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+{"text": "Numerous studies have investigated mortality during a heatwave, while few have quantified heat associated morbidity. Our aim was to investigate the relationship between hospital admissions and intensity, duration and timing of heatwave across the summer months.st, 2003), and is subdivided in seven provinces with 60 hospitals and about 20000 beds for acute care. Five consecutive heatwaves (three or more consecutive days with Humidex above 40\u00b0C) occurred during summer 2002 and 2003 in the region. From the regional computerized archive of hospital discharge records, we extracted the daily count of hospital admissions for people aged \u226575, from June 1 through August 31 in 2002 and 2003. Among people aged over 74 years, daily hospital admissions for disorders of fluid and electrolyte balance, acute renal failure, and heat stroke , respiratory diseases (RD), circulatory diseases (CD), and a reference category chosen a priori  were independently analyzed by Generalized Estimating Equations.The study area  holds 4577408 inhabitants  and 5% (p < .0001) with each additional day of heatwave duration. At least four consecutive hot humid days were required to observe a major increase in hospital admissions, the excesses being more than twofold for HD (p < .0001) and about 50% for RD (p < .0001). Hospital admissions for HD peaked equally at the first heatwave (early June) and last heatwave (August) in 2004 as did RD. No correlation was found for FF or CD admissions.The first four days of an heatwave had only minor effects, thus supporting heat health systems where alerts are based on duration of hot humid days. Although the finding is based on a single late summer heatwave, adaptations to extreme temperature in late summer seem to be unlikely. Numerous studies have investigated mortality during a heatwave, while there have been comparatively few studies that have quantified heat associated morbidity. Only a single paper examined the whole burden of heat-related illness by analyzing hospital discharge diagnoses according to International Classification of Diseases, 9th Revision (ICD-9): during the 1995 heatwave in Chicago there were 35% more than expected admissions among patients aged >65 years, mainly due to dehydratation, heat stroke, heat exhaustion and acute renal failure , a recenIn examining the effect of heatwaves, the timing of these episodes across the summer months must be considered. It has been reported that heat waves occurring in the spring or early summer often resulted in more deaths than heatwaves occurring later in the summer -7. SimilA study found a significantly increased mortality with heatwave duration and timing within the summer season, but not with intensity of hot weather . A similThis would suggest that individual hot days are less significant than consecutive hot days, in which individuals do not receive sufficient respite from the heat to recover physiologically.In this report, we examine all of these issues. In order to show the relationship between hospitalization pattern and, on the other hand, intensity duration or timing of hot spells, we compare five consecutive heatwaves (one occurred in 2002 and four in 2003) and the associated hospital admissions in people aged 75 and more years, residents in the Veneto Region, North-Eastern part of Italy.st, 2003), and is subdivided in seven provinces with 60 hospitals and about 20000 beds for acute care. From the regional computerized archive of hospital discharge records, we extracted the daily count of hospital admissions for people aged \u226575, from June 1 through August 31 in 2002 and 2003. A satisfactory level of accuracy of primary discharge diagnoses provided by this archive has been verified for circulatory disease [The study area holds 4577408 inhabitants . All hospital discharge records were anonymized and handled according to national and regional rules on confidentiality.Humidex is a measure of perceived heat that results from the combined effect of excessive humidity and high temperature. The Weather Service of Environment Canada  uses HumWe obtained from the seven weather stations located in each provincial capital of our region the daily values of maximum temperature (\u00b0C) and humidity occurring at the same hour, from which we calculated Humidex according to Masterton and Richardson . Since tEpidemiological evaluation of the data was undertaken using both simple analysis and multivariate models. Analysis was carried out for the entire period of the study, subsuming 184 days, 92 for summer 2002 and 92 for summer 2003. Ours is therefore an ecologic study of time relation between hospital admissions and heat, day being the standard statistical unit. In the univariate analysis, scatter diagram was produced showing the values for each cause of hospitalization and heatwave intensity, duration, and timing within summer. Data included outcome variables , daily value of heatwave intensity and duration and day of week in order to account for the weekly cyclical pattern of admissions . In the multivariate analysis, the parameters of each one of the above variables were estimated through Generalized Estimating Equations (GEE). Modeling the relation of hot weather with hospital admissions was performed using the statistical package STATA 8  through st day) through n (last day) within a heatwave, and 0 otherwise;\u2022 \"duration\", which was 1  limited to the heatwave period, by allowing the response of other days to have a separate estimate .In order to disentangle the effect of heatwave duration from that of timing within summer period, we built another GEE model that included nine dichotomous variables  to take into account five consecutive heatwaves:\u2022 heatwave 1-2002, June 16 through 24, 2002: we separately coded the first (4 days) and second part (5 days) of the heatwave;\u2022 heatwave 1-2003, June 10 through 16, 2003: we separately coded the first (4 days) and second part (3 days);\u2022 heatwave 2-2003, June 23 through 26, 2003 (4 days)\u2022 heatwave 3-2003, July 21 through 23, 2003, (3 days)\u2022 heatwave 4-2003, August 3 through 14, 2003; we separately coded the first (4 days), the second (4 days) and the last part (4 days).In the whole observation period, the mean (standard deviation) of daily Humidex values was 36.5 (4.2) \u00b0C. Means (standard deviations) of daily hospital admissions were: 5.7 (4.3) for Heat Diseases (HD); 39.7 (11.5) for Respiratory Diseases (RD); 124.6 (29.1) for Diseases of the Circulatory System (CD) and 11.7 (3.4) for Fractures of Femur (FF).The daily values of heatwave duration, humidex and admissions for HD or RD are shown in Figure If Disorders of Fluid and Electrolyte Balance (276) and Acute Renal Failure (584) were excluded from the corresponding Main Groups, the latter  as well as all other nosologic groups and FF did not show any relationship with heatwave characteristics, using information obtained in the graphical representations (data not shown).Table Heatwaves 1-2002, 1-2003, and 4-2003 greatly influenced hospital admissions for HD and RD in their last part, while in their first part (four days) they had only a minor effect, like did heatwaves 2-2003 and 3-2003 which persisted for 4 and 3 days, respectively. Duration was thus confirmed as major determinant of hospitalization. Furthermore, early occurrence of heatwave did not result in higher hospital admissions than that of the late event within the summer 2003. Again, FF and CD did not show any consistent pattern of correlation  declare that they have no competing interests.GM contributed to conception and design of the study. UF and GM participated in the analysis and interpretation of data. CV extracted hospital admissions data from the regional archive of discharge records. EF and PS participated in drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "In December 2004, coral populations were devastated by the deadly tsunami that swept across the Indian Ocean. A new survey by the World Conservation Society found high densities of baby corals in 60 tsunami-ravaged locations in Aceh, Indonesia. The coral comeback is being linked to natural colonization by more resilient coral species along with a decline in drastic fishing practices such as the use of dynamite. In addition, transplanting baby corals from healthy wild coral has proved to be a more effective strategy than direct seeding for restocking coral-depleted areas.On 29 December 2008, Agriculture Secretary Ed Schafer announced the U.S. Department of Agriculture will, as directed by the 2008 Farm Bill, form a new Office of Ecosystem Services and Markets and create a federal Conservation and Land Management Environmental Services Board. These entities will assist the department in guiding the implementation of market-based approaches to conservation, such as compensating farmers, ranchers, and forest landowners for providing wildlife habitat, carbon storage, and scenic landscapes. The first service to be examined by the new office will be carbon sequestration.Despite concerns by domestic and international groups about the endocrine-disrupting effects and persistence in the environment of endosulfan, Australia will continue to allow the restricted use of this insecticide. This announcement was made in January 2009 by the Australian Pesticides and Veterinary Medicines Authority. In December 2008 Australia\u2019s closest neighbor, New Zealand, joined 54 other countries in banning endosulfan. The Stockholm Convention on Persistent Organic Pollutants will determine in October 2009 whether to do a final assessment on endosulfan, which could result in a global ban by 2011.In 2008, the Consumer Product Safety Improvement Act was enacted to bolster efforts to protect children from lead and phthalates in products such as toys and clothing. In response to concerns expressed by thrift store owners and purveyors of handmade toys, the Consumer Product Safety Commission voted in January 2009 to refine the act\u2014for example, by relieving thrift shops of the need to test used goods for lead and phthalates. However, resellers and parents alike should avoid selling or buying used products that are likely to contain lead or phthalates.The U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has endorsed two standardized, oral, cell-based tests to help companies determine the potential hazards of chemicals. The new methods measure cell death and can determine the first dose to test in animals. Acute oral toxicity tests are currently the most commonly used toxicity tests used in animals worldwide, and the new tests could greatly reduce the numbers of lab animals needed for such tests. ICCVAM is seeking incorporation of the test recommendations in guidance to be issued by the Organisation for Economic Co-operation and Development (OECD), which will go to OECD\u2019s 30 member countries.In a move that could dramatically curb carbon emissions and European household energy costs, the European Union (EU) is now moving forward with formal legislation to ban low-efficiency light bulbs by 2012. The first phase of the legislation will begin in September 2009, when bulbs with a light output of 100 watts or more must achieve at least a C-class efficiency rating\u2014a requirement that incandescent bulbs of this wattage will be unable to meet. The regulations will continue to be implemented in stages until all classes of bulbs have at least a C-class rating. According to an EU technical briefing on the new legislation, lighting can represent up to one-fifth of a household\u2019s electricity consumption."}
+{"text": "It is built from carefully selected data sets stemming from other curated databases and the biomedical literature. To date, CellFinder describes 3394 cell types and 50 951 cell lines. The database currently contains 3055 microscopic and anatomical images, 205 whole-genome expression profiles of 194 cell/tissue types from RNA-seq and microarrays and 553 905 protein expressions for 535 cells/tissues. Text mining of a corpus of >2000 publications followed by manual curation confirmed expression information on \u223c900 proteins and genes. CellFinder\u2019s data model is capable to seamlessly represent entities from single cells to the organ level, to incorporate mappings between homologous entities in different species and to describe processes of cell development and differentiation. Its ontological backbone currently consists of 204 741 ontology terms incorporated from 10 different ontologies unified under the novel CELDA ontology. CellFinder\u2019s web portal allows searching, browsing and comparing the stored data, interactive construction of developmental trees and navigating the partonomic hierarchy of cells and tissues through a unique body browser designed for life scientists and clinicians.CellFinder ( Therein, CellFinder provides a tool for harvesting the large scientific body of data, allows efficient data browsing and searching at the spatial  and temporal  level together with ontology-based semantic data integration and expandability to new data types. It was designed for easy usage by life scientists and clinicians. Presently, its features are best exemplified for human kidney and liver, yet other tissues and organs are being added at constant pace. Data retrieval functions are demonstrated in a screencast at http://www.cellfinder.org/help/screencast/.The need for cell-focused information resources is accompanied by technological advances that facilitate the analysis of cells in even more detail, which leads to the generation of an enormous amount of cell-related data encompassing expression patterns, function, plasticity, potency, shape, intracellular structures, developmental stage and interactions with their environment. However, world wide-generated cell-, cell line- or tissue-related data are spread over a multitude of heterogeneous resources and partly only available in scientific publications, thus \u2018hidden\u2019 from conventional ways of computer-based processing. Existing integrative databases often focus on singular aspects cell-specific data. These include general gene-centered expression data repositories such as the Gene Expression Omnibus , Array ECell: Expression, Localization, Development, Anatomy) (in vivo and in vitro: Cell Ontology (CL)   was deveogy (CL) , Cell Liogy (CL) , Experimogy (CL) , Human Dogy (CL) , Foundatogy (CL) , Adult Mogy (CL) , Gene Onogy (CL)  and the ogy (CL) . In CELDogy (CL)  and the ogy (CL) . Furtherogy (CL)  was usedogy (CL) , it adheBy incorporating these ontologies and the supplementation with additional data not present elsewhere, CellFinder is capable of hosting the description of cell types based on species, gender, anatomical location, subcellular structures, developmental origin and molecular composition . The conftp://ftp.nextprot.org/pub/current_release/controlled_vocabularies/cellosaurus.txt). CellFinder includes currently 3394 cell types distinguished by ontological terms and species (including 1058 distinct human and 489 murine cell types). A total of 1032 of these cell types have been derived through manual expert selection. Moreover, CellFinder considers 50 951 cell lines, of which 14 346 cell lines have been supplemented from literature, Cellosaurus and hESCreg, whereas the remaining is from CLO and CL.The dictionary of distinct cell types used in CellFinder is thus derived from several ontologies, including those designated for cell lines (CL), anatomical , organism  and developmental information (EHDAA). However, the set of cell types, defined as phenotypically distinct cells, is still incomplete in these databases, and new cell types are continuously defined due to improved characterization methods. Therefore, we use expert knowledge to select and integrate missing tissue and developmental stage-specific cell types from literature and existing databases such as the Characterization Tool , hESCregCellular phenotypes are characterized by classical descriptors such as morphological features, shape, nucleus/cytoplasm ratio, an increasing number of intracellular components  and moreFor the exploitation of expression data, it is important to annotate analyzed samples to cell types, e.g. for the identification of reliable cell- or tissue-associated markers or gene/protein functions. Although there is an increasing amount of available gene and protein expression data available, many data sets are small scale, use specific protocols, which hinder their comparability and are often of unknown quality with respect to the cellular source. Therefore, the high-quality data sets, which were incorporated into CellFinder were selected by an expert committee of biologists and bioinformaticians, taking into account the original publications, acceptance by the community and scientific impact. Several data sets were selected to build comprehensive panels for profiling tissues and cells.Supplementary Table S1. CellFinder is designed to allow researchers to find markers associated to particular cell types and of expression profiles for given genes or proteins, which can be useful when studying the function of a gene. Two precomputed analyses of differential expression were integrated in CellFinder: gene expression in murine samples from stem cells and derivatives from StemBase (Supplementary Methods for details). These analyses are accessible via the \u2018Compare\u2019 button in the CellFinder home page. In addition, the tissue-specific expression levels for genes are provided through the search option and semantic body browser (SBB) (Supplementary Use Cases).Most of the transcriptome data included in CellFinder are derived from microarrays, with the focus on tissues, normal tissue-specific cells and pluripotent stem cells, but also including some cancer cells. The transcriptome data were supplemented with RNA-seq profiles from RNA-seq Atlas . ProteinStemBase , and proSupplementary Figure S1). It is composed of the following steps: triage , preproneRanker ]. The pihttp://commons.wikimedia.org). CellFinder contains in total 3055 images of 1790 cells, tissues and organs. High-resolution microscopy images are available for 85 distinct cells and tissues.Imaging is an important method in cell biology to provide histological, cytological and morphological information on the cellular phenotype. A multitude of different techniques are available to generate images of cells and subcellular structural components, molecular composition and dynamics of cells and tissues. A problem for assessing and analyzing image data is the proprietary file formats that also contain the metadata defining the experimental and acquisition parameters. To store, organize and display images including their metadata in CellFinder, the Open Microscopy Environment (OMERO)  server whttp://jena.apache.org/) and translated into a relational database for the purpose of speed. Access to the ontological data is provided via web services. CellFinder has been carefully implemented to run on a large number of devices and different screen resolutions, although some restrictions still exist (Supplementary Table S2). The backend, integration and access components of CellFinder are summarized in The CellFinder Web site is implemented using PHP, MySQL, Java and JavaScript on a Linux server. CELDA is implemented with the JENA framework . The results list shows basic information as well as an overview of the available data per hit. Exclusion and filtering of certain types of data from the results list is possible  and connects the outcomes with a graphical interface . In a first analysis of 241 complementary DNA microarrays from the StemBase database  to characterize cells derived by in vitro stem cell differentiation by applying the SBB and (ii) to identify and characterize cell differentiation derivatives during renal differentiation with the help of the Developmental Tree.To demonstrate CellFinder data usage, use cases are provided in the CellFinder is a data portal, which provides a unified resource of diverse data on cells. All the data available in CellFinder are of public origin and can be accessed freely using a convenient and intuitive web application.Its implementation as an ontology-based platform allows for further expansion. For instance, we are currently working on integrating the recently developed cell phenotype ontology (CPO) . FurtherThe platform is constantly expanded with further pre-analyzed data and tools for analyzing its content in a convenient manner, for instance to identify genes or protein markers that are expressed in a precise cell type and not in proximally or developmentally related ones. The need for a fully comprehensive cell type catalog and definition of classification standards is emphasized by different numbers of human cell types provided by CellFinder (1058) versus the 2260 suggested by CELLPEDIA by a combination of conventional taxonomy with physical mappings . To the Supplementary Table S3). The CellFinder database has been registered at the BioDBcore catalog (http://www.biosharing.org/biodbcore) to improve its visibility to the community in support of this process.CellFinder was initially focused on a few organs such as kidney and liver because of the required extensive manual data curation and data selection. Work is currently ongoing for other clinically relevant tissues, namely, the cardiovascular and hematopoietic systems. Expansion into more organs and organisms will develop through establishment of a dynamic curation process between experts and users .Conflict of interest statement. None declared."}
+{"text": "World Allergy Organization Journal or the WAO Journal. We envision this journal to be a clinically oriented communication, blending excellent reviews with clinically applied research. We have established an excellent international editorial board, contracted with Lippincott to publish the journal, and will fund the initial distribution, starting with volume 1, January 2008.The world has gotten smaller, and continues to shrink! What goes on in Beijing or Cairo needs to be communicated to New York and Paris. To establish a rapid, efficient, and cost-effective communication system, the World Allergy Organization (WAO) has created a new unique on-line journal, the WAO Journal will immediately reach more allergists than any other publication in the world. We can count on this immediate and extensive impact because our biweekly e-newsletter is a huge success: it has a vast global readership and is translated into 7 languages. We are actively soliciting both high-quality reviews and translational research articles for publication, and greatly look forward to receiving your submission for review.The WAO has made a major commitment to the success of this journal, which will be distributed to more than 25,000 allergists worldwide, ensuring that the Allergy and Clinical Immunology International (ACII), the journal of the World Allergy Organization, has been discontinued. This journal was fun to read and was always interesting. However, it never achieved the readership it deserved or required for continuation, nor was it included in any of the citation indices. We thank those of our colleagues who supported ACII, particularly the past editors, Alain de Weck and Allen Kaplan, and the publisher Hogrefe and Huber. The WAO is giving a special recognition award to Rob Dimbleby of Hogrefe and Huber for his highly valued support.After 18 years, the WAO Journal, joined by Lanny Rosenwasser as coeditor. They have assembled a group of world-renowned allergists as regional editors and an excellent editorial board.Johannes Ring, the current editor of ACII, will transition to the editorship of the Thus, the WAO recognizes the responsibility it has to facilitate excellence in allergy. We are committed to providing education through our lecture series , Web site-related materials and case reports, our new webinars on immunology , a new series of on-line Continuing Medical Education programs based on clinical case histories in allergy, and the Emerging Societies Program .The WAO is now taking this next step to help the world of allergy communicate by sponsoring the new on-line journal. We promise you that it will maintain the highest standards with respect to both review articles and original research. We promise that the subscription cost of the journal will be inexpensive, and that the journal will arrive on-line and on time on your computer. Publication will be rapid and will focus on clinically relevant materials. The WAO journal will continue to be complemented by the biweekly e-newsletter, where important relevant articles will be summarized with commentary and news, and announcements from around the world will be communicated. Thus, you will be receiving continued on-line information from WAO, and the opportunity to communicate observations or information from wherever you work to the rest of world will achieve a new level of availability.Yes, the world is shrinking, we are all working together at a new level of communication, and the WAO journal is an important cog in the wheel that will take us forward. We hope that you love and use this new journal. We all need to work together to ensure its success.Michael A. Kaliner, MDPresident, WAO, 2005-2007"}
+{"text": "The dipole interaction decreases the MR ratio requirement when the elements are properly spaced.The most promising approach to attain a narrow linewidth and a large output power simultaneously in spin torque oscillators is self-phase-locking of an array of oscillators. Two long range coupling mechanisms, magnetostatic interaction and self-induced current, are explored. Synchronization occurs with MR ratio ~14% and volume ~2.1\u2009\u00d7\u200910 The prediction of magnetization manipulated through a spin polarized electric current1\u03bcm. Thus, the most promising approach is a long range coupling through STNOs\u2019 self-emitted microwave currents, predicted by Grollier, Cros and FertThe system including a single STNO has been studied extensively experimentally410In this paper, we use the Landau-Lifschitz-Gilbert equation to describe the dynamics of each individual oscillator and study in detail the MR ratio threshold necessary to achieve phase-locking under two common magnetic nonuniformities: variation in the anisotropy and saturated magnetization magnitude. We check the effect of thermal fluctuation on the required minimum MR ratio. The interaction between the microwave current coupling and the long range coupling mechanism provided by the magnetostatic field is also studied. The interaction effect on the MR ratio threshold is obtained.an\u2009=\u2009Han0\u2009+\u2009(i\u2009\u2212\u20091)/(N\u2009\u2212\u20091)\u2009\u00d7\u2009\u03b4Han (or MS\u2009=\u2009MS0\u2009+\u2009(i\u2009\u2212\u20091)/(N\u2009\u2212\u20091)\u2009\u00d7\u2009\u03b4MS), with i varying between 1 and 10. \u03b4Han(\u03b4MS) is defined as the amount of anisotropy(saturation magnetization) difference between consecutive free layers. Under both conditions, the synchronization process begins when the MR ratio is above 5%. The MR ratio threshold MRth to achieve the completely synchronized state is 8%, while below this threshold, the total oscillation state is partially synchronized with individual adjacent peaks merged into several multiple peaks. The frequency increases monotonically in the MR ratio range where the synchronization mechanism occurs. This trend may be caused by the positive dc component in the self-generated microwave current. In the synchronized state, the peak is similar to a delta function in that its linewidth is nearly zero. The amplitude has a small oscillation with MR ratio caused by the finite integration time (8.4\u2009ms). The time scale of the dynamics for the transition from the static state to synchronization is about one nanosecond for the different MR ratios above the threshold. We also check the MRth as a function of \u03b4Han and \u03b4MS as shown in \u03b4Han and \u03b4MS. The frequency range \u0394f, defined as the intrinsic frequency difference of N oscillators, is induced by the non-uniform properties of Han or MS: it is found that the MRth is also linearly dependent on the \u0394f. Although the coupling appears to be more difficult under the condition of saturated magnetization variation, considering its influence on both the excited oscillation energy and the microwave feedback, the MRth can be viewed as determined only by the resulting frequency range. Based on this prediction, the observed experimental frequency dispersionS and anisotropy dispersion, as shown in \u22125\u2009\u03bcm3). The MRth for \u03b4Han/Han0\u2009=\u20091.8 and \u03b4MS/MS0\u2009=\u20090.03 is 13% and 14% respectively at room temperature 300\u2009K. We inquire into the peak profile at these threshold points and compare the phase-locked state of N oscillators with one single oscillator, in While the noise in the magnetic system caused by thermal fluctuations is detrimental to phase locking, the self-generated ac current reduces the incoherent phase found in multiple oscillator states. This helps to enhance the peak height and reduce the peak linewidth, as shown in the inset of \u03b4Han/Han0\u2009=\u20091.8 at T\u2009=\u20090\u2009K. When only microwave current is included, the MR ratio threshold is 24% in Magnetic feedback from the time dependent dipole field between the STNOs could enhance the coupling at a proper distance and reduce the MR ratio requirement for the phase locked state. We use the finite difference integration technique (more than 7000 elements per oscillators) to calculate the magnetostatic interaction. We study the coupling between two non-identical oscillators with icos(\u03c6i), risin(\u03c6i), miz), where ri and \u03c6i is the amplitude equal to iz is a constant. By Fourier transform of ix\u2009+\u2009imiy, we obtain the polarization of the out-of-plane oscillation mode. The polarization is defined as +1 for counterclockwise rotation, and the polarization is \u22121 for the converse case. We find that for small output power, the two oscillators are in the same polarizations, while for large power, the two oscillators have opposite polarizations, shown in ix oscillation is out of phase, as shown in the insets. We examine the phase difference \u03c61\u2009\u2212\u2009\u03c62 and it closely equals to \u03c0 when the two oscillators have the same polarization in \u03c61\u2009+\u2009\u03c62 is approximately 0 in ix component is in phase and the miy component is out of phase.To analyze the complex oscillatory behavior, we investigate the out-of-plane oscillation modes i injected into the individual oscillator from the feedback mechanisms is computed in one period. Here, the spin torque field produced by the ac current is neglected, because its amplitude is less than 0.01 of the dipole field To illustrate the synchronization via the ac dipole field and self-generated microwave current, we study the phase dynamics of the STNOs in the extended Kuramoto model6Tx and Ty are the diagonal elements of the magnetostatic tensor. The sign of \u00b1 corresponds to the opposite (same) polarization. Following references Ei could be treated as a perturbation, compared to the excited state energy Ei0\u2009~\u20092\u03c0(MSmiz)2. The modified frequency \u03b4fi due to Here, fres. Thus the coupling parameter, which controls the frequency range consistent with synchronization, isFor the synchronized STNO array, both oscillators are in resonance frequency Ty and Tx. So if the intrinsic frequency difference \u0394f\u2009=\u2009f2\u2009\u2212\u2009f1 exceeds the coupling strength for opposite polarization, the same polarization is chosen to achieve the resonant but anti-phase state. Tx completely cancels Ty. We find that in the resonant state of two STNOs of circular shape, anti-phase out-of-plane oscillation is always preferred.f. The approximate intrinsic frequency fi equals to Tz is the diagnonal element of the magnetostatic tensor and negative. If the z components of two STNOs have opposite signs, which represent the out-of-plane direction of the oscillation modes, \u0394f equals to miz, \u0394f equals to m unchanged in both polarization states in the perturbation scheme. Thus the oscillation state with the same sign of miz has larger intrinsic frequency difference. From the simulation results, \u0394f under the same sign of miz is in the range of 0.65 to 0.67\u2009GHz and needs more coupling to lock phase, compared with \u0394f under the case of the opposite signs in the range of 0.61 to 0.63, in miz chooses the polarization state directly to be the same or opposite. This connection is verified for both conditions of fixed distance or fixed MR ratio, as shown in miz evolution is determined interactively by magnetostatic and current feedback mechanism and is difficult to predict. Therefore device design may require a technique for selecting the starting point in order to guarantee the same phase locked state for each use.To confirm the coupling parameters analysis, we estimate the intrinsic frequency difference \u0394\u03c0 and 0 phase when the dipole field or MR ratio is varied. The proper coupling interaction benefits the synchronization so that the required MR ratio for synchronization is below the value for coupling by current alone.In conclusion, we obtain the minimum MR ratio requirement for synchronization subject to the two long range coupling mechanisms of self-induced microwave current and dipole field. The MR ratio threshold is determined by the frequency dispersion (caused by non-uniform properties). It increases linearly as thermal fluctuations induce chaos in the serial array of STNOs. At room temperature, the MR ratio requirement is achievable in GMR devices under the experimental frequency dispersion. When the interaction between self-induced dipole field and microwave current is included, the set of oscillators shows oscillatory phase behavior between Magnetization dynamics in the nanoscale can be accurately described using the Landau-Lifschitz-Gilbert equation.\u03b3, \u03b1, and f. The third term on the right is the spin torque generated by the spin current. The current is polarized by the fixed layer magnetization \u0127 is Plancks constant, e is the magnitude of the electron charge, P is the polarization constant and MS is the free layer saturation magnetization. IS(t) includes the dc source current and ac self-generated current. Under the first order estimation of the circuitwhere \u03b8i is the angle between the magnetization in the free layer and fixed layer. Rload is 50\u2009\u03a9 for the bias tee in the circuitP\u2009+\u2009RAP)/2 is the average resistance of the parallel and anti-parallel states of a single STNO where RP\u2009=\u200910\u2009\u03a9. The material parameters for Co in the following calculations are \u03b1\u2009=\u20090.007, P\u2009=\u20090.35, MS\u2009=\u20091352\u2009emu/cm3, Han\u2009=\u2009500\u2009Oe. Hext\u2009=\u20092000\u2009Oe and the current is 9\u2009mA, that the current density is in the order of 107\u2009A/cm2 to 108\u2009A/cm2.N is the number of oscillators. N, where N is the number of oscillators. When the magnetization in FM2 layer is excited to a stable oscillation state, FM1 functions as both a polarizer of injected charge current depolarized by NM2 and as an analyzer to record the generated voltage signal. Positive injected dc current is defined as electrons flowing from the free to the fixed magnetic layer. The free layer is assumed to be a typical Co thin film with a thickness of 3\u2009nm and an elliptical shape of dimensions 130\u2009nm\u2009\u00d7\u200970\u2009nm, shown in \u03bcW, for a reasonable MR range of 1\u2009~\u200910%.The geometry of serially connected STOs could be implemented in the type of nanowires which have been developed for CPP-GMR experimentsHow to cite this article: Qu, T. and Victora, R.H. Phase-Lock Requirements in a Serial Array of Spin Transfer Nano-Oscillators. Sci. Rep.5, 11462; doi: 10.1038/srep11462 (2015)."}
+{"text": "Angelica gigas Nakai extract (AGNE) through the mitogen-activated protein kinases (MAPKs)/NF-\u03baB pathway using in vitro and in vivo atopic dermatitis (AD) models. We examined the effects of AGNE on the expression of proinflammatory cytokines and chemokines in human mast cell line-1 (HMC-1) cells. Compound 48/80-induced pruritus and 2,4-dinitrochlorobenzene- (DNCB-) induced AD-like skin lesion mouse models were also used to investigate the antiallergic effects of AGNE. AGNE reduced histamine secretion, production of proinflammatory cytokines including interleukin- (IL-) 1\u03b2, IL-4, IL-6, IL-8, and IL-10, and expression of cyclooxygenase- (COX-) 2 in HMC-1 cells. Scratching behavior and DNCB-induced AD-like skin lesions were also attenuated by AGNE administration through the reduction of serum IgE, histamine, tumor necrosis factor-\u03b1 (TNF-\u03b1), IL-6 levels, and COX-2 expression in skin tissue from mouse models. Furthermore, these inhibitory effects were mediated by the blockade of the MAPKs and NF-\u03baB pathway. The findings of this study proved that AGNE improves the scratching behavior and atopy symptoms and reduces the activity of various atopy-related mediators in HMC-1 cells and mice model. These results suggest the AGNE has a therapeutic potential in anti-AD.We investigated the cellular and molecular mechanisms mediating the effects of After the plates had been vortexed, they were centrifuged at 14,500\u2009r/min. The supernatants (cytosolic fraction) were removed and the pellets (nuclear fraction) were resuspended with 300\u2009\u03bcL buffer B . After centrifuging, the supernatants (nuclear fraction) were separated.The nuclear and cytosol extracts were isolated to detect NF-Male BALB/c and ICR mice  were purchased from Daehan Biolink Animal Facility  and housed in clean cages at Daegu Haany University during the 7 days of the experiment. Five mice were placed into each cage under controlled conditions with specific light/dark cycles twice a day. The mice were maintained at 23 \u00b1 2\u00b0C and 55 \u00b1 10% temperature and humidity, respectively, and were fed standard diet with water. All animal experimental protocols were approved by the Animal Ethics Committee of Daegu Haany University (DHU2016-044).\u03bcg/kg) was subcutaneously injected in ICR mice. An acute toxicity test of AGNE determined that the LD50 was >2000\u2009mg/kg. The dosage used in our experiment was determined based on the dosage verified in a previous study of the positive effects of AGNE on ulcerative colitis [Compound 48/80, a reagent which locally induces itching/scratching, was used to verify the anti-itching effect of AGNE on ICR mice. AGNE  and terfenadine (10\u2009mg/kg) as a positive control were orally administered 1\u2009h before compound 48/80  and terfenadine (10\u2009mg/kg) dissolved in PBS were administered orally for 2 weeks. Six symptoms  were assessed using a numerical scale of 0\u20133 based on the severity  at intervals of 1 week.The dorsal hair of the BALB/c mice was removed, and they were left for 24\u2009h to enable the dorsal skin to heal. Then, DNCB solutions (0.5 and 1%) were prepared by dilution with an acetone-olive oil mixture (3\u2009:\u20091). To induce AD, 150\u2009\u03b1, IL-1\u03b2, IL-4, IL-6, IL-8, and IL-10 concentrations of the serum, skin tissues, or cell culture medium were measured using ELISA methods as described. Briefly, 96-well plates were coated with monoclonal antibodies (100\u2009\u03bcL) diluted in carbonate coating buffer (Na2CO3/NaHCO3) pH 9.5 for 12\u2009h at 4\u00b0C. The plates were washed with PBS (0.05% Tween), blocked with PBS containing 1% bovine serum albumin (BSA), 5% sucrose, and 0.05% sodium azide (NaN3). After washing the plates, serum was added for 2\u2009h at 37\u00b0C, the plate was cleaned, and then biotin combined with the second antibody was added for 2\u2009h. After washing the plates, avidin peroxidase was added for 30\u2009min at 37\u00b0C, and then the reaction solution was measured using an ELISA reader at 405\u2009nm.The IgE, TNF-\u03bcm sections, which were mounted on glass slides. The mounted sections were subsequently deparaffinized by treatment with xylene and stained with hematoxylin and eosin (H&E). After the washing and dehydration processes, the specimens were observed using a microscope .The mice were anesthetized with ketamine, and then the skin tissue was excised to examine the histological changes. The skin tissue samples were fixed with 10% formalin solution for 24\u2009h, washed several times, and dehydrated, and embedded in paraffin solution for the preparation of paraffin blocks. The blocks were sliced to obtain 0.5\u20130.6-6 cells/dish), treated with various concentrations of AGNE  for 1\u2009h, followed by PMACI (50\u2009\u03bcM PMA + 1\u2009\u03bcg/mL CI A23187) for 24\u2009h (COX-2), 2\u2009h /p38), and 30\u2009min (NF-\u03baB p65 and I-\u03baB\u03b1). Following treatment, the ICR mice were euthanized, and the dorsal skin samples were isolated. The cells and tissues were lysed with radioimmunoprecipitation assay (RIPA) solution . The protein concentration of the nuclear extracts, cell lysates, and tissue lysates was analyzed using the bicinchoninic acid protein assay . An equal amount of protein (20\u2009\u03bcg) of each sample was heated with 5x sample buffer  at 100\u00b0C for 5\u2009min. The samples were separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane  using a semidry transfer system obtained from Bio-Rad . For measuring the expression of target proteins, the PVDF membrane was blocked in 5% nonfat milk solution for 1\u2009h and then incubated overnight at 4\u00b0C with primary antibodies against COX-2, GAPDH, p-ERK, ERK, p-JNK, JNK, p-p38, and p38. After hybridization of the primary antibody, the membrane was washed twice with 0.01\u2009M Tris-buffered saline (pH 7.2) containing 0.1% Tween 20 (TBST) for 15\u2009min, incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-goat) for 2\u2009h, and then washed five times with TBST for 5\u2009min. The protein expression was quantified using a western blot analysis system , followed by final detection using enhanced chemiluminescence (ECL) western blotting reagents from Santa Cruz Biotechnology Inc. .HMC-1 cells were seeded into six-pi dishes . The significant differences were determined using an ANOVA with Student-Newman-Keuls test for the multiple comparison. A \u03bcg/mL, which showed a 25.6% higher decrease than was observed in PMACI-induced HMC-1 cells  compared to the control treatment, but AGNE pretreated cells showed a more significant dose-dependent inhibition of cytokine production than PMACI-induced HMC-1 cells did. In particular, a high concentration (10\u2009\u03bcg/mL) of AGNE showed inhibition rates of 81.9, 41.1, 49.1, 44.4, 34.6, and 39.8% for IL-1\u03b2, IL-4, IL-6, IL-8, IL-10, and TNF-\u03b1, respectively.We investigated the effect of increasing concentrations of AGNE on histamine release from PMACI-induced HMC-1 cells. Histamine release was enhanced in PMACI-stimulated cells compared to that of the control cells, but AGNE-treated cells showed a higher decrease in histamine release than PMACI-induced HMC-1 cells did. The inhibition of histamine release by AGNE was significantly dose-dependent, especially at 10\u2009-1 cells . We exam\u03b1 level increased in DNCB-induced BALB/c mice. The serum IL-6 level of mice induced with DNCB alone was 1.20 \u00b1 0.050\u2009ng/mL, which was decreased to 0.63 \u00b1 0.016, 0.58 \u00b1 0.015, 0.55 \u00b1 0.037, and 0.59 \u00b1 0.031\u2009ng/mL following treatment with AGNE 10, 20, and 40\u2009mg/kg, and terfenadine, respectively , the IL-6 level was similar to those observed in the treatment with terfenadine (38.12 \u00b1 4.375\u2009pg/mL). The cytokine production in the serum showed the same results in the skin tissue.The IgE level is a very important index of stimulated mast cells and alleviation of the allergic response. To investigate the effect of AGNE on IgE production, a critical factor in allergy induction, we collected blood samples and analyzed the levels of serum IgE using ELISA. As shown in ectively . These lectively . The lev\u03bcg/kg) was intradermally injected. The scratching behavior was counted as one incident of scratching for 30\u2009min. The number of scratching behaviors was significantly increased by compound 48/80 compared to that of the control group, but AGNE dose-dependently and significantly blocked the number of scratches elicited by compound 48/80. In addition, terfenadine showed the strongest inhibition of scratches. The inhibition rates of total scratches in the AGNE groups were 41.2, 57.6, and 62.1%, respectively, while that of the terfenadine group was 72.5% compared with the compound 48/80-induced ICR mice  and terfenadine were orally administered 1 week prior to the end of the experiment. We visually monitored the dorsal skin of the mice for induced atopy, and the incidence was numerically expressed using the scoring AD (SCORAD) index. The DNCB group exhibited markedly severe wounds, erosion, keratinization, and exfoliation, but the AGNE and terfenadine groups showed a reduction of these symptoms. AGNE, especially, showed a marked, dose-dependent improvement in the atopy compared with that of terfenadine (\u03bcm) exhibited marked hypertrophy of epidermal thickness, but the AGNE 40\u2009mg/kg group (2.409 \u00b1 0.490\u2009\u03bcm) showed an inhibition of these symptoms and improved inflammatory responses  and terfenadine as the positive control were orally administered 1\u2009h before compound 48/80 , decursinol, and nodakenin similar to coumarin, especially D, DA, and nodakenin, which are known to have anti-inflammatory effects. In addition to the complex medicinal herbs included in AGN, Gamisasangja-tang or decursin derivatives suppress pruritus and atopic skin inflammation in AD mouse models \u201327. Howeodakenin . The par\u03b2, TNF-\u03b1, IL-6, and IL-8) and release of IgE and histamine in mast cells. Compound 48/80 is widely used in animal and tissue models as a \u201cselective\u201d mast cell activator and can induce scratching behavior in mice [ in vitro and in vivo and specifically induced each biomarker related to the AD model from 2- to 10-fold compared to the control. These results are a similar fold to that of the control of other studies using the same models [\u03b1 and IL-6 [AD is a chronic, allergic inflammatory skin disease characterized by eczema and scratching behavior, which can induce skin injury. PMACI has been evaluated as allergen related to the production of various cytokines  in PMACI-induced HMC-1 cells and TNF-\u03b1 and IL-6 in DNCB-induced mice. Increased IL-4 synthesis is induced by IgE in B cell after overproduction of IL-4 and IL-10 by functional reduction such as inhibition of Th2 cell proliferation when some antigen exposure occurs in the blood of patients [\u03b2, a cytokine that induces inflammation, is produced by macrophages, neutrophilic, leukocytes, epithelial cells, and endothelial cells and is increased by other cytokines such as TNF-\u03b1 or bacterial lipopolysaccharides [In this study, we examined the potential modulatory effect of AGNE on histamine release in activated HMC-1 cells and DNCB-induced mice. The results demonstrated that AGNE relieved itching and heat by inhibiting histamine release. In addition, we investigated the inhibitory effect of AGNE on inflammatory cytokines (TNF-patients . AGNE docharides . IL-6, acharides . IL-8, a Angelica gigas Nakai extract inhibits Th2 cell differentiation [\u03b1 controls epidermal growth and induces the activation of the signaling molecule NF-kB to stimulate Th2 cytokines such as IL-4 and IL-6 and subsequently increase IgE switching. This increases the level of IgE and the subsequent symptoms of AD, such as hyperplasia and acanthosis in AD mice model. It is also known to accelerate mast cell degranulation [Taken together, the deterioration of skin lesions observed in DNCB-induced mice model is due to increased IgE production in serum and skin tissue, as well as degranulation of mast cells caused by an increased mast cell population . A previntiation . TNF-\u03b1 cnulation .\u03baB by phosphorylation of MAPKs [\u03baB is considered a critical transcription factor that is regulated by initial inflammatory reactions due to the expression of genes such as chemokines and adhesion molecules for transporting immune cells or inflammatory cytokines such as TNF-\u03b1, IL-1\u03b2, IL-6, and IL-8. I\u03ba-B\u03b1 binds to and inhibits NF-\u03baB by sequestering it in an inactive state in the cytoplasm, and NF-\u03baB subsequently translocates to the nucleus when I\u03ba-B\u03b1 is isolated from NF-\u03baB. In this study, we demonstrated that AGNE effectively blocked the migration of NF-\u03baB to the nucleus by isolating I\u03ba-B\u03b1 in the cytosol. These results suggest that AGNE suppressed inflammatory responses in AD by reducing the secretion of inflammatory cytokines such as TNF-\u03b1, IL-1\u03b2, IL-6, and IL-8 blocking the NF-\u03baB pathway. MAPKs play an important role in the induction of inflammatory mediator production after activation by external infection. JNK and p38 kinase induce biological reaction such as apoptosis, cell differentiation, and inflammation mediated by inflammatory cytokines such as IL-1\u03b2 and TNF-\u03b1 ERK is involved in signal transduction, which promotes the differentiation or growth of cell. In this study, as the production of cytokines decreased, phosphorylation of MAPKs decreased following AGNE treatment.COX-2, a protein related to inflammation and immune function, is involved in the arachidonic acid cascade and is converted to prostaglandin and leukotriene . In AD, of MAPKs , 45. NF-\u03baB, and I\u03ba-B\u03b1 levels through MAPK signaling pathways. Therefore, AGNE may be an ideal oral anti-AD agent for treatment.In conclusion, our findings showed that AGNE reduces the production of histamine and atopy-related cytokines in HMC-1 cells and histamine, serum IgE, and proinflammatory cytokines in mice model. Also, our data suggest that AGNE mitigates the scratching behavior and atopy symptoms and inhibits the histologic changes. These effects are mediated by the suppression of COX-2, NF-"}
+{"text": "A heat exchange mechanism in the head of kissing bugs helps to prevent stress and regulate their temperature while they feed on warm blood. Related research article Lahond\u00e8re C, Insausti TC, Paim RMM, Luan X, Belev G, Pereira MH, Ianowski JP, Lazzari CR. 2017. Countercurrent heat exchange and thermoregulation during blood-feeding in kissing bugs. eLife6:e26107. doi: 10.7554/eLife.26107Over 14,000 species of insects, arachnids and other arthropods feed on the blood of vertebrates. This blood-feeding lifestyle appears to have evolved independently at least six times since the Jurassic and Cretaceous periods, and perhaps up to 20 times . Blood iAcquiring a blood meal is laden with considerable risks before, during and after feeding . For exaThe heat of the vertebrate represents a frequently overlooked stress associated with blood ingestion even though the temperature of blood-feeding arthropods may increase by up to 15\u00b0C in less than one minute during their meal . It is kAnopheles stephensi (Arthropods use a range of different mechanisms to reduce heat stress during blood feeding: some open small holes called spiracles on their surface to increase heat loss from breathing; the mosquito tephensi  retains tephensi . Now, intephensi .Heat dissipates quickly from the head of a kissing bug while it feeds, allowing the rest of the body to remain at ambient temperature. This is in stark contrast to what happens in many of the other blood-feeding arthropods examined by Lahond\u00e8re et al. (most of the body increases to a temperature near that of the host .Using histology, micro-computed tomography and X-ray synchrotron imaging, Lahond\u00e8re et al. \u2013 who are based at Tours, the Universidade Federal de Minas Gerais, the University of Saskatchewan and the Canadian Light Source Inc. \u2013 noted the close proximity of the circulatory and ingestion systems in the head. They suggested that the flow of the kissing bug\u2019s blood toward the head could help to cool the blood meal as it moves through the ingestion system, before it reaches the thorax and abdomen. This is an example of a countercurrent heat exchanger \u2013 a system where two fluids that flow in opposite directions act to reduce the temperature difference between them.To test this hypothesis Lahond\u00e8re et al. conducted a set of experiments in which they stopped blood circulating around the kissing bug by severing the dorsal vessel. This intervention caused the abdominal temperature of the bug to soar to near that of the host. This triggered the production of more heat shock proteins in the bug, and demonstrates that the head thermal exchanger plays a critical role in dissipating heat from the blood meal.When an arthropod ingests a blood meal, there are a multitude of stresses that must be prevented or tolerated. As well as heat stress, arthropods must eliminate large amounts of excess water and ions , detoxif"}
+{"text": "Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional , antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants.The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains ( Mushrooms are fleshy fungi and have become attractive as a source for the development of several extracellular enzymes with agricultural and biotechnological significance. Worldwide, they are well recognized as supplementary food due to their high nutritional values , 2 and mEven today, most of the tribal people in rural areas use wild mushrooms as their mainstay which sometimes leads to fatality due to the lack of knowledge in differentiating between edible and non-edible/poisonous mushrooms . MoreoveCurrently, amplification of the Internal Transcribed Spacers region of ribosomal DNA (ITS rDNA) has been proven as a powerful tool for the identification and phylogenetic analysis of mushrooms \u201318. In tPa\u201d and sold them at local market for the betterment of their family. Hence, the present work was focused on the collection of wild fruiting bodies from two protected forest areas of Mizoram, Northeast India and was morphologically and molecularly identified by the amplification of the Internal Transcribed Spacer (ITS) region of ribosomal RNA (rRNA). Mycelial cultures were raised and screened for their antimicrobial activities. Furthermore, the identified mushrooms were genetically characterized by using RAPD and ISSR molecular markers. All the collected specimens were deposited in the Directorate of Mushroom Research, Solan, India. We hypothesized that the present study in general will help the local mushroom hunters to differentiate between edible and poisonous fruiting bodies. Overall the selected potential isolates can be exploited for the development of effective antimicrobials.Northeastern India is well known for its rich biodiversity and for its un-tapped bioresources which fallsunder Indo-Burma Mega biodiversity Hot Spot . Though,The following protected forest areas were investigated inthe present study:2 (Dampa Tiger Reserve (DTR) , which is one of the largest wildlife sanctuaries in Mizoram. It covers an area of \u2248 500 km2  and the 2 (Murlen Nation Park (MNP)  is located close to China Hills and covers an area of \u2248 100 km2 . Altitudin situ if possible or else the specimens were retained for later identification. The samples were collected in sterile paper bags and brought into the laboratory. Morphological parameters  were assessed from fresh samples as per Largent and Stuntz [Selected forests were visited for three consecutive years (2013\u201315) during rainy and winter sessions (June to December). Permission to collect wild mushrooms from the protected forests was obtained from the Chief Wildlife Warden, Environment and Forest Department, Government of Mizoram, India issued by Mr. LiankimaLailung, Conservator of Forest (WL), Mizoram, India. The wild mushrooms were identified d Stuntz . DigitalThe collected fruiting bodies were used to raise mycelial culture as per the modified method of Bolla et al. . BrieflyStaphylococcus aureus: MTCC-96; Escheridia coli: MTCC-739; Pseudomonas aeruginosa: MTCC-2453), three fungal pathogens  and a yeast pathogen . All the tested pathogens were obtained from the Microbial Type Culture Collection (MTCC), Chandigarh, India and were maintained at Molecular Microbiology and Systematics Laboratory, Department of Biotechnology, Mizoram University on specific media as per instructions.Antimicrobial activities were tested using three bacterial pathogens  of cell free extractof mushroom isolate and the plates were incubated at 37\u00b0C for 24 h. All experiments were carried out in triplicates and the zone of inhibition was measured in mm.Antibacterial activity was determined by modified agar well diffusion method . BrieflyTCCGTAGGTGAACCTGCGG-3\u2019) and ITS4 (5\u2019-TCCTCCGCTTATTGATATGC-3\u2019) [2, 2.5mm dNTPs, 10 pmols of each primer, 50 ng of template DNA and 1 unit of Taq DNA polymerase. The PCR was carried out in the Applied Biosystems thermal cycler with an initial denaturation at 95\u00b0C for 5 min, followed by 30 cycles of denaturation at 95\u00b0C for 1 min, annealing at 57\u00b0C for 1 min, extension at 72\u00b0C for 2 min and a final extension at 72\u00b0C for 7 min. The amplified PCR product was separated on 1.5% agarose gel and visualized under Bio-Rad Gel Documentation System XR+ . The products were purified by using the Pure-link PCR Purification Kit (In-vitrogen) and sequenced commercially at Sci-Genom Labs Pvt. Ltd, Cochin, India.Total genomic DNA was extracted from the freshly grown mycelial mat as described by Cenis  with minATGC-3\u2019) . PCR rea2, 0.5 mMdNTPs, 5 pmols of each primer, 1 unit of Taq DNA polymerase and 50 ng template DNA. Negative control reaction without DNA was included in each set. The PCR amplification was carried out with the following conditions: initial denaturation at 95\u00b0C for 5 min, followed by 35 cycles of denaturation at 94\u00b0C for 35s, annealing at 32\u00b0C for 45 s, extension at 72\u00b0C for 2 min and a final extension of 10 min at 72\u00b0C. The amplified PCR product was separated on 1.5% agarose gel using TAE buffer and examined under gel documentation system as described earlier. Ten ISSR primers were selected  Ltd,  were useselected  and ampl2, 2.0 \u03bcl of 2.5 mM dNTPs, 0.5 \u03bcl of 10 pmol ISSR primer,1 \u03bcl of DMSO (100%), 0.5 \u03bcl of BSA (10 mg/ml) and 2U Taq DNA Polymerase.The amplification conditions were as follows: initial denaturation at 94\u00b0C for 4 min followed by 35 cycles at 94\u00b0C for 50 s, at 45\u00b0C for 50 s, and at 72\u00b0C for 2 min with a final extension step at 72\u00b0C for 7 min. The amplified fragments were separated on 1.2% agarose gel using 1X TAE buffer and the banding patterns were examined under gel documentation system asmentioned above.The PCR reaction mixture was performed in 25 \u03bcl total volume, containing 50 ng of genomic DNA, 2.5 \u03bcl of 10X Taq Buffer with 15mM MgClSolanumkurzii(KC535794) as an out group. The robustness of the phylogenetic tree was tested by bootstrap analysis using 1,000 replicates using p-distance model [The obtained ITS sequences were compared with type strains retrieved from GenBank/NCBI database using BlastN search program and were aligned using Clustal W . Evolutice model . Trees wSYS version 2.2).Amplified fragments of RAPD and ISSR were scored as present (1), or absent (0), the fragments produced were numbered sequentially and entered into a binomial matrix . Only frSamples (1 g each) were dried for overnight in the oven maintained at 105\u00b0C and weighed, then placed at 575\u00b0C for 5 h. Then, the samples were weighed for final mass and difference between the mass at 105\u00b0C and 575\u00b0C was taken as ash content .Carbohydrate content was estimated by phenol sulphuric acid method with glucose as standard .Protein content was estimated by Bradford method using BSA as standard .The fat was extracted by Chloroform-methanol extraction and estimation method .The lyophilized powder (1.5 g) was extracted by stirring with 40 mL of methanol (25\u00b0C at 150 rpm) for 2 h and subsequently filtered through Whatman No. 4 paper. The residue was then extracted with 20 mL of methanol (25\u00b0C at 150 rpm) for 2 h. The combined methanolic extracts were evaporated at 40\u00b0C  to dryness and stored at 4\u00b0C until analysis .2CO3; finally 4.3 mL of distilled water was added to the solution. The reaction mixture was incubated for 60 min in the dark at room temperature and then the absorbance was measured at 725 nm using a microplate spectrophotometer UV-vis . Total phenolic content was expressed as mg of Gallic acid equivalents (GAE) per gram of dry sample (mg GAE/g). The measurements were done in triplicate.Total phenolic content was measured according to the method given by Singleton et al. . Briefly\u2122 GO, Thermo Scientific, MA, USA) after incubation for 30 min in darkness at room temperature. The flavonoid content was calculated in mg Quercetin equivalents per g of dried sample (mg QAE/g).The flavonoids content was determined by the calorimetric method of aluminium chloride according to methodology previously described by Chang et al . In thisO+) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate  and allowing the mixture to stand in the dark at room temperature for 12\u201316 h before use. The ABTSO+solution was diluted with water to an absorbance of 0.70 (\u00b10.02) at 734 nm. The reaction mixture consisted of 0.07 mL of extract and 3 mL of the ABTS radical. After incubation for 6 min, absorbance was determined in spectrophotometer at 734 nm. The antioxidant activity was calculated by using the following equation.ABTS radical scavenging activity was determined according to Re et al  with somcontrol = Absorbance of negative control at the moment of solution preparationAsample = Absorbance of sample after 45 minA50 values were calculated as the concentration of the sample (mg) required to scavenge 50% of the ABTS free radical. The IC50 is often used to express the amount of concentration of extracts need to scavenge 50%of the free radicals.The IC-1 and 64 scans per second were recorded [The selected wild mushroom samples were dried and mixed with FT-IR grade potassium bromide . The samples were grounded in agate pestle and mortar for obtaining pellets by hydraulic press. The absorbance Fourier transform infrared (FT-IR) spectra of the samples were recorded using JASCO FT-IR 6800 within the scanning range of 400\u20134000 cmrecorded .http://bioinformatics.psb.ugent.be/webtools/Venn/).The data were calculated in mean \u00b1 SD of the values found in triplicates from antimicrobial screening and analysed by one way of analysis of variance (ANOVA) to compare statistical significant differences (P<0.05) between antimicrobial activities of all isolates using Duncan\u2019s multiple range test of SPSS software version 6.0. All the experiments were done in triplicates and the mean was taken. Relative abundance of mushroom isolates was compared between the selected natural forests by using Sigma Plot 12.0 version. Venn diagram was prepared online by using Bioinformatics and evolutionary genomics site .Lactarius, Trametes, Calocybe, Agaricus, Pleurotus, Schizophyllum, Cantharellus, Russula, Boletus, Termitomyces, Pholiota and Lentinus. Relative abundance of the wild mushrooms was compared between the two selected forests at the genus level (Boletus was the most abundant genus at DTR and MNP with 20.16 and 12.1% respectively. Genus Russula(10.48%) and Amanita (6.45%) were the most dominant genus in DTR as compared to MNP with 9.34% and 3.73% respectively. At the same time, genus Mycena and Trametes were dominant in MNP with 2.8% and 5.6% respectively. However, 19 genera were found in common among both the studied locations  and Ascomycota . Anatomical characteristics were also recorded of all collected fruiting bodies . Some ofus level  and the ocations . Moreovein vitro antagonistic activity against the selected fungal plant pathogens by dual culture plate assay and showed diverse antagonistic effects. Out of 76, 45 isolates showed positive growth inhibitory activity against atleast three fungal pathogens, with the percentage of inhibition ranging from 25% to 85% . Five isolates BPSM  exhibited positive antagonistic activity against all the tested pathogens. Among them, isolate BPSM30 indicated highest antifungal activity (78%) against F. proliferatum whereas isolate BPSM41 showed maximum antifungal activity (80%) against F. oxy f. sp. ciceri. The strongest antagonistic activity was found in BPSM33 isolate against F. oxysporum with 85.71% inhibition as compared to control.Out of 231, 76 isolates could be grown on artificial media and were screened for their antimicrobial potential. All isolates were tested for their % to 85% . All isoP. aeruginosa, S. aureus and E. coli. Out of 76 isolates, 37 isolates showed positive effects against two out of three pathogens and all isolates showed positive effect against P. aeruginosa except isolates BPSM17 and BPSM32. E.coliis the strongest pathogen against all the tested isolates except BPSM . The result showed that isolate BPSM01 displayed greatest inhibitory effect (9.63 mm) followed by BPSM48 (9.6 mm) and BPSM47 (9.56 mm) against P. aeruginosa. The result indicated that isolates BPSM01 and BPSM47 had the most significant effect against S. aureusgrowth with 9.6 mm and 9.46 mm respectively. Isolate BPSM03 exhibited broad spectrum antibacterial activity against E. coli(5.36 mm) and S. aureus (4.36 mm), therefore it can be considered as the most potent isolate for further studies  followed by Xylaria , Schizophyllum , Pleurotus , Marasmiellus , Microporus , Auricularia , Polyporus , Lentinus , Bjerkandera , Panus , Gymnopus , Pholiota , Fomitopsis , Hymenopellis  and Lentinula . The phylogenetic tree was constructed based on the neighbor-joining method with Kimura 2-parameter model in accordance with lowest BIC and highest AICc values using Mega 5.05. Transition/Transversion values were also estimated using Mega 5.05 (R = 1.75). ITS rRNA gene analysis classified the isolates into 16 genera and 11 families  with a bootstrap value of 75%  to maximum polymorphic of 78.8% (R5) was observed with an average of 56.8% polymorphism. Two primers showed more than 46% polymorphism. The dendrogram based on RAPD data was constructed by UPGMA analysis. The dendrogram grouped the 45 isolates into two clusters (A & B) with similarity coefficient ranges from 0.58 to 1.00. Cluster A was the largest cluster comprised 39 isolates and divided into two sub clusters A1 and A2. Cluster A1 consist of 25 isolates i.e.  similar .Trametes. Though, the dendrogram was similar with RAPD analysis, there was a slight difference from RAPD analysis. For example, Schizophyllum sp. BPSM  had the closest relationship with Pleurotus sp. BPSM  in the RAPD analysis, while it had similarly found in ISSR analysis where Schizophyllum sp. had the closest relationship with Pleurotus sp. Cluster B showed all the isolates belongs to Trametes in the RAPD analysis, while cluster B showed highly similar result in ISSR analysis  while the minimum percentage of polymorphic was observed using primer P2 (38.6%) with an average of 72.5%. Two primers showed more than 52% polymorphism. Dendrogram was constructed by UPGMA on the basis of the distance matrix by ISSR analysis. It divided the isolates into two clusters (A & B) with similarity coefficient ranges from 0.59 to 1.00. Cluster A was the largest cluster containing 42 isolates. Cluster A divided into two sub cluster A1 and A2. Cluster A1 consist of 34 isolates and Cluster A2 contain 8 isolates. Cluster B was composed of 3 isolates all belong to genus analysis .Pleurotusdjamor BPSM41  showed the highest level of ash content among the mushrooms studied. Three out of six mushrooms  showed almost the same level of proteins 30.3, 34.3 and 35.59 g/100 dw respectively. Carbohydrates content is highest in PanusgiganteusBPSM27 (80 g/100g dw), on the other hand the level of carbohydrates is extremely lower in the other samples ranging from 20\u201380 g/100g dw. Regarding fat contents, Lentinula sp. BPSM45 showed the highest fat content (6.26 g/100g dw) and Pleurotussp. BPSM34 revealed the lowest fat content (2.32 g/100g dw).Nutritional value and energetic contribution of the selected six wild mushrooms is presented in Bioactive properties of the selected six edible mushrooms were assessed by three different assays measuring total phenolic contents, total flavonoids content and scavenging activity of ABTS radical . As showS. commune BPSM01 exhibited the highest TPC value among the studied mushrooms (55.19 mg GAE/g) followed by Pleurotusdjamor(BPSM41) and Pleurotussp. (BPSM34) with 52.27 and 41.44 mg GAE/g, respectively. The lowest TPC value was recorded in Lentinussp. (BPSM37) with 12.76 mg GAE/g. Total flavonoid content (TFC) was quantified as milli-gram of Quercetin equivalent per gram of extract ranges from 0.373 to 0.744 mg of QAE/g. P. djamor BPSM41 showed the maximum TFC (0.744 mg QAE/g) which is followed by Lentinula sp. BPSM45 and Pleurotus sp. BPSM34 containing 0.652 mg QAE/g and 0.528 mg QAE/g, respectively. Panusgiganteus BPSM27 (0.373 mg QAE/g) and Lentinussp. BPSM37 (0.382 mg QAE/g) showed the lowest values of TFC among the mushrooms studied.50 values range from 3.25 to 11.43 mg/ml. The lower the IC50 value of mushroom extract, the higher was their free radical scavenging activity. Among the mushroom extracts we have studied, S. commune BPSM01 with an IC50 of 3.25 mg/ml and Pleurotusdjamor BPSM41 with an IC50 of 3.73 mg/ml indicates a significant antioxidant activity in the methanolic extract of these mushrooms.In this study for scavenging activity of ABTS radical, the IC-1 with functional group of ketone/aldehyde C = O stretch was greatly reduced in P. giganteus BPSM27 when compared to S. commune BPSM01, it shows that solubilization of the hemicellulose has taken place in P. giganteus BPSM27. The hemicellulose peak is totally absent in Pleurotus sp. BPSM34, Lentinussp. BPSM37 and Lentinula sp. BPSM45; it might be due to the complete solubilization of the hemicellulose part present in the samples. Reduction in lignin peak intensity of S. commune BPSM01 of wave number 2937 cm-1 with a functional group of C-H stretching was observed when compared to sample the rest of the studied mushrooms. Similarly reduction in peak intensity for lignin of wave number 1639 cm-1 and 1556 cm-1 was noticed in S. commune BPSM01 and Lentinussp. BPSM37 when compared to P. giganteus BPSM27, Pleurotussp. BPSM34, P. djamor BPSM41 and Lentinula sp. BPSM45. Functional groups representing cellulose and hemicellulose were observed at wave numbers 1200 cm-1 for P. djamor BPSM41 and Lentinula sp. BPSM45. Similarly for S. commune BPSM01, cellulose and hemicellulose peak with different functional group of glycosidic linkage was noticed at the wave number of 930 cm-1. Reduction in peak intensity for Lentinussp. BPSM37 and increase in P. djamor BPSM41 of wave number 1035 cm-1 with a functional group of C-O, C = C, and C-C-O stretching representing cellulose, hemicellulose and lignin was observed respectively, when compared to S. commune BPSM01, P. giganteus BPSM27, Pleurotussp. BPSM34 and Lentinussp. BPSM37.Mushroom samples were analysed in ATR-FT-IR  and different wave numbers, functional groups and their corresponding polymer of mushroom sample were presented in Agaricusbisporus[Volvariellabombycina [Pleurotussajor-caju [Pleurotuspulmonarius [Lycoperdonperlatum [Lentinus tuber-regium [Lentinussquarrosulus [Schizophyllumradiatumin the extracellular biosynthesis of silver nanoparticles and showed antimicrobial activity against gram positive and gram negative bacteria with anti-oxidative properties under submerged fermentation. Moreover, Ramberg et al. [Trameteshas immunomodulatory and anticancer effects.More than 200 different mushroom species have been identified from different parts of the world. Previous researchers have shown the importance of mushrooms for the production of novel secondary metabolites having antimicrobial potential , 49. In sbisporus, Volvariombycina , Pleurotjor-caju , Pleurotmonarius , Lycoperperlatum , Lentinur-regium  and Lentrrosulus . Recentlrrosulus  reportedg et al.  suggesteAmong the studied locations, it was revealed that the diversity of wild macrofungi was higher in DTR than in MNP. It is likely that the variation of wild macrofungi in different ecosystems is governed by different climatic conditions such as temperature, pH of soil, physiognomy, succession and geography. This finding was similarly reported by Angelini et al. , where sPleurotus pulmonarius (BPSM36) showing antibacterial activities against P. aeruginosa (9.4 mm) and S. aureus (7.7 mm). These findings were similarly reported by Chowdhury et al. [P. ostreatus has broad spectrum antibacterial activity. Isolate BPSM45 identified as Lentinula sp. showed antibacterial activity against all tested bacterial pathogens which was in concordance with the findings of Ishikawa et al. [Lentinula edodes has antibacterial action against B. cereus, S. aureus and E. coli. We found that all isolates were highly resistant against E. coli and C. albicans. Previous researchers have similarly reported that both the pathogens were found to be strongly effective against mushrooms [All the wild macrofungi tested were found to have various degrees of antimicrobial effects against gram positive and gram negative bacterial pathogens. Similarly, a previous study has also reported antimicrobial activity from the extract of mushrooms . In our . aureus .7 mm. Tha et al.  who showushrooms . Four stushrooms , Ramesh ushrooms , Chowdhuushrooms  who provin vitro antagonistic activity against three fungal pathogens and 45 isolates (19.48%) showed strong antimicrobial activity against at least two out of three tested pathogens. Interestingly, five isolates  found to inhibit all selected fungal pathogens, similar findings were reported by Jagadish et al. [Lentinula sp.Exhibited the highest percentage of inhibition (55.3%) against F. oxysporum, which is significantly higher than the finding reported by Osman et al. [F. oxysporumby Lentinula edodes LC202 and Lentinula edodes LC2141. Most of the selected isolates in the present study showed antagonistic potential against F. oxysporum f. sp. ciceri, a potent pathogen causing diseases in several crops. The potential isolates  have shown strong antifungal activity which has the potential to develop as a biocontrol agent. Park et al. [TCCGTAGGTGAACCTGCGG-3\u2019) and ITS4 (5\u2019-TCCTCCGCTTATTGATAGC-3\u2019) primers to amplify the ITS region to identify isolates of medicinal mushroom Hericium erinaceum whereas Dentinger et al. [All the isolates were screened for their h et al.  and Udu-h et al.  who demon et al.  who showk et al.  has usedr et al.  have user et al. , and pror et al. , primarir et al. ,71 and sPleurotus sp. (BPSM10 and BPSM34) has been closely clustered together. This finding was similarly reported by Yin et al. [P. eryngii clustered together based on RAPD grouping. It proved that effectiveness of the molecular marker in the genetic diversity analysis of mushrooms at the genus level. The results obtained in the present study provided information regarding the genetic diversity of the strains by combined RAPD/ISSR analysis. In the present study, both RAPD/ISSR fingerprinting analysis clearly showed closely related Schizophyllum sp. strains and formed a mono-subclade at the coefficient of 90% and 95% respectively. Similarly Auriculariapolytricha  clustered together in both RAPD and ISSR technique, which further proved that the selected markers can be used to differentiate at the genus level. The RAPD technique has been successively used to differentiate among different strains of Agaricus bisporus [Ganoderma lucidum complex [Lentinula edodes[Agaricusbisporus[Trametes strains were clustered together and showed high levels of genetic diversity in ISSR markers, and been used in the identification and taxonomy for more than two decades [Lentinula edodes[Cordycepssinensis[Auriculariapolytricha[Agaricus bisporus[The analysis of genetic diversity is important to understand the genetic variation among the closely related organisms based on their genetic makeup. In RAPD analysis, both n et al.  and Ro en et al.  who repobisporus , Ganoder complex , Lentinula edodes; Agaricusbisporus. RAPD hasbisporus. Tramete decades . It is ala edodes, Cordycessinensis, Auriculolytricha and Agar bisporus.Lentinula sp. BPSM45 showed the highest ash content 21.406 g/100g. On the other hand, our study revealed the highest energetic contribution of Panusgiganteus BPSM27 among the selected mushrooms, due to the highest content of carbohydrates and proteins with low fat content. The level of antioxidant activity in mushrooms is largely varied from one species to another and the solvent used for extraction. This could be due to their capability to extract bioactive substances [Among the nutritional contribution of the selected six mushrooms we have studied, bstances . Apart fbstances ,85,86.S. commune 25.03 mg GAE/g where as the TPC of S. commune BPSM01 was 55.19 mg GAE/g which is the highest TPC in this study. Flavonoids were not detected in P. Ostreatusin the findings of Matilla et al [Phenolic group is the major antioxidant compound found in mushrooms, . There ala et al  and Chowla et al . It is rS. commune BPSM01 with an IC50 of 3.25 mg/ml which is the most significant antioxidant activity in the methanolic extract of these mushrooms.The DPPH and ABTS assays are known for their reproducibility; ease of application and low cost  The ABTSMicroporus sp. and Schizophyllum commune respectively. Further testing of these strains for the discovery of novel and efficient antimicrobials should be considered. Microporus sp. strain BPSM 33 showed the highest antagonistic potential against the phytopathogen F. oxysporum with 85.71% of inhibition and could be used for the development of biocontrol agent against F. oxysporum. Furthermore, the genetic variation among the potential strains showing antimicrobial potential was considered by using RAPD and ISSR molecular markers. We reported that the selected markers can be used for assessing genetic diversity of wild mushrooms, considering both marker systems gave similar results in the present study. We also conclude that the wild edible mushroom strains does not have only nutritional values but also can be used as an accessible source of natural antioxidants.The macrofungal diversity from two protected forest areas in India was undertaken and their antimicrobial potential was investigated. For the first time 231 wild macrofungi were collected and documented from the selected locations. The significant antifungal and antibacterial activity was found in"}
+{"text": "Developed societies with advanced economic performance are undoubtedly coupled with the availability of electrical energy. Whilst industrialized nations already started to decrease associated carbon emissions in many business sectors, e.g., by substituting combustion engines with battery-powered vehicles, less developed countries still lack broad coverage of reliable electricity supply, particularly in rural regions. Progressive electrification leads to a need for storage capacity and thus to increasing availability of advanced battery systems. To achieve a high degree of sustainability, re-used batteries from the electromobility sector are appropriate, as they do not consume further primary resources and still have sufficient residual capacity for stationary electrical storage applications. In this article, a blueprint for the electrification of a remote region by utilizing second-life lithium ion traction batteries for an integrated energy system in a stand-alone grid is presented and the implementation by the example case of a Tanzanian island in Lake Victoria is demonstrated. First, economic potentials and expected trends in the disposability of second-life lithium ion batteries and their foreseeable costs are outlined. Subsequently, key decision variables are identified to evaluate logistic aspects and the feasibility of the implementation of an off-grid electrical system in remote areas for economically and geographically unfavorable environments. The practical realization is pictured in detail with a focus on technical performance and safety specificities associated with second-life applications. Therefore, a new type of battery management system is introduced, which meets the special requirements of climate compatibility, low maintenance, enhanced cell balancing capability and cell configuration flexibility, and combined with a fiber-optical sensor system, provides reliable status monitoring of the battery. By carrying out on-site measurements, the overall system efficiency is evaluated along with a sustainability analysis. Finally, the socioeconomic and humanitarian impact for the people on the island is debated. Great efforts are being made in all parts of the world to replace fossil fuels for power generation and in the transportation sector by renewable energy sources. The storage of electrical energy plays a central role in this global endeavor. Batteries are evolving into a key component capable of fulfilling the associated requirements. Notably, the automotive industry has become one of the major drivers of innovation in the battery manufacturing segment over the past five years. Currently, the predominant technology is lithium ion batteries. No other type of commercial energy storage reaches comparable energy densities with a concomitant high-performance and cyclization stability. However, the major disadvantages of the lithium ion battery include high purchase price, uncertain availability of rare raw materials required for production and unclear recycling potential. Consequently, it is expedient and sustainable to use a battery storage of this technology over a prolonged lifespan before disposal. In particular, electric vehicle applications place high demands on the traction battery. After the end of the life cycle, which is usually specified at a state of health of 80%, these components still provide enough residual electrical capacity to be transferred into a further application. Practically each sold electric vehicle leads to a return flow of a traction battery, which turns its respective manufacturer or owner into a potential supplier of energy storage devices or pre-used cells. Accordingly, applications with less stringent requirements for performance and gravimetric energy density are demanded, which, however, have a clear benefit for technological advantages and are likewise sensitive in terms of the purchase price. Stationary energy storage systems are particularly appropriate for the utilization of second-life lithium ion batteries, because they supply and store electricity as needed or provide additional power for the local grid, for example for fast-charging stations of electric vehicles or frequency control tasks, without strict requirements on volume and weight specifications.A specific scenario of developmental interest emerges for stand-alone power grids that are separated from the national grid and exist where a deliberate disconnection is preferred because self-production is more cost-efficient or where no connection is economically or technically feasible for the geography under consideration, as it is often applicable in tropical or remote regions. Operators of these stand-alone grid networks are thus potential customers for second-life battery storages. Lithium ion batteries are well suited for rural power supply due to their cycle stability without a significant loss of capacity over a long service life and the accompanying low maintenance effort. However, it must be taken into account that even brand-new lithium ion cells, as a result of their high specific energy density, pose a major hazard potential, which can result in devastating accidents in the event of a malfunction or during an operation outside of the specification limits. For this reason, the monitoring of the operating conditions is of central importance when batteries of this cell chemistry are applied. To accomplish this, a battery management system is deployed, which records the state variables voltage, current and temperature and adjusts the operating mode based on the condition analyses and predefined system parameters. As even tougher safety requirements apply to second-life batteries, since degradation processes that have already occurred during the first life cycle are often unknown, a battery management system with specific attributes and an operating mode that can be individually tailored to the intended purpose is indispensable. In addition to the integrability of supplementary sensing technologies, such as fiber-optical measuring systems for a redundant determination of the state of charge and state of health, furthermore, an intelligent malfunction detection and the capability to combine different cell classifications and types are essential, making the battery management system the key component for a safe and efficient operation for pre-used, and if necessary, refurbished battery storages.The implementation of energy storage systems in an off-grid operation mode in remote regions requires, in general, a high planning and development effort due to many unknown accompanying parameters and the prevailing system individuality. Moreover, although the realization of such projects is technologically feasible, in many cases it cannot be financed by the users itself. Plant sites also perchance are located in hardly accessible areas with potential safety concerns. However even if the assembly and operation of energy systems in rural areas may not be economically viable without supporting subsidies, they, in turn, constitute an enormous development potential for the regions concerned and represent major humanitarian progress, above all, through the support for the healthcare and education sectors. In view of this fact, the aspiration of this article is to present a comprehensive approach for the construction and subsequent realization of stand-alone power grids with second-life lithium ion batteries as energy storages in tropical and remote regions, using the concrete example of the island of Kibumba in the southwestern Lake Victoria in Tanzania. In the following As previously indicated, the automotive industry is a major force in the further development of lithium ion batteries. For the application of vehicle traction, particularly battery cells with a large capacity and gravimetric energy density are appropriate. In the three prevailing structural shapes, pouch cell, prismatic cell and round cell, single-cell capacities up to 96 Ah and an energy density of 280 Wh/kg are achieved ,2. ReliaIn 2016, the total production capacity of this battery storage type was 198 GWh . By 2025The electric vehicle market is predominated by the USA, Europe and China. The worldwide sales of these vehicles in 2020 are expected to be ten million units, in 2025, 31 million units, and in 2030, 54 million units. Of this total, in 2020, one million units will be allocated to the USA, two million units to Europe and six million units to China, in 2025, five million units to the USA, seven million units to Europe and 14 million units to China, and in 2030, ten million units to the USA, eleven million units to Europe and 23 million units to China . Based oIn the implementation of battery storages assembled from pre-aged lithium ion cells, there are a variety of both technical and economic questions. For a sufficient performance of the system, it is desirable to merge battery cells of the same type and uniform degradation level. Consequently, it is essential either to know the event history during the first life cycle or to have the capability to perform rapid tests on the state of health on a single cell level. For the latter topic, there is a whole range of research work addressing, for example, impedance spectroscopy , voltageEven if pre-aged traction batteries return from their first use, they might be cumbersome to purchase for external third-party storage providers, as the original equipment manufacturers are likely to remain in the possession of the product and will refrain from transferring their technology into inscrutable re-use with security and reputational risks. Accordingly, they will strive to realize their own second-life storage projects autonomously. In fact, major manufacturers have already implemented a number of joint projects and built up the corresponding know-how. In Regardless of the aspirations described above, the vast majority of battery systems for stationary applications assembled so far are still based on the established lead-acid battery technology. The regarding cells are comparatively inexpensive to purchase at a price of 100 \u20ac/kWh  and alsoFocused in this work are battery storage systems in off-grid operation without access to the public grid infrastructure. Although such systems have very high electricity generation costs, a stand-alone grid is most likely appropriate in case the installation of the infrastructure in relation to the expected demand is out of balance, for example, if a natural barrier exists or extremely long distances must be overcome. Under preferable environmental conditions, usually combined with a governmental price incentive scheme, a selective decoupling from the power grid by consuming the self-generated electricity can also be economically worthwhile. Tanzania is a country that is particularly interesting for off-grid projects. Especially in the northern regions with Lake Victoria, dense forest areas and mountain ranges, there are many obstacles, which can only be accessed with significant effort. While 65% of the population in Tanzania lives in remote areas, by 2016 only 7% of this group had access to electricity . The govFor the construction of an off-grid power network, essentially three major components are needed. In addition to the actual electrical cable infrastructure between the supplier and the consumer, a power generator, a network converter and an energy storage device are required. Without a connection to the main power grid, solar energy is the most appropriate source of electricity in Tanzania\u2019s climate. Also conceivable and widespread are diesel generators. The supply of fuel, however, is a problem, particularly for remote regions. Besides, this type of production causes local noise and exhaust emissions. For this reason, it is desirable to use diesel generators only to bridge electricity shortages. The power converter, in accordance with the grid type in a single-phase or three-phase design, regulates the current flows in the system and generates the alternating current for the power grid. DC power grids are also feasible, but only for short transmission paths in the regarding voltage range. In addition, their operation leads to difficulties in the use of commercial electrical equipment. Since generation and consumption, especially in off-grid networks, must correlate at any time, it is necessary to integrate a power storage unit. As described before, lithium ion batteries are particularly suitable for this purpose, if technological and economic hurdles can be overcome. In the following, it is demonstrated which parameters within a geographical balance limit are applied in order to evaluate decoupled regions according to their potentials for electrical infrastructure development.Due to the tremendous divide in infrastructural development between densely populated and remote areas, in Tanzania specifically, places that are far or isolated from major cities have the potential for electrification with rural power supply. This involves primarily islands and some of the areas in the mainland blocked by a natural barrier. Due to limited budget and expert resources, the national supply utility company focuses on highly populated areas closer to the existing grid. In contrast, rural regions are not as profitable for a connection to the national power network ,33. Electrification can be economically appropriate through independent initiatives if external funding or technical equipment are provided and supported by a specialized organization for rural energy supply. Whether projects are realized or not is evaluated by means of key decision variables, which are defined by these organizations. In general, they assign higher priority to commercial activities over individual demands. Accordingly, an assessment is initially conducted to identify the ratio between private and business development opportunities. Another factor is the number of inhabitants or the number of people who live temporarily or permanently in the region concerned as well as the expected population growth. Furthermore, there is an analysis of the people\u2019s daily activities like employment status, economic performance or commuting. The decision is also influenced by the number of houses and a determination of their condition. Likewise, the distribution of houses is implemented. A large spread of houses is accompanying with an increased material consumption for electrical exploitation, which has an adverse effect on the decision. From a technical point of view, primarily, the energy system\u2019s expected lifespan, operational reliability, maintenance effort, safety and installation costs as well as the technology-specific funding eligibility are decisive. If the rural power supply organization evaluates projects based on the aforementioned aspects exclusively, soft factors such as nature conservation beyond legal requirements, development aid and knowledge from test facilities in realistic environmental conditions as well as humanitarian aspects like medical and educational development can be undervalued.In case of a decision in favor of an electrification project, exclusive contracts are concluded with the local authorities for most commonly 30 years, which limit the activities of other investors in the area concerned. An exemplary player in the field of specialization for electrification and power supply in remote regions in Tanzania is Jumeme Ltd., which also acted as a partner for the project presented here. Apart from this, the organization is currently implementing 14 individual projects. Among them are ten villages in the Kigoma region alongside Lake Tanganyika, which will be electrically connected to a stand-alone grid as the national power network is not available. The greatest development potential is found in the southern regions of Lake Victoria. There is a vast number of small but inhabited islands without power connection. Jumeme Ltd. applied the aforementioned economical specifications and four of the islands are now in the process of electrification with a stand-alone grid. Due to the lack of technological resources, such as monitoring electronics and operating knowledge, all of these systems will be equipped with lead-acid batteries for energy storage. As a result of the cooperation, a technology transfer is possible that represents a clear economic advantage in the business environment of the organization. In the following section, the key decision variables are applied to the concrete example of Kibumba Island.The archipelago Nazinga in Lake Victoria consists of eight individual islands. Thereof, two were positively evaluated due to their high population and economic activity in the fish processing industry. In contrast, the island of Kibumba , addressed in this article, was given a negative rating because of the comparatively small number of 150 inhabitants and the lack of production. In terms of business activity, only a store, a food stand, a bar and fishery exist. Aggravating the initial situation, the housing arrangement is exceedingly sprawled, which leads to correspondingly complex infrastructure construction.Despite these disadvantages, Kibumba has a significant humanitarian impact on the whole archipelago. According to the Ujamaa society model, each village community was obligated to organize self-sufficiently with a minimum use of resources. Consequently, only one school and one infirmary in this region exist, which are both located on the centrally positioned Kibumba Island and are a focal point for all residents of the Nazinga archipelago. An exclusion of electrical power for these vital facilities would be a significant barrier for the development opportunities of the entire region. By including these humanitarian factors in the key decision variables, electrification of Kibumba proved to be most impactful. With Jumeme Ltd. it had been agreed that, without any share of expenses, an energy system based on second-life lithium ion batteries would be built on the island as a pilot plant by our group. In return, the partner obtains the complete system and manages the installation of power cables between consumers, subsequent system maintenance and any customer care.After extending the key decision variables resulting in a favorable evaluation for electrification of the island Kibumba, first, the development of the electricity demand was analyzed for the following five years. The focus was on the two before mentioned objects, school and infirmary, as well as on the existing fishing lodge, that each was electrically implemented in parallel to the energy system. In order to provide enhanced access to education and facilitate administrative work, the school has been provided with power cables, sockets and lighting by means of an associated charity project. In this context, computers and printers were also procured. In a similar way, this was realized for the infirmary. By equipping the building with an electrical connection, a cooling system for medicines has already been set up and in the future, it will be possible to operate electrical health screening equipment. The third focus was on the fishery, the only economic sector in the region. Through electrification and the organized ice cube machine, it is now possible to freeze the fish catch and transport it without interruption of the cold chain to the markets on the mainland. Since the electricity offered is very expensive in relation to the average income of a private person, the connection to ordinary households is expected with considerable delay and is therefore subordinate to the main project objectives. The price structure for the electricity is staggered into three parts. For public institutions, like the infirmary and the school, as well as private households the tariff is 1.35 \u20ac/kWh. In contrast, a private individual has an estimated buying power of 1\u20131.5 \u20ac per day. Commercial customers, like shops and other small businesses, have to pay 0.96 \u20ac/kWh and for manufacturing facilities with machinery, such as mills or processing plants, the price level is 0.29 \u20ac/kWh with an additional weekly connection fee of 3.83 \u20ac. However, the latter group is not represented on Kibumba. Based on the estimated customer potential, a maximum requested power supply capacity of 3.125 kW and maximum daily energy demand of 18.75 kWh were assumed. In accordance with this presupposition, the individual main technical components were designed or assembled as described in the next section.To simplify the traceability of the operating principle, the structure and operation of the entire energy system are graphically schematized in 2 and an effective active material area of 64.25 m2. Divided into four parallel strings, the modules were connected in series, resulting in an average string working voltage of 328 V. The orientation of the plant was made in a southerly direction at an inclination angle of 5\u00b0.For electricity production, a photovoltaic system was selected exclusively. Pre-owned modules  with a residual capacity of approximately 90% were therefore utilized. Compared to the brand-new condition, the efficiency had dropped from the original 15% to 13.5%. Taking into account the condition of pre-aging and an estimated total internal system efficiency of 90%, electric power of 6.8 kW must be available for a maximum charging power of the off-grid converter of 5.5 kW. During preliminary measurements at the site, it had become apparent that on average two-thirds of the maximum location-specific solar energy was available due to weather phenomena. Thus, a solar system with a maximum power output of 10.2 kW was required, which corresponded for the respective modules to a number of 44 units with a total module area of 71.71 mIn order to convert the DC supply for the grid into AC power, two inverters , each with two connected photovoltaic strings, were integrated into the system , which have already been used in a previous application in the field of vehicle traction, in the specific case for trucks for the autonomous transport of shipping containers. Each cell has a capacity of 320 Ah at a weight of 9.3 kg and a normal voltage of 3.7 V. The rated charge and discharge current is 160 A. The entire battery of one vehicle, consisting of 192 cells, was completely disassembled to the cell level initially and subjected to intensive status condition testing. At the time of the examination, the battery had an age of four years. Almost all of the cells had a residual capacity of 93%, which was a very high value for a second-life application, as the automotive industry typically assumes a residual value of 80% for the end of the first life cycle. In the solar pre-measurements on site, the average daily energy input was 5.08 kWh/mThe battery was regulated and main-monitored by the battery management system. For the operation of second-life battery storages, special requirements are involved, which result from the different degree of cell aging and the increased risk of malfunction. In addition, in the present case, difficult climatic conditions and the isolated location of the system with the obligation of a maintenance-free operation were aggravating factors. For these reasons, a completely novel and innovative battery management system, with key features that were presented in the following, was developed specifically for the mentioned demands. In principle, the management system was configured in a master/slave structure. Each of the six battery strings was equipped with a slave system and the first string was additionally provided with the master unit, which gathered all status data as well as communicated via Ethernet with the off-grid converter and a central measuring computer and also switched the main relay. Basically, any master/slave configuration could be realized, so that necessary flexibility was given to build second-life batteries of different energy and performance classes with a matched serial and parallel circuit topology. Due to the increased safety requirement, a three-level protection system was implemented. First, the slaves were verified by the master through a permanent information query function with an embedded logical calculation task. Furthermore, regardless of its own condition after a malfunction, each slave could be put into a safe state from the previous one by switching off the string relay control current, resulting in an open string circuit; the switched-off relay thus defined the safe state. Third, all integrated circuits were provided with a watchdog timer, so that a program error of a device led to an auto reboot, independent of external control units. Another special development feature was that the management system had a very high cell balancing capability of 1.08 A per cell, which corresponded to a total balancing power in each string of 55 W at a maximum cell voltage of 4.2 V. Due to the usage of pre-aged cells with potentially greatly varying residual capacities and the very high single cell capacity of 320 Ah, this high power requirement was placed on the balancing system. Under the terms of a maintenance-free system with as few electronic components as possible, a passive balancing unit with ceramic load resistors with a component-specific maximum power per cell of theoretical 7 W was implemented. In order to meet the climatic conditions and extend their lifetime, these parts were equipped with active cooling. In operation, the slaves measured the minimum and maximum voltage values and recognized differences in the state of charge and state of health of cells within a string and compensated them during standby mode by the balancing procedure. For the state calculations, the battery management system used an intelligent and pre-implemented charging detection, so that no external information or supply sources were needed and thus an autonomous operation was enabled.In addition to the differently implementable master/slave configurations, the free programmability was a decisive target for the development. Therefore, the prerequisites were created to integrate and combine cells of different types and reaction chemistries as well as to implement new analysis algorithms and safety systems. These included, as a complement to the standard monitoring values current, voltage and temperature on a cell as well as string level, for example, fiber-optical sensors, which detect, irrespective of electrical and magnetic interferences, measurement data such as cell expansion, pressures and temperature fields. Moreover, system behavior could be influenced elementarily by the free configuration of trigger values.Due to the unfavorable accessibility of the plant and lack of broadband network, data storage and adapted transmission were of crucial importance. The master unit was provided with a memory card that could store internal data at a general memory clock rate of, for instance, 60 s over several decades. In each of the six battery strings, the twelve single cell voltages, with an absolute measurement error of 1.2 mV, as well as the total string voltage, with a total accuracy of <0.1 V, were recorded. Besides, the string current, with an accuracy of 0.1 A, and four temperature points, using thermistor sensors with a resolution of 0.1 K, were monitored. The temperature working range was designed for the widest possible field of application and was between \u221240 \u00b0C and +85 \u00b0C. The value query could be performed up to 20 Hz, depending on the desired resolution. At the highest possible resolution of all measured values, the clock rate was 2 Hz. The external data storage was accomplished on the one hand via Ethernet to the central measuring computer and on the other hand, data records were sent via the global system for mobile communications (GSM) worldwide to a selected recipient even in poor reception conditions. Due to the redundant data acquisition and accurate recording of the cell\u2019s operating history for a status analysis, the state of health could be precisely reconstituted so that the battery cells could be potentially transferred to a third life cycle in later use. For a better understanding of the configuration and functioning, a circuit diagram of the battery management system is attached as Since increased safety concerns prevail for second-life batteries due to their often unknown pre-aging background, additional state monitoring sensors are beneficial for the operation. For this reason, the first string of the energy storage . For this type of data transmission, however, a sufficient mobile radio standard must be available. Since there was no, or only an unreliable, UMTS connection at the location of the energy system on Kibumba Island, the independent state value transmission function of the battery management system was of particular importance. Besides the radio antenna, a solar meter  for the measurements of the solar radiation power, which was used for the system design and efficiency determination, was also connected to the central measuring computer. For a high-performance operation of the system with low losses due to heat generation or fault currents, suitable and generously dimensioned cables were a deciding factor. In order to minimize these losses, all electric cable types and diameters as well as screw connections and contact pressures were selected in accordance with the directive DIN VDE 0276-604. Furthermore, system components were arranged in such a way that only the shortest possible cable lengths were required. This was particularly important for the DC transmission between the photovoltaic installation and the off-grid converter. In the next section, necessary work steps and required expenses for the system built-up process were described.The construction and implementation of such a plant in the prototype stage were very complex and a multi-faceted endeavor that could not be entirely captured in this section. This particularly concerned the development, work and travel effort of the employees involved in the project. Although that component undoubtedly represented the greatest expense, it had to be assessed individually as a result of the specific circumstances and was consequently not considered in the following. Devices available on the market, such as an off-grid converter (2750 \u20ac), the two DC/AC inverters (each 1100 \u20ac) and photovoltaic modules, could be simply accounted for. Since the latter were pre-used modules in the present case, no residual value could be specified; new solar components of these performance class would cost approximately 3500 \u20ac. An indication of the value of the battery was problematic because during the use as a vehicle traction battery it was already a prototype. The current target cost of second-life battery storage was given in the introduction as 88 \u20ac/kWh. With the existing battery capacity, this equated to a price of 7000 \u20ac. In addition, there were costs for electronic small parts and power cables as well as for the individual frame of the photovoltaic system of 1500 \u20ac, so that the total value of the system located on Kibumba amounted to approximately 17,000 \u20ac. In the following The sole weight of the cells was 670 kg, the total for the battery was 1350 kg and with included packaging as well as additional components it was 1500 kg. Furthermore, the solar panels together with the foundation (900 kg), fixing material and power cables, especially those between photovoltaic system and inverter (400 kg) as well as tools and equipment (200 kg) resulting in a total freight weight of 3000 kg. Especially the reloading of the goods from the container to the truck, from this to the transport boat and from the landing place to the location of the system setup were not to be underestimated logistical challenges. In this specific case, as a result of a total lack of technical support, up to eight casual workers were employed at the various sites with a daily wage of 4 \u20ac/person. The focus of this pilot plant was on the demonstration of the technical and organizational feasibility as well as the acquisition of research data. On the basis of the knowledge gained, the derived blueprint would offer the possibility to carry out future, more economically oriented projects efficiently. Savings potentials for these projects constituted economies of scale in logistics processes and cost advantages in purchasing, most notably for pre-used batteries, and transporting large quantities of the equipment in general. Furthermore, the installation by trained local personnel following standardized process steps could be more cost-efficient leaving only the commissioning to the system engineers. Finally, the use of the specially designed resilient battery management system and outlined installation tailored to tropical or remote regions represents a major saving for future projects. After the setup and commissioning of the energy system, various experiments were carried out on the performance of the components. In the following The aim of the investigation was to examine the efficiency and reliability of the system as well as to validate the previously made assumptions and performed measurements. The testing presented in the following takes place within a period of two days. The battery storage was shipped to Tanzania with a state of charge of 33%, which decreased until the finalization of the plant through consumption during assembling to approximately 26%. On the first day, the battery was charged throughout the entire sunshine duration with photovoltaic power; meanwhile, the state of charge increased to 80%. At the same time, the radiation power prevailing at the solar modules was continuously measured utilizing the solar power meter. After sunset, three halogen lamps, each with a rated power of 1.15 kW, were connected to the system as consumers. With sunrise the next day, the consumers were completely turned off. Subsequently, the recharge of the battery storage was monitored for several hours before the measurement was terminated. The data obtained from the initial measurement of the solar irradiation energy are graphically represented in 2 was in good accordance to the pre-measurements, which were taken as the basis for the system design. Taking into account the effective solar area of 64.25 m2 as well as the efficiencies of the modules of 13.5% and the system of 90%, feed-in energy of 42.31 kWh was available. At a maximum approximated solar energy of 8.86 kWh/m2, up to 69.16 kWh could be fed into the system, which was 1.63 times the actual measured value. During periods without connected loads, the maximum storable power was limited by the off-grid converter\u2019s rated charging current of 115 A, which corresponded to a power of 5.11 kW at a standard battery voltage of 44.4 V. Including the system parameters explained above, this limitation occurred with a solar radiation output of 654 W/m2. According to the rated power of the converter and based on the maximum battery voltage of 50.4 V, up to 5.80 kW could be processed for a short time with a converter\u2019s maximum temporal charging current of 140 A even 7.06 kW, which corresponded to a solar radiation of 743 W/m2 and 904 W/m2, respectively. The latter value fitted well with the measured solar radiation profile of 99% or 41.92 kWh of the total energy of the sun, theoretically convertible by the system, could be transformed into a usable form. Based on the rated charging current of the converter and the standard battery voltage, 90% or 38.14 kWh could be stored by the system without any connected loads. When electricity was consumed in addition, the two DC/AC inverter fed directly into the stand-alone grid with a total rated power of 10.00 kW, so that, if the grid system was sufficiently efficient, the entire solar energy could be transmitted, also in the case of an already fully charged battery.The measured total energy on experiment day 1 of 5.42 kWh/m2 as shown in 2 was available. The base load range, up to a set power level of 225 W/m2, covered a daily capacity of the energy of 2.35 kWh/m2. After completion of the solar measurements, the DC/AC inverters were switched off and controlled discharge of the battery was initiated. The logged measuring data during this process are shown in Distinctive in the measured solar radiation profile were the strong power gradients due to shading. Even during the period of maximum insolation, the solar radiation dropped into the intermediate load range, which was classified up to 390 W/mIn order to preferably carry out the discharge experiment in the linear nominal voltage range of the battery, an initial state of charge of 80% was appropriate. After switching on the three bulbs as a load, a current of 73 A was provided by the battery, which corresponded to a power of 3.40 kW. At 23:10 there was a failure of one lamp, so that the current dropped to 50 A, and thus, with the preceding battery voltage of 45.7 V and a state of charge of 73%, a power of 2.29 kW was delivered. On experiment day 2 at 05:10 the second lamp failed at a state of charge of 54%. As a result, at a voltage level of 44.2 V, the current supply decreased to 29 A with a remaining power of 1.28 kW. By the end of the discharge experiment at 08:15, a total energy of 29.03 kWh was removed from the battery with an average power of 2.29 kW over a period of twelve hours and 40 min. Meanwhile, the state of charge dropped from 80% to 42% by 38 percentage points. Based on the discharged energy and the variation in the state of charge, an actual battery energy content of 76.39 kWh results, which was 4% less than assumed for the system design. Possible reasons for this were internal losses, self-consumption of the monitoring or progressive aging during the transportation due to unfavorable ambient conditions.2. With regards to the effective solar module area, this corresponded to a total radiation capability of 33.41 kW. At this measuring point, a battery voltage of 44.2 V and a current of 72 A occurred, resulting in a charging power of 3.17 kW. Together with the 0.68 kW base load, there was a power supply by the system of 3.84 kW from the solar feed, which resulted in an efficiency of 11.5%. At 11:00 at 699 W/m2 with a power capability of 44.91 kW, the second operation point was defined. The battery had a voltage of 44.4 V and a charging current of 93 A at this moment. The power feed was 4.14 kW for the battery and 0.68 kW for the base load inside the grid, corresponding with an overall system efficiency of 10.7%. The last considered operation point was at 11:20 at a solar radiation of 767 W/m2 with a capability of 49.28 kW. At this moment, for the first time, the battery charging current reached its maximum with 115 A at a voltage level of 44.6 V and a power of 5.13 kW. Taking into account the prevailing base load of 0.68 kW, a total efficiency of 11.8% resulted. In this consideration, no internal losses of the battery were assumed because lithium ion battery cells generally had a very high degree of efficiency. For verification, based on the experiment depicted in The second part of the experiment started at 09:15 of the second day with the activation of the DC/AC inverters. The battery charging current varied significantly with the fluctuations of the solar radiation. Over a period of five hours and 20 min, the battery storage was supplied with an energy of 20.11 kWh, whereby the state of charge increased from 42% to 68%; this corresponded to a theoretical storage energy content of 77.36 kWh. The overall efficiency of the installed power system depended on solar radiation, converter operating mode, battery state of charge and ambient temperature. Hitherto, the efficiency of the system in charging operation mode was assumed to be 12.2%, composed of 86.5% loss on the solar modules and 10.0% loss due to conversion. For the analysis, exemplarily three characteristic operating points throughout the second experiment day were selected to approximate the actual efficiency. In order to demonstrate the system behavior in a realistic application scenario, the ice cube machine installed in the fishery was started at 08:35 in continuous operation as a base load with a DC-side power consumption of 0.68 kW. The first operation point considered was at 10:30 at a solar radiation of 520 W/mWhile the second determined efficiency deviated perceptibly, the other two outcomes were in good accordance with the initial assumptions, so that the previous considerations of the implemented energy system\u2019s processable solar radiation power were applicable. A certain degree of additional losses had to be expected since self-consumption of controlling components, active device cooling, system monitoring programs and cable resistances were not included in the valuation. The plant did not show limitations in performance and component alignment and thus met the imposed requirements. Furthermore, no abnormalities, faults or other problems could be located. Conclusively, an examination of selected temperature courses during the discharge and charge experiment was carried out. The respective measuring data are plotted in The battery powerhouse was built of solid stone to achieve the lowest possible temperature fluctuations over the course of the day. The ambient temperature of the system was 26.5 \u00b0C at the beginning of the discharge experiment at 19:30. At 03:00 the temperature dropped to 26 \u00b0C according to the lower nighttime temperatures and temporarily rose to 26.5 \u00b0C at 09:00 due to activities in the house. Starting at 10:15, the value continued to increase, initially stable to 26.5 \u00b0C and from 12:45 more clearly in a short step sequence up to 28.0 \u00b0C until 14:30. A similar course was shown by the cell interspace temperature. Due to the cell activity in the supply of electricity, the temperature rose from the initial 26.2 \u00b0C to 27.1 \u00b0C at 01:00. Due to the dropping ambient temperature and the lower power demand from 23:00 through the failure of one load, again the value decreased slightly to 26.8 \u00b0C. From 12:00, the cell interspace temperature rose significantly to 28.5 \u00b0C, because on the one hand, with high power, the photovoltaic current was stored, and on the other hand, the ambient temperature increased accordingly.The cell\u2019s current collector temperature is one of the crucial state values for the battery management system calculations. For enhanced safety, which is a particular requirement of second-life batteries, the cells were equipped with both typical resistant-electrical and fiber-optical sensors for temperature monitoring. Caused by the high discharge rate at the beginning of the experiment, the cell temperature rose significantly from 28.3 \u00b0C to 30.8 \u00b0C  or from 29.4 \u00b0C to 31.6 \u00b0C , respectively. Subsequently, with the decrease of the ambient temperature, a drop was detected, which accelerated after 23:00. At 09:00 the minimum temperature of 25.5 \u00b0C  or 27.4 \u00b0C  was reached. It was noticeable that the negative temperature trend recorded to this point was markedly more pronounced in the case of the electrical sensor and had less continuity with an additional temporary maximum at 04:30. After 09:00, with the exception of a temporary maximum at 11:00, the value recorded by the electrical sensor increased largely constantly until 14:30 to 33.0 \u00b0C. Over the same period, the data acquisition of the optical sensor indicated a similarly rising cell temperature. At 11:20, when the maximum charging current was reached for the first time, the optically detected temperature increased markedly from 28.2 \u00b0C to 30.5 \u00b0C. After a temporary drop to 29.8 \u00b0C, the temperature rose again significantly to 35.8 \u00b0C at 14:30, which was a noticeably higher value compared to the electrical sensor. One reason for this could be the sunrays entering the house through ventilation slits, which have a different strong effect on the measuring principles.Finally, the internal temperature profile of the battery management system was examined. With the gradual termination of the charging experiment on day 1 at 18:00, the device went into balancing mode. Due to charge balancing, ohmic losses occurred so that the temperature at the main monitoring device increased. With the start of the discharge, the balancing operation ended, which is why the measured temperature initially dropped from 34.5 \u00b0C to 30.2 \u00b0C. Due to a higher system temperature during discharging, at 23:00 a temperature increase of the management system to 30.5 \u00b0C was measured. Subsequently, the temperature dropped overnight to 27.5 \u00b0C and increased again with the start of charging. Meanwhile, with the failure of the second lamp at 05:00 on the second day of the experiment, a short-term rise in temperature by about 0.5 K could be detected. From 13:00 a much stronger increase occurred, ending at 14:30 at a temperature of 33.0 \u00b0C.Despite the high load of the battery storage unit under high ambient temperatures, no abnormalities in the temperature measurement, neither on the cells nor in the monitoring electronics, could be determined. The maximum value recorded was 35.8 \u00b0C, which was within the system specifications of the battery and electronics, that ensured a safe operation up to 60 \u00b0C or 85 \u00b0C, respectively. Due to the low impedance of the battery, the rated current was 160 A per single string and thus 960 A for the entire storage, which was both in the charging and discharging mode that was by far more efficient than required by the system parameters. Batteries suffer thermal losses especially in the range of the charge and discharge cut-off voltage. In particular, lithium ion-based systems are most susceptible to degradation at very high or low state of charge levels. In the use as stationary energy storage, second-life lithium ion batteries should preferably be operated in a limited voltage range. After the termination of the test series, the cell threshold voltage values were set to 3.2 V and 4.0 V to achieve a prolonged second life cycle. As consumer demands increase, the voltage boundaries can be reset.Based on the peak-load tests carried out, it was shown that the installed energy system, on the one hand, meets the electrical performance requirements, and on the other hand, did not exceed any specification limits during operation. It was also demonstrated that the design and the dimensioning of the components were consistent as well as that assumptions about efficiencies and states of health were correct. Since no realistic consumer profiles due to the deferring connection of loads were available up until now, no appropriate estimation of the expected service life could be obtained. In the following The government of a country has the fundamental task of ensuring the best possible development opportunities in its own territory. Since the availability of electricity is a basic prerequisite for this, in most cases a national supply utility company exists, which is responsible for the overall connectivity and public power provision. With limited available governmental budget resources, those areas to be electrically supplied will be explored first and foremost, which are located close to already installed infrastructure and thus are easily accessible; this procedure represents the first implementation scenario in electrification. Rural regions, further away from the national grid, may be economically attractive to access with electrical infrastructure if there is a clear economic potential that can develop after connection. This second implementation scenario requires specialized organizations that hold the necessary know-how for the occurring characteristic circumstances. Furthermore, there are areas that are both remote and lack economic potential. Despite this, under certain preconditions, it is feasible to carry out electrification, as a high humanitarian impact would prevail. For this third implementation scenario, the applied key decision variables must be supplemented by non-economic factors, so that a power grid connection can still be assessed positively if specific location factors, such as health care, education, nutrition or business start-up opportunities appear. As demonstrated by the concrete case of the island of Kibumba, the existence of a school and an infirmary with catchment areas extending beyond the territory of the island itself, as well as the potential to establish a commercial fishery led to a positive evaluation under the terms of the diversified key decision variables, although economic location factors did not sufficiently prevail.Already the impact of the availability of electrical light was a central point in society\u2019s development possibilities since private and business activities were no longer limited by daylight hours and also safety-related issues could be overcome. The access to internet and the use of computers, facilitated by electricity, significantly increased the educational standards of the inhabitants. A future-oriented apprenticeship of the students was an essential contribution to equality of opportunity. In addition to education, a reliable health care system was the basic prerequisite for the emergence of a prosperous society. The first step that this project had initiated, was the possibility of cooling the most important medicines and lighting for a 24-h physical examination. On the basis of this, medical equipment for an advanced treatment could now be procured. Although, surprisingly, it does not appear to be widespread regionally on Kibumba, malaria is the dominant disease throughout the lake and coastal areas. Additionally, bilharzia and otherwise contaminated water pose a major health risk for the inhabitants. Despite its location in the middle of a freshwater reservoir, the supply of drinking water, which is largely collected by roof drainage cisterns, remains a serious challenge. Accordingly, polluted and contaminated lake water is used for cooking and washing, which is particularly critical in the shore area due to the higher temperatures and the lower water change. The lack of sanitation is an added negative factor. Water can be treated thoroughly by boiling and filtering with an electrical appliance, which should be considered in the continued aid to this region. The third principal factor for livability is nutrition. The main supply is contributed due to the geographical location by fishing. This activity is moreover the only opportunity for economic development. The installed refrigeration system with icemaker, which allows the fish catch to be cooled overnight or for longer journeys to lucrative marketplaces without interruption of the freezing chain is therefore very important for this potential industry.As discussed above, the energy system that was assembled within the framework of this project, together with the additional equipment procured in the parallel sub-projects, generated a clear and multifaceted humanitarian impact. In Due to the deliberately robust design of the electronic components, the system was not expected to fail abruptly but degrade gradually. A period of ten years was specified as the minimum service life. The commercially purchased components such as the off-grid converter and the DC/AC inverters meet this requirement according to general experience. The solar modules aged by 10% during their first decade of existence. It was assumed that the linear degradation of 1% per year will continue at a constant level so that after ten years an efficiency of 80% would remain, which corresponded to a remaining daily feed-in capacity of 33.85 kWh taking into account the further previously determined system efficiencies. For the battery, a prognosis is very difficult, because the demand profile had completely changed compared to the first life cycle. However, the cells had lost 7% capacity in four years, which corresponded to an annual rate of 1.75%. As a result, the battery capacity would still be 64.18 kWh after ten more years of service with a constantly progressing degradation. There was no forecast for the consumer profile after the next decade, which was why it was presumed that the consumption anticipated during the system design of 18.75 kWh would be steady after five years. Unlike in the automotive industry, no conclusive storage capacity wear limit could be specified for stand-alone power systems, as it is largely dependent not only on customer behavior but also on the type of supply contracts, customer flexibility and structure as well as the weather situation. From the installer\u2019s point of view, the system is no longer sufficiently efficient as soon as the electricity requirement can no longer be covered for one daylight and two night periods without additional solar supply. Over these 36 h, consumption of 28.13 kWh is expected. Based on the aging process described above, the feed-in capacity of the solar modules would drop to this value after a further 30 years of operation in the system. By then, the battery would still have an energy storage capacity of 40.38 kWh. Nevertheless, a lifetime prognosis of more than ten years is theoretical due to the complexity of the installation. Of great relevance is the disposal of the individual components, especially the photovoltaic panels and the lithium ion battery. As pointed out, in a stand-alone grid even electricity from second-life batteries was virtually unaffordable for the average citizen in the area under consideration. However, there are multiple application possibilities without significant power demands, which would already contribute substantially as improvements in everyday life, for example through LED lighting or mobile phone charging. After the end of its current use, the second-life battery system can therefore be transferred into several individual systems in their third life cycle; the necessary parameter settings and operating modes are already integrated into the developed battery management system presented here. Irrespective of the possible continued use, the disposal case will occur, and in all circumstances, it must be prevented that components of the battery get in contact with the ground or lake water. It is essential, for this reason, to hold the battery cells in the possession of a responsible organization and to lend them exclusively to private individuals by means of a deposit system. The disposal, and in the preferable case, the recycling of the lithium ion battery is a potentially developing industry due to containing raw materials. Generally, the disposal of discarded objects on the African continent is considered critical. All the more important is the specification of governmental framework conditions, which have to be enforced accordingly. In the case of the pilot project, the electricity supplier and operator of the energy system had assured that the batteries would be recycled by certified companies when their useful life ends. This is also an important prerequisite for future projects in order to ensure a sustainable and safe goods cycle. On the northern shore of Lake Victoria, in the Ugandan area around Kampala, several companies, e.g., Asante Waste Management Ltd. and Bodawerk International Ltd., already exist that have specialized in the recycling of batteries. Furthermore, clear regulations for storage and disposal exist, so that good cooperation between battery power suppliers and waste disposal companies in geographical proximity can be established on this basis.For the research institutions involved, this project is about the outline of a blueprint as well as the implementation of a pilot plant, which is not exclusively seen as a contribution to the development of a remote region, but also as an establishment of a quantifiable expertise and the creation of practical test opportunities for second-life batteries in tropical environments in a stand-alone power network operation. The technical and organizational knowledge gained in this way can be directly incorporated into the process efficiency in the realization of future projects, thus significantly improving their cost\u2013benefit ratio. For example, in Lake Victoria alone there are numerous islands with parameters similar to those of Kibumba. Through less cumbersome adaptation without entirely newly engineered solutions and organizational pioneering work, a system implementation can be beneficial even from an economic point of view. The cost factors listed in"}
+{"text": "The pregnant rats were obtained and had a cesarean-section to get at gestational ages of 8.5, 13, and 21 days, following blood samples and skeletal muscle were obtained from fetal rats. Levels of thyroid hormone, insulin, mitochondrial protein, and adipokines were detected using ELISA. Western blotting was performed to analyze mitochondria and insulin signal transduction-related protein in fetal rat skeletal muscle. Immunostaining of Periodic Acid-Schiff (PAS) and Oil Red O was used to observe the accumulation of muscle glycogen and lipid in the fetal rat. The results showed that the levels of thyroid hormone, insulin, insulin signal transduction-related protein, mitochondrial, and adipokines increased with the fetus developed, but had no statistical differences in the PTU group compared with the normal group. In conclusion, pregnant rats with hypothyroidism had no influence on insulin resistance (IR), lipid accumulation, and mitochondrial dysfunction in skeletal muscle of the fetal rats.The present study aimed to investigate the effect of maternal hypothyroidism during pregnancy on thyroid function of the fetal rat. Female Sprague\u2013Dawley rats were randomized into two groups. Propylthiouracil (PTU) group received PTU in drinking water for 6 weeks ( Hypothyroidism is the most common thyroid disease, which is characterized by disordered circulating levels of thyroxine T4), tri-iodothyronine (T3), and thyroid-stimulating hormone (TSH). Thyroid hormones are involved in regulating various physiological functions and pathological processes in the body, including changes in metabolism of carbohydrate, protein, and lipids, influence cell proliferation and development, and modulate response to other hormones . Thyroid, tri-iodThyroid dysfunction may affect the action of adipose tissue, and induce other metabolic dysregulation, such as IR , which iHowever, it is not clear whether pregnant rats with thyroid dysfunction could induce fetal thyroid dysfunction. Hence the present study was undertaken to explore the effect of pregnant rats with hypothyroidism on IR, lipid accumulation, and mitochondrial dysfunction in skeletal muscle of fetal rats. In our study, a stable rat model of hypothyroidism was first established by the treatment of propylthiouracil (PTU). We next evaluated serum levels of thyroid hormone in fetal rats and levels of maldondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), adiponectin, leptin, visfatin, resistin, Irisin and, TNF-\u03b1 in gastrocnemius muscle homogenates, as well as proteins involved in mitochondrial oxidation and insulin signal transduction were detected. Furthermore, in fetal rat skeletal muscle, contents of muscle glycogen and lipid deposition were observed.n=90) by the maintenance of 0.05% PTU in drinking water for 6 weeks (PTU group); control rats (n=50) receive an equivalent amount of water . After 6 weeks, rats in each group had free access to water. Sampling blood was taken from the orbital venous plexus of rats (n=6 each group) randomly selected from PTU group and normal group. Thyroid hormone was analyzed to validate the feasibility of the model. Then female rats with hypothyroidism and control group were kept in separate cages with mature male rats, and the day when a pessary was found was taken as day 0 of gestation (E0). In addition, pregnancy and spontaneous abortion rates were observed. After the mothers were fasted for 12 h, the embryo was taken out at gestational age of 8.5, 13, and 21 days  using a cesarean section. After a 12-h fast, blood sample was taken from fetal rats  to isolate serum and gastrocnemius muscle samples were collected snap-frozen in liquid nitrogen and stored at \u221280\u00b0C or fixed in 4% paraformaldehyde for follow-up study.In the experiment, all Sprague\u2013Dawley  were obtained from Experimental Animal Center of Anhui Medical University . All experimental protocols were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Anhui Medical University. The female rats were randomly divided into two groups. Hypothyroidism was induced in female rats (Homeostatic model assessment of IR (HOMA-IR) was used for assessment of IR by Matthews et al.  method. Serum levels of fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), and free fatty acids (FFA) were evaluated using an automatic biochemical meter .Blood samples from female and fetal rats were collected for serum hormonal analyses. The serum levels of total T3, T4, TSH, and insulin were measured by using ELISA kits . The mitochondrial proteins and adipokines in gastrocnemius muscle homogenates  were evaluated using rat-specific commercially available ELISA kits according to the manufacturer\u2019s instructions. The kits were purchased from the following companies respectively: SOD, GSH-Px, MDA, and CAT ; adiponectin ; leptin, TNF-\u03b1, resistin, visfatin ; Irisin .Total protein (20 \u03bcg) from gastrocnemius skeletal muscle of PTU group and normal group was used for Western blot. Then equal protein samples were separated by SDS/PAGE (12% gel), transferred on to PVDF membranes, and blocked with 5% skim milk powder. Then, the membranes were incubated with primary antibodies as follows: rabbit polyclonal anti-peroxisome proliferator-activated receptor-\u03b3 co-activator-1\u03b1 , rabbit anti-carnitine palmitoyltransferase 1 , anti-glucose transporter 4 , rabbit anti-insulin receptor (IRC) substrate-1 , rabbit anti-IRC , rabbit anti-phosphoinositide 3-kinase , \u03b2-actin  were added at room temperature for 1 h. The results were quantitated with the Enhanced Chemiluminescence-Plus Western Blotting Detection System . The optical density of each band was normalized by \u03b2-actin optical density.Immunohistochemistry was performed on the muscle samples from fetal rats. Tissue sections (5-\u00b5m-thick) were dewaxed, rehydrated, and repaired, then endogenous peroxidase activity was quenched using 3% hydrogen peroxide in methanol for 10 min. The sections were stained with Periodic acid-Schiff (PAS)  or Oil Red O  according to manufacturers\u2019 guidelines. Histology pictures were captured using a microscope with a digital camera .ttest or by one-way ANOVA, followed by Dunnett\u2019s test. P<0.05 was considered statistically significant.All data were expressed as mean \u00b1 S.D. Statistical analyses were performed using the SPSS 19 statistical software program. Statistical significance was evaluated with an unpaired two-tailed Student\u2019s n=6; normal group, n=6) are shown in P<0.05). Levels of TSH were significantly increased in PTU groups compared with the normal group (P<0.05). These results suggested an animal model of hypothyroidism was set up successfully. Examination of pregnancy rate of female rats was not markedly different between each group , whereas a significant increase in miscarriage rate of PTU group was observed . Meanwhile, we found that PTU rats had an obviously increased in adipokines-adiponectin  but no statistical differences in E13 group compared with E8.5 group. Levels of GSH-Px, SOD, and CAT were higher in E13 and E21 group (P<0.05 compared with E8.5 group). In addition, there were no statistical differences in levels of MDA, GSH-Px, SOD, and CAT between the PTU groups and the normal groups in each gestational age.The important components of the antioxidative enzymes play a key role in endogenous defense mechanism, such as SOD, CAT, GSH-Px . To explP<0.05). However, there was no significant difference in the levels of adipokines between the PTU and normal group.It is reported that thyroid dysfunction effect on metabolism of adipokines . We nextP<0.05, P<0.05, It is reported that PGC-1\u03b1 regulates lipogenesis, lipid accumulation, and substrate oxidation in skeletal muscle . CPT defTo analyze the contents of muscle glycogen and lipid deposition, immunohistochemistry stain of PAS and Oil Red O was performed on the muscle samples of fetal rats. PAS stain showed that the PTU groups and the normal groups had glucogen within the cytoplasm at E8.5, E13, and E21, and the positive rate of PAS stain was increased as the growth of fetuses A. BesideThyroid dysfunction results in alterations of glucose and lipid metabolism which causes a series of pathophysiological changes. It is reported that mothers of congenitally hypothyroid infants had a lower T3 resin uptake (T3RU) concentrations . In our An increasing number of researches suggest that maternal hypothyroidism could increase the risk of fetal thyroid dysfunction . It has Various studies reveal that patients with hypothyroidism have an increased level of the hormone insulin . In addiThe abnormal composition and the transport of lipoproteins have been reported in thyroid diseases ,28. T4 tThe research indicates that TH could alter muscle performance and heat production, and thus affect whole-body glucose homeostasis . The rolThyroid hormones have been demonstrated to be a major regulator of mitochondrial bioactivity ,32. It iIn conclusion, we have described an increased insulin and lipids levels, as well as enhancive mitochondrial function as growth of the fetal. But all the data suggest that maternal hypothyroidism during pregnancy is not influence upon IR, lipid accumulation, and mitochondrial dysfunction in skeletal muscle of fetal rats."}
+{"text": "Worldwide, sickle cell disease (SCD) poses a significant public health concern. It causes recurrent morbidity, and premature death is a distinct possibility, especially in Nigeria, which bears half the world\u2019s burden of SCD patients. Nigeria has yet to establish a newborn screening program; consequently, most affected children are diagnosed between one and three years of age when a health problem arises.\u00a0Parents are unprepared to identify SCD and seek comprehensive management early enough for the best outcome. Awareness of carrier status and knowledge of SCD would ensure informed reproductive choices.Questionnaires were employed to conduct a prospective, cross-sectional study of the occurrence of missed carrier status of one biological parent of children enrolled at the pediatric sickle cell disease clinic of a teaching hospital. The institution\u2019s ethics committee approved the study.Of 133 respondents, 53% of the parents were unaware of being sickle cell carriers and did not expect to have children with SCD. Among families in which one or both parents proactively sought to know their genotype, 35% of all parents received incorrect genotyping results regarding the SCD risk of their offspring. The majority of incorrectly assigned hemoglobin AA results occurred in private laboratories. More than 60% of the respondents\u00a0reported gaining awareness regarding SCD\u00a0through antenatal care (51%) and schools (17%), as opposed to public awareness campaigns (8%).Many parents in our study population were unprepared for their children\u2019s SCD diagnosis. Awareness campaigns need to be revisited\u00a0as a major potential\u00a0modality to increase\u00a0outreach about SCD. Standardization and improved quality control of laboratory testing involving screening\u00a0of the sickle cell trait could reduce the frequency of wrong genotype assignment. Sickle cell disease (SCD) is a global health problem affecting over 100 million people \u00a0and is mSCD is an autosomal recessive disorder and can be clinically expressed either by receiving two copies of hemoglobin (Hb)\u00a0S (sickle cell anemia) or one copy of HbS plus another \u03b2-globin variant  . Unlike The presence of the HbS gene can be detected either prenatally or postnatally ,9. PrenaSCD interferes with many aspects of the patients' and their family members\u2019 lives, including psychosocial development, education, marital condition, employment, and overall quality of life . Genetic2\u00a02% to 3%, HbF\u00a00.8% to 2%, HbS\u00a00%, and HbC\u00a00%. For infants and children, the Hb consists of the following types:\u00a0HbF (newborn)\u00a050% to 80%, HbF (six months)\u00a08%, and HbF (over six months)\u00a01% to 2%. Abnormal laboratory results signify the presence of possibly significant levels of abnormal HbS, which may indicate SCD and other hemoglobinopathies [The most common technique used to diagnose SCD and to identify sickle cell trait carriers is Hb electrophoresis . In geneopathies -13.The average age at which the confirmation of the Hb genotype in children with SCD in Lagos, Nigeria, occurs is around 27.33 months. There are several essential implications associated with knowing one\u2019s sickle cell trait status, ranging from ethical considerations (in vitro and in vivo genetic testing) to those concerning societal prejudices . Due to The aim of our study was to determine the prevalence and correlation of missed sickle cell disease\u00a0carrier identification among parents of children with SCD.Study designA\u00a0prospective, one-center, cross-sectional study was performed at a pediatric sickle cell clinic. The clinic was in a tertiary health institution and is one of the main referral facilities providing both general and specialist pediatric care for inhabitants of one of the largest cities in West Africa. Our study was conducted\u00a0over three months from January 2014 to March 2014. A semi-structured questionnaire,\u00a0developed by the study team, was administered to 133 parents of children registered at the pediatric sickle cell clinic who were enrolled in the study.Data regarding parents' age, level of education, family size, whether the parent\u2019s genotype was known before the birth of their child with SCD, and the effect of having a child with SCD on various aspects of daily life, including finances, education, and family interaction, was collected. The biological parent(s) of the child(ren) presenting at the sickle cell clinic and those who have or had a child with SCD who is or was tested and registered at the sickle cell clinic were included in the study. The parents of the child(ren) with SCD presenting with life-threatening medical emergencies were excluded. During the appointments at the sickle cell clinic, the parents were explained the study and consent was taken.Sample size calculation2 p(1-p)/d2, where \u201cN\u201d is the minimum sample size, \u201cZ\u201d is the critical value in the two-tailed test, \u201cp\u201d is the expected proportion of wrong carrier statuses in the population, and \u201cd\u201d is the permissible standard error. Here, Z=1.96, p=8% and d=0.05. Therefore, N=1.962 x 0.08(0.092)/0.0025=113.096. If 10% is added for attrition, sample size becomes 124 patients. One hundred and thirty parents of children with SCD were studied.\u00a0The calculations were based on a\u00a0brief pilot study conducted by the authors at the same site.N=ZStatistical methodsContinuous variables were reported as mean (standard deviation) and range\u00a0and categorical variables as number (percent). The Chi-square test was used to analyze between-group differences for the categorical variables. A p-value of <0.05 was considered statistically significant. All the analyses were done using SAS 9.4 .Age, sex, religion, and marital status of parents of children with SCDA total of 133 parents of children with SCD were assessed during the study period. They consisted of 125 mothers (96%) and eight fathers (4%). The ages of the parents ranged from 23 to 55 years with a mean of 37.3 years. Ninety (67.7%) of the parents were Christian, and 43 (32.3%) were Muslim. None of the parents reported traditional practices as a religion. A majority of the parents interviewed were married (93.23%), three were widowed (2.3%), and two were single (1.5%).Level of education and monthly income of the parentsA total of 52 parents 39.1%) had completed secondary education, 41 parents (30.8%) received tertiary level education, 18 parents (13.5%) had completed primary education, and 19 parents (14.3%) had completed a level of education considered as other. The majority of the parents  reported a monthly income of 10-30,000 naira, 32 parents (24.1%) reported 30-60,000 naira, 24 parents (18%) reported 60-100,000 naira, 16 parents (12%) reported more than 100,000 naira, and 14 parents (10.5%) reported less than 10,000 naira  was almost evenly split with those parents who learned about SCD after the birth of a child with SCD . A majority of the parents  learned about SCD via the hospital, 27 parents (17.3%) learned about SCD via school, 26 parents (16.7%) learned via friends, neighbors, etc., 11 parents (7.1%) learned from family, 11 parents (7.1%) learned from media, and two parents (1.2%) learned about SCD from church SCD campaigns.Reason parents got their initial genotype testedTwenty-three\u00a0parents (33.8%) initially did their genotyping due to school requirements, 14 (20.6%)\u00a0due to an awareness of the disease, 13 (19.1%) due to premarital counseling, 10 (14.7%) as part of their\u00a0antenatal care (ANC), four (5.9%) due to a family history of SCD, and four (5.9%) because of church/mosque SCD campaigns.Currently reported genotype of the respondent parentAll 133 respondents reported a current genotype of sickle cell trait (AS) (100%), with 70 (52.6%) tested in a private lab\u00a0while 63 (47.4%) were tested in a public lab.Currently reported genotype of the second parentThe respondents also reported on their spouse\u2019s/second-parent genotype status. The majority  were reported to be AS, one parent HbC trait (AC) (0.75%), one parent sickle cell HbC disease (SC) (0.75%), and one parent sickle cell anemia (SS) (0.75%). Sixty-nine of those (51.9%) were reported to have done their genotype in a private lab\u00a0while 64 parents (48.1%) reported having completed their genotype in a public lab.Genotype initially assigned to respondentA total of 63 respondents (47.4%) reported to have known their correct genotype, 18 respondents (13.5%) reported to have been misidentified as normal Hb (AA); of those, three respondents (2.3%) had their genotype done in a public lab, and 15 respondents (11.3%) had their genotype done in a private lab. A total of 69 respondents\u00a0(51.9%) genotypes were unknown before the marriage or conception of their child with SCD.Genotype initially assigned to the second\u00a0parentA total of 31 spouses/second parents (23.3%) were reported to have known their correct genotype initially; 33 parents (24.8%) were misidentified as AA; of those, three parents (2.3%) carried out the genotype in a public lab while 30 parents (22.6%) conducted their exam in a private lab. Over half of the spouses/second parents  were reported not to have known their genotype before marriage or conception of their child with SCD  reported that they did not consider their genotype nor their partners' before entering into marriage or conceiving their child with SCD while 38 did Table .Genetic counselingThe majority of parents 117, 88%) reportedly received genetic counseling after discovering their child had SCD while 16 (12%) did not . Parents and caregivers of children with SCD are faced with several challenges, which can be costly for the family and can result in a\u00a0subpar health-related quality of life due to the parents\u2019 ill-preparedness to finance a child\u2019s healthcare .Early diagnosis of SCD provides the opportunity to deliver early treatment and appropriate and timely vaccination and management . A randoMissed identification poses a significant impact on the type of care received. Several interventions introduced in early childhood, including penicillin prophylaxis, transcranial Doppler scans, and pneumococcal vaccination, have decreased the mortality rate . The impSCD has a considerable influence on the socioeconomic status of a family. Twenty-two percent of respondents reported having marital discord due to the stress of having a child with SCD; marital discord can further negatively influence the quality of care of their child with SCD due to potentially creating stressors at home for the child with SCD. Parents also reported a high incidence of their other children showing resentment towards their child(ren) with SCD (19%), again potentially increasing the stressors the child with SCD has to deal with it. Nevertheless, many parents also reported that having a child with SCD has brought the family closer together (47%), thus demonstrating that families can rally together to serve as a support system for one another. Respondents also frequently reported the frequent absences their child with SCD had from school. However, parents did not let it affect the school attendance of their other children (78.2%).As mentioned earlier, SCD can have a significant impact on the finances of a household. Most of the parents reported a monthly household income of 10-30,000 naira. Though a majority of the respondents stated that they spend less than 2,000 naira/month for the general care of one child with SCD, financial hardship can easily arise when the child falls ill or needs to be hospitalized. According to respondents, most hospitalizations of a child with SCD cost more than 20,000 naira (60%), thus a large majority of the respondents of this study spend a range, from 60% of their monthly income to all of their income, with the need to also take out loans (43%) per hospitalization. Financial problems are compounded because many parents also have unstable employment. Though a majority of respondents reported that there was no influence on their employment due to having a child with SCD, 14.5% of respondents stated that they were voluntarily unemployed to care for a child with SCD, further reducing the income the household earns/month.The study demonstrated that knowledge of SCD correlates with educational level and financial status. More-educated parents knew about SCD before having an affected child. The highest risk of being misidentified as a non-carrier of a hemoglobinopathy trait lies with those in a higher income bracket  and those with higher levels of formal education. Respondents in these classifications were less likely to have initially unknown genotypes and had an increased likelihood of using a private lab facility. Utilization of private lab facilities proves to be a precarious option due to the lack of oversight and quality control, which may contribute to the significantly higher rate of misidentification of genotypes (88%) as compared to those that occur in public facilities (12%). Inversely, it can be inferred that the lack of education and funds inadvertently protected from incorrect identification. However, the same group is shown to be at an increased risk due to ignorance and lack of preparation.The majority of respondents who knew about SCD before the birth of their child with the disorder reported learning about the health condition through the hospital (50%), followed by school (17%). Surprisingly, media  was the second least cited source of knowledge about the disorder (7%). Most respondents stated that school (34%) was the reason for their initial (before having a child with SCD) genotype testing, followed by awareness of the disorder (20%) and marital counseling (19%). This demonstrates the significant public health role that hospitals and school systems play in educating and mobilizing the public about the disorder. It is also important to note that many respondents quoted \u201cantenatal care\u201d (14.7%) as a reason for getting initial SCD genotyping done. This finding is significant, potentially correlating to the reason why more mothers than fathers are genotyped before having an affected child, also capturing another prospective technique to educate the population further.LimitationsOne of the limitations of the study was the inability or lack of access to interview both parents of each child, which resulted in questionable data regarding the income reported by the mother. Of the parents interviewed, mothers were the majority in the study; as a result, there is a biased motherly view of the data collected. Also, the educational status of the spouse was unable to be assessed, which inhibited the ability to categorize the social class of each participant. Another limitation of the study was the \u201cNigerian parlance,\u201d encountered while administering the questionnaire. Due to cultural reasons, many parents would not want to speak negatively, as they do not want their statements to come to fruition as a result. Instead of answering questions truthfully, or candidly, parents would give answers such as \u201cGod will provide,\u201d \u201cIt is well,\u201d \u201cThe child is strong,\u201d and \u201cGod forbid.\u201d This occurrence slightly disturbed the data collection during the study.RecommendationsThe government should initiate an SCD awareness campaign, utilizing more media sources to reach the population, creating awareness\u00a0and providing knowledge of SCD. A push to have schools and antenatal care providers strengthen SCD awareness and educative programs would also be a great benefit to society. Lastly and most importantly, the government should help standardize and improve the quality control of laboratory practices, especially in the private sector, by implementing policies and establishing oversight over the industry. This project\u00a0would serve as a great pilot for a\u00a0multicenter study\u00a0to increase the power and reduce variance.SCD presents significant challenges to both the families of affected children and the country\u2019s healthcare system. There is a high rate of missed information and misinformation about the risk of having children with SCD, leaving parents ill- or unprepared. Currently, private hospitals are less accurate at identifying hemoglobin genes relevant to SCD. Additionally, antenatal care and schools are great platforms to promote the testing and awareness of the disease. This study is the first to address the issue of missed identification and incorrect identification of sickle cell trait carriers in Nigeria. Our results demonstrate that there is room for cost-effective, public-health-oriented contributions to increase the awareness and implementation of screening for SCD. Furthermore, a greater push in ensuring that citizens receive accurate and affordable SCD genotyping results by establishing quality control standards. These measures can help combat the prevalence of the disease\u00a0and empower potential future families to make informed decisions."}
+{"text": "The study aimed to investigate the effectiveness of Apadent Pro (Sangi) Nanohydroxyapatite (nHAP) dental cream to relieve Dentin Hypersensitivity (DHS), compared with a positive control cream containing 20% pure silica (Silica).P<0.05).In this double-blind, randomized, parallel-group clinical trial, patients diagnosed with DHS and qualified to participate were randomized into two groups, nHAP (n=25) and Silica (n=26). Subjects\u2019 baseline and posttreatment sensitivity were assessed using two pain scales, a four-point Dental Pain Scale (DPS) followed by a linear Visual Analog Scale (VAS), after the application of ice-cold and air stimuli. Subjects used custom-fabricated trays to apply their respective cream for 5 minutes once daily following brushing with standard fluoride toothpaste. Posttreatment sensitivity (efficacy) was assessed every 2 weeks for 8 weeks. Mean treatment outcomes (percentage change from baseline) at each time point were compared using the Tukey HSD test for multiplicity (P<0.001) reduction in DHS at each time point with either nHAP or Silica. Comparing pain scales, VAS showed no significant difference in DHS reduction between the products with either air or cold. However, with DPS, DHS reduction was significantly (P<0.05) better with Silica than with nHAP at all time points with cold, and at 2, 4, and 8 weeks with air.With either air or cold stimulus, VAS and DPS indicated a significant (Both Apadent Pro nHAP and Silica dental creams are effective at promoting the relief of DHS symptoms. When comparing the efficacy of the two compounds to relieve DHS, results of the two pain scales were conflicting. These stThe reported prevalence of DHS is higher in females than in males. In a United Kingdom study of 18 general dental practitioners, the ratio of female to male patients diagnosed with DHS was 1.5 to 1 . The mosThe most widely held theory for DHS is the hydrodynamic theory, which postulates that when dentin tubules are open to the oral cavity due to enamel and/or cementum loss and a trigger stimulus occurs, fluid movement within the dentin tubules stimulates nerve receptors within the pulp, resulting in short, sharp pain .The primary therapeutic approaches to DHS are desensitization through chemical agents that suppress or modify the nerve impulse, or occlusion of the dentin tubules to decrease dentin permeability and thereby prevent fluid movement in the dentin tubules . PotassiOcclusion of the dentin tubules is the most common therapeutic approach . Many ofin vitro studies that established the potential of nHAP to occlude dentin tubules as a surrogate measure of its ability to relieve DHS symptoms [Nanohydroxyapatite (nHAP) is another agent with the potential to treat DHS. Studies have shown that nHAP has the ability to remineralize caries when applied in toothpaste or topical cream form -38. It isymptoms -42. Thessymptoms . HoweverIt is pertinent to mention that DHS is a form of pain. Pain is a personal psychological experience, and its severity is known only to the sufferer, and an observer can play no legitimate part in its direct measurement . For assThis study tested six null hypotheses. The first null hypothesis was that VAS scores of the patients treated with Apadent Pro were not significantly different from those before treatment. The second null hypothesis was that DPS scores of the patients treated with Apadent Pro were not significantly different from those before treatment. The third null hypothesis was that VAS scores of the patients treated with pure silica were not significantly different from those before treatment. The fourth null hypothesis was that DPS scores of the patients treated with pure silica were not significantly different from those before treatment. The fifth null hypothesis was that there was no statistically significant difference between the VAS scores of patients treated with Apadent Pro and pure silica in relieving DHS. The sixth null hypothesis was that there was no statistically significant difference between the DPS scores of patients treated with Apadent Pro and pure silica in relieving DHS.2This was a double-blind, randomized, parallel-group, single-center clinical trial to test the ability of dental cream containing 20% nHAP to relieve the symptoms of DHS, comparing it to that of a positive control cream containing 20% pure silica. The primary outcome to be examined was the percent change from baseline to air and cold stimulation at 2, 4, 6, and 8 weeks on a linear Visual Analog Scale (VAS) and a four-point Dental Pain Scale (DPS).The study was conducted at the clinical research facility of the University of Texas Health Science Center at San Antonio (UTHSCSA) School of Dentistry. The UTHSCSA Institutional Review Board (IRB) approved the study (protocol #HSC20150221H), and the study was conducted in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki and its later amendments. The study was registered with ClinicalTrials.gov (NCT02918617). Written informed consent was obtained from all participants prior to their participation in the study. The majority of participants were recruited from among patients receiving treatment in the dental school clinic and from among employees of UTHSCSA.2.1Subjects aged 18 to 80 years were given a screening examination that included a medical history, oral examination, and DHS evaluation of teeth with exposed buccal cervical dentin. DHS was assessed by a positive response to a 2-second blast of air from an air-water syringe. To be eligible for enrollment the subject must have recorded a response of 20 mm on a 100-mm VAS scale on at least one tooth with exposed buccal cervical dentin.Exclusion criteria were a history of adverse effects with the use of any oral hygiene products; use of over-the-counter desensitizing products within the previous 3 months; use of medications that could interfere with the perception of pain including chronic use of anti-inflammatory, analgesic, anticonvulsant, sedative or other psychotropic drugs; and pregnancy or breastfeeding. Subjects were also excluded from participation if the sensitive tooth was associated with periodontal abscess, mobility >1, or pain from periodontal-related causes or occlusal trauma, or if the sensitive tooth was restored in the previous 3 months or served as an abutment for a fixed or removable dental prosthesis.2.2Subjects who satisfied enrollment criteria were given a commercially available standard fluoride dentifrice  and an adult soft-bristled toothbrush to use for a period of 1 week. Subjects were instructed to use the dentifrice and toothbrush for 2 minutes twice a day in place of their normally used dentifrice and toothbrush. Subjects were given no further instructions regarding oral hygiene during the washout period.At the time of enrollment (beginning of washout period), impressions of the arch(es) containing the identified sensitive tooth or teeth were made using alginate impression material  and disposable plastic trays. Impressions were poured with yellow dental stone , and spacer was applied to the dental casts on the buccal surface of identified sensitive teeth to create room for the test or control cream. Custom trays were fabricated using a vacuum former  and 0.9-mm soft tray material . Subjects were asked to return after 1 week.2.3At the end of the washout period, patients returned to the clinic for baseline sensitivity measurements by a clinical examiner (KL or SS). Ice-cold stimulus was applied using refrigerant spray  on a cotton pellet for 1 second. Air stimulus was applied using a dental air-water syringe for 2 seconds. Patient response to ice-cold and air stimuli were recorded on the VAS and DPS pain scales for each sensitive tooth identified at the time of enrollment. To ensure accurate interpretation and analysis of the sensitivity response, the primary (KL) and secondary (SS) examiners were trained and calibrated by a benchmark examiner (the primary investigator BA) on DHS assessment techniques. The first five subjects that were recruited into the study were used for the calibration exercise. These were patients who had been diagnosed as having DHS. The sensitivity evaluation data recorded during calibration comprised four measurements: Air VAS, Air DPS, Cold VAS, and Cold DPS. The agreement between the two examiners\u2019 objective evaluation of sensitivity (interexaminer agreement) and each examiner\u2019s individual sensitivity evaluation (intraexaminer agreement) was evaluated using the unweighted kappa (\u03ba) statistic.Patients were then randomized by the study coordinator to a group to use either dental cream containing 20% nanohydroxyapatite (\u2018nHAP\u2019) or dental cream containing 20% pure silica, in the form of standard pure silica particles combined with hydrated nanosilica (\u2018Silica\u2019). However, to ensure that both the operators and the patients were blinded as to product assignment, all dental cream tubes were packaged identically and coded (P or Q) by the manufacturing/ packaging company, who retained the code until the completion of the study and data interpretation. The custom trays were tried in place and assessed for fit and comfort.e.g., mouthwash, chewing gum) or tooth-whitening product for the duration of the study.Patients were instructed to continue brushing with the standard fluoride toothpaste and toothbrush for 2 minutes in the morning and 2 minutes before bed, followed on each occasion by rinsing with 10 mL of water for 10 seconds. Patients were instructed to apply their respective cream for 5 minutes once daily following the before-bed brushing using a ribbon of cream in the custom tray, followed by expectoration of the cream without rinsing. The first cream-use occasion took place at the clinical research facility and was supervised by the study coordinator. Subjects were instructed not to take any food or drink for 30 minutes after cream use. As a method of monitoring compliance, a calendar was provided to each subject for recording of daily cream use, and in addition, dental cream tubes were weighed at the time of randomization and at each study visit. Subjects were instructed to maintain their normal dietary habits and were prohibited from using any other oral hygiene product  as described above, and a clinical examiner screened the subjects for adverse events.2.4t-test. The primary analysis was a linear mixed effect model, which should achieve similar or greater power as the two-sample t-test. The primary outcome to be examined in the present study was disease treatment, i.e., relief of DHS.The sample size calculations, which were based on a power analysis, were performed using nQuery Advisor software . Based on previous studies, , 47 and 2.5t test between treatment groups using final (8 weeks) data. A P value of < .05 was considered significant. Statistical analyses were conducted using the R environment for statistical computing (v3.1).The VAS and DPS values as well as their respective percent changes compared to baseline were treated as continuous variables and assessed separately using a random-intercept mixed model analysis that accounted for the correlation among outcomes from the same patient. The primary test of the treatment effect was the treatment by time interaction. Additionally, all the mean treatment outcomes at each time point were compared with the Tukey Honestly Significant Difference (HSD) test for multiplicity. The secondary test of the treatment effect was a 31). Of the 56 subjects recruited for this trial, 4 declined to participate further during the washout period. One subject declined to participate further after randomization but prior to the first posttreatment sensitivity measurement. Fifty-one healthy adults  with a mean (SD) age of 45.5 (13.0) years, from different ethnic origins and socioeconomic statuses, participated in this study (Table 1). The number of participants enrolled in each group were as follows: nHAP (n = 25) and Silica (n = 26). The mean sensitivity at baseline for each measurement  is shown in Table 1 for the nHAP and Silica groups. No incidences of adverse effects were reported by subjects or ascertained clinically.A flow diagram of participation in the trial is shown in Fig.  for the four sensitivity measurements for the primary (KL) and secondary (SS) examiners respectively were 0.80/0.88 (Air VAS), 0.87/0.89 (Air DPS), 0.91/0.94 (Cold VAS), and 0.90/0.89 (Cold DPS). The mean interexaminer reliability values for the four sensitivity measurements comparing the benchmark examiner versus each of the two examiners (KL and SS) were 0.72 (primary) and 0.80 (secondary). These values met the targets set for acceptability prior to the training and calibration.2-5) and in Table 2. With either air or cold stimulus, VAS and DPS indicated significant (P < .001) reduction in DHS at each time point with either nHAP or Silica. VAS showed no significant difference between nHAP and Silica in DHS reduction with either air or cold , although at 8 weeks, Silica had an 11.68%  larger Air VAS change from baseline and a 4.46%  larger Cold VAS change from baseline than nHAP. However, when measured with DPS, DHS reduction was significantly (P < 0.05) better for the subjects using Silica cream than for those using nHAP cream at all time points with cold , and at 2, 4, and 8 weeks with air . At 8 weeks, Silica had an 11.07%  larger Air DPS change from baseline and a 10.27%  larger Cold DPS change from baseline than nHAP.The percent change from baseline over time for the four sensitivity measurements is shown in Fig. . This may be explained by the restrictiveness [The findings of the present study rejected the null hypotheses that VAS and DPS scores of the patients treated with Apadent Pro or pure Silica were not significantly different from those before treatment. However, it failed to reject the null hypothesis that there was no statistically significant difference between the VAS or DPS scores of patients treated with Apadent Pro and pure silica in relieving DHS. The percent change from baseline for any sensitivity measurement following 8 weeks of product use ranged from 15.30% (Cold DPS) to 38.99% (Air VAS) for nHAP, and from 25.57% (Cold DPS) to 50.67% (Air VAS) for Silica. Using the VAS pain scale, no significant difference in DHS reduction between the two creams with either air or cold was observed. Using the DPS pain scale, although DHS reduction was significantly better with Silica than nHAP at all time points with cold, the difference was significant only at 2, 4, and 8 weeks with air. It should be noted that the percent change from baseline was smaller at every time point for both air and cold when recorded using the DPS scale as compared to VAS  should not be considered a limitation of the study. The most common reason for failure to qualify for study enrollment was lack of exposed buccal cervical dentin among potential participants who self-identified as having tooth sensitivity.The use of the DPS pain scale may represent a limitation of the present study. The DPS is a four-point verbal descriptor scale, and it has been noted that the averaging of responses on categorical scales may not be meaningful . The perin situ study nHAP caused the remineralization of early caries lesions [Moreover, the effectiveness of the 20% nHAP cream relative to other currently marketed desensitizing agents was not assessed in this study, which showed that Apadent Pro nHAP dental cream and a pure silica positive control cream were equally effective at promoting relief of DHS symptoms. However, nHAP has been shown to have additional properties that may benefit oral health. Nanoparticulate hydroxyapatite may act as a calcium and phosphate reservoir , maintai lesions .Furthermore, nHAP-containing paste has been shown clinically to decrease the duration of postbleaching sensitivity , a commoP<.001) reduction in DHS to air or cold stimuli with either nHAP or Silica. The VAS scale showed that Apadent Pro dental cream containing 20% nHAP and a 20% pure silica positive control cream were equally effective at promoting relief of DHS symptoms. In contrast, the DPS scale showed statistically superior results for the pure silica positive control cream over 20% nHAP. Within the limits of this study, it can be concluded that 20% nHAP dental cream is an effective method to promote the relief of DHS symptoms when applied daily.Both VAS and DPS pain scales indicated significant ("}
+{"text": "Frailty is increasingly recognised as a public health priority due to the associated demand for acute and longer term health and social care support, and the impact on the lives of individuals, caregivers and families. Integrated care is widely considered to be most effective when applied to an older population, but there is limited data on outcomes and costs from studies of integrated care to prevent and manage frailty. This paper describes work by the ADVANTAGE Joint Action (JA), co-funded by the European Union and 22 Member States, to develop a common European approach to the prevention and management of frailty. The authors reflect on the emerging evidence and experience of implementing integrated care for frailty, and invite readers to participate in ongoing dialogue on this topic through the ADVANTAGE JA website and IFIC Academy activities. Multimorbidity, disability and frailty are distinct clinical entities that are related, often associated, and may overlap . Frailty356Frailty shares many features of a chronic condition: a dynamic syndrome that cannot be cured but may be prevented and better managed in primary care through an interdisciplinary chronic disease management approach that anticipates and proactively manages episodes of deteriorating function . IntegraA Joint Action (JA) is a grant for actions co-financed with Member States, or other countries participating in the Programme and the European Commission, to allow nominated authorities to take forward work on jointly identified issues that have a clear added value for the European Union under the third Health Programme 2014\u20132020. For ADVANTAGE JA partners, the shared issue is a belief that we must urgently review our policies on ageing and design more integrated models of health and social care to better meet the complex and changing needs of the growing number of adults at risk of, or living with, frailty.We conducted a systematic search of peer-reviewed medical literature published from 2002 to 2017, combining the concepts of frailty and models of care. This search yielded 1065 potential articles. Articles on a specific disease, process or intervention without considering the approach to service delivery were excluded leaving 163 abstracts and 43 full papers for analysis. We also reviewed information on frailty projects funded by the European Union or registered with the European Innovation Partnership on Active and Healthy Ageing, and grey literature (including good practices) identified by ADVANTAGE partners.A synthesis of 18 comprehensive programmes encompassing various components of the Chronic Care Model for people with multimorbidity or frailty reported some evidence of improved health-related quality of life, function, and satisfaction with care but no reduction in health services utilisation or costs . These pAn important distinction between chronic disease and frailty is that frailty is more often associated with functional impairments and physical inactivity that require a restorative or enabling approach beyond the scope of a traditional chronic care model. A restorative approach to care for frail older people living at home has significant advantages over the traditional model of home care maintenance and support . Timely Comprehensive assessment, individualised care plans, and coordination of tailored interventions are the essence of Comprehensive Geriatric Assessment (CGA): a highly evidence based approach that improves care for frail older people in hospital and increases the likelihood of patients living in their own homes at three to 12 months after discharge . There h1416Varying results of community based studies of integrated care for frailty may be due to different sample size, casemix, fidelity, staff expertise, access to care out of hours and duration of study and follow up. Most were small scale demonstration projects that have yet to spread across larger geographic areas, or population groups. In our experience, large scale integrated care models require a favourable political and funding context that may only be achieved through changes in legislation, policy, funding and delivery arrangements as we have recently observed in France, Spain, and Scotland 19.The evidence of cost effectiveness from well executed economic evaluations of models of care is limited. Economic benefits of implementing system-level changes at scale are well described in the Program of Research to Integrate the Services for the Maintenance of Autonomy (PRISMA) in Quebec . HoweverWe believe that more large-scale studies with longer intervention and follow up periods are needed to evaluate system outcomes and costs of integrated care to prevent and manage frailty. Moreover, studies to date have generally focused on traditional healthcare outcomes. Our vision is that future models of care should be designed around outcomes that matter for individuals and their caregivers as well as health and care system and societal impact. We suggest that a focus on patient, client or user-defined goals and outcomes should capture care experience, quality of life and participation outcomes in addition to function and traditional health and social care metrics.Current evidence supports the case for a holistic response to frailty that blends a chronic care approach with education, enablement and rehabilitation to optimise function, particularly at times of a sudden change in health status, or when moving between home, hospital or care home. In all care settings, these approaches should be supported by comprehensive assessment and multidimensional interventions tailored to modifiable physical, psychological, cognitive and social factors and appropriate to the goals and circumstances of the individual and their caregiver.We suggest that model of care for frailty should incorporate the following components:a single entry point in the community \u2013 generally in Primary Careuse of simple frailty specific screening tools in all care settingscomprehensive assessment and individualised care plans \u2013 including for caregiverstailored interventions by an interdisciplinary team \u2013 both in hospitals and communitycase management and coordination of support across the continuum of providerseffective management of transitions between care teams and settingsshared electronic information tools and technology enabled care solutionsclear policies and procedures for service eligibility and care processes.www.advantageja.eu along with a State of the Art on Frailty Report that has information on frailty definition and diagnosis; epidemiology, population screening, monitoring and surveillance; prevention, clinical management; education/training of the workforce; and research. ADVANTAGE JA partners will facilitate a series of sessions on frailty at ICIC18, Utrecht 23\u201325th May, under Theme 5: Vulnerable populations and populations at risk.More details on our review of integrated care for frailty are available at the JA website The aim will be to:raise awareness of Frailty as a high impact priority for integrated carehighlight examples of good practice in preventing and managing frailtyextend our ADVANTAGE community of practiceestablish an IFIC Academy special interest group for frailtyAnne.hendry@lanarkshire.scot.nhs.uk.For further information on ADVANTAGE JA and IFIC special interest group please contact the authors at the address below"}
+{"text": "It was found that the combination of both aromatic \u03c0\u2013\u03c0 stacking and boron\u2013oxygen ligand interactions affords supramolecular arrays of dendrons. Furthermore, the use of boric acid makes this dendritic catalyst a good choice, from corrosion, recyclability and cost points of view. The catalytic activity of Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2, as an efficient magnetically recoverable catalyst, was investigated for the synthesis of polyhydroacridines (PHAs) as well as polyhydroquinolines (PHQs) via one-pot multicomponent reactions of dimedone and/or ethyl acetoacetate, different aldehydes and ammonium acetate in EtOH under reflux conditions. Very low loading of the catalyst, high to quantitative yields of the desired PHAs or PHQs products, short reaction times, wide scope of the substrates, eliminating any toxic heavy metals or corrosive reagents for the modification of the catalyst, and simple work-up procedure are remarkable advantages of this green protocol. An additional advantage of this magnetic nanoparticles catalyst is its ability to be separated and recycled easily from the reaction mixture with minimal efforts in six subsequent runs without significant loss of its catalytic activity. This magnetic and dendritic catalyst can be extended to new two- and three-dimensional covalent organic frameworks with different applications.A new multifunctional dendritic nanocatalyst containing boric acid and 1,3,5-tris(2-hydroxyethyl)isocyanurate covalently attached to core\u2013shell silica-coated magnetite (Fe In this manner, nanomaterials can play a vital role9. One of the emerging fields for the preparation and fabrication of new nanomaterials is dendrimer chemistry which has been recently expanded as two- or three-dimensional covalent organic frameworks (COFs). These strategies afford multifunctional materials which demonstrate synergistic effects and hence, higher performance and efficacy as well as newer and more specific properties than previous counterparts24. In addition, dendrimers can encapsulate and consequently, stabilize metallic catalytic active nanoparticles28. Furthermore, the properties of new materials can be modified and improved by their immobilization onto the surface of magnetic nanoparticles (MNPs), especially in the case of heterogeneous catalysis36. These improvements include better separation using an external magnetic field40, enhancement of the reaction rates by MNPs via local heating through induction and increasing the surface area as well as synergistic effects in conjunction with other catalytic species or centers due to the catalytic performance of magnetic materials, including Fe, Ni, Co or Ce-based ones43. Hence, active catalytic species or centres supported onto the surface of MNPs have received much attention in the field of heterogeneous catalysis for promoting organic reactions in recent years46.New materials are required to be developed for the modern science and technology. These new materials are used for different applications such as drug delivery, medical diagnosis, reinforced composites, semiconductors, electronics, optics, sensors, sorbents, CO57. On the other hand, heterogenization of the active sites of usual dendritic catalysis has been pursued by either attaching the catalyst covalently within the dendrimer core or at the branch termini as well as through supramolecular interactions such as metal\u2013ligand, hydrogen bonding, aromatic \u03c0\u2013\u03c0 stacking, hydrophobic and van der Waals forces61. Therefore, design and preparation of new magnetic dendritic catalytic systems by appropriate application of dendron segments which can be expanded to 2D or 3D covalent organic frameworks (COFs) is still in high demand.As a particular type of magnetic nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs) are more widely available than other MNPs due to advantages such as biologically well-accepted constituents, established size-selective preparation, diminished agglomeration, ease of preparation, and lower cost77. On the other hand, boric acid and its derivatives have achieved specific attention, as appropriate catalysts, in organic synthesis due to their advantages including high solubility in water, easy handling, low prices, and environmentally friendly and commercial availability87. In an attempt to indicate how applying SPIONs would affect the dendrimer bearing tridentate and thermally stable isocyanurate moiety as well as boric acid catalytic activity, this study reports the use of multifunctional dendritic nanocatalyst containing boric acid and 1,3,5-tris(2-hydroxyethyl)isocyanurate covalently attached to core\u2013shell silica-coated SPIONs (Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2, 1), as a novel and efficient supramolecular heterogeneous catalyst, in the one-pot synthesis of polyhydroacridines  and polyhydroquinolines  through multicomponent reaction (MCR) strategy  moiety has received significant attention in polymer and material chemistry due to its numerous industrial applications, particularly in the field of low toxic drug-delivery agents, tensioactive building blocks and nonlinear optical properties, foams, surface coatings, films, paints, fibers, selective anion receptors and preparation of periodic mesoporous organosilica90. Thus, MCRs are usually a good alternative for the sequential multistep synthesis, especially for useful heterocyclic scaffolds such as Hantzsch esters including 1,4-dihydropyridines (DHPs), PHQs and PHAs in organic synthesis and medicinal chemistry96. Generally known as one of the main groups of nitrogen heterocycles, polyhydroquinolines (PHQs) and polyhydroacridines (PHAs) have become considerably interesting due to their significant therapeutic and pharmacological properties100. Indeed, they are used as antimalaria, calcium \u03b2-blocker, antioxidant, antimicrobial, antifungal, vasodilator, anticancer, bronchodilator, antiatherosclerotic, geroprotective, hepatoprotective and antidiabetic agents as well as in the production of laser colors, radical reservoirs and safe hydrogen transfer agents111. First introduced by Arthur Hantzsch in 1882, Hantzsch reaction is an MCR that contains the combination of a \u03b2-dicarbonyl compound, an aldehyde and a source consisting of ammonia 112. However, catalytic systems are required to accelerate this multicomponent reaction. Here are some recent reported catalysts in this area: Mn@PMO-IL103, vanadium ion doped titania nanoparticles113, Lewis acidic mesoporous material (TUD-1) containing Fe114, magnetite nanoparticle-supported ceria41, silica-coated magnetic nanoparticles with tags of ionic liquid115, Boehmite silica sulfuric acid (Boehmite-SSA) 116, PMO-ICSPrSO3H117, Fe3O4@B-MCM-41118, PS/PTSA119, PdRuNi@GO13, 1,3,5-tris(2-hydroxyethyl) isocyanurate covalently functionalized MCM-41120, alginic acid122 and glycine nitrate (GlyNO3) ionic liquid123.MCRs are one-pot reactions that involve more than two substrates demonstrating convergence as well as very high atom efficiency and bond-forming-index (BFI)3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2, 1) was characterized using different spectroscopic or analytical methods. As it has been shown in FT-IR spectrum isocyanurate attached to the silica-coated SPIONs (Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2 (1) proved that the magnetic catalyst functionalized with dendrons containing 1,3,5-tris(2-hydroxyethyl)isocyanurate and boric acid has been functionalized properly due to the presence of Fe, Si, O, C, N and B elements. The percentages of elements were measured to be B (1.96), C (6.99), N (2.50), O (63.58), Si (12.33) and Fe (12.65), respectively. It can be deduced from the absence of Cl and Br elements that terminal chloride groups of the 3-chloropropyl trimethoxysilane (3-APTS) linker as well as terminal bromide groups of the 1,3-dibromopropane linker have been completely replaced by covalent bonding  spectrum of Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2 (1) exhibited the phase structure and crystallization of the magnetic nanomaterials  with the reference card numbers in the X'pert software, the crystal network of Fe3O4, SiO2 and B(OH)3 correspond with 072\u20132303, 082\u20131572 and 030\u20130199 card numbers, respectively.The X-ray diffraction (XRD) pattern of Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2 catalyst (1) was investigated by nitrogen adsorption\u2013desorption isotherms 3OB(OH)2 catalyst (1) according to the data obtained by DTA  and differential thermal analysis (DTA) measurements were carried out under air atmosphere by heating the sample at the rate of 10\u00a0\u00b0C\u00a0min\u00a0\u00b0C Fig.\u00a0. The firDTA Fig.\u00a0b.Figure 1) at room temperature 3OB(OH)2 was measured to be 47.9 and 35.2\u00a0emu/g, respectively. Indeed, the reduction of saturation magnetization of Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2 shows that the dendritic catalyst has been formed. However, the observed saturation magnetization of catalyst (1) is enough and hence, it\u00a0can be easily separated by an external magnetic field.Vibrating sample magnetometry (VSM) technique was used for measuring the magnetic properties of catalyst 3OB(OH)2 catalyst (1), field emission scanning electron microscopy (FESEM) technique was used 3OB(OH)2 catalyst (1), the Hantzsch reaction for the synthesis of polyhydroacridine and polyhydroquinoline derivatives was chosen to examine the catalytic activity of Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2 (1). For this purpose, the condensation of 4-chlorobenzaldehyde , dimedone (3), NH4OAc  and/or ethyl acetoacetate  were selected as the model reactions, for the synthesis of polyhydroacridine 5a and polyhydroquinoline 7a, respectively. The reactions were optimized considering different parameters such as the amount of catalyst loading, solvents and temperature. The results are reported in Table H,5H)-dione (5a) or ethyl 4-(4-chlorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (7a) were trace in the absence of any catalyst in EtOH at room temperature (entry 1). However, low yields of the desired products 5a and 7a were obtained under reflux conditions (entry 2) after long times. Interestingly, the yields were improved significantly in the presence of dendritic Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2 catalyst . Further optimization of the reaction conditions illustrated that EtOH is the best solvent to promote the reaction with high efficiency for the synthesis of the desired products 5a or 7a (entries 6\u201312). The results of optimizing of\u00a0the model reactions demonstrated that the optimal conditions for the reaction are 10\u00a0mg catalyst 1 loading in EtOH under reflux conditions. On the other hand, both boric acid and Fe3O4@SiO2@PTS-THEIC, as the components of the catalyst 1, afforded moderate yields of the desired products 5a and 7a at same catalyst loading under optimized conditions (entries 13 and 14). Finally, hot filtration test (the Sheldon test) was performed to prove the heterogeneous nature of the catalyst 1. During this test, the solid catalyst 1 was removed from the mixture of model reaction for producing 7a by filtration after 10\u00a0min using an external magnet. Then, the obtained mixture was heated again for 10\u00a0min. The result showed that after removal of the magnetic catalyst 1, the model reaction did not proceed significantly. Indeed, only 48% of the desired product 7a was isolated after 1\u00a0h 3OB(OH)2 nanocatalyst (1) was further expanded to several other aromatic or heterocyclic aldehydes for the synthesis of other derivatives of PHAs 5a\u2013o and PHQs 7a\u2013u. As it is shown in Tables 6 or 8 were good to excellent in all studied cases under the optimized condition of reaction. In most cases, the products were obtained in similar periods of time and yields compared to the model reaction. Indeed, aldehydes including aromatic carbocyclic or heterocyclic substrates well survived under optimized conditions without formation of any by-products. It is noteworthy that aldehydes bearing electron-withdrawing groups or six-membered heterocycles almost reacted faster than substrates having electron-donating groups or five-membered heterocycles. This trend of reactivity was observed in both symmetric and asymmetric Hantzsch reaction to afford PHAs 5a\u2013o or PHQs 7a\u2013u derivatives, respectively. Furthermore, the \u03b1,\u03b2-unsaturated cinnamaldehyde (2q) or aliphatic butyraldehyde (2r) reacted in longer reaction times and afforded lower yields. These may be due to resonance and electron-releasing of the double bond and alkyl groups, respectively. All of these findings, led us to purpose a plausible mechanism depicted in Scheme After finding the optimal conditions, the catalytic activity of Fe1) beside easy separation\u00a0from the reaction mixture is its recyclability. After the reaction was completed, the catalyst was separated and washed by acetone and hexane, respectively. Then, it was dried and reused in the model reactions for the next runs. The obtained results have been summarized in Fig.\u00a03O4@SiO2@CPTS-THEIC-(CH2)3OB(OH)2 nanocatalyst (1) and the recycled sample after six consecutive runs for the synthesis of 5a demonstrated that their structures are almost similar 3OB(OH)2. These data clearly demonstrate that the nanocatalyst 1 is more active than other previously reported catalytic systems in terms of catalyst loading, product yield, required reaction time and avoiding the toxic solvents.Table 1 was obtained using an equipment ASAP 2020 Micromeritics. Magnetic susceptibility measurements were taken out by using a Lakeshore VSM, 7410 series. Melting points were determined using an Electrothermal 9100 apparatus and are uncorrected. 1H NMR (500\u00a0MHz) spectra were obtained using a Bruker DRX-500 AVANCE spectrometer in CDCl3 at ambient temperature. Analytical TLC was carried out using Merck 0.2\u00a0mm silica gel 60 F-254 Al-plates and n-hexane: EtOAc,  as eluent. All products are known and their structures were established by comparing the physical constants as well as FTIR and NMR spectroscopic data with authentic samples141.All chemicals and reagents were provided by Merck or Aldrich chemical companies and used as received without any further purification, except for benzaldehyde which was used as a fresh distilled sample. FTIR spectra were recorded using KBr pellets on a Shimadzu FT IR-8400S spectrometer. Energy dispersive spectroscopy (EDS) was recorded on a SAMx instrument. The X-ray powder diffraction (XRD) data were collected on an X'Pert MPD Philips diffractometer with Cu radiation source (\u03bb\u2009=\u20091.54050\u00a0\u00c5) at 40\u00a0kV voltage and 40\u00a0mA current. Field emission scanning electron microscopy (FESEM) images were obtained using a MIRA3 instrument of TESCAN Company, Czech Republic. Thermal gravimetric analysis (TGA) and differential thermal analysis (DTA) were performed by means of a Bahr company STA 504 instrument. The BET specific surface area of the catalyst 3O4@SiO2@CPTS materials were prepared according to the reported methods in literature with a slight modification56.The Fe3O4@SiO2@CPTS (1\u00a0g) was dispersed in toluene (30\u00a0ml) and KI (1.66\u00a0g) was added to the obtained mixture with the mechanical stirring at 80\u00a0\u00b0C for 1\u00a0h. Then, K2CO3 (1.38\u00a0g) and tris-(2-hydroxyethyl)-1,3,5-triazinane-2,4,6-trione (1\u00a0g) were added to the mixture and it was heated under reflux conditions for 8\u00a0h. The obtained solid was filtered off and washed with EtOH (5\u00a0ml) and then dried in an oven for 2\u00a0h.Fe3O4@SiO2@CPTS@THEIC (1\u00a0g) and 1,3-dibromopropane  was added to toluene (15\u00a0ml) and heated at 40\u00a0\u00b0C for 12\u00a0h. The obtained solid was filtered off, washed with toluene (5\u00a0ml) and then dried in a vacuum oven at 60\u00a0\u00b0C for 2\u00a0h. The as-prepared solid and H3BO3 (1\u00a0g) were mixed in EtOH (30\u00a0ml) and the obtained mixture was stirred at room temperature for 18\u00a0h. After completion of the process, the obtained brown solid was filtered off and washed with EtOH (5\u00a0ml) on a Buchner funnel and then kept in a vacuum oven at 60\u00a0\u00b0C for 12\u00a0h. The complete procedure for the preparation of catalyst 1 has been represented in Scheme A mixture of Fe2a\u2013o, 1\u00a0mmol), dimedone , NH4OAc  and Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2  were added to EtOH 96% (2\u00a0mL). The obtained mixture was stirred under reflux conditions for the times indicated in Table 1 was easily separated by an external magnet and the solution was filtered. The filtrate was kept at room temperature and the crystals were\u00a0collected by filtration to afford 1,8-dioxoacridindione derivatives 5a\u2013o in high purity.In a 5\u00a0mL round-bottomed flask, a mixture of aldehyde , dimedone , NH4OAc , ethyl acetoacetate  and Fe3O4@SiO2@PTS-THEIC-(CH2)3OB(OH)2  were added to EtOH 96% (2\u00a0ml). The obtained mixture was stirred under reflux conditions for times indicated in Table 1 was easily separated by an external magnet and the solution was filtered. The filtrate was kept at room temperature and the crystals were collected by filtration to afford polyhydroquinoline derivatives 7a\u2013u in high purity.In a 5\u00a0mL round-bottomed flask, a mixture of aldehyde (\u22121): 3282, 3176, 3060, 2954, 2875,1650, 1608, 1492, 1365, 1220, 1147, 1089, 1014, 840, 761, 597, 526; 1H NMR : \u03b4 (ppm): 0.98 , 1.10 , 2.19\u20132.37 , 5.06 , 7.17 , 7.28 , 6.97 .Pale yellow solid; m.p.\u2009=\u2009310\u2013312\u00a0\u00b0C; FT-IR : 3604, 3519, 3440, 3284, 2875, 1637, 1600, 1477, 1365, 1218, 1139, 995, 744, 563; 1H NMR : \u03b4 (ppm): 0.98 , 1.07 , 2.12\u20132.46 , 5.22 , 7.51\u20137.58 , 8.41 , 6.97 .Pale yellow solid; m.p.\u2009=\u2009284\u2013286\u00a0\u00b0C; FT-IR : 3278, 3203, 3076, 2956, 1699, 1604, 1496, 1379, 1276, 1218, 1070, 1031, 842, 765, 536; 1H NMR : \u03b4 (ppm): 0.92 , 1.04 , 1.20 ), 2.11\u20132.28 , 2.33 , 3.71 , 4.03\u20134.07 ), 4.98 , 6.43 , 6.71\u20136.73 , 7.21 .Pale yellow solid; mp.: 255\u2013260\u00a0\u00b0C; FT-IR : 3276, 3193, 2964, 1703, 1604, 1490, 1379, 1278, 1215, 1143, 1070, 1022, 829, 754, 690, 507; 1H NMR : \u03b4 (ppm): 0.93 , 1.09 , 1.19 ), 2.13\u20132.40 , 4.03\u20134.07 ), 5.15 , 5.98 , 7.35\u20138.10  3OB(OH)2) was prepared and properly characterized for the first time. It was found that the combination of both aromatic \u03c0\u2013\u03c0 stacking and boron\u2013oxygen ligand interactions affords supramolecular arrays of dendrons. The use of boric acid makes this dendritic catalyst a green choice from corrosion, recyclability and cost points of view. The magnetic dendritic catalyst was used, as a mild and recyclable catalyst, for the one-pot efficient synthesis of polyhydroacridines and polyhydroquinolines through MCR strategy in EtOH as a green solvent. Indeed, very low catalyst loading, short reaction times, mild reaction conditions, high to excellent yields, reusability of the catalyst, ease of separation by an external magnetic field, and the use of nontoxic materials for the preparation of the catalyst are among other advantages of this protocol. Further exploring of this magnetic dendritic magnetic catalyst for other organic transformations is underway in our research lab and would be presented in due course.In conclusion, the multifunctional dendritic nanocatalyst containing boric acid and 1,3,5-tris(2-hydroxyethyl)isocyanurate covalently attached to core\u2013shell silica-coated magnetite (FeSupplementary Information 1."}
+{"text": "The development and use of mobile health  apps for asthma management have risen dramatically over the past two decades. Asthma apps vary widely in their content and features; however, prior research has rarely examined preferences of users of publicly available apps.The goals of this study were to provide a descriptive overview of asthma mobile apps that are publicly available and to assess the usability of asthma apps currently available on the market to identify content and features of apps associated with positive and negative user ratings.Reviews were collected on June 23, 2020, and included publicly posted reviews until June 21, 2020. To characterize features associated with high or low app ratings, we first dichotomized the average user rating of the asthma app into 2 categories: a high average rating and a low average rating. Asthma apps with average ratings of 4 and above were categorized as having a high average rating. Asthma apps with average ratings of less than 4 were categorized as having a low average rating. For the sentiment analysis, we modeled both 2-word (bi-gram) and 3-word (tri-gram) phrases which commonly appeared across highly rated and lowly rated apps.Of the 10 apps that met the inclusion criteria, a total of 373 reviews were examined across all apps. Among apps reviewed, 53.4% (199/373) received high ratings (average ratings of 4 or 5) and 47.2% (176/373) received low ratings (average ratings of 3 or less). The number of ratings across all apps ranged from 188 (AsthmaMD) to 10 (My Asthma App); 30% (3/10) of apps were available on both Android and iOS. From the sentiment analysis, key features of asthma management that were common among highly rated apps included the tracking of peak flow readings (n=48), asthma symptom monitoring (n=11), and action plans (n=10). Key features related to functionality that were common among highly rated apps included ease of use (n=5). Users most commonly reported loss of data (n=14) and crashing of app (n=12) as functionality issues among poorly rated asthma apps.Our study results demonstrate that asthma app quality, maintenance, and updates vary widely across apps and platforms. These findings may call into question the long-term engagement with asthma apps, a crucial factor for determining their potential to improve asthma self-management and asthma clinical outcomes. Asthma is the leading chronic disease in children and adolescents and one of the most common among adults, with more than 25 million Americans impacted . NationaMany factors combine to contribute to poor asthma rates and worsen outcomes. Provider practice behaviors, suboptimal access to health care, lack of patient knowledge regarding proper medication use, and patients\u2019 difficulty adhering to medical regimens contribute to poor asthma outcomes -8. With Mobile health apps and devices to monitor and track health-related questions are a rapidly growing field within the public health, data science, and technology sectors -16, and However, promotion of new technologies is predicated on the hypothesis that enabling people to quantify their own behaviors will drive health behavior change through contextualization and goal setting . Mobile In recent years, there has been a proliferation of new mobile apps for the self-assessment and self-management of asthma ,25. HoweThe aims of this study were (1) to provide a descriptive overview of asthma mobile apps that are publicly available and (2) to assess the usability of asthma apps currently available on the market to identify content and features of apps associated with positive and negative user ratings.asthma management, asthma game, asthma quiz, peak flow, and asthma education. All apps were evaluated by the research team based on inclusion criteria; of the 51 apps initially reviewed, 29 apps had been updated in the last 5 years. Of the 29 apps recently updated, 19 were excluded based on having less than 10 written reviews available. The final list of 10 apps included in the analysis is presented in To examine the average user rating and reviews for asthma apps, the following inclusion criteria were applied for our search: apps must be available for download on Android or iOS platforms, written in English, available within the United States, able to be downloaded onto a smartphone or tablet, and had at least one update within the last 5 years. Apps must also have a primary focus on asthma, with respect to either asthma education or asthma self-management. Because of the potential for bias in ratings due to small sample size, only apps that had more than 10 written reviews were included. Apps were not excluded based on cost for use or intended audience. We started with a comprehensive list of apps that strictly matched our inclusion criteria. The list was expanded by using the following keywords commonly associated with asthma education or asthma management to identify additional asthma apps: To identify specific sentiments within language characteristics of user reviews that are associated with high or low app ratings, we first dichotomized the average user rating of the asthma app into 2 categories: a high average rating and a low average rating. Asthma apps with average ratings of 4 and above were categorized as having a high average rating. Asthma apps with average ratings of less than 4 were categorized as having a low average rating.N-gram is a contiguous sequence of n items from a piece of article, sentence, or speech. An N-gram model is a probabilistic language model for predicting the next item given the sequence of (n\u20131)\u2013gram, which simulates characteristics of the language used in a certain corpus. Its simplicity and scalability enable its application in sentiment analysis of the reviews [peak flow meter in both the good and bad reviews. The bi-grams extracted from the model showed distinct patterns in good reviews and bad reviews. As such, results from bi-gram models for highly rated reviews and lowly rated reviews are discussed in the next section. We removed the stop words in the preprocessing step, and separated the reviews into good reviews (ratings >3) and bad reviews (ratings \u22643). Reviews were collected on June 23, 2020, and contain reviews from January 08, 2010, to June 21, 2020. Of the 10 apps that met the inclusion criteria, a total of 373 reviews were examined across all apps.An  reviews . For thiOf the 10 apps included in the analysis, 6 were available at no cost to users and 4 apps were available for purchase, with a maximum cost of US $2.99; 50% of apps (n=5) were updated in the past 2 years.As shown in Stream graph of the number of review counts from 2010 to 2020 is shown in Distinct patterns of language were observed when comparing highly rated and poorly rated apps. Among the highly rated reviews, bi-grams emerged with respect to features as well as functionality of the app. Among poorly rated asthma apps, bi-grams  and 5 prThis study analyzed ratings and features of publicly available asthma apps to identify user preferences. Our descriptive results confirmed those of prior studies which observed the limited availability of publicly available, up-to-date asthma apps on the market . In termFurthermore, our study results demonstrated that asthma app quality varied widely, ranging anywhere from an average user rating of 1.5 out of 5 to 5 out of 5. It also appeared that individual asthma apps varied in their user rating between Android and iOS platforms. These findings may call into question the long-term engagement with asthma apps, a crucial factor for determining their value ,33. In oN-grams model, we were able to capture several characteristics related to features and functionality that users like or dislike about the apps; for example, peak flow and action plan were frequently mentioned among features, whereas easy to use and doesn\u2019t work were frequently mentioned around functionality. Previous studies of asthma apps confirmed peak flow tracking, symptom tracking, and medication tracking as some of the most common functions in highly rated apps [With the ted apps . Additioted apps ,26,35. Ated apps -40.One potential way to improve long-term engagement, which has been successfully applied to physical activity, is interactions with virtual coaches ,42. ThusOur study did have several limitations, which should be considered when interpreting its findings. The popularity of an asthma app may not yield information regarding the quality of the asthma content presented in the app with respect to the clinical or scientific literature. Prior studies have observed variability in the quality of asthma information with respect to its consistency with National Asthma Education and Prevention Program guidelines . Future Limitations of sentiment analysis include its sensitivity to review content; as such, user ratings may not always correspond to descriptive reviewer feedback. Further, displays of reviews were limited to downloads with a minimum threshold of 10 reviews. However, sentiment analysis has been increasingly applied across a variety of health outcomes -52. FinaOur study extends previous research in this field by focusing on the experiences and reviews of asthmatics\u2019 interactions with publicly available asthma apps. Our study results have several implications with respect to informing the development of asthma mobile apps and their recommendation for clinical use. As use of asthma apps have been found to have an impact on several clinical outcomes, including but not limited to control, quality of life, medication adherence, and patient-reported outcomes, improvements to asthma apps should include a focus on user-centered design and experiences. Combining big-data analytic approaches with qualitative data from users may yield additional insights to improve usability and long-term engagement with asthma apps. Further, collaborations between asthma app developers, clinicians, and researchers should include considerations regarding data security, privacy features, and sharing of personal health information which would also increase patient and provider confidence regarding recommending the use of asthma mobile apps to improve asthma self-management."}
+{"text": "Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment\u2010friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100\u00a0ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250\u00a0ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon\u2122S was effective in inhibiting the growth of the mycelium . Actidrox\u00ae and Detarox\u00ae AP showed MIC\u00a0=\u00a05,000 and 1,000\u00a0ppm, respectively, while MLCs were 10\u2010fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.Oomycetes of the genus  The active ingredient of Pyceze\u00ae is bronopol , a broad\u2010range biocide used as a preservative in pharmaceutical and cosmetic industry  for egg disinfection in aquaculture. From 1 January 2016, formaldehyde has been classified as a category 1B carcinogen  eggs  tested as a prophylactic measure permitted to increase the hatching rate in salmonids; however, the required concentrations (20\u201030\u00a0g/L) are hardly applicable in intensive farms . In vitro results showed inhibition of the mycelium growth at concentrations above 0.6\u00a0g/L, while in vivo tests allowed a high survival rate after continuous (0.2\u20101.4\u00a0g/L) and intermittent (1\u20104\u00a0g/L) exposure of eggs and larvae. These results suggest that boric acid could be safely used in aquaculture although its environmental impact must be carefully investigated.Ali, Thoen, Evensen, and Skaar  tested bSaprolegnia spp. in order to provide information for the selection of safer and more environmentally friendly alternative treatment of saprolegniosis in aquaculture. The activity of new molecules is compared to the effectiveness of two compounds  used in the past to control saprolegniosis in aquaculture.The aim of this work was to perform an in vitro screening of promising molecules and commercial products against 22.1Saprolegnia strains: one reference strain of S.\u00a0parasitica  isolated in Holland from northern pike (Esox lucius), one field strain of S.\u00a0parasitica (ITT 320/15/20) isolated in Italy from brown trout  and one field strain of Saprolegnia delica (ITT 290/15/15) isolated in Italy from rainbow trout (Oncorhynchus mykiss). Each strain has been tested in triplicate per each concentration.Tests were carried out on three 2.2Saprolegnia spp. strains were maintained with periodic subcultures on glucose\u2010yeast (GY) agar medium (5\u00a0g D\u2010(+)\u2010glucose, 1\u00a0g yeast extract, 12\u00a0g agar in 1L deionized water) supplemented with 6\u00a0mg/L of penicillin and 10\u00a0mg/L of oxolinic acid (GY\u00a0+\u00a0P\u00a0+\u00a0Ox) . Malachite green was used as a reference compound, and copper sulphate was added because it is widely used to control saprolegniosis in aquacultured fish.Products under test include ten molecules  belonging to different chemical classes and three commercial products  at concentrations of 0.1; 1; 5; 10; 50; 100; 250; 500; 1,000; 5,000\u00a0ppm.2.4Tests were performed following protocols I and II according to Alderman .protocol I, different concentrations of the products under test were added to sterilized liquid GY agar at a temperature of 49\u00b0C. Mixtures were then distributed in six\u2010well plates (\u00d8 35\u00a0mm), allowing to test five different concentrations and one negative control. Following overnight solidification, a 5\u2010mm\u2010diameter well was excised in the centre of the agar using a sterile glass cannula. The well was then filled with a standard 5\u2010mm inoculum, culture surface uppermost. Plates were incubated at 18\u00b0C and checked after 24, 48, 72\u00a0hr and 6\u00a0days, determining the colony diameter of the growing mycelium as average of two axes measured at 90\u00b0 from each other. The minimum inhibitory concentration (MIC) was defined as the lowest concentration inhibiting the growth of the mycelium after 6\u00a0days of incubation.For protocol II, polycarbonate filter membranes  were sterilized by autoclaving and then placed on the surface of 92\u2010mm petri dish (six per each petri dish). Filters were used as a support for the inoculum, obtained by placing a 4\u00a0\u00d7\u00a04\u00a0mm piece of agar with growing mycelium. The inoculum was placed, inverted, at the centre of the filter. The dishes containing the filters were then incubated for 24\u00a0hr, until the resulting mycelial growth had almost reached the edge of the filters. The agar attached to the original inocula was then clipped off using hot forceps tips. The mycelia together with their supporting filters were lifted off the agar surface and placed in sterile 92\u2010mm petri dishes containing different concentrations of the products under test  was confirmed in both tests , with radial mycelial growth considerably slowed down at 50\u00a0ppm Figure\u00a0d, followa Figure\u00a0e,f, but S.\u00a0parasitica strain CBS 223.65, while for the other two strains the MLC was set at 500\u00a0ppm.For acetic acid, the MIC in agar resulted in 250\u00a0ppm  only, while the other two tested strains showed a MLC\u00a0=\u00a05,000\u00a0ppm.Minimum lethal concentration of tartaric acid was not determined at tested concentrations, whereas oxalic acid in water was lethal at 1,000\u00a0ppm for one Sodium percarbonate was effective only in slowing down the mycelium growth at the highest tested concentrations , but at 48\u00a0hr the diameter of mycelium was equal to the control Figure\u00a0g; therefHydrogen peroxide showed equal values of MIC and MLC  for all tested strains, but slower growth was observed at 250\u00a0ppm after 24\u00a0hr  cannot be excluded.Sodium percarbonate is an environmentally safe compound that has been successfully tested in vitro against developmental stages of \u2122S), performed differently in protocols I and II  and two commercial products  resulted considerably more effective in water.For Actidrox\u00ae, the different performances observed in the two protocols are possibly linked to the characteristics of the product itself, which releases peracetic acid (effective against a wide range of microorganisms) when solubilized in water. A similar behaviour was observed for Detarox\u00aeAP, an acidic sanitizer with oxidant properties, formulated with stabilized peracetic acid and hydrogen peroxide, that is widely used in the food industry for the disinfection of production equipment. Also for Detarox\u00aeAP, active compounds showed greater effectiveness in the presence of water. Both peracetic acid and hydrogen peroxide show a wide\u2010range antimicrobial activity Baldry,  and low Oncorhynchus tshawytscha ). It is included among the disinfection by\u2010products (DBPs) that could be produced in raw water after disinfection process. Furthermore, studies are in progress to evaluate whether subtoxic doses in water could represent a carcinogenic risk for humans , and be possibly close to values reported in previous investigations . In aquaculture, Virkon\u2122S is used at a concentration of 1% w/v for the disinfection of ponds and farm equipment  during in vitro trials, in which higher concentrations were needed to inhibit S. delica. This oomycete species is widely distributed in natural freshwater systems , remarkably different behaviours were observed between the two Saprolegnia spp., although at different concentrations. Among these, benzoic acid and iodoacetic acid showed the lowest MIC/MLC, respectively; however, acetic acid and peracetic acid\u2010based products, particularly in combination with hydrogen peroxide, represent promising candidates for controlling saprolegniosis in aquaculture, due to their effectiveness associated with low environmental impact.In conclusion, the in vitro tests performed here show that, with the exception of sodium percarbonate, the compounds/products tested are effective against Saprolegnia , their possible cytotoxic effects, and ultimately their safety and efficacy in vivo.In order to assess the possible field application of these most promising compounds, further tests will be necessary to evaluate their efficacy on different developmental stages of The authors declare that there is no conflict of interest."}
+{"text": "Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes dorsalis (Meigen), Aedes notoscriptus (Skuse), Aedes vigilax (Skuse), Anopheles quadrimaculatus (Say), Culex pipiens (L.), Culex quinquefasciatus (Say), and Culex tarsalis (Coquillett). Male and female mosquitoes were placed in cages and allowed to feed on liquid diets under controlled environmental conditions for 1 wk. For seven of the nine species, adult survival was significantly higher in the presence  versus the absence  of sugar, with no indication that salt had any effect on survival. Anopheles quadrimaculatus showed intermediate survival in Sugar + Salt to either Sugar Only or no sugar diets, whereas Aedes dorsalis showed low survival in Salt Only versus other diets. Based on our data and coupled with the fact that mosquitoes have physiological and behavioral adaptations that allow them to avoid or process excess salt , we conclude that there is no scientific foundation for salt-based control methods of mosquitoes.Various products and insecticides are available that purport to reduce wild populations of adult mosquitoes. Recently, several manufacturers and general public comments on the internet have promoted devices that claim that ingestion of salt will significantly reduce populations of wild mosquitoes to near zero; there are no known scientific efficacy data that support these claims. We tested the survival of nine mosquito species of pest and public health importance across four adult diets: Water Only, Sugar Water Only (8.00%), Salt Water Only (1.03%), and Sugar + Salt Water. Species included the following:  The pest and public health risks associated with mosquitoes are significant and are often perceived to be greater where individuals are exposed to mosquitoes in and around residential properties . As a result, there continues to be demand for commercial products designed for mosquito control around the home. However, the effective control of mosquitoes has had mixed success. The complex biology and ecology of mosquito species presents challenges in finding an effective and sustainable broad ranging control strategy and the realm of mosquito control has remained relatively static over the past few decades . AlthougThere is a clear demand among the public for affordable and effective mosquito control, and many do-it-yourself approaches have proven popular on internet and social media sites . Many of the more widely shared approaches include mixing of various household products alleged to have value as a mosquito attractant, repellent, or control agent. Several devices make claims that salt-based solutions are active killing agents, with some videos describing this approach having millions of views on social media. Within the past few years, several companies have begun to produce devices in the United States that make the claim that salt feeding by adults will reduce mosquito numbers in the wild. These devices include the Spartan Mosquito Eradicator , the Mosquito XT , the Skeeter Eater , Mosquito Dynamite , and Donaldson Farms \u2013 Mosquito Eliminator . These devices generally contain some combination of dried salt, sugar, and yeast, which is mixed with warm water by the purchaser and then placed outdoors to either attract mosquitoes who then drink the fluid and are claimed to die from the salt, or who are repelled by the action of other additives like various essential oils. There are no data that have tested the effectiveness of salt as a substance to kill mosquitoes.Aedes aegypti (L.) and Aedes albopictus (Skuse) show egg hatchings even at 2.0% salt concentrations  and Aedes taeniorhynchus (Wiedemann), are known to have a tolerance to salt  , Ae. albopictus (Skuse) (MS), Ae. notoscriptus (Skuse) (AU), Ae. dorsalis (Meigen) (UT), Ae. vigilax (Skuse) (AU), Anopheles quadrimaculatus (Say) (MS), Culex pipiens (L.) (UT), Cx. quinquefasciatus (Say) (CA), and Cx. tarsalis (Coquillett) (CA). All species are important known vectors of pathogens or nuisance-biting pest species and often are a main focus of vector control and suppression.This study represents the combined contributions of five laboratories who tested species available in their area, and although the methods for the experiments were fundamentally the same, there were slight differences that we highlight by laboratory location . The nine species included the following: Aedes dorsalis) were from colonies maintained in each laboratory using similar rearing and husbandry protocols (Aedes aegypti (LA) were reared under 28\u00b0C. Aedes dorsalis were wild caught females that were trapped in field cages using CO2 as bait and supplied overnight with water but no sugar until the next day when trials began. Aedes vigilax were reared in diluted seawater with deionized water to a salinity of ~16 ppK. Anopheles quadrimaculatus were purchased as eggs from Benzon Research, Inc. .Adult mosquitoes used in all experiments , adults were fed on a 10% sucrose solution ad libitum under similar conditions as the feeding trials. Female Aedes dorsalis were subjected to feeding trials the day after they were collected from the wild.All locations used similar environmental conditions for larval rearing and feeding trials (unless noted), with feeding trials conducted either in walk-in or separate smaller environmental chambers kept at 27\u00b0C (28\u00b0C in the case of LA) on a 12:12 light:dark cycle . Humidity was maintained between 50 and 75%. Cages were approximately 30 \u00d7 30 \u00d7 30 cm and were of either a metal or plastic frame with mesh covering all sides, with the exception of LA who used one quart paper cylindrical containers . Into each cage, we added 20 adult mosquitoes  each 1\u20137 d old. Based on the availability of adults, UT used 17\u201323 total adults for trials, although an approximately equal sex ratio was still maintained and for For the feeding trials, we used four no-choice diets : Water Only (negative control), Salt Water Only , Sugar Water Only , and Sugar + Salt Water . Percentages used were based on the product description from the most widely available commercial product (Spartan Mosquito Eradicator) but are similar to other available products. These percentages reflect those found after filling the container with fluid per the manufacturer\u2019s directions and not the percentages of dried product. Mosquitoes are commonly fed a 10% sucrose solution in colonies under laboratory conditions. We replicated each diet three times for each species. Liquid for each diet was added fresh on day 1 of the experiment and replaced on day 4. Diets were added to vials with an exposed cotton wick. The trials ran for 7 d and on each day, we recorded the number of dead mosquitoes of each sex. Within each laboratory, all diets were run concurrently for each species.P value to account for multiple comparisons (P = 0.05/6 contrasts = 0.008). We did not analyze sex as a separate factor given that none of the claims made by any of the manufactures of the devices mentioned above suggest sex-specific results of salt feeding, nor did we expect that males and females would differ in their tolerance to ingestion of salt water.Survival analyses were conducted for each species, separately, using PROC PHREG in An. quadrimaculatus, which showed intermediate survival in Salt + Sugar compared to either Sugar Only , or to Salt Only or Water Only   , and forer diets . Howeveron day 7 . Among tperiment , F, wherperiment .An. quadrimaculatus, 40% of adults were still alive at the end of the experiment. For Aedes aegypti from the LA colony, the difference in Salt Only and Salt + Sugar diets was only apparent on the last day of the trial. For all but one species, a diet with only saltwater did not lead to higher mortality rates than water alone, and thus this strongly suggests that salt by itself is not a detrimental substance for the mosquito digestive system. Aedes dorsalis did show that a salt only diet led to higher mortality compared to all other diets ; however, on the very first day of the experiment, this diet had the lowest overall survival across all species tested, and we note that survival overall was low across all diets . 2 had higher median rejection thresholds by adults compared to other salts and other chemicals . This suggests that mosquitoes, in this case Ae. aegypti, reject sucrose solutions that contain high concentrations of salts relative to natural sugar meals. However, there are also data to suggest that salts may be an important stimulatory factor for adult feeding. For Cx. pipiens, NaCl at 150 mM acted as a phagostimulant (Anopheles stephensi (Liston), An. freeborni (Aitken), and An. dirus (Petron and Harrison) all elicited greater feeding, indicating that these chemicals were phagostimulatory . Our data specifically addressing the effect of salt ingestion appear to support the conclusion of Although devices currently marketed on social media and by some manufacturers would appear to be, \u201ctoo good to be true,\u201d consumers have already spent millions of dollars purchasing them, perhaps at the expense of known effective approaches to killing adult mosquitoes. Recent work by As in many instances state and federal laws do not require efficacy data to support claims made by these devices, it is important to evaluate individual claims to better inform the public and ensure that limited public health dollars are not needlessly wasted on approaches that do not effectively control mosquitoes . We woulBased on the response of nine medically important species of mosquitoes to different diets, and the substantial literature on the physiological and behavioral ways that mosquitoes deal with salt in nature, we can conclude that there is no scientific or experimental evidence to support the claims that salt-based approaches are effective for mosquito control as currently formulated. As adult mosquitoes do not appear to suffer mortality from ingesting low doses of salt in their diet, and higher concentrations of salt can be detected and avoided by adults, we conclude that salt is ineffective for the control of mosquito populations by individual consumers, regulatory agencies, or mosquito control districts."}
+{"text": "Sport psychology research has long sought to uncover the determinants of the optimal psychological state for peak performance. Persistent inquiries in this work include whether there is a set of ideal psychological and emotional factors that are required to achieve optimal performance and, if there are, what are they and how are they related to optimal performance. To answer these questions, the current study aimed to identify potential profiles of personality and emotional traits based on a sample of professional Taekwondo athletes from China. In addition, the study also aimed to examine the utility of the profiles in predicting successful athlete performance. Using multidimensional scaling (MDS) profile analysis, two latent profiles of personality and emotional traits were identified that indicate four subtypes of athletes. Regression analyses were conducted to examine how the identified profiles were associated with performance success. The results seemed to suggest that Taekwondo athletes with more performance success were more likely to have a profile of positive personality and emotional traits, while athletes with less performance success were likely to have somewhat elevated levels of self-control, extraversion, and aggression. Knowledge of athletes\u2019 personality profiles will help sport psychologists develop suitable interventions to enhance athletes\u2019 performance success. In concluding, the results are discussed in the context of athlete psychosocial development. The study added further evidence about the association between psychological and emotional factors and successful Taekwondo athletes. All athletes strive to achieve peak performance, particularly during competition. For our purposes, it is important to note that \u201cpeak performance\u201d is the state in which athletes can achieve superior functioning at their optimal psychological and physical levels, leading to outstanding outcomes . The seaSome persistent questions in sport psychology include: whether there is there a set of ideal psychological and emotional factors for optimal performance? If there is, what are these ideal factors? Moreover, what are their links with optimal performance  More recTheoretically, if we assume that the zone of zero gravity is necessary for optimal performance and likely to be experienced by successful athletes, then it is worth investigating whether athletes with certain personality and emotional profiles are more likely to achieve this state. The earliest investigation into this question was done by Morgan and his colleague , 1980. TMeta-analyses have shown that personality traits  are associated with various personal, interpersonal, and social behaviors e.g., . One wouAnother interesting issue in sport psychology important to note for our purposes is athlete aggression and mental health. Reviewed literature suggested that antisocial behaviors in athletes, such as aggression, may influence emotion and performance during competition . For insAccording to a review by This study contributes to sport psychology literature in three ways. First, although personality and emotional traits are known to be associated with sport performance, our knowledge in this regard is still incomplete and gaps in literature remain. For example, SD = 2.97) years. Experience in the sport ranged from 1 to 20 years, with the mean 5.81 (SD = 3.48) years. The weight category level ranged from 42 kg to 87 kg. Among the participants, 14 were elite athletes at an international level, 85 were elite athletes at a national level, 143 were first-level athletes at a national level, 55 were second-level athletes at a national level, and 35 were professional athletes without rank. All participants had not previously used psychological services.The data for the study included 332 professional Taekwondo athletes from different regions in China . Participant age ranged from 16 to 30 years old, with mean age 19.32  to 5 (agree strongly). A high score indicates a high level of the personality trait. Reliability, as assessed through Cronbach\u2019s alpha, was 0.78 for extraversion, 0.79 for agreeableness, 0.81 for conscientiousness, 0.76 for openness, and 0.77 for neuroticism in the current sample.The big five personality traits were assessed using the Big Five Inventory \u2013 44 . The meaextremely uncharacteristic of me) to 5 (extremely characteristic of me). A high score indicates a higher tendency of aggression. Reliability, as assessed through Cronbach\u2019s alpha, was 0.86 for physical aggression, 0.83 for verbal aggression, 0.77 for anger, and 0.87 for hostility in the current sample.Athlete aggression tendency was assessed through the Aggression Questionnaire . The meararely or none of the time) to 4 . Examples items are, \u201cI had trouble keeping my mind on what I was doing\u201d and \u201cI talked less than usual.\u201d Reliability, as assessed through Cronbach\u2019s alpha, was 0.91 in the current sample.Depression was assessed through the Center for Epidemiologic Studies Depression Scale (CES-D) . It is aI often feel this way), S (I sometimes feel this way), R (I rarely feel this way), or N (I never feel this way). Examples items are, \u201cMy interests and ideas are not shared by those around me\u201d and \u201cNo one really knows me well.\u201d Reliability, as assessed through Cronbach\u2019s alpha, was 0.91 in the current sample.Perceived loneliness was assessed through the UCLA Loneliness Scale (version 3) . It is acompletely disagree and 5 indicating completely agree. Example items are, \u201cI generally feel inferior to others\u201d and \u201cIt is so frustrating to see some people always having a good time.\u201d High scores indicate a high level of envy. Reliability, as assessed through Cronbach\u2019s alpha, was 0.78 in the current sample.A 7-item questionnaire was used to assess participants\u2019 feelings of envy . Items acompletely not describe me and 5 indicating completely describe me. High scores indicate a high level of self-control over emotions. Reliability, as assessed through Cronbach\u2019s alpha, was 0.85 in the current sample.Athlete self-control was assessed through 24 items designed to measure athletes\u2019 emotional regulation during competition . Examplenot at all to 5 = very much), with a high score indicating a high level of impulse control. Reliability, as assessed through Cronbach\u2019s alpha, was 0.76 for impulse control, 0.81 for work/study ethic, 0.77 for health habits, and 0.80 for deliberate action in the current sample.Impulse control was assessed using a revised impulse scale  based onrarely, to 5, indicating always. Example items are, \u201cI can cope and deal with any difficulties during the competition\u201d and \u201cI have confidence to beat stronger opponents during training.\u201d Higher scores indicate a high level of self-efficacy. Reliability, as assessed through Cronbach\u2019s alpha, was 0.96 in the current sample.Self-efficacy was assessed using the Sport Self-efficacy Questionnaire . It compmost elite or more performance success and 5 indicating non-elite standing or less performance success.We used athlete performance success as a correlate of athletes\u2019 profiles of personality and emotional traits. To assess athlete performance success, participants were asked to report on their performance rankings, and we used this information to approximate the performance success or elite standing among professional athletes. For example, if an athlete had won a medal  at an international competition, we considered him or her to have more performance success compared to those who had won a medal at a national competition. We considered participants\u2019 standing or performance success on a continuum scale, with 1 indicating Given the exploratory nature of the study, the primary analysis method used was multidimensional scaling (MDS) profile analysis . Since tTraditionally, MDS models have been mainly employed for data reduction, wherein the researcher sought to reduce complex interrelationships between stimuli to a simpler and more understandable form. This use is quite similar to that of factor analysis, and the two techniques can be used to study similar issues. However, in MDS profile analysis, we are interested in identifying latent profiles reflecting patterns of co-occurrence in the personality and emotional traits under inquiry, rather than homogeneous constructs. The MDS profile model represents a subgroup of athletes on a continuous basis, with athletes showing different degrees of \u201cmatching\u201d prototypical personality and emotional trait patterns, rather than aligning with a simple \u201cyes or no\u201d category. Moreover, MDS profile analysis is based on the framework of prototype theory  in whichSpecifically, in the MDS profile model, dimensions are represented as prototypical profiles  and the fundamental estimates are scale values for these variables. One of the major goals of MDS profile analysis is to determine the number of prototypical profiles expressed in scale values, which indicate a particular arrangement of variables in the profiles. The idiographic aspect of the model can be assessed using individual profile match indices (PMIs). Each individual\u2019s PMIs are equal in number to the number of profiles. These PMIs reflect the degree to which a person\u2019s observed data tends to match the identified prototypical profile\u2014the closer to the core, the more prototypical. Thus, a positive profile match index indicates that the individual\u2019s observed data tend to manifest the personality and emotional trait patterns represented at the positive end of the profile, while a negative profile match index indicates that the individual\u2019s data tend to exhibit a pattern opposite to the profile. These PMIs are useful for individuals because (1) by correlating these indices with other variables, one can study, for example, whether profiles can predict performance success and (2) one can examine a within-person pattern in that a participant may be more likely to manifest one profile than another; this means that one could evaluate how individuals perceive themselves with respect to these profiles.A word of caution is warranted: in MDS profile analysis, the actual appearance of a particular profile depends on how variables are listed. Since the positioning of each variable is arbitrary, the physical appearance of the profile can be arbitrarily changed without affecting the level, dispersion, or shape of the profile. As a result, some researchers call the positive end of a profile a \u201cprofile\u201d and the low points in the profile a \u201cmirror image of the profile\u201d . In addiStress value (ranging from 0 to +\u221e)  , with 0 0 to +\u221e) , a commo0 to +\u221e)  was alsoDuring the national Taekwondo competition period, the first author asked coaches to administer batteries of questionnaires comprising the above-mentioned measures to Taekwondo athletes during a break. Participation in the study was voluntary and athletes could withdraw from the study at any time. The study was approved by the university committee on human research (Protocol ID 2019157).Stress values. The Stress values were 0.13, 0.09, 0.07, and 0.06 for the one-, two-, three-, and four-profile solutions, respectively. The difference in the Stress value between the two- and three-profile solutions was 0.02. It seemed that the data could be approximated by two profiles without being overfitted. Therefore, we chose a two-dimensional solution as our best model of the data for interpretation and subsequent analysis.When studying profiles of personality and emotional traits, an explicit theory for predicting trait patterns among professional athletes is often not available or only prematurely specified. Under such circumstances, it can be beneficial to derive profiles of personality and emotional traits without specifying how these trait patterns may differ from one another. In the current study, we compared models of one-profile to four-profile solutions and compared the The results of the scale values from a two-profile solution are shown in The scale values were used to define profiles, which reflected patterns of co-occurrence among personality and emotional traits under inquiry, rather than homogenous constructs. Since the dimensional solution was standardized, we regarded any variable with a scale value greater than 0.5  as a salient variable that defined a profile. For example, in The scale values in As shown in Profile 2 was defined by athlete self-efficacy, extraversion, verbal aggression, and physical aggression at the positive end, and by conscientiousness, depression, loneliness, neuroticism, and impulse control at the negative end see . ProfileIn sum, the above results show some possible prototypical types of professional Taekwondo athletes with particular personality and emotional traits. As prototype theory suggests  there arb = \u22120.11, p = 0.31, partial h2 = 0.003) nor was there any interaction between Profile 1 by sex  and Profile 2 by sex . Given these results, we dropped the variable of sex from subsequent analyses, using two profiles as the predictors across sex.To investigate the association between profiles and performance success, we conducted a regression analysis with performance success as a criterion variable and derived profiles as predictors. First, we examined whether the results differed across sex; that is, whether the effect of profiles on performance success was moderated by sex. The results showed that there was no significant main effect for sex  = 4.55, p = 0.011]. Performance success was not statistically significantly associated with Profile 1 , but Profile 2 significantly predicted performance success . Specifically, the results seemed to suggest that athletes who resembled the positive end of Profile 2 were more likely to have less performance success, while athletes who resembled the negative end of Profile 2 were more likely to have more performance success.The results of subsequent regression analysis showed that the two profiles explained 3% of the variance [In order to get a better picture of what these results indicated, we further performed a quantile regression analysis in which the dependent variable of performance success was modeled at quantiles that corresponded to performance success standing. Specifically, we modeled performance success at quantiles 4 , 32 (corresponds to Level 2), 76 (corresponds to Level 3), and 94 (corresponds to Level 4). As shown in b = 0.40\u20130.55, p = 0.00\u20130.023 at quantiles 4, 32, 76, and 94), as shown in In contrast with the above, the results of quantile regression analysis showed that well-performing athletes were more likely to be associated with the negative end of Profile 2 , while poorly performing athletes were more likely to be associated with the positive end of Profile 2   to identify personality and emotional trait profiles based on a sample of professional Taekwondo athletes and (2) to investigate the association between the identified profiles and performance success. The study was motivated by the questions of (1) whether an athletic personality exists  and (2) The main findings from our MDS profile analysis seem to support the presence of salient personality and emotional trait profiles among professional Taekwondo athletes. Two profiles were identified, with Profile 1 more pronounced than Profile 2 in terms of the magnitude of scale values. More specifically, Profile 1 revealed two sub-types of athletes, one with salient positive personality and emotional traits  and another with negative ones . Interestingly, Profile 1 did not have a statistically significant association with athlete performance success or standing. However, the trend in In contrast, Profile 2 represents profile patterns with a modest level of mixed positive and negative emotional traits, as indicated by the smaller scale values used to define the profile. Patterns of Profile 2 showed a statistically significant association with Taekwondo athletes\u2019 performance success. In particular, athletes who resembled the pattern at the positive end of Profile 2  were more likely to be associated with less performance success, while those resembling the pattern at the negative end of Profile 2  were more likely to be associated with more performance success. One reason for the inclination toward more success among athletes resembling the pattern at the negative end may be that they showed modest levels of conscientiousness and impulse control. A modest level of conscientiousness, along with a modest level of impulse control, may effectively enable a balanced mix of planned, focused, flexible, and spontaneous behaviors among athletes , which mThe study provides a more comprehensive view of the personality and emotional trait profiles of a sample of professional Taekwondo athletes. This information may help sport psychologists design a better intervention to enhance performance success or avoid wasting limited time and resources. For example, the study found that openness and agreeableness did not form part of the profiles, suggesting that these traits may not be central to sport competition. However, positive personality and emotional traits such as athlete self-efficacy, deliberate action, extraversion, health habits, impulse control, work ethic, and conscientiousness may be necessary for an optimal psychological state for performance success. Thus, practitioners may have to enhance these traits among athletes, since they are challenged with providing interventions that improve core psychological functioning relevant to individual athletes. Evidence from this finding may provide information for designing better intervention strategies.As another example, the results showed the combination of anger, loneliness, depression, and aggressive behaviors as a pronounced profile among some Taekwondo athletes. This finding is consistent with those suggesting that mental health issues, particularly depression, are a common concern affecting both the general and sporting population . Some stThis study had several limitations that should be addressed in subsequent studies. First, the study used 18 personality and emotional trait measures as inputs for the profile analyses. The profiles will necessarily be influenced by the specific variables included in the study. There is a potentially rich array of personality and emotional traits not captured in the study, and the results may be determined by the variables used as inputs. Thus, the study results may not adequately capture the content domain of possible personality and emotional traits.Second, two prototypical profiles of personality and emotional traits were identified based on the available measures. However, other possible profiles could be identified. The question of whether a two-profile structure actually exists in the professional Taekwondo athlete population cannot be unequivocally answered based on the current data, and the best solution is to determine this through replication studies.Furthermore, the study was exploratory in nature, and the prototypical profiles were selected on the basis of statistical grounding, rather than theoretical consideration. Thus, researchers should interpret their findings in light of the possibility that other hypothesized profiles of data might exist. In addition, the measures were self-reported, thereby suggesting biases and inaccuracy in the reports.Third, in the present study, performance success was operationalized as whether Taekwondo athletes had won medals at international or national competitions. To be sure, there may be better ways of defining performance success. Future studies may thus want to explore other key performance success indicators in examining the relationships between performance success and its antecedents. In addition, the data were collected  without any supervision and without the presence of any researchers. Thus, the participants may not have given the questionnaires their best efforts, which may have impacted the quality of the data.Despite these limitations, as far as we know, the present study is the first to provide a comprehensive picture of how a set of personality and emotional traits co-occur among professional Taekwondo athletes. These limitations do not necessarily invalidate the potential utility of the current study in that the purpose of identifying personality and emotional trait profiles is to examine the potential psychological patterns typical in certain subgroups and not to eliminate all possible mathematically equivalent or even similar profiles. Nevertheless, it is important to recognize that athletes may develop personality and emotional states in ways that are not described here. It is also important for future studies to investigate the association between prototypical profiles of these traits and other important outcome variables .lb1127@swu.edu.cn.The datasets analyzed in this article are not publicly available in order to protect the participants\u2019 confidentiality. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by The Research Ethics Committee of College of Sports Sciences. The patients/participants provided their written informed consent to participate in this study.BL and CD conceptualized the framework of the study and wrote the manuscript. BL, FF, HS, LG, and FY help collect the data and provide feedback on the manuscript.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": "Sphingosine\u20101\u2010phosphate (S1P) is a natural bioactive lipid molecule and a common first or second messenger in the cardiovascular and immune systems. By binding with its receptors, S1P can serve as mediator of signalling during cell migration, differentiation, proliferation and apoptosis. Although the predominant role of S1P in bone regeneration has been noted in many studies, this role is not as well\u2010known as its roles in the cardiovascular and immune systems. In this review, we summarize previous research on the role of S1P receptors (S1PRs) in osteoblasts and osteoclasts. In addition, S1P is regarded as a bridge between bone resorption and formation, which brings hope to patients with bone\u2010related diseases. Finally, we discuss S1P and its receptors as therapeutic targets for treating osteoporosis, inflammatory osteolysis and bone metastasis based on the biological effects of S1P in osteoclastic/osteoblastic cells, immune cells and tumour cells. The whole cycle is divided into three phases: (a) the interaction of osteoclast precursors (OCPs) and osteoblasts (OBs) on the bone surface, including OCP recruitment, RANKL\u2010RANK binding and adhesion to the bone surface; (b) the differentiation of osteoblast precursors (OBPs) is facilitated by various cytokines from mature osteoclasts; and (c) the apoptosis of osteoclasts (OCs), as well as mineralization of the bone matrix.Sphingosine\u20101\u2010phosphate (S1P) is a natural bioactive lipid molecule and a common first or second messenger in the cardiovascular and immune systems.S1P is derived from ceramide, which is transformed into sphingosine primarily in vascular endothelial cells or circulating red blood cells/platelets. In addition, S1P is phosphorylated by sphingosine kinase 1/2 (SPHK1/2) and transported into blood vessels in different manners. While several transporters of S1P have previously been identified, spinster homologue 2 (SPNS2), a member of a large family of non\u2010ATP\u2010dependent organic ion transporters, has recently attracted much attention as an S1P transporter. Moreover, S1P is also exported from mast cells independently of their degranulation in a manner that is mediated by ATP\u2010dependent ABC transporters. Interestingly, the remaining S1P in different cells participates in several cellular behaviours via many other intracellular signalling pathways.Edg1, 5, 3, 6 and 8.i/o proteins. S1PR2 and S1PR3 are less widely distributed and are coupled to G12/13, as well as to Gq, Gs and Gi. The expression of S1PR4 and S1PR5 is even more restricted and only detected in specific tissues. As the research on S1PRs is insufficient, especially in skeletal systems, there is an urgent need for more studies to investigate this field. Hence, this review will focus on how S1PR1\u20103 influences bone metabolism and how to use these receptors as novel drug targets for bone\u2010related diseases. Because a multitude of S1PR agonists or antagonists are recognized and used in the laboratory to determine which S1PR is involved in certain functions, the development of our understanding of S1P has largely broadened.At present, five different S1P receptors (S1PR1\u20105) have been identified and are encoded by 2As a dynamic metabolic system, the skeletal system undergoes degradation and renewal. Healthy people stably maintain this cycle, while patients with bone disorders suffer from disequilibrium of bone formation and resorption, leading to increased or decreased bone mass. As S1P has been discovered to play an essential role in bone metabolism, targeting S1PRs on osteoclastic and osteoblastic cells is a novel direction of scientific research. More importantly, S1P cooperates with RANKL to participate as a coupling factor in osteoclast\u2010osteoblast crosstalk. Thus, the clinical value of S1P involves the whole bone regeneration cycle.2.1Osteoclasts are derived from the monocyte/macrophage lineage and fuse into multinuclear cells after stimulation with RANKL and macrophage colony\u2010stimulating factor (M\u2010CSF). These cells are responsible for bone matrix demineralization by secreting cathepsin K (CTSK) and HCl.12/13/Rho signalling axis is downstream of S1PR2.\u2212/\u2212 mice showed a significant attenuation in osteolysis in vivo but not in vitro.Some chemokines determine the direction of OCP migration, which play important role in bone resorption. S1PR1 and S1PR2, which were originally thought to be correlated with cell migration in lymphocytes, are more highly expressed than other S1PRs, indicating the impact of S1P on OCP migration.+, cathepsin K and matrix metalloproteinases (MMPs). The markers of osteoclast formation and activation, including NFATc1, CTSK, acid phosphatase 5 (ACP5), osteoclast\u2010associated receptor (OSCAR), dendritic cell\u2013specific transmembrane protein (DC\u2010STAMP) and osteoclastic cell\u2013specific transmembrane protein (OC\u2010STAMP), sharply increase after S1PR2 stimulation.Based on previous studies, RANKL and M\u2010CSF play important roles in osteoclast differentiation. In addition, S1P participates in OCP differentiation in an indirect manner. Usually, S1P impacts this process by regulating RANKL expression or its downstream signalling pathway. The existence of SPHK1, which is responsible for S1P production, sharply reduces osteoclastogenesis in BMM cultures by blocking p38 MAPK, c\u2010Fos and NFATc1 and augmenting ERK.2.2Osteoblasts, unlike osteoclasts, are derived from mesenchymal stem cells (MSCs).Similar to OCPs, S1P regulates OBP mobilization and recruitment through S1PR1/S1PR2 downstream signals. S1PR1 stimulates the JAK/STAT signalling axis, while S1PR2 activates the FAK/PI3K/AKT signalling axis, both of which promote MSC migration.In addition to osteoblast location, S1P is also crucial for OBP differentiation. S1PR1\u20103 is largely expressed in osteoblastic cells. In contrast to the decrease in S1PR1 and S1PR2 expression, S1PR3 expression increases sharply during OBP differentiation. Moreover, when S1PR3 is knocked out, bone matrix mineralization is not impacted by S1P, which means that S1PR3 regulates osteoblastogenesis. Other studies also found that S1PR1 and S1PR2 participate in osteoblastogenesis despite their decreased expression during differentiation. Bone morphogenetic protein 2 (BMP2), a crucial protein for OBP differentiation, is the target for S1PR1/2 downstream signalling pathways. The phosphorylation of Smad1/5/8, as well as ERK1/2, and the expression of Runx2 increase dramatically after activation of S1PR1 and, to a lesser extent, S1PR2.i were identified as downstream signalling pathways of S1P in the proliferation process.2+] but not PKC was required for S1P\u2010induced p42/44 MAPK activation, indicating that PKC exerts a proliferative effect without activating MAPK.2+] plays an essential role in bone metabolism and metastasis.2+] in bone homeostasis. Furthermore, pertussis toxin (a Gi inhibitor), LY294002 or wortmannin (PI3K inhibitors) and calphostin C (a PKC inhibitor) treatment were used on osteoblasts to determine which downstream signal of S1PR impacts osteoblast apoptosis. The results revealed, as expected, that Gi proteins and PI3K are the downstream signals of S1P for anti\u2010apoptotic effects in osteoblasts.Since mature osteoblasts still undergo mitosis, osteoblast proliferation and survival are crucial for osteogenesis. Various studies have found that S1P facilitates osteoblast proliferation and survival.2.3As previously stated, S1P is crucial for the differentiation, proliferation and migration of both osteoclasts and osteoblasts. For cell migration of osteoclasts and osteoblasts, the same S1PR (S1PR1/2) may exert the same effect. Therefore, by selectively activating or blocking specific S1PRs on specific cells or by impacting S1PRs on different cells to different degrees we can selectively regulate the migration of OCPs or OBPs individually. In addition, it has been gradually accepted that osteoclasts and osteoblasts are not completely antagonistic cells. In contrast, they are tightly coupled and interact with each other to maintain homeostasis and bone turnover  transcription, which enhanced the release of S1P from osteoclasts.By modulating S1PRs on osteoclasts or osteoblasts, their effects against osteoporosis were discovered. For example, S1PR1 agonists or S1PR2 antagonists decrease osteoclasts in the bone matrix, while S1PR3\u2010specific agonists are responsible for osteoblast differentiation. These agonists or antagonists serve as effective and valuable drugs for osteoporosis. However, the most promising S1P\u2010related therapeutic targets for osteoporosis at present are the enzymes involved in S1P metabolism. Weske et al provided a potential anabolic therapy for bone loss by targeting S1P lyase.\u2212/\u2212 osteoclasts resulted in enhanced SPHK1, demonstrating that deletion of CTSK enhances bone formation in vivo by increasing the generation of osteoclast\u2010derived S1P.Moreover, S1P also regulates the function of hormones associated with osteoporosis. Calcitonin (CT) was found to be involved in bone loss in a non\u2010canonical manner, which abolished the secretion of S1P from osteoclastic cells to inhibit osteoblast differentiation.4Inflammatory osteolysis is a common but severe disease, causing millions of people around the world to suffer from bone density loss. It is the result of bone infection and is characterized by overactivated osteoclasts and an imbalanced bone remodelling cycle.\u2212 bacteria infect bone tissue, their cell wall component LPS is released to the bone matrix, which activates macrophages by binding to Toll\u2010like receptor 4 (TLR4). Activated macrophages, called M1 macrophages, release a variety of pro\u2010inflammatory cytokines, such as tumour necrosis factor\u2010\u03b1 (TNF\u2010\u03b1), interleukin\u20101\u03b2 (IL\u20101\u03b2) and interleukin\u20106 (IL\u20106), and enhance the expression of inducible NO synthase (iNOS), C\u2010C motif chemokine receptor 7 (CCR7) and CD86.et al examined the expression of pro\u2010inflammatory cytokines induced by LPS. Not only COX\u20102 and iNOS but also IL\u20101\u03b2, IL\u20106 and TNF\u2010\u03b1 were inhibited after treatment with the S1PR3 inhibitor TY52156, demonstrating the pro\u2010inflammatory effect of S1PR3 i/o/PI3K/JNK signalling axis in liver inflammation. However, whether this signalling pathway is involved in inflammatory osteolysis is still unclear.When GMacrophage\u2010produced cytokines not only directly or indirectly impact the formation and activation of osteoclastic cells but also act as inflammatory mediators or chemokines to recruit lymphocytes to execute adaptive immunity reactions. Surprisingly, the S1P gradient between the blood/lymph and other organs is also responsible for lymphocyte trafficking from the thymus to inflamed sites and is mediated by S1PR1 signalling.Inhibiting inflammatory osteolysis through the intervention of S1P and lymphocyte recruitment has great advantages compared with those of formal surgical methods . Thus, there is a strong need to investigate the mechanisms by which S1P affects inflammation and lymphocyte recruitment.5Cancer is regarded as the top killer among patients worldwide and is characterized by uncontrolled growth, infiltration and metastasis. Tumour metastasis is the leading cause of cancer death, and bone is the third most common site for tumour metastasis after the lung and liver.reg cells in immune tolerance. Tumour\u2010specific Treg cells were thought to egress from bone marrow to tumour tissue in breast cancer patients by the stimulation of S1PR1, according to Rathinasamy et al This group also found that S1PR1 expression in Treg cells was induced after binding with antigen.reg cell\u2013mediated immune tolerance. However, a brain\u2010specific mechanism by which tumours escape immunosurveillance though the loss of S1PR1 on T cells was discovered.Recently, various studies have found a link between S1P and bone metastasis, providing us with a novel drug target for bone metastasis. At the early stage of metastasis, S1P participates in the metabolism and migration of primary tumour cells in the tumour microenvironment by cooperating with tumour\u2010associated macrophages (TAMs). TAMs act to eliminate tumour cells and are recruited to the tumour microenvironment via S1P\u2010mediated \u2018find me signals\u2019.Migration of tumour cells through blood and lymph vessels is a key step in tumour metastasis, and S1P seems to participate in this process via various mechanisms. On the one hand, the function of cytotoxic T cells is attenuated after binding to circulating S1P, promoting immune tolerance and preventing the elimination of tumour cells.Finally, OB/OC\u2010secreted S1P recruits certain tumour cells by enhancing the connection between tumour cells and the osteoclastic/osteoblastic niche, which is called bone colonization Figure\u00a0. An earl6Due to the predominant role of S1P receptors in bone\u2010related diseases have been widely noted, it is necessary to develop and test drugs that target S1P receptors in patients with bone\u2010related diseases. Among the several kinds of different drugs targeting one or more S1P receptors, fingolimod, ponesimod, siponimod, ozanimod and other related drugs are participated in clinical application and further development. Fingolimod (FTY720) acts as an agonist of the S1P receptor (S1PR). It could induce internalization and subsequent degradation of the receptor and consequently render lymphocytes to physiological S1P stimulation upon binding to the S1PR expressed on lymphocytes.7Although scientific research on the relationship between S1P and bone biology is much more restricted than that of the cardiovascular and immune systems, S1P still has tremendous clinical value in bone pathology and will become a promising target for the treatment of many bone\u2010related disorders, such as osteoporosis, inflammatory osteolysis and bone metastasis.The authors declare no conflict of interest.Shiwu Dong and Yueqi Chen conceived the idea. Lincheng Zhang, Wenhui Hu and Yueqi Chen wrote and edited the manuscript. Lincheng Zhang, Yutong Dong and Yiran Wang drew the figures and organized the table. Shiwu Dong and Yueqi Chen approved the final version of the manuscript. All authors critically revised the manuscript."}
+{"text": "There exists a lack of an understanding of how to facilitate knowledge sharing (KS) behaviors in healthcare organizations. This study is among the first to specifically address this issue through synthesizing psychological ownership (PO), self-determination theory, and psychological empowerment (PE) theory. This study developed a research model that described the impact of the psychological and motivational facilitating factors, including autonomous motivation, user PE, and PO on knowledge sharing intention (KSI) and knowledge sharing behavior (KSB). Data collected from 343 healthcare professionals were analyzed using the technique of partial least squares (PLS) to validate the research model. The results indicated that user PE, organization-based PO, and autonomous motivation all had significant direct/indirect positive effects on KSI and KSB as we hypothesized. Surprisingly, knowledge-based PO had a significant positive effect on KSI, which contradicted our original hypothesis. The implications for theory and for practice, limitations, and future research directions are discussed accordingly. Knowledge has long been recognized as the primary source of organizational growth and sustainable competitive advantage . OrganizHowever, even though the factors affecting KS have been extensively investigated, gaps remain in the existing research on knowledge sharing behavior (KSB). First, a significant amount of research has explored the factors affecting KSB based on the social exchange theory, which proposes that human behavior is driven by individual expectations of the costs and benefits inherent in interpersonal exchanges . HoweverSecond, many studies have investigated individual motivation that leads to KSB, including intrinsic motivation , extrinsic motivation . Nevertheless, it may be difficult to understand the overall quality of motivations in terms of the level of autonomy involved. This study is an attempt to close this gap in the literature by focusing on the controlled-to-autonomous continuum of motivations proposed in SDT (self-determination theory) , rather Third, in addition to attempting to explore the proactive motivation toward KS, it is important to understand the barriers to KS. Based on the nature of self-interest, employes may be afraid of losing their knowledge power and their unique value within an organization ; they maIn summary, to bridge these gaps, this study adopted SDT as the main underlying theory and integrated PE and PO theories to develop a research model that present the relationships among KS motivations, knowledge sharing intention (KSI), and KSB. Focusing on the potential psychological and motivational factors, including user PE (intrinsic task motivation), autonomous motivation , PO , KSI  to explore the effects of these factors on KSB among healthcare practitioners, as well as the moderating role of autonomous motivation in order to advance our understanding of KS and provide useful insights into initiating appropriate measures to achieve effective KS practices.Knowledge will become more valuable through sharing. KS not only allows personal knowledge to accumulate, but also enhances the competitiveness of enterprises. KS plays a crucial role in the knowledge-based economy era . It is aExisting literature has indicated that KS is a process of multi-directional exchanges of individual knowledge . It is aAccording to the theory of reasoned action (TRA) and theory of planned behavior (TPB), behavioral intention  is considered to be the most important determinant of behavior  and an iMany prior studies have provided empirical evidence supporting a positive correlation between KSI and KSB , as showH1: KSI positively influences KSB.Psychological ownership is based on the psychological theory of possession, which involves feelings of psychological possession of objects . The senPsychological ownership is a key emerging construct in the area of organizational behavior , but litResearch findings have suggested that the cognition of OPO is an essential part of the employe-organization relationship , reflectH2a: OPO positively influences employe KSI.Relative to OPO, few studies have examined the antecedents and consequences of employe perceptions toward KPO. Excessive feelings of possession may trigger negative sides of possessiveness, in turn inhibiting the implementation of KSB . EmployeH2b: KPO negatively influences employe KSI.The empowerment theory provides a comprehensive theoretical foundation that pictures how organizations build a helpful environment  . EmpowerBased on PO, empowered employes have a high sense of self-efficacy and self-identify, in turn giving them the ability to have a significant impact on tasks and circumstances with the use of HISs and to be proactive in terms of fulfiling their responsibilities , which iH3a: User PE positively influences OPO.H3b: User PE positively influences KPO.Many studies have validated the positive impact of PE on employe behavior  in the workplace . KS is aH3c: User PE positively influences employe KSI.At present, SDT  is a popSelf-determination theory spotlights how different types of motivation reflect levels of autonomy, where motivation can be divided into amotivation (lack of motivation) or a controlled-to-autonomous continuum, which ranges from extrinsic motivation (controlled) to intrinsic motivation (autonomous) . ExtrinsPrevious research indicates that more attention should be paid to the degree of influence of the controlled-to-autonomous continuum . To reduA higher RAI score indicated that the individual\u2019s behavior is autonomously motivated to a greater degree, whereas a lower the score indicated that the individual\u2019s behavior is more controlled.Psychological empowerment can be considered a motivational cognitive state, representing employes\u2019 motivational orientation along with the authoritative power required to perform work . HISs suH3d: User PE positively influences autonomous motivation.Motivation stems from personal expectations of beneficial outcomes, which encourages willingness to engage in certain behaviors , such asH4a: Autonomous motivation positively influences employe KSI.H4b: Autonomous motivation positively influences employe KSB.This study investigates the potential antecedents of KSB, including user PE, PO, autonomous motivation, and KSI. However, based on a review of previous research, the existing research has not shed much light on the interactions among these predictors. This research is intended to fill the gap by exploring autonomous motivation as a strategic instrument playing a moderating role. When serving as a moderator, autonomous motivation moderates the relationship between PO and KSI depending on the level of autonomous motivation, as well as whether employes react differently when experiencing OPO or KPO, and in turn determines their level of KSI. Employes with higher levels of autonomous motivation may have a greater sense of freedom and discretion at work, and can thus control the manner in which they complete tasks. They have confidence related to controlling their surrounding environment and thus develop a sense of responsibility and accountability. In this situation, we believe that autonomous motivation has the potential to strengthen the relationship between PO (including OPO and KPO) and KSI, because it cultivates the awareness of PO by satisfying the need for efficacy, self-identity, and belongingness , 2003. TH4c: Autonomous motivation positively moderates the positive relationship between OPO and KSI.H4d: Autonomous motivation positively moderates the negative relationship between KPO and KSI.Based on the discussion above, the proposed research model is shown in To conduct an effective survey, 48 items related to the six constructs of the research model were adapted and refined from the existing literature according to the specific focus of this study to ensure content validity  showed that all first-order constructs reached the recommended level of 0.7 . Thus, nData for the study were collected from the employes of three large teaching hospitals that were under the supervision of a prestigious research-centered medical university in Taiwan, and all these three hospitals have implemented HIS to support the provision of various healthcare services. We focused on investigating KSB of the employes with experience with HIS usage, and we thus randomly distributed 450 questionnaires with the consent of the supervisor of the employes in these three hospitals. Participation in the survey was voluntary, and the confidentiality of the identities of the survey respondents was ensured. The survey respondents were requested to answer questions based on their experience with using KM-related modules of the HIS . At the end of the data collection process, 430 responses were received. Eventually, 87 responses were excluded because some respondents failed to answer all the questions in the survey, had a systematic answering pattern, or failed to pass the check of the reverse questions that were deliberately included in the questionnaire. Finally, a total of 343 valid questionnaires were obtained, achieving a valid return rate of 79.8%. t-test was performed on the demographic variables  by comparing early and late responders. The results showed that these two data sets had no statistically significant differences with respect to gender (p = 0.31), age (p = 0.37), education (p = 0.81), and tenure (p = 0.47), suggesting that the non-response bias was unlikely to be a serious problem in this study.To assess for potential non-response bias, an independent samples A partial least squares (PLS) analysis, which is a variance-based structural equation modeling (SEM) technique, was used to validate our research model. Based on the flexibility of the PLS terms of model measurement, it can handle reflective and formative types of latent variables  and alsoConvergent and discriminant validity were first evaluated to validate the research model. Convergent validity can be assessed by inspecting the factor loadings of the indicators, composite reliability (CR), and average variance extracted (AVE) from the measures . Three iFinally, discriminant validity (Fornell-Larcker criterion) was assessed by examining the square roots of the AVE statistics . The resp < 0.001). To evaluate for multicollinearity, we conducted a variance inflation factor (VIF) test to ensure the validity of the formative construct. The results indicated that the VIFs for the first-order indicators of user PE (ranging from 1.32 to 1.96) were less than the cutoff value of 3.3  bias. In this approach, all survey items were loaded into the principal component factor analysis using an unrotated factor solution. The results showed the presence of 12 distinct factors with eigenvalues >1.0, rather than a single factor, with the first factor explaining only 29.6% of the variance. Thus, CMV was not considered a serious issue in this research.2) for endogenous constructs  indicated that KPO has a positive effect on KSI. Meanwhile, it was found that autonomous motivation had neither a significant moderating effect on the relationship between OPO and KSI (p > 0.10) nor did it have a moderating effect on KPO and KSI (p > 0.10). Thus, H4c and H4d were rejected. The results of our hypotheses testing are presented in These results showed that the hypotheses were largely supported by the data, except for H2b, H4c, and H4d. Contrary to our expectations, the statistically significant result for H2b  were calculated using 5,000 bootstrapping samples drawn from 343 cases  with replacements.Furthermore, we also examined the mediating effects of the influencing factors on KSI and KSB by adopting the method of Sobel test using the bias-corrected (BC) bootstrapping approach The results indicated the indirect effect of user PE on KSI via autonomous motivation to be positive (0.13) and that the 95% CI did not contain zero (ranging from 0.09 to 0.18), confirming the significance of this mediating effect. Similarly, user PE can indirectly influence KSI via KPO and OPO. t = \u22120.96), age , education , and tenure  did not significantly affect KSB (p > 0.05), indicating that our hypotheses testing results remained the same when controlling for the effects of age, gender, education, and tenure.Regarding the influences of the control variables, the results showed that gender . Specifically, KPO was found to significantly affect KSI, which was contrary to the findings of a previous study . A plausFinally, we observed the interactions between these influencing factors. This finding unexpectedly showed that autonomous motivation does not amplify the impact of OPO on KSI. This was attributed to the fact that employes who have high feelings of OPO may have a deep affectional investment in the organization, reflecting a sense of belonging, responsibility, and accountability to the organization . This caIn addition, the results show that autonomous motivation is directly positively associated with KSI and KSB, as expected. This implies that fostering autonomous motivation in employes is critical for facilitating KSB because when they sense a high level of autonomy, they are likely to inherently value KSB and thus engage in KSB.This study contributes to the KS and IS literature in several significant ways, as summarized below. First, this study links employes\u2019 psychological motivation, cognition, and behavior by integrating the PE and PO perspectives with SDT, which offers a broader perspective on KSB in healthcare organizations. This offers a comprehensive understanding of how numerous variables  affect employes\u2019 KSB, and remedies the lack of theoretical understanding of the interactions among these predictors, which can serve as the foundation for future theorizing initiatives for KSB.Second, the effect of user PE on KS is another unique aspect of this study and contributes to the application of PE theory to the IS domain. HISs comprise one of the most important ISs in healthcare organizations. They provide support that helps employes efficiently perform tasks and improve the quality and efficiency of healthcare services. This research conceptualized user PE as the extension of a specific type of PE in the context of HIS use. It reflects employes\u2019 motivational orientations toward the use of HISs at work and the authority necessary to exert the full potential of working systems. User PE plays an important role in increasing feelings of PO, including OPO and KPO, and nurtures the KSI of employes. We provided evidence of the significant effect of autonomous motivation on KS, and found that increased levels of user PE can lead to a greater degree of autonomous motivation of healthcare professionals regarding KSB, which has not been empirically examined in the existing literature. These findings shed additional light on active motivational orientations  that are beneficial to successful KS and conducive to developing new insights into the motivational mechanisms that support the applicability and extension of PE theory and SDT.Third, this study enriches our understanding of PO (OPO and KPO) regarding KSB in healthcare organizations and contributes to the KS literature by addressing existing gaps in previous studies where the effect of KPO on KSI remains mixed. We provided evidence supporting the positive relationship between PO and KSI. Specifically, we found the positive side of KPO in healthcare organizations, which reflected the unique characteristics of healthcare professionals. There may be differences in this issue across various workplace contexts. This empirical finding supports future theoretical development intended to deepen the understanding of employes\u2019 behavioral patterns regarding KS in healthcare organizations, which has not been adequately addressed in the literature.Finally, this research goes beyond the traditional dichotomous view of motivation  by adopting the autonomous motivation construct. The measurement was represented by a compound RAI that encompasses motivation on a controlled-to-autonomous continuum, rather than using proxies such as altruism or financial rewards. This construct can clearly reflect the relative autonomy associated with individual behavioral motivation  and makeIn the following, we provide important managerial implications related to efforts to enhance employe KSI and lead to effective KS practices in healthcare organizations.First, in a patient-centered care environment, relying solely on single-professional care may not be sufficient to address all patient issues. KS and integration across different specialties are key issues. However, user PE and autonomous motivation are effective in promoting voluntary behavior. Managers should promote multi-professional knowledge integration to obtain well-balanced inter-professional collaboration through empowerment and autonomy. Multi-specialist teams can be empowered by giving them increased responsibility and autonomy by which to devise healthcare plans for patients with high homogeneity and establish clinical pathways through discussion and high levels of involvement. This may enhance user competence related to user PE and exert and impact from HIS usage that will improve HIS functions so they meet task requirements, in turn generating higher levels of mutual knowledge and experience sharing among employes.Second, these findings indicated that PO, whether OPO or KPO, encourages employe KSI, making KSB smoother. We suggest building a favorable work environment that leads to satisfaction of efficacy, self-identity, and a sense of belongingness to cultivate the sense of PO. For example, employes can take advantage of on-the-job education and training, as well as seminars, thus increasing opportunities for learning and absorbing new knowledge and improving professional competence and confidence in their ability to respond to the rapidly changing healthcare environment. Moreover, publicly announcing a list of honors for KS contributors who are recognized and praised may lead to their being respected and highly accomplished, in turn strengthening their sense of self-identity. Also, more opportunities for participation and communication can be offered to encourage interaction among employes through various activities, such as health promotion, patient safety awareness, and healthcare quality improvement campaigns. This may build consensus and promote feelings of responsibility for the targets of PO, result in an increased sense of belonging. Additionally, by enhancing an intimate mentor-protege relationship through close interaction, the transformation that takes place due to experience, knowledge, and emotional care will help form a positive teaching and learning environment, in turn promoting KS practices.Finally, self-efficacy and enjoyment will have a significant effect on the use of electronic knowledge repositories , and an This study provides empirical evidence by integrating the PE and PO approaches with the SDT, with a focus on KS in healthcare organizations, which has not been heavily studied. We explored the intensity and significance of numerous factors, including user PE , OPO, KPO, autonomous motivation, and the effects of KSI on KSB, to better understand the entire employe KSB picture, in order to find new ways to boost, support, and improve KSB in the workplace. These findings revealed a positive and significant impact of user PE, PO , and autonomous motivation on KSI and KSB, offering valuable insights that will help managers facilitate effective, efficient KS practices. Although these results contribute to theory and practice, there are still some limitations that suggest possible directions for further research.First, the survey data in this study were collected from employes of healthcare organizations in Taiwan, which may have a limitation related to the regional culture, thus affecting the generalization of the results. Depending on the cultural context, feelings of PO may vary . Thus, iSecond, this study categorized PO into OPO and KPO, which are fruitful contributions to advancing an understanding of PO and its effects, but may have limited the scope of the investigation of PO. Further studies are suggested to examine these targets of PO toward different foci beyond employe knowledge and organizations, such as projects, specific roles, and workspaces, so as to enrich the empirical research in the PO literature.Third, these findings reveal that user PE has significantly positive effects on autonomous motivation, PO, and KSI, thus playing an important role in KSB. PE theory was applied to the context of HIS usage in healthcare organizations, where user PE was viewed as a specific type PE in order to deepen the understanding of KS practices among healthcare professionals. However, different industries may implement different IS to improve performance. Future research could apply PE to different scenarios due to different industry characteristics.Finally, this study investigated the factors influencing KSB in healthcare organizations, and the results indicated positive effects on KSB. It is unclear whether the factors that boost KS may also decrease the likelihood of employes withholding knowledge. For future researchers, this may be a good topic to advance the understanding of the opposite sides of KSB.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.S-YW contributed to develop the theoretical framework, data collection, data analysis, and manuscript writing. W-TW contributed to design the theoretical framework, review and edit the manuscript, and overall design. M-HH contributed to conduct data collection and data analysis processes. All authors contributed to the article and approved the submitted version.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": "Octogenarians and beyond have often been neglected in the populational study of disease despite being at the highest point of non-modifiable disease risk burden and the fastest-growing age group for the past decade. This study examined the characteristics and in-hospital management of octogenarian patients with acute coronary syndrome (ACS) in a multi-ethnic, middle-income country in South East Asia.n\u2009=\u20093162) were identified. Demographics, in-hospital intervention, and evidence-based pharmacotherapies over the 10-years were examined and compared across groups of interests using the Chi-square test. Multivariate logistic regression was used to calculate the adjusted odds ratio of receiving individual therapies according to patients\u2019 characteristics.This retrospective study utilised the Malaysian National Cardiovascular Disease- ACS (NCVD-ACS) registry. Consecutive patient data of those \u226580\u2009years old admitted with ACS at 24 participating hospitals from 2008 to 2017 (p\u2009<\u20090.001). Within the 10-year, there were positive increments in cardiovascular intervention and pharmacotherapies. Only 10% of octogenarians with ACS underwent percutaneous coronary intervention (PCI), the majority being STEMI patients . More than 80% were prescribed aspirin (91.3%) either alone or combined, dual antiplatelet therapy (DAPT) (83.3%), anticoagulants (89.7%) and statins (89.6%), while less being prescribed angiotensin-converting enzyme inhibitors/angiotensin receptor blockers (47.6%) and beta-blockers (43.0%). Men were more likely to receive PCI than women (adjusted Odds Ratio (aOR): 0.698; 95% CI: 0.490\u20130.993). NSTEMI  and unstable angina (UA)  were less likely to receive PCI but more likely given anticoagulants  than STEMI. The presence of cardiovascular risk factors and comorbidities influences management.Octogenarians made up 3.8% of patients with ACS in the NCVD-ACS registry  from 2008 until 2017. The largest ethnic group was Chinese (44%). Most octogenarians (95%) have multiple cardiovascular risk factors, with hypertension (82%) being the main. Non-ST-elevation myocardial infarction (NSTEMI) predominated (38%, Octogenarians with ACS in this country were mainly treated with cardiovascular pharmacotherapies. As the number of octogenarians with ACS will continue to increase, the country needs to embrace the increasing use of PCI in this group of patients.The online version contains supplementary material available at 10.1186/s12877-021-02724-7. The ageing population are increasing globally, a phenomenon known as the \u2018silver tsunami\u2019. An estimated 125 million individuals reaching 80\u2009years and older were considered octogenarians in 2018 . The preMalaysia is a multi-ethnic, upper-middle-income country in South-East Asia with a 32 million population . MalaysiThough healthcare services are available for all, demographic variation may influence patient management. Variations in intervention and pharmacotherapies for ACS patients in multi-ethnic Malaysia have previously been described , 16. LesThis cross-sectional descriptive study is a part of research approved by the Medical Review and Ethics Committee (MREC), Malaysian Ministry of Health (MOH), with approval code of NMRR-19-4066-52,389 (IIR). MREC waived informed consent for NCVD.Anonymised patient data were obtained from the NCVD-ACS registry, which forms part of the NCVD registry. This registry was endorsed by the Ministry of Health, Malaysia, and co-sponsored by the National Heart Association of Malaysia (NHAM) . The NCVThere was no patient or public participation in the development of this study\u2019s research question and outcome. All consecutive patient data from the year 2008 to 2017 were obtained retrospectively from the Malaysian NCVD-ACS registry.n\u2009=\u20093162). Patient information obtained were demographics, risk factors, and co-morbidities, summarised in Table\u00a0Malaysian citizens aged 80 or older (known as octogenarian) diagnosed with ACS  based on the American College of Cardiology/American Heart Association (ACC/AHA) definitions  were ideIn-hospital interventions included in this study were coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI), both emergency and non-emergency. Further details of the PCI intervention are available in the Malaysian NCVD-PCI registry . The phap) value of less than 0.05 is considered statistically significant. Statistical analysis was performed using Statistical Package for Social Science (SPSS) software .Continuous variables (age) are presented as mean\u2009\u00b1\u2009standard deviation (SD), while categorical variables are presented as frequencies and percentages. Patient characteristics were compared across gender using Student\u2019s t-test and chi-square test as appropriate. Choice of in-hospital intervention and evidence-based pharmacotherapies were analysed as cumulative, comparing the different ACS stratum using the chi-square test. The trends over the years were analysed using the linear trend test. Variations of in-hospital management for different groups of interest according to demographics, risk factors and co-morbidities were analysed using multivariate logistic regression, presented as adjusted odds ratio (aOR) with a 95% confidence interval (CI). The choice of covariates was based on factors influencing ACS management in the literature and included gender, ethnicity, types of ACS, risk factors and co-morbidities . A probap\u2009<\u20090.001) .A total of 3162 octogenarians  was admitted with ACS from 2008 to 2017. Octogenarians made up 3.8% of the total ACS patients cumulatively during the study period and have increased significantly over the years from 3.7% in 2008 to 17.2% in 2017 (Most patients (95%) already had at least one CV risk factor at presentation. Hypertension was the highest recorded risk factor at 82%, followed by DM at 47%. There were significant differences in the CV risk factors profile between gender, with women having higher rates of hypertension, DM, and dyslipidaemia Table . In compp\u2009<\u20090.001).Cumulatively, aspirin (93.5%), DAPT (85.1%), anticoagulants (81.7%) and statins (89.6%) were prescribed at near maximal levels, whilst about half were prescribed ACEIs/ARBs (51.5%) and beta-blockers (54.9%) in the 10-years as presented in Fig.\u00a0PCI intervention was performed at greater frequencies than CABG in all ACS stratum, Table\u00a0. Less thThis study found an increasing trend of octogenarians with ACS in this multi-ethnic country, with NSTEMI presentation predominated. The Swedish National registry reported that the cumulative incidence of MI for those aged 80\u2009years old and above are the highest amongst other age groups . Thus, oIn contrast to the ACS trends in the younger age group , 25, theThere is a high prevalence of octogenarians with multiple CV risk factors, affecting the presentation, severity, and complexity of managing ACS. Hypertension remains the major risk factor. Thus, advocating evidence-based management to treat hypertension may result in a more favourable outcome . More thThere was an increasing trend of PCI in octogenarians with STEMI, despite the rates being relatively low compared to what is being practised in other countries . The advThe prescribing of pharmacotherapies in the octogenarians with ACS were in accordance with international standard . Recent We need to bridge the knowledge gap on the effectiveness and risks of current management by performing clinical studies in the local setting. Physicians need to be aware of age-related physiological changes that may affect the pharmacokinetics and pharmacodynamics of these drugs in octogenarians while considering the immediate and long-term benefits. An example that could be modelled is Finland\u2019s follow-up accumulation of risk factor study  and the This study uses data from a nationwide cardiology registry obtained from 24 voluntary hospitals to represent an unselected group of octogenarian patients with ACS in a real-world setting in this country. The database is well maintained, and training is provided regularly for those involved to ensure data quality . A one-tThis registry only captures data of those reported to the participating hospitals; thus there may be a selection bias. Information that measures socioeconomic status that may influence demographics presentation, such as occupation and educational levels, were also unavailable. Elderly patients have varied tolerability towards intervention and pharmacotherapies . The speThe variations in PCI and evidence-based pharmacotherapies may impact short- and long-term outcomes in this group of patients , 47. ThiAs octogenarians with ACS continue to increase, the country needs to plan for disease management and prevention. Despite being the most vulnerable age group, octogenarians were not receiving aggressive treatment as younger patients. The use of evidence-based pharmacotherapies was maximised instead. It is expected that ACS management will continue to improve with an increasing number of octogenarians, better technologies, enhanced cardiologist skills and knowledge of disease in this age group.Additional file 1."}
+{"text": "Achieving malaria elimination requires the targeting of the human reservoir of infection, including those patients with asymptomatic infection. The objective was to synthesise evidence on the accuracy of the rapid-onsite diagnostic tests (RDTs) and microscopy for the detection of asymptomatic malaria as part of the surveillance activities in Asian countries.This was a meta-analysis of diagnostic test accuracy. Relevant studies that evaluated the diagnostic performance of RDTs and microscopy for detection of asymptomatic malaria were searched in health-related electronic databases. The methodological quality of the studies included was assessed using the QUADAS-2 tool.Plasmodium falciparum, pooled sensitivity by RDT  or microscopy  were almost comparable. For detection of Plasmodium vivax, pooled sensitivity of RDT  had also the comparable accuracy of microscopy . Of note are the wide range of sensitivity and specificity.Ten studies assessing RDT and/or microscopy were identified. The diagnostic accuracies in all these studies were verified by PCR. Overall, the pooled sensitivities of RDT, as well as microscopy for detection of any malaria parasites in asymptomatic participants, were low, while their pooled specificities were almost ideal. For the detection of Plasmodium infections. Other methods including a combination of PCR-based strategies, Loop-Mediated Isothermal Amplification (LAMP) technique must be considered to target these infections, in order to achieve malaria elimination. However, more data is needed for the wide acceptance and feasibility of these approaches. Studies to explore the role of asymptomatic and sub-patent infections in the transmission of malaria are of critical importance and are recommended.The findings of this meta-analysis suggest that RDTs and microscopy have limited sensitivity and are inappropriate for the detection of asymptomatic The online version contains supplementary material available at 10.1186/s12936-022-04082-0. Many malaria endemic countries are now entering the pre-/elimination phase of malaria control. The elimination phase aims to bring local transmission to zero indigenous cases of a specified malaria parasite , 2. TherRelevant studies were searched electronic databases of Medline, EMBASE, Web of Science, the Latin American and Caribbean Health Sciences Literature (LILACS) and African Journals Online (AJOL) for relevant studies published in English until May 2021. The search was conducted using keywords and Boolean operators:  AND (\u201cdipsticks\u201d OR \u201cRDT\u201d OR \u201crapid diagnosis\u201d OR \u201crapid onsite diagnosis\u201d OR \u201cICT\u201d OR \u201cimmunochromatographic\u201d) OR (\u201cmicroscopy\u201d OR \u201cPCR\u201d). We also checked the references of retrieved articles and relevant reviews manually for any additional studies. A search strategy in PubMed, as an example was (((((\"diagnosis\"[MeSH Terms] OR \"diagnosis\"[All Fields] OR \"diagnostic\"[All Fields]) AND accuracy[All Fields] AND rapid[All Fields] AND (\"research design\"[MeSH Terms] OR (\"research\"[All Fields] AND \"design\"[All Fields]) OR \"research design\"[All Fields] OR \"test\"[All Fields])) OR (\"microscopy\"[MeSH Terms] OR \"microscopy\"[All Fields])) AND ) AND asymptomatic[All Fields]) AND (\"epidemiology\"[Subheading] OR \"epidemiology\"[All Fields] OR \"surveillance\"[All Fields] OR \"epidemiology\"[MeSH Terms] OR \"surveillance\"[All Fields]).Human studies based on the guideline for diagnostic test accuracy (DTA) review  were selAny study design was included, if it had evaluated RDT/microscopy in participants residing in Asian countries.Asymptomatic cases living in the malaria-endemic countries, detected by active case detection (ACD), reactive case detection (RCD) or survey. Asymptomatic cases were as defined in the primary study.Any type of RDTs or routine microscopy for diagnosis of malaria,There was either no test or test with alternative RDT/microscopy.Human asymptomatic malaria cases, regardless of parasite species.PCR.These were the sensitivity and the specificity of the diagnostic test of interest. Sensitivity refers to the probability that the index test result will be positive in an infected case. Specificity refers to the probability that the index test result will be negative in a non\u2010infected case .Studies that that focussed on symptomatic cases, pregnant women or travellers were excluded. Moreover, studies without sufficient data had to be excluded as well.Two investigators  individually checked the titles and abstracts, and then selected the relevant full-text articles, according to the inclusion criteria. The two investigators independently extracted information from each included study, using a pre-tested data extraction form prepared for this review. Information collected comprised author, year of publication, country, participant characteristics , study design characteristics , diagnostic tests\u2019 characteristics  and the test performance results . Disagreements between the two investigators were resolved by consensus.Two investigators  independently assessed study quality following the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool , approprStudy-specific test accuracy was estimated in terms of sensitivity and specificity along with their 95% confidence intervals (CIs). TP, TN, FP, and FN represent the number of true positives, true negatives, false positives and false negatives, respectively.The pooled estimates of sensitivity and specificity were calculated using a random-effects bivariate model. These bivariate random-effects modelling of sensitivity and specificity produced the pair of performance measures that are interdependent \u201314. SepaI2 test. The I2 test values describe the percentage of total variation across studies that is attributable to the heterogeneity rather than chance. I2 value greater than 50% is regarded as substantial heterogeneity [Heterogeneity was statistically assessed using the ogeneity .Analysis was stratified by type of index test, either RDT or microscopy. It was planned to do subgroup analyses to assess the effect of age group, the methodological quality of studies, RDT brands and study country. However, due to the small number of studies or limited data in the studies, it was not possible to do so.Midas\u2019 programme in STATA 14  and the QUADAS-2 tool was with RevMan 5.3 .As there is no validated method for publication bias in test accuracy reviews available at present , this waFigure\u00a0The characteristics of the included studies are presented in Table The methodological quality of each study included in this review is presented in Additional file P. falciparum in asymptomatic participants varied widely, being from 19 to 100% and the summary estimate was 54% (95% CI: 11\u201392%). This was with substantial within-study heterogeneity (I2% 88.2%) .Overall, there was a greater variation in sensitivity than specificity in the diagnostic tests evaluated. Sensitivity with the use of microscopy for detection of 2%) Fig.\u00a0. There wP. vivax in asymptomatic participants varied from 9 to 100% and the summary sensitivity was 54% (95%CI: 11\u201392%). This was with substantial within-study heterogeneity (I2% 80%) . There was substantial within-study heterogeneity (I2% 84%) This review consists of ten individual studies, in which blood samples were collected from 10,945 participants across six malaria-endemic countries in Asia.(ii)The true positive rate of RDT, as well as microscopy, was relatively low in the detection of asymptomatic malaria cases, while the true negative rates were close to an ideal situation.The major observations are as follows.P. falciparum in symptomatic malaria cases. This was not found in the present study that assessed asymptomatic malaria. This contradiction could be related to variations in parasite densities in symptomatic cases and asymptomatic cases. RDTs have a limit of detection around 200 parasites/\u03bcL [According to the Global Technical Strategy (GTS) 2016\u20132030 . The majsites/\u03bcL , methodssites/\u03bcL  and objeP. vivax and 45% for P. falciparum compared with PCR. A systematic review of sub-microscopic P. vivax infection reported that PCR detects 67% more P. vivax infections than microscopy in surveys of endemic populations [P. falciparum compared with microscopy in the present meta-analysis study was likely to be due to persistent antigenaemia, rather than to the presence of current infections among these participants.The present meta-analysis found that microscopy could miss 46% of the asymptomatic malaria infections in endemic areas for ulations . The theulations . Moreoveulations . Additioulations . The higI2 test is that it does not inherently depend on the number of studies in the meta-analysis [I2 values for both RDT and microscopy as shown in this study indicated the variability of diagnostic accuracy among different populations. As mentioned above, factors such as sample type (volume of blood samples), age of patients and immune status of the individuals/community were likely to impact the heterogeneity of the results. Due to a paucity of data, we were unable to quantify the impact of these factors.Concerning the methodology, the advantage of the analysis . The subTargeted screening for asymptomatic infections may have a potential role in accelerating elimination and reducing the probability of reintroduction and resurgent transmission in these populations . Across P. falciparum\u00a0specimens occurred at low, medium and high parasite densities proving that parasitaemia did not influence the false negativity [Due to the limited data available in the studies included, and the scope of this review, it is not possible to provide that additional information from the current analysis. But it is to be noted that FN- RDT results have severe consequences in malaria-endemic settings because negative RDT results are not routinely confirmed by any other diagnostic tests . FP resugativity .Ultrasensitive RDTs (uRDTs) have been proposed as a tool to detect malaria infections even at low-density infections (below 100\u2013200 parasites/\u00b5L) through mass test and treat (MTAT) campaigns, or proactive or reactive testing and treating . HoweverIn malaria hotspots, it is important to be able to identify and treat these infections . A combiPlasmodium infections. The substrate can come from dried blood spots (DBS), venous blood samples, or in the case of high-volume PCR a cryoprecipitate The lower limit of detection varies by orders of magnitude. This information was not described in many of the studies included. Hence, it is not possible to stratify by a different form of PCR used.There are several limitations to the present study. The current results are limited by the quality of primary diagnostic test accuracy studies. As most individual studies included in this review did not report on whether they had performed a blinded index/reference test interpretation, over/underestimation of true accuracy is a concern. With regard to the reference test PCR, a common concern is the potential influence of contamination in these low-transmission areas as the contamination rates of 0.7%\u201310% has been reported for the laboratories encountered . MoreovePlasmodium malariae is increasing in the proportion of all malaria and Plasmodium ovale is also identified in some areas, additional tests that detect P. malariae and/or P. ovale are needed as a priority in the malaria elimination context. The increasing role of Plasmodium knowlesi as a contributor towards fever due to zoonotic malaria may require the availability of field-ready diagnostics specific to this species.As Plasmodium infections. Other methods including a combination of PCR-based strategies, Loop-Mediated Isothermal Amplification (LAMP) technique must be considered to target these infections, in order to achieve malaria elimination. However, more data is needed for the wide acceptance and feasibility of these approaches. Studies to explore the role of asymptomatic and sub-patent infections in the transmission of malaria are of critical importance and are recommended.The findings of this meta-analysis suggest that RDTs and microscopy have limited sensitivity and are inappropriate for the detection of asymptomatic Additional file 1: Table S1. PRISMA-DTA Checklist.Additional file 2: Table S2. Summary of excluded studies.Additional file 3: Figure S1. Summary of the methodological quality assessment across all studiesAdditional file 4: Figure S2. Forest plot of sensitivity and specificity of microscopy for P. vivaxAdditional file 5: Figure S3. Forest plot of sensitivity and specificity of RDT for P. vivax"}
+{"text": "Many structural patterns have been found to be important for the stability and robustness of mutualistic plant\u2013pollinator networks. These structural patterns are impacted by a suite of variables, including species traits, species abundances, their spatial configuration, and their phylogenetic history. Here, we consider a specific trait: phenology, or the timing of life history events. We expect that timing and duration of activity of pollinators, or of flowering in plants, could greatly affect the species' roles within networks in which they are embedded. Using plant\u2013pollinator networks from 33 sites in southern British Columbia, Canada, we asked (a) how phenological species traits, specifically timing of first appearance in the network and duration of activity in a network, were related to species' roles within a network, and (b) how those traits affected network robustness to phenologically biased species loss. We found that long duration of activity increased connection within modules for both pollinators and plants and among modules for plants. We also found that date of first appearance was positively related to interaction strength asymmetry in plants but negatively related to pollinators. Networks were generally more robust to the loss of pollinators than plants, and robustness increased if the models allow new interactions to form when old ones are lost, constrained by overlapping phenology of plants and pollinators. Robustness declined with the loss of late\u2010flowering plants, which tended to have higher interaction strength asymmetry. In addition, robustness declined with loss of early\u2010flying or long\u2010duration pollinators. These pollinators tended to be among\u2010module connectors. Our results point to networks being limited by early\u2010flying pollinators. If plants flower earlier due to climate change, plant fitness may decline as they will depend on early emerging pollinators, unless pollinators also emerge earlier. In plant\u2013pollinator networks of southern British Columbia, we found that networks were generally more robust to the loss of pollinators than plants. Robustness declined with the loss of late\u2010flowering plants, and with the loss of early\u2010flying or long\u2010duration pollinators. Many species attributes contribute to their roles within a network, including traits like flower color and feeding preferences  How do date of first appearance in a network, and length of activity during the season, affect individual species interaction patterns? We predict that species whose date of first appearance is earlier, and that are active longer in the season, should be less specialized, have greater within\u2010module degree, greater among\u2010module connectivity, and have higher values of interaction asymmetry (they affect their partners more than the reverse). (b) How robust are networks to removal of species due to varying phenological \u201ctraits\u201d ? Networks should be more robust to losing species whose date of first appearance is later in the season, and species that are active during less of the season.22.1A total of 33 mutualistic plant\u2013pollinator networks were studied in British Columbia, Canada: oak savanna (12 networks), shrub\u2013steppe (eight networks), and restored hedgerows (13 networks). These three vegetation types comprised three different studies. See Table\u00a02.2Data were collected for two of three vegetation types using the plot method and for the third using the transect method. Plots are generally more appropriate when the plant species in the community are very patchily distributed , we calculated Pearson's correlation coefficient for log10\u2010transformed variables for each of plants and pollinators separately. We found that the two variables were moderately to weakly correlated , meaning that if a species has a very late First Julian day it cannot, by definition, be present many days in the network. In contrast, if a species has an early First Julian day, it can be present many days, or few days.We collected the following phenological species variables for every pollinator and plant species in each network: (a) first Julian day observed interacting in the network, and (b) number of days observed in the network (last date observed\u2013first date observed). We treat each of the 33 networks as replicates and calculate both phenological variables for all species within each network. This means that there is no single value for first day observed or total days observed for any particular species across networks. Phenological variables were calculated from the observation of the interactions Figure . While w2.4Before calculating network metrics, we normalized network matrices by dividing each cell value by the number of days the community was observed (zeros remain zeros). We did this because there was unequal sampling effort across studies, even though there was equal effort across networks within studies. Normalizing resulted in noninteger values in some cases, but standardized individuals observed per unit time; therefore, the network is weighted by the frequency of interactions for a given monitoring time. This normalization allowed us to control for differences in sampling effort between studies but it did not control for differences in the ecosystems themselves. We account for this variation between ecosystems in the analyses as explained below.d\u2032) following Bl\u00fcthgen et\u00a0al.\u00a0(d\u2032), on the other hand, takes into account how \u201cevenly\u201d species interact with their partners; therefore, for the same number of interacting partners, utilizing one partner 99% of the time versus as they are available will result in different levels of specialization. d\u2032 is based on Shannon's entropy; therefore, it can be interpreted as the deviation from the null distribution of interaction frequencies that assumes that all partners are used in proportion to their availability. We also calculated (b) interaction strength asymmetry (ia), which measures the average mismatch between a focal species' effect on its interaction partners and the effect of the interaction partners on the focal species  standardized specialization for each species , and (d) among\u2010module connectivity . We chose these because we were interested in how phenology affected network metrics, so needed to use metrics that were quantified at the species level (where phenology was varying). In addition, within\u2010module degree (z) and among\u2010module connectivity (c) characterize the roles that species play in a network, providing a rich way of understanding networks . To calculate the species\u2010level modularity metrics (z and c), we used a modularity\u2010detecting algorithm, which maximized modularity using simulated annealing (SA) implemented in the command line function netcarto_cl in the C program Rgraph  and phenological variables using lme4, lme4Test , we used linear mixed effects models, with two phenology variables (date of first appearance and days observed) as fixed effects and network nested within study as a random effect (1|study/network). We also included a random effect for taxonomic group at the family level. This random effect was included as some groups will tend to have a longer duration in the season; for example, bumble bees have multiple generations throughout a season and will therefore be present for a longer period. Including these random effects allows different families to vary either in the intercept or the slopes. We compared five models for each predictor where we varied whether the family random effect was: (a) only for the intercepts (1|family), (b) a random slope for the number of days but no covariance in between the intercept and slope (0\u00a0+\u00a0days|family), (c) a random slope for the first Julian day with no covariance between the intercept and slope (0\u00a0+\u00a0first Julian|family), (d) a random slope for days with covariance between the intercept and slope (1\u00a0+\u00a0days|family), and (e) a random slope for first Julian day with covariance between the intercept and slope (1|first Julian|family). For c and d\u2032, variables that are proportions , we used nonlinear mixed effects models, with the same formula as above, but specifying a binomial distribution. Since the proportions in these variables are not derived from counts, a more appropriate distribution is the beta distribution. We tried to fit these classes of models using a beta distribution; however, due to poor convergence we could not fit all of the models presented above. We fit the simplest random effect structure, and the coefficients are similar in magnitude and direction as those obtained from the binomial distribution. Therefore, we present the results obtained using the binomial distribution. We selected the best fitting model for the family random effect using AIC  on the interaction with the removed species, and the average dissimilarity between the removed species j and all other species. Demographic dependence (Ri) determines how strongly a plant or pollinator species depend on their interacting species for their growth. For example, if a plant is able to produce viable seeds through selfing or apomixis, then its demographic dependence will be lower. Based on the species i susceptibility to extinction, a Bernoulli trial will decide whether the species i indeed goes extinct. If the species i is determined to go extinct based on the Bernoulli trial, the algorithm will then deal with this situation in two ways. First, if the species i that is susceptible to extinction only interacted with the removed species j, the species i that is susceptible to extinction can only persist if it creates new interactions. The algorithm only allows species that have a susceptibility less than the extinction threshold to create new interactions. The creation of new interactions depends on the similarity between the removed species j and the others in the network; in this analysis, it included similarity of the interaction matrix or overlapping phenology (see description below). The maximum number of new interactions is constrained to be the original degree of the species i(n) plus one (n\u00a0+\u00a01). Second, if the species i that is susceptible to extinction also interacted with other species, then the algorithm will enhance the interaction strength with these other species only if the susceptible species is allowed to persist following a Bernoulli trial.To measure network robustness , the interaction matrix, and the dissimilarity between species of the same type (plants or pollinators). Following Baumgartner et al. , we usedearliest date of the season have a higher probability of being removed first (earliest date first); (b) species are removed from a network according to the reverse order of activity\u2014that is, the species that were first active on the latest date have a higher probability of being removed first (latest date first); (c) species are removed from a network according to total duration of activity during the season\u2014that is, the species active the least number of days have a higher probability of being removed first (shortest duration first); and (d) species are removed from a network according to reversed order of total duration of activity during the season\u2014that is, the species active the most number of days have a higher probability of being removed first (longest duration first). In total, we ran 90 different parameter combinations for 33 networks.As mentioned above, the algorithm randomly removes species at the beginning of the run in order to cause the extinction cascade. In order to test the effect of removing species based on phenological order , we modified the algorithm so that the original extinction was not random but instead proportional to these phenological variables. We were interested in four scenarios with respect to network robustness: (a) species are removed from a network according to when they are first active\u2014that is, the species that were first active during the To calculate the probability of secondary extinction, the algorithm was run 1,000 times, and we counted the number of times that a secondary extinction occurred. To differentiate between the secondary extinctions caused by the removal of a pollinator or a plant, we divided the algorithm runs on whether the original extinction was that of a pollinator or a plant. Since the algorithm does not draw the original extinction based on total species richness, but instead it first does a binomial draw between plant and pollinator, and the number of original extinctions of plants and pollinators was roughly equal.3The networks in oak savanna had on average 19.8 plant species (2.96 sd) and 61.8 pollinators species (13.8 sd), in shrub\u2013steppe 22 plant species (6.09 sd) and 83 pollinator species (25.2 sd), and in hedgerow sites 13.8 plant species (6.18 sd) and 22 pollinator species (9.54 sd). All networks except one in a hedgerow site had more pollinator species than plant species.3.1z was 1\u00a0+\u00a0log10(first_Julian)|FamilyName, for c and d\u2032 was 0\u00a0+\u00a0log10(days)|FamilyName, and for ai was 1\u00a0+\u00a0log10(days)|FamilyName. Therefore, the slopes for z with first Julian varied between families, while for the other metrics, the slopes with the duration activity varied between families. For pollinators, the best model for z, d\u2032, and ia was 0\u00a0+\u00a0log10(days)|FamilyName and for c was 1\u00a0+\u00a0log10(days)|FamilyName. Therefore, for pollinators all of the slopes varied within families, but for c, the intercepts and the slopes were correlated while for the other metrics, it was not. This result highlights that the relationship between species roles in a network and their phenology varies between families. On average, species\u2010level network metrics were positively related to phenology variables. For both plants and pollinators, within\u2010module degree (z) and among\u2010module connectivity (c) were positively related to the number of days in a network (plant and pollinator p\u00a0<\u00a00.001), such that plants and pollinators that were active longer in the season were more connected within the module and among modules  for plants and pollinators was negatively related to the number of days in a network , such that plants and pollinators that were active longer in the season were more generalized. For pollinators, the degree of specialization (d\u2032) was negatively related to the day of first appearance (p\u00a0=\u00a00.035), such that pollinators that appeared later in the season were more generalized , such that plants and pollinators that were active longer in the season had a greater effect on the species they interacted with, compared with the effect those species had on them. The date of first appearance was positively related to interaction asymmetry for plants (p\u00a0=\u00a00.019) and negatively related to pollinators (p\u00a0<\u00a00.001). Plants that appeared later in the season had a stronger effect on pollinators they interact with, while pollinators that appeared early in the season had a stronger effect on the plants they interact with. On average, pollinators were more strongly impacted by the plants they interacted with  had a higher probability of secondary extinction when t Figure\u00a0. On the t Figure\u00a0.Allowing new interactions to occur based on how different species are in their phenology reduced the probability of secondary extinctions compared with allowing new interactions based on the current interaction matrix Figure\u00a0.The probability of secondary extinction depended on the extinction threshold and the demographic dependence between the species (Ri). This result confirms the model is working as expected. We found that as the extinction threshold increased, the probability of secondary extinctions decreased Figure\u00a0 and, as 44.1Cornus stolonifera (Family: Cornaceae), Symphoricarpus albus (Caprifoliaceae), and Ranunculus repens (Ranunculaceae). Ranunculus is a weedy forb while Symphoricarpus and Cornus are long\u2010lasting shrubs. These species additionally have radially symmetrical flowers allowing many types of pollinators to access the flowers , Sphaerophoria weemsi (Syrphidae), and Lasioglossum pacatum . Bumble bees (Bombus) tend to be highly generalized in floral visit patterns , Lythrum salicaria (Lythraceae), and Polygonum persicaria (Polygonaceae); these are all non\u2010native herbaceous species with multi\u2010flower inflorescences that may provide a large resource pulse. In contrast, we found that for pollinators, the interaction strength asymmetry decreased as species emerged later . Some of the pollinator species that emerged were solitary mining bees, such as Andrena nigrihirta, Andrena merriami, Andrena sladeni, Andrena trizonata, and Andrena porterae (Andrenidae). In two of the ecosystems studied, oak savanna and shrub\u2013steppe, we observed that at the beginning of the season, many plant species bloom in high density, but temperatures are not yet reliably warm enough for consistent insect activity. This may lead to plant reproduction being pollinator limited  and interaction strength asymmetry. Plant species that emerged later in the season  had a higher interaction strength asymmetry. That is, species that emerged later in the season affected their interacting partners more strongly than their partners affected them. Some of the plant species that emerged later in the season were 4.2We observed that networks were on average more robust to removal of pollinators than to removal of plants. This makes sense because plants more often link together the plant\u2013pollinator network (hubs organize around plants), whereas pollinators are less often important hubs  resulted in more robust networks than removing plants and pollinators in order from the most to least days observed (longest duration first). This is consistent with our previous results, which found that plants and pollinators that are active the most number of days were more connected both within and among modules, were less specialized, and were affected more strongly by interacting partners. Therefore, removing plants and pollinators that are well connected  results in less robust networks. While a phenological change is distinct from a complete removal, a species phenological change can result in species flowering or emerging earlier, but also have a shorter duration of their activity . Both the shift, but also the reduction in activity, have been shown to reduce the robustness of networks . Plants that appeared later in the season were more important for network robustness than the plants that appeared earlier in the season. On the other hand, removing pollinators according to the first Julian date of appearance in a network (earliest date first) resulted in less robust networks than removing plants in order from the last Julian date of appearance (late date first). These results are consistent with our results for plants and pollinators relating interaction strength asymmetry to the first Julian day of appearance. The mechanism is hypothesized to be the same that different resources are limiting for plants versus pollinators at different times over the flowering/flight season (see above). Our results are consistent with Ramos\u2010Jiliberto et\u00a0al.\u00a0(Removing plants according to the first Julian date of appearance in a network (earliest date first) resulted in o et\u00a0al.\u00a0, who fouAllowing networks to produce new interactions based on phenology increased the robustness of the networks, comparing to the robustness when new interactions were produced solely based on the pattern of current interactions, consistent with what Vizentin\u2010Bugoni et al.  found. TBombus spp.; Williams & Osborne,\u00a0Collinsia parviflora has shifted its phenology to earlier in the season in dry sites that truncate the length of the flowering season . Another caveat of these results is that we used the first and last day as estimates of phenology. These types of estimates can be biased by outliers ; Methodology ; Software ; Visualization ; Writing\u2010original draft ; Writing\u2010review & editing . Scott A. Chamberlain: Conceptualization ; Formal analysis ; Methodology ; Software ; Visualization ; Writing\u2010original draft . Elizabeth Elle: Conceptualization ; Data curation ; Funding acquisition ; Methodology ; Project administration ; Resources ; Writing\u2010original draft ; Writing\u2010review & editing .Supplementary MaterialClick here for additional data file."}
+{"text": "Compound\u2013protein interaction prediction is necessary to investigate health regulatory functions and promotes drug discovery. Machine learning is becoming increasingly important in bioinformatics for applications such as analyzing protein-related data to achieve successful solutions. Modeling the properties and functions of proteins is important but challenging, especially when dealing with predictions of the sequence type.We propose a method to model compounds and proteins for compound\u2013protein interaction prediction. A graph neural network is used to represent the compounds, and a convolutional layer extended with a bidirectional recurrent neural network framework, Long Short-Term Memory, and Gate Recurrent unit is used for protein sequence vectorization. The convolutional layer captures regulatory protein functions, while the recurrent layer captures long-term dependencies between protein functions, thus improving the accuracy of interaction prediction with compounds. A database of 7000 sets of annotated compound protein interaction, containing 1000 base length proteins is taken into consideration for the implementation. The results indicate that the proposed model performs effectively and can yield satisfactory accuracy regarding compound protein interaction prediction.The performance of GCRNN is based on the classification accordiong to a binary class of interactions between proteins and compounds The architectural design of GCRNN model comes with the integration of the Bi-Recurrent layer on top of CNN to learn dependencies of motifs on protein sequences and improve the accuracy of the predictions. Compound\u2013protein interaction (CPI) is important in the design of new compounds for the pharmaceutical industry. Proteins consist of large of small units called amino acids, which forms long chains that regulate specific functions of the human body. In humans, 20 types of amino acids are combined to form proteins. An amino acid sequence is structured into a three-dimensional complex, and its surface has a pocket that interacts with a compound through a specific combination of amino acids. In the framework of modern pharmaceutic research, the relationship between a compound and a protein can be depicted as a network, in which each node represents a compound or a protein, and an edge indicates a CPI.Based on this paradigm, many methods based on in silico networks have been introduced to predict CPIs \u20133. NeverThe discovery of new drugs is time-consuming and costly, usually taking over 10\u00a0years in development to then conduct clinical trials for their profound study and ensure compliance with safety standards. The recording and sharing of drug information has greatly accelerated discovery and production, further facilitating the search for new interactions of drugs that can bind to more than one protein. Experimental wet laboratory experiments are available to predict interactions of known drugs, but they require considerable effort and time to set up and implement. The need for faster results has triggered the development of accurate and powerful analytical tools. Although such tools have been experimentally implemented in previous decades, current technologies and data availability have enabled the analytic process of drug development to be driven by machine learning and artificial intelligence.Bioinformatics and data science have been combined to develop solutions based on various methods and algorithms, especially for CPI prediction , 8. ConvMachine learning has been used to construct strong and sustainable drug delivery pipelines in a shorter time compared with the use of conventional methods . Such piArtificial intelligence involves various machine learning methods, with the most prominent being deep neural networks (DNNs), which provide state-of-the-art solutions in many applications, such as speech recognition and visual object recognition . DNNs haUnlike conventional methods, a feature vector allows to automatically extract features from data without requiring expert knowledge or the three-dimensional structure of objects/proteins. Jacob et al.  applied This study contributes to the development of an end-to-end learning framework based on chemical information using a graph representation of a compound and a sequence of a protein by combining neural networks to identify the existence of CPIs. We represent compound and protein complexes as feature vectors and apply a learning algorithm to train a classifier for CPI prediction. This method, called graph convolutional recurrent neural network (GCRNN), uses protein analysis based on a CNN after a max-pooling layer followed by a bidirectional LSTM layer. The integration of recurrent layers into a CNN for protein modeling improves the representation of protein functions that dictate interactions with a compound and promote accurate results in real laboratory experiments. In addition, we integrate a recurrent layer after the max-pooling layer because protein functions follow patterns that represent specific biological arrangements, and the integration increases the detection probability and provides memory for capturing long-term dependencies , 18.The remainder of this paper is organized as follows. Section\u00a0After selecting the features for analyzing the data, despite factors such as data size or complexity, the performance is essential to choose the appropriate machine learning model. In addition, the selected model should deal with factors such as linearity, numbers of parameters and features of the data bank, training time, and accuracy. This work mainly measures the performance based on the classification accuracy. Also, to be noticed that the study conducted in this paper is compared with accuracy of replicated work of Tsubaki et al.  the modeThe open source genomic and protein data were retrieved from respective data repositories, for chemical structure of the compound from the PubChem database and protein sequences from the Protein Data Bank . ProteinThe GNN uses a chemical input given by the simplified molecular-input line-entry system, which provides molecular encoding sequential strings. The system uses RDkit  to obtair-radius number of 2 to represent molecules as vectors. For proteins, the CNN takes the original amino acid sequence and passes through a three-layer structure with 320 convolutional kernels and a window size of 30 with random initiation based on a similar model [This study use a three-layer GNN with an ar model . The pooThis study implemented the experiments in the PyTorch  using a CPI analysis requires wet laboratory experiments, but we only considered the data bank in this study assuming that the protein and interaction information is approved before the data were recorded. In addition, the bidirectional LSTM or bidirectional GRU after the max-pooling layer affects the CPI prediction performance. Thus, we obtained a high accuracy on the data, as shown in Fig.\u00a0The results in Fig.\u00a0Compared with the GCNN, our GCRNN shows a small improvement in the overall performance, as listed in Table Visualization tools provide insights on the medical outcomes expected for patients with a high accuracy to predict effects while reducing the time and setup workload of wet laboratory experiments for producing a specific drug. With the advancement of research, data banks will become larger, increasing our ability to understand CPI patterns for healthcare, and patients will be treated with specific drugs related to their health condition.This research is limited to the analysis process of the framework, even why several implementations are performed, a confident discussion of compound protein interaction requires wet laboratory experiments to be associates with, but this work will focus only on the database supposing that protein and interaction information is approved before when data are recorded.This work proposes GCRNN to identify CPIs using high-end machine learning methods. Also, emphasize the end-to-end representation learning of a GNN and a CNN with bidirectional LSTM/GRU to predict CPIs. Experimental results demonstrate that a relatively low-dimensional end-to-end neural network can outperform various existing methods on both balanced and imbalanced data.This study provides new insights on CPI prediction to construct general machine learning methods in bioinformatics rather than using feature engineering. Unlike existing structure-based computational approaches, the proposed GCRNN shows high performance using only protein primary structure information instead of three-dimensional structure information. Nevertheless, a deep learning model is usually considered a black box. Consequently, it is difficult to interpret the features that the model learns for CPI prediction. Improving the prediction performance on the validation and test sets would provide a starting point for subsequent research. In future work, this study will evaluate the model learning and performance considering comparisons with the results from wet laboratory experiments.Machine learning and computational methods are enhancing data analysis on a large scale and providing faster solutions, impacting research on biology and pharmaceutics. Biological data have been collected in data banks with plenty of information about genome and proteins being available for researchers to obtain reliable results in areas such as healthcare.This work address CPI prediction, an important aspect for drug discovery and development. Figure\u00a0A normal or abnormal condition carries information in the genome sequence, which can be translated into a protein sequence that interacts with a compound. The interactions can be stored continuously by using machine learning to determine the effective compound and protein for a specific disease. Then, laboratory experiments provide accurate results for clinical trials, and the resulting compound extends the dataset for new cases and developments.Deep learning techniques provide state-of-the-art performance and high accuracy for handling protein sequences and modeling molecules. Among the available models and architectures, we combine three powerful methods for CPI prediction, namely, GNN, CNN, and bidirectional RNN, as shown in Fig.\u00a0ni\u2009\u2208\u2009N represents atom i of a molecule, and eij\u2009\u2208\u2009E represents the bond between atoms ni and nj.The GNN can provide the low-level error vector of a molecular chart. We use the GNN to represent a molecular embedding that maps a graph into a vector through transformation and output functions. In the GNN, the transformation function updates the node values related to the neighboring nodes and edges, and the output function describes the nodes as vectors. In the graph structure, G\u2009=\u2009, where N is the set of nodes, and E is the set of edges that connect neighboring nodes. We consider undirected graph G, in which a node r-radius subgraphs [r-radius subgraph, for graph G\u2009=\u2009, the set of all nodes within a radius r of node i are represented as S, and the subgraph of r-radius nodes ni is defined asr-radius edges is defined asConsidering molecules as graphs simplifies the representation by defining few types of nodes and bonds and few parameters to learn. We also adopt ubgraphs  that outr-radius node and r-radius edge, which are randomly initialized, and backpropagation is used for training. To update the node information with respect to its neighborhood, the transition functions in Eqs.\u00a0 represents a node in Eq.\u00a0(e(t) represents an edge in Eq.\u00a0(f being a ReLU (rectified linear unit) activation function [neighbor and bneighbor being a weight matrix and bias vector, respectively, at time step t. In the same iteration, the edges are updated as follows:q is the neural network model for the edges and An embedded vector is assigned for the  in Eqs.\u00a0 and 4)  r-radiuse in Eq.\u00a0 and e(t)function  and \\docfunction  and Wnein-gram (n\u2009=\u20093) technique allows to represent amino acids as words. The group of three overlapping amino acids makes a word and represents input sequence P. The convolution for input protein sequence P is defined asi is the index of the output position, k is the index of the kernels, and Wk is an M\u2009\u00d7\u2009N weight matrix with M windows and N input channels. Then, a max-pooling layer reduces the size of the input or hidden layers by choosing the maximally activated neuron from a convolutional layer. Accordingly, for the CNN to be independent of the length of the protein sequence, max-pooling is applied when the maximally activated neuron is selected from the convolutional layer. Consequently, the number of hidden neurons generated by the convolution filter is the same as that of filters and not affected by the length of the input. For input Q, pooling is defined asCNNs are DNNs that also effective for analyzing protein sequences. As a CNN uses a weight-sharing strategy to capture local patterns in data, it is suitable for studying DNA (deoxyribonucleic acid) because convolution filters can determine functions of protein sequences that are short repeating patterns in DNA that may have a biological function. The proposed deep CNN is characterized by sequential interactive convolutional and pooling layers that extract features form sequence at various scales, followed by a fully connected layer that computes the whole-sequence information to extract protein features. Each CNN layer undergoes a linear transformation from the previous output. Then, it is multiplied by a weight matrix and proceeds with a nonlinear transformation. To minimize prediction errors, the weighted value matrix is learned during training. The CNN model base layer is a convolutional layer that calculates the output of a one-dimensional operation concerning a specific number of kernels (weight matrices later transformed by ReLU activation). The CNN for proteins maps sequence P into vector y with multiple filter functions. The first CNN layer is applied to proteins, where the An RNN is another type of DNN. Unlike a CNN, the connections between the RNN units form a directed cycle that creates an internal state of the network to exhibit a dynamic temporal or spatial behavior. A bidirectional LSTM is a variant of the RNN that combines the outputs of two RNNs to process a sequence both from left to right and from right to left. Instead of regular hidden units, the two proposed RNNs contain LSTM layers, which are smart network units that can remember a value over an arbitrary period. A bidirectional LSTM can capture long-term dependencies and has been effective for various machine learning applications. Bidirectional gated recurrent units (GRUs) are an alternative to bidirectional LSTMs to constantly represent sequential input without using separate memory units . We use v depend on forward and backward processing as follows:Features V provided from the pooling layer form a sequence t,\u2009\u2192\u2009and\u2009\u2190\u2009indicate the calculation direction, i is the input gate, f is the forget gate, o is the modulate gate, h is the hidden state at time t, Wi, WF, Wo, and Wc are weight matrices for their corresponding gates, and r is the reset gate and z is the update gate. The LSTM/GRU encodes t. As both the left and right directions are important for the global structure of proteins, we use a bidirectional LSTM . The bidirectional layers encode each position into leftward and rightward representations. H is the output, which is the sum of the results along both directions:At time Out[The vectors are concatenated to obtain output vector"}
+{"text": "Drosophila melanogaster. This study shows that fruit flies from a population living in a boreal and relatively unpredictable climate have more variable variable phototactic biases than fruit flies from a more stable tropical climate, consistent with bet-hedging theory. We experimentally show that phototactic variability of D. melanogaster is regulated by the neurotransmitter serotonin (5-HT), which acts as a suppressor of the variability of phototactic choices. When fed 5-HT precursor, boreal flies exhibited lower variability, and they were insensitive to 5-HT inhibitor. The opposite pattern was seen in the tropical flies. Thus, the reduction of 5-HT in fruit flies\u2019 brains may be the mechanistic basis of an adaptive bet-hedging strategy in a less predictable boreal climate.When organisms\u2019 environmental conditions vary unpredictably in time, it can be advantageous for individuals to hedge their phenotypic bets. It has been shown that a bet-hedging strategy possibly underlies the high inter-individual diversity of phototactic choice in  Life in the natural environment exposes organisms to a number of abiotic and biotic factors. The stability of environmental conditions differs across latitudes, geographic locations and time, resulting in diverse patterns of fluctuation in ecological interactions, population sizes, sex and age ratios, parasite burdens, range shifts and extinction. To predict the direction and magnitude of responses to fluctuating environments, it is important to understand the strategies that facilitate the survival of organisms under environmental change  and between Turku and Tampere (Finland). Local wild flies were caught using banana and yeast baited traps or collecting the pupae from compost piles in April 2017, in Kenya and in August 2017 in Finland. In either country, we collected the flies from five locations separated by a 5-km distance at least.Drosophila media .For behavior experiments, parental flies were kept in vials for 24-h to oviposit. When their progeny enclosed, they were removed in the first 5\u20137 h post-eclosion, anesthetized, and separated by sex. In this study, we tested flies grown on Formula 4\u201324 instant We studied the variability of phototaxis behavior in F1 flies. The FlyVac apparatus allowed us to measure the startled phototaxis behavior of many individual fruit flies simultaneously  grown without any drugs, flies grown on food supplemented with 5-HTP and flies grown on food supplemented with \u03b1MW  generalized linear model (GLM) using positive light-choice count as a dependent variable and geographic location , treatment groups  and sex as predictors with interaction (sex \u00d7 treatment groups). After determining that sex was not a significant factor, we excluded it and fitted the final GLM using geographic location, group and an interaction between geographic location and treatment group. We assessed the variation of phototactic choices by calculating the variability beyond expectation  and, therefore, we pooled sexes in the further analyses of LCP. We found that geographic location was a significant predictor of light-choice probability . Flies were photopositive both in Kenya and Finland, choosing the light 80% and 68% of the time , however, there was significant interaction between geographic location and treatment group . Feeding Kenyan flies, \u03b1MW (a serotonin synthesis inhibitor) increased their LCP significantly while feeding 5-hydroxytryptophan, a serotonin precursor (5-HTP) reduced LCP  either in Finland or Kenya  and sexes were pooled in the further analyses of VBE. Finnish fruit flies had significantly higher VBE than flies in Kenya . In Kenyan flies, feeding \u03b1MW significantly increased VBE, while 5-HTP did not affect their VBE , and more consistent across individuals (lower VBE) than flies from the northern climate. Conversely, flies from the higher latitude site were less attracted to light and less consistent across individuals. Bet-hedging theory predicts that higher phenotypic diversity may be adaptive under conditions of less predictable climate. Kain et al.  developeDrosophila behaviors including locomotor bias , dopamine, and histamine (Borycz et al., white mutants (Borycz et al., white mutants. For example, 1118w mutants chose light 61% of the time and their VBE is ~0.87, values which are closer to those in Finnish flies than, for example, flies of the standard lab wild type strain Canton-S (76% and 0.56, respectively; Kain et al., We found that Finnish flies exhibited high VBE for phototaxis, similarly to what was observed in a laboratory strain, 1118w flies. We found that 5-HTP significantly affected VBE of Finnish flies, while it had no effect on VBE of Kenyan flies. The same pattern was observed in 1118w and Canton-S flies, respectively (Kain et al., Common factors may be responsible for the behavioral metrics of Finnish and Theory predicts that the relative stability of the local climate in Kenya should favor heritable and lower variability phototactic preferences, i.e., a strategy with less stochastic bet-hedging Hopper, . In suchIn a population utilizing a bet-hedging strategy, individuals exhibiting a wide variety of preferences are born continuously across a season. If the summer is cooler, spring-adapted individuals will survive, while summer-adapted flies will survive if the summer is hot and long (Bergland et al., While Kain et al.  observedWhite mutants have reduced concentrations of dopamine in the brain (Borycz et al., white mutants, they may also have lower dopamine levels. This in turn might explain their lower LCP, while lower serotonin could explain their higher VBE. Kain et al. (white mutants. However, it has been shown that dopamine affects the production and release of melatonin (Gonz\u00e1lez et al., It is possible that differential levels of serotonin do not explain the mean LCP of these strains, since serotonin or its precursor 5-HTP have been previously reported to decrease photopositivity in larval bryozoans (Pires and Woollacott, n et al.  did not Importantly, 5-HT is a precursor of melatonin (Richter et al., Flies that follow a bet-hedging strategy might only attain environmental conditions well-matched to their behavioral biases if they live through long periods of poorly matched conditions. Thus, there is likely an interplay between generation/lifespan length and the timescale of environmental fluctuations. Indeed, modeling suggests that bet-hedging is an adaptive response to environmental fluctuations at specific timescales roughly corresponding to the lifespan (Krams et al., Proving that phenotypic variability reflects a bet-hedging strategy is a tall order. A formal approach to this question Simons, , stipulaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.IK, TK, RK and BB conceived and designed the study. TK, RK, IK, GT, SP, PJ, MM and MR performed the study, collected and extracted data. GT, DE, RK and IK analyzed the data. JM, SP and IK built the equipment. BB, TK, MR, PJ, SP, JM and MM participated in data analyses, results interpretation, and drafting the manuscript. All authors contributed to the article and approved the submitted version.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": "Inheritance of genetic material following chromosome duplication in eukaryotic cell division is coordinated by the spindle apparatus. The spindle is a highly interconnected network of microtubule filaments that are cross-linked by different types of molecular motors. How the different motors cooperate to organize the spindle network is not understood. Here, we show that an asymmetric cross-linker design can confer bifunctionality to a mitotic motor in the presence of other motors. The asymmetric motor supports both extensile and contractile microtubule network behaviors as observed in different parts of the spindle. These findings define rules controlling the generation of active microtubule networks and allow us to better understand how motors cooperate to organize the correct spindle architecture when a cell divides. During cell division, cross-linking motors determine the architecture of the spindle, a dynamic microtubule network that segregates the chromosomes in eukaryotes. It is unclear how motors with opposite directionality coordinate to drive both contractile and extensile behaviors in the spindle. Particularly, the impact of different cross-linker designs on network self-organization is not understood, limiting our understanding of self-organizing structures in cells but also our ability to engineer new active materials. Here, we use experiment and theory to examine active microtubule networks driven by mixtures of motors with opposite directionality and different cross-linker design. We find that although the kinesin-14 HSET causes network contraction when dominant, it can also assist the opposing kinesin-5 KIF11 to generate extensile networks. This bifunctionality results from HSET\u2019s asymmetric design, distinct from symmetric KIF11. These findings expand the set of rules underlying patterning of active microtubule assemblies and allow a better understanding of motor cooperation in the spindle. The mitotic spindle, which segregates the chromosomes during eukaryotic cell division, is a paradigm for self-organizing active filament networks. It is composed of dynamic microtubules, interconnected by motile cross-linkers, forming an active gel continuously driven by internal stresses while maintaining steady-state shape \u20135. The mIn vitro reconstitutions of self-organizing microtubule networks with purified cross-linking motors have provided insight into the basic properties of active microtubule-based networks , 6, 8\u2013168Moreover, natural motors have distinct designs. Kinesin-5 is a plus end\u2013directed, symmetric motor that can cross-link two microtubules using pairs of motor domains present at either end of the tetrameric molecule . It slid19Here we perform in\u00a0vitro experiments with mixtures of both mitotic kinesins and microtubules nucleating in solution. We find that cross-linker design plays a particularly important role when motors with opposite directionality work together to generate an active network. Kinesin-14, mostly known as an aster-forming motor, promotes radially polar network formation when dominant but is surprisingly also able to act cooperatively with kinesin-5 to generate nematic networks. Computer simulations demonstrate that this collective behavior is a consequence of kinesin-5 being a stronger cross-linking motor than kinesin-14, which on the other hand is still an efficient microtubule bundler. Our study illustrates how the design of mitotic motors is optimized for their combined action in the spindle and provides insight into the principles of active multimotor network organization.Methods)  . Under tRight). Asters became centered between the top and bottom of the experimental chamber and HSET accumulated in the middle of the asters, indicating that the minus ends were gathered (Left). Asters that came into contact fused (AMovie S1). We explored the phase space of network organization by HSET . The overall contractile nature of HSET/microtubule networks is due to the relatively slow growth speed of microtubule minus ends compared to the HSET motor speed, allowing HSET to gather microtubule minus ends over a wide range of conditions  (Left)  (vie S1B) . The micvie S1B) E, Left  (Left) \u201316, 19. ) (Left) F. At low) (Left) G, i  (Left) , 19. BunThe organizational capacity of the two motors at low tubulin concentrations is similar, but diverges at higher tubulin concentrations. This can be understood as a consequence of the opposite directionalities of the motors and the different growth speeds at the two microtubule ends. When overall microtubule growth is faster, it is more difficult for KIF11 to accumulate at the faster-growing plus ends, whereas HSET can still accumulate at the slower-growing minus ends , 30.Next, we set out to determine the network patterns formed in the presence of both motors.AMovie S2). Initially, microtubules appeared to be heavily cross-linked into dense clusters without obvious orientation or polarity. Over time, these clusters reorganized into asters with central HSET accumulation, revealing that when in excess HSET can outcompete KIF11 under aster-promoting conditions . KIF11 resided at the center of the assembled structures, indicating that it had successfully gathered the plus ends and dominated the contractile network behavior. HSET was relegated to the periphery where it bundled microtubules, but did not appear to efficiently gather minus ends , the addition of a 10-fold lower concentration of KIF11 completely inhibited aster formation or any local accumulation of HSET (Right). Instead, a cross-linked mesh of curved microtubule bundles formed, characteristic of extensile network behavior. These results show that in dense networks with higher microtubule growth rates, in which HSET forms asters only within a narrow range of concentrations . We found that the addition of an equimolar amount of HSET resulted in weak nematic network formation with some extensile activity . Increasing the HSET concentration twofold and eightfold produced increasingly active nematic networks with long, dense microtubule bundles . This is remarkable because HSET on its own is unable to promote nematic network formation in these experimental conditions .Next, we investigated more closely the high-tubulin concentration regime, in which the independent organizational capacities of the two motors diverge, with HSET forming contractile and KIF11 extensile networks . Keepingnditions C. HSET wnditions A, iv. InMovie S5) which otherwise promote contractile activity with either motor alone , indicating KIF11-driven microtubule sliding. Some seeds displayed saltatory motility, indicative of a \u201ctug of war\u201d in some parts of the network , mimicking the behavior of the docetaxel-stabilized microtubules in the experiments. KIF11 was modeled as a symmetric complex consisting of two plus end\u2013directed and processive motor units mechanically connected by a Hookean spring with nonzero resting length (Methods). In contrast to previous microtubule network simulations, we explicitly considered here the asymmetric nature of HSET. It was modeled as an asymmetric cross-linker with one minus end\u2013directed, nonprocessive motor unit and one diffusive unit (the HSET tail domain)  (Methods).To pursue our investigation of these mixed motor microtubule networks, we turned to computer simulations. Microtubules were modeled in a thin three-dimensional space as diffusing bendable lines repelling each other via soft-core interactions , 35. Theg length A 34). K. KSI App domain) A. The dwdetails) . Both HSSI Appendix, Fig. 3B). At a microtubule density of 0.8 microtubules/\u00b5m2, KIF11 organized microtubules into bundles that continuously extended, broke apart, and reformed again (AMovie S6), similar to experimentally observed active nematic networks driven by KIF11 at high tubulin concentrations (Methods). In KIF11-driven networks, the average microtubule sliding speed increased with the number of microtubules becoming incorporated in the mixed polarity bundles (BMovie S6) leading to immobile microtubules , similar to experimentally observed HSET asters (Inset), further validating the HSET model.We first examined networks driven solely by HSET and KIF11. In the absence of any motor, microtubules remained randomly oriented (trations E. To chaotubules B. This sotubules . CompareLeft and AMovie S7). Adding a low amount of HSET cross-linkers promoted bundle formation (Middle and BMovie S7). Visualizing only some of the microtubules in the simulated bundles revealed mostly KIF11-driven microtubule sliding and occasionally saltatory movement, resembling experimental observations (Movie S8)  as a consequence of the diffusive binding mode of its tail (Right and C and CMovie S7), again similar to the experiments ..Left andovie S8) , despiteits tail . Neverthits tail C. Furtheits tail C and at eriments A. Thus, SI Appendix, Fig. 6A, i), similarly to normal HSET, but leading to an increased speed of plus end\u2013directed sliding. Further increasing the concentration of this \u201cplus HSET\u201d caused the formation of inverted asters with focused plus poles . These results demonstrate that the directionality of the poorly processive HSET motor does not determine its ability to assist KIF11 in extensile bundle formation, but determines aster polarity when HSET is dominant. Another hypothetical HSET, now with normal minus directionality but with a nondiffusive microtubule binding unit, also assisted KIF11-mediated extensile bundle formation . This nondiffusive HSET variant, however, slowed down KIF11-driven microtubule sliding more than normal HSET . Microtubule sliding was faster when the unbinding rate of the hypothetical nondiffusive HSET tail was increased together with the number of nondiffusive HSETs to maintain a similar number of cross-links . This shows that the diffusive nature of the HSET tail allows it to be bound to the microtubule for some time without presenting too much resistance to KIF11-driven microtubule sliding.To understand which properties of HSET make it able to either synergize with or antagonize KIF11, we simulated variations of the HSET model. Low amounts of a hypothetical plus end\u2013directed HSET variant assisted low amounts of KIF11 to generate extensile bundles  were equally efficient as the normal HSET in helping KIF11 to organize microtubules into bundles D, i. TheSI Appendix, Fig. 6, Right). KIF11-driven plus-end aster stabilization by double-tail HSET required the ability of the diffusive unit to hold on to microtubule ends. When the diffusive units in the model were allowed to unbind from microtubule ends, KIF11-driven extensile bundles formed instead of plus-end asters , also helped KIF11 to form microtubule bundles D, ii. BuNeither of these simulated symmetric HSET variants replicated the full range of extensile and contractile microtubule networks driven by HSET in the presence of KIF11. Therefore, the asymmetric design of HSET is key to its microtubule organizing properties. First, the diffusive tail domain allows sustained tethering to the microtubule lattice and, coupled with a nonprocessive motor domain with rapid binding/unbinding rates, this facilitates bundling and presents low effective friction to KIF11-driven sliding. Second, these same features can also promote gathering of minus ends in local contractile structures due to effective minus end\u2013directed movement of the microtubule-tethered HSET motors. Therefore, the design of the asymmetric cross-linker HSET combines properties that favor context-dependent microtubule bundling or aster formation. This unique set of properties makes HSET multifunctional, supporting either plus motor\u2013driven nematic network formation when present in lower numbers or minus-end aster formation when dominant.We observed both contractile and extensile network states in active microtubule networks generated by motors with opposite directionality, depending on system composition. Due to the asymmetric design of the minus motor HSET and the symmetric design of the plus motor KIF11, the two motors behaved differently. They do compete, but especially in the higher microtubule density regime, HSET can also assist KIF11 in nematic network formation, effectively creating a cross-linker with two functionalities depending on context.HSET\u2019s asymmetric design, characterized by a nonprocessive, minus end\u2013directed motor that is diffusively anchored to a second microtubule for relatively long times, allows efficient aster formation in the absence of other motors, although it does not focus microtubule minus ends into very tight poles. Against an opposing motile cross-linker like KIF11 with two processive motors, HSET\u2019s weak force-producing capacity as a consequence of its tail\u2019s diffusive binding  requiresWe previously demonstrated how the characteristically asymmetric properties of microtubule growth affect the generation of active networks by a single type of motor. Fast growth of microtubule plus ends favors nematic network formation by plus end\u2013directed cross-linkers, whereas slow (or no) minus end growth favors the formation of asters with a minus pole by minus end\u2013directed cross-linkers . This raThe specific design of these two motors appears to be optimized for their function in the mitotic spindle. HSET is distributed over the entire spindle , 36, 37,40Biological components with designs optimized for different tasks in living cells offer a variety of \u201ctools\u201d for integration into biomimetic materials, enriching the rulebook by which we understand macroscopic self-organized states, while giving rise to surprising new behaviors \u201316. HereI.N-ethylmaleimide  tubulin was prepared from purified tubulin as described previously  before adjustment, final ultracentrifugation, and aliquoting . Aliquoted tubulin was then snap-frozen and stored in liquid nitrogen.Tubulin was initially purified by two rounds of polymerization and depolymerization from porcine brain as described previously . N-ethyleviously  without eviously . Final c5A-HSET (pJR291) and StrepTagII-KIF11-A3G5-monomeric green fluorescent protein (mGFP) (pJR303) have been described previously . This construct was used for polarity-marked microtubule seed assays as an alternative to mCherry-HSET for use with the AlexaFluor568-labeled seeds\u2014the biotin tag was unused and did not interfere with HSET activity.Insect cell expression constructs for StrepTagII-mCherry-Geviously . To geneeviously , HSET waRecombinant motor proteins were expressed in cultures of Sf21 insect cells (Thermo Fisher) according to the manufacturer\u2019s protocols. Cell pellets from 500 mL of culture were snap-frozen and stored at \u221280\u2009\u00b0C.SI Appendix, Fig. 7A and B).mCherry-HSET and KIF11-mGFP were purified by affinity chromatography using Strep-Tactin resin (Cytiva) as described previously . StrepTa2, 1 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 5 mM \u03b2-mercaptoethanol (\u03b2-ME), and 0.5 mM adenosine triphosphate (ATP), pH 7.5), supplemented with DNaseI (Sigma-Aldrich) and protease inhibitors . Lysate was homogenized in a glass douncer on ice, clarified by centrifugation at 50,000 rpm in a Ti70 rotor (Beckman Coulter) at 4\u2009\u00b0C for 45 min, and filtered using disposable syringe filters . Biotin from the media was removed by buffer exchange over HiPrep desalting columns (Cytiva) equilibrated in lysis buffer. The lysate was loaded onto immobilized monomeric avidin beads (Pierce/Thermo Fisher), and bound protein was washed with lysis buffer, HSET gel-filtration buffer  supplemented with 5 mM ATP, and finally, HSET gel-filtration buffer. After eluting protein with HSET gel-filtration buffer supplemented with 5 mM biotin, biotin was again removed by running the eluate over a HiPrep desalting column equilibrated in HSET gel-filtration buffer. The protein was gel-filtered using a Superose 6 10/300 GL column (Cytiva), concentrated to 1 mg/mL, clarified in a table-top ultra-centrifuge, snap-frozen in 5-\u03bcL aliquots, and stored in liquid nitrogen .Cell pellets expressing BAP-mBFP-HSET and BirA were resuspended in ice-cold lysis buffer  against a bovine serum albumin standard (Pierce/Thermo Fisher) on snap-frozen and thawed protein aliquots. Reported concentrations refer to monomers based on their predicted molecular weights.Polyethylene glycol (PEG)\u2013passivated glass was prepared as described previously ; however2, and 1 mM EGTA, pH 6.8; prepared as a 5\u00d7 stock and diluted to 1\u00d7 before use) was used to resuspend \u03b2-casein , glucose oxidase , and catalase . Resuspended proteins were centrifuged at 80,000 rpm in a TLA 100 rotor (Beckman Coulter) at 4\u2009\u00b0C for 15 min, aliquoted, snap-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C. ATP and guanosine triphospate (GTP) were dissolved in MilliQ water to 100 mM each, adjusted to pH 7 using KOH, and filtered  and aliquots were stored at \u221280\u2009\u00b0C. Docetaxel (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) to 1 mM and filtered (0.22-\u03bcm pore size), and aliquots were stored at \u221280\u2009\u00b0C. Glucose was dissolved in MilliQ water to 1 M, filtered (0.22-\u03bcm pore size), and stored at 4\u2009\u00b0C.Freshly prepared, ice-cold BRB80 buffer , generating a chamber about 5 mm wide and 7 mm long. Chambers for visualizing motion of polarity-marked seeds in self-organized networks were assembled using two layers of 10-\u03bcm-thick tape (Nitto Denko). With appropriately sized cover-glasses, multiple experiments could be performed in tandem. The assembled chambers were kept for up to one day at room temperature (RT) and placed on a 33\u2009\u00b0C heat block just before preparing the assay.2, 1 mM EGTA, 5 mM \u03b2-mercaptoethanol, and 0.1 mM ATP, pH 7.5; KIF11 gel-filtration buffer: 50 mM Na-phosphate, 300 mM KCl, 2 mM MgCl2, 10 mM \u03b2-mercaptoethanol, and 0.1 mM ATP, pH 7.5). Aliquots of catalase and glucose oxidase were mixed and diluted with BRB80 to make the oxygen scavenger mix containing 18.1 mg/mL catalase and 11.8 mg/mL glucose oxidase. The oxygen scavengers were centrifuged at 80,000 rpm in a TLA 100 rotor at 4\u2009\u00b0C for 15 min, along with thawed \u03b2-casein to get rid of aggregates. The oxygen scavengers and \u03b2-casein were stored on ice.On each day of experiments, BRB80 was freshly diluted from 5\u00d7 stock (stored for up to two weeks at 4\u2009\u00b0C) and kept on ice. HSET and KIF11 gel-filtration buffer stocks without ATP or \u03b2-ME (stored at 4\u2009\u00b0C) were supplemented with ATP or \u03b2-ME and kept on ice .Buffers and protein solutions were kept on ice, except for thawed docetaxel aliquots, which were kept at room temperature. Fresh KIF11 and HSET protein aliquots were thawed each experimental day, diluted appropriately in their respective gel-filtration buffers. Labeled and unlabeled tubulin aliquots were thawed before each experiment, mixed for a final labeling ratio of 3.5% CF640R, and diluted appropriately in BRB80. The final experimental solution contained 19.2 \u03bcL tubulin mix, 5.76 \u03bcL KIF11 dilution or KIF11 gel-filtration buffer, 5.76 \u03bcL HSET dilution or HSET gel-filtration buffer, 2.12 \u03bcL \u03b2-casein, and 2 \u03bcL oxygen scavenger mix, with 18.3 \u03bcL \u201cdocetaxel mix\u201d to bring final buffer concentrations to 35 mM PIPES, 65 mM KCl, 1.68 mM MgClThe sample was clarified by spinning at 13.3K rpm in a 4\u2009\u00b0C table-top centrifuge for 5 min. The supernatant was transferred to a new tube on ice. The prewarmed flow chamber was washed, using blotting paper to draw the solution through the chamber, by flowing 50 \u03bcL of a \u201cwash\u201d solution with the same components as the final experiment solution, but KIF11, HSET, tubulin, or oxygen scavengers were replaced by their respective storage buffers. The wash solution was replaced by flowing through 50 \u03bcL of the sample solution. The chamber was sealed with silicone vacuum grease, then transferred to an inverted spinning-disk confocal microscope with an incubator at 33\u2009\u00b0C. After locating the center of each chamber (between the tape and glass boundaries), imaging was initiated between 3 and 4 min after initial temperature shift. Exposure times for each laser line were 200 to 300 ms, with images taken at intervals of 1 min.n = 747).Stable polarity-marked microtubules, with a bright minus end and a dim plus end, were prepared using AlexaFluor568 tubulin. Polarity labeling was performed as previously described . First, n = 35) and 97% in the dim (plus) direction in HSET gliding assays (n = 36). Additionally, microtubules with a repeating bright-dim segment pattern were also moved toward the bright end by KIF11, indicating that annealed polarity-marked seeds retained directionality.Microtubule gliding assays were performed as described previously . Flow chWhen polarity-marked seeds were to be added to active microtubule networks, they were first diluted 1 in 10 in BRB80 (to 1 \u03bcM docetaxel). The experimental solution for polarity-marked seed embedded networks contained 14.2 \u03bcL tubulin mix, 5,76 \u03bcL KIF11 dilution, 5.76 \u03bcL HSET dilution or HSET gel-filtration buffer, 2.12 \u03bcL \u03b2-casein, 2 \u03bcL oxygen scavengers, and 18.3 \u03bcL docetaxel mix on ice. The seeds were further diluted 1 in 3 in RT BRB80 directly prior to mixing the final sample to avoid the addition of excess docetaxel while minimizing depolymerization. After spinning and before being added to the prewarmed, washed flow chamber, the solution was transferred into a RT tube, supplemented with 4.7 \u03bcL of the dilute polarity-marked seeds, and mixed gently before flowing into the chamber and imaging as described earlier.Drosophila melanogaster kinesin-1 construct, with a point mutation that prevents ATP hydrolysis and promotes strong microtubule binding (Kinrigor). Then 50 \u00b5L of a sample was washed over the Kinrigor surface; after washing out unbound microtubules with 50 \u00b5L of the assay buffer, the chamber was sealed with vacuum grease and imaged. Microtubules in a field of view were counted and measured by manually tracing them using Fiji (Chambers were prepared from one piece of biotin-PEG-passivated coverglass and one glass slide treated only with poly(L-lysine)-PEG (SuSoS), assembled with 80 \u00b5m double-stick tape (Teva). Microtubules were nucleated by docetaxel in a solution prepared as if for self-organization microscopy assays, without the inclusion of motor proteins. The sample tube was incubated at 33\u2009\u00b0C using a heat block. Samples were taken at the indicated intervals and diluted 100 (for 15 \u00b5M tubulin) or 500 times (for 35 \u00b5M tubulin). For each sample, a rigor-mutant kinesin surface was prepared as described  by washiing Fiji .https://gitlab.com/f-nedelec/cytosim), an open source project. Cytosim solves the Langevin equation of motion of bendable microtubules with cross-linkers in viscous medium  were chosen near the experimentally determined values or, when experimental data were missing, chosen to best match the network behavior observed experimentally in this study. Simulator and simulation configuration files are available on Zenodo, to allow reproducibility.Simulations were performed with Cytosim , corresponding to experimental time scales.SI Appendix, Fig. 1). The time-dependent growth rate is expressed as t, and Microtubules interact with each other via Hookean soft-core repulsive forces directed perpendicular to the filament axis so as to minimize sliding force parallel to the microtubule axis. Microtubules have discrete lattice binding sites covering 8 nm to which the cross-linker units can bind. Microtubules were nucleated from randomly distributed \u201cseeds\u201d at a rate such that most microtubules were nucleated at the very beginning of the simulation, similar to the experiment. Once nucleated, the plus end of the microtubule grows at a gradually decreasing speed, to mimic the limited availability of soluble tubulin pool as in the experiments  as |1\u2009+\u20090.25 N|. The resulting speed distribution is centered near To mimic the broadly distributed microtubule lengths in the experiment, we introduced variability in growth speed for each microtubule. We assigned a random speed sf is the stall force, and mv is the unloaded motor speed. Speed is thus decreased by antagonistic force as KIF11 was modeled as a symmetrical cross-linker with a pair of motor units connected by a Hookean spring with nonzero rest length. A freely diffusive motor in the medium can bind to a microtubule at a constant rate if it is within a specified binding distance. A bound motor can unbind with a force-dependent rate. Once bound, the motor walks stochastically on the one-dimensional lattice toward the plus end in a processive manner at a speed linearly dependent on the load. The velocity of a motor varies with force As shown in previous simulations , microtuHSET was modeled as an asymmetric cross-linker with one motor unit and one diffusive unit. The motor is minus end directed and nonprocessive. When bound, the diffusive subunit steps stochastically to neighboring lattice sites on the microtubule. When the motor is not bound, the force is null and the diffusible subunit undergoes unbiased diffusion. However, when the motor is bound, the force would bias the plus and minus end\u2013directed hopping rates in a thermodynamically consistent manner, as described previously , 50. UnlTo calculate the overall speed of microtubule motion in To calculate the size of bundles and asters as shown in Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File"}
+{"text": "Although older adults are at a high risk of severe or critical Covid-19, there are many cases of unvaccinated centenarians who had a silent infection or recovered from mild or moderate Covid-19. We studied three Brazilian supercentenarians, older than 110 years, who survived Covid-19 in 2020 before being vaccinated.Despite their advanced age, humoral immune response analysis showed that these individuals displayed robust levels of IgG and neutralizing antibodies (NAbs) against SARS-CoV-2. Enrichment of plasma proteins and metabolites related to innate immune response and host defense was also observed. None presented autoantibodies (auto-Abs) to type I interferon (IFN). Furthermore, these supercentenarians do not carry rare variants in genes underlying the known inborn errors of immunity, including particular inborn errors of type I IFN.These observations suggest that their Covid-19 resilience might be a combination of their genetic background and their innate and adaptive immunity.The online version contains supplementary material available at 10.1186/s12979-022-00310-y. The emergence of the Covid-19 pandemic resulted in more than six million deaths worldwide, with a higher risk for older adults and people with comorbidities to develop severe cases of the disease . Covid-1One of the leading hypotheses for the higher Covid-19 severity in older people is a decrease in the immune response that occurs with aging . The immThe chronic physiological stimulation of the immune system during life can establish the inflammaging phenomenon, characterized by a progressive and continuous increase of circulating levels of pro-inflammatory mediators , 17. ThiIn addition, recent studies reported the presence of pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs in patients with life-threatening Covid-19 pneumonia, which block the antiviral activity of correspondent type I IFNs against SARS-CoV-2 . As thesDespite that, worldwide reports of unvaccinated centenarians and supercentenarians (105 years or older) recovered from Covid-19 with mild or moderate symptoms called our attention \u201329. UndeCCR5 gene [HLA-B [Host genotype influences how an individual responds to viral infections. For example, natural resistance to HIV-1 infection has been associated with a specific mutation in the CR5 gene . In conte [HLA-B , 33. In e [HLA-B . On the Here, we present three cases of Brazilian supercentenarians who recovered from Covid-19 before the vaccination onset, including a 114 years old woman, the second oldest person in the world in this condition. Covid-19 in these three volunteers occurred in 2020 before new SARS-CoV-2 variants were reported in Brazil . Aiming to enhance our comprehension of the underlying factors contributing to their resistance to the disease, we performed a comprehensive immunogenetic assessment and whole-exome sequencing.Serological assays for SARS-CoV-2 RBD IgA, IgG, and IgM were performed through enzyme-linked immunosorbent assay (ELISA) for the Receptor-binding domain (RBD) of the Spike protein, and Nucleocapsid (NP) protein, at least four weeks after Covid-19 initial diagnosis. IgG seroconversion was detected for RBD and NP for all three individuals  \u03b1-CoV 229E, \u03b1-CoV NL63, \u03b2-CoV OC43, and \u03b2-CoV HKU1. All supercentenarians displayed IgG antibodies for all four HCoV tested, presenting higher titers for NL63 and OC43  neutralizing type I IFNs assays showed that none of the three supercentenarians had IFN-\u03b1, IFN-\u03b2, and/or INF-\u03c9 auto-Abs, based on negative results of luciferase-based immunoprecipitation (LIPS) assay. Table\u00a0For label-free quantitative proteomics analysis, we compared the 3 supercentenarian\u2019s plasma samples with 3 healthy subjects older than 95 years-old (non-infected). A total of 702 proteins were identified among all the samples analyzed. We performed a t-test to obtain the differentially expressed proteins between these two groups  and negative (183 metabolites) ionization modes. The proteomics statistical analysis was also applied to metabolomics  and interferon regulatory factor 7 (IRF7) dependent type I IFN immunity, which underlease life-threatening Covid-19 pneumonia , 40. AlsTable S1).Due to its central role in the adaptive immune response, genes from the MHC, especially those in the Human leukocyte antigen (HLA) system, are likely candidates to influence infection outcomes. Some HLA alleles were previously associated with severe Covid-19. Here we described three centenarians that recovered from Covid-19, which is a rare condition. Therefore, the small sample size does not allow an in-depth analysis of associated polymorphism. Nevertheless, since these data might be important to understand the mechanisms underlying HLA associations, and other groups evaluating centenarians might be interested in this data, we report the HLA alleles observed for all these centenarians as supplementary data  than in younger individuals living in the same environment .Neutralization analysis revealed that the asymptomatic individual produced lower titers than the two symptomatic ones. At first glance, this seems contradictory, but only a few asymptomatic individuals produce detectable neutralization titers  even witWe also assessed pre-existing antibodies to RBD of the four common seasonal HCoVs \u03b1-CoV 229E, \u03b1-CoV NL63, \u03b2-CoV OC43, and \u03b2-CoV HKU1. All supercentenarians displayed IgG antibodies for all four HCoV tested, presenting higher titers for NL63 and OC43. Interestingly, normalized values were generally lower for SARS-CoV-2 compared to HCoV. IgG for SARS-COV-2 RBD did not correlate with neutralization titers among them. The one who presented the highest titer of RBD was not the one with the highest virus neutralization titer.The literature on the influence of pre-existing humoral immunity to HCoVs in SARS-CoV-2 infection is still controversial. Some studies have pointed out that immunity to the HCoVs has a protective effect on Covid-19 . In contIt is very likely that supercentenarians have been exposed to various pathogens in their life even when children and acquired active immunity making them more prone to defeat SARS-CoV-2. In this sense, it is tempting to speculate that the 1918 H1N1 influenza virus immunity could confer some protection against SARS-CoV-2 infection . The hypBesides, a specific and unique subset of CD4 T cells has cytotoxic features in supercentenarians. These cells were accumulated during life exposition to pathogens. They might be considered an adaptation to aging since the immune system needs extra support to eliminate abnormal and infected cells. Such observation corroborates the hypothesis that the original antigenic sin phenomenon  could plIt has been reported that circulating auto-Abs neutralizing type I IFNs (IFN-\u03b1 and/or -\u03c9) were distinctly found in elderly patients with severe Covid-19 and rarely detected in asymptomatic, benign infectious, or healthy individuals. They account for about 20% of critical Covid-19 cases in people over the 80s and total fatal Covid-19 cases , 24. IntRegarding the plasma proteomic and metabolomic analyses, we were able to compare these 3 supercentenarians with a control group of 3 individuals with comparable age, albeit no older than 110-years, whose serology was negative for Covid-19. Proteomics analysis showed that glycolytic proteins were more abundant in the supercentenarians compared to the control group, which is associated with the infection phisiopatology \u201357. KrisOn the other hand, the metabolomics approach showed some up-regulated metabolites in the supercentenarians\u2019 plasma related to fatty acid metabolism, especially the biosynthesis of unsaturated fatty acids. These molecules have a central role in modulating the immune pathways and inflammatory responses . Some stThe genetic analysis indicated that all these three supercentenarians do not present variants associated with inborn errors of type I IFN immunity (IEI) genes, which is not surprising considering their advanced age without associated diagnosis. Because of the small sample size, conducting an in-depth analysis of polymorphisms associated with their Covid-19 resistance phenotype is not feasible. Nevertheless, their DNA WES data is available to the community for joint efforts to detect variants related to Covid-19 resistance.Furthermore, the HLA alleles that each of these centenarians carry were described, being likely candidates to influence infection outcomes \u201370 and lIn the present study, we investigated three Covid-19 recovered supercentenarians (older than 110-years-old) who displayed robust IgG levels and neutralization titers against SARS-CoV-2. An enrichment of plasma proteins and metabolites related to innate immune response and host defense was observed. Despite their advanced age, none of them had neutralized auto-Abs against the type I IFNs. Also, they do not carry variants associated with inborn errors of type I IFN immunity (IEI) genes. They belong to a selected group of individuals with a long lifetime of pathogens\u2019 expositions, immunity training, and genetic factors that lead them to develop mild symptoms not only against Covid-19 but also for several other diseases. Understanding the underlying mechanisms may be important to protect us from future pandemics.Three Brazilian unvaccinated supercentenarians who recovered from Covid-19 were contacted by our Human Genome and Stem Cell Research Center (HUG-CELL) research group following their report in national media: a 114-years-old woman (ID 01) and two men aged 111 (ID 02) and 110 ID 03) years-old, respectively, at blood collection time. To our knowledge, these were the longest-lived people who recovered from Covid-19 in South America before the vaccination started and new SARS-CoV-2 variants emerged. RT-PCR tests confirmed the previous diagnosis of Covid-19, and all relevant clinical data related to the disease episode and comorbidities were collected from clinical reports and interviews. Baseline characteristics of the three supercentenarians are shown in Table\u00a0 years-olWe collected peripheral blood samples of the volunteers from 30 to 120 days after the reported viral infections. For DNA extraction, samples were taken in vacutainer tubes with ethylenediaminetetraacetic acid - EDTA . Plasma and Serum were obtained by centrifugation for 10\u00a0min at 2000 x g at room temperature within 30\u00a0min after venipuncture. Then, the supernatant was transferred in aliquots of 1.5 mL into cryovials . Samples were transferred to a -80\u00a0\u00b0C freezer until the moment of use.oC with 4\u00a0\u00b5g/mL of SARS-CoV-2 RBD, 0.8\u00a0\u00b5g/mL of the RBD of HCoVs, and 2\u00a0\u00b5g/mL NP . Volunteers\u2019 plasma samples were incubated at 56\u00a0\u00b0C for 30\u00a0min, diluted at 1:100, and run-in triplicates in ELISA. Results were given as the ratio of participant sample/average of a set of 20 control pre-pandemic samples. An antibody ratio of \u2265\u20091.2 was considered positive.The humoral immune response was analyzed by ELISA for IgA, IgM, and IgG-binding antibodies against the receptor-binding domain of Spike protein and NP protein of SARS-CoV-2. Besides, RBDs from human seasonal coronaviruses (HCoV) HKU-1, OC43, NL63, and 229E were also tested. RBDs from HCoVs were expressed in HEK293T cells, which plasmids are described in . ELISA wThe detection of auto-Abs neutralizing type I IFNS  in plasma samples of the supercentenarians was assessed by LIPS assay, as described in . BrieflyNeutralization titers were measured in a pseudovirus assay adapted from , only chPlasma proteomics and metabolomics analyses from the supercentenarians were performed using tandem mass spectrometry. Detailed protocols concerning both analyses and data processing are available in Supplementary Methods. Three individuals older than 95 years-old who were not infected by SARS-CoV-2 and displayed a negative COVID-19 serology were included as the control group.Whole-exome sequencing (WES) was performed in peripheral blood DNA with the Illumina NovaSeq platform at HUG-CELL facilities. Sequencing data were analyzed following bwa-mem and GATK Best Practices workflow, quality control, and annotation were performed as previously described . HLA genThe inference of genetic ancestry was performed in ADMIXTURE v1.36 , in supeBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2"}
+{"text": "Conclusions: The smaller small-sided game tested promotes greater within-player variability in locomotor demands while promoting smaller within-player variability heart rate responses. Possibly, 5v5 is more recommended to stabilize the locomotor demands, while the 3v3 is recommended to stabilize the heart rate stimulus.Background: Small-sided games (SSGs) are drill-based and constrained exercises designed to promote a technical/tactical and physiological/physical stimulus on players while preserving some dynamics of the real game. However, as a dynamic game, they can offer some variability making the prediction of the stimulus hardest for the coach. Aim: The purpose of this study was to analyze the between-session and within-player variability of heart rates and locomotor responses of young male soccer players in 3v3 and 5v5 small-sided game formats. Methods: This study followed a repeated-measures study design. Twenty soccer players were enrolled in a study design in which the SSG formats 3v3 and 5v5 were performed consecutively across four days. Twenty under-17 male youth soccer players (16.8 \u00b1 0.4 years old) voluntarily participated in this study. Participants were monitored using a Polar Team Pro for measuring the heart rate mean and maximum, distances covered at different speed thresholds, and peak speed. Results: Between-players variability revealed that maximum heart rate was the outcome with a smaller coefficient of variation , while the distance covered at Z5 . The repeated measures ANOVA revealed that the four games tested were different in the within-player variability considering the maximum heart rate ( Small-sided games (SSGs) are a widespread form of soccer practice given their capability to simulate both physical and technical\u2013tactical demands at least in part similar to those required in official matches ,2,3. In Two common manners to check the consistency/inconsistency of SSG responses consist in determining their within- and between-session variability, and looking for capturing the possible day and seasonal oscillations through repeated measures, respectively. Importantly, the SSG variability data obtained in senior standards cannot be extrapolated to teenagers owing to the discrepancies previously detected ,11. ThisResearch conducted on soccer players  indicateIt is recognized that combining data collection and analysis of player effort, skill outputs, and psychological aspects  is important in promoting a holistic approach to understanding SSGs ,17,18,19This study followed a repeated-measures study design. Twenty soccer players were enrolled in a study design in which the SSGs formats 3v3 and 5v5 were performed consecutively across four days . The stuParticipants were enrolled based on convenience sampling. The sample size was calculated a priori in G*power (version 3.1.9.6.). For a power of 0.80 and an effect size of 0.5 (medium), the recommended total sample was 21. From the initial recruitment of twenty-four players, twenty male youth soccer players  belonging to the same team competing in the regional/national level voluntarily participated in this study. Four players from the initial twenty-four were excluded due to them being goalkeepers (n = 3) and missed one assessment (n = 1). The players were typically involved in five training sessions per week, plus one official match. Each training session lasted about 90 min. The eligibility criteria for being enrolled in the study were: (i) an outfield player; (ii) not injured in the last month before the experimental data collection and not present any signals or reports of injury or illness on the days of data collection; (iii) did not miss any of the data collection moments or repetitions performed; and (iv) did not take any drugs or energy drinks or changed any of their daily dietary routines.The participants were preliminarily analyzed for their final locomotor profile. In the week before starting the observation, they were tested for the maximal sprint speed in a 30-m sprint test and for their ability to perform repeated efforts in the 30\u201315 Intermittent Fitness test. The data collection was preceded by a 24-h rest and occurred in the afternoon (5 pm). The players performed a standardized warm-up protocol (FIFA 11+). After that, they rested for 3 min and started the 30-m sprint speed assessment. They started with a split position (using the preferred leg in front), and the peak speed was assessed using a Polar Team Pro GPS  which was confirmed for the validity and reliability to estimate their peak speed in the previous study. After performing two trials of the 30-m sprint (interspaced by 5 min rest), they recovered for 3 min and followed the 30\u201315 Intermittent Fitness tests. The original protocol was implemented, and the final velocity completed was registered as the main outcome. For the case of maximal sprint speed, the best speed in the two trials was considered the main outcome.The formats 3v3 and 5v5 without a goalkeeper were implemented based on the characteristics presented in The heart rate and locomotor demands were monitored using the Polar Team Pro  which is confirmed for its reliability to measure the demands analyzed ,22. The p < 0.05.The descriptive statistics were presented in form of mean and standard deviation. The coefficient of variation (expressed as a percentage) was calculated to analyze within-player variability (variability of the player across different days for the same condition) and between-player variability (heterogeneity of the responses for a specific game and condition). The normality of the sample was not verified in all the measures; however, since the data were greater than 30, we have assumed normality by the Central Limit Theorem. A repeated measures ANOVA was performed to compare the within-players variability obtained in each game (format and pitch dimension). Moreover, for each format of play and pitch dimension, a repeated measures ANOVA was tested . Partial eta squared was used to calculate the effect size. Bonferroni was used as a post hoc test in the pairwise comparisons. The statistical procedures were executed in the SPSS  for a The physical fitness assessment revealed that players presented a maximal speed sprint of 28.7 \u00b1 2.8 km/h and a final velocity in the 30\u201315 Intermittent Fitness test of 15.2 \u00b1 1.3 km/h. Descriptive statistics of between-training sessions variability can be found in Similarly, the maximum heart rate presented a smaller coefficient of variation in 5v5 played at 50 \u00d7 31 m (6.6% to 16.9%) and 40 \u00d7 25 m (7.0% to 15.2%) and a greater coefficient of variation in distance covered at Z5 in 5v5 played at 50 \u00d7 31 m (103.7% to 221.1%) and 40 \u00d7 25 m (94.0% to 156.8%).p = 0.032; p < 0.001; p < 0.001; p < 0.001; p < 0.001; p = 0.001; p = 0.023; p = 0.005; p = 0.001; p = 0.007; p = 0.006; p = 0.002; p < 0.001; p = 0.003; p = 0.007; p = 0.001; p = 0.009; p = 0.037; The between-session analysis for each game was performed using a repeated measures ANOVA. In the case of format 3v3 played at 39 \u00d7 24 m , signifi2 \u00d7 19 m , signifip < 0.001; p < 0.001; p = 0.008; p < 0.001; p = 0.012; The between-session analysis for each game was performed using a repeated measures ANOVA. In the case of format 5v5 played at 50 \u00d7 31 m , no signThe current research reveals that locomotor demands present greater between-session and within-player variability than heart rate responses. Additionally, it was found that 3v3 small-sided game formats promote smaller within-player variability in heart rate responses, yet promote greater locomotor demand variability than the 5v5 format. One of the concerns in training prescription is projecting the dose for the players. The act of designing small-sided games is complex since many task constraints can be used in combination and some of them can contribute to changing behaviors that are naturally associated with variation in players\u2019 responses . In our On the other hand, locomotor demands presented great levels of within-players variability. While the total distance covered presented coefficients of variation between 18% and 35% in the games tested, the distances covered by different speed thresholds revealed a progressive increase of within-player variability. The distance covered at running intensity Z5 was about 113% and 171% in the games tested. These values and tendencies are aligned with previous studies ,27 whichA descriptive observation allowed also us to identify the heterogeneity level in which the game (between players) was smaller in heart rate responses and greater in locomotor demands. Using small-sided games as the format of exercise for prescribing physical stimulus is challenging since the game is dynamic. The heterogeneity between players is expectable based on the dynamic of the game as well. Between-player variability varied from 3.2% to 11.1% in the worst case for the heart rate responses in the 3v3 games performed, while it varied from 7.0% to 19.7% in the 5v5 format. This means that 5v5 may induce a greater inter-player variability. This can be caused by the greater number of players participating in the game and by the fact that tactical positions assumed by the players may interact to explain these varieties in the physiological stimulus. However, greater variabilities occur in locomotor demands as in the case of distance covered at Z5 in 3v3 in which variabilities between players achieved 245% of the coefficient of variation, while in the 5v5, dropped to a maximum of 221.1%. Coaches must be attentive to this between-player variability since locomotor demands are commonly considered for the identification of neuromuscular stimuli. Considering that Z5 corresponds to the sprint zone, and keeping in mind that sprint is one of the most important neuromuscular stimuli for players, some players can suffer in small-sided games by under or over-stimulation.This study presents some limitations. The small sample included is one of the limitations that should be considered as a potential bias. Another limitation is convenience sampling. All the players were part of the same team, which should be faced as a bias for generalization of the evidence. Thus, any conclusion should not be observed as an irrefutable finding and requires more observations to be confirmed. Moreover, this study tested repeated measures on consecutive days which, in fact, can interfere with the way players respond day-by-day. However, this is also an issue for the practice in which specific formats are applied in different contextual scenarios. Another limitation is that some thresholds  are associated with physical fitness, which makes it harder to detect stable values. For example, speed thresholds are player-dependent which means that some thresholds (exempla as sprint) may not start at the same velocity for all. Future research should consider increasing the number of weeks observed . Moreover, increases in the number of players will favor the generalization of the findings. Finally, other measures that can better describe the anaerobic contribution as blood lactate concentrations must be considered in future research to identify the influence of the most intense SSGs.The current research may provide some practical applications. In this case, the 3v3 format must be used by coaches for an aerobic power stimulus, since it seems to be more stable in terms of stress on heart rate. On the other hand, in the case of aiming to focus on locomotor demands, possibly adjusting the format for medium-sided games (as in 5v5) can be better, since the values are less variable across the sessions. The current research suggests that within- and between-player variability occurs in small-sided games; however, they are associated with the formats and task constraints implemented. The results indicate that heart rate responses are less variable in 3v3 formats, while locomotor demands are less variable in 5v5 formats. Additionally, it is observed that between-player variability is smaller in heart rate responses than in locomotor demands. The results come from a small sample and convenience sampling. Thus, these results are context-dependent which should be faced as a limitation for the generalization of the findings. Future studies should be conducted to confirm or refute the evidence. The next studies must include more players to increase the sample size. Additionally, different teams and populations must be included for a more robust analysis. Finally, consideration for moderators and mediators such as training status, skill level, and moment of the season must be also included."}
+{"text": "The effect of prolonged, high-intensity endurance exercise on myocardial function is unclear. This study aimed to determine the left ventricular (LV) response to increased exercise duration and intensity using novel echocardiographic tools to assess myocardial work and fatigue.LV function was assessed by echocardiography before, immediately, and 24 h after a cardiopulmonary exercise test (CPET) and a 91-km mountain bike leisure race. Cardiac Troponin I (cTnI) was used to assess myocyte stress.p < 0.0001): Race/CPET: exercise duration: 230 /43  minutes, mean heart rate: 154 \u00b1 10/132 \u00b1 12 bpm, max cTnI: 77 /12  ng/L. Stroke volume and cardiac output were higher after the race than CPET (p < 0.005). The two exercises did not differ in post-exercise changes in LV ejection fraction (LVEF) or global longitudinal strain (GLS). There was an increase in global wasted work (p = 0.001) following the race and a persistent reduction in global constructive work 24 h after exercise (p = 0.003).59 healthy recreational athletes, 52 (43\u201359) years of age, 73% males, were included. The race was longer and of higher intensity generating higher cTnI levels compared with the CPET (Increased exercise intensity and duration were associated with increased myocardial wasted work post-exercise, without alterations in LVEF and GLS from baseline values. These findings suggest that markers of myocardial inefficiency may precede reduction in global LV function as markers of myocardial fatigue. Increased physical activity increases health benefits \u20134. HowevNumerous studies have reported on post-exercise LV function assessed by echocardiography . A largeA novel echocardiographic method has been developed to assess non-invasive parameters of myocardial work (MW) \u201316. MW uThis study aimed to determine the LV response to increased exercise workload by comparing two exercises of different exercise intensity and duration, using both traditional and MW echocardiographic parameters before and after exercise.Study participants were recruited among healthy, prior participants in the NEEDED 2013 and 2014 studies , 20. ExcThe present study was conducted in 2018. Echocardiography was acquired before exercise, immediately after, and at 24 h following two episodes of high-intensity endurance exercise in 2018 . The firGE Vivid E 95 ultrasound systems and 4V probes  were used for all echocardiographic assessments. Three medical doctors were responsible for acquiring images in relation to the CPET, and six medical doctors worked on parallel stations in relation to the cycle race to acquire images immediately after finishing the race. Comprehensive imaging protocols were applied. Appropriate frame rates were applied, to allow later high-quality post-processing, including speckle-tracking strain analysis.All echocardiographic analyses were performed offline on EchoPAC V202  by a researcher blinded to clinical data and exercise information. All echocardiographic parameters were calculated according to the European Association of Cardiovascular Imaging , 23. TheMyocardial work (MW) calculation was performed offline using EchoPAC software version 202 . A commercially available algorithm was used to calculate four MW parameters (see below). The LV pressure curve was estimated using an empiric reference pressure curve that was adjusted according to the duration of the isovolumic and ejection phases. SBP was measured with a brachial cuff with study subjects sitting. The opening and closure of the aortic and mitral valves were determined manually in the three- and four-chamber apical views based on Doppler signals and visualization of valve opening and closure.Pressure strain was generated in each myocardial segment by the EchoPAC software, and global values were calculated as mean values of all segments. The global work index (GWI) parameter is the total work performed by the left ventricle, using the area of the pressure-strain loop between the mitral valve closure and opening. Global constructive work (GCW) is the sum of positive work due to myocardial shortening in systole and negative work due to myocardial lengthening during isovolumetric relaxation. Global wasted work (GWW) is the sum of myocardial lengthening in systole and shortening in isovolumetric relaxation, reflecting the work that does not contribute to LV ejection. Global work efficiency (GWE) is constructive work divided by the sum of constructive work and wasted work.The High sensitivity cTnI assay (STAT) from Abbott Diagnostics was used for the measurement of cTnI, before exercise and at 3 and 24 h following exercise. The assay was analyzed on an Architect SR2000i . Overall 99th percentile is 26 ng/L (men: 34 ng/L and women: 16 ng/L) .U test or a Student T-test was used to compare groups, as appropriate. Spearman analysis was used to calculate bivariate correlations. A two-tailed p-value < 0.05 was considered significant. SPSS version 26.0.0.1 was used for statistical analyses.Normally distributed continuous variables are reported as mean \u00b1 SD, while continuous variables with markedly skewed distributions are reported as the median and interquartile range . The Shapiro\u2013Wilk test was used to test for normality. For continuous variables, The Mann\u2013Whitney A total of 59 healthy, well-trained, recreational athletes, 52  years of age, 73% males, without obstructive coronary artery disease (> 50% stenosis) verified by CT scans, were included in the study . The stu2 and a mildly elevated median LV end-diastolic volume index of 82  mL/m2 compared to reference values (Before CPET (baseline), the mean SBP (systolic blood pressure) was 136 \u00b1 16 mmHg, and the heart rate (HR) was 59 \u00b1 10 bpm. Echocardiographic recordings at baseline showed a mean LV mass index of 87.5 \u00b1 14.5 g/me values . All othBoth CPET and the race exercises were of high physical intensity . There w\u2035, E/E\u2035, LV GLS, GWI, and GCW  decreased, but HR increased, and so did cardiac output (CO) and mechanical dispersion . Most ec and GCW . There wp 0.001) compared with the pre-exercise value and global work efficiency (GWE) was lower (p 0.006), this was not observed following the CPET. Twenty-four hours after the race GWW and GWE returned to baseline values.Global wasted work (GWW) was significantly higher after the race . Post-race HR was only higher than CPET immediately after the race returning to baseline values, equal to the CPET values, at 24 h following the race (p < 0.05), with a similar reduction after both exercises. LVEF did not change after the CPET nor after the race.The race was of higher workload and longer duration than the CPET, resulting in higher post-exercise cTnI values . Immediathe race . At all the race , 4. Therp = 0.05 and p = 0.03). There was also a positive correlation between LV GLS immediately after the race and cTnI 3 h after the race  and 24 h after the Race . There were no significant correlations between cTnI, LVEF, diastolic parameters, and MW parameters.There were no significant correlations between echocardiographic parameters and cTnI after the CPET. There was a weak positive correlation between cTnI and end systolic- and diastolic volumes 24 h after the race  higher pre-exercise SBP than the CPET, but no difference in heart rate. A large portion of the echocardiographic findings before the race differed from pre-CPET findings (p = 0.005), LV SV (p < 0.001), E\u2035(p < 0.001), GWI (p < 0.001), GCW (p = 0.006), GWE (p = 0.024), and post systolic shortening index (p = 0.015). The following values were lower: ESV (p = 0.018), E/E\u2035 (p = 0.048), and post-systolic shortening (p = 0.009).Comparing the pre-exercise assessment before the CPET and the race revealed several significant differences , 4. Befofindings , 4. The p = 0.025), and there was a significant persisting reduction in GCW 24 h following the race (p = 0.015). The difference in many pre-exercise parameters before the CPET and the race indicates the presence of significant confounders complicating a direct comparison between the race and the CPET. In order to reduce the impact of baseline confounders, the difference in post-exercise parameters between the race and the CPET was assessed by the percentual change from each pre-exercise value . When usIntra- and inter-observer variability analysis confirmed high reproducibility of the parameters LV GLS, four-chamber strain, three-chamber strain, and two-chamber strain analyses, and the myocardial work parameters GWI, GCW, GWW, and GWE .This is the first study to provide evidence suggesting myocardial inefficiency as an early indicator of exercise-induced myocardial fatigue. The study is based upon multiple echocardiographic assessments before and after exercise comparing two different workloads, in well-trained middle-aged athletes, without obstructive coronary artery disease verified by CT scans. The study found that increased exercise workload was associated with increased post-exercise wasted myocardial work without alterations in LVEF and GLS adjusting for baseline values. These findings suggest that markers of myocardial inefficiency may precede reduction in global LV function as markers of myocardial fatigue.Exercise-induced cardiac fatigue is a reversible myocardial dysfunction caused by strenuous exercise , 25. A lMyocardial work by echocardiography combines strain analysis with SBP providing a less load-dependent and more comprehensive assessment of LV systolic function . MyocardMuscular inefficiency occurs in skeletal muscle when workloads exceed maximal oxygen consumption intensity . The proOur echocardiographic findings suggest that there may be a similar relation between workload and myocardial inefficiency, as previously described in skeletal muscle. In our study, the increased workload and the duration of work during the race  were assOur study also found increases in other parameters of myocardial inefficiencies, such as post-systolic shortening (PSS) and early systolic lengthening (ESL) immediately after both exercises. Post-systolic shortening relates directly to LV systolic function . Myocard\u2035), similar to the findings in the present study. At the end of systole and during isovolumic relaxation, myocytes are still active and exert the force to oppose the ventricular pressure and residual elastic forces due to the twist, which is the highest at the apex. One may speculate that the faster the untwisting, the more negative intraventricular pressure is generated, thus unloading the myocytes, which can eventually result in the myocyte shortening during isovolumic relaxation and increased global wasted work (GWW). However, the exact mechanisms and consequences for the observed reduced cardiac inefficiency are unknown, and further work regarding the mechanisms underpinning the reduced efficiency following intense exercise is required.Left ventricular (LV) rotation and twist are important factors of LV systolic and diastolic performance. During diastole, the untwisting results in the abrupt release of the energy stored in elastic components, resulting in a negative intraventricular pressure gradient that facilitates LV filling at low filling pressure , 41. ParIn line with previous exercise studies, there were significant post-exercise reductions in 3D-derived LV end-diastolic, left atrial volumes, and alterations in diastolic function , 8, 43. The present study used two different workloads to assess the post-exercise response. The CPET was used to define the reference LV response to standardized exercise intensity, ensuring that all study individuals were exposed to an exercise of similar duration with the same metabolic demands (lactate threshold and max V02). Furthermore, the CPET allowed interpretation of the implications of the work performed during the race using power meters. The power meters indicated a substantial increase in the exercise above max VO2 level during the race, increasing the likelihood of developing myocardial inefficiency.As indicated by the significant increase in blood pressure the day before the race, the race and the preparation for the next day may have induced a stress response in the race contenders. To adjust for this increased stress, echocardiographic parameters were corrected by using the pre-exercise measurements as a baseline, and the percentual change from each pre-exercise value assessed the difference in post-exercise parameters.The present study demonstrates pre-exercise stress\u2019s impact on assessing resting echocardiographic parameters. SBP was higher before the race compared with the CPET. This increase in SBP affects the MW parameters, causing increased GWI and GCW compared with the assessment before the CPET. The increased blood pressure is most likely due to the mental stress caused by the competitive event, resulting in increased myocardial work before the race. The present study\u2019s findings underscore the impact of emotional stress on myocardial work and the need to consider the influence of emotional stress when baseline MW parameters are interpreted.The study provides insights into the longitudinal cardiac morphology changes and measures after prolonged exercise in middle-aged recreational athletes. The present study addresses morphological and functional changes in a population different from younger athletes, and the current findings may not apply to a younger population with a higher functional capacity. When calculating MW, we used SBP as a substitute for force; therefore, the use of pressure and strain does not provide a direct measure of work . Calculation of work is underestimated in dilated ventricles because of higher wall stress at any given LV pressure, and this could also be the case in our study, where study subjects had mildly dilated ventricles. Calculating the myocardial work parameters relies on global longitudinal strain measurements, systolic blood pressure, and valvular events. Small changes in the timing of valvular events could potentially lead to significant differences in the work parameters, especially the global wasted work and global work efficiency parameters. When assessing and evaluating these parameters, it is essential to be aware of these challenges.Changes in left ventricular function, consistent with exercise-induced fatigue, were seen after both exercises with the varying workload. However, increased endurance exercise duration and workload, as performed at the race, were associated with decreased myocardial efficiency following exercise. When correcting for alterations in baseline parameters, there was no difference in LVEF and GLS comparing the two exercise workloads and durations. These findings suggest that myocardial work parameters may be more sensitive measures of myocardial inefficiency than traditional markers of systolic myocardial dysfunction.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 the Regional Ethics Committee (REK nr 2013/550). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CE and S\u00d8 drafted the manuscript. CE, \u00d8K, MB-B, VF, MC, LK, LD, TM, \u00d8S, and S\u00d8 contributed to the data collection. CE and BA performed the statistical analyses. All authors contributed to drafting and critically revising the manuscript and approved the submitted version."}
+{"text": "Cu2O NPs exhibited excellent peroxidase-like activity and served as a matrix for LDI MS analysis, achieving visual and accurate quantitative analysis of glucose in serum. Our proposed method possesses promising application values in clinical disease diagnostics and monitoring.Glucose is a source of energy for daily activities of the human body and is regarded as a clinical biomarker, due to the abnormal glucose level in the blood leading to many endocrine metabolic diseases. Thus, it is indispensable to develop simple, accurate, and sensitive methods for glucose detection. However, the current methods mainly depend on natural enzymes, which are unstable, hard to prepare, and expensive, limiting the extensive applications in clinics. Herein, we propose a dual-mode Cu Glucose plays important role in the human body, providing energy for metabolism and normal operation of various organs . As is r2O2) and gluconic acid with the help of glucose oxidase (GOD). The generated H2O2 catalyzes the substrates to colored products in the presence of peroxidase, which could be analyzed by electrochemical sensors or optical sensors  has been widely applied in biomolecules analysis . Compare2O nanoparticles (NPs) were facilely prepared and testified the peroxidase-like activity, achieving visual detection of glucose combined with GOD. Meanwhile, Cu2O NPs were utilized as a matrix for LDI MS analysis of small molecules (e.g. glucose) with good salt tolerance. By adding internal standard, glucose level could be quantitative analysis by Cu2O NPs assisted LDI MS. Notably, the dual-mode Cu2O NPs based biosensor was applied to detect glucose from serum and a consistent result was obtained, demonstrating that the method could be reliable in glucose analysis for clinical diseases diagnostics and monitoring.Herein, we propose a copper-based biosensor for dual-mode glucose analysis based on colorimetric assay and LDI MS . Cu2O na4 5H2O (98%), sodium hydroxide , D-(+)-glucose , ethylene glycol (99%), D-(+)-cellobiose , L-arginine , d-phenylalanine (98%), anhydrous ethanol , sodium acetate (98.5%), choline , ascorbic acid , d-galactose , and dopamine  were acquired from Inno-chem Co., Ltd. . Polyvinylpyrrolidone , L-aspartic acid , D-(-)-fructose , L-alanine , 3,3\u2032,5,5\u2032-tetramethylbenzidine , and acetic acid (99%) were ordered from Sigma, United States. Dimethylsulfoxide  was bought from Aladdin Reagent Co., Ltd. . Phosphate-buffered saline  and Sodium acetate (98.5%) were ordered from Tokyo Chemical Industry Co., Ltd. . Glucose oxidase (GOD) was acquired from Shanghai Macklin Biochemical Co., Ltd. . All chemicals were used without any further purification unless otherwise stated. Deionized (DI) water (18.2\u00a0M\u03a9\u00a0cm) was prepared by a Milli-Q water purification system .CuSO\u03bb = 0.154\u00a0nm). The materials absorption spectra were obtained on an Ultraviolet-visible (UV-Vis)  spectrophotometer and MD SpectraMax i3x using water suspension at room temperature . The LDI MS analysis was performed on Matrix-Assisted Laser Desorption/ionization Time of Flight Mass Spectrometry  with the Nd: YAG laser  and smart beam system. The acquisitions were conducted in positive reflector ion mode with the repetition rate of 1\u00a0kHz and an acceleration voltage of delayed extraction set as 20\u00a0kV. Optimized delay time for this experiment to 250\u00a0ns and laser shots of 2000 per detection was applied throughout LDI MS analysis. For the calibration of each spot, dropped mixed small molecules for the accurate measurement of mass spectrometry (<20\u00a0ppm). Each sample detection performed five independent experiments and all spectra results were used for analysis directly without any smoothing procedures.Transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), high-angle annular dark-filed (HAADF), and elemental mapping images were collected using a JEOL JEM-2100F instrument by depositing materials on a copper grid with a mesh size of 200. Scanning electron microscopy (SEM) images were recorded on Hitachi S-4800 by dropping the ethanolic material suspensions on aluminum foil. The crystal structure of the wide-angle powder pattern was analyzed through X-ray diffraction  with Cu K\u03b1 radiation  were added and stirred at RT for 10\u00a0min. After that, 25\u00a0ml of glucose (1.3\u00a0M) were added with slowly stirring for 15\u00a0min and the obtained mixture was heated at 80\u00b0C for 1\u00a0h in a water bath. After the resulting solution cooled to RT naturally, the orange sediment was collected and washed several times thoroughly with DI water and EtOH by centrifugation. Finally, the Cu2O NPs were obtained by drying for 5\u00a0h in a vacuum oven at 55\u00b0C.The Cun method . Briefly2O NPs have the property of peroxidase, which was verified by the oxidation of TMB in the presence of H2O2. In a typical procedure, 100\u00a0\u00b5l of TMB , 100\u00a0\u00b5l of H2O2 (5\u00a0mM) and 5\u00a0\u00b5l of Cu2O NPs (7\u00a0mM) were added in 400\u00a0\u00b5l of HAc-NaAc buffer (pH 3.0). After incubation at 37\u00b0C for 5\u00a0min, the color of the solution turned blue and its absorption at 652\u00a0nm was measured by UV-vis spectrophotometer.The Cu2O NPs (7\u00a0mM), 100\u00a0\u00b5l of TMB (20.8\u00a0mM), and 400\u00a0\u00b5l of acetate buffer (pH 3.0) were added to the above solution and incubated for 10\u00a0min for absorption measurement. For serum glucose detection, instead of glucose solution, a 20\u00a0\u00b5l serum sample was performed with the same experimental steps as mentioned above and the absorption values reflected the glucose concentration in serum samples due to the linear curve.A series of concentrations of glucose (20\u00a0\u00b5l) and GOD  were mixed in PBS and incubated at 37\u00b0C for 60\u00a0min, respectively. Then, 5\u00a0\u00b5l of Cu2O NPs were dropped on the plate and analyzed by LDI-MS after drying. Cu2O NPs were dispersed in DI water at the concentration of 2.9\u00a0mM for use as a matrix. For LDI-MS analysis of glucose in serum, typically, the liquid of serum sample was mixed with equal volume cellobiose solution (2.9\u00a0mM) and spotted on the plate (1.5\u00a0\u00b5l) until dried in air at 25\u00b0C, followed by adding 1.5\u00a0\u00b5l of matrix and also dried in the same condition for LDI MS analysis.One milliliter of glucose with concentrations ranging from 0.56 to 5.6\u00a0mM was mixed with an equal volume of cellobiose solution (2.9\u00a0mM), respectively. Then 500\u00a0nL of mixture solution and 500\u00a0nL of Cu2O NPs was characterized by SEM and TEM. As shown in 2O NPs exhibited a uniform spherical morphology with an average diameter of about 200\u00a0nm. Meanwhile, TEM images in 2O NPs . As shown in 2O as (111), (200), (220), and 311) (JCPDS card No. 5-0667), respectively  was selected  by OH radicals produced from H2O2 and the solution will turn to blue . Then, the in situ generated H2O2 was subsequently utilized to oxidize substrate TMB to chromogenic ox TMB with the Cu2O NPs in acetate buffer (pH 3.0). And the discoloration degree was recorded by microplate reader at the absorbance of 652\u00a0nm, which could reflect the concentrations of glucose. Using this method, a series of concentrations of glucose were detected and the results were displayed in R2 = 0.999) between glucose concentration and the absorbance intensity of 652\u00a0nm was obtained (2O NPs-TMB system exhibited a broad linear range (0.28\u20132.8\u00a0mM) and the limit of detection (LOD) of the assay was 1.37\u00a0\u00b5M (S/N = 3), which is comparable or even superior to many recently reported nanocomposites in the literature  to verify the selectivity of the assay for glucose detection . As show2O NPs could not only be used as peroxidase mimic but also be employed as the matrix for LDI MS analysis. To testify the LDI MS performance based on Cu2O NPs, the typical small molecules were analyzed containing alanine, glucose, cellobiose . From the mass spectra in +, [Glu + Na]+ and [Cel + Na]+, respectively. Notably, the background signal from Cu2O NPs could not affect the detection of the above molecules.LDI MS presents high sensitivity, accuracy, resolution, and throughput in molecular analysis, especially for metabolites at the low molecular weight . However2O NPs based LDI MS for analysis of the mixing liquid in water and salt solution. As shown in +, [Asp + Na]+, [DA + Na]+, [Vc + Na]+ and [Glu + Na]+, respectively, even in the interference of salt. Such results demonstrated the capability of multiple metabolites analysis and the good salts tolerance using Cu2O NP assisted LDI MS, which is hopefully applied in practical applications without pretreatment . Firstly, different concentrations of glucose ranging from 0.56 to 5.6\u00a0mM were mixed with cellobiose (2.9\u00a0mM) and analyzed by Cu2O NP based LDI MS. The results in I203/I365) was increased as the concentration of glucose increased and a good linear relationship (R2 = 0.997) between the I203/I365 and the concentration of glucose from 0 to 3.36\u00a0mM was obtained in Encouraged by the good performance and salt tolerability of Cu2O NPs shown in Considering that glucose level in serum is associated with various diseases in the clinic, quantitative evaluation of glucose from serum is meaningful. To demonstrate the feasibility of dual-mode quantitative of glucose from serum, 31 serum samples were prepared and analyzed by both colorimetric and LDI MS assay based on Cu2O NPs based biosensor for glucose analysis by integrating colorimetric assay and LDI MS. The Cu2O NPs were simply prepared and low-cost, which presented excellent peroxidase-like activity and assisted LDI MS analysis of metabolites. The Cu2O NP based dual-mode biosensor was successfully applied in visual and quantitative analysis of glucose from serum, demonstrating the potential values in clinical diseases diagnostics and monitoring.In summary, we developed a dual-mode Cu"}
+{"text": "Bi-layer lattice-filled sandwich structures have good application prospects for multi-physics problems; however, high-precision numerical analysis methods are lacking. Recently, the newly proposed asymptotic homogenization method called the novel numerical implementation of asymptotic homogenization (NIAH) was further developed based on the Mindlin plate theory, which is a potential method for overcoming the above limitation. This study investigates the feasibility of this method for Bi-layer lattice-filled sandwich structures. The obtained results are compared to those from homogenization methods developed based on the Kirchhoff theory, and accordingly, the influence of the shear effect on the accuracy of the structural responses of the considered structures is studied. Subsequently, the impacts of the size effect, macrostructure type, and lattice type are also considered. The analysis results showed that, for most cases, the NIAH method can yield high-precision results for Bi-layer lattice-filled sandwich structures. When the number of lattice cells is insufficient or different layers of the lattice have excessive differences in their stiffness, the accuracy of the results obtained using the NIAH method is degraded. Lattice-filled sandwich structures have been extensively studied and used in applications owing to their advantages, such as ultrahigh specific stiffness and specific strength ,2,3,4,5.In a gradient lattice-filled sandwich structures, the lattice shows regular changes in the thickness direction, which may be variations in the lattice structure or the lattice size, whereas the lattice remains unchanged in the plane direction. Research on gradient lattices has been conducted in many fields ,10,11. ZIn uniform lattice-filled sandwich structures, this problem has been overcome using homogenization analysis methods. Homogenization methods treat a uniform lattice-filled sandwich structure as a solid structure with material parameters that depend on the used lattice structure. However, most conventional homogenization methods use the Kirchhoff theory, and thus, lack the consideration of the shear effects. Concurrently, significant shear deformation occurs in the structural response of a gradient lattice structure. For a gradient lattice sandwich plate, the lattice structure changes in the thickness direction. Thus, the gradient lattice plate structure no longer strictly satisfies the assumption of a straight line. If the Kirchhoff plate assumption is still adopted, the structural responses would be smaller than the true responses because of the overestimation of the shear stiffness. Thus, the Mindlin plate theory is expected to be relatively more suitable for gradient lattice-filled sandwich structures.With the recent developments in the novel implementation of asymptotic homogenization (NIAH) method, shear stiffness can be introduced in homogenization analysis, providing a potentially effective method for solving the above-mentioned problem. The NIAH method was first proposed by Cheng  and is bz direction but also dictate the size of each layer of the lattice uniform due to Bi-layer so that it can easily be analysed and manufactured. Therefore, it is often used in practical engineering applications. This study takes the Bi-layer lattice-filled sandwich structure as an example to investigate the feasibility of the NIAH method for the structural analysis of gradient lattice structures, which solves the problem of the lack of efficient and high-accuracy analysis methods for these structures. The effectiveness of the proposed method to solve the structural equivalence analysis of a Bi-layer lattice-filled sandwich structure is verified based on various examples. Compared to gradient lattice structures, Bi-layer lattice-filled sandwich structures, as shown as The remainder of this paper is as follows. x direction and y direction, and the aperiodic boundary is S.For a Bi-layer lattice-filled sandwich plate structure, as shown in Using the NIAH method, the equivalent stiffness of one unit cell of the Bi-layer lattice-filled sandwich plate can be obtained in three steps.x and y directions, respectively, In the first step, the displacement field, which generates the unit generalized strain field, is calculated. For the periodic plate structure, the unit generalized strains are defined asi = 1, 2, 3) represents the node displacement along the x, y or z direction and i = 1, 2, 3) represents the nodes angle of rotation along the x, y or z direction. The corresponding characteristic displacement fields in the unit cell equivalent to the six generalized unit strains can be obtained using\u03b1 = 1, 2, \u2026, 6) from the structural analysis are the results that are to be obtained from these structural analyses. The governing equations of these structural analyses are expressed in Equation (3).In the second step, six independent structural analyses must first be performed on a unit lattice cell using the six displacements obtained in the first step, respectively. The node forces Y as constant. The second denotes tractions on nonperiodic boundaries S as zero. Others are the periodic boundary conditions of force and displacement on the two-direction boundary in the plane. Subsequently, the node force \u03b1 = 1, 2, \u2026, 6) is computed by applying the boundary conditions of the displacement and the force and restricting the rigid body displacement. Subsequently, the displacement field is reloaded on the unit cell to obtain the corresponding node forces, \u03b1 = 1, 2, \u2026, 6). The first equation of Equation (3) denotes body force in the unit cell domain D, is determined by solving Equation (4). A flowchart of the NIAH method for solving equivalent stiffness D is shown in Finally, in the third step, the equivalent stiffness, For a Bi-layer lattice-filled sandwich plate, the mode of bending deformation does not typically strictly follow the assumptions of a straight line, as shown in The calculation steps of the NIAH method based on the Mindlin plate theory (S-NIAH) includes those of the NIAH method, as shown in D1, D2, and D4 represent the in-plane stiffness, coupling stiffness, and bending stiffness, respectively; N and M are the in-plane internal force and the bending moment corresponding to strain Q is the shear force and corresponds to the shear deformations of the plate K is the equivalent shear stiffness. Note that in Equation (5), only the shear stiffness matrix, K, still has no calculation formula, and the other terms can be obtained using the equations presented in It is assumed that a Bi-layer lattice-filled plate satisfies the matrix form of the constitutive equationK, is solved by macroscopic and microscopic strain energy equivalence. To construct the macroscopic stress\u2013strain state related to the equivalent shear stiffness, it is assumed that in-plane internal force is N = 0 first; thus,In the S-NIAH method, the matrix, L1 \u00d7 L2 is chosen, and a strain field of linear curvature is defined as Equation (6) can be rewritten asEquation (8) can be brought back to Equation (6) and the equilibrium equation to obtain Equation (9).The macroscopic strain energy at this strain state by Thus, at the microscopic level, a linear displacement field x direction. To satisfy the condition that the external force equals to zero and the continuity of the displacement and the force in the periodic boundary condition, the displacement field, Defining b1 and b2 are determined using Equation (18).The solution of the displacement field, Similar to the above steps (Equations (8)\u2013(19)), Equation (20) is obtained for the strain state of linear curvature K11 and K22 can be solved. The FE forms of Equations (19) and (20) are expressed in Equation (21), and the process of derivation is seen in the literature [K is shown in Combining Equations (19) and (20), the equivalent shear stiffness coefficients terature . A flowcD, and the equivalent shear stiffness, K, can be obtained based on the flowcharts shown in In this study, we performed equivalent analysis of a Bi-layer lattice-filled structure using ANSYS, in which the lattice unit cell, as shown in r1, are set as 1.0 mm each and in the dense layer, r2, are set as 0.5 mm each. The thickness, t, of the plates on the upper and lower bounds is 2.0 mm, and the thickness, tm, of the inner plates is also 2.0 mm. The material parameters of aluminium are used with Young\u2019s modulus is 71,000 MPa and Poisson\u2019s ratio is 0.33.Body centred cubic (BCC) lattices as a positive Poisson\u2019s ratio structure show high strength over a wide range of temperatures and large strain states. The considered example is a single Bi-layer lattice unit cell, as shown in D, of the unit cell can be calculated following the steps given in z = 0, there is no coupling stiffness and each element of D2 is zero, and there is no tensile-shear coupling or bending-torsional coupling, so Using the NIAH method, the equivalent stiffness, Following the computing process shown in The remainder part of this section discusses the effects of the unit cell parameters on the equivalent structural stiffness of the Bi-layer lattice cell.D1 versus various unit cell parameters are shown in h, upper and lower bound plate thickness t, inner plate thickness tm, radii of the robs in the spare layer r1, and radii of the robs in the dense layer r2 are considered. It can be seen that h, r1, and r2 have a slight effect on the values of the coefficients in D1, whereas t and tm show large effect on them. This is because D1 represents the tensile stiffness, and changing t and tm directly changes the bearing area of the tensile force; thus, the variation in the thickness of the plates directly affects the values of the coefficients in D1. In contrast, it can also be observed that the changes in the values of the coefficients in D1 are linear, which is consistent with the correlation of the bearing area and the tensile stiffness.The variations in tensile stiffness D4 versus various unit cell parameters are shown in h and t have large effects on the values of the coefficients in D4, whereas other parameters show little effect. D4 represents the bending stiffness, and changing h and t directly changes the bearing area or the moment of inertia; thus, the variation in the lattice height thickness of the upper and lower bound plate directly affects the values of the coefficients in D4. The difference is that the influence of t is linear, whereas that of h is nonlinear.The variations in tensile stiffness K versus various unit cell parameters are shown in K11/t increases, the shear stiffness shows little increase, whereas the ratio K11/K11/h or t increases, indicating an increase in the necessity of considering the shear deformation.The variations in shear stiffness n \u00d7 n lattices, where n is the number of lattice extensions along x and y direction. The plate structure and the adopted lattice are shown in n = 40. The considered Bi-layer lattice-filled sandwich plate consists of n. In the table, wFEM represents the results from the finite element modelling analysis and wS-NIAH and wNIAH represent those using S-NIAH and NIAH, respectively. wS-NIAH and wNIAH with respect to wFEM versus n.To show the shear effect on the structural responses of the lattice structure, the structural responses are also obtained using the NIAH method. n is small. The results obtained using NIAH show a large error when n is small, which keeps decreasing as n increases. The value of n is the number of lattice extensions in the structure, which affects the size of the plate. Because the parameters of the lattice are assumed to be constant, the length of the plate increases when n increases, leading to an increase in the length to thickness ratio. Therefore, the contributions of the shear effect to the global structural responses decreases. However, it can be seen that when n = 80, the error of wNIAH is still much larger than that of wS-NIAH; thus, the shear effect should be concerned in Bi-layer lattice-filled plates.It can be found that the results determined from S-NIAH are identical to those from the finite element modelling analysis even when n. The errors of wS-NIAH are in the range of engineering errors, whereas the errors of wNIAH are far large to meet the engineering requirements. Therefore, for the Bi-layer lattice-filled sandwich plate structure, under the condition of limited number of extensions, the equivalent analysis considering the equivalent shear stiffness can yield higher prediction accuracy than the other method.The analysis results, considering the equivalent shear stiffness, are far superior to the equivalent analysis results without considering the equivalent shear stiffness under the same n is set as 40.This section studies the effects of other parameters on the errors of the results obtained from different NIAH methods. The value of h. The relative errors of the different methods are listed in wS-NIAH has high accuracy, whereas wNIAH has low accuracy. It also can be seen that the value of h has little effect on the accuracy of the results.The first considered parameter is the lattice height, t. The results obtained from S-NIAH still demonstrate good performance, whereas those from NIAH still show poor performance. The results show that the thickness of the bound plate of the sandwich structure has a significant influence on the errors of the NIAH method. As t increases, the errors of the results from NIAH continuously increase. Specifically, as the thickness of the upper and lower plates increases, the contribution of the shear deformation to the global deformation increases.tm, r1, and r2, respectively. The trends are almost same as described above.The above results again highlight the importance of the shear effect for the high-precision structural analysis of Bi-layer lattice-filled plates. However, as shown in In summary, the shear stiffness is mainly provided by the lattice core layer. By changing the parameters of the lattice core layer, the difference in shear stiffness between the sparse and dense layers is remarkable when the mass ratio of the sparse and dense layers increases. In view of the phenomenon that the disparity of the mass ratio increase causes the prediction error of the displacement response to increase, the mass ratio should be controlled to ensure the accuracy of the equivalent prediction in the structural analysis.To further investigate the effectiveness of S-NIAH for Bi-layer lattice-filled structure, this section considers four different Bi-layer lattice cells, as shown in r1 is 0.9438 mm and the radius of the dense layer r2 is 0.4674 mm. For FCC, the radius of the sparse layer r1 is 0.9036 mm and the radius of the dense layer r2 is 0.4518 mm. For FCCZ, the radius of the sparse layer r1 is 0.8546 mm and the radius of the dense layer r2 is 0.4273 mm.The results show that the stiffnesses differ remarkably in terms of the different types of lattice cells. Thus, the unit cell configuration has an important effect on the structural performance. FCC has greater tensile and bending stiffness than BCC, although the shear resistance is weaker compared to BCC. By adding supports in the Z-direction, the tensile and bending stiffnesses are slightly enhanced, whereas its shear stiffness is reduced and the shear resistance is weakened.The cylinder structure is widely used in engineering. Bi-layer lattices can help the cylinder structure to achieve high specific stiffness and high thermal insulation. However, owing to a lack of high-precision analysis methods, the structural design of gradient lattice-filled cylinders is difficult.m on the circumference shown in A cylinder with an aspect ratio of 1:2 and a unit cell number wFEM, are compared to the mid-point deflection values considering the equivalent shear stiffness, wS-NIAH, and without considering the equivalent shear stiffness, wNIAH, under different m. For the BCC-filled Bi-layer lattice sandwich cylinder, the deflections at the mid-point of cylinder versus m from finite element modelling analysis, S-NIAH and NIAH, are shown in The node deflections along the thickness direction at an intermediate point with different lattice cells, As shown in It can be seen that the difference in the lattice structure leads to the difference in structural responses by For gradient lattice-filled sandwich structures, there is a lack of high-precision numerical analysis methods. This study investigates using the newly proposed asymptotic homogenization method, NIAH, to overcome this problem. The Bi-layer lattice-filled sandwich structures are considered in numerical examples. The necessity of considering the shear effect is studied by comparing the accuracies of the structural responses obtained using the S-NIAH method developed based on the Mindlin plate theory and the NIAH method developed based on the Kirchhoff theory. The effects of the size effect, macrostructure type, and lattice type are also considered in the numerical examples. The numerical results show that the analysis results obtained from the S-NIAH method have good performance, based on accuracy analysis. From the examination of the structural parameters of the lattice proposed in this paper, it can be seen that when the mass ratio of the sparse and dense layers increases, the error in the prediction of the displacement responses increases. The verification of other lattice configurations confirms the applicability of the NIAH method considering the shear stiffness to the equivalent analysis of the displacement response of a Bi-layer gradient lattice structure.In addition, based on this study, we find that for the considered Bi-layer lattice sandwich structure, an increase in the number of cells can reduce the error between the equivalent analysis results and the finite element modelling analysis results. In addition to increasing the number of extensions, another approach to reduce the error is to improve the unit cell shear resistance. When the ratio of the shear stiffness to bending stiffness is large, the unit cell has a strong shear resistance and the shear deformation does not have an extremely significant effect on the deformation mode of the structure. Conversely, the shear stiffness has a greater effect on the analysis results. Therefore, in engineering applications, the design parameters need to be adjusted when performing equivalently analysis because more lattice cells are filled on the macroscopic level. Moreover, the shear resistance of the unit cells should be improved on the microscopic level to ensure the accuracy of the equivalent analysis results. In addition, we will manufacture gradient lattice structures based on 3D printing technology and design experiments to further verify the accuracy of this method. We can also try to develop this method on the nano or micro scale and explore the performance of this method in the trans-scale field."}
+{"text": "Thioredoxin reductase 1 (TXNRD1) is one of the major redox regulators in mammalian cells, which has been reported to be involved in tumorigenesis. However, its roles and regulatory mechanism underlying the progression of HCC remains poorly understood. In this study, we demonstrated that TXNRD1 was significantly upregulated in HCC tumor tissues and correlated with poor survival in HCC patients. Functional studies indicated TXNRD1 knockdown substantially suppressed HCC cell proliferation and metastasis both in vitro and in vivo, and its overexpression showed opposite effects. Mechanistically, TXNRD1 attenuated the interaction between Trx1 and PTEN which resulting in acceleration of PTEN degradation, thereby activated Akt/mTOR signaling and its target genes which conferred to elevated HCC cell mobility and metastasis. Moreover, USF2 was identified as a transcriptional suppressor of TXNRD1, which directly interacted with two E-box sites in TXNRD1 promoter. USF2 functioned as tumor suppressor through the downstream repression of TXNRD1. Further clinical data revealed negative co-expression correlations between USF2 and TXNRD1. In conclusion, our findings reveal that USF2-mediated upregulation of TXNRD1 contributes to hepatocellular carcinoma progression by activating Akt/mTOR signaling. Liver cancer is the seventh most frequently occurring cancer and the second leading cause of cancer-related deaths in the world , 2. The Akt signaling is an evolutionarily conserver kinase cascade pathway, whereas dysregulation of Akt signaling contributes to cancer development. Typically, Akt is one of the major downstream effectors of PI3K, and its modification is sufficient to activate the mammalian target of rapamycin complex (mTOR) . DisturbThe thioredoxin system, which comprises thioredoxin (Trx), thioredoxin reductase (TXNRD), thioredoxin-interacting protein (TXNIP) and NADPH, is indispensable for retaining harmony and regulation for the redox status in cells . Our preThis study focuses on the role of TXNRD1 in HCC progression, which also sheds light on the regulatory mechanism of TXNRD1 in HCC. Here, we identified TXNRD1 as a tumor promoter that stimulates HCC proliferation and metastasis through activating AKT/mTOR signaling, and Upstream transcription factor 2 (USF2) was a major transcriptional repressor of TXNRD1 expression.A total of 112 paired HCC tumor tissues and adjacent normal tissues were collected and subjected to Western blotting analysis. Tissue microarray plates containing another 115 HCC cases were constructed from paraffin-embedded HCC tissues for IHC and clinical analysis. Tissue samples were obtained from resected specimens at Tongji Hospital, Wuhan, China. The diagnosis of HCC was based on the pathological examination. Informed consent for data analysis was obtained from each patient, and the study protocol was authorized by the Medical Ethics Committee of Tongji Hospital and complied with all relevant ethical regulation.Male BALB/c nude mice (5 weeks old) were purchased from HUAFUKANG  and fed under the specific pathogen-free condition at optimal temperature and humidity. All animal experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Tongji Hospital of Tongji Medical College.6 cells were subcutaneously injected into the dorsal part of mice. Tumor size were recorded every three days from day 7 to 9. When tumors grew to 3\u20135\u2009mm in diameter, the mice were peritoneally treated with Auranofin . The mice were euthanized 5 weeks after injection, and tumors were excised and weighed. Mice bearing a tumor with a tumor size large than 15\u2009mm in any direction were euthanized. Tumor volume was calculated by length \u00d7 width 2 /2. The excised tumors were embedded in paraffin for IHC analysis.For subcutaneous tumorigenicity assay, 1\u2009\u00d7\u2009106 cells were injected subcutaneously into the flank of nude mice. When the subcutaneous tumor reached approximately 1\u2009cm in length, it was removed, minced into small pieces of equal volume (1\u2009mm3), and transplanted into the left liver lobe of nude mice. Mice were euthanized and the liver tissue specimens were collected after 6 weeks. The incidence of liver tumors was recorded.For the liver in-situ xenograft model, 5 \u00d7 106 cells were injected into tail vein of nude mice. The mice were sacrificed at 8 weeks after injection, their lungs were removed and fixed with paraformaldehyde (4%) and embedding in paraffin. Paraffin sections were stained with H&E according to standard protocol and metastatic nodules were calculated with a microscopy to evaluate the development of lung metastasis.For lung metastasis assay, 1 \u00d7 105 cells per well in 24-well plates and allowed to settle for 24\u2009h. Each well was transiently transfected TXNRD1 firefly luciferase reporter construct and the pRL-TK renilla luciferase vector for normalization of transfection efficiency together with pcDNA3.1 vector or pcDNA3.1-USF2 using Lipofectamine 2000 transfection reagent in triplicate. After 8\u2009h, the transfection media was replaced with fresh complete DMEM.The TXNRD1 promoter regions 2000bp from the transcription start site (\u22122000bp-+1\u2009bp) were cloned into pGL4.17 firefly luciferase reporter vector. The promoter truncations and E-box mutant was constructed by site-directed mutagenesis. Cells were seeded at a density of 10The cocultures were lysed 48\u2009h post-transfection and were assayed sequentially for Firefly and Renilla luciferase using the Dual-Luciferase Reporter Assay System  with a Glo/Max 20/20 Luminometer (Promega) according to the manufacturer\u2019s instructions. Relative light units were calculated as the ratio of Firefly luciferase activity to Renilla luciferase activity as described previously .7 cells were harvested and crosslinked with 1% formaldehyde for 10\u2009min at room temperature. After glycine quenching, samples were lysed with sodium dodecyl sulfate buffer and sonicated to shear DNA. Fragmented chromatin extracts with an average of 250-1000\u2009bp were incubated with anti-USF2 antibody and normal mouse IgG at 4\u2009\u00b0C overnight with rotation. Immunoprecipitated products were captured by incubating with Protein G Magnetic Beads followed by washes. Elutes were subjected to reverse cross-linking and then digested with RNase A and proteinase K. Immunoprecipitated DNA and input were purified for qPCR analysis with primers specifically targeting the TXNRD1 promoter region that encompassed the USF2-binding E-box site. Primer sequences were listed in Supplementary Table Both Huh7 and Bel-7402 cells were subjected to chromatin immunoprecipitation analysis using the SimpleChIP Plus Sonication Chromatin IP Kit  following the manufacturer\u2019s instructions. Briefly, 1\u2009\u00d7\u2009102 or Fisher\u2019s exact test. Correlations were determined by Pearson\u2019s correlation. Cumulative survival curves were calculated according to the Kaplan-Meier method and compared by the log-rank test. Statistical significance was determined as indicated in the figure legends, set to P\u2009<\u20090.05 and represented as *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001.Statistical analyses were performed using SPSS software  or the GraphPad Prism software . Values were expressed as the mean\u2009\u00b1\u2009SD or mean\u2009\u00b1\u2009SEM from at least three independent experiments. Quantitative variables were compared using Student\u2019s t-test or Mann-Whitney-U test when applicable. Also, One-way ANOVA followed by a Turkey post hoc test was performed for multigroup comparison. Categorical variables were compared with Pearson\u2019s \u03c7Additional materials and methods can be found in n\u2009=\u200937) and high TXNRD1 group (n\u2009=\u200978). The detailed clinicopathological features is summarized in Table P\u2009=\u20090.028). Regarding the correlation of TXNRD1 expression with postoperative outcomes, Kaplan-Meier\u2019s analysis revealed that patients with high TXNRD1 expression demonstrated significantly shorter overall survival (OS) and disease-free survival (DFS) than those with low expression  of promoter analysis combined with luciferase reporter assays. USF2 belongs to the basic helix-loop-helix-leucine zipper transcription factor family and act as either homodimer of heterodimer by binding to E-boxes of the DNA-core sequence (5\u2019-CANNTG-3\u2019) in their target genes , 45. AltUSF2 has been reported to implicate in several cellular processes, such as embryogenesis, metabolism and cancer development. Interestingly, the data with respect to the role of USF2 in tumor development are conflicting suggesting that it has a dual role as either tumor promoter or suppressor. Earlier result revealed that lack of USF2 function caused increased cell growth in Saos-2 osteosarcoma cells . MoreoveIn summary, we provide novel evidence that USF2-mediated upregulation of TXNRD1 contributes to hepatocellular carcinoma progression by activating Akt/mTOR signaling. A better understanding of the roles and mechanisms of TXNRD1 in HCC may lead to new therapeutic strategies in HCC treatment.Supplementary TablesSupplementary materials and methodsSupplementary Figure legendsFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Original Data File"}
+{"text": "Correction: BMC Nutr 9, 58 (2023)10.1186/s40795-023-00708-xFollowing publication of the original article , the autTables\u00a04 and 5th column, 6th row (% of energy intake in MPA adolescents who did not meet RV) is 90.3 and not 9.3.The original article  has been"}
+{"text": "The purpose of this systematic review was to research the difference between root-filled teeth (RFT) and vital pulp teeth (VPT) in orthodontically induced external apical root resorption (EARR) and to offer suggestions for clinicians on therapeutic sequence and timing when considering combined treatment of endodontic and orthodontic.An electronic search of published studies was conducted before November 2022 in PubMed, Web of Science and other databases. Eligibility criteria were based on the Population, Intervention, Comparison, Outcome, and Study design (PICOS) framework. RevMan 5.3 software was used for statistical analysis. Single-factor meta-regression analysis was used to explore the sources of literature heterogeneity, and a random effects model was used for analysis.This meta-analysis comprised 8 studies with 10 sets of data. As there was significant heterogeneity among the studies, we employed a random effects model. The funnel plot of the random effects model exhibited a symmetrical distribution, indicating no publication bias among the included studies. The EARR rate of RFT was significantly lower than that of VPT.In the context of concurrent endodontic and orthodontic treatment, priority should be given to endodontic therapy, as it serves as the foundation for subsequent orthodontic procedures. The optimal timing for orthodontic tooth movement post-root canal therapy is contingent upon factors such as the extent of periapical lesion resolution and the degree of dental trauma sustained. A comprehensive clinical assessment is essential in guiding the selection of the most suitable approach for achieving optimal treatment outcomes. Tooth root resorption is a complex and unpredictable pathological process that relates to cementum, root dentin or apex. Resorption can even cause irreversible loss of tooth structure. Orthodontically induced external apical root resorption (EARR) is a common and deleterious adverse consequence of inflammation-driven tooth movement . HistoloRoot resorption in both physiologic and pathologic instances involves a coordinated interaction among osteoblasts and osteoclasts as well as odontoblasts and odontoclasts that are regulated . Under a. found that there was no significant difference in the degree of EARR between RFT and VPT [Influencing factors of EARR include patient age, duration of treatment, the magnitude of orthodontic force, and type of orthodontic devices \u201310. Publ and VPT . Bellini and VPT . As for  and VPT . Souza e and VPT . This maOrthodontically treated teeth sometimes need endodontic treatments, and endodontically treated teeth may also need orthodontic treatments, so this can be a common multidisciplinary problem. However, the possibility of root resorption after orthodontic movement remains controversial . When coPreferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [This meta-analysis was conducted according to the tatement . The proPopulation: Patients of any age after endodontic and orthodontic treatment were included.Intervention: Teeth that have undergone endodontic and orthodontic treatment were the studied objects.Comparison: Contralateral teeth with vital pulp that underwent orthodontic treatment were used for comparison.Outcome: Comparison of orthodontic root resorption was the outcome of interest.Study design: The article type was limited to clinical trials. Prospective, retrospective, and cross-sectional studies were reviewed.Eligibility criteria were based on the Population, Intervention, Comparison, Outcome, and Study design (PICOS) framework:An electronic search was conducted for studies published up to November 2022 in the PubMed, Scopus, MEDLINE (Ovid), Web of Science, and Cochrane Library databases. There were no restrictions on publication year, language or status. A search was performed using the following keywords:  AND (\u201croot resorption\u201d) AND (\u201corthodontic\u201d). These search keywords were originally created for PubMed and modified appropriately for the other databases.Clinical studySample size givenImaging techniques were used to assess root resorption outcomesRaw data on root resorption before and after orthodontic treatmentInclusion criteria included the following:Studies conducted using primary teeth or animal modelsSample with a particular disease Raw data measures were not standardizedExclusion criteria included the following:Titles and abstracts were screened according to inclusion and exclusion criteria. For potentially useful studies, the same criteria were used for full-text screening. 2 reviewers made separate research selections and extracted data independently according to the predesigned items. A third reviewer was involved when there was a disagreement. Cohen's kappa was used to evaluate the consistency of research qualifications and data extraction among reviewers.The following data were extracted from each included study: study characteristics , sample characteristics , treatment characteristics , and result characteristics .The risk of bias was assessed using RevMan 5.3 software . Since none of the included studies were randomly assigned to the intervention control group, the Risk Of Bias In Non-randomized Studies of Interventions tool  was used to assess the estimated risk of bias for the relative effectiveness of the intervention . 2 reviePublication bias was assessed by visual funnel plot asymmetry. When a funnel plot made it difficult to judge whether publication bias existed subjectively, Egger\u2019s linear regression quantitative test was used.The quality of evidence included in the study was evaluated, and the quality level of relevant evidence was finally determined , 28. Coh2 statistic were used to test interstudy heterogeneity. When I2\u2009<\u200950% and p\u2009>\u20090.1, the fixed effects model was selected for the analysis because the heterogeneity among studies was small. When I2\u2009\u2265\u200950% and p\u2009\u2264\u20090.1, the heterogeneity between studies was considered to be large, and the random effects model was selected for the analysis. The differences in EARR and different intervention characteristics were expressed by the standard mean difference (SMD) and 95% confidence interval (CI). The heterogeneity test level was set as \u03b1\u2009=\u20090.1, and the remaining test levels were set as \u03b1\u2009=\u20090.05.The Excel data were imported into RevMan 5.3 software in the \".xls\" format using the meta-analysis module for statistical analysis. A mixed effects model was selected to test the main effect through a heterogeneity test. Single-factor meta-regression analysis was used to explore the sources of heterogeneity in the literature. The Q test and IIn addition, the effect size of removing individual studies was analyzed to evaluate the robustness and reliability of the combined results.Initially, the search yielded 109 records , therefore, a random effects model was used for analysis. The results of the main effect test showed an SMD\u2009=\u2009-0.45, 95% CI , p\u2009=\u20090.002, indicating that the EARR of RFT was significantly lower than that of VPT , p\u2009=\u20090.03, so it could be considered that the EARR of RFT was significantly lower than that of VPT , The standardized effect size was used as the Y variable, and the study characteristics such as treatment duration were coded and then set as the X variable for single-factor meta-regression analysis. The age and treatment duration of the subjects could not be considered sources of interstudy heterogeneity.p\u2009=\u20090.002. When 1 study was excluded, the result showed SMD\u2009=\u2009-0.52\u2009~\u2009-0.38. There was no statistically significant change in the total effect after removing a certain group of data, and the result was relatively robust.Ten sets of data are covered in 8 studies, analysis of the included literature showed SMD\u2009=\u2009-0.45, 95% CI , The results indicated that EARR was significantly less in RFT than in VPT. This conclusion supported the views obtained in the previous systematic review and meta-analysis , 22. In Regarding the influencing factors of EARR, the age of patients and the time of orthodontic treatment were not found to be correlated with EARR in this study. Many studies have shown that the degree of EARR is related to tooth position, but there is no consensus on the order of how much the EARR of each tooth position is. The majority of studies showed that the EARR of the anterior teeth is greater than that of the posterior teeth , 36. In There are still no clear conclusions about the timing of orthodontic treatment after root canal treatment. Lee and Castro et al. confirmed that EARR was significantly greater in RFT with periapical pathosis before orthodontic treatment from a controlled clinical study , 39, whiAccording to Consolaro et al. and Pustu\u0142ka et al., teeth with mild trauma and an intact periodontal membrane may require a waiting period of 3\u20134\u00a0months to allow for the restoration of normal periodontal tissue and structure before orthodontic movement is initiated. This waiting period is necessary to prevent potentially adverse effects on the healing process and minimize the risk of further damage to the traumatized tooth , 40. ModIn the absence of original and high-quality randomized controlled trials, the results of the current study should be interpreted with caution. At present, there are many controversies about the risk factors for orthodontic root resorption, which may be due to the different measurement methods (CBCT results are considered more reliable) or the limitations in the study sample size . PanoramClinicians must carefully consider the safety of orthodontic movement following the essential endodontic treatment. In cases where both endodontic and orthodontic therapies are required, we recommend that root canal therapy should take priority. In the event of severe EARR during orthodontic treatment, force application should be discontinued, and an endodontic consultation should be sought to determine the need for root canal therapy, particularly in relation to anterior teeth.Orthodontic forces can be applied within a few days after root canal therapy if there is no sign of periapical lesions and traumatic source of pathogenesis. If there is a large periapical radiolucency, orthodontic treatment should be delayed by 6\u00a0months, and apical radiolucency reduction should be determined before treatment.Teeth that have been treated with root canal therapy after trauma need to be considered carefully. For teeth with mild trauma, 3\u20134\u00a0months after root canal treatment must pass before orthodontic treatment. For teeth with moderate damage, a year must pass until the periapical X-rays show normal. In more severe cases, 2\u00a0years or more must pass. According to the degree of tooth injury, follow-up should be carried out for 3 to 12\u00a0months as recommended by the clinician.Aidos et al. have shown that orthodontics movements promote external resorption and endodontic treatment is mandatory in severe EARR . BioceraRFT shows relatively lower EARR after orthodontic treatment, and orthodontic movement after root canal treatment can be considered a relatively safe treatment by clinicians.Endodontic treatment should be carried out first when both endodontic and orthodontic treatment is needed. The timing of orthodontic treatment should be determined according to the healing degree of periapical lesions and the severity of trauma if the teeth were injured, and it is best to start when the periapical X-rays show normal.Studies referring to questions such as the optimal timing of endodontic-orthodontic treatment considering the effect of different periapical infection states and the size of apical radiolucency on orthodontic EARR should be conducted in the future."}
+{"text": "Given the urgency due to the rapid emergence of multidrug-resistant (MDR) bacteria, bacteriophages (phages), which are viruses that specifically target and kill bacteria, are rising as a potential alternative to antibiotics. In recent years, researchers have begun to elucidate the safety aspects of phage therapy with the aim of ensuring safe and effective clinical applications. While phage therapy has generally been demonstrated to be safe and tolerable among animals and humans, the current research on phage safety monitoring lacks sufficient and consistent data. This emphasizes the critical need for a standardized phage safety assessment to ensure a more reliable evaluation of its safety profile. Therefore, this review aims to bridge the knowledge gap concerning phage safety for treating MDR bacterial infections by covering various aspects involving phage applications, including phage preparation, administration, and the implications for human health and the environment. The re-emergence of bacteriophages (phages) for the treatment of chronic or difficult-to-treat infections and diseases has been extensively observed in recent years. Phages are viruses that specifically target and kill host bacteria. They serve as a possible alternative to antibiotics in the treatment of bacterial infections, especially in this era where there is a lack of new and effective antibiotics to combat the rise of multidrug-resistant (MDR) bacteria. The term \u201cMDR\u201d refers to organisms that are resistant to at least one antibiotic agent from three or more antibiotic classes . ExtensiThe development of phage therapy as an alternative to traditional antibiotics has gained substantial interest over the years. However, its ultimate success in clinical use relies on evidence that proves phage therapy is safe and non-toxic for humans. A major safety concern is the disruption of the body\u2019s microbiome, which is a key regulator of human health. Another concern is the impact of phage on the immune system, which may ultimately lead to chronic inflammation and other immune reactions. As phages are prepared by co-culturing with bacteria, it is crucial to consider the potential risk of endotoxin contamination that could trigger the inflammatory cytokine response, resulting in serious health issues such as toxic shock . AdditioRecent studies have shown that phages are generally safe and do not produce any adverse effects when used in animals or humans ,6,7,8. NIn this review, we discuss the advancement, importance, and current applications of phages. Phage applications in clinical settings, the food and agricultural sector , and environmental control  are reviewed. It also focuses on the safety concerns and challenges of translating phages from the bench to clinical bedside applications. The topics that are included are the disruption of the microbiome, immunological responses, induction of phage resistance in bacteria, lysogeny, and contaminants  associated with phage preparation. The environmental safety issue related to phage release is also briefly discussed.Acinetobacter baumannii such as pneumonia, meningitis, and sepsis have traditionally been treated with beta-lactams [A. baumannii strains, including those resistant to beta-lactams, are increasingly reported; hence, last-line antibiotics such as polymyxins are prescribed for their treatment [A. baumannii is listed by the World Health Organization (WHO) as a priority pathogen for which new antibiotics are urgently needed, along with other bacteria such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, collectively referred to as the \u2018ESKAPE\u2019 pathogens [Ever since the discovery of the \u201cwonder drug\u201d penicillin, antibiotics have been the first line of defense against bacterial infections. Indeed, virtually everyone alive today has grown up in the antibiotic era. However, the overuse of antibiotics has inadvertently led to the emergence of MDR bacteria. For example, infections caused by -lactams ,13. Howereatment ,15. Thusathogens . As MDR athogens , there iEscherichia coli and can be used to reduce contamination in beef, whereas S5 and vB_EcoM_swi3 are polyvalent and target both Salmonella and E. coli [Phages are the most abundant and diverse biological entities that are present on Earth . Phages  E. coli ,20,21. P E. coli . Worth n E. coli .Ever since the discovery of phages, researchers have taken advantage of their abundance and infection specificity to combat pathogenic bacteria, mainly for treating bacterial infections, preventing food contamination, and for environmental control. With the emergence of MDR bacteria, research examining phages as an alternative to antibiotics has once again begun in earnest, with the current application of phages against these \u201csuperbugs\u201d briefly discussed below.When phages were first identified and described in the early 20th century, they were used mainly for therapeutic purposes against infections such as dysentery, diarrhea, furunculosis (hair follicle abscess), urinary tract infections, and respiratory tract infections. For example, d\u2019 Herelle utilized phage therapy in the treatment of severe dysentery in children (1919) and cholera in India (1931) ,24,25. HP. aeruginosa [Salmonella spp., suggesting they might be potential therapeutic agents for the treatment of salmonellosis [There has been an increasing focus on phage therapy in recent years due to the alarming increase of MDR bacteria. For example, phages AP025 and AP006 showed good infectivity rates when used against 51 clinical strains of MDR ruginosa . Furthernellosis ,28.A. baumannii, which is known to cause pneumonia, urinary tract infection (UTI), sepsis, and wound infection [A. baumannii infection [P. aeruginosa lung infection [K. pneumoniae post-heart transplant infection [A. baumannii ventilator-associated pneumonia in COVID-19 [Mycobacteroides abscessus chronic lung disease [The good efficacy of phage therapy has also been illustrated using in vivo models. Utilizing a mouse model, phages have been found to be effective in eradicating MDR nfection ,29. Phagnfection , MDR P. nfection , MDR K. nfection , carbapeCOVID-19 , and Myc disease .Campylobacter, Salmonella, Escherichia, and Listeria that are capable of causing disease when ingested have been found in water [Since foods of animal origin are frequently contaminated with MDR pathogens, MDR bacterial infections are no longer only associated with hospital-acquired infections and have started to pose a risk to the general public. This is said to be due to the excessive use of antibiotics in feedstock and food preparation ,36. MDR in water ,38,39, min water ,41,42, vin water ,44,45, ain water .E. coli [S. aureus (MRSA) [Salmonella strains [Vibrio spp., which are known to easily gain resistance against antibiotics [E. coli in meat products, and ShigaShield\u2122, which is designed to treat foods with a high risk of Shigella contamination, such as meat, poultry, seafood, vegetables, and dairy [Phages can be applied for pre-harvest and post-harvest pathogen control. Studies have been conducted to evaluate the effectiveness of phage therapy against MDR bacteria in livestock. Naghizadeh et al. (2019)  demonstrE. coli ,49, meths (MRSA) , and MDR strains . In aquaibiotics ,53. Phagibiotics , potatoeibiotics , onions ibiotics , and Geribiotics  to prevend dairy ,59. OverBacillus spp. isolated from soil exposed to insecticides were found to be resistant to chloramphenicol, ampicillin, cefotaxime, and streptomycin [E. coli and MRSA, from peat soil. Recent research has also found that the soil microbial community is able to disseminate antimicrobial resistance through horizontal gene transfer, hence contributing to the abundance of MDR bacteria in the environment [Bacteria are capable of surviving in the environment, including soil. These bacteria often thrive in soil supplied with insecticide or decomposed organic matter. Importantly, some of these bacteria are reported to exhibit the MDR phenotype. For example, ptomycin . In addiptomycin  isolatedironment .Phages are able to act as bioindicators to detect the presence of MDR bacteria in the environment as well as biocontrol agents. As feces are often a source of MDR bacterial infection , early warning of sewage contamination can be detected by phage fecal indicators. Yahya et al. (2015)  demonstrDespite successful cases of treatment with phage therapy, the safety and potential side effects pose a significant challenge to its broader application, especially for clinical use. The concerns associated with the human body include the impact of phage on the microbiome, lysis-induced endotoxin release, immune activation, and increased bacterial resistance. Phage usage may also have environmental implications.Candida albicans yeast infections and Clostridium difficile colitis [E. coli (UPEC) with minimal microbiome impact compared with antibiotic treatment. After receiving antibiotics (5 mg/mL streptomycin sulfate and 1 mg/mL kanamycin sulfate), 50 gut microbiome genera were reduced and 14 had increased, whereas with phage therapy, only 11 species were observed to have reduced in abundance while 21 had increased [Many broad-spectrum antibiotics are prone to inducing superinfections such as MDR  colitis . In cont colitis . The gut colitis  and Febv colitis  reduced ncreased . On the ncreased .E. coli strains that colonize the gut, contributing to the dissemination of drug resistance among gut bacteria [E. coli. In-depth investigation is required to evaluate the impact of phage-mediated horizontal ARG transfer on human health.Careful consideration is essential when determining phages for therapeutic use, as certain phages have the ability to transmit genes to bacteria, particularly antibiotic resistance genes (ARGs) . ARGs onbacteria . Howeverbacteria . It was Phage cocktails  have been commonly employed to increase the efficiency and/or broaden the coverage of targeted bacteria. The increased number of phages used may potentially affect non-targeted bacteria, even though the impact may not be as significant as antibiotics .P. aeruginosa [Given that effective phages can lyse bacteria within minutes of commencing therapy , phage truginosa  and reprruginosa . Cytokinruginosa . When thruginosa . The limS. aureus phages were found to suppress lipopolysaccharide (LPS)-induced inflammation in mammalian epithelial cells and induce pro- and anti-inflammatory responses in peripheral blood mononuclear cells [Phages may stimulate both innate immunity (by triggering phagocytosis and cytokine production) and adaptive immunity (by influencing the synthesis of antibodies and effector polarization). Utilizing in vitro cell culture models, ar cells ,82. In iar cells ,84. On tar cells  found thV. parahaemolyticus infection and S. aureus bacteremia [There are conflicting reports on phage-induced antibody production. While an increase in the production of IgG and IgM was reported following intraperitoneal administration of phages for treatment of MDR cteremia ,86, somecteremia ,78,87. Acteremia . Attribucteremia .Apart from the phage\u2019s inherent characteristics, the safety of the phage product can be influenced by the presence of bacterial and chemical contamination during phage preparation.During phage amplification, a large amount of endotoxins is released as the bacteria undergo lysis. These endotoxins may be difficult to remove completely via purification and may induce infusion-related reactions . Thus, tcis-element to modify host gene activity, activate proto-oncogenes, silence tumor suppressor genes, and control pathways involved in cancer [Other than endotoxins, contaminants such as bacterial DNA may also be present in phage preparation and hinder therapy results. Generally, the insertion of bacterial DNA into the human genome may work as a n cancer . Howevern cancer  and McCan cancer .50 of 20 ug/kg and an ED50 of 400 ng/kg [S. aureus is known to secrete alpha hemolysin, which would generally cause pneumonia, skin and soft tissue infections, sepsis, arthritis, and abscess formation [Another potential contaminant during the preparation of phages is bacterial toxin. Staphylococcal enterotoxin B is a potent bacterial superantigen capable of causing significant inflammatory responses and potentially being lethal at an LD00 ng/kg ,98. Alth00 ng/kg  and lipo00 ng/kg . S. aureormation . Lipoteiormation .Phage purification traditionally involves polyethylene glycol (PEG)-based precipitation, followed by caesium chloride (CsCl) density gradient centrifugation and a final dialysis step to remove CsCl. CsCl is highly toxic to cells when present in high concentration, causing symptoms such as hypotension, numbness, and gastrointestinal discomfort. However, it was said that the residual CsCl levels in the phage products would typically be insignificant . Over thLytic phages are unanimously preferred over temperate phages for therapy purposes. The administration of temperate phages may not be favored due to their intrinsic nature, which favors the lysogenic cycle, in which their genomes are integrated into the bacterial genome and do not lead to immediate bacterial killing. Importantly, bacterial lysogens often exhibit resistance to subsequent infections by phages . SuperinTemperate phages have the ability to obtain ARGs and virulence genes from the infected host and subsequently transfer them to other bacterial hosts. This process can inevitably lead to the widespread distribution of these factors among bacteria, contributing to the development of difficult-to-treat MDR bacteria. Hence, it is advisable to refrain from using temperate phages in phage treatment until more comprehensive research is available to guide their application. In fact, researchers have been exploring phage engineering techniques to modify temperate phages, making them safe and effective for use in phage therapy.Bordetella spp. that prevents infection from phage BPP-1 by suppressing the expression of the phage receptor pertactin autotransporter (Prn) [Erwinia carotovora subspecies atrosepticawa was found to consist of a toxI gene that encodes for an antitoxin that neutralizes ToxN [Bacillus cereus consists of six-gene cassettes called BREX defense systems that are subject to substantial horizontal gene transfer and offer total phage resistance to a variety of phages, including lytic and temperate phages [As phages are increasingly employed for the treatment of bacterial infections, it has unavoidably led to the emergence of phage-resistant bacteria. It has been reported that bacteria can develop phage resistance through various mechanisms, which include receptor alterations, abortive infection systems, bacteriophage exclusion (BREX), and quorum sensing defense . An examer (Prn) . The abozes ToxN ,108. Thee phages . A summaThe application of phage therapy may directly or indirectly affect ecosystem biodiversity as phages are released into the environment; however, there is limited research on the environmental impact.Research has been conducted to compare the disposal of therapeutic phages and broad-spectrum chemical antibiotics in the environment. There has been an increasing focus on antibiotic surveillance and investigations into strategies for appropriate antibiotic disposal . The preAlthough phages are naturally present in the environment, the release of high phage concentrations as a result of phage therapy could potentially cause an imbalance in the ecosystem by disrupting the natural microbial balance . Hence, Several animal studies ,8,83, caMurine and sheep are often used as animal models for studying phage safety. The use of the sheep model provides an advantage over the more commonly used murine model as it is one of the most significant representations of human organ systems, providing a more accurate assessment of phage safety in humans.E. coli, P. aeruginosa, S. aureus, and A. baumannii in sheep and mouse models [E. coli infection without inducing an innate inflammatory response [A. baumannii in mice without eliciting any cytotoxic effects [P. aeruginosa infection resulted in no significant adverse effects, such as loss of appetite, fever, or other signs of systemic illness [S. aureus-associated rhinosinusitis in sheep with the phage cocktail NOV012 was not associated with tissue damage or inflammatory infiltration [Phage therapy has been demonstrated to be safe and effective in treating pathogenic e models ,83. Util illness . Similarltration . DetailsMultiple case studies have included phage safety monitoring. Liu et al. (2021) have conducted one of the first systematic literature reviews on phage therapy clinical cases where phage safety monitoring alongside treatment is included . These cAs endotoxins are a major source of phage contamination that may lead to adverse effects during therapy, several case reports have reported the endotoxin level of the phage products used for clinical treatment. While most of the studies reported endotoxin levels within the FDA limits ,114,115,In one study, inflammatory changes were assessed following IV and/or intraarticular (IA) administration of SaGR51\u03a61, along with antibiotic administration. This study reported no significant inflammatory effect except for a transient, reversible transaminitis . Most caOverall, it is crucial that standardized therapeutic guidelines are made available to promote consistency and uniformity in monitoring the safety profile of phage therapy, enabling a more reliable conclusion to be drawn.Enterococcus spp., E. coli, Proteus mirabilis, P. aeruginosa, Staphylococcus spp., and Streptococcus spp.), patients were subjected to either receiving Pyo phage , placebo, or antibiotics [P. aeruginosa, Biophage-PA significantly reduced the bacterial load, and no phage-related adverse effects were observed [Clinical studies that evaluate the safety aspects of phage therapy are scarce. In a clinical trial to test the efficacy and safety of phage therapy for treating urinary tract infections caused by MDR uropathogens , hematology lab assessment , and immunological response evaluation  . The avaP. aeruginosa and observed that combining piperacillin and ceftazidime with P. aeruginosa phage KPP22 showed the strongest PAS [of K. pneumoniae [Phages are often used in combination with antibiotics to achieve better antibacterial activity ,87,117, gest PAS . Understeumoniae . It shoueumoniae . OverallIn order to further advance the field of phage therapy, it is essential to establish comprehensive phage libraries consisting of a diverse array of phages that are known to be safe and effective against bacterial infections. By having such accessible repositories of phages, the potential for finding suitable phages can be significantly enhanced.Since their discovery, phages have been extensively utilized in the agricultural and environmental sectors to ensure food safety and effective pathogen control. Phages have also re-emerged as an alternative to antibiotics due to the emergence of MDR bacteria. As phage therapy research against MDR bacteria advances quickly, ensuring its safety in clinical applications becomes an absolute priority.The recent success of in vivo studies, case reports, and clinical trials has demonstrated that phages exhibit a relatively safe profile and are typically tolerable when administered to animals and humans. These encouraging findings solidify the foundation for the broader application of phage therapy as a safe and well-tolerated treatment modality. Given existing knowledge gaps and limited data on the potential health implications of phage therapy, it will be imperative to address and overcome the safety concerns discussed in this paper. It is also vital to establish and implement standardized safety assessments. By adhering to consistent and rigorous safety evaluation protocols, researchers can effectively address any uncertainties and validate the reliability of phage therapy, reinforcing its position as a trustworthy medical approach.Nonetheless, ongoing research in the phage field enables researchers to enhance their comprehension of the safety aspects surrounding phage therapy for combating MDR bacteria. While there remains a considerable journey before phage therapy becomes an established standard of clinical care, it is crucial to continue expanding the knowledge base and facilitating the translatability of phages from bench-side to clinical bedside applications."}
+{"text": "Phages possess the ability to selectively eliminate pathogenic bacteria by recognizing bacterial surface receptors. Since their discovery, phages have been recognized for their potent bactericidal properties, making them a promising alternative to antibiotics in the context of rising antibiotic resistance. However, the rapid emergence of phage-resistant strains  and the limited host range of most phage strains have hindered their antibacterial efficacy, impeding their full potential. In recent years, advancements in genetic engineering and biosynthesis technology have facilitated the precise engineering of phages, thereby unleashing their potential as a novel source of antibacterial agents. In this review, we present a comprehensive overview of the diverse strategies employed for phage genetic engineering, as well as discuss their benefits and drawbacks in terms of bactericidal effect. The World Health Organization (WHO) has proposed that antimicrobial resistance is one of the greatest threats to global public health in the 21st century. According to WHO, approximately 700,000 individuals succumb to antibiotic resistance annually . MeanwhiBacteriophages (phages), also known as bacterial viruses, exhibit a high specificity towards particular bacterial species, with fewer off-target effects on intestinal microbiota compared to antibiotics ,8,9. AltGenetic engineering and biosynthesis technology hold great promise for advancing phage therapy and overcoming the challenges associated with bacterial infections. This paper aims to present a comprehensive review of the latest advancements in phage engineering and biosynthesis technology, as well as their diverse applications in the treatment of bacterial infections.Recent advances in phage engineering and biosynthesis have led to the development of phage-based therapeutics with has improved efficacy and safety profiles. These include host-mediated homologous recombination, in vivo recombineering, BRED, yeast-based assembly of phage genomes, L-form bacteria, and CRISPR-Cas. Phage engineering and biosynthesis strategies hold great promise for the development of novel antibacterial agents to combat antibiotic-resistant bacteria.Homologous recombination (HR) is a highly versatile strategy for phage genome engineering, which can occur between two DNA fragments that share only limited regions of homology . To execHR is a rare occurrence in most organisms, and attaining the desired recombinant through endogenous recombination machinery is frequently challenging. To circumvent this issue, an improved editing technology termed recombineering has been devised. It employs the recombination system harbored by phage genomes to increase the frequency of HR, thereby enabling the generation of gene knockouts, deletions, and point mutations  20]. . 20]. Recombineering relies on linear donor DNA and heterologous proteins, including Gam, Bet, and Exo, which are expressed from the phage genome. These proteins protect linear dsDNA from intracellular degradation and promote recombination between the linear donor DNA and the injected phage genome. This process increases the frequency of recombination and reduces the length of homologous regions to as little as 50 bp . The enhM. smegmatis), recombinant phages can be retrieved through PCR-based plaque recovery following cell lysis and infection of susceptible hosts. The benefits of BRED include the absence of a need for constructing intricate cloning systems and a selectable marker, as well as the ability to introduce mutations into any region of the phage genome . Be. Be23]. e genome . BRED ofe genome .At present, the progress of BRED is primarily hindered by challenges in attaining sufficiently high transformation efficiencies when working with the extensive genomes of phages in various host organisms . In ordeSaccharomyces cerevisiae, a yeast species, to recombine linear double-stranded DNA fragments into a single genome facilitates genetic modifications and empowers the generation of engineered phages [S. cerevisiae, where gap repair enables a recombination-mediated joining of all phage genome segments , and size . This fiThe CRISPR-Cas system is a naturally occurring adaptive defense mechanism present in numerous prokaryotes, which confers sequence-specific protection against invasive nucleic acids ,37,38. TE. coli has been found to exhibit glucosyl DNA hypermodifications as well as DNA recombination and repair mechanisms. These mechanisms serve to safeguard the phage against the DNA-targeting type I and II CRISPR-Cas systems, thereby leading to a diminished efficacy in counter-selecting the wild-type T4 [The utilization of CRISPR-Cas-based phage engineering techniques is constrained to bacteria that possess a well-defined native CRISPR-Cas system or have the ability to undergo transformation in order to facilitate the expression of an operational heterologous CRISPR-Cas system. This can pose a significant constraint for the manipulation of bacteriophages targeting bacteria that lack genetic manipulability. Furthermore, phages have developed various mechanisms to resist targeting by CRISPR-Cas, including the concealment of their DNA through covalent modifications of nucleotides or the utilization of anti-CRISPR proteins (Acrs) . The ext-type T4 ,45.Phage therapy has emerged as a promising approach to address the global rise of antibiotic resistance among bacterial pathogens. Advancements in biosynthesis and genetic engineering technologies have greatly facilitated phage genome engineering. Significant progress has been made in engineering phages to restore the sensitivity of drug-resistant bacteria, reduce the minimum inhibitory concentration (MIC) of antibiotics, target the deletion of essential genes of host bacteria, and provide crucial therapies for patient treatment . NotablyE. coli EV36. Distinctive trafficking pathways between the two phages were also observed: K1F-GFP-EGF entered cells through the endolysosomal pathway by inducing the EGF receptor (EGFR), while K1F-GFP entered cells and underwent degradation through LC3-assisted phagocytosis. This enabled K1F-GFP-EGF to accumulate rapidly within different human cell lines, thereby enhancing its efficiency in locating its intracellular host. The bactericidal efficacy of natural phages varies depending on the specific phage and can be enhanced through the development of genetically modified phages. A phage K1F was genetically modified using the homologous recombination technique to incorporate fluorescence and facilitate the expression of epidermal growth factor (EGF) derived from the ErbB family of tyrosine kinases . The modE. coli lysogenized with the engineered \u03bb prophage was deleted, resulting in a restored antibiotic sensitivity. In another study, Edgar R.\u2019s group employed homologous recombination to introduce a streptomycin-sensitive gene into a phage genome [E. coli, resulting in a significant reduction of the MIC of antibiotics from 100 to 12.5 mg/mL. The same strategy restored the sensitivity of E. coli to naphthyridic acid, resulting in a twofold reduction in the MIC value.RNA-guided nucleases (RGNs), derived from Type II CRISPR-Cas systems, are programmable endonucleases that enable precise genome editing. RGNs comprise two essential components: a guide RNA (gRNA) of approximately 100 nucleotides, which utilizes 20 variable nucleotides at its 5\u2032 end to form base pairs with a specific genomic DNA sequence, and a nuclease  that cleaves the target DNA . Severale genome . This enThese strategies could be utilized to treat hospital surfaces and hand sanitizers, specifically targeting the skin microbiota of medical personnel. Unlike antibiotics and disinfectants, which promote the growth of resistant pathogens, this proposed treatment enriches and selects for susceptible pathogens. Additionally, these strategies promote the growth of pathogens that are unable to acquire or transfer resistance determinants horizontally, potentially reducing the spread of antibiotic resistance. The enriched susceptible population may also prevent the establishment of newly introduced resistant pathogens by outcompeting them for their ecological niche.Vibrio cholerae by fusing the minor coat-encoding gene pIII from fd with a sequence of the orfU gene from CTX\u03c6, another filamentous phage [One major limitation of phage-based applications is their narrow and specific host range. The natural host range of phages is insufficient to cover all pathogenic microbial strains. To circumvent this challenge, genetic engineering techniques have been adopted to expand or alter the host range of phages . Severalus phage . HoweverE. coli-specific phage T7 was genetically modified to incorporate the biofilm-degrading enzyme dispersin B (DspB) during phage infection [Bacterial growth within biofilms is frequently associated with the pathogenesis of numerous clinically important infections . Biofilmnfection . DspB exnfection . These fBacillus anthracis was cloned into the T7 phage, resulting in the creation of an engineered phage named T7aiiA. The biofilm formation was significantly inhibited by phage T7aiiA, resulting in a remarkable reduction in biomass by 74.9%, whereas the T7control phage only caused a reduction of 23.8% [Acyl-homoserine lactone (AHL) is a well-known quorum-sensing signal that plays a crucial role in the formation of biofilm. The activity of AHLs can be effectively inhibited by AHL-lactonases, which catalyze the cleavage of the lactone bond in AHLs, thereby preventing biofilm formation . Accordiof 23.8% .This design obviates the necessity of expressing, purifying, and delivering high doses of enzymes to hard-to-reach infection sites, thereby enhancing the effectiveness of phage therapy in biofilm removal. The cost-effectiveness of genome sequencing and synthetic biology technologies, such as phage genome refactoring and large-scale DNA synthesis, should facilitate the production of engineered enzymatic phages and expand the limited range of biofilm-degrading phages isolated from the environment.E. coli through the PhoE signal peptide, thus resulting in the formation of a lipid coating on the surface of T7 phage. The lipid-coated engineered T7 phage exhibited a 100-fold increase in stability compared to the wild-type phage T7 in the gastrointestinal tract of animals, representing a promising candidate for orally delivered phage therapy. Compared to other methods, such as microencapsulation, this approach offers the advantages of process simplicity, requiring significantly fewer optimization steps and a straightforward scale-up process (only the phage amplification is necessary). Importantly, this research demonstrates the feasibility, simplicity, and cost-effectiveness of phage engineering as a means of enhancing phage properties for oral administration in animals.Despite the clinical success of some antimicrobial treatments, the widespread application of phage therapies has been hindered by several severe physiochemical obstacles that phages encounter in the digestive or circulatory systems . It is nPseudomonas aeruginosa strain PAO1 by 99% upon infection, comparable to that of the parental phage [600 value of PAO1 infected with Pf3R remained constant for 7 h, and endotoxin levels in the supernatant were not significantly increased. In an animal trial, the survival rate of mice treated with a Pf3R phage was significantly higher than that of mice treated with a lytic phage. The improved survival rate observed in that study was associated with the attenuation of the inflammatory response induced by the Pf3R treatment, rather than the lytic phage itself.Treatment with lytic phages can elicit rapid cell lysis, which may subsequently lead to the release of cell debris and toxins, thereby triggering an adverse immune response. To address this limitation, phages have been modified as nonreplicative variants. For instance, an engineered nonlytic phage (Pf3R) was constructed to minimize endotoxin release by replacing ORF40 with an endonuclease gene in the phage genome. The bactericidal efficacy of Pf3R remained unchanged, and it could effectively reduce the colony-forming units (CFUs) of al phage . HoweverStaphylococcus aureus, was genetically modified to create a lysis-deficient variant (known as P954R) by replacing an endolysin-encoding gene with the chloramphenicol acetyl transferase (cat) gene through HR [Staphylococcus aureus (MRSA) infections in mice, highlighting its potential as a promising therapeutic intervention.The phage P954, which targets rough HR . The phaThese studies highlight the potential of endolysin gene disruption in reducing the number of phages released by their lytic parent phage following infection. In clinical settings, this approach offers the benefit of a precise dosage, addressing a key concern regarding phage therapy. Additionally, it may result in a reduced immune response and endotoxin release when targeting Gram-negative bacteria.Phages can also be utilized as nanocarriers for the targeted elimination of pathogenic microorganisms and tumor cells through genetic manipulation or chemical modification . These mP. aeruginosa is recognized as a pulmonary pathogen, but drug-resistant P. aeruginosa is also a significant pathogen in chronic wounds [This approach proposed herein is most appropriate for treating tissues or surfaces that are directly accessible. In the short term, this technique could be used for localized topical therapy, particularly for wound infections or the colonization of medical devices, where engineered phages can be directly applied to the biofilm. For instance, c wounds , surgicac wounds . These wM. abscessus, resulting in the development of cystic fibrosis in the transplanted lungs [In 2019, the application of genetically modified phages in clinical treatment was initially observed. In this particular instance, a 15-year-old recipient of a bilateral lung transplant was afflicted with an infection caused by ed lungs . Three mGD40 and BPs\u039433HTH_HRM10, were administered intravenously to a 26-year-old patient suffering from severe cystic fibrosis caused by drug-resistant M. abscessus in pulmonary infections [GD40 is an HRM of D29 isolated on an M. abscessus strain GD40, which does not resemble the D29 parent\u2019s effects on severe M. abscessus clinical isolates. The M. abscessus isolate was successfully eradicated through incubation with BPs\u039433HTH_HRM10 and D29_HRMGD40, alone or in combination, across a diverse range of bacterial and phage concentrations. Following the administration of the treatment, the patient\u2019s lungs were effectively cleared of the drug-resistant mycobacterium abscesses, leading to the successful completion of a lung transplantation procedure. This case represents a significant milestone in the field of medical research as it marks the initial application of engineered phage therapy in the treatment of drug-resistant M. abscessus. In a similar study, two types of mycobacteriophages, namely, D29_HRMfections . BPs\u039433HM. abscessus, suggesting that the three-phage cocktail may not be universally effective. The intricate nature of cases such as this one poses challenges in accurately evaluating the efficacy of phage therapy. As there have been no prior reports of successfully treating pulmonary M. abscessus infection with phages, it is challenging to ascertain whether the outcomes observed in this singular case can be extrapolated to other patients undergoing this therapy. Additionally, there may be unidentified factors that have differentially influenced the treatment response.However, the phages Muddy, BPs, and ZoeJ exhibited limited efficacy in eradicating other clinical isolates of Compared to natural phages, genetically modified phages offer stronger advantages in combating bacterial infections. As stated in While engineered phages offer numerous benefits, there are also potential consequences that need to be considered. Firstly, there is no doubt that the safety of utilizing engineered phages in human applications is of paramount concern. While phages are generally regarded as safe, rigorous testing is imperative to ensure that engineered phages do not elicit any deleterious effects on the overall health of patients. Additionally, like antibiotics, phages can also develop tolerance in host bacteria. Furthermore, there are concerns regarding the potential for phages to trigger bacterial tolerance similar to that observed with antibiotics. While certain treatment approaches, such as cocktail therapy, have been shown to mitigate bacterial tolerance to phages, the sustained efficacy of phage therapy may still be constrained by the existence of phage tolerance . FinallyWhile there are numerous unresolved issues surrounding the application of engineered phages, it is indisputable that, given the pervasive issue of antibiotic resistance, they continue to represent a crucial area of current research. Engineered phages possess immense potential for diverse applications, owing to their ability to be designed and modified to meet specific requirements for each application. Hence, they are emerging as a novel category of biological agents with extensive potential."}
+{"text": "The Belgian Society for Viruses of Microbes (BSVoM) was founded on 9 June 2022 to capture and enhance the collaborative spirit among the expanding community of microbial virus researchers in Belgium. The sixteen founders are affiliated to fourteen different research entities across academia, industry and government. Its inaugural symposium was held on 23 September 2022 in the Thermotechnical Institute at KU Leuven. The meeting program covered three thematic sessions launched by international keynote speakers: (1) virus\u2013host interactions, (2) viral ecology, evolution and diversity and (3) present and future applications. During the one-day symposium, four invited keynote lectures, ten selected talks and eight student pitches were given along with 41 presented posters. The meeting hosted 155 participants from twelve countries. Staphylococcus aureus phages and the first experimental therapeutic use of phages by Richard Bruynoghe and their student Jos\u00e9ph Maisin, both in 1921 [www.bsvom.be, accessed on 20 May 2023) on 9 June 2022, with its associated articles being published in the Belgian official gazette. The BSVoM aims to sustain and improve the dense Belgian research and development network on viruses of microbes by bringing together all stakeholders from academia, government and industry. Moreover, this national society is formally associated with the International Society for Viruses of Microorganisms . In spite of the small geographical size of Belgium, at least fourteen entities are currently active in the field of viruses of microbes, all represented by at least one co-founder , began with a keynote lecture by Prof. Edze Westra of the University of Exeter, Exeter, UK, who discussed their lab\u2019s research into the ecology and evolution of bacterial defense systems against phage predation. Westra has studied both the molecular and evolutionary aspects of bacteria\u2013phage interactions, focusing on the CRISPR-Cas adaptive immune system acting as a genetic memory to detect and destroy re-infecting phages that carry a cognate target sequence. Westra\u2019s lab combines genomics, mathematical modelling and experimental analyses of P. aeruginosa. Schroven\u2019s research involves performing several high-throughput analyses using a library of individually expressed proteins from lytic phages to identify potential effectors targeting virulence factors of P. aeruginosa. Schroven discovered three different phage ORFans that specifically and significantly attenuate key virulence factors of the pathogen, including the type IV pili, the type 2 secretion system and ExoS (a type 3 secretion system product). The identified phage-encoded virulence attenuators expand the diversity of regulatory mechanisms encoded by phages to impact bacterial physiology and may serve as a source for diverse biotechnological applications [The next speaker was Kaat Schroven of KU Leuven , who presented research on lytic phages as a treasure trove for virulence attenuating proteins against ications ,8.Streptococcus (GAS), specific phages and the human host. Utilizing RNA-Seq, they deciphered phage\u2013GAS interactions and found a reprogramming of up to 50% of the transcriptome of an M25 strain during infection by the virulent phage A25. The most downregulated genes belonged to the fatty acid synthesis (FASII) pathway. They also found that serum protects GAS from phage infection, while the addition of human serum albumin and fatty acids increases bacterial clearance. The topic of the first session switched towards Gram-positive host cells with the talk of Dr. Lionel Schiavolin, affiliated with Universit\u00e9 Libre de Bruxelles. They discussed the tripartite interactions of Group A P. aeruginosa\u201d, they discussed the problem of clinical strains of P. aeruginosa, which are equipped with multiple phage defense systems. Their research group demonstrated that the major determining factor for the host spectrum of phages specific to clinical strains is the intracellular defense system. Multiple systems can provide complementary and overlapping phage protection, resulting in panphage resistance. They also discovered that mobile genetic elements with phage defense systems can be exchanged via horizontal transfer, resulting in up to 19 phage defense systems per single P. aeruginosa clinical isolate. The data are available as a preprint under the same title [An important topic from the phage therapy point of view was covered by Prof. Stan Brouns . With their lecture entitled \u201cAccumulation of defense systems drive panphage resistance in Klebsiella pneumoniae, were the subject of collaborative research between Ghent University (Belgium) and the University of Wroclaw (Poland). Dr. Agnieszka Latka\u2019s presentation focused on phage KP32 receptor-binding proteins with depolymerizing activities. A crystal structure analysis revealed a multimodular structure with a central enzymatic domain, two additional C-terminal domains, a carbohydrate-binding module (CBM) and a lectin-like domain (LD) [Phages specific to another ESKAPE pathogen, ain (LD) . C-termiClostridium difficile phages, an important pathogen causing infectious diarrhea, with the view to developing them for next generation therapeutics. Their work has included unraveling phage diversity using computational, structural and phenotypic approaches and understanding the resistance rates towards phages compared to antibiotics. Furthermore, they have optimized ex situ models to study C. difficile\u2013phage interactions in epithelial cells and optimized biofilm, Galleria mellonella and artificial gut models to better understand application regimens, dosages and efficacies [The second session of the conference, chaired by Prof. Annika Gillis, focused on viral ecology, evolution and diversity. The keynote speaker was Prof. Martha Clokie, a renowned expert in bacteriophage biology from the University of Leicester in the United Kingdom, who is devoted to developing phages as therapeutics for humans and animals. Their work uses genomic and structural approaches to identify traits associated with phage efficacy and to study phage\u2013bacterial interactions in relevant models. Martha Clokie has pioneered research into ficacies ,12,13. ANext, Daan Jansen from KU Leuven presented a study to use community-typing as a tool to explore virome compositional changes in inflammatory bowel disease (IBD) patients (a group of chronic inflammatory diseases of the gut) . Viral mThe second scientific session was concluded with a presentation by Prof. Evelien Adriaenssens from the Quadram Institute Bioscience in the United Kingdom, who discussed the importance of phages in the healthy human gut microbiome. The advances in sequencing technology and bioinformatics have enabled the reconstruction of complete phage genomes, unlocking the investigation of phage diversity . EvelienBacillus cereus host. Vincent De Maesschalck  discussed the development of a bioluminescent ex vivo wound model to characterize novel phage-inspired enzybiotics [Escherichia coli K1 [A pitch session chaired by Dr. Pieter-Jan Ceyssens was organized in the middle of the symposium and preceding the poster session with the particular goal to give a first-stage experience to junior researchers and to attract attention to their poster . In totaybiotics . Sam van coli K1 . The besK. pneumoniae and P. aeruginosa). In her keynote lecture, she discussed the role of phage-borne depolymerases in bacteriophage\u2013bacteria\u2013host interactions. Bacterial surface glycans serve as molecular patterns, recognized by the immune system. Via glycan digestion or modification, bacteria can become sensitive to host immune responses. Prof. Zuzanna Drulis-Kawa demonstrated how depolymerases can be exploited for new diagnostics and therapeutic approaches against infectious diseases. She emphasized the need to deepen the knowledge of the phage enzyme structure and domain build-up, as well as in vitro and in vivo enzyme specificity and stability. Prof. Dr. Willem-Jan Metsemakers talked about bacterial infections as a major complication in orthopedics. Their incidence continues to rise due to higher numbers of both elective joint replacements and operatively treated fractures. Biofilms on the surface of implanted devices, intracellular bacteria and bacteria within canaliculi are known to cause infections. To address this, antibiotic therapy has been utilized; however, this is associated with side effects and the development of antimicrobial resistance. Investigations into the use of phage therapy for musculoskeletal infections are sparse; most of the knowledge coming from the Eliava Institute of Bacteriophages, Microbiology and Virology . Metsemakers is involved in multiple research projects on the topic of implant-related infections and reported a rising number of studies/instances in recent years [The third session, focusing on the present and future applications of phages, was chaired by Dr. Jean-Paul Pirnay from the Queen Astrid Military Hospital (Belgium). In this session, two keynote lectures were presented: the first by Prof. Zuzanna Drulis-Kawa , an executive committee member of the ESCMID Study Group for Nontraditional Antibacterial Therapy\u2013ESGNTA and current president of the International Society for Viruses of Microbes, and the second by Prof. Dr. Willem-Jan Metsemakers, a trauma surgeon at the Department of Trauma Surgery of University Hospitals Leuven  and associate professor at KU Leuven. Prof. Zuzanna Drulis-Kawa\u2019s research focus is the antibacterial activity of phages and phage-derived depolymerases and endolysins specific to ESKAPE pathogens  discussed the triple helix model of collaboration between industry, academics and government. They explained that the three players have synergistic functions: universities engaging in basic research, companies producing commercial goods and governments regulating markets. This model stimulates innovation by valuing the role of universities as creators of knowledge and enabling entrepreneurial activity by capitalizing on knowledge. Knowledge transfer between universities and industry is a critical aspect, for example, through technology transfer offices and joint projects. Governments act as a facilitator, providing funding and policy-making tools for economic growth and regional development. This form of innovation is most successful in a knowledge-based society. In the context of phage therapy, V\u00e9sale Biosciences has collaborated with LabMCT , signing a Triple Helix agreement in 2020 with the main goals of commercializing the personalized phage therapy model, developing a value chain from diagnostic to production and distribution and working closely with the academic and government sectors.Erwinia amylovora. Field trials at different locations within the EU have yielded promising efficacy data, with more than 90% efficacy being reported under natural infection pressure in open field conditions, while studies under greenhouse conditions with artificial high-dose E. amylovora inoculation were more challenging, requiring an appropriate multiplicity of infection (MOI). However, current EU plant protection regulations pose a challenge to registration due to the narrow host range of bacteriophages. A flexible regulatory framework that allows variability in the composition of a bacteriophage cocktail is needed to ensure its efficacy against any isolate of the bacterial target. This could provide a sustainable and biological alternative to chemical control of diseases.Plant-related phage application was discussed by Dr. Gil Luypaert and Anneleen Volckaert (De Ceuster Meststoffen nv (DCM), Belgium). They presented that DCM is developing a phage-based plant protection product to control fire blight caused by Pseudomonas aeruginosa PAO1 biofilms, grown on titanium coupons and treated with phages in combination with ciprofloxacin, confirmed these findings. It was concluded that a combination of phages and antibiotics is more efficient than either type of agent alone. Furthermore, the sequence of therapy (phage treatment first or antibiotic treatment first) resulted in the same reduction in infecting cells; however, a simultaneous combination therapy of both phages and antibiotics further diminished the number of pathogens present. Further research on optimizing the conditions of exposure is ongoing.The final talk of the conference was given by Steven De Soir from UCLouvain and Queen Astrid Military Hospital on the potential of a novel treatment modality for bacterial-biofilm-related infections on orthopedic implants. They studied the synergy between de novo isolated phages and routinely used antibiotics, such as ciprofloxacin, meropenem and ceftazidime. Significant reductions in CFU counts and biofilm biomass were seen when applying antibiotics in combination with bacteriophages. Scanning electron microscopy of the The BSVoM was founded to bring together the Belgian microbial virus community active in academia, industry, clinics and government. The enthusiasm present at its inaugural symposium was encouraging and underlines the benefits and opportunities of this initiative. A community that wishes to grow invests in its junior researchers. This goal was addressed by the pitch session, the numerous posters and follow-up initiatives such as a phage hands-on workshop in the Queen Astrid Military Hospital under the supervision of Dr. Jean-Paul Pirnay, Dr. Tea Glonti and Dr. Maia Merabishvilli. In addition, the \u201cMy hero and me\u201d webinar series was launched, giving junior researchers the opportunity to present a back-to-back webinar with their scientific heroes in the phage field that excel in their domain. The society further capitalized on its strong triple helix interactions already pre-existent to the foundation. Bringing all stakeholders together in a single community and symposium stimulates fruitful interactions and new initiatives. We are looking forward to the second edition of the BSVoM symposium, which will be held on 8 September 2023 at the University of Li\u00e8ge, Belgium."}
+{"text": "Phage therapy is a medical form of biological control of bacterial infections, one that uses naturally occurring viruses, called bacteriophages or phages, as antibacterial agents. Pioneered over 100 years ago, phage therapy nonetheless is currently experiencing a resurgence in interest, with growing numbers of clinical case studies being published. This renewed enthusiasm is due in large part to phage therapy holding promise for providing safe and effective cures for bacterial infections that traditional antibiotics acting alone have been unable to clear. This Essay introduces basic phage biology, provides an outline of the long history of phage therapy, highlights some advantages of using phages as antibacterial agents, and provides an overview of recent phage therapy clinical successes. Although phage therapy has clear clinical potential, it faces biological, regulatory, and economic challenges to its further implementation and more mainstream acceptance. Phage therapy employs viruses that specifically infect bacteria to combat bacterial infections, including those infections that do not respond to treatments using traditional antibiotics. Despite being developed 100 years ago, phage therapy is currently experiencing a resurgence in interest, as outlined in this Essay. To explore why that is so, this Essay begins by introducing basic bacteriophage biology, some of the post-1921 history of phage therapy, and also multiple advantages associated with using phages as antibacterial agents. We then turn to the growing catalog of recent clinical phage therapy successes, discussing the general nature of these studies in particular, as well as important future directions. Despite these successes, multiple obstacles to the further development, acceptance, and approval of phage therapy continue to exist, which we differentiate into biological hurdles to contrast with those we dub instead as \u201csocietal.\u201d Overall, although we highlight the increasing potential for phages to serve as alternatives or adjuncts to antibiotic therapy for bacterial diseases, we emphasize the remaining challenges to making this promising technology more clinically available. For a complementary recent review emphasizing multiple additional aspects of phage therapy not covered here, we point the reader to Strathdee and colleagues .\u201c\u2026le microbe antidysent\u00e9rique est un bact\u00e9riophage obligatoire.\u201d [atoire.\u201d Bacteriophages, first and foremost, are viruses of Bacteria, sharing the world with viruses of Archaea and viruses of domain Eukarya. An alternative ecological categorization separates those that infect primarily \u201cmacro\u201d-organisms  from those that infect microorganisms  ,18. For Phages recently have been differentiated into numerous taxa\u2014particularly families and genera but also subfamily ranks\u2014as based on genomic similarities between isolates . More trThe virion-productive life cycle of all tailed phages ends in lysis of the host bacterium, initiating an extracellular search for new bacteria to infect  Fig 1)..Fig 1). Escherichia coli O157:H7) [During lysogenic cycles, temperate phages protect their bacterial hosts from being lysed by other, related phages. This type of protection is known as superinfection immunity ,38. AlthO157:H7) ,43.Even though many or even most viruses of archaea are also tailed , archaea\u201cSoon after F\u00e9lix d\u2019H\u00e9relle discovered bacteriophages in association with diarrheal illnesses, he speculated that phages were responsible for the usual recovery from such disease through their antibacterial action in vivo.\u201d [n vivo.\u201d Historically, the translation of phage therapy from the bench to the clinic has happened at a rapid pace. This is in part because systems for testing new therapeutics were not as well developed 100 years ago as they are today. In addition, at that time, there were few alternative approaches to responding to the great deal of morbidity and mortality associated with bacterial infections . It was We can consider the historical development of phage therapy in terms of phases or periods. Especially in North America, we can distinguish these different phases into what Summers  describeMore recently, bacteriophage-derived antibacterial enzymes, also called \u201cenzybiotics,\u201d have raised interest due to their typically rapid and unique modes of action along with their high specificity ,83. TheyThe inherently bactericidal nature of especially obligately lytic phages is not the only attribute that makes phages useful as antibacterial therapeutic agents \u201389. Curt\u201cThe excitement about the prospects of phage therapy (PT) has been growing worldwide, fueled by the recent reports of its successful application in severe cases of bacterial infections.\u201d [ctions.\u201d A rise in phage therapy clinical reports is noticeable starting from 2018. This represents a landmark year for the growing implementation of modern phage therapy (\u201crecent interest\u201d), resulting in important new English language evidence of clinical phage therapy efficacy. Numerically, while only 2 clinical reports were published in 2015, 1 in 2016, and 5 in 2017, this rises to 13 in 2018, 16 in 2019, and 11 in 2020 . Our as-Many of these newer, clinical phage therapy reports have been case studies or case series conducted as compassionate treatments ,97,98. TBearing in mind these limitations, as well as the small sample sizes of these studies\u2014a majority are single case reports and only a few are case series\u2014and also the nature of the studies , we discuss below representative reports of clinical improvements during and after use of phage therapy, highlighting in s; [The concept of personalized or bespoke phage therapy may be viewed as \u201ca paradigm shift in the development and application of infectious disease therapeutics\u201d . Indeed,s; ), a new,s; , which ms; .Excellent consensus documents have also recently emerged from Europe  and the While important hurdles remain to be addressed, the current use of phage therapy and still somewhat informal but improving evidence of its efficacy suggest that phage therapy likely will be introduced to the mainstream especially as a personalized medicine. Employing the abovementioned standardized and multidisciplinary approaches will help identify and address major scientific and treatment hurdles for furthering the acceptance of phage therapy in the context of personalized medicine, but also help to improve the design of randomized controlled trials.Engineering can enhance the therapeutic potential of bacteriophages ,131\u2013133.Mycobacterium abscessus. For this case study, 1 naturally obligately lytic phage (phage Muddy) and 2 temperate phages (BP and ZoeJ) were identified that could effectively kill the clinical isolate, starting from a library of over 1,800 phages. To generate a therapeutic cocktail, the authors employed a Bacteriophage Recombineering of Electroporated DNA (BRED) technique [A breakthrough study by Dedrick and colleagues , in 2019echnique  to removechnique . This stechnique .Mycobacterium) and used for compassionate care . CarefuWhile the safety of phage therapy has been demonstrated in more than 50 studies conducted since 2000 , systemaS. aureus bacteremias, including infective endocarditis [In 2020, a study conducted as a single-arm, noncomparative trial explored the safety and tolerability of phage therapy in 13 severely ill patients with carditis . A systeMycobacterium infections reported safety and tolerability of phage therapy, using the noted natural and genetically modified phages [In 2022, a case series involving 20 patients with non-tuberculosis d phages . The aut\u201cNotwithstanding the extensive need, interest, experience and reported successes of phage therapy, typical western approaches to biomedical research and implementation are poorly adapted to motivate, regulate or assess such nonstandard approaches to antibacterial therapy.\u201d [herapy.\u201d Phages not only offer numerous advantages as antibacterial therapies but also, despite their ongoing and increasing use for treating bacterial infections for which antibiotics are not or no longer useful, present a number of challenges to their increased application. These challenges include issues of limitations to phage host ranges and thereby spectra of activity, the potential for development of bacterial resistance to phages, possible negative impacts of antibiotics on phage functionality, treatment phage-mediated transduction of bacterial DNA, interactions with the immune system, regulatory issues, unusual pharmacology, insufficient awareness of phages as therapeutic antibacterial agents, and the potential for phage therapy skepticism . We diffAlthough obligately lytic phages are inherently bactericidal, that does not mean that their existence as potential antibacterial agents is without limitations. Many of these issues, along with phage-induced bacterial lysis, are aspects of the pharmacodynamics of phage therapy, particularly treatment-phage impact on bacteria but also their effect on treated bodies . Though The host ranges of phages tend to be relatively narrow, typically consisting of only a subset of strains within a single bacterial species ,150. IndA partial answer at least to the first concern , but in some cases to the second as well , is the development of what are known as phage cocktails \u2013157. CocIt is the nature of genetic entities to evolve, if they can, in response to environmental conditions. This is abundantly true in response to chemotherapies, whether what is being treated is abnormal cell growth within bodies or bacterial populations being exposed to antibiotics. The same is true for bacteria and phages . We can Mutations to phage resistance, in particular, can result in antagonistic pleiotropies (trade-offs) . These, Limitations to phage host ranges are a corollary of phage resistance, and both can be addressed by the use of phage cocktails. This usually is accomplished by combining phages with complementary host ranges to increase empirical coverage for a single targeted bacterial species. Application of novel combinations of antibiotics can also be justified toward increasing empirical coverage . The genTo employ phage cocktails to limit bacterial capacities to evolve resistance, it is generally necessary to include within cocktails not just multiple phage types possessing complementary host ranges but also more than 1 phage host range type that is able to infect the targeted bacterial strain, and for which bacterial mutation to cross-resistance to both (or more) cotargeting phages is of low probability. In other words, to reduce the potential for bacteria to evolve resistance to phages by employing phage cocktails, it is necessary that cocktails possess a sufficient \u201cdepth\u201d of antibacterial activity , with a As antibiotics represent the standard of care for treatment of bacterial infections, it is inevitable that phage therapies will often be undertaken as antibiotic cotherapies . As is tAlternatively, combining antibiotic and phage treatments can increase overall effectiveness. In order of increasing effectiveness, this can variously be described as involving facilitating, additive, or synergistic interactions . These aWe can also differentiate whether phages in combination with antibiotics are more effective at simply clearing susceptible bacteria in the near term or, instead, are more effective at interfering with the evolution of phage or antibiotic resistance over longer time frames. Near-term improvements could be associated with PAS but can also result from simply additive interactions, as well as the noted facilitation. Longer term, the typical lack of cross-resistance to phages and antibiotics should increase the number of bacterial mutations required to achieve full resistance to a combined treatment ,185. ThiTransduction is phage virion-mediated movement of non-phage DNA between bacteria . It is ppac sites and headful genome packaging, both in combination with phages not substantially degrading the DNA of their host bacterium in the course of lytic cycles [It is also possible to simply avoid using phages that are capable of transducing bacterial DNA, i.e., by not using so-called \u201ctransducing phages.\u201d The latter include temperate phages, with their ability to move small numbers of bacterial genes in what is known as specialized transduction . Of probably greater relevance to phage therapy is avoidance of phages that package their DNA using c cycles . Phages Of the biological challenges facing phage therapy, the potential for transduction nevertheless is often considered a lesser concern . This isIn clinical settings, phage\u2013bacteria dynamics develop in conjunction with pressure from the mammalian host immune system, and this has often been stated as a substantial concern regarding the clinical implementation of phage therapy. In particular, there are concerns that a patient\u2019s immune status will influence the effectiveness of phage therapies, but also that phage\u2013immune system interactions might be harmful to patients. Alternatively is the concept of \u201cimmunophage synergy,\u201d as presented by Roach and colleagues , where bS. aureus infections [Phages can be found, in particular, in the human gastrointestinal tract, including, e.g., those infecting Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria ,209,210.fections . Even iffections ,212. Phafections ,213,214.fections  as it alfections ,202.An additional issue during phage therapy is that treatment of gram-negative pathogen infections with high phage dosages may lead to a synchronized lysis of large numbers of bacterial hosts along with concomitant release of endotoxin. This triggers inflammation, typically via Toll-like receptor 4 pathways, as well as fever and may cause local pain . NeverthWidespread implementation of nonstandard treatments by physicians requires not just evidence of efficacy but also regulatory approval, successful marketing, and a willingness of those involved to undertake such treatments. In this section, we consider these additional, societal challenges to broader adoption of phage therapy, especially by Western medicine.In the former Soviet Union, phages were mass produced for therapy and have long been available, even over the counter ,216, witTo move forward, either current regulations will need substantial modification or new legislation will need to be proposed to cover aspects of phage therapy that differ from those of traditional antibiotics. Regulations, in particular, need to accommodate phage genetic malleability , their narrow host range, and their unusual pharmacological properties. Both US and European regulatory authorities at least agree, however, that therapeutic phages should be classified as biological therapies , requiriBecause existing regulations have been developed for industry-scale production of medicines, they are less well suited to the more personalized approaches of phage therapy . InsuffiAn important component of successful regulatory approval of drugs is a robust characterization of their associated pharmacology. Study of the pharmacology of phages has been somewhat neglected, however, owing to how long historically phages have been used to treat bacterial infections, which largely has predated the development of modern pharmacological study, and also, arguably, due to the relative safety of phage use as antibacterial agents, which have made concerns over potential toxicities somewhat moot . In addiPharmacology can be considered, in particular, along the traditional divisions of PK and pharmacodynamics (PD), and this is true for phage therapy pharmacology as well ,224,225.max/MIC), and the fraction of time between dosings over which in situ drug concentrations exceed MIC (abbreviated as t>MIC). These key PK identifiers, however, have not been as well defined for phages as they have been for antibiotics. This is, at least in part, because although multiple groups have used checkerboard-type assays to describe phage MICs [A drug\u2019s PK can be broadly defined by a handful of key identifiers. These include area under the curve (in situ concentration as a function of time) divided by minimum inhibitory concentration (AUC/MIC), maximum drug in situ concentration also divided by MIC  may be useful toward achieving treatment success [Difficulties in defining phage therapy PK as well as MICs stem largely from the potential for phages to proliferate during treatments. Compounding this complication, the extent of this in situ phage proliferation will tend to vary with phage properties (particularly as impacting the productivity of their infections), the properties of targeted bacteria, and the properties of the treated infection itself . Another confounding variable is the noted PAS ,182,183, success ,58,225 a1/2) also may vary with routes of phage administration [Especially when phage proliferation is less influential, then AUC and thereby phage therapy PK should be more similar to that of traditional pharmaceuticals. This should be seen particularly with so-called passive treatments ,231,232 stration ,237.The stage that an infection is treated is another significant factor affecting phage therapy PK. Biofilm-resident bacteria, which are often found later during chronic bacterial infections , can be While phage therapy PK can be somewhat more difficult to define than those of antibiotics, the in situ phage proliferation underlying much of that difficulty, i.e., phage auto-dosing, is likely also a particular advantage of phage therapy. Nevertheless, to achieve consistently effective phage treatments, routes and dosages of phage administration must be evaluated and standardized to each specific phage\u2013host-infectious disease combination ,244,245.Trust in phages and phage therapy remains strong in former Soviet Union republics, particularly Georgia and Russia ,216. In Phage Companies.There has been a corresponding increase in funding for phage research and initiation of a number of controlled clinical trials in the field ,147,249.Physicians appear to be playing less of a role in driving the resurgence of interest in phage therapy. A survey from 2019 conducted in the largest Belgian hospital and biggest phage therapy center in Western Europe  indicated that more than 70% of phage therapy requests came from patients themselves or their family members and only one-third or so from treating physicians . This waA somewhat unique challenge to the use of phages as antibacterial agents is a combination of their long history and insufficiently well-documented efficacy. The former allowed for the initial \u201centhusiasm\u201d for phage use. Particularly, their ability to target bacteria but not our own tissues (selective toxicity) was at the time (in the 1920s and 1930s) without peer among readily available medicaments. This, however, also provided little incentive to study the clinical use of phages rigorously. In addition, many successful anecdotal case studies were poorly documented, and it is thought that many treatment failures were a consequence of poorly formulated or applied therapies. Phage therapy\u2019s early challenges to rigorously establish itself also stemmed from a dearth of well-controlled clinical trials  in Together, these issues seem to have resulted in long-standing cultural impediments to phage therapy implementation . SkepticFurther complicating the broader implementation of phage therapy are economic uncertainties associated with phage therapy development. While biotech companies often succeed in translating basic research into profitable clinical applications , investmNaturally occurring, i.e., not genetically engineered phages, by contrast are overwhelmingly what have been used in the development as well as clinical implementation of phage therapies. But without robust patentability, there is less financial incentive to invest in the kind of vigorous research necessary to overcome phage therapy skepticism, particularly including the funding of clinical trials, and also toward financing phage therapy commercial development more broadly. The fragile patentability of naturally occurring phages for phage therapy thus may represent the greatest societal challenge to phage therapy and, indeed, challenge to phage therapy generally in those many countries where phages are not yet regulatorily approved as antibacterial treatments. We return to yet further considerations of phage therapy economic issues in the following, final section of this Essay.\"Although more translational research is needed before the clinical implementation of phage therapy is feasible, phages may be pivotal in safeguarding the overall health of humans in the near future.\" [future.\" Phage Directory facilitate connections between phage suppliers, such as from academic research laboratories, with possible phage clinical users, i.e., doctors [There is a wealth of preclinical as well as more basic science-derived data supporting the potential for phage therapy use clinically. At the other extreme, various phage therapy centers and initiatives , compani doctors .Overall, then, there are 4 general routes to human phage therapy. The first is administration without clinical supervision, which is possible in places where phages are available over the counter, particularly Georgia and Russia . The secLastly are clinical trials, which are necessarily limited in scope and expensive to run, but which are essential for the explicit proof of phage safety and efficacy that regulators, prescribers, and consumers need. To date, a handful of phage therapy clinical trials have been published in English language journals . ThoughA final and nontrivial problem is the reduced commercial enthusiasm to bring new antibacterial agents of any kind from the laboratory to the clinic. There is a general expectation that antibacterial agents will not only be used for only short periods of time  but will be relatively inexpensive during that ideally somewhat brief use . Togethe"}
+{"text": "The discovery of antibiotics has revolutionized medicine and has changed medical practice, enabling successful fighting of infection. However, quickly after the start of the antibiotic era, therapeutics for infectious diseases started having limitations due to the development of antimicrobial resistance. Since the antibiotic pipeline has largely slowed down, with few new compounds being produced in the last decades and with most of them belonging to already-existing classes, the discovery of new ways to treat pathogens that are resistant to antibiotics is becoming an urgent need. To that end, bacteriophages (phages), which are already used in some countries in agriculture, aquaculture, food safety, and wastewater plant treatments, could be also used in clinical practice against bacterial pathogens. Their discovery one century ago was followed by some clinical studies that showed optimistic results that were limited, however, by some notable obstacles. However, the rise of antibiotics during the next decades left phage research in an inactive status. In the last decades, new studies on phages have shown encouraging results in animals. Hence, further studies in humans are needed to confirm their potential for effective and safe treatment in cases where there are few or no other viable therapeutic options. This study reviews the biology and applications of phages for medical and non-medical uses in a narrative manner. In the fight against infectious diseases, the application of hand hygiene and the discovery of antibiotics are the two most important innovations that have radically changed the course of history through the reduction of infection incidence as well as through the reduction of morbidity and mortality associated with infectious diseases. However, right after the discovery of penicillin by Fleming and the widespread use of antibiotics for medical purposes in the 1940s, it became quite evident that inappropriate use of these agents could lead to the development of resistant microorganisms . This phAcinetobacter baumannii is a microorganism classically involved in hospital-acquired infections that has gained considerable antimicrobial resistance during the last decades, and this has left few therapeutic options, namely colistin. On the other hand, the development of PDR strains left no clear therapeutic option, leading to the use of antimicrobial combinations of two or three drugs, usually including colistin, tigecycline, or other antimicrobials, in a method of bending the mechanisms that render this pathogen resistant [For many decades, antibiotic development surpassed antibiotic resistance. Hence, the problem remained dormant. However, in the last decades, the antibiotic pipeline slowed down . This waesistant ,13,14. Iesistant . Of noteesistant . Hence, esistant ,18,19. Desistant .Based on these data, it becomes clear that there is an imperative need for the development of new approaches either in terms of classical antimicrobials or in terms of novel compounds such as antimicrobial peptides or bacteriophages that could be used in the fight against infectious diseases and, more specifically, against drug-resistant microorganisms. The aim of the present study is to review the history, the present condition, and the future implications of phage therapy against infectious diseases.Mycobacterium abscessus, a collection of more than 10,000 phages that were isolated using Mycobacterium smegmatis needed to be screened to identify just three useful phages [Bacteriophages (phages) are viruses with a size of 20 to 200 nm that infect bacteria with very high specificity . For exal phages . In the l phages ,24,25. Dl phages ,27,28,29Caudovirales, which are tailed viruses (cauda is the Latin word for \u201ctail\u201d) and, according to the Baltimore classification scheme, are group I viruses with double-stranded DNA (dsDNA) for their genome, with a length of 3400 to 500,000 base pairs [Phages were discovered independently by two scientists: the British bacteriologist Frederick William Twort in 1915 and the French-Canadian microbiologist Felix d\u2019Herelle in 1917. D\u2019Herelle noticed that these entities had the ability to kill bacteria and named them \u201cbacteriophages\u201d . Phages se pairs . Other tse pairs .Escherichia coli through two sets of tail fibers [Bacillus subtilis or the baseplate proteins used by some Siphoviridae phages to initiate the infection of Lactococcus lactis [The life cycle of a phage involves its binding to a receptor of a bacterium that leads to releasing of the genetic content of the phage into the bacterium. This binding occurs after the recognition of a receptor of the bacterium that allows the phage to attach to and enter the bacterial cell. These receptors are commonly proteins or sugars on bacterial cells recognized by proteins of phages that mediate their adhesion on the bacterial cell, leading to the entry into the bacterial cell . An examl fibers . Other es lactis ,35. Seves lactis ,36. Untis lactis . Currents lactis .After the binding of the phage to the bacterial receptor and the genome injection into the cell, the genetic material of the phage takes advantage of the molecular machinery of the host bacterium and undergoes transcription, translation, and replication, leading to the formation of the required components of new phages that are, further on, released with concomitant lysis of the bacterium. Then, this process is further repeated by the new viral particles after the infection of new bacterial cells. Hence, using phages as an antibacterial treatment has the theoretical advantage of using a self-amplifying therapeutic modality, contrary to the therapeutic mode of classical antibiotics that require repeated doses and, often, prolonged treatment for achieving the required clinical result . HoweverStaphylococcus aureus or Escherichia coli [Phages have been long used for prophylaxis and treatment in cattle, where the most common infectious diseases are mastitis, metritis, or respiratory tract infections, and most of them are due to bacterial causes . For a lhia coli ,41,42,43Foodborne illnesses result in hundreds of millions of human infections and hundreds of thousands of deaths every year, posing a significant public health issue ,45. DecoCampylobacter jejuni or Salmonella enterica serovar Enteritidis, the phage titers increased, and the pathogen bacterial load was reduced [Salmonella flexneri, Salmonella dysenteriae, and Salmonella sonnei was found to effectively reduce the likelihood of contamination of ready-to-eat chicken products by Shigella spp. [Listeria monocytogenes [E. coli O157:H7; SALMONELEX  targeting Salmonella spp.; and Listex P100 , which consists of a single phage targeting L. monocytogenes, are currently available [As shown by several studies, the direct application of lytic phages to food that is ready to be used can reduce the number of potentially hazardous foodborne bacteria in a significant manner ,51,52,53lla spp. . The U.Sytogenes . Other svailable .Moreover, phages can be of great help in food safety if used in biosensors to detect pathogens that may contaminate food. For example, current methods for the diagnosis and analysis of food samples are more laborious and time-consuming than the application of biosensors that could occupy specific phages to detect food contamination by specific bacteria. These could take the form of biosensors that could use bio-probes and transducers in the biosensor to allow timely identification of food contamination ,56.E. coli O157:H7, L. monocytogenes, or Salmonella [L. monocytogenes on non-food equipment, as they prevent the formation of biofilm and may even help to eliminate it if it has been previously formed [(a)Novel Food Regulations: Many countries have regulations governing the approval and use of novel food ingredients, which can include phages. These regulations are designed to ensure the safety and proper labeling of new or non-traditional foods. Phages used in food applications may need to undergo a regulatory approval process to demonstrate their safety and efficacy before being permitted for use;(b)Risk Assessment and Safety Evaluation: Regulatory authorities typically require a thorough risk assessment and safety evaluation for novel food ingredients, including phages. This evaluation may include determining the potential for adverse effects on human health, assessing the likelihood of gene transfer or antibiotic resistance development, and evaluating the stability and persistence of the bacteriophage in the food environment;(c)Codex Alimentarius: The Codex Alimentarius Commission is an international body that develops food standards, guidelines, and codes of practice. Codex standards provide a reference for national regulatory authorities when developing their own regulations. The use of phages in food safety may be subject to Codex guidelines or specific regulations implemented by individual countries in line with Codex recommendations;(d)Labeling and Consumer Information: Proper labeling and consumer information are important aspects of food regulations. Regulatory authorities may require clear and accurate labeling of foods treated with phages, including information on the presence of phages, their specific targets, and any necessary handling or storage instructions;(e)Country-Specific Regulations: Each country has its own regulatory framework for food safety, including the use of novel ingredients such as phages. The requirements and approval processes can differ significantly between countries due to variations in risk assessment methodologies, regulatory structures, and levels of acceptance for novel technologies ,67,68,69Another aspect regarding food safety has to do with biofilms. Biofilms are complex structures that are hard to remove or eradicate with the use of disinfectants, and they have inherently very high resistance to antimicrobials, as they have several layers of microorganisms with different levels of metabolic dormancy as well as a rich extracellular matrix that also poses a barrier for the diffusion of antimicrobials. Biofilms pose a threat to consumers of dairy products. Phages could also be of use for decontamination of inanimate surfaces that may host biofilms, thus reducing the likelihood of disease transmission across the dairy chain ,40,57. Ilmonella ,59,60,61y formed . Howevery formed ,63. The Ralstonia solanacearum, a phage cocktail, P1, that contained six phages was used in preventing potato bacterial wilt by decontamination of sterilized soil spiked with R. solanacearum or by injecting the phage cocktail into the plants [R. solanacearum on a tomato rhizosphere led to complete inhibition of bacterial wilt occurrence, while amendment of the surfactant Silwet L-77 at 0.1% to the phage suspension did not affect its disease control activity [Xanthomonas campestris pathovar vesicatoria and Pseudomonas syringae that are responsible for causing disease in tomatoes and peppers, or AgriPhage-Fire Blight , which was approved for use against fire blight (caused by Erwinia amylovara) in pears and apples [Some plant pathogens may cause disease leading to severe financial losses in agriculture by affecting the quality of products and reducing the production yield. Phages could be an option in agriculture for allowing the reduction of plant pathogens and reducing their hazardous effects . Indeed,e plants . In anotactivity . Currentd apples . These pd apples .Lactococcus, Vibrio, Pseudomonas, and Aeromonas have the potential to cause disease in cultured fish and shellfish, while they could also, under specific circumstances, cause disease in humans as well [Edwardsiella tarda phage (ETP-1) that was isolated from marine fish-farm water was found able to control infection by the pathogenic MDR E. tarda in zebrafish aquaculture [Some species of the genera  as well . To that as well ,76. Seve as well ,82,83,84Vibrio, E. coli, Shigella, and Salmonella are of particular concern for human health, and their control in this setting is a priority. Hence, the likelihood of human disease can be reduced without affecting antimicrobial resistance [Contamination of wastewater plants by waterborne bacteria is of global concern due to the associated morbidity and mortality as well as the high costs required for these plants\u2019 disinfection. Some bacteria such as sistance . In partsistance ,86. Howesistance .S. aureus and, more specifically, methicillin-resistant (MRSA) strains that are of particular concern due to difficulties in treatment as well as Pseudomonas aeruginosa and E. coli, which are among the most common causes of HAIs and are also associated with particularly problematic antimicrobial resistance patterns [Hospital-acquired infections (HAIs) are a leading cause of morbidity and mortality in hospitals and are closely associated with antimicrobial resistance ,90,91,92patterns ,99,100. patterns ,102,103.patterns ,104,105.E. coli, Salmonella, MRSA, or A. baumannii, and their results are indeed promising [A. baumannii along with standard cleaning procedures, the occurrence of HAIs due to this microorganism was substantially reduced, implying that phage decontamination could be of practical use in the fight against HAIs [There are studies assessing the effect of phages against pathogens that could be isolated from hospital surfaces, such as romising ,106,107.romising ,106. Intnst HAIs .Bacillus genus in an effort to control the microbiome of the hospital [Staphylococcus species in bathrooms, which are the most heavily contaminated areas of hospitals, showed that daily sanitation by the combination led to a rapid and significant decrease in the levels of Staphylococcus spp. on the surfaces where it was applied, and it was 97% more effective as compared to the use of PCHS alone [In an effort to promote clever and eco-friendly cleaning by respecting non-pathogenic bacteria on hospital surfaces, phages were evaluated in addition to eco-friendly detergents (probiotic cleaning hygiene system (PCHS)) containing non-pathogenic probiotic bacteria of the hospital ,110. To hospital ,113,114.hospital ,90,115. hospital ,109,116.P. aeruginosa showed that phages can be successfully combined with chemical disinfectants such as benzalkonium chloride and sodium hypochlorite to increase the efficacy of wet biofilm and bacterial spot removal on surfaces and also reduce the likelihood of biofilm regeneration [Notably, phages can be used not only for the disinfection of hospital surfaces and medical equipment from free bacteria but could also be of use for disinfection from biofilms . For exaneration .Moreover, phages could be also used in medical applications in non-human settings if used in biosensors, as in food safety, since in that case biosensors could use bio-probes and transducers to allow timely bacterial contamination in critical specimens and surfaces ,56. FiguSalmonealla gallinarum with success [Shigella dysenteriae [Immediately after the discovery of phages by Twort and d\u2019Herelle, d\u2019Herelle postulated that phages could have therapeutic applications. In 1919, he used phages to treat chickens that were infected by  success ,120. Basenteriae ,121. Furenteriae .After these encouraging results in animals and humans, other scientists recognized phages\u2019 potential in prophylaxis and treatment and started targeting other infections, although with partial success. Beyond criticism regarding the quality and the design of these studies in humans, an important drawback at that time was the phages\u2019 high specificity against certain bacteria, implying that previous recognition of the pathogen was absolutely necessary to allow successful treatment by phages . In 1923E. coli, showing the greatest results in in vitro virulence [A series of well-designed experiments by Smith and Huggins helped to rediscover phage therapy, as they assessed several issues that had been identified as limitations in the previous sets of experiments in the first half of the 20th century and also confirmed that phage treatment is safe and efficient in animal models . In the irulence . In the irulence . Furtherirulence .E. coli diarrhea model in calves. They developed a multi-phage treatment plan to combat the development of resistance during treatment. Before experimenting in vivo, they performed in vitro studies. Hence, they chose lytic phage B44/1, which had the ability to infect only K85+ strains of E. coli. Then, they isolated bacterial mutants that were resistant to that phage in vitro. Subsequently, they chose another phage, namely B44/3, which was able to infect bacteria resistant to the initial B44/1 phage that they used. Additionally, they selected bacterial mutants resistant to B44/3 but susceptible to B44/1. By evaluating the dynamics of phage resistance before using them in vivo, they were able to use this double-phage approach to overcome the problem of resistance evolution to phages [In another set of experiments, Smith and Huggins investigated the issue of bacterial resistance to phages, choosing an o phages . Smith ao phages . Thus, to phages .P. aeruginosa or A. baumannii [E. coli provided 100% protection to mice, while treatment with phage not targeting K1 led to a mortality of 60%. A comparison with streptomycin revealed that the phage targeting K1 led to 9% mortality, while treatment with streptomycin was associated with 54% mortality, thus confirming the previous findings by Smith and Huggins [New experiments from other research groups have further elucidated questions on phage biology and therapeutics. A more recent study showed that even though rapid clearance of phages in the blood was considered a drawback, it is possible to select phage variants that have longer blood half-lives . Other eaumannii . Moreove Huggins .The 21st century offers many more tools than the previous one, such as high-throughput methods for efficiently screening thousands of samples at the same time, affordable whole-genome sequencing, automated technology for microbiological techniques, etc. On the other hand, clinical studies have shifted to a different level, with a need for more adequate design for power, double-blind and randomized settings, as well as higher standards regarding safety for participants. Current technology allows deeper investigations at the time of the study or even after that, addressing clinical and biological questions that may have to do with the in vitro as well as the in vivo interaction of the phage with the target bacterium and the immune system of the human host .Mycobacterium smegmatis was needed for screening to allow the identification of just three useful phages that were used for the treatment of a young boy with cystic fibrosis and infection by Mycobacterium abscessus. Two of them were engineered to allow appropriate bacterial targeting [Importantly, the well-known physiological property of very narrow targeting of phages that could be considered a limitation in some instances could be addressed with the current technology. The work by Dedrick et al. showed an example of how narrow the targeting of phages is and how phage engineering could be elaborated. In this study, a collection of\u2009more than 10,000 phages isolated using argeting . Nowadayargeting ,138.Regarding the development of resistance to phages by bacteria, modern technologies of evolutionary biology could provide insights about appropriate phage selection. In particular, the selection of a phage targeting a specific molecule as a receptor leads to the selection of bacterial strains that do not express it. Hence, therapeutic use of phages could take advantage of the receptors that are concomitantly virulence factors. For example, the selection of strains that do not produce the virulence factor that is the target receptor for the phage might lead to bacterial survival; however, their virulence would be lower, leading to better infections outcomes ,139,140.Currently, the application of artificial intelligence in the field of phage therapeutics is a trending topic since it can facilitate the selection of phages depending on the specific characteristics of the target pathogens and the host\u2019s profile as well ,148,149.Pseudomonas sepsis, 67% survival was noted after oral administration of phage therapy one day after the infection of mice [Enterococcus faecium resistant to vancomycin [Vibrio vulnificus, successful treatment was noted only when the administration of phage therapy was performed at the same time as the infection by the bacteria [Phages have been found to be effective in the management of systemic infections in several animal models. In a gut-derived model of  of mice . Phage dncomycin . In anotbacteria . Thus, tP. aeruginosa in mice treated at the same time with phages showed 92% survival [S. aureus leading to the formation of abscesses, administration of phages at the same time as the pathogen prevented the formation of abscesses; meanwhile, when phage treatment was given 4 days after the bacterium inoculation, a single dose of phage treatment led to 100-fold reduction of the bacterial load, and when multiple doses of phage treatment were applied, 10,000-fold reduction was observed [The use of phages for the treatment of localized infections such as an abscess, an ear infection, or a burn has been proven very efficient. An intraperitoneal model of infection by survival . In anotobserved .E. coli to the gut of mice led to reduced bacterial colonization and was associated with a reduced likelihood of colitis development [Clostridioides difficile colonization showed that prophylactic treatment with a phage two hours before inoculation of bacteria led to 100% survival. Simultaneous administration of phage and bacteria led to a 72% survival, while phage treatment two hours after inoculation of bacteria led to 30% survival [The use of phages against pathogens infecting the gastrointestinal tract may eradicate the pathogenic bacterium without altering the normal gut flora. Phage treatment four days after inoculation of adherent-invasive elopment . A studysurvival .P. aeruginosa showed that intranasal treatment with two doses of phages led to complete eradication of the bacterium when administered 24/36 or 48/60 h after initiation of the infection, while treatment at 144/156 h after an infection led to complete eradication of the infection in 70% of the animals and a significant reduction of the bacterial load in the lungs of the rest [Treatment of lung infections with phage therapy could help people with chronic lung infections, such as those with cystic fibrosis, who are also at particular risk of colonization and developing infections by antibiotic-resistant microorganisms. An experiment in mice using Campylobacter jejuni in chicken [Salmonella enterica serovar Enteritidis in chicken [S. enterica serovar Typhimurium in weaned pigs [S. aureus in bovine mastitis [S. aureus in lactating dairy cattle, among others [Phage therapy may also have application in veterinary medicine, with phages being used against a variety of pathogens and in a variety of hosts. Hence, phages have been effectively tested against  chicken ,160,161, chicken ,163,164,ned pigs ,166, agamastitis ,168, andg others ,170.E. coli, treatment with fluoroquinolone (enrofloxacin) led to a reduction in mortality from 68% down to 3%, while treatment with phages led to a reduction in mortality to 15%. Combination treatment with fluoroquinolone and phage simultaneously led to 0% mortality [P. aeruginosa, combination treatment of ciprofloxacin and phage led to a 10,000-fold reduction of bacterial load compared to treatment with ciprofloxacin or phage alone; meanwhile, the same combination showed synergy in the killing of P. aeruginosa both in vitro and in vivo [There are several studies evaluating the effect of phage treatment on specific infections. However, there are few studies evaluating the effect of the combination treatment of phages with antibiotics, with some of them being promising. Hence, in a study of broiler chickens infected with ortality . Another in vivo . Thus, eP. aeruginosa bacteremia and failed treatment with anti-pseudomonal antimicrobial agents was treated with two-phage combination with temporary blood sterilization. However, after the end of this treatment, the blood cultures became positive again [The use of phages in humans for the treatment of infectious diseases in the modern era is limited. However, there are studies evaluating their efficacy and safety ,173. Theve again . It is oP.-aeruginosa-infected aortic graft, complicated by aorto-cutaneous fistula with purulent discharge, failed very prolonged treatment with several antibiotics. Since, at that point in time, the patient was not a candidate for a new surgery, a phage active against P. aeruginosa that was shown to have synergy with ceftazidime and was screened for lytic activity against the causative organism was applied locally in the exit point of the fistula, along with systematic administration of ceftazidime [In another case report, a patient with a tazidime ,175. One9 PFU for two weeks was shown to be a safe and well-tolerated treatment, while the preliminary efficacy observations were promising [Recent clinical trials in humans often show contradictory findings . Howeverromising . This stP. aeruginosa were recruited in French and Belgian burn centers and received treatment after randomization with either standard treatment involving 1% sulfadiazine silver emulsion cream or a cocktail of 12 natural lytic anti-P.-aeruginosa phages, and the time to an adequate sustained reduction of bacterial burden was assessed. The phage cocktail was found to decrease the bacterial burden in burn wounds at a slower pace compared to the standard of care, implying that future studies with higher concentrations of phages are required [The PhagoBurn trial was a randomized phase I/II trial where adult patients with confirmed burn infection by required . Table 1Nowadays, FDA approval has not yet been granted to phage treatment, and this could be due to concerns regarding approving a medication that is \u201calive\u201d and difficult to standardize. Thus, since concerns still exist and hinder the approval of this modality, other approaches such as developing recombinant phage-derived proteins could be elaborated ,189. EveAntimicrobial resistance has emerged as an enormous problem threatening millions of lives and causing thousands of infections in patients, most commonly in hospitalized ones. Since many pathogens have multiple mechanisms of resistance and have few viable therapeutic options, alternative treatments for infectious diseases are warranted. To that end, the use of phages, which were discovered at the beginning of the previous century and have shown some clinical success in early trials at that time, could now improve the treatment of patients suffering infections from difficult-to-treat pathogens. Until now, there has been plenty of evidence from animal models about the safety and efficacy of phage therapy, and phages are used broadly in agriculture, aquaculture, food safety, and wastewater plant treatment and as hospital environment sanitizers. However, there is a lack of high-quality evidence regarding phage therapy in clinical practice, with most studies being case reports or non-controlled trials often with contradictory results. Further well-designed studies with randomized, blind, controlled designs and with careful and thoughtful selection of phages and respect to global standards set by regulatory authorities are needed to identify the role of this promising treatment in the future of infectious diseases. Until then, phage use in clinical practice remains non-approved by the FDA and the EMA."}
+{"text": "Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR)\u00a0assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR\u2010Cas9\u2010assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection. Approximately 0.7 million people die across the globe annually due to AMR pathogens, and it is estimated that this figure could outnumber cancer\u2010driven mortality by 2050.Bacteriophages (phages) are viruses that attack specific bacteria and archaea.Escherichia coli phage for targeting Yersinia and Klebsiella bacteria by changing the tail fiber and other associated genes.Following the discovery of antibiotics,Recent developments in synthetic phage bioengineering techniques improved the arduous task of phage hunting by effectively tailoring interactions between bacteria and phages. The important factor for successful application of phage bioengineering is efficiency of genome editing. Classical homologous recombineering approaches or more innovative type I\u2010E clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR\u2010associated protein (Cas)\u2010based counter selection or yeast\u2010based reconstruction of phage genomes have been useful in the generation of recombinant phages.One of the promising leads in development of bioengineered phages against MDR pathogens is a CRISPR\u2010Cas system, which employs a successful genome editing tool in eukaryotic and prokaryotic systems.2In the type II CRISPR system, the first 10\u201312 nucleotides in the 3\u2032 region of the spacer sequence  are referred as the seed sequence. Mismatch in the seed sequence with the other DNA sequences being targeted would not allow the endonuclease to perform its activity. However, in DNA sequences with close homology, binding might occur, but cleavage is rarely observed. Thus, in the Cas9\u2010gRNA system, binding the complex to a sequence homologous with the seed sequence could lead to off\u2010target effects.33.1\u22124 to 10\u221210).Engineering a phage genome by homologous recombination within its bacterial host is a well\u2010established method, and it is one of the most commonly used engineering techniques. Homologous recombination occurs naturally between homologous DNA sequences and enables the introduction of heterologous DNA into the phage genome.3.2Escherichia and Salmonella species.Bacteriophage recombineering of electroporated DNA (BRED) is a phage engineering technique first developed by Marinelli et al.3.3et al. reported the first usage of a type I\u2010E CRISPR\u2010Cas system for improved engineering of T7 phage in 2014.Streptococcus thermophiles, S. pyogenes, Listeria monocytogenes, and Staphylococcus epidermidis. The CRISPR\u2010Cas system from S. pyogenes is most often employed for phage bioengineering as it can efficiently target a wide range of phage genomes to generate recombinant viral progeny.Recent developments in CRISPR\u2010Cas\u2010based genome engineering have significantly improved phage bioengineering. Kiro nluc) or nluc fused with carbohydrate binding module into hoc and soc genes of the T4 phage genome.One of the major rate limiting factors for CRISPR\u2010Cas\u2010based phage engineering is the selection of appropriate gRNA.sacB and a 30\u2010bp target sequence with a PAM motif. This is introduced into a host along with a prophage and another vector facilitating homologous recombination.In addition to engineering the phage via CRISPR\u2010Cas technology, strategies have been designed and demonstrated for incorporation of CRISPR\u2010Cas9 systems into the phage genome for therapeutic applications.Like any genome editing tool, the use of CRISPR\u2010Cas system has disadvantages. One of the risks is the off\u2010target activity of the Cas endonuclease; however, this can be overcome by screening through a sequencing approach.3.4Saccharomyces cerevisiae. This host has been used to modify the genomes of phages such as K11 and T7.S. cerevisiae eliminates the phage toxicity to allow more stable maintenance of the phage genome and provides more efficient homologous recombination machinery, resulting in higher recombination rates.Propagating phage genomes in a bacterial host can be toxic to the host, potentially limiting some engineering methods,3.5Staphylococcus species.Phages are highly specific toward their bacterial host via tail fibers that attach to specific receptors on the bacterial surface.S. cerevisiae cells. The assembled genomes were transformed into E. coli cells, producing hybrid T7 phage particles that were able to infect different hosts, such as Yersinia and Klebsiella strains.A modular engineering approach in yeast has been used for swapping the tail fibers from different phages to enable infection in\u00a0different hosts.44.1Gregory P. Smith and his partners first introduced phage display by successfully expressing recombinant peptides exposed with capsid proteins of filamentous phages, which constitute the foundation of this powerful tool.pIII or the major pVIII gene, to expose its products on the exterior of the phage coat while the DNA remains inside. The preparation of phage display libraries is the first step in the procedure and is known as \u201cbiopanning.\u201d By immobilizing the target proteins on the surface of a plate or bead, phages that display the protein that binds to one of those targets remain bound after washing the plate, while others are removed.E. coli strains, to enrich the pool of specifically binding phages. After several cycles, the corresponding DNA is sequenced to identify the target proteins.In phage display techniques, the DNA encoding interested proteins or peptides is ligated with phage coat\u2010protein genes, either the minor The phage display technique focuses on building a library of peptides or antibody variants, which are then selected based on their binding characteristics.4.2Phage display is essential in the field of bioengineering and immunology and is also used for development of vaccines and drug molecules.An important step of vaccine development is to select the appropriate antigen and adjuvant. Phage\u2010based vaccination is achieved by exposing multiple foreign antigen copies on the capsid surface of immunogenic particles to obtain an effective immune response. The combinatorial peptide libraries of the phage display techniques also help to identify promising vaccine candidates against different diseases, particularly against microbial infections. Based on an affinity selection strategy, these libraries are screened to select mimotopes with high antigenicity and immunogenicity.+ and CD8+ T lymphocytes and induces strong cytotoxic responses.Cancer immunotherapy is considered a promising approach to treat cancer as an alternative to or in addition to chemotherapy and radiation because of its ability to minimize systematic side effects.N\u2010acryloyl\u20103\u2010aminophenylboronic acid moieties to expose dynamic covalent binding to the surface of bacterial cells.Acinetobacter baumannii and S. aureus. Thus, this phage display platform supports rapid identification of peptide probes of the specific pathogen converted into bactericidal agents with high specificity.In the field of infectious diseases, an ideal treatment of an infection involves a specifically targeted antibiotic to kill the pathogen instead of broad\u2010spectrum ones, as broad\u2010spectrum antibiotics could lead to antibiotic resistance. McCarthy and partners constructed a phage library incorporating 5As a proof of principle, the use of CRISPR\u2010Cas9 alone or in combination with phages for eradication of several MDR pathogens has been successfully shown in multiple studies. A brief description of such studies is listed in Tables\u00a05.1E. coli is a well\u2010known prokaryotic model organism that is frequently used for the study of genetics, physiology, metabolism, and biochemistry. Due to its readily available molecular biology toolbox, it has emerged as a bacterium that is easy to manipulate. However, this gut symbiont is not only a \u201claboratory workhorse,\u201d but is also known to cause intra\u2010intestinal and extra\u2010intestinal diseases such as bacteremia and urinary tract infection (UTI).E. coli. This is concerning because colistin is considered the last line of antibiotic treatment.Nature BiotechnologyE. coli and other microorganisms were used for experimental demonstrations.blandm\u20101), \u03b2\u2010lactamase (blashv\u201018), intimin (eve), and DNA gyrase A (gyrAD87G) could result in MDR E. coli growth inhibition.gyrAD87G confering quinolone resistance. The same study also investigated a phage system enabling the user to modulate the bacterial population in the consortium by selectively knocking down the genetic signature of the targeted strain using CRISPR\u2010Cas technology.E. coli in in vitro and in vivo mouse skin and intestinal infection models. The lysogeny phage equipped with the CRISPR\u2010Cas9 cassette in the genome was used for sensitization of drug\u2010resistant bacterial cells in a PRESA strategy.E. coli to control the infection.E. coli type I\u2010E CRISPR system and CRISPR array sequence, together targeting ndm\u20101 and Cefotaximase\u2010Munich (ctx\u2010M\u201015), resulted in removal of the plasmid\u2010based resistant bacteria but maintained the sensitive bacteria.E. coli by targeting GFP via oral administration of M13 bacteriophage loaded with phagemid facilitating CRISPR\u2010Cas9 expression.E. coli and S. aureus that resulted in 2\u2010 to 3\u2010fold greater bactericidal activity compared to that of Cas9 nuclease. The same system also demonstrated in vivo therapeutic effects when tested in Galleria mellonella as a model organism.In a quest for a new therapeutic strategy against MDR pathogens, a study demonstrated that M13 bacteriophage loaded with phagemid facilitating CRISPR\u2010Cas9 expression that targets genes such as New Delhi metallo\u2010\u03b2\u2010lactamase\u20101 blandm\u20101, E. coli against antibiotics. For example, a CRISPR\u2010Cas9 system was developed for knocking out the antibiotic\u2010resistant gene to resensitize E. coli that were further targeted with antibiotics for growth inhibition and control of infections. The system was designed in a manner that it targets a conserved sequence shared among >1000 extended\u2010spectrum \u03b2\u2010lactamase mutants.mcr\u20101, which was discovered in China (2016), was efficiently knocked out in E. coli using the CRISPR\u2010Cas9 system, and proven to be an important anti\u2010resistant strategy.E. coli to colistin using a CRISPR\u2010Cas9 system by targeting mcr\u20101 in plasmids has been reported in several other studies.E. coli for reduction of MDR; here, Class I integrons are primarily responsible for AMR transfer among bacterial cells.E. coli by targeting virulence factors such as fimbrial adhesion (papG).E. coli using CRISPR\u2010Cas9 for efficient control and management of infections. Phage is an option for packaging of CRISPR\u2010Cas9 tool for specific delivery in bacterial pathogens and removal of the same from a mixed population.Many similar studies have been conducted where CRISPR\u2010Cas9 was used to resensitize 5.2K. pneumoniae is an opportunistic pathogen that causes a wide range of nosocomial infections such as pulmonary pneumonia, UTIs, bacteremia, meningitis, and liver abscesses.K. pneumoniae (CRKP).Enterobacteriaceae with carbapenem\u2010 or cephalosporin\u2010resistant profiles (including K. pneumoniae) among critical pathogens requiring urgent action. In addition, a pressing need has arisen to control and eradicate K. pneumoniae, exhibiting resistance towards\u00a0several antibiotics including colistin.K. pneumoniae has become a serious problem in clinics and requires immediate measures.Siphoviridae family is capable of both reducing K. pneumoniae growth and efficiently reducing K. pneumoniae biomass in biofilms during its post\u2010 or pre\u2010treatment. This phage also happens to be stable at 37 \u00b0C at pH 7. Likewise, bacteriophage SU503, SU552A, \u03d5Kp16, and \u03d5Kp27, and bacteriophage \u03d5Kp34 and \u03d5Kp24 belonging to Autographiviridae and Myoviridae families, respectively, have been characterized and are found to be able to kill clinical isolates of K. pneumoniae.K. pneumoniae bacteriophage phiKpS2 has been reported.K. pneumoniae infections.K. pneumoniae have been characterized and its genome engineering strategy has been developed, the use of phages for delivering CRISPR\u2010Cas9 vectors to target K. pneumoniae is a missing piece in the literature. However, several studies have reported the use of CRISPR\u2010Cas9 systems to target MDR K. pneumoniae. For example, Wang et al. designed a plasmid expressing CRISPR\u2010Cas9 for targeting the fosfomycin resistance gene (fosA) in K. pneumoniae 5573 that enhanced their susceptibility toward fosfomycin. Two plasmids, namely, pCasKP and pSGKP were used wherein, the former facilitated CRISPR\u2010Cas9 expression, while the latter enabled \u03bb Red recombination. When used together, the system results in highly efficient DSBs. In the same study, Cas9 nickase conjugated with the murine cytidine deaminase rAPOBEC1 was used to create premature stop codons in the fosA gene of K. pneumoniae 5573.K. pneumoniae KP_CRE23 to carbapenems by targeting the carbapenemase gene, blaKPC\u20102, and two of the extended spectrum \u03b2\u2010lactamases genes, blaSHV and blaCTX\u2010M\u201065.Enterobacteriaceae (CRE) to carbapenems. Genes such as blaKPC, blaNDM, and blaOXA\u201048 within the clinical isolates of carbapenem\u2010resistance K. pneumoniae, E. coli, Enterobacter hormaechei, E. xiangfangensis, and Serratia marcescens have been targeted, leading to a promising result of 94% curing efficiency.K. pneumoniae bacteriophage could be engineered for CRISPR\u2010Cas9\u2010mediated gene targeting with promising therapeutic results. However, before reaching any concrete conclusions, in vitro and in vivo studies are necessary.Phages have the potential to replace antibiotics and have recently attracted much scientific and public attention for treating MDR bacterial infections.5.3Mycobacterium tuberculosis is a single infectious agent causing tuberculosis (TB), which is among the top nine causes of death across the globe. M. tuberculosis is a great threat to human society due to its high pathogenesis.M. tuberculosis is based on reprograming host macrophages that help them to evade elimination, formation of granulomas which assist pathogen survival and lastly conversion of M. tuberculosis to a dormant state that resists host defense mechanisms.Mycobacterium strains.M. tuberculosis to target rpoB genes of the same organism. The phage could be engineered to deliver mini\u2010CRISPRs, which are DNA sequences compatible with endogenous CRISPR\u2010Cas systems, for processing the mini\u2010CRISPR into crRNA to target the desired gene.M. tuberculosis cells. In another report, the CRISPR associated proteins1 (Cas1) from the Mycobacterium type III\u2010A CRISPR family has been highlighted as a potential candidate for M. tuberculosis treatment. The study reported the presence of disrupted Cas1 protein in 57.14% of clinical isolates. Further investigation identified the role of Cas1 in increasing the sensitivity of pathogens against anti\u2010tuberculosis drugs during drug treatment.M. tuberculosis physiology and antibiotic resistant mechanisms. The system encompasses RecX, to suppress RecA\u2010dependent DNA repair system; NucSE107A, to suppress NucS dependent DNA repair system; the Cas9 nickase fusion protein of cytidine deaminase, and uracil DNA glycosylase inhibitor for efficient base pair conversion in the M. tuberculosis genome.M. tuberculosis has been constructed.As a recent development in gene editing technology, CRISPR\u2010Cas has the potential to neutralize antibiotic\u2010resistant genes in a specific targeted MDR bacterial population or kill them without affecting the beneficial wild\u2010type populations.Mycobacterium infections as well. As per the report, a patient suffering from cystic fibrosis due to M. abscessus showed clinical improvement when using genetically modified bacteriophages as therapeutic agents.M. abscessus was detected from the sputum or serum of the patient after phage injection, and no adverse effect of phage treatment was reported. However, even though the treatment showed promising results, the study should be expanded to similar patients to develop a more thorough understanding. Recently, the lytic phages based therapeutic intervention have been used to treat 20 patients suffering from nontuberculous Mycobacterium infection. No adverse reactions were reported in any of the patients treated with the phages and 11 patients displayed a favorable clinical outcome. Antibodies against phages were reported in few of the patients that were intravenously injected with phages, however no phage resistance was reported in 11 patients treated.Mycobacterium genome editing have been used in several studies. However, the use of this in M. tuberculosis for resensitization against antibiotics, target\u2010specific killing, or phage\u2010mediated CRISPR\u2010Cas delivery is yet to be explored.The potential of using bacteriophages as a therapeutic tool has been evaluated for other 5.49 plaque\u2010forming units/mouse was very effective in protecting the mouse against Salmonella infection. The study suggested that the isolated bacteriophage could be a potential candidate for therapeutic purposes and might help prevent foodborne illnesses. A study has demonstrated that the phage PA13076 protects mice from a lethal dose of S. enteritidis 13076 by reducing the concentration of bacterial cells in blood and various organs such as the intestine, liver, spleen, and kidney.S. enteritidis infections. Another study has comprehensively analyzed the phage susceptibility variation in the two strains of S. enterica serovar Typhimurium DT104 and DT104b.Worldwide, salmonellosis is a very common foodborne disease. It has been associated with outbreaks in several countries, resulting in high morbidity and mortality.5.5S. aureus is a Gram\u2010positive pathogenic bacterium that is highly resistant to antibiotics. Methicillin\u2010resistant S. aureus (MRSA) is the prime culprit of global S. aureus bacteremia and causes metastatic or other infections such as endocarditis and sepsis.S. aureus infection from MEDLINE, Cochrane Database of Systematic Reviews and Embase databases (1991 to May 2021), one in every four patients dies within 3 months due to S. aureus bacteremia.S. aureus infections. As an alternative to antibiotics, phages have shown potential to treat S. aureus infections. Recently, a clinical trial has shown significant reductions in the staphylococci without any adverse effects after intravenous administration of Myoviridae bacteriophages (AB\u2010SA01) in patients.Aph\u20103) and penicillin binding protein (mecA) genes in drug\u2010resistant isolates of S. aureus.S. aureus strains and inhibit their growth. The same system also reported promising results when \u03a6NM1\u2010carrying constructs were used to treat S. aureus skin infectioremn in a CD\u20101 mouse model.nuc) and type VII secretion system extracellular protein A (esxA) was integrated into the genome of a \u03c6SaBov temperate phage. In the study, S. aureus ST1, ST5, ST8, and ST36 and CTH96 were used to test the potential of the engineered phages as therapeutics.Staphylococcus (1\u2009\u00d7\u2009105\u2009CFU) due to engineered phages compared to a native unmodified phage treatment (1\u2009\u00d7\u2009109\u2009CFU). This study was extended in an in vivo C57BL/6 mouse model to treat skin infection. Remarkably, the study demonstrated a significant bacteriophage bactericidal activity in both in vitro and in vivo.S. aureus. The study focused on targeting the nuc gene of S. aureus ATCC 6538\u2010GFP by integrating a programmed CRISPR\u2010Cas9 system into a noncoding genomic region of \u03c6SaBov phage. The engineered phage was also challenged in vitro to treat S. aureus biofilm. Intriguingly, in vitro qualitative fluorescent imaging showed significant anti\u2010biofilm activity compared to fosfomycin and vancomycin antibiotics, whereas quantitative anti\u2010biofilm effects gradually increased over time for phage, fosfomycin, and phage\u2010fosfomycin treatments delivered via alginate hydrogel.mecA) of S. aureus USA300 was demonstrated. The study highlighted the superior bactericidal activity of Cas13a compared to Cas9 nuclease. Furthermore, the proposed system demonstrated remarkable therapeutic effects in a G. mellonella model.S. aureus infections have entered into clinical trials, and notably phages carrying a CRISPR\u2010Cas system have shown potential as bactericidal agents for resensitization and eradication of S. aureus infections. Nevertheless, there are a few shortcomings that need to be addressed before applying phage\u2010based CRISPR\u2010Cas to combat S. aureus infections such as generalized transduction of virulent genes and the narrow host range. However, genetic engineering resources are emerging6Myoviridae family and two phages CX5 and P\u2010PSG\u201011 of the Podoviridae family, were used for adaptive laboratory evolution to improve their stability at elevated temperature.Phage stability and effectiveness are the major factors for proficient phage therapy. Natural phages are sensitive to several environmental factors such as temperature, solute present in the sample, and ultraviolet (UV) light. A simple solution to overcome this hurdle could be adaptive laboratory evolution, a method to improve the evolutionary fitness and adaptability of organisms in changing environments.7Mycobacterium, namely, M. smegmatis was used for directed evolutionary studies. In the study, directed evolution was used as a tool for increasing the lytic activity and infectivity of mycobacteriophage. The study investigated the effect of phage inoculum size to achieve desired adaption.M. smegmatis and M. tuberculosis, the same study could be expanded using M. tuberculosis as a host for enhancing the potential of phage as a therapeutic.trxA mutant, where trxA encodes thioredoxin, which is an essential subunit for phage DNA polymerase. This setup allowed directed evolution of the phage for narrowing the host range by recognizing specific forms of LPS present on the cells while avoiding the others. The resulting evolved phage had mutations on tail genes 11 and 12 and on tail fiber gene 17 that altered their specificity. The experimental set up used in this study could be channeled for bacteriophage\u2010mediated elimination of bacterial serotypes in a mixed population.E. coli B strain REL606 for better therapeutic applications. Phages were trained for 28\u2009days and were able to suppress the bacteria with 1000\u2010fold higher efficiency for 3\u20138 times longer compared to their ancestor strain. Intriguingly, it only took one mutation step of the bacteria to become resistant to the untrained phage, whereas it took multiple mutations in bacteria to achieve the same for laboratory evolved phages.Directed evolution is a method mimicking the natural selection process for genes and their corresponding proteins toward a user\u2010defined goal. Directed laboratory evolution is similar to adaptive laboratory evolution; however, the aim in directed laboratory evolution is to drive the protein toward improved functionality.8Bacteriophages have immense potential as antibacterial candidates in the upcoming post antibiotic era, as is evidented by in vitro as well as clinical studies. It is crucial to ensure that the bacteriophages are delivered at the site of infection for the treatment to be effective, failing to which the treatment could be ineffective.Salmonella that were encapsulated by cationic liposomes protected them against simulated gastric fluid (SGF) of pH\u00a02.8. In addition, encapsulation has been shown to improve the retention of phages in chicken intestinal tracts.Liposomes are enclosed lipid bi\u2010layered nanostructures, spherical in shape, and hollow, allowing them to carry aqueous solutions. Bacteriophages enclosed by liposomes have been shown to protect the phages against host environmental insults such as acidic pH and degrading enzymes found in the stomach and gut, respectively.Salmonella spp.K. pneumoniae using burn wound mice models have been examined. Higher reduction of bacterial load was reported in blood and other organs of mice when treated with encapsulated phages compared to phage delivered freely. In addition, encapsulated phages offered higher retention values and greater specificity to cure the infection. Moreover, phages delivered through liposomal preparation protected the mice from death even if the treatment was delayed for 24\u2009h.S. aureus infection.K. pneumoniae residing in the macrophage, and this result was in contrast to free bacteriophages that were unable to penetrate eukaryotic cells.K. pneumoniae biofilms. Using the clinical achievable antibiotic concentration increased the efficacy of the liposomal encapsulated phage for reducing the bacterial load in young as well as mature biofilms. In the same study, the synergistic effect for reduction of bacterial load via free bacteriophages was not significant in mature biofilms.In addition to stability, liposomes have also been investigated for their therapeutic value in different in vivo models. For instance, enhanced efficacy of encapsulated bacteriophages is observed compared to freely delivered phages for protecting broilers against S. aureus phage K stability in SGF of pH\u00a02.5. The encapsulation via alginate hydrogel microspheres improved the phage stability compared to that of free phages in SGF, and these\u00a0microspheres further improved phage survival when calcium carbonate microparticles were included\u00a0in the formulation.E. coli bacteriophage ZSEC5 against acid stress. The same encapsulation has been highlighted to prevent phage degradation at elevated temperature.3 encapsulated cocktails of three phages against Salmonella were tested for their ability to be orally administered in chickens. The alginate/CaCO3 preparation allowed 100% encapsulation of phage cocktail, were stable in the stomach, and retained in the intestines during in vivo studies. A high antibacterial activity of encapsulated phage cocktail was reported against Salmonella infections.S. aureus mediated osteomyelitis and soft tissue infection. As per the study, alginate encapsulated phages gave therapeutic effect comparable to high dose of fosfomycin for skin infection models, however the same was not effective for rat models with bone infections.Hydrogel can be defined as a 3D cross\u2010linked network of hydrophilic polymers with the capacity to retain a large amount of water.Proteus mirabilis colonize and form a biofilm that results in an increase in the pH of the surrounding area. The increase in pH acts as a stimulus for the \u201ctrigger layer\u201d that leads to release of the phage from the lower \u201creservoir layer\u201d of the hydrogel. In a study of an in vitro bladder model, phages entrapped in a pH\u2010responsive hydrogel made from poly(methylmethacrylate\u2010co\u2010methacrylic acid) delayed blocking of the catheter due to biofilm formation.N\u2010isopropyl\u2010acrylamide is an example of a thermo\u2010responsive polymer used as nanospheres to entrap S. aureus phage K and added to a nonwoven fabric for usage in adhesive bandages.Hydrogels have also been used to devise smart systems based on pH responsive surface coatings for long term catheters that release phages during infection. At the time of infection, pathogens such as Pseudomonas bacteriophage on its surface. The resulting biomaterial was effective until 25\u2009cycles of washing.Apart from the liposomes and hydrogels, phages bound to fibers have also been studied for their therapeutic effects. Phages immobilized on fibers are a simplistic yet effective strategy for topical administration of phages during wound dressing or in bandages.The recent increase in number of clinical trials using intravenous or oral phages provides an evidence of growing global interest in phage therapy. However, large\u2010scale utilization of phages in clinical settings are limited by various factors such as need for repeated administration, narrow host range and loss of activity in physiological condition. Another limitation is in vivo decay of bacteriophage lytic activity by physiological factors such as change in pH or serum inactivation. Therefore, sustained release of phages from biomaterials, which are locally implanted at the site of infection, might prolong its residing time for better treatment and improve its therapeutic efficacy.9With continued excessive use of antibiotics, human society is approaching a post\u2010antibiotic era where the antibiotics are turning ineffective. Immediate action is needed or a common infection or minor injury could prove to be fatal. Phage therapy could be an alternative approach for controlling rapidly evolving MDR pathogens that are difficult to treat with existing antibiotics. Diversity and adaptability are advantages to using phages to treat an infection. Furthermore, literature shows successful use of phages as therapeutics. With the growing genome engineering tools and techniques and increasing knowledge of phage biology, it is possible to engineer a phage with desired characteristics. These techniques and tools have enabled an increase in the overall fitness of the phage, significantly influencing its therapeutic ability. Furthermore, integrating CRISPR\u2010Cas technology into the phage has shown potential for target\u2010specific removal of pathogens from a mixed population. The therapeutic effect of this technology could be different from the currently available broad\u2010spectrum antibiotics. One of the drawbacks of broad\u2010spectrum antibiotics is the spread of resistant genes across bacterial species, and alteration of the host microbiome. If the pathogen behind infectious diseases is known, CRISPR\u2010assisted phage therapy could help to overcome some of these issues. CRISPR\u2010Cas has also enabled strategies such as PRESA, which has shown promise as compared to lytic phages for therapeutics. The use of phage acquired CRISPR could alter the global genetic landscape of the bacterial population. However, a large proportion of this change relates to antimicrobial\u2010resistant genes of the targeted pathogens, which is also the need considering the current global crisis. For successful bacteriophage therapy, encapsulation of a phage is necessary for stabilization during storage and treatment. Encapsulation has also enabled researchers to construct smart phage release strategies. Over the years, much attention has been given to formulating various recipes for phage encapsulation, and this could be key for its clinical success. In summary, phages integrated with a programmable endonuclease are a promising therapeutic candidate to combat MDR pathogens. However, rigorous assessment and data from clinical trials using genetically engineered or evolved phage as therapeutics are lacking. Until data regarding safety are gathered, phages, whether wild type, laboratory evolved, or genetically engineered, could be the last option when other treatments fail.Khushal Khambhati: Writing \u2013 original draft (lead). Gargi Bhattacharjee: Funding acquisition ; writing \u2013 original draft (supporting). Nisarg Gohil: Funding acquisition ; writing \u2013 original draft (supporting). Gurneet K. Dhanoa: Writing \u2013 original draft (supporting). Antonia P. Sagona: Conceptualization ; writing \u2013 review and editing (supporting). Indra Mani: Writing \u2013 original draft (supporting). Nhat Le Bui: Writing \u2013 original draft (supporting). Dinh\u2010Toi Chu: Conceptualization ; writing \u2013 review and editing . Janardhan Keshav Karapurkar: Writing \u2013 original draft (supporting). Su Hwa Jang: Writing \u2013 original draft (supporting). Hee Yong Chung: Supervision (supporting); writing \u2013 review and editing . Rupesh Maurya: Writing \u2013 original draft (supporting). Khalid J. Alzahrani: Funding acquisition ; writing \u2013 review and editing . Suresh Ramakrishna: Conceptualization ; funding acquisition ; supervision (lead); writing \u2013 review and editing (supporting). Vijai Singh: Conceptualization ; funding acquisition ; supervision (lead); writing \u2013 review and editing .The authors have no competing interests to declare.https://publons.com/publon/10.1002/btm2.10381.The peer review history for this article is available at"}
+{"text": "Regarding deep learning networks in medical sciences for improving diagnosis and treatment purposes and the existence of minimal resources for them, we decided to provide a set of magnetic resonance images of the cardiac and hepatic organs.http://databiox.com.The dataset included 124 patients (67 women and 57 men) with thalassemia (THM), the age range of (5\u201352) years. Patients were divided into two groups: with follow-up (1\u20135 times) at time intervals of about ( Thalassemia (THM) is one of the most inherited hemoglobinopathies and the most common monogenic disorder worldwide. Every year, approximately 300\u2013400 thousand fetal-affected types with anemia are born , 2. Due Methods include a hepatic biopsy, serum iron and ferritin levels, transferrin saturation, and magnetic resonance imaging (MRI) were used to calculate IrO. Although serum ferritin level estimation is inexpensive and the most accessible method for assessing the body\u2019s iron concentration because it shows the short-term total iron of the body, this test has low precision , 6. Non-Magnetic resonance T2-star (T2* MRI) and T2 (T2 MRI) are methods used to assess the enhancement of tissue relaxation caused by high molecular weight iron complexes such as ferritin and hemosiderin . In thesThe ability of artificial intelligence to facilitate diagnosis and treatment procedures and the need for data related to the same domain for training neural networks highlight the importance of preparing datasets with the aim of speeding up the work. This study is the first to simultaneously prepare a set of cardiac MR images (CMR) and hepatic MR images (HMR) for the analysis and image processing of THM patients.All T2* MR images were obtained using GE Healthcare . Cardiac gating MRI examination was performed using a single mid-papillary ventricular short-axis slice in the supine position with a single breath-hold using a torso phased-array body coil, and a multi-echo gradient-echo sequence (12 echoes) was obtained. The field of view (FOV) in CMR extends caudally from the carina to the lower renal pole (40\u2009\u00d7\u200940) cm, matrix size (128\u2009\u00d7\u2009116), slice thickness 10\u00a0mm, 12 different TEs  ms, TR (31.3) ms, and bandwidth 1562 Bw/pixel. A cardiac T2* value calculated by T2 mapping with a time of more than 20 ms is considered normal for Iron Overload (IrO). A T2* value between 15 and 20 ms is considered mild, between 10 and 15 ms is moderate, and less than 10 ms is indicative of severe myocardial siderosis.0. Hepatic T2* values of less than 30 ms indicate hepatic abnormal IrO: mild (>\u20096/2) ms, moderate (3.1\u20135.2) ms, severe (2.1-3. 1) ms.For the HMR, a single breath-hold technique using a multi-echo gradient-echo in 12 different TEs  ms, FOV (40\u2009\u00d7\u200940) cm, matrix size (128\u2009\u00d7\u2009116), voxel size should have 3.1 * 3.4\u00a0mm in-plane resolution, slice thickness 8\u00a0mm, 120 ms, and bandwidth 1736 BW/pixel. The flip angles for both CMR and HMR were 20To determine iron concentration, we used a GE workstation software tool workstation following acquisition using a multi-echo T2 star. To eliminate bias and computation errors, we followed these steps:The intensity correction filter was disabled prior to data acquisition.Exclude vein, artery, and hepatic duct for computation.A radiologist marked Regions of Interest (ROIs) on the mid-liver slice and mid-ventricular short-axis slice of the generated R2* map and computed the average R2* value for liver and cardiac, respectively.After the third step, the R2* value was converted to liver iron concentration (LIC) using the formula LIC\u2009=\u20090.0254 \u00d7 (R2*)\u2009+\u20090.202 as described by Wood et al. .For calibration, we calculate mean liver iron concentration (LIC) using liver R2 values and a calibration curve determined through a needle biopsy as described by Tim St Pierre et al. .The current dataset included 210 MRI image files (every file different series) of 124 patients with THM, including 67 women and 57 men age range (5\u201352) years. The data were divided into two groups with 75 patients with follow-up (Between 1 and 5 times) in time intervals of about  was provided. Figure\u00a0The sex separation of the patients as well as their cardiac and hepatic IrO conditions are shown in detail in Figs.\u00a0To date, the only dataset provider study for patients with THM was conducted by Shiae et al. in Mashhad, Iran, between February 2016 and January 2019, in the form of open-source CMR images of 50 subjects, including 37 THM patients and 13 healthy subjects, with clinical and echocardiographic data, such as clinical signs of heart failure, shortness of breath, decreased activity, hand and foot swelling, round the eye and chest pain, and arrhythmia. All images were 16-bit grayscale with a resolution of (192\u2009\u00d7\u2009256) pixels, stored in DICOM format, and finally compressed and saved in RAR format [However, present dataset including a larger number of patients, has two sections with and without Follow-up to perform more various studies such as disease prediction. Also, we mentioned the status report of hepatic IrO in the dataset, which can be used in some hepatic studies.The provision of Echocardiography data of patients is also another noteworthy point of this study, which is in Excel format in dataset. The details of this file include \u201cEjection Fraction (EF), Left Ventricular Size (LVSize), (TGR), Pulmonary Artery Pressure (PAP), Tricuspid Annular Plane Systolic Excursion (TAPSE), and ECG Report \" which does not include the with Follow-up section of the patients and is for Without Follow-up and the first stage with Follow-up."}
+{"text": "The functional traits of species depend both on species\u2019 evolutionary characteristics and their local environmental conditions and opportunities. The temperature-size rule (TSR), gill-oxygen limitation theory (GOLT), and temperature constraint hypothesis (TCH) have been proposed to explain the gradients of body size and trophic level of marine species. However, how functional traits vary both with latitude and depth have not been quantified at a global scale for any marine taxon. We compared the latitudinal gradients of trophic level and maximum body size of 5,619 marine fish from modelled species ranges, based on (1) three body size ranges, <30, 30\u2013100, and >100 cm, and (2) four trophic levels, <2.20, 2.20\u20132.80, 2.81\u20133.70, >3.70. These were parsed into 5\u00b0 latitudinal intervals in four depth zones: whole water column, 0\u2013200, 201\u20131,000, and 1,001\u20136,000 m. We described the relationship between latitudinal gradients of functional traits and salinity, sea surface and near seabed temperatures, and dissolved oxygen. We found mean body sizes and mean trophic levels of marine fish were smaller and lower in the warmer latitudes, and larger and higher respectively in the high latitudes except for the Southern Ocean (Antarctica). Fish species with trophic levels \u22642.80 were dominant in warmer and absent in colder environments. We attribute these differences in body size and trophic level between polar regions to the greater environmental heterogeneity of the Arctic compared to Antarctica. We suggest that fish species\u2019 mean maximum body size declined with depth because of decreased dissolved oxygen. These results support the TSR, GOLT and TCH hypotheses respectively. Thus, at the global scale, temperature and oxygen are primary factors affecting marine fishes\u2019 biogeography and biological traits. Latitudinal diversity gradients (LDGs) integrate over the local and regional patterns where species have evolved and survived on both ecological and evolutionary time scales . In contA positive relationship between body size and latitude has been demonstrated in freshwater and marine ectotherms . The pheMore marine herbivorous or omnivorous fish exist in the low than high latitudes, and few herbivores in areas with an annual average temperature of below 20 \u00b0C . This maTherefore, the hypotheses TSR and TCH directly, and GOLT indirectly, suggest that temperature is the primary driver to constrain the body size and thereby influence the trophic level of fish, as supported for the effect of paleoclimate on body size evolution of fishes . In thisAnother environmental gradient of interest regarding the diversity pattern is depth. The deep sea below 200 m depth is dark, cold, and with low-dissolved-oxygen across all latitudes . TherefoThis study describes the gradients of body size and trophic level of marine fish among latitudes in different depth zones, and along depth zones with latitude, globally. The relationship between functional traits and environmental variables is presented. We hypothesize that fishes with smaller body size and lower trophic levels dominate warmer waters , and the reverse in cooler waters .https://doi.org/10.6084/m9.figshare.19314317.v3). AquaMaps models field observations with environmental variables to predict the environmental niche and thus geographic ranges of species , AquaMaps limits species ranges to their known occurrence in FAO regions  in these size categories was 62%, 32% and 6% respectively, our data were more inclusive of larger fish species  (low-trophic-level omnivores); 2.81\u20133.70 for omnivores with a preference for animals but feeding on diverse prey  (high-trophic-level omnivores); and >3.70 for piscivores and carnivores with a preference for large decapods, cephalopods and fish (carnivores)  . The pronivores) .The mean and standard error of body size and trophic level for all fish was calculated in 5\u00b0 latitude bands between 75\u00b0S and 75\u00b0N and four depth zones: whole water column, surface (0\u2013200 m), middle , and deep ; reflecting the photic, mesophotic and aphotic zones of light penetration respectively . This stThe mean body size and trophic level were correlated with the long-term decadal averages of monthly salinity , and sea surface and bottom temperature  and dissolved oxygen  in 5-degree latitude bands. The environmental variables were obtained from the Global Marine Environmental Datasets . Raster 2, were used to assess distributional and smoothing assumptions  plot, minimized Generalized Cross-Validation (GCV) scores, maximized deviance explained and maximized adjusted rumptions . The devMean maximum body size of fish species was smaller in the Southern Ocean, tropics and sub-tropics (30\u00b0S and 30\u00b0N) . Thus, tHigh-trophic-level omnivore and carnivore species occurred across all latitudes . In contOverall, we found that the diversity of body sizes and trophic levels were highest in the tropics and subtropics, but the mean body sizes and trophic levels in this area were smaller and lower  and 2. TThe distribution of body size and trophic groups in the surface zone were similar to the pattern described in the whole water column because over 73% of marine fish occurred shallower than 200 m depth  and 4. CAcross 100 m depth bands, mean maximum body size decreased with depth, and below 1,200 m fish species were below average in size . HoweverThe depth ranges of almost all (99%) of herbivores and detritivores, and 98% of low-trophic-level omnivores, included the surface zone . Only onMean sea surface temperature (SST) declined from the tropics to the poles with peaks at 10\u00b0N and 5\u00b0S . The narThe latitudinal gradient of mean sea bottom temperature (SBT) contrasted with SST because mean SBT was less than 6\u00b0C in all latitudes because of the dominating effect of the large area of deep sea in each latitude. SBT ranges were widest in the tropics to subtropics, and narrower at high latitudes because SBT varied more from shallow to deep sea in the tropical areas .Mean sea surface dissolved oxygen (SDO) decreased from the poles to the tropics , being lMean sea surface salinity varied between 28 to 35 psu with latitude, which is within the tolerance of marine organisms . HoweverAll environmental variables were significantly correlated with gradients in maximum body size and trophic level . Mean SSMean SDO was 6 ml/l and mean BDO was 5 ml/l . As withMean sea surface salinity was the least influential factor compared to temperature and dissolved oxygen. Most species, regardless of body size and trophic level, were living in salinities between 33 and 35 psu. Only a few species with larger body size and higher trophic level live in lower salinity water .Our results showed that the diversity of fish species\u2019 body size and trophic level were highest in the tropics and subtropics (between 30\u00b0S and 30\u00b0N). This may be because the tropics and subtropics also have a high diversity of associated predator, prey and competitor richness and habitats . The warOur finding of a paucity of herbivores outside the tropics and subtropics supports the Temperature Constraint Hypothesis (TCH) that posits that the efficiency of digestion for plant materials is compromised in cooler environments . Howeveri.e., deeper than 200 m)   and 4. Tn 200 m) .Across 100 m depth bands, body size may be expected to be larger and the trophic level higher in the deep sea because the temperature is lower in the deep than shallow depths and food supply is largely dependent on secondary production in shallower waters. However, the results showed that deeper than 200 m, mean body size decreased and mean trophic level stayed at near 3.70 to 2,300 m depth, and then decreased deeper than 2,300 m. Thus, the depth gradient of mean body size did not follow the temperature-size rule (TSR) but did follow the gill-oxygen limitation theory (GOLT) because the dissolved oxygen (DO) decreased with depth .Fewer than 40 species in our dataset occur deeper than 2,300 m, and most of them were high-trophic-level omnivores, with less than 10 of these species being carnivorous . WithoutAlthough the primary trend for mean body size decreased with depth and mean trophic level stayed stable at 3.7 till 2,300 m, there were some depths where values rose or fell. These depths were at 100, 500, 900, 1,400, 1,800, 2,300, and 3,000 m, respectively  and 6A, Fish\u2019s body size and trophic level were smaller and lower in the warmer and low DO latitudes (tropics and subtropics) but larger and higher in the cooler and high DO latitudes (temperate areas and the Arctic Ocean but not the Southern Ocean). These results may support hypotheses of temperature-size rule, gill-oxygen limitation theory, and temperature constraint hypotheses. As warmer temperature decreases aerobic capacity, fish with larger body size may be limited by oxygen supply , and thuBoth the Arctic and Antarctic are polar environments with near freezing temperatures but high DO  and relaThis study found that mean body sizes and mean trophic levels of marine fish were lower in the tropics and sub-tropics, between 30\u00b0S and 30\u00b0N, than high latitudes, and less in the deep-sea and Antarctica. The flatter latitudinal gradients of these traits in the deep sea reflect its more homogenous environment. While mean body size generally increases at colder temperatures, the reverse was the case with depth. Body size and trophic level decreased with depth not because of temperature, but because of the low dissolved oxygen and scarce food resources in the deep sea. Thus, the latitudinal gradients at the surface zone support the Temperature-Size Rule, Temperature Constraint hypothesis, and Gill-Oxygen Limitation Theory while the depth gradient only supports the latter. Therefore, our global scale analysis shows that in the surface zone, temperature is the primary and dissolved oxygen is the second factor influencing the biogeography of fish body size and trophic level, whereas oxygen and food supply limit these traits in the deep-sea and Antarctic species.10.7717/peerj.15880/supp-1Supplemental Information 1Click here for additional data file."
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