trends in research activities in psychology. We believe that a text mining approach with topic modeling for research abstracts is advantageous in clarifying past and present trends of research topics in different geographic regions and in providing important insights to aid future research activities in psychology.rvest and tidyverse  2020 APA, all rights reserved”). To preprocess the text data, we used the R package tm . All captopwords . In addi, and each word in each document (dnw) is drawn from one of the vocabularies of the topics that appear in the document. The presence of topics follows the dθ distribution. Topic modeling is used to identify the latent topics in a document and to overview the topics in collections of documents. We performed LDA, which is a popular topic modeling and unsupervised machine learning method, to determine the underlying semantic structure of the documents including text data . LDA assk = 1, …, KStep 1. For topic kφ ~ Dirichlet (β)Generate a distribution over words d = 1, …, DStep 2. For document dθ ~ Dirichlet (α)(a) Generate a distribution over topics n = 1, …, N(b) For word dnz ~ Multinominal (dθ) (i) Generate a topic dnw ~ Multinominal (dnφz) (ii) Generate a word topicmodels (ldatuning (ldatuning and compared the contents of words in each topic among the different models. We conducted LDA topic modeling with the following parameters: method = Gibbs (the method to be used for fitting is Gibbs sampling); iteration = 5,000; burn-in = 2,000; thin = 100; seed = 123 (to replicate the results of machine learning); the other parameters were on the default setting of topicmodels.LDA was performed using the R package icmodels . It is cicmodels . In thisdatuning  to deterdatuning . Howeverα and β, is that STM contains prior structure information on generalized linear models with covariates of each document, Χ and U, as parameters ; the other parameters were on the default setting of stm. Then, to examine the effects of each metadata type on the topic proportions, we estimated a regression using the function estimateEffect, where documents were the units, the outcomes were the proportions of each document about topics in STM, and the geographic regions and publication periods were metadata.We performed STM using the R package stm  with R s0 topics . Conside® Xeon® Gold 5,118 CPU (2.30 GHz) and 128 GB of RAM.We ran the R-program for topic modeling on a Windows computer , with an Intelldatuning). LDA for the Stroop test with five topics identified topics referring to “test,” “patient,” “function,” “execut,” “memori,” “age,” and “impair.” However, different fields were grouped together in the same topic  = 3.49, p < 0.001] and Asia . On the other hand, Topics 13 and 18 related to clinical research, such as “patient,” “cognit,” “diseas,” “depress,” and “schizophrenia,” were more likely to appear in Europe and Asia than in North-Central America .Next, we describe the results of visual search with three geographic regions as the metadata of STM. Overall, we observed that the expected proportions of Topic 4 related to the Stroop test experiments were high in North-Central America, whereas topics related to visual search experiments did not exhibit regional differences. Moreover, topics related to clinical research were high in Europe irrespective of whether they were related to the Stroop test or visual search. More importantly, the pattern of the main results using the Stroop test abstracts was observed on both STM with 80 topics  and STM In this section, we examined whether keywords of manuscripts show the same patterns of regional differences as those found using STM for the abstracts. We calculated the frequencies and percentages of keywords of manuscripts related to the Stroop test and visual search among the three geographic regions  = 12.09, p = 0.017, N = 10,748, Cramer’s V = 0.02) and residual analysis for keywords of the Stroop test, the frequencies of keywords corresponding to the words of Topic 4 reflecting the Stroop test experiments were significantly lower than the expected value (809.21) in North-Central America (p = 0.015) and significantly higher than the expected value (1162.62) in Europe (p = 0.002). Furthermore, the frequencies of keywords corresponding to the words of Topic 18 reflecting clinical research were significantly lower than the expected value (1931.39) in Europe (p = 0.012). Moreover, as a result of the chi-square test (χ2 (4) = 15.45, p = 0.004, N = 4,898, Cramer’s V = 0.04) and residual analysis for keywords of visual search, the frequencies of keywords corresponding to the words of Topic 4 indicating clinical research were marginally higher than the expected value (477.37) in Europe (p = 0.081). The frequencies of keywords corresponding to the words of Topic 14 indicating visual search experiments were significantly lower than the expected value (403.16) in Europe (p = 0.004).Overall, the frequencies of keywords corresponding to clinical research were low in Europe, and those corresponding to the Stroop test experiments were low in North-Central America. Thus, the frequency of keywords cannot capture the trends in STM of the manuscript abstracts.estimateEffect: t(7506) = −20.33, p < 0.001), that of Topic 11  has increased . In contrast, the proportion of Topic 19  did not differ across the publication periods . Furthermore, the proportions of both Topics 1  and 3 , which consisted of words related to neuroscience, have increased as a function of publication periods , those of Topic 5  have increased . Moreover, the expected proportions of Topic 16  did not differ across publication periods , whereas those of Topic 11  have increased [t(5406) = 2.42, p = 0.015; Next, we describe the results of visual search with publication periods as the metadata of STM. Taken together, although the trends of the topic proportions related to the Stroop test and visual search experiments differed depending on the contents of observed topics, the proportions of topics related to neuroscience have increased as a function of the publication periods. Again, the pattern of the main results using the Stroop test abstracts was observed on both STM with 80 topics  and STM Psychological Science from 1990 to 2020, with publication periods as metadata of STM. We selected Psychological Science as a target journal for examination because it is a general-interest and influential journal in psychology. We obtained 4,112 abstracts of manuscripts published in Psychological Science and the corresponding publication years from Scopus database on February 15, 2021. They did not include Erratum, Corrigendum, Reply, Commentary, Retraction, and Editorial items. We conducted the same preprocessing as in Study 1. Again, we created nine publication periods so that each period had the number of abstracts as close to 200 as possible: before 1994 was defined as 1993 (206 abstracts), 1994–1997 as 1997 (256 abstracts), 1998–2000 as 2000 (265 abstracts), 2001–2003 as 2003 (292 abstracts), 2004–2005 as 2005 (294 abstracts), 2006–2007 as 2007 (326 abstracts), 2015–2016 as 2016 (328 abstracts), 2017–2018 as 2018 (312 abstracts), 2019–2020 as 2020 (262 abstracts). We used each publication year from 2008 to 2014 separately  and 10  reflecting human visual attention and cognition have decreased as a function of publication periods [t(4110) = −7.58, p < 0.001 and t(4110) = −3.05, p = 0.002, respectively; t(4110) = 5.33, p < 0.001, t(4110) = 5.50, p < 0.001, t(4110) = 5.24, p < 0.001, and t(4110) = 8.14, p < 0.001, respectively; t(4110) = −0.568, p = 0.570; First, in Study 1, we observed not only topics consisting of basic words related to experiments involving both the Stroop test and visual search, but also those consisting of words referring to clinical, developmental, aging, and neuroscience research. These results suggest that topic modeling for psychology manuscript abstracts retrieved from a research database, such as PubMed, is effective for overviewing the trends of research themes.More importantly, in Study 2, STM for the abstracts related to the Stroop test with geographic regions as metadata showed that the proportions of topics consisting of “word,” “color,” and “effect” were higher in North-Central America than in Europe and Asia, whereas those of other topics related to the Stroop test and visual search experiments did not differ among the three geographic regions. In contrast, the proportions of topics related to clinical research were higher in Europe than in North-Central America, irrespective of the use of the Stroop test or visual search. To the best of our knowledge, this is the first quantitative study to provide evidence of diversity of geographic regions in research activities in psychology. It is possible that the observed regional differences are related to the history of psychology in that region. In the United Kingdom, Charles Myers established the National Institute of Industrial Psychology, which engaged in applied psychology in industrial and clinical sites . FurtherNote that the regional differences found in STM for the manuscript abstracts were not observed by comparing the frequencies of the corresponding keywords. This is because the authors may intentionally select more global words in psychology as keywords. In contrast, topic modeling of abstracts reflecting the manuscript research contents could capture regional differences in research activities. We therefore emphasize that topic modeling for manuscript abstracts can capture the implicit characteristics of regional differences in the research activities in psychology that the frequencies of keywords might miss.Next, STM for abstracts related to the Stroop test with the publication periods as metadata showed that the proportion of topics consisting of “word,” “color,” and “interfer” has decreased as a function of publication periods, whereas that of topics consisting of “conflict,” “trial,” and “respons” has increased. In contrast, the proportion of topics consisting of “task,” “control,” and “perform” did not differ over time. Similarly, STM for the abstracts related to visual search showed both universal features and patterns of diversity. Furthermore, the proportions of topics related to neuroscience have increased as a function of publication periods, both for the Stroop test and visual search. Although these results are consistent with Psychological Science. Thus, the increasing trend of topics related to neuroscience is not due to an increase in the number of accepted papers related to neuroscience research in a specific journal. Instead, it may be due to advancements in measurements and an increasing number of publication venues. Notably, STM showed a consistent increase in the proportions of topics related to cultural and social psychology as a function of publication periods .The authors declare that the research was conducted in the absence of any commercial or financial relationship 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.


Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies. Kidney transplantation (Ktx) is the most frequent transplantation worldwide, but transplant survival is limited by a high incidence of late antibody-mediated rejection (AMR) ,2. KTRs Mechanisms and causes of AMR have been investigated by plasma cell and biopsy analyses ,3,4. IdeConsequently, numerous approaches for non-invasive, non-DSA-based diagnostic strategies have been explored in recent years. Among the diverse immunological biomarkers that could be relevant, many non-invasive biomarkers have shown potential in preliminary studies. The chemokines CCL3, CCL4, CXCL9, CXCL10 and CXCL11 have recently been identified as markers for inflammation of the microvascular blood vessels ,20,21,22Translation of these biomarkers to the clinic requires robust validation studies and standardized, commercially available assays that need to be paralleled, but not solely depending on increased serum creatinine levels , which mSuch sandwich assays usually apply antibodies, but new test setups are being sought in the field of LFAs as the use of antibodies has some disadvantages and limitations: commonly generated in animals or cells, batch-to-batch variations, limited shelf life, difficult to label at a specific location and sensitive to temperature, possibly even irreversible denatured. In comparison, aptamers, which are single-stranded DNAs or RNAs, can be chemically synthesized , modifieIt was the aim of this study to select AMR biomarkers and to investigate their diagnostic potential at the day of biopsy to develop an LFA for straight forward and early detection in urine of patient with AMR after Ktx.Patient samples and data were derived from two independent cohorts of KTRs from a protocol biopsy program, collected within a defined biomarker study  at the day of a biopsy. The 1st cohort included 120 KTRs, aged between 18 and 75 years, transplanted between 1990 and 2012; the 2nd cohort included 78 partially pediatric KTRs, aged between 5 and 75 years, transplanted between 1986 and 2017. Patients of the 1st cohort had a relatively decreased transplant age as compared to patients of the 2nd cohort. In addition, patients of the 2nd cohort more often had a tacrolimus/mycophenolate/steroid-based immunosuppressive regimen . In patients with repeated biopsies, biopsies as well as urine and serum samples were analyzed only in case of a histological diagnosis of rejection. Eight sets of data out of the 1st cohort were excluded for biomarker determination due to diagnoses other than rejection . Whole-urine samples used were free of leukocytes or macro-proteinuria. Kidney graft function and further rejection episodes were followed up for 4.5 years in the 1st and for 9 months in the 2nd patient cohort. Identification of single DSAs in AMR was performed ±3 months around the biopsy  as described before . Anti-HLChemokines, cytokines or growth factors as possible biomarkers must be stable enough to be detected in patients’ urine despite ongoing immunosuppression comprising different individual drug regimens and individual immunity conditions. Therefore, we chose an approach with a comprehensive panel of 65 markers in different sample cohorts (cohorts of patients with TCMR or AMR or a biopsy finding unsuspicious for any rejection). The markers were comparatively tested using a Luminex-based multiplex assay in plasma of patients from the 1st cohort, comprising 16 KTRs with AMR. A selected panel of 14 markers  was testUsing the method of neural net analysis, 65 biomarkers and four clinical parameters  were scored. As the clinical parameters serve as confounders, markers that are clinically relevant for rejection were involved . Two-layer feed-forward networks with 5–20 neurons in the hidden layer were used. In total, 250 feature selection runs were carried out per neuron. A ranking order for the various markers was calculated by evaluation of score values. For this classification tasks with a given set of classes, supervised learning schemes were used. A set of training samples contained input values together with the desired output. In combination with an error function, the training set was used to adjust the internal parameters of the network. For classification, this function mapped the values of the parameters to ’rejection´ or ´non-rejection´, while ’rejection’ contained values of all rejection subgroups (T-cell mediated rejection (TCMR), borderline-TCMR and AMR). Among the best-scored 15 biomarkers, biomarker values of all rejection subgroups were compared using Wilcoxon test versus the KTR group with non-rejection, the level of statistical significance was indicated by asterisks. ROC analyses were performed to display the area under the curve (AUC) as well as the sensitivity and specificity of the LFA as compared to eGFR at the day of biopsy.−1) as a positive control for 1 h and secondly with a range of CXCL9 of different concentrations (0 to 20 ng∙mL−1). Supernatant with self-made CXCL9 used was produced by γ-interferon-stimulated human macrophages according to the method by Sudan et al. [Antibody generation was performed by Helmholtz Munich  based upon standardized immunization of rats with commercial CXCL9  and using the mouse myeloma cell line P3X63-Ag8.653 (ATCC CRL-1580) as described elsewhere . 19 rat n et al. . The ELIThe sequences of applied ssDNAs and their modifications are summarized in ® CN95 was kindly provided by Sartorius Stedim Biotech . Gold nanoparticles (AuNP) were kindly provided by Fassisi GmbH . Commercial CXCL9 (Cat# TP720369) was acquired from OriGene . Trehalose was purchased from Fluka BioChemika . Sucrose and bovine serum albumin (BSA) were obtained from Sigma-Aldrich GmbH . Tween20 was purchased from PanReac AppliChem . Phosphate buffered saline  was prepared . Blocking buffer was based upon PBS supplemented with 10% FCS . PBS was prepared with 137 mM sodium chloride, 2.7 mM calcium chloride, 10.1 mM disodium hydrogen phosphate and 1.8 mM calcium hydrogenphosphate . Solution pH was adjusted to 7.4. Sodium borate buffer was prepared with 2 mM sodium tetraborate  and set to pH 9. Sodium borate buffer was prepared with 2 mM sodium tetraborate . Binding buffer (BB) is PBS based and contained 138 mM sodium chloride, 5 mM potassium chloride, 8.1 mM disodium hydrogen phosphate and 1.5 mM potassium dihydrogen phosphate, 1 mM magnesium chloride, 1 mM calcium chloride, 170 mM urea, 7 mM ammonium acetate. BB (pH 6.5) was PBS-based with its content adjusted to urine milieu, supplemented with 0.25% BSA (Sigma-Aldrich GmbH), 0.1 mg∙ml−1 salmon sperm DNA  .Nitrocellulose membrane UnisartPreparation of AuNP-G123 was performed according to an adjusted protocol of Phung et al. . Conjuga−1) and control zone were automatically applied onto the nitrocellulose membrane , in some cases by hand (0.2 µL). Beforehand, streptavidin  and the same volume of biotinylated oligonucleotide solution (20 µM in PBS) was mixed and incubated for 2 h at 500 rpm to prepare the solution for the control zone. Test and control zones were dried for 3 h at 50 °C, cut into strips of 4 mm and placed in an airtight bag under exclusion of light.The hybrid-LFA was composed of a sample pad  and an absorbent pad  glued to a backing card  with 2 mm overlap to the centered nitrocellulose membrane. Sample pads were incubated for 2 h with 100 mM Tris (pH 8.0) supplemented with 1% BSA, 1% sucrose and 0.05% Tween 20 and dried at RT overnight before they were applied on the backing cards. Solutions for test  were placed at RT in a well of a 96-well plate. A total of 1 µL AuNP-G123 was applied onto the conjugate pad. Concentrations of purified CXCL9 were diluted in BB  or in BB combined with pooled patient negative urine samples (spiked samples). Pooled urine samples derived of four KTRs with non-rejection proven by biopsy. All patient samples were diluted in BB (ratio 1:1). LFA strips were placed in a sample well and scanned (Epson V370) after 15, 30 and 45 min before quantitative analysis by eye as well as by ImageJ-estimation of scans .Two independent patient cohorts were included in this work. The 1st cohort, recruited between 2011 and 2012, served for biomarker selection (see methods) and here 112 data sets, plasma samples of 112 KTRs and 2nd biopsies of 44 KTRs out of these group were available. There was a slightly higher percentage of protocol biopsies 55.4%) in the 1st cohort. According to the BANFF-classification at that time , biopsy 5.4% in tImmunosuppressive therapies were frequently minimized or calcineurin-inhibitors were replaced by an mTOR-inhibitor in AMR and borderline-TCMR-patients of the 1st cohort as well as in 50% of patients with borderline-TCMR and TCMR and ~30% of patients with AMR of the 2nd cohort. 12 children, who were included in the 2nd cohort, received steroid-sparing therapy .Serum creatinine was significantly higher in all groups with graft rejection of the 1st cohort A, possibA general screening for HLA I or II DSAs was positive in all AMR-patients of the 1st and 2nd cohort . At the In total, 65 biomarkers consisting of growth factors, chemokines and cytokines were mostly known from literature to be characteristic for the inflamed endothelium within rejection . These wTo evaluate the presence in the renal cortex of the kidney graft, selected markers  were detLuminex-based multiplex assay was performed within 78 whole-urine samples of the 2nd cohort using the 14 candidate biomarkers identified by neural network analysis . In accoFour antibodies with high affinity for CXCL9 were initially determined with the self-designed ELISA  and thenTo determine CXCL9 in patient samples, conjugates of AuNPs and CXCL9-binding aptamer (G123) were prepared. If successful, conjugation of aptamers to the AuNP surface exhibited an increased diameter of nanoparticles without signs of agglomeration. The altered size of nanoparticles was verified using DLS and UV-vis . Here, tInstead of conventionally applied paired antibodies, the presented LFA was designed as a sandwich assay with a capture antibody  and different running times (15 to 45 min). An LOD of 10 pg·mL−1 was determined with a running time of 45 min  and non-rejection (25 samples). Scans after a running time of 15 min showed distinct red lines in the according zone for patient samples . LFAs wiSince AMR is a major risk for late transplant function loss ,49, reliThe aim of this work was to identify potentially suitable immunological biomarkers in plasma that may reveal AMR in an early stage of this condition. To this end, a set of 65 parameters including cytokines and chemokines ,51 were CXCL9 has previously been shown to have diagnostic potential as urine biomarker within TCMR ,55,56,57CXCL9, mainly produced by human macrophages in response to interferon-γ ,61,62,63As limitation of the work presented here is the fact, that despite a positive screening for the presence of HLA I-or II-DSAs, not in all patient samples tested with the hybrid-LFA, single antigen HLA-DSAs could be identified. Non-HLA-antibody screening was not available at that time. At the time of recruiting the 1st cohort, the presence of DSA was not yet an obligatory criterion for the diagnosis of chronic AMR, whereas it was postulated for this diagnosis according to the BANFF Classification 2015  for patiAlternative biomarker or tests for these cases are highly warranted and qualitatively differing assays have recently been evaluated. However, neither autoantibodies caused by organ-derived autoantigens after surgical trauma or transplant ischemia ,68 nor cSince in this work, CXCL10 was only significantly increased at the time of chronic AMR diagnosis in biopsy lysates C, but noDespite the promising findings in AMR-patients or patients with a kidney graft highly suspicious for AMR, further studies are required to narrow the value of this hybrid-LFA. Its suitability should also be clearly differentiated against non-rejection-related immunological diseases such as a systemic inflammation or a specific graft or urogenital infection. In addition, studies using such an easy-to-use-CXCL9-POCT could clarify the precise onset of CXCL9 production within AMR in correlation with biopsy findings.


Kaposi’s sarcoma is an uncommon complication in renal transplant patients, and typically presents with cutaneous lesions on the lower extremities. Penile involvement has been reported only rarely. Management of cutaneous-limited disease is primarily reduction of immunosuppression and conversion to an mTOR-inhibitor, whereas the treatment of disseminated disease in transplant patients is more variable.A 75-year-old male, originally from Somalia, received a deceased-donor kidney transplant for diabetic and hypertensive nephropathy. Seven months post-transplant he presented with lower limb lesions, oedema and bilateral deep vein thromboses. He then developed a fast-growing painful lesion on his penile shaft. A biopsy of this lesion confirmed KS, and a PET scan demonstrated disseminated disease in the lower extremities, penis and thoracic lymph nodes. His tacrolimus was converted to sirolimus, and his other immunosuppression was reduced. He was treated with single agent paclitaxel chemotherapy in view of his rapidly progressing, widespread disease. The penile lesion completely resolved, and the lower extremity lesions regressed significantly. His kidney allograft function remained stable throughout treatment.This case illustrates a rare presentation of an uncommon post-transplant complication and highlights the need for a high index of suspicion of KS in transplant patients presenting with atypical cutaneous lesions. It serves to demonstrate that the use of single agent paclitaxel chemotherapy, switch to an mTORi and reduction in immunosuppression where possible produces excellent short-term outcomes, adding to the body of evidence for this management strategy in disseminated Kaposi’s sarcoma. Kaposi Sarcoma (KS) is a low-grade multi-centric tumour composed of endothelium-lined vascular spaces and spindle cells . It was The case was a 75-year-old male of Somali heritage, who received a deceased donor kidney transplant for presumed diabetic and hypertensive nephropathy. The donor was marginal but immunologically low-risk, being a 4/6 HLA mismatch with a single weak class II donor-specific antibody. At the time of transplant, he received our unit’s standard induction immunosuppression with basiliximab and methylprednisolone, then commenced maintenance immunosuppression with tacrolimus, everolimus, and prednisone. A denovo mTOR inhibitor (mTORi) was chosen in place of mycophenolate mofetil due to the recipients older age, pre-existing leukopenia and known vascular disease in the donor. The transplant was complicated by delayed graft function necessitating haemodialysis on day one. A renal biopsy on day nine demonstrated borderline acute T-cell mediated rejection; as such, the everolimus was replaced with mycophenolate mofetil. The graft function subsequently improved to a baseline creatinine of 170 µmol/L (eGFR 34 ml/min) with minimal proteinuria . A protocol biopsy at eight weeks showed no features of rejection.Seven months post-transplant the recipient presented with bilateral lower limb pain and swelling, and dark macular skin lesions on his legs. He underwent a duplex ultrasound which demonstrated bilateral proximal deep vein thromboses. Anticoagulation with apixaban was commenced. Soon after he presented with a painful papular lesion on his penile shaft, which began as a small macule and had grown rapidly Fig. . There wA biopsy of the penile lesion demonstrated diffuse involvement of the dermis by nodules of mitotically active spindled cells, with irregular slit-like spaces, which stained positive for HHV-8, diagnostic of KS Fig. .Fig. 2BiA PET scan demonstrated diffusely increased metabolism in the skin and subcutaneous tissues of the feet and lower legs, and over the surface of the penis. There was also abnormally increased metabolism in the paratracheal lymph nodes, with no increased metabolism in the inguinal, femoral, pelvic or abdominal nodes nor visceral organs Fig. a.Fig. 3aIn view of the rapidly progressive disseminated disease, the recipient commenced weekly single-agent paclitaxel chemotherapy with a plan for twelve weeks in total. His immunosuppression was reduced, with the mycophenolate ceased, prednisone dose tapered to 5 mg daily, and the tacrolimus switched to sirolimus, with a dose of 1.5 mg daily maintaining a level of 5-8 µg/L.The recipient responded well to chemotherapy and reduced immunosuppression. After four weeks of paclitaxel the penile lesion resolved completely with no residual scarring, and the lower limb lesions significantly regressed. A progress PET scan Fig. b demonstThroughout the treatment the patient’s transplant function remained stable, with no evidence of rejection despite the reduction in immunosuppression. He is now 18 months post-transplant, with the most recent pathology demonstrating a creatinine of 162 µmol/L, with no proteinuria (ACR 1.3 mg/mmol).. Immunosuppression is hypothesised to have a carcinogenic effect through impaired immunosurveillance mechanisms and direct damage to host DNA, as well as the potentiation of proto-oncogenic viruses  6]. [6].5] [. [6].5]  viruses .Of all p viruses  to 61 in viruses .Four distinct clinical subtypes of KS are described: classic; organ-transplant associated; epidemic (AIDS-related); and endemic .HHV-8 has been causally associated with all forms of KS . The serIn more than 90% of cases, KS presents as angiomatous cutaneous lesions in the lower extremities. Associated lymphedema is common, and often precedes the appearance of cutaneous lesions. Disseminated visceral involvement occurs in approximately 25% of kidney transplant-associated KS, with higher rates seen in liver and heart transplant recipients  14]. Th. Th14]. Genital KS is rare and reported almost exclusively in AIDS-associated KS, where the incidence is approximately 3% . Only a Reduction in immunosuppression is the cornerstone of management in transplant-associated KS and is often adequate in treating cutaneous-limited disease. However, any reduction in immunosuppression must be balanced with the risk of allograft loss from rejection. Switching the more oncogenic immunosuppressants  for an mTORi has become a commonly accepted management strategy. The efficacy of this approach was demonstrated in a 2005 case series of 15 patients, where cessation of cyclosporine and commencement of sirolimus was associated with complete resolution of cutaneous KS in all patients, with no reports of graft dysfunction [The management of disseminated KS in transplant recipients is not well described in the literature. In 2019 a retrospective analysis was published describing the management strategies of 145 organ transplant recipients with KS across different European centres . Of thesThere are no guidelines recommending which chemotherapeutic agent to use in disseminated transplant-associated KS. Treatment decisions are typically based off safety and efficacy data extrapolated from AIDS-related and classic KS cohorts with disseminated disease. In a 2020 update on therapy for Kaposi sarcoma, bleomycin and vinblastine were identified as the most widely employed single-agent therapies . HoweverManagement of KS in transplant recipients is particularly complex due to the risk of rejection and graft loss with immunosuppression reduction, and the increased risk of infection when already immunosuppressed patients are treated with chemotherapy. Outcomes for disseminated disease have traditionally been poor. In 1997 Penn reported a mortality rate of 57% in those with disseminated disease . In a 20In summary, we present a case of a very rare manifestation of KS in a kidney transplant recipient. Penile lesions in immunosuppressed men should prompt consideration for KS and early referral for biopsy. Given the rarity of this disease, trials in this area are not feasible. There are no clear guidelines for optimal management of disseminated KS in transplant patients, and prognosis for allograft function and mortality are poor. This case serves to demonstrate that treatment with single agent taxane chemotherapy, switch to an mTORi, and reduction in immunosuppression where possible produced excellent short-term outcomes, and thus should be considered as a potential management strategy in similar patients.


Background: Prokineticin family correlates with important roles in several biological processes, including homeostasis. We discovered novel functions of prokineticin1 (PROK1) in lymphangiogenesis and lymphnode metastasis in colorectal cancer.Materials and Methods: We examined changes in the number of lymphatic endothelial cells after PROK1 stimulation. PROK1 protein was stimulated with subcutaneously implanted in mice. Also a high-PROK1-expressing colorectal cancer cell line and anti-PROK1 antibody(Ab) were subcutaneously implanted in mice, and then examine lymphangiogenesis. PROK1 expression and the number of lymph vessels were examined in the primary lesion of 391 patients whose colorectal tumors had been resected.Results: When PROK1 was used as a stimulus, the number of lymphatic cells increased compared to unstimulated cells. And the number of lymph vessels in the skin of mice increased compared to mice implanted without PROK1. The number of lymph vessels in the primary tumor tissue increased when PROK1 was highly expressed compared to cases with non-detectable PROK1 expression. When PROK1 was expressed in human colorectal tumors, the rate of lymphnode metastasis was significantly higher than that in cases with non-detectable PROK1 expression.Conclusions: PROK1 is a lymphangiogenic factor involved in the formation of new lymph vessels and lymphnode metastasis in human colorectal cancer. The morbidity rate for colorectal cancer is very high in Japan and in western countries –2. WhileProkineticin-1 (PROK1) was cloned by Ferrara, and characterized as an angiogenic growth factor that selectively acts on the endothelium of endocrine cells. The gene encoding PROK1 is positioned at chromosome 1p21, with the coding region consisting of 105 amino acids . The matIn the present study, we observed that PK-R1 and PK-R2 were expressed in normal lymphatic endothelial cells and that proliferation of lymphatic endothelial cells and lymphangiogenesis are induced by PROK1 stimulation. Furthermore, we identified a new relationship between PROK1 expression and lymphatic formation, invasion, and metastasis in human colorectal cancer.Immunohistochemical analyses using anti-PK-R1 and anti-PK-R2 antibodies demonstrated the expression of PK-R1 and PK-R2 in a human lymphatic endothelial cell line .The number of lymph vessels was 12.5 per visual field in the skin tissue adjacent to the subcutaneously implanted chamber containing no PROK1; however, the number of lymph vessels significantly increased to 19.8 per visual field when PROK1 was added to the chamber .The number of lymph vessels was 19.5 per visual field in the skin tissue adjacent to subcutaneously injected high-expressing-PROK1 LoVo colorectal cancer cells. However, the number of lymph vessels decreased to 13 per visual field with the addition of the anti-PROK1 antibody. The number of lymph vessels was 20.5 per visual field in the skin tissue adjacent to subcutaneously injected high-expressing-PROK1 DLD1 colorectal cancer cells, and there were 13.5 per visual field when the anti-PROK1 antibody. The number of lymph vessels was significantly suppressed by the addition of the anti-PROK1 mAb .PROK1 expression was found in the primary lesion of 142 of 391 (36%) colorectal cancer patients who had undergone resection in our department. Lymph node metastasis was observed in 100 of 249 (40.2%) colorectal cancer patients with negative PROK1 expression in the primary lesion, whereas 96 of 142 (67.6%) patients with positive PROK1 expression, demonstrating a significant increase in lymphatic metastasis with PROK1 expression in the primary lesion .Under normal healthy conditions, lymph vessels, which are networked throughout the body, play the significant role of drawing tissue fluid leaking from blood vessels and returning the tissue fluid to the blood vessel . HoweverLymphangiogenesis is a sequence of lymph vessel formation initiating from migration and proliferation of lymphatic endothelial cells. VEGF-C, PDGF-BB, FGF-2, angiopoietin-1, HGF, and other angiogenesis-related proteins have been reported as factors for lymphangiogenic activity , 37, 38,The tumor microenvironment has recently been considered to be significant for growth and progression of tumor cells. It consists of fibroblasts, inflammatory cells, immunocompetent cells, blood vessels, and lymph vessels, each of which plays important roles in tumor development –47. The The role of PROK1 in the tumor microenvironment has been revealed by the present study, which leads the development of a new therapy for colorectal cancer by providing basic insight for the molecular mechanism of invasion and metastasis.The antibodies were used: anti-mice LYVE-1 Ab , anti-human D2-40 Ab , anti-human PK-R1 Ab  and anti-human PK-R2 Ab , anti-human PROK1 Ab (prepared in our department) .The human colon cancer cell lines, DLD-1 and LoVo (American Type Culture Collection. ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL streptomycin and 100 U/mL penicillin  at 37°C in 5% CO2 . The autThe human Dermal Lymphatic Microvascular Endothelial Cells  were cultured in EGM™-2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit™ (CC3202) medium .2 cells) were seeded onto a well (96 well plate), and were incubated with PROK1 protein (20 ng/ml) at 37°C for 72 hours. The nucleus of cells were stained by using DAPI (diamidino-2-phenylindole) (Sigma-Aldrich. USA). The cell number were counted at magnification 200×.The lymphatic vessel cells  was filled with Phosphate buffered saline (PBS) plus PROK1 protein was implanted under the skin into the dorsal side of six-week-old female SHO nude mice . At 7 days after implantation, a rectangular incision was made in the skin on the dorsal side .6 cells in 0.1 mL of matrix gel . Two groups of mice were tested. Group A was injected with non-stimulated colon cancer cells (DLD-1 and LoVo) and normal mouse IgG. Group B was injected with colon cancer cells and the anti-PROK1 mAb (5 μg). After 21 days after implantation, the tumor resection was made in the skin on the dorsal side. The skin was embedded in OCT compound  [Six-week-old female SHO nude mice  were subcutaneously injected in the the dorsal side with 1.0 × 10, Japan) . Four-μm, Japan) .Primary colorectal cancer tissues and adjacent normal colorectal tissues were obtained from surgical resection from 391 patients with sporadic primary colorectal cancer in the First Department of Surgery, University of Fukui, Japan between 1990 and 2007. According to the TMN classification , 76, 109The eligibility criteria were as follows: (1) a histopathological findings confirmed primary colorectal cancer; (2) resection of colorectal cancer with extended (D2 or D3) lymphnode dissection ; (3) hisParaffin sections (4 μm thick) were deparaffinized with xylene and dehydrate through a graded ethanol series. Endogenous peroxidase activity was blocked by incubation for 30 minutes with 1% hydrogen peroxidase in methanol. These hydrate sections were incubated in a dilution of normal goat serum at room temperature for 20 minutes to reduce nonspecific staining, and incubated with anti-PROK1 mAb or anti-D2-40 Ab for 1 hour. After washing with TBS, and analyzed for the expression of or D2-40 protein by the ChemMate method using the EnVision system . Finally, the slides were lightly counterstained with hematoxylin. The expression was interpreted as positive when the protein was expressed in more than 30% of all cancer cells.t-test or the χ2 test using Stat Mate IV . P value of < 0.05 were considered statistically significant.Statistical significance was determined by student To our knowledge, the present study was the first to identify PROK1 as a lymphangiogenic factor, and the molecular mechanism of the process from lymphangiogenesis through to lymph node metastasis was newly discovered in human colorectal cancer. Our findings indicate the potential of development of a new therapy.


The evolutionary stability of photosymbiosis differs among lineages of reef-building corals. More than half of reef-building corals (Scleractinia) participate in a nutritional symbiosis, known as photosymbiosis, with photosynthetic dinoflagellates that ranges from obligate to facultative dependence. Fitting hidden-rates models allowing among-lineage variation in the rate of trait evolution to supertree and molecular phylogenies of Scleractinia, we reconstruct the history of photosymbiosis within Scleractinia and characterize its evolutionary stability. We find that most lineages of scleractinians are extraordinarily stable for the trait, evincing no instances of loss, but that in some clades photosymbiosis is more labile, thus providing a framework for comparative studies to further our mechanistic understanding of the factors that shape the evolutionary fates of scleractinian photosymbiosis. Symbioses are an important source of evolutionary novelty , there will be a “slow” rate category in which transition rates are relatively low and a “fast” rate category in which transition rates are relatively high. Fitting HRMs with one to four rate categories across the supertree phylogenies, we found that the model that best explains the evolution of photosymbiosis is an HRM with three rate categories  weight of 98.27%; We found that the model that best explains the evolution of photosymbiosis across the molecular phylogenies is an HRM with two rate classes . Of the 767 zooxanthellate species in the supertree phylogeny, 721 were estimated to be in the Stable rate category . Of the 450 zooxanthellate species in the molecular phylogeny, 416 were estimated to be in the Stable rate category .The Zoox Stable state has multiple, independent origins in the supertree and molecular phylogenies and once gained is never lost . Within Madracis + Palauastrea (sister lineages to the Zoox Stable members of the Pocilloporidae), Oulastrea + Heterocyathus, Meandrinidae, and Oculinidae. Across the molecular phylogenies, the same clades were reconstructed as Zoox Labile with the exception of the genera Oulastrea and Heterocyathus. Across the supertree phylogenies, nine scattered species are inferred to be Zoox Volatile in Complexa (table S1): Stephanocoenia intersepta (Astrocoeniidae), Helioseris cucullata (Agariciidae), and five species within Dendrophylliidae—Balanophyllia  europaea, Duncanopsammia axifuga, Dichopsammia granulosa, Heteropsammia eupsammides, and Heteropsammia cochlea. Across the molecular phylogenies, two species were inferred to be Zoox Labile in Complexa (table S2): H. cucullata (Agariciidae) and H. cochlea (Dendrophylliidae). Of the 767 zooxanthellate species in the supertree phylogeny, 33 were estimated to be in the Volatile rate category . Of the 450 zooxanthellate species in the molecular phylogeny, 16 were estimated to be in the Labile rate category . Notably, in both supertree and molecular phylogenies, nearly all facultative species were estimated to be in unstable rate categories (tables S1 and S2). Across the molecular phylogenies, all facultative species were estimated to be in the Labile rate category . Analogous to these results, across the supertree phylogenies, all facultative species were estimated to be in the Volatile rate category [Pr(Zoox Volatile) > 0.75] with the exception of D. granulosa . To verify that facultative corals were not driving the reconstruction of Volatile clades in the supertree and Labile clades in the molecular phylogeny, we repeated the analyses with facultative corals pruned from 100 randomly subsampled phylogenies and obtained the same results (figs. S4 and S5). Across the supertree phylogenies, the Azoox Volatile state was not reconstructed confidently at any internal nodes . Only 10 species were estimated to be in the Azoox Volatile state . All 10 species represent losses of photosymbiosis and were found in clades reconstructed as Zoox Volatile.Several small clades were found to have high rates of gain and loss of photosymbiosis. Across the supertree phylogenies, five small clades within Robusta were reconstructed as Zoox Volatile : AstrocoFitting HRMs to both supertree and molecular phylogenies, we found that the rate of evolution of photosymbiosis varies among lineages. The best fit HRMs contain multiple rate categories to account for variability in the evolutionary stability of photosymbiosis, illuminating differences in the probability of a lineage to gain, retain, or lose this trait. Using rates estimated under the HRMs, we reconstructed the ancestral states at interior nodes across the posterior sets of the supertree  and 2A. et al.  before acquiring photosymbiosis . Within We suggest that the transitions to Azoox Labile indicate the time points in the evolutionary history of corals during which the molecular and cellular mechanisms evolved that allow mediation of the coral host-photosymbiont relationship. The processes mediating coral photosymbiosis overlap with innate immunity pathways . Stable photosymbiosis has evolved independently multiple times within both Complexa and Robusta. Almost all families in Zoox Stable clades appear in the fossil record by the late Cretaceous , 16S rDNA, adenosine 5′-triphosphate synthase subunit 6, cytochrome c oxidase subunit 1, control region, cytochrome b, and NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 5] and partial nuclear DNA  markers   isabellae] lacked data on symbiotic state and were pruned from the phylogenies.Each species is scored as being azooxanthellate, obligately zooxanthellate, or facultatively zooxanthellate (tables S3 and S4) based on data obtained from the Coral Trait Database , HRM + 2, and HRM + 3] using 100 trees subsampled from both the supertree and molecular phylogenies. We then performed ancestral state reconstruction under the best-fit model for supertree and molecular phylogenies. Ancestral state reconstructions as summarized across the posterior set samples of the supertree and molecular phylogenies are shown in figs. S4 and S5, respectively. The removal of facultative corals from the phylogenies did not substantially alter the results or their interpretation.


Plants have amazing regenerative properties with single somatic cells, or groups of cells able to give rise to fully formed plants. One means of regeneration is somatic embryogenesis, by which an embryonic structure is formed that “converts” into a plantlet. Somatic embryogenesis has been used as a model for zygotic processes that are buried within layers of maternal tissues. Understanding mechanisms of somatic embryo induction and development are important as a more accessible model for seed development. We rely on seed development not only for most of our caloric intake, but also as a delivery system for engineered crops to meet agricultural challenges. Regeneration of transformed cells is needed for this applied work as well as basic research to understand gene function. Here we focus on a MADS-domain transcription factor, AGAMOUS-Like15 (AGL15) that shows a positive correlation between accumulation levels and capacity for somatic embryogenesis. We relate AGL15 function to other transcription factors, hormones, and epigenetic modifiers involved in somatic embryo development. Somatic embryogenesis (SE) is a powerful resource and biotechnological tool used for plant improvement, micropropagation, and clonal regeneration  has been used for much of this work . Arabidopsis zygotic embryogenesis has a predictable pattern and basic body plan from early division      , and Gossypium hirsutum (cotton), while mutants (loss-of-function) of both AGL15 and AGL18 (closely related to AGL15) significantly reduce SE in Arabidopsis —This involves morphological stages of somatic embryogenesis analogous to zygotic embryo development including establishment of shoot and root apical meristems. During this step, auxin is often removed from the media. For instance, often in indirect systems, SEs are only obvious upon removal or a decrease in the concentration of plant growth regulators (PGR). It is thought molecular patterns supporting development to the globular stage are established on the high PGR medium, and the decrease in exogenously added PGRs is needed to continue development —This stage involves mild drying that can switch the embryo from development to germination or for storage purposes.In vitro germination (radicle elongation).Conversion (establishment of the new plant).AGL15 has been shown to promote SE in several plants  as SE tissue. Loss-of-function or dominant negative forms of AGL15 reduce initiation of this tissue. Because AGL15 transcript accumulation is induced in response to auxin treatment . The findings show that 35S:AGL15 produced approximately twice the frequency of seedlings with SAM SE compared to Col, wt . Genes encoding orthologs of AGL15 from soybean (GmAGL15) and the closely related MADS-factor that has redundant functions (GmAGL18) were cloned and introduced into SE tissue by biolistic transformation. We found that increased expression of these genes via a 35S promoter could 1. Enhance recovery of transgenic lines, presumably by promoting regeneration of transformed cells by SE and 2. Produce transgenic plants that have enhanced ability to form SE tissue. Meanwhile, Yang et al.  showed no such increase. Ghadirzadeh-Khorzoghi et al. 6GG, but AGL15 can also bind variants of this motif with a longer A/T rich core  and transcriptome studies in Arabidopsis and soybean , where addition of EP3 can rescue the production of SE in a SE-defective line of carrot  embryos, but rather in cells surrounding the embryos and are thought to have a nursing function, in part by cleavage of AGPs, some of which have stimulatory and others inhibitory effects on SE  showed direct expression in response to AGL15 accumulation, with one additional (FLA10) showing reduced transcript in both comparisons.A number of gene ontology (GO) categories are overrepresented among the direct (as determined by ChIP-chip and/or ChIP-seq) AGL15 targets that are expressed or repressed. Select categories are shown in , Coffea canephora , is a key marker for cells able to form somatic embryos, and, when expressed via a strong constitutive promoter, can enhance SE production in multiple species. SERK1 transcript accumulation is increased by auxin and stress  regulated by WUS , and BBM showed a positive correlation of transcript accumulation over the 2,4-D concentrations used, including sub-optimal (0.1 μM) that led to shoot development, 5 μM , and supra-optimal (20 μM) that produced non-embryogenic callus, whereas AGL15 and WUSCHEL (WUS) had very little or no transcript accumulation other than at the SE optimal 2,4-D concentration. Grzybkowska et al.  encoded proteins bind to the Auxin Responsive Factors (ARFs), which, in turn, are associated with DNA via auxin response elements on auxin-responsive genes ; however, too high auxin concentration may hinder the induction of embryogenic cells in the SE culture. It has been observed that cells that are resistant to auxin in that they maintain cell-cell adherence and do not elongate as much, may be the particular cells capable of developing as embryos Emons, . In the YUCCA gene family are induced by LEC1 and LEC2), overexpression of YUCCA's alone cannot induce SE TIR1 ARF6 and (Gm)ARF8 AGL15 (here (Gm)AGL15 is meant to indicate similar observations for soybean and Arabidopsis AGL15; AGL15 is meant to indicate the observation in Arabidopsis to date) directly represses a gene encoding an auxin receptor agl15 agl18 double mutant, although the 35S:AGL15 did not show increased accumulation of this transcript. ARF5, when present as a loss-of-function, had the largest effect on SE efficiency and productivity, but overexpressing this gene did not produce any SE for the few explants that could be obtained; again, indicating that restrictions on auxin response may be important for SE AGL15 regulated members may promote auxin response via other family members. For instance, there is data supportive for direct expression of ARF5/MONOPTEROS (MP) by AGL15 with Zheng et al.  data sho35S:AGL15 may reduce the biologically active GA, thereby promoting SE. Our prior work corroborated this hypothesis as we found that AGL15 directly up regulates a GA catabolism gene GA2ox6 in both Arabidopsis and soybean  was repressed by AGL15 , a gene encoding a GA biosynthetic enzyme , that encodes an α class of pyridoxal-50-phosphate (PLP) dependent enzymes AGL15 impacts ethylene biosynthesis and perception, which is relevant to SE  transcription factors that was shown to be required for somatic embryogenesis. This gene is induced by ethylene and responds to auxin and cytokinin , and BRASSINAZOLE-RESISTANT 1 (BZR1), were found to bind directly to AGL15's promoter region. Furthermore, inhibition of BR biosynthesis chemically or through mutants can promote SE development on seedlings of the single mutant val1 that normally only shows this phenotype in combination with val2 mutants (discussed below), whereas dominant forms of bes1-D and bzr1-1D can rescue normal seedling development. AGL15 is involved in the production of the SEs as shown by double and triple mutants with an agl15 loss-of-function mutant. Ruan et al. (While Brassinosteroids (BR) have been reported to enhance SE  function is critical during embryogenesis through various cellular, physiological, and developmental processes. MiRNAs target transcripts from genes that include those with a regulatory role such as TFs that can mediate stress adaptation, developmental regulation, and hormone response  with a characteristic hairpin structure that are processed by a complex that includes DICER-LIKE1 (DCL1) to generate a pre-miRNA. This pre-miRNA duplex structure is further processed, producing the mature single stranded miRNA, which is incorporated by ARGONAUTE1 (AGO1) to form an active RISC  compared to 35S:AGL15. This indicated a disconnect between production of the pri- and mature miRNA suggesting regulation of miRNA processing components. They found a negative correlation between AGL15 accumulation and transcript accumulation from the miRNA biogenesis genes DCL1, SERRATE, and HUA-ENHANCER1 (HEN1). While they did not perform ChIP for AGL15 for these genes, DCL1 had been identified as a direct target of AGL15    using chromatin immunoprecipitation (ChIP) assays  were more abundant in microspores before SE induction, suggesting it might increase transcriptional activity and regulate cellular reprogramming and embryo development .  and VAL2, are also known as HIGH-LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE2 (HSI2) and HSI2-LIKE1 (HSL1), respectively. VAL genes encode B3 domain transcription factors similar to LEC2, FUS3, and ABI3 , and histone deacetylases (HDACs) are the complexes that control the acetylation state , suggesting expression of these genes. Zheng et al.  and by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project (SP) under accession number 1013409.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.


Staphylococcus aureus was predominant (54%), followed by Escherichia coli (19%). The diagnostic yield was low (4.9%). Only 6.3% of isolates were identified. AST was performed on 70% (18/26) of identified AMR priority isolates, and only 40% of these tests adhered to recommended standards. Interventions are needed to improve laboratory BC practices for effective patient management through targeted antimicrobial therapy and AMR surveillance in Uganda. Further research on process documentation, diagnostic yield, and a review of patient outcomes for all hospitalized febrile patients is needed.Blood culture (BC) processes are critical to the utility of diagnostic testing, bloodstream infection (BSI) management, and antimicrobial resistance (AMR) surveillance. While Uganda has established BC guidelines, often laboratory practice does not meet the desired standards. This compromises pathogen recovery, reliability of antimicrobial susceptibility testing, and diagnostic test utility. This study assessed laboratory BC process outcomes among non-malarial febrile children below five years of age at five AMR surveillance sites in Uganda between 2017 and 2018. Secondary BC testing data was reviewed against established standards. Overall, 959 BC specimens were processed. Of these, 91% were from female patients, neonates, infants, and young children (1–48 months). A total of 37 AMR priority pathogens were identified;  Salmonella enterica (58.4%), Streptococcus pneumoniae (18.3%), Staphylococcus aureus (9.5%), and Escherichia coli (7.3%) . How. How9]. Uganda’s Clinical Guidelines (2016) and National Action Plan for AMR (2018–2023) envision timely and reliable microbiology laboratory services, including BC, as an essential component for guiding targeted antimicrobial therapy and AMR containment. However, AMR surveillance capacity-building programs in Uganda suggest recurrent BC laboratory shortfalls ,13. ThisA cross-sectional study was conducted at the five national AMR surveillance sites using secondary data from routine laboratory BCs from October 2017 to September 2018.Uganda is a low-income country in East Africa, with approximately 41.6 million people . In 2020Five regional referral public hospitals with onsite microbiology laboratories were supported to initiate AMR surveillance in 2016, through provision of technical assistance to ensure the necessary infrastructure and resources were put in place. At a minimum, each AMR surveillance site has access to a network of microbiology laboratories that offer a limited range of diagnostic services including bacterial culture, pathogen identification, and AST using disk diffusion techniques. Peripheral laboratories linked to national reference laboratories (NRLs) provide specialized testing, including advanced/confirmatory pathogen identification and quantitative AST. NRLs also provide technical support to lower-level laboratories through external quality assessments, in-service training, mentorship, and laboratory supervision by subject matter experts and implementing partners.The study was conducted at the five regional referral hospitals (RRHs) in Arua, Mbale, Kabale, Jinja, and Mbarara that have participated in a national AMR surveillance program since 2016 . These hStaphylococcus aureus, Escherichia coli, Klebsiella spp., Acinetobacter spp., Pseudomonas Aeruginosa, and Salmonella spp.The AMR surveillance program involves the use of standardized guidelines. Aggregate AST data is reported to the national AMR coordination office to inform AMR containment strategies, and subsequently to WHO via the GLASS portal. Each AMR surveillance site serves a population of at least 500,000 people in its catchment area, with approximately 50 pediatric beds and an annual caseload of 5000 febrile children, under five years of age, admitted. Each surveillance site has access to onsite BC and AST services. All surveillance site laboratories receive technical support from government and health partners to ensure AMR priority pathogens are identified, and AST is performed following CLSI guidelines . TechnicAt each surveillance site, specimens from hospitalized NMF children less than five years old were collected in BACTEC™ Plus Aerobic/F bottles and sent to the laboratory for testing. Blood culture bottles were processed onsite using the BD BACTEC™ 9050 automated system as per the manufacturer’s instructions. When bacterial growth was detected, a preliminary Gram stain was performed to determine and report a Gram-stain reaction to guide initial antimicrobial therapy. Consecutively, an inoculum was sub-cultured for species identification and AST, following CLSI guidelines. For quality assurance purposes, each positive blood culture isolate was referred to the national AMR reference laboratory for parallel testing. In addition, the designated AMR reference laboratory performed confirmation of any resistance patterns.Laboratories were required to report preliminary results (Gram stain) and deliver all culture and AST results (pathogen identification and resistance patterns) to the clinic care unit within a turnaround time of one day from completion of testing. Samples that showed no sign of any growth after seven days were declared negative. All laboratories had a quality control system and were enrolled in an external quality assessment program administered by the reference laboratory.Laboratory AMR surveillance data were captured in microbiology test request forms, specimen reception logbooks, and WHONET, a free Windows-based database software. Routine AST data generated at the facility level was subsequently submitted to the national AMR surveillance coordination center for validation, use, and reporting to GLASS, in support of the global actions for AMR containment.The study reviewed and analyzed secondary data from anonymized laboratory blood cultures of hospitalized, NMF children less than five years old collected from October 2017 to September 2018 at the five selected AMR surveillance sites.At each AMR surveillance sentinel site, aggregate AST data were retrieved from standard WHONET files and submitted monthly to the national AMR coordination center. This data, by the BC testing process is summarized in Routine AST data captured by surveillance sites in the WHONET software were exported and analyzed using STATA™ version 15. Categorical data were presented as frequencies and proportions in tables and figures.p-value < 0.001). Neonates, infants, and young children (1–48 months) were the main contributors of processed specimens, accounting for about 91% compared to 9% from those aged 49–59 months, p-value < 0.001). Patient age was not documented for three of the 959 (0.3%) specimens processed. Initial antibiotic exposure before specimen collection was recorded for only 53% of the specimens. Data on dates of fever presentation were not available.A total of 959 blood culture specimens were collected from hospitalized, NMF children less than five years old at five public RRHs in the national AMR surveillance program. Jinja had the highest number of BC specimens collected, followed by Arua, Mbarara, Kabale, and Mbale, respectively . During A total of 959 BC specimens were routinely collected from hospitalized, NMF children less than five years old during the study period. All of the 959 specimens did not have data on specimen volume, time of collection, and culture contamination. All laboratories did not track all prescribed quality indicators related to specimen collection and transportation.All of the 959 collected specimens underwent a complete laboratory workup to identify organisms. Of these, only 4.9% showed clinically significant growth (diagnostic yield) . BC contStaphylococcus aureus was the most predominant isolate (54%), followed by Escherichia coli (19%), Salmonella spp. (11%), Streptococcus pneumoniae (11%), and Klebsiella spp. (5%).Staphylococcus aureus, Escherichia coli, Klebsiella spp., and Salmonella spp. were resistant to two or more antimicrobials. Streptococcus pneumoniae isolates were susceptible to all tested antimicrobial agents. Overall, only 40% of these tests adhered to recommended CLSI guidelines. Areas of non-conformity included the following:(1)none of the recommended antimicrobial agents were tested (28%),(2)some of the recommended antimicrobial agents were tested (22%), and(3)incorrect pathogen–antimicrobial agent combination was used (10%).AST was performed on only 70% of the identified AMR priority pathogens; non-AMR priority pathogens were excluded from our AST analysis. During the study period, all the surveillance site laboratories did not have patient result delivery data (from the laboratory to patient care points/wards).This study reported findings of BC testing process outcomes, including sampled patient profiles, AMR priority pathogen etiology, quality management, and compliance to recommended AST guidelines at five RRHs, after the initiation of AMR surveillance. We identified areas for further strengthening and research. There were substantial deficits in BC laboratory process outcomes, potentially limiting blood culture test utilization in routine patient care and introducing bias regarding AMR surveillance data reported nationally.The high number of hospitalized febrile neonates, infants, and young children (1–48 months) whose BC were processed were consistent with studies in Uganda, Switzerland, and the United States of America ,12,21,22Staphylococcus aureus and Escherichia coli as the predominant AMR priority pathogens identified, and their resistance patterns were not surprising. These results were similar to prior blood culture studies [Salmonella spp. isolated in Arua RRH, which likely was due to differences in underlying patient conditions, predisposing factors, specimen collection, and laboratory techniques.The low diagnostic yield/pathogen recovery . These findings are consistent with previous reports in Uganda ,24. This studies ,21,28,29Salmonella spp. isolates from blood. Other factors that could influence the recovery of Salmonella spp. include circulating bacterial load, specimen collection timing, volumes of blood collected, and exposure to amicrobial agents before specimen collection [Jinja RRH recovered a wider variety of AMR priority pathogens as it had the largest number of specimens processed. Arua RRH was the only surveillance site that recovered llection ,24.Our study highlights considerable gaps in quality of laboratory performance. Most surveillance sites were unable to adhere to basic standards for culture and pathogen identification. There were variations in the identification of bacterial pathogens. The choice of antimicrobial agents for AST was not consistent with national guidelines (40% level of compliance). Laboratory performance on key quality indicators such as specimen volume, turnaround time, and specimen contamination rates was not routinely documented and monitored. Reporting of preliminary Gram-stain results for positive BC specimen was not adequately documented and tracked. Both pediatric and adult studies evaluating laboratory BC culture practices in Uganda, the Democratic Republic of Congo, Nigeria, Nepal, India, Switzerland, and the United States of America similarly reported shortfalls in microbiology laboratory quality capacity as major bottlenecks to BC execution ,23,27,30Challenges related to supply chain reliability for critical laboratory commodities (including BC specimen collection supplies), personnel technical competence, and equipment functionality were potential impediments to the inadequate quality and poor compliance to standard testing guidelines. These observations are comparable to previous studies that reported laboratory supplies, personnel technical competence, and equipment functionality as major impediments to quality test utilization by clinicians ,16,31. CTo our knowledge, this study is one of the first blood culture testing cascade-analyses at AMR surveillance sites in Uganda. Strengths of our study included the inclusion of regional referral hospitals that pioneered the national AMR surveillance in 2016 and received external technical assistance and resources to enhance AST practices in support of a national AMR surveillance program. This approach minimized bias due to restrictive sampling. A systematic approach to reviewing the entire blood culture process against established technical and quality standards revealed laboratory technical challenges and their impact on patient care and AMR surveillance.This study was limited by missing data for crucial patient demographics, clinical characteristics, and diagnostic microbiological variables. Whereas specimen collection, transportation and handling, communication of results , and patient outcomes are important factors in blood culture execution, this study did not assess them due to incomplete documentation and missing variables. Because of the low diagnostic yield and number of recovered pathogens (< 20 for the most predominant pathogen), this study did not perform statistical analysis comparing patient demographics and pathogens and conclude on resistance patterns to minimize potential bias. Lastly, our study focused on hospitalized non-malaria febrile children below five years of age, limiting the generalizability of pathogen etiology and their resistance patterns.The quality of AST data generated and reported to inform treatment guidelines and AMR control strategies nationally and globally was inadequate. Site level capacity-building interventions premised on implementing laboratory procedures and practice changes could substantially improve diagnostic test utility for patient management and AMR surveillance.Staphylococcus aureus, followed by Escherichia coli, were the most predominant AMR priority pathogens. Laboratory BC testing process outcomes did not conform to established testing and quality standards. Interventions to improve BC testing processes, including specimen collection, bacterial culture, AST, and quality indicator monitoring at AMR surveillance sites are needed. If these processes are improved, routine microbiological diagnostic data can adequately guide targeted antimicrobial therapy for patient management, and reliably contribute to AMR surveillance in Uganda. Other studies with improved documentation of routine BC processes, a higher diagnostic yield, and analysis of patient outcomes for all hospitalized febrile patients will be necessary to further assess specimen collection practices and priority pathogen resistance patterns at AMR surveillance sites.This study has provided insight into BC testing process indicators, including specimen collection and transportation, bacterial culture, AST, and quality indicator monitoring at the five RRHs, after the initiation of AMR surveillance. Blood culture was mostly performed in neonates, infants, and young children (1–48 months).


Mycobacterium bovis infection. The pathogenesis of bTB, and thereby the process of bovine tuberculoid granuloma development, involves the recruitment, activation, and maintenance of cells under the influence of antigen, cytokines and chemokines in affected lungs and regional lymph nodes. The granuloma is key to successful control of bTB by preventing pathogen dissemination through containment by cellular and fibrotic layers. Paradoxically, however, it may also provide a niche for bacterial replication. The morphologic and cellular characteristics of granulomas have been used to gauge disease severity in bTB pathogenesis and vaccine efficacy studies. As such, it is critical to understand the complex mechanisms behind granuloma initiation, development, and maintenance.The bovine tuberculoid granuloma is the hallmark lesion of bovine tuberculosis (bTB) due to  In bTB, more work at the individual granuloma level will be needed to advance our understanding of bovine tuberculoid granuloma development, maintenance and bacterial killing capacity.The bovine tuberculoid granuloma is a dynamic physical and functional structure that directly reflects the host: pathogen interface . It is h imaging ,128, whi


Vibrio vulnificus is a pathogenic marine bacteria associated with high mortality. Changes in climate and the global seafood trade have increased the prevalence of marine and freshwater systems affected by V. vulnificus. As a result, the incidence of land animals, plants, and insects contacting V. vulnificus and acting as disease vectors is on the rise. We report the case of a 53-year-old male who was infected with V. vulnificus as the result of a bee sting. The patient had no history of contact with the sea or fresh water or aquatic organisms or products. Due to bacterial pathogenicity and the patient’s underlying diseases, his condition deteriorated rapidly and eventually resulted in death. Here, we review the pathogenic mechanisms and treatment of V. vulnificus. We determined that V. vulnificus has spread from seawater to freshwater and that individuals may become infected from insects, even in the absence of direct contact with infected water. This case report will inform clinicians about the possible sources of V. vulnificus infection and indicates the possibility that more insects may transmit V. vulnificus in the future. Vibrio vulnificus is a marine pathogen associated with high mortality. The primary clinical manifestations of V. vulnificus infection include acute fever, chills, haemorrhagic bullous lesions, and septic shock. Severe cases of V. vulnificus infection can quickly lead to death. Even in the United States, the mortality of V. vulnificus infection is as high as 50%, according to the FDA [V. vulnificus, and the mortality rate of patients without proper treatment within three days can reach 100% [V. vulnificus is a halophilic and thermophilic bacteria; when the water temperatures exceed 18°C, V. vulnificus propagation increases, with an optimum growth temperature of 26°C [V. vulnificus is expanding annually [V. vulnificus in freshwater environments [V. vulnificus colonizes freshwater environments, the possibility of terrestrial animals, plants, and insects being exposed to V. vulnificus and potentially becoming disease-transmitting vectors concomitantly increases. The atypical clinical signs of V. vulnificus infection along with increasingly unclear exposure risk factors have caused difficulties in its early diagnosis and treatment. the FDA . Patientach 100% . V. vuln of 26°C . As a reannually . Additioronments . As V. vGanoderma lucidum on a trip through Huadu. He was questioned to assess his recent exposure to possible infective agents and denied transport or exposure to aquatic products, oceans, lakes, etc. His medical history included hepatitis B cirrhosis, hyperthyroidism, chronic gastritis, and renal calculi. His highest recorded body temperature was 39.8°C, and it was accompanied by chills, vomiting, and diarrhoea. Physical examination revealed swelling of the left lower limbs and a wound from a bee sting without blisters or ulcers. Preliminary blood tests (26 September 2020 noon and afternoon) revealed significantly elevated procalcitonin (PCT), creatine kinase (CK), and CK-MB; significantly decreased platelet count (PLT), and gradually increasing blood urea nitrogen (BUN) and serum creatinine (Cr)  . Neverthine (Cr) . Based o2, 98%. Three hours later, the patient developed shortness of breath and lost consciousness. At this time, the following vital signs were observed: blood pressure, 80/50 mmHg; respiration, 30 breaths/min; heart rate, 122 beats/min; and SpO2, 98%. Physical examination revealed that the patient’s skin was cold and wet. Swelling, ecchymosis, and blistering of the left lower limb continued to aggravate (9/L) and PLT (12 × 109/L) and a continuous increase of PCT (166.54 ng/mL), indicating that the infection was further aggravated and unaffected by the antibiotic treatment. BNP was as high as 8591 ng/mL, transaminase was increased, and Cr and BUN continued to rise, indicating that the patient was experiencing multiple organ dysfunction syndrome affecting the heart, liver, and kidney. , causing iron overload [V. vulnificus [al areas . The higoverload . Iron ovlnificus .V. vulnificus sepsis. Antibacterial treatment should follow the principle of early, combined, and sufficient loading dose [Early diagnosis and early start of multi-disciplinary treatment are the key to the treatment of ing dose . In seveing dose . Therefoing dose ,15.V. vulnificus was evident. Humans infected with V. vulnificus generally have exposure histories to water systems or aquatic products. We have repeatedly confirmed the relevant exposure history with the patient and his family, but they always gave a denial reply. These exposure histories are not shameful or interest related. They have no motive and reason to conceal. We could not identify other possibilities leading to V. vulnificus infection. Therefore, the focus of this discussion concerns the transmission of V. vulnificus through insects such as bees.Since both the patient and his family denied any history of contact with seawater, freshwater, or marine organisms, and the patient only had an acute history of bee sting, the early diagnosis was misjudged as bee sting poisoning and allergies. However, through the results of post-mortem blood cultures and a review of the patient’s condition, infection of V. vulnificus can survive in freshwater environments [V. vulnificus in fresh water products from Guangdong, Guangxi, Beijing, Shenzhen and other places in China, and found that V. vulnificus infection was present in multiple species and was characterized by easy infection and seasonality [V. vulnificus in freshwater fish in Shenzhen, and the detection rate in some freshwater fish was up to 38.6%, which is higher than that of seafood. The positive detection rate in fresh water was 19.9% [V. vulnificus infection after exposure to fresh water. Zhongshan Hospital of Fudan University reported a case of V. vulnificus infection related to freshwater shrimp stings [V. vulnificus, which is far more serious than expected. Through infected freshwater or aquatic product, V. vulnificus can infect human beings.Some subspecies of ronments . Lei et sonality . In Guanas 19.9% . There aPseudomonas aeruginosa arthritis and osteomyelitis of the foot in a boy whose foot had been stung by a bee [Insect bites can lead to severe infections. Olivares-Becerra et al. reported the first case of the association between spleen abscess and bee bite . Richardby a bee . Howeverby a bee .V. vulnificus and adsorb bacteria onto their bodies through static electricity. When attacking a human, V. vulnificus can then enter the body through the bee sting site. In addition, patients with liver diseases who accumulate large amounts of iron in the liver are at risk for worsened V. vulnificus infection. Under these conditions, V. vulnificus grows rapidly through iron overload and causes a series of bodily injuries and may eventually lead to death.Bees collect gum, water, and inorganic salts in addition to pollen and nectar during foraging. Bees forage on a relatively large range, about 1 km in radius, but in the absence of adequate nectar sources, this range can reach 4 km . Bees buV. vulnificus has spread from seawater to freshwater and that individuals may become infected even in the absence of direct contact with infected water. Clinicians treating high-risk patients should enhance vigilance and consider atypical routes of V. vulnificus infection, especially in patients likely to develop severe cases. In suspected cases of V. vulnificus, early intervention with appropriate intravenous antibiotics is critical in order to reduce the mortality and disability rate.This unique case indicates that