The data were collected with the acquisition system RESON SeaBat 7150 (12.5 kHz), using the integrated navigation system Applanix POS MV, and processed using QINSy software. The multibeam bathymetry data are presented in tabular format (ASCII-table (*.txt), spreadsheets (*.xlsx)), and as a digital elevation model (DEM) of the bottom relief. The DEM was created using ArcGIS software in ESRI-ASCII-grid (*.asc) and GeoTIFF raster (*.tif) formats with a spatial resolution of 100 m. We provide processed data that can be used directly for further studies. The dataset is available with the article.We present a multibeam bathymetry dataset of Discovery Gap; a narrow gap in ridge of the Azores-Gibraltar Fracture Zone (eastern North Atlantic). The gap serves as gateway for the exchange of deep water between the Madeira and Iberian abyssal plains. A bathymetric survey was carried out during the 43 Specifications table•We present new multibeam swath bathymetry data of the abyssal ocean gateway. Ocean gateways play an important role in basin evolution and the exchange of water, sediment, and biota from one basin to another. Despite of their importance, little is known about these bathymetric features. The presented data can significantly contribute to the understanding of the morphology and sedimentation regimes of ocean gateways.•The DEM reveals a well-detailed topographic image of Discovery gap. It allows to define various morphological features of the gap. The most prominent are deeps, sills, ridges, topographic highs, and flattened areas. Erosive and accumulative features caused by action of bottom currents can also be distinguished.•The dataset could be used for the geomorphological study of the area, sedimentological studies of abyssal gateways, numerical modeling of deep ocean circulation, and habitat mapping.•The dataset can be used to improve multibeam survey methods in areas of abyssal ocean gateways.1Discovery Gap is a narrow gap located between the Azores-Gibraltar Fracture Zone and the Madeira-Tore Rise in the eastern North Atlantic . DiscoveDiscovery Gap serves as a gateway between the Madeira and Iberian abyssal plains. Ocean gateways play an important role in water mass exchange and transport of sediment and biota between basins. Previous studies have shown that near-bottom currents are intensified through ocean gateways. This promotes the erosion and accumulation of sediment forming a variety of sea-floor bedforms rd cruise of R/V Akademik Nikolaj Strakhov in October 2019.The multibeam data were collected during integrated oceanographic studies in Discovery Gap that were held by the Atlantic Branch of the Shirshov Institute of Oceanology, Russian Academy of Sciences (Russia). Measurements were conducted during the 43According to the collected data, the gap is presented by a variety of pronounced morphological features: deeps, sills, ridges, topographic highs, and flattened areas . The dep-An ASCII-table (*.txt) of processed bathymetry data collected by multibeam echosounder Reson SeaBat 7150;-Spreadsheets (*.xlsx) of processed data, divided into four parts;-An ESRI-ASCII-grid (*.asc) DEM with a scale 1:100 000, spatial resolution of 100 m in the UTM Zone 28N projection,WGS84;-A GeoTiff raster (*.tif) of filtered DEM (low pass 3-by-3 filter)  with a sThe multibeam bathymetry data are fully processed and presented here in tabular format and as a digital elevation model of the bottom relief. The presented dataset includes:The structure of the ASCII-table (*.txt) and MS Excel spreadsheets (*.xslx) is presented in 2Akademik Nikolaj Strakhov. The acquisition system includes: a multibeam echosounder RESON SeaBat 7150 ; a navigation system Applanix POS MV with two-antenna GPS receivers  and a motion-control sensor; and the data acquisition software, QINSy.The bathymetric survey was carried out by deep sea multibeam echosounder (MBE) system mounted onboard of R/V A single swath of MBE data covered a 10 km area, the mean distance between swaths was 80-90 m, the distance between depth points in a single swath was approximately 80 m. The spatial resolution of generated DEM is 100 × 100 m. Any current and wave effects were removed by using a motion-control sensor of Applanix POS MV. The tidal corrections were not applied.Sound velocity in the water column was calculated from temperature, salinity, and pressure data measured by the CTD-probe SBE19v2+ at the five oceanographic stations in the survey area. Sound velocity profiles were used to correct the multibeam data in the post-processing stage. The multibeam echosounder calibration (Patch Test) was performed.Data post-processing included the following successive steps: filtration by on-line filters, processing in QINSy, export in ASCII-table, creation of DEM, and conversion to GeoTIFF raster. The depth artifacts were filtered during the survey by the on-line filters in QINSy software . Data processing in QINSy included: manual filtering of errors, corrections according to the sound velocity in the water column, calculation and application of correction coefficients including the Refract filter for correction of beam refraction, removal of acoustic noise by the off-line filters  and data filtration in the Qloud software (Qlean++ filter). Then we exported the data of each survey line in XYZ format in ASCII-table. The next step was creation of DEM by triangular method with a spatial resolution of 100 m using QINSy processing manager. Finally, we applied the “Low Filter” function by the module “Spatial Analyst” and converted DEM to GeoTIFF raster using ArcGISsoftware. The resulting bathymetry is presented in UTM 28 zone projection, datum WGS84.The presented data don't need any pre-processing and may be used directly.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Background: Although hen's egg (HE) allergy was thought to be usually resolved by late childhood, majority of HE allergy patients with a high level of egg white (HEW)-specific IgE could not acquire tolerance for HE by age 8 years.Objective: The aim is to investigate whether the avoidance of HE until 6 years of age increased the risk of heated HE allergy at age 6 years.Methods: This was a retrospective case-control study. The HE tolerance children (n = 17) and children with low-dose HE reactor [a positive reaction to ≤ 4 g of heated HEW in oral food challenges (OFCs)] children (n = 26) were included based on the results of OFC at 6 years old. Multivariate logistic regression analysis was applied to examine the associations between HE avoidance until age 6 years and HE allergy status confirmed by OFC, adjusting the level of ovomucoid-specific IgE (OM-sIgE) during early infancy.Results: A lower proportion of strict avoidance of HE was observed in the HE tolerance group than in the low-dose HE reactor group . OM-sIgE levels in children younger than 2 years old were significantly higher in the low-dose HE reactor group than those in the HE tolerance group . The avoidance of HE until 6 years of age increased the risk of heated HE allergy even after adjusting OM-sIgE levels.Conclusions: The long-term avoidance of HE from infancy increased the risk of heated HE allergy confirmed by OFC at age 6 years. Food allergy is one of the major global children's health issues, and hen's egg (HE) is the most common allergenic food among Japanese children . Ohtani Du Toit et al.  noted thWe hypothesize that HE-allergic infants who are highly sensitized to HE in infancy may not be able to achieve oral tolerance for HE by long-term avoidance based on the presence of IgE sensitization. Furthermore, we believe that strict avoidance of HE can delay oral tolerance, thereby resulting in an increased risk of persistent HE allergy. This study aimed to investigate whether the strict avoidance of HE until 6 years of age in HE-allergic children with positive HE sIgE during early in life could increase the risk of a persistent HE allergy in clinical practice.Our study was designed as a retrospective single-center case-controlled study using the data from the children's medical charts. This study was approved by the ethics committee of the National Center for Child Health and Development (2019-066).n = 17) included children who could tolerate 35–40 g of heated HEW  in the OFCs at 6 years of age as controls, while the low-dose HE reactor group (n = 26) included children who showed a positive reaction to ≤ 4 g of heated HEW (low dose ≤ 1/10 of one HEW) in OFCs at 6 years of age as cases. We defined a case as children with low-dose HE reactor  because mild or moderate HE allergy  could naturally induce oral tolerance to HE. In both groups, two children  were excluded because data on OM-sIgE levels prior to 2 years of age were missing. In the low-dose HE reactor group, two children were excluded because they had no previous reactions to HE.We defined cases as OFC positive children and controls as OFC negative children at 6 years of age. Inclusion criteria were as follows: (i) sensitization to HE before 2 years of age  system and defined lower than 0.35 kUA/L as non-sensitized); (ii) children had undergone OFCs at age 6 years for heated HE white (HEW); and (iii) positive reaction to ≤ 4 g of heated HEW  or tolerance to 35–40 g of heated HEW . We reviewed the records of children who underwent HEW OFCs at the National Center for Child Health and Development (NCCHD) between November 2013 and July 2019 and who tested positive for OM-sIgE or EW-sIgE before 2 years of age . Among 2We performed OFCs  followinAll the OFC data, history of avoidance, allergic comorbidity , EW-sIgE levels, OM-sIgE levels , and totU-test was and the Fisher's exact test were used to analyse the variables between the two groups. Multivariate logistic regression analysis was applied to both patient groups as dependant variables, while OM-sIgE levels at <2 years of age or the peak egg white IgE levels >50 UA/ml and OIT were considered covariates (explanatory variables). A p < 0.05 was considered statistically significant. All statistical analyses were performed using the JMP®14 software .For statistical analysis, the Mann Whitney-p = 0.006, see p = 0.024, see Sex, prevalence of atopic dermatitis, prevalence of allergic rhinitis, and total IgE values at <2 years of age were not different between the two groups, but the prevalence of asthma was significantly higher in the HE tolerance group than in the low-dose HE reactor group see . All chiOur study provided evidence that a higher proportion of experiencing strict avoidance was observed in school-aged children with low-dose HE reactor than in those who achieved HE tolerance. Our findings also showed that strict avoidance of HE intake for a long period from early infancy might increase the risk of a persistent HE allergy in school-going children.Our results show that although OM-sIgE levels in children younger than 2 years and the peak egg white IgE levels >50 UA/ml were significantly higher in the low-dose HE reactor group than in the HE tolerance group, multivariate logistic regression analysis revealed that avoidance of HE was associated with low-dose HE reactor even after adjusting the OM-sIgE levels at <2 years of age or the peak egg white IgE levels >50 UA/ml, respectively. Our findings are consistent with the previous study revealed that patients with an egg white-specific IgE (EW-sIgE) level of >50 kU/L increased the risk of low-dose HE reactor in school-age children . DespiteTo the best of our knowledge, this study is the first to demonstrate the risk of strict avoidance of HE for a long period in allergic children in a real-world setting. We used heated HEW, which was similar to baked egg, instead of raw HE in our study. Baked egg tolerance was previously observed in 64–80% of children with HE allergies , 12. HowThe strength of this study is that the HE allergy outcomes were confirmed by the gold standard method of OFC. In Japan, majority of children tend to undergo OFCs at 6 years of age before being elementary schoolers. Age of 6 years is very important point for food allergy patients and their families because elementary schools in Japan provide mandatory school lunch for children. As mentioned earlier, our previous RCT (PETIT Study) demonstrated that early introduction of HE  was effective in preventing HE allergy development at 12 months of age, although 80% of participants at 6 months of age were already sensitized to HE. Our study results were consistent with the PETIT study  guidelines  state thThere are two limitations to this study. First, this study was an observational, therefore, its evidence level is lower than that of an RCT. Second, this study included a case from a single facility.In summary, our study showed that strict avoidance of HE intake for a long period from early infancy increased the risk of low-dose HE reactor in school-age children. We reiterate that the recommendation to avoid an allergenic food item should not be decided based on a simple finding of IgE sensitization. Even in children who are already sensitized and have developed an HE allergy, we believe that introduction of low-dose HE might resolve HE allergy as a safe method of OIT. Our findings significantly highlight the risk of avoidance of HE with children with HE allergy by high EW- and OM-sIgE levels.The datasets presented in this article are not readily available due to ethical considerations and the privacy laws of Japan.The studies involving human participants were reviewed and approved by the ethics committee of the National Center for Child Health and Development (2019-066). Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.YMiyag managed and analyzed the data. KY-H and YO obtained the funding [Grant-in-Aid for Scientific Research (B) 19H03569], contributed to the conception and design of the study, and interpretation and discussion of the results. All authors read and approved the final 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.


The hypothesis of the Great Evolutionary Faunas is a foundational concept of macroevolutionary research postulating that three global mega-assemblages have dominated Phanerozoic oceans following abrupt biotic transitions. Empirical estimates of this large-scale pattern depend on several methodological decisions and are based on approaches unable to capture multiscale dynamics of the underlying Earth-Life System. Combining a multilayer network representation of fossil data with a multilevel clustering that eliminates the subjectivity inherent to distance-based approaches, we demonstrate that Phanerozoic oceans sequentially harbored four global benthic mega-assemblages. Shifts in dominance patterns among these global marine mega-assemblages were abrupt  or protracted (mid-Cretaceous 129 Ma), and represent the three major biotic transitions in Earth’s history. Our findings suggest that gradual ecological changes associated with the Mesozoic Marine Revolution triggered a protracted biotic transition comparable in magnitude to the end-Permian transition initiated by the most severe biotic crisis of the past 500 million years. Overall, our study supports the notion that both long-term ecological changes and major geological events have played crucial roles in shaping the mega-assemblages that dominated Phanerozoic oceans. Rojas et al. present a new multi-scale model that reveals the three major biotic transitions of the Phanerozoic fossil record. This new model supports the hypothesis that both long-term ecological changes and major geological events played crucial roles in shaping ocean mega-assemblages through the Phanerozoic. Sepkoski formulated this three-phase model based on a factor analysis of family-level diversity2, which became a framework-setting assumption in studies on the evolution of marine faunas and ecosystems6, changing our view of the Phanerozoic history of life. However, while the hypothesis continues to serve as a conceptual platform for many recent studies9, some analyses question the validity of Sepkoski’s hypothesis10. Moreover, because Sepkoski’s study predicts unusual volatility in the Modern evolutionary fauna starting during the mid-Cretaceous, a three-phase model fails to capture the overall diversity dynamics during long portions of the Mesozoic11. Whether such mid-Cretaceous radiation12 represents an intra-faunal dynamic or a biotic transition from Sepkoski’s Modern evolutionary fauna towards a neglected mid-Cretaceous-Cenozoic fauna remains unexplored.Sepkoski’s hypothesis of the Three Great Evolutionary Faunas that sequentially dominated Phanerozoic oceans represents one of the foundational concepts of macroevolutionary research. This hypothesis postulates that the major groups of marine animals archived in the Phanerozoic fossil record were non-randomly distributed through time and can be grouped into Cambrian, Paleozoic, and Modern evolutionary faunas13, provided the first global assessment based on a qualitative approach. However, empirical estimates of the macroevolutionary pattern depend on several methodological decisions, including background assumptions, statistical threshold, and hierarchical level11. The choice of input data also matter: Sepkoski’s compendia and benthic taxa from the Paleobiology Database15 generate different results. These limitations raise two fundamental questions: How can we identify global-scale mega-assemblage shifts without relying on critical methodological decisions? And given the underlying Earth-Life System, how should we represent the paleontological input data to accurately capture complex interdependencies? These limitations result in methodologically volatile and often inconsistent estimates of large-scale macroevolutionary structures, thus obscuring the causative drivers that underlie biotic transitions between successive global mega-assemblages. As a result, whether abrupt global perturbations, such as large bolide impacts or massive volcanic eruptions17, and long-term ecological changes18 both operate at the higher levels of the macroevolutionary hierarchy remains unclear19.It has been long recognized that Phanerozoic diversity is highly structured22. Because the input network can capture the complexity inherent to the underlying system, network analysis has become an increasingly popular alternative to the typical procedures used in almost every area of paleontological research25. However, as might be expected of an emergent interdisciplinary field, methodological inconsistencies and conceptual issues in network-based paleobiology research make it difficult to compare outcomes across studies. Also, the rapid development of the broader field of network science demands a major effort from paleobiologists working across disciplinary boundaries. Current network-based approaches in paleobiology26 use standard network representations based on pairwise statistics that do not explicitly represent higher-order relationships27 in the paleontological data. In addition, clustering methods are limited to a single scale of analysis and do not capture the multiscale dynamics28 of the underlying Earth-Life System.Our understanding of the macroevolutionary dynamics of Phanerozoic life is being transformed by network-based approaches29. In practice, we created an input network that explicitly represents different geological stages30 as ordered layers assembled into a multilayer network. With multilevel hierarchical clustering28, we tested for the major biotic transitions in the Phanerozoic fossil record of the benthic marine faunas22. This multilayer network approach is transforming research on higher-order structures in both natural and social systems31, and can help us to understand the metazoan macroevolution. We demonstrate that Phanerozoic oceans sequentially harbored four global mega-assemblages that scale up from lower-scale biogeographic structures and are defined by shifts in dominant faunas across the major biotic transitions in Earth’s history. We found that abrupt global perturbations and long-term changes both played crucial roles in mega-assemblages transitions. Our study sheds light on the emergence of large-scale macroevolutionary structures32. For example, we show that biogeographic structures underlie the marine evolutionary faunas and that long-term changes controlled the shift to the modern mega-assemblage, which first emerged during the early Mesozoic but did not become dominant until the mid-Cretaceous. Our multilayer network approach provides an integrative framework of the metazoan macroevolution for future research.Here we implemented a multilayer network framework that captures higher-order temporal structures that emerge in multilayer networks describing temporal data25. In recent years, similar standard network representations, including bipartite networks .We use the map equation multilayer framework28 to understand the benthic marine fossil record provides a more reliable coarse-grained description of the macroevolutionary patterns in two ways: first, we provide a more realistic input network that explicitly represents different time intervals in Earth’s history and better captures the relationship in the underlying paleontological data. Second, we employ a clustering approach that operates directly on the multilayer network, delineates taxonomically overlapping assemblages, and automatically defines the number of assemblages or evolutionary faunas, which eliminates the subjectivity of the distance-based approaches. As a result, the macroevolutionary pattern revealed using multilayer networks integrates two major paleobiological hypotheses—the Mesozoic Marine Revolution and the Sepkoski’s Great Evolutionary Faunas—into a single macroevolutionary story.In summary, our implementation of higher-order network modelsWe found that the assembled multilayer network is best described by four significant modules at the first hierarchical level, which correspond to Phanerozoic marine mega-assemblages of highly interconnected marine benthic taxa and geographic cells .The mid-Cretaceous mega-assemblage shift is protracted, representing a gradual shift in dominance among two mega-assemblages that share more taxa (Jaccard similarity index = 0.11) and exhibit substantial overlap in geographic space Fig. C. The pr1, which postulates the gradual diversification of the Modern evolutionary fauna during the Cretaceous12. Sepkoski’s simulations arguably anticipated the Mesozoic transition11 delineated in the multilayer network analysis presented here.In this way, the quadripartite structuring of the Phanerozoic marine fossil record captured by a multilayer network analysis couples the Three Great Evolutionary Faunas and the Mesozoic Marine Revolution hypothesis47. Although this punctuated recovery pattern should be further explored at finer temporal resolutions, our results suggest that full biotic recovery of the global biosphere from the end-Permian crisis was completed by the Early Jurassic. At this point, the Triassic-to-lower-Cretaceous mega-assemblage (Tr-lKr) became robust: the probability of retrieving this globally dominant mega-assemblage across all bootstrapped solutions reaches high values (>0.9). Nevertheless, because both the Late Triassic and Early Jurassic extinctions should have contributed to the observed instability of the Tr-lKr mega-assemblage . A mass extinction event initiated the biotic transition at the end-Permian, but more studies are required to understand the subsequent evolutionary history of the marine mega-assemblages.Alternative solutions reproduce either a four-phase model with a younger Cretaceous biotic transition, Sepkoski’s three-phase model with biotic transitions occurring at era boundaries, or a three-phase model with a mid-Cretaceous but not end-Permian biotic transition. Regardless of the number of mega-assemblages delineated, these alternative solutions demonstrate that the major biotic transitions in Earth’s history occurred across the end-Cambrian, end-Permian, and mid-Cretaceous boundaries. However, network clustering shows instability of the marine mega-assemblages at the geological stages following the Permian-Triassic boundary Fig. . The sigage Fig. , our res50—could have been less severe than previously proposed41. Regardless of the estimated magnitude of extinction, we demonstrate that the reduction in generic richness associated with such catastrophic events has little effect on the temporal arrangement of the global-scale evolutionary faunas , end-Devonian (Frasnian), and end-Cretaceous (Maastrichtian) mass extinctions, all considered within the major post-Cambrian biodiversity and ecological crises in Earth’s history25 that change over geological time , control second-level but not first-level structures in this macroevolutionary hierarchy  using the Hexbin R-package (http://github.com/edzer/hexbin). Aggregated fossil data are provided in the Supplementary Information , size (5∘–10∘ latitude–longitude), and coordinate system of the grid used to aggregate data53.We used resolved genus-level occurrences derived from the Paleobiology Database 28, where layers represent ordered geological stages30 and nodes represent taxa and geographic cells25 . Specifically, for weight (wki) between geographic cell k and taxa i, we divided the number of collections at grid cell k that register taxa i by the total number of collections recorded at geographic cell k. A similar link standardization has been employed in previous studies25. We combined the last two Cambrian stages, that is, Jiangshanian Stage (494–489.5 Ma) and Stage 10 (489.5–485.4 Ma), into a single layer to account for the lack of data from the younger Stage 10 and to maintain an ordered sequence in the multilayer network framework. However, our results show that the Cambrian to Paleozoic mega-assemblage shift occurred before the gap, and they are not directly related. The assembled network comprises 23,203 nodes (n), including 4906 spatiotemporal grid cells and 18,297 genera, joined by 144,754 links (m), distributed into 99 layers (t) , and it moves guided by the weighted links in both the current and adjacent layers with a probability r. Consequently, the random walker tends to spend extended times in multilayer modules of strongly connected taxa and geographic cells that correspond to Phanerozoic marine mega-assemblages. Following the methodology of previous studies, we used the relax rate r = 0.25, which is large enough to enable interlayer temporal dependencies but small enough to preserve intralayer geographic information55. We tested the robustness to the selected relax rate by clustering the assembled network for a range of relax rates and compared each solution to the solution for r = 0.25 using Jaccard Similarity (Supplementary Fig. We used the flow-based map equation multilayer framework with the search algorithm Infomap to cluster the assembled multilayer network40. We resampled taxon occurrence at a given geographic cell using a truncated Poisson distribution with mean equal to the number of taxon occurrences. The truncated distribution has all probability mass between one and the total number of collections in the grid cell, thus avoiding false negatives. We obtained a resampled link weight for each link in the assembled network by dividing the sampled number by the total number of recorded collections in the grid cell. We included MATLAB code showing how we created bootstrap networks in the 56. We visualized the modular descriptions with an alluvial diagram of several bootstrapped networks and the reference solution (Fig. We employed a parametric bootstrap to estimate the significance of the four Phanerozoic mega-assemblages delineated in the reference solution. This standard approach accounts for the uncertainty in the weighted links connecting taxa to geographic cells due to numerous biases39. To visualize and compare the multilevel network partitions, we used the Alluvial Generator and Network Navigator apps available on https://www.mapequation.org.We clustered the networks into multilevel partitions with The Infomap Software PackagePeer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


Effective therapies and prophylactic vaccines for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is causing the coronavirus disease 2019 (COVID-19) pandemic are urgently needed. The scientific community is rapidly testing antivirals that target the ability of SARS-CoV-2 to infect and replicate including protease inhibitors, polymerase inhibitors and antimalarials [Irrespective of the type of therapy, a suite of bioanalytical assays is required to characterize efficacy and safety. Even repurposed therapies need testing with new assays specific for SARS-CoV-2. The time needed to develop these assays and their reagents is one of the major bottlenecks for SARS-CoV-2 trials. However, we also cannot sacrifice quality, and assays must be properly designed and validated for their intended use because of the immense public health implications they carry. This article provides a snapshot of the assays needed for SARS-CoV-2 with suggestions for accelerating development and implementation of these assays within analytical and logistical challenges.At the same time, COVID-19 will likely forever change the field of drug development and bioanalysis. Looking forward, we propose ideas to prepare for a new future where assays must regularly be developed rapidly, and on a global scale.in vitro assays showing neutralization of the virus are needed. Importantly, the components of the assay  and validation of the assay must be tailored to the use of the assay. For each of the assays described below, their intended use determines whether the assay should be quantitative versus quantitative or which components of the virus-host response need to be measured.Assays and biomarkers are needed for COVID-19 trials for the purposes of patient stratification and enrollment, determining pharmacokinetics (PK), characterizing mechanism of action and measuring therapeutic effect. Human sample assays to detect biomarkers, antibodies and therapeutic responses in patients and The presence of active SARS-CoV-2 infection must be assessed for enrolling patients who are positive for the virus in trials and assess prevention or improvement in the infection by measuring viral load . Similar to diagnostic tests, quantitative polymerase chain reaction (qPCR) should be used to detect SARS-CoV-2 RNA. The virus nucleocapsid primers (N1 and N2), noninfectious positive control and human specimen RNA extraction control available from the Centers for Disease Control and third-party vendors, are crucial components of this assay. The US FDA, based on recent evidence, also believes a validated single viral target SARS-CoV-2 assay could provide an acceptable performance.The design of the assay will depend on the context of use of the assay. RNA measurement for patient enrollment and screening of clinical staff can be qualitative, while assays trying to show reduction in viral load with therapy should be semiquantitative with a synthetic standard of viral genes containing a known quantity of viral RNA copies.Key considerations for viral load measurement of SARS-CoV-2 include proper collection, transport, storage and extraction of RNA. For swab methods, a nasopharyngeal collection is preferred over throat swabs because higher viral loads are seen sooner after symptom onset in the nose than in the throat  and sputRegardless of the specimen chosen, samples should be placed in recommended transport mediums and stored as recommended . Options for mediums to store samples include purchased or in-house prepared viral transport mediums and phosphate-buffered saline. Due to the need of rapid results, extraction of viral RNA should be performed with automated methods.Special considerations should also be given to the accuracy of testing assays and the false negative rate. Suggestions to decrease false negative rate include rigorously standardizing sampling and transport procedures, and the use of TRIzol™  to stabilize the RNA while inactivating the virus. Experts also recommend combinatorial testing with different or repeated viral load assays, different anatomic site sampling such as sputum or bronchoalveolar lavage fluid (BALF) and serology testing for SARS-CoV-2 antibodies .in silico and in vitro tests to demonstrate analytical specificity and exclusivity for molecular experiments. Software such as basic local alignment search tool queries are necessary to generate primers without false positives.We should also prepare to employ the rapid deployment of qPCR testing currently being used, for future infectious diseases or the evolution of SARS-CoV-2 over the next few months. This requires readiness of properly designed primers and the availability of reagents described above. Bioanalytical scientists should be familiar with Detection of antibodies against SARS-CoV-2 with immunoassays is used qualitatively to determine active or past infection (immunized) necessary for patient selection and quantified for determining if a therapy or vaccine produces antibodies against the virus. Antibody assays must not be used alone for diagnosis and patient enrollment.Capture ligand design for the immunoassay depends on intended use. Basing the capture antibody on the entire S-spike protein will increase sensitivity of the assay but decrease specificity due to homology with other coronaviruses. On the other hand, using a peptide sequence specific to SARS-CoV-2 will likely miss too many positive antibodies. Using the receptor binding domain (RBD) of the S-spike protein that binds to human ACE2, is likely the best balance . An assaThe type of antibodies detected will depend on the time course of infection and can be selected with different secondary antibodies. IgM and IgG antibodies can be detected approximately 4 days after SARS-CoV-2 infection as a marker of active infection followed by IgA antibodies .The positive antibody controls required to validate antibody assays should use human serum. Controls for system suitability, and day-to-day monitoring can use animal antibodies produced by immunizing against recombinant full-length S-spike protein. In assay validation to determine sensitivity and specificity, human serum from at least 30 patients with past infection should be used. We recommend the use of serum taken before December 2019, if possible, as negative samples.E. coli or cell lines in approximately 2 months. Batch-to-batch consistency and antibody sequencing prevents the need to revalidate assays – a common occurrence when using animal antibodies. Antibodies can be customized with human Fc regions so a single detection antibody can be used for both human serum and animal antibody controls. Making these technologies widespread will make us better prepared for the next pandemic.Generation of antibody reagents in animals for serology assays usually takes 4–9 months. Recombinant antibody library generation can produce scalable antibodies in in vitro cell-based assays to shorten the time and increase throughput needed for these assays.For both vaccines and therapeutics, the antibodies produced are tested for their functional efficacy to neutralize the target virus . Modern neutralizing assays employ a two-part method with: ligand-binding assays using human serum; and For SARS-CoV-2, ligand-binding competitive ELISA methods identify ‘positive’ samples that prevent RBD and ACE2 binding with increasing serum concentrations. Cell-based assays with infectious viral particles are then used to determine if positive serum neutralizes virus entry and replication. Separating the ligand binding and cell-based steps is logistically beneficial as the functional neutralizing assay requires a biosafety level three laboratory, while a screening ligand-binding assay does not.For vaccine trials, the viral component antigen in the vaccine must be measured after dosing as a measurement of PK. Current components for vaccines under development include whole live attenuated virus, protein subunits of S-spike protein or the RBD and DNA/RNA vaccines . The assPrimary potency measurements for vaccine trials include antibody titers against vaccine antigens and determination of antiviral neutralizing activity. These assays can employ strategies for anti-SARS-CoV-2 antibodies and neutralizing antibodies described above. Using the vaccine component as the capture ligand enables detection of relevant antibodies induced by vaccine components.The release of cytokine biomarkers after presentation of SARS-CoV-2 viral particles on antigen presenting cells initiates a cytokine storm likely responsible for the respiratory complications of the disease . StudiesIn SARS-CoV-2 trials, cytokine biomarkers can be monitored for patient enrollment, showing mechanism of action (particularly for anti-inflammatory therapies) and monitoring treatment effect in contexts of use. In previous viral challenge trials with neutralizing antibody therapies, only IP-10 and IFN-g showed significant changes after drug dosing . The spein vitro assay with recombinant furin would show a decrease in furin cleavage of S protein after development of neutralizing antibodies that block furin cleavage.Several other assays can be used as biomarkers to support the mechanism of action of therapies and vaccines. Many viruses use human endogenous proteases/convertases  to cleave the surface glycoproteins for entry into a cell. The SARS-CoV-2 strain, uniquely uses furin expressed highly in the lung to cleave S-spike protein into functional S1 and S2, which binds to ACE2 . IntraceThe antibody responses measured in the assays above characterize B-cell humoral response to infection and vaccination. Cell-mediated immunity should also be characterized for drug development as T-cell release of cytokines after an infection or vaccination promote B-cell maturity. T-cell responses to past coronaviruses have been assessed with enzyme-linked immune absorbent spot (ELISpot) assays . ELISpotAssays for COVID-19 must be developed quickly and scaled to laboratories worldwide, while maintaining rigorous quality because of the implications of the test results. Scientists must therefore ensure reagents such as antibodies are specific for SARS-CoV-2 and be able to source enough quantities needed for the high demand.Determining whether assays are detecting antibodies against SARS-CoV-2 and not other coronaviruses is crucial, because studies show there is limited cross-reactivity between antibodies for SARS-CoV and SARS-CoV-2 even though they share the same ACE2 binding domain . We shouWith the urgent need for therapeutics, laboratories characterizing SARS-CoV-2 therapies should understand our responsibility in developing assays with wide implications for individual patients and the public. We encourage following guidelines from worldwide regulatory considerations such as public health authorities, existing FDA Bioanalytical Method Validation guidelines, FDA guidelines for clinical trials during the COVID-19 outbreak and having discussions with regulators when needed .There is also ongoing discussion of whether assays to measure biomarkers for drug development should be performed in a Clinical Laboratory Improvement Amendments lab or a Good Laboratory Practice (GLP) lab. Current guidance formed at the 2019 Workshop for Recent Issues in Bioanalysis indicate biomarkers must be tested under CLIA regulations when intended for individual patient treatment , but the approach should be reviewed with regulatory agencies . Biomarkin vitro assays  presented above may streamline the time and cost of bioanalytical testing to support therapeutic development. The urgent worldwide need for therapeutics will require a sustained capacity of many laboratories to perform these assays.At a time when the world is looking to scientists to alleviate the COVID-19 pandemic, bioanalytical scientists can play a pivotal role in developing assays rapidly to bring these therapies to patients faster. The combination of human sample  and in vitro assays such as those suggested in this article into trial designs. We should also embrace novel technologies such as recombinant antibodies and combinatorial antibody libraries to reduce lead time for antibody generation.Beyond COVID-19, we as a community must adapt for a future where drugs must be developed rapidly. It is a matter of when, not if, another pandemic occurs requiring rapid assay development. This requires adopting more biomarkers and Finally, we should develop novel surrogate end points for clinical trials, especially vaccine trials that currently can take years to show an end point of population immunity. We can learn from recent history when the incorporation of CD4/CD8 cell ratios and HIV viral load as surrogate end points for HIV successfully accelerated antiviral therapy approval . Validat


Syphilis has been associated with an increase in HIV RNA and a temporary decline in CD4 T cell counts in people living with HIV who are not receiving antiretroviral treatment (ART), and may be associated with a transient HIV RNA rebound in those who are receiving ART. Our case is the first to highlight the risk of a multidrug-resistant HIV viral rebound during the course of early syphilis even if antiretroviral drug concentrations are within the therapeutic range.This 50-year-old HIV-1-positive male patient with concomitant early syphilis presented with an HIV RNA rebound (8908 copies/mL) during a scheduled visit to our clinic. He was receiving a stable ART regimen consisting of darunavir/cobicistat plus dolutegravir, and had a 15-year history of viral suppression. Good short-term drug adherence could be inferred as liquid chromatography tandem mass spectrometry showed that his trough antiretroviral drug concentrations were within the therapeutic range: darunavir 2353 ng/mL (minimum effective concentration > 500 ng/mL) and dolutegravir 986 ng/mL (minimum effective concentration > 100 ng/mL). A plasma RNA genotype resistance test revealed wild-type virus in the integrase region and protease region (PR), but extensive resistance in the reverse transcriptase (RT) region . Phylogenetic analysis of next-generation sequences , the PR and RT sequences from plasma HIV RNA and pro-viral DNA extracted from peripheral blood mononuclear cells during the viral rebound, and a Sanger sequence obtained during a previous virological failure suggested clonal viral expression because the previous PR resistance mutations had been lost or had not been archived in pro-viral DNA.This case shows that early syphilis may cause an HIV RNA rebound in patients under stable virological control with the potential of transmitting an extensively drug-resistant virus. There has recently been an increase in the incidence of sexually transmitted diseases (STDs) among men who have sex with men (MSM), whether they are people living with HIV/AIDS (PLWHAs) or the recipients of pre-exposure prophylaxis (PrEP) , 2. SyphWe here describe the case of an HIV-infected MSM with early syphilis experiencing a viral rebound and showing extensive resistance in the reverse transcriptase (RT) region. This is the first case to highlight the risk of an HIV multidrug-resistant viral rebound during early syphilis even when antiretroviral drug concentrations are within the therapeutic range.During a scheduled visit in September 2018, a 50-year-old MSM with early syphilis infection, who had tested positive for clade B HIV-1 in December 1991 and had subsequently been regularly followed up at our centre, was found to be affected by an HIV RNA rebound (8908 copies/mL). Figure At the time of the viral rebound, the patient’s syphilis was considered to be in an early stage as a rapid plasma reagin (RPR) test within 12 months of the index event was negative after treatment with benzathine penicillin for primary syphilis infection (RPR titre down from 1:32 to < 1:1 in March 2015), but the titre had increased to 1:4 in December 2017. Moreover, the patient reported that he had had multiple condomless, insertive/receptive anal intercourses in the previous months.After being purified by means of solid-phase extraction, the plasma sample used to quantify his HIV RNA viral load (handled on ice and stored at − 20 °C) was used to determine trough antiretroviral drug concentrations by means of liquid chromatography tandem mass spectrometry. The results indicated good short-term adherence to the prescribed ART as the trough concentrations were within the therapeutic range . Furthermore, all of the previous assessments of the trough concentrations of both drugs  had always shown levels within the therapeutic range, which provides indirect evidence of long-term treatment compliance.Viral rebound was assessed by means of genotype resistance tests . The test of plasma RNA showed wild-type virus in the PR and integrase region, but extensive resistances in the RT region , and the test of plasma DNA showed the same resistance profile. In order to investigate the presence of minor viral variants, next-generation sequencing analysis (NGS) of the same samples  revealed the presence of PR variants in < 1.0% of the viral population in the plasma RNA sample (DNA data are not available because of a failure in the amplification process). Phylogenetic analyses of Sanger PR and RT sequences obtained from plasma HIV RNA and pro-viral DNA extracted from peripheral blood mononuclear cells during the viral rebound, and a sequence obtained from the same patient during a previous VF in 2002 revealed close correlations, especially between the RNA and DNA samples simultaneously collected in 2018  is increasing among MSM worldwide , 2, and Conversely, Grewal et al. did not observe an increased risk of VF in a Canadian cohort of PLWHA MSM with acute syphilis who were receiving effective ART: the overall incidence of VF was 3.5/100 person-years  and, as there was no statistically significant association with acute syphilis, the authors concluded that ART may still reduce the risk of transmitting HIV to sexual partners . HoweverOur phylogenetic analysis reasonably excluded the possibility that the mutations observed during our patient’s viral rebound were due to HIV super-infection , 14, butAt the time of the viral rebound, our patent had trough concentrations of antiretrovirals within the therapeutic range. As therapeutic drug monitoring (TDM) can only provide evidence of short-term compliance to treatment, we cannot exclude the possibility that he may not have been compliant in the weeks before the event. However, he was regularly followed up at our clinic and all of the previous TDM assessments (made at least twice a year) also showed therapeutic drug concentrations, thus indirectly suggesting long-term adherence.Our case suggests that early syphilis can cause an albeit time-limited rebound of HIV viremia in patients with self reported good adherence to ART and antiretroviral trough concentrations within the therapeutic range. The HIV RNA levels reached during the rebound are consistent with a high rate of HIV transmission , and the


Neurolymphomatosis is rare. Neoplastic lymphocytes are seen to invade nerves , nerve roots or other related structures in patients with hematological malignancy. It is a separate entity from central nervous system lymphoma. Neurolymphomatosis has most commonly been described in association with B-cell non-Hodgkin lymphoma. Neurolymphomatosis in the context of Burkitt lymphoma and the post-renal transplant setting has not been described before.We report for the first time in the Arabian Gulf countries and nearby Arab states four cases of neurolymphomatosis  occurring between 2012 and 2017 involving the median nerve, optic nerve, nerve root and cauda equina in patients with Burkitt lymphoma, Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma.Neurolymphomatosis is rare and can be difficult to diagnose by biopsy but reliably confirmed by a combined imaging approach. Prior treatment with high-dose dexamethasone might suppress 18F-fluorodeoxyglucose (FDG) activity and decrease the sensitivity of positron emission tomography/computed tomography (PET/CT). The prognosis is generally poor but using high-dose methotrexate as well as high-dose chemotherapy and autologous stem cell transplantation may be an effective way to treat neurolymphomatosis. Neurolymphomatosis (NL) is the term for nerve infiltration by neurotropic neoplastic cells in non-Hodgkin lymphoma (NHL) or acute leukemia . It is rHere we describe four cases with NL with disparate presenting features that together demonstrate different approaches to the diagnosis and treatment of NL as well as different outcomes.A 33-year-old Filipino man was diagnosed at our center with Burkitt lymphoma in July 2011. Disease staging revealed an Ann Arbor stage IA disease. The patient had undergone kidney transplantation in 2007 for renal failure secondary to chronic glomerulonephritis and had been on immune suppressive treatment with sirolimus 2 mg daily since that time. The patient was treated with a methotrexate-free regimen consisting of six cycles of R-CHOP and achieved a complete remission (CR). Three months post-CR, the patient presented with pain in the right axilla, extending to the thumb, index, middle fingers and median half of the ring finger. Hyperesthesia was present in the palm of the right hand, along the sensory distribution of the median nerve. The pain rapidly worsened and hampered daily activities involving the right upper limb. Clinical examination revealed the presence of an elongated hard mass, located on the proximal medial side of the right upper arm. There was also right thenar eminence atrophy, defective apposition of the right thumb and incomplete flexion of the index and middle fingers. The patient underwent a full-body PET-CT scan. This revealed abnormal FDG-avid activity in the anatomical location of the median nerve in the right upper arm  did not provide additional benefit. Following treatment, the patient decided to temporarily move to his home country and was lost to follow-up for 3 months. On his return, he presented with generalized lymphadenopathy and deteriorating general health. The patient and his family were informed of the rapid disease progression and limited therapeutic options. Two months after beginning palliative measures, the patient died.A 40-year-old Egyptian man presented in October 2010 with generalized lymphadenopathy, hepatosplenomegaly and marked leukocytosis . Bone marrow showed a preponderance of BCR-ABL-positive lymphoblasts , consistent with pre-B acute lymphoblastic leukemia. The patient refused to receive a standard induction chemotherapy regimen and was initially treated with prednisolone and imatinib. A subsequent bone marrow reassessment showed no cytogenetic response. Three months post-diagnosis, the patient agreed to receive additional chemotherapy with Hyper-CVAD  together with dasatinib, a second-generation tyrosine kinase inhibitor. The patient achieved complete cytogenetic response after 3 months, but then refused any further chemotherapy except for oral dasatinib. Molecular remission was achieved after 6 months. CSF cytology was consistently negative, and no clinical finding suggesting central nervous system (CNS) involvement was present. In November 2012, the patient developed right eye swelling and blurred vision. MRI revealed a prominent right optic nerve with abnormal enhancement of the optic sheath involving the intra-orbital and intra-canalicular segments of the optic nerve . Eight years later, he relapsed with biopsy-proven DLBCL in the initial anatomical site and stomach fundus. The patient received six R-CHOP cycles with prophylactic intrathecal methotrexate. Following the second complete remission, he received rituximab maintenance for 1 year. Three months following maintenance completion, the patient developed lower limb motor and sensory impairment as well as urinary retention. MRI revealed cauda equina nerve root thickening and enhancement, both intra- and extradural . MRI head was normal, and cerebrospinal fluid cytopathology was negative for malignant cells. The patient achieved complete remission after six cycles of R-CHOP chemotherapy plus intrathecal methotrexate followed by two consolidation courses of high-dose methotrexate and radiotherapy to the paranasal sinuses and both testicles. The patient presented again in May 2017 with right-sided low back pain and severe right lower limb pain. MRI spine showed thickening and enhancement of the right L4 nerve root highly suggestive of infiltration by a neoplastic process  in our study. Three patients did not have a biopsy; two refused the procedure (patients 3 and 4), and in one patient the risk of permanent optic nerve injury from the procedure was deemed too high patient 2). This is in keeping with previous reports where nerve biopsy is only available in about half of cases [ in our s. This isMRI and PET/CT can be useful tools to confirm clinical suspicion of NL when a biopsy cannot be obtained. The sensitivity of those two techniques has been compared in previous studies. The International Primary CNS Lymphoma Collaborative Group has shown similarly high sensitivity for MRI and PET/CT  . MRI wasCSF involvement is not required for a diagnosis of NL. In our report CSF cytopathology findings supported the diagnosis of NL in one case (patient 2) by showing leukemic cells. One further case (patient 3) had increased T lymphocytes in the CSF without any other explanation. All four patients initially relapsed with NL after initial treatment despite complete remission from the original disease.et al. [The initial presenting complaints were varied, including painful peripheral neuropathy patient 1), cranial nerve palsy patient 2) and nerve root compression (patient 4) similar to previous descriptions , cranial. In our  and nervNeurolymphomatosis has not been described before in a patient with post-transplant lymphoproliferative disorder (PTLD). PTLDs are themselves rare complications of transplantation and NL rarer still. PTLDs arise in patients who are chronically immunosuppressed, with an incidence of 1–5% in solid organ transplant recipients and 1% in allogeneic hematopoietic stem cell transplant (HSCT) recipients –35. Most2 that penetrate the central nervous system can be given, based on experience gained in treating primary central nervous system lymphoma (PCNSL) [Currently, there is no standard treatment for NL; steroids alone provide only short-lived symptom relief . Most pa (PCNSL) . Three oWe conclude that NL is a rare but increasingly recognized entity that can be difficult to diagnose by biopsy but reliably confirmed by a combined imaging approach. However, prior treatment with high-dose dexamethasone with or without local radiotherapy might suppress FDG activity and decrease the sensitivity of PET/CT. The prognosis is generally poor but using high-dose methotrexate as well as high-dose chemotherapy and ASCT may be an effective way to treat NL Table .Table 1P


Genome-wide association studies (GWAS) have identified 113 single nucleotide polymorphisms (SNPs) affecting the risk of developing ankylosing spondylitis (AS), and an on-going GWAS study will likely identify 100+ new risk loci. The translation of genetic findings to novel disease biology and treatments has been difficult due to the following challenges: (1) difficulties in determining the causal genes regulated by disease-associated SNPs, (2) difficulties in determining the relevant cell-type(s) that causal genes exhibit their function(s), (3) difficulties in determining appropriate cellular contexts to interrogate the functional role of causal genes in disease biology. This review will discuss recent progress and unanswered questions with a focus on these challenges. Additionally, we will review the investigation of biology and the development of drugs related to the IL-23/IL-17 pathway, which has been partially driven by the AS genetics, and discuss what can be learned from these studies for the future functional and translational study of AS-associated genes. Ankylosing Spondylitis (AS) is a common form of immune-mediated arthritis that predominantly affects the sacroiliac and spinal joints and can result in excessive ossification of the affected tissues. Over the past decade the successful introduction of new treatments for AS -α and interleukin (IL)-17A) has highlighted some of the important pathological pathways involved. However, <50% of patients achieve good response (ASAS40) to either TNF-α or IL-17A blockade . More imThe value of genetics in drug discovery is increasingly appreciated , 4. The The functional investigation of genetics association currently encountered a number of challenges: (1) difficulties in determining the causal genes regulated by disease-associated SNPs, (2) difficulties in determining the relevant cell-type(s) that causal genes exhibit their function(s), (3) difficulties in determining appropriate cellular contexts to interrogate the functional role of causal genes in disease biology. This review will discuss recent progress and remaining challenges. While appreciating the importance of identifying causal SNPs, limited by the length of this mini-review, we choose to refer readers to recent review rather than discuss this topic here . Followi*27 as a strong genetic risk factor in 1973 , promoter capture Hi-C (PCHi-C), and HiChIP constitute key advances for the prediction of causal genes through the annotation of genetic risks .Expression quantitative trait loci (eQTL) identifies genomic variants that contribute to altered expression levels of mRNAs. eQTL have been carried out using various primary human immune cells  in different cellular contexts (resting and activation) –21. ThesThe development of chromosome conformation capture (3C) and its related techniques, such as Hi-C, has allowed the detection of long-range regulatory DNA interactions , 27. To disease-relevant cell-types for functional investigation is a key challenge impeding the translation of genetic findings to new biology and therapeutic options. Some causal genes identified by eQTL or chromatin looping datasets will be limited in their action to specific cell-type(s), guiding the selection of cells to be investigated . This is particularly important for genes where existing knowledge of function is limited, a common situation for GWAS hits. To this end, transcriptional data from patient-derived cells and/or particular disease-related cell-types, such as Th17 cells in AS, would be of great use. For example, if a causal gene is elevated in Th17 cells, one would predict it to be a possible Th17 regulator and test its function in a Th17 functional cellular assay. Single cell RNA sequencing (scRNA-seq) would excel here in the provision of gene abundancy data for multiple cell subsets in patient blood or synovium. However, a well-known drawback of scRNA-seq is its inability to detect genes in low abundance. Antibody (CITE-seq) or oligo (BD Rhapsody)–based tagging of genes of interest might go some way to solve this problem. In addition, for genes with available antibodies, mass cytometry or CyTOF is an alternative approach to acquire the expression profile of a gene at the protein level.eQTLs are frequently different in different cell types. For example, the eQTL link of GAB2 gene with rs2511162 is found for naïve B cells and T cells, but not monocytes . Even wiEvidence suggesting key roles for rare immune cell populations in AS has recently emerged,. For example invariant NK cells (iNKT) and γδ T cells have recently been reported to be a major source of IL-17 in the inflamed joint . These iEven within one cell-type, specific subsets might be highly relevant to the pathogenesis of human diseases. For example, using single cell RNA sequencing (scRNA-seq), MerTK+ synovial tissue macrophages have recently been shown to be key for the remission of rheumatoid arthritis after treatment cessation . Thus, sMost functional genomics data are at the DNA or RNA level. This does not invariably relate to cellular and cell surface protein expression. Advances in quantitative MS might allow QTL at the protein level. Indeed, quantitative proteomics has been utilized to advance knowledge in biology, such as the dynamic protein landscape of human Th17 differentiation , and theIt will also be important to contextualize the anatomical location of immune cells and their detailed functional interactions. The human tissue atlas will provide a framework and detailed spatial transcriptomic and protein expression studies of diseased tissue including entheses will undoubtedly enrich current knowledge. Without doubt the greatest knowledge gains will flow from the study of cells from inflamed tissues. We believe that obtaining these from human diseased tissues will be more informative given the limitations of current animal models and the rapid advances in single cell technology.Moving from tissue level understanding to whole organism will be a further challenge. Animal models of AS have proved useful for studying specific pathogenic processes and offer opportunities for intervention. The HLA-B27 transgenic rat and the SKG mouse have both provided key insights, with the former model confirming the role of HLA B27, myeloid cells and gut flora in disease and the latter confirming the key role for the IL-23-17 pathway (see below). Considering both animal models and human studies it will also be important to distinguish the relative roles of tissue-resident and tissue-specific cells from those of circulating cells. We believe that using animal models to label leucocytes present in the gut mucosa  and then follow their potential movement to joints and other inflammatory sites is likely to offer major insights into disease pathogenesis. Ultimately human experimental medicine studies will prove the key arbiters of target selection and will provide a rich source of data.IL-23 is formed by P19 and P40 subunits with the later, along with P35, also forming IL-12 . IL-23 sMore than 90% of genetic risk SNPs are present in non-coding regions. Thus, IL23R, where genetic risk loci reside both within coding (the cytoplasmic tail) and non-coding regulatory regions, represents the exception rather than the norm. The genetic association of IL23R loci with AS was first reported in 2007 , the firAntibodies blocking cytokines or receptors related to this pathway have been extensively tested in AS. IL-17A blockers have demonstrated efficacy and been approved for the treatment of AS , 55. In The success of IL-17A blocking and failure of IL-23 inhibition in AS suggested that IL-23 might not be the main driver of IL-17A production in AS. In human, IL-1β and IL-6 are required for the differentiation of Th17 cells . Of inteThe therapeutic development of inhibitors targeting the IL-23/IL-17 pathway in AS highlights the notion that genetic risk alone is not necessarily the ideal guide to drug target identification and that downstream protein(s) might be better therapeutic options in some cases. Indeed the association of genetic risk with drug success in trails is substantially enhanced when proteins interacting with these risk-associated gene products are included . ConsideExciting progress has been made in the genetics of AS, resulting in identification of over one hundred genetic variants that affect the risk of disease development. Entering the post-GWAS era, we have encountered multiple challenges and bottlenecks in the translation of GWAS findings to new biology and drug targets. With the rapid development of functional genomic techniques/methods and transcriptomic and phenotypic profiling of primary cells at single cell resolution, it is now possible to predict both causal genes and their relevant cell-type. This will allow us to more rigorously investigate the cellular contexts of disease pathogenesis and to functionally validate therapeutic targets. However, disease-specific functional genomic datasets and those for rarer immune cells are currently not available, representing opportunities for future research. The successful development of drugs targeting the IL-23/IL-17 axis for conditions genetically associated with IL23R is a great example demonstrating the value of genetics in drug development. We also learned that causal genes are not always the best drug targets, highlighting the importance of establishing downstream pathways. Thus, an in-depth understanding of causal gene-related biology is absolutely crucial for the development of novel treatment options.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Excitatory synapses in the mammalian cortex are highly diverse, both in terms of their structure and function. However, relationships between synaptic features indicate they are highly coordinated entities. Imaging techniques, that enable physiology at the resolution of individual synapses to be investigated, have allowed the presynaptic activity level of the synapse to be related to postsynaptic function. This approach has revealed that neuronal activity induces the pre- and post-synapse to be functionally correlated and that subsets of synapses are more susceptible to certain forms of synaptic plasticity. As presynaptic function is often examined in isolation from postsynaptic properties, the effect it has on the post-synapse is not fully understood. However, since postsynaptic receptors at excitatory synapses respond to release of glutamate, it follows that they may be differentially regulated depending on the frequency of its release. Therefore, examining postsynaptic properties in the context of presynaptic function may be a useful way to approach a broad range of questions on synaptic physiology. In this review, we focus on how optophysiology tools have been utilized to study relationships between the pre- and the post-synapse. Multiple imaging techniques have revealed correlations in synaptic properties from the submicron to the dendritic level. Optical tools together with advanced imaging techniques are ideally suited to illuminate this area further, due to the spatial resolution and control they allow. Synapses in the brain are diverse, plastic structures with distinct morphologies . Even wiUltrastructural studies help illustrate the extent of synaptic variability. They have revealed that spine head volume in the hippocampus can vary by ∼180 fold, postsynaptic density (PSD) area by ∼70 fold, spine length by ∼10 fold, and synaptic vesicle number by ∼500 fold . The ricThe functionality of synapses is related to their structure and molecular composition. For example, one highly reproduced finding that mirrors the presynaptic structural diversity of synapses is that the probability of neurotransmitter release [P(r)] at central synapses is highly variable. The diversity of P(r) has been measured using a variety of methods including the progressive block by the use-dependent NMDA receptor (NMDAR) antagonist MK-801 , the actWhen the post-synapse is studied, a similar correspondence between structure and function is observed. A precise relationship exists between the molecular complement of spines and their geometry. For example, the PSD length and basal AMPAR expression are positively correlated, with functional AMPARs expressed at a similar density across different spines . In addiMany factors influence the relationship between postsynaptic structure and function. For example when specific proteins such as PSD-95 are knocked out, there is a resultant increase in silent synapses on mature spines . AdditioUltrastructural studies suggest that numerous features of the pre- and post-synapse are correlated. These include relationships between the PSD size and the active zone size, and between the postsynaptic spine head volume and the number of vesicles in the presynaptic varicosity  Harris , 1999. TConversely, knocking down AMPARs reduces presynaptic functionality, suggested by reduced mEPSC frequency and reduced uptake of antibody directed against the vesicular protein synaptotagmin. When other measures of presynaptic activity were examined, by measuring paired pulse facilitation and MK-801 block, there was no difference detected. This led the authors to conclude that a subset of synapses had become inactive, leaving the remaining synapses with unaltered P(r) . Two typThe ability to image synaptic proteins using super resolution imaging has resulted in the discovery of subsynaptic domains. These are ∼70–80 nm domains enriched with PSD-95, Homer, and AMPARs at the post-synapse of excitatory synapses . These dExamining synapses using 3D stochastic optical reconstruction microscopy (3D-STORM)  has reveStimulated emission depletion (STED) microscopy  has reveIn order to study pre-synapse function, imaging techniques have proved invaluable. Styryl dyes such as FM1-43 or FM4-64 have proved useful as they offer the benefits of being amenable to measuring P(r) of evoked neurotransmission directly, and can be used in semi-intact  preparations. FM dyes were used originally to study vesicle recycling at the frog neuromuscular junction  and the These amphipathic dyes are incorporated into biological lipid membranes due to their short hydrophobic tail, but do not pass all the way through the membrane due to the highly charged hydrophilic group at the opposite end of the molecule  Betz et. When apFM dye uptake has been used in two ways to assess presynaptic activity. The first involves loading FM dye into presynaptic vesicles using a low number of electrical stimulations, a protocol that results in individual peaks in the fluorescence intensity frequency histogram that likely represent the fluorescence from individual vesicles . With reA functional correlation between pre- and post-synaptic activity has been observed under a variety of conditions. An initial connection was observed by relating the intensity of GluA1 immunofluorescence staining in dissociated culture to the uptake of antibody directed against the vesicular protein synaptotagmin . HoweverLive cell imaging aimed at understanding the AMPAR trafficking that contributes to the expression of hippocampal synaptic plasticity has helped shed light on the activity-dependent mechanisms that may underlie the correspondence between pre- and post-synaptic properties. One imaging method involves using super ecliptic pHluorin (SEP), a pH-sensitive variant of Green Fluorescent Protein that yields greater fluorescence at neutral pH compared to acidic pH. SEP expression allows the preferential imaging of GluA2 at the cell surface where pH is ∼7, rather than in the endocytic pathway where pH is ∼5. Versions of this fluorophore were initially developed, and have been used extensively, to study presynaptic secretion . This meWe have recently combined the use of SEP-GluA2 and FM4-64 to investigate the relationship between postsynaptic AMPAR trafficking and P(r) at individual hippocampal synapses . In partWhere manipulations have been performed that modulate P(r), for example, changing the calcium to magnesium ratio, the magnitude of DHPG-LTD and the extent of AMPAR trafficking is altered in a way that is consistent with this mechanism. For example, increasing the calcium to magnesium ratio results in higher P(r) and DHPG-LTD and SEP-GluA2 trafficking are both reduced; if the ratio is decreased to lower P(r) then LTD and GluA2 trafficking are both increased .high P(r) synapses theta burst stimulation (TBS) induces downregulation of mGluR1 resulting in lower basal mGluR1 levels (low P(r) synapses because this is where the trigger, mGluR1, is expressed most highly. As theta burst activity develops over the course of development (Why does DHPG-induced AMPAR trafficking occur predominantly at low P(r) synapses? DHPG-LTD can be triggered by either mGluR1 or mGluR5 . The tri1 levels   was found to enhance the trafficking of SEP-mGluR1 suggesting that the selective effect of theta bursts at high P(r) synapses is likely because the greater frequency of glutamate release at these synapses results in more spillover of glutamate to peri-synaptic areas where mGluRs are expressed . ConsistWhat are the possible functional consequences of a relationship between P(r) and AMPAR trafficking? Here we put forward a few suggestions.We found that TBS induces SEP-GluA2 trafficking in synapses of intermediate P(r) via a mechanism that requires mGluR1 activation  Sanders. That thin vitro application of a GABAA antagonist such as bicuculline results in decreased synaptic transmission due to the cell wide downregulation of surface AMPARs, a form of homeostatic plasticity termed synaptic scaling-down  Homer 1a  Brakema. An incring-down . The exping-down . Homer 1ing-down   synapses , it coulOf significant interest is whether the mechanisms revealed by making these experimental manipulations are engaged in endogenous physiological processes. One exciting possibility is that Homer 1a-induced synaptic scaling-down is engaged during sleep. Synaptic Homer1a is upregulated during sleep where it orchestrates synaptic downregulation as a result of group I mGluR-induced AMPAR trafficking and dephosphorylation . These fin vivo in response to sensory experience  is an input non-specific way through heterosynaptic signaling. If LTP is induced at a cluster of synapses, as has been observed to occur perience , neighboperience . This doInterestingly, using inducible presynaptic expression of tetanus toxin light chain to suppress transmitter release, the IEG Arc was found to be trafficked specifically to synapses with reduced activity, in a process termed inverse synaptic tagging . The traIn addition to the mechanisms detailed above, signals that involve retrograde messengers may coordinate the pre- and post-synapse. For example, these may be of relevance to the increase in presynaptic activity induced by overexpression of postsynaptic scaffolding proteins . In partThe adaptations in response to postsynaptic receptor blockade typically occur over the time frame of hours . HoweverThere is evidence that mGluR-LTD is expressed via both pre- and post-synaptic changes , with prIn addition to diffusible signaling molecules, precise co-ordination between the pre- and post-synapse occurs via direct physical interactions. The matching of AMPARs with the presynaptic release machinery in trans-synaptic molecular “nanocolumns”  depends In summary, there appear to be multiple signaling mechanisms and dedicated molecular machinery that could result in coordination between changes at the pre- and post-synapse.Above we have summarized data indicating that the pre- and the post-synapse are structurally and functionally correlated, that multiple mechanisms may bring this about including mGluR signaling, and that the expression of mGluRs may play a role in patterning the modulation of synapse strength in several physiological situations. It is not clear how synapses with certain physiological characteristics, including P(r), are arranged on the dendritic trees of excitatory neurons. Are dendrites structured such that synapses with certain properties are located in specific locations? A range of studies have addressed this question and found that synapses diminish in size toward the ends of basal and apical oblique dendrites , while iin vitro and in vivo  using FM dyes has been used to reveal that neighboring synapses on the same dendritic branch have very similar P(r) and that the P(r) is set by the local activity level . This re in vivo . It is tin vivo that occurs in response to sensory experience , since when astrocytic function is perturbed the P(r) of heterosynaptic inputs become less divergent, implying that astrocytes play a role in maintaining heterogeneity of P(r) over the entire cell . The invA major area of neuroscience research is aimed at understanding the processes involved in synaptic plasticity, the most extensively studied of which is NMDAR-dependent LTP . AlthougAt certain other synapses, such as the parallel synapses between granule cells and Purkinje cells, mGluR1 is the trigger for LTD in adult tissue  and postIn summary, rapidly coordinated changes in pre- and post-synaptic activity, mediated by the actions of the neurotransmitter itself, are likely to impact on many facets of synaptic transmission and plasticity in health and disease. These are areas ripe for future investigation.The review was written by TS and edited by JG and GC.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.


The Australian/Canadian Osteoarthritis Hand Index (AUSCAN), the Patient-Rated Wrist/Hand Evaluation (PRWHE) and the Thumb Disability Exam (TDX) are patient-reported outcome measures (PROM) designed to assess pain and hand function in patients with hand arthritis, hand pain and disability, or thumb pathology respectively. This study evaluated the content validity of AUSCAN, PRWHE and TDX in people with hand arthritis.This study enrolled participants with hand arthritis to rate the items of all 3 PROM in terms of relevance and clarity. The Content Validity Index (CVI) was computed for each item in each scale (I-CVI) as well as for the overall scale (S-CVI). Kappa was used to determine the inter-rater agreement among the raters.Overall, 64 individuals with hand arthritis  participated in the study. The I-CVI for all items and all scales were very high (I-CVI > 0.76) and the modified Kappa agreement among the raters demonstrated excellent agreement (k > 0.76). The S-CVI for all PROMs was very high for relevance  and for clarity , respectively.This study demonstrated very high content validity indices for the AUSCAN, PRWHE and TDX; with strong consensus across raters. This augments prior studies demonstrating appropriate statistical measurement properties, to provide confidence that all three measures assess important patient concepts of pain and disability. Hand osteoarthritis (OA) is one of the most common musculoskeletal diseases and a leading cause of disability with an increasing prevalence mainly attributed to increased life expectancy , 2. ClinPatient-reported outcome measures (PROMs) are often administered to assess any health-related changes that may have occurred as a consequence of health-management interventions , 8. ManyThe Australian/Canadian Osteoarthritis Hand Index (AUSCAN) , the PatTo evaluate the Content Validity Index (CVI) of the Australian/Canadian Osteoarthritis Hand Index (AUSCAN), the Patient-Rated Wrist/Hand Evaluation (PRWHE), and the Thumb Disability Exam (TDX) in patients with hand arthritis.This study was a cross-sectional design that investigated the content validity of patient-reported outcomes  for hand arthritis. Ethical approval was granted from the Hamilton Integrated Research Ethics Board (HiREB).The participant was able and willing to provide informed consentParticipants were between 18-85 years oldThe participant had hand arthritis.The participant can read and write English.Hand pathologies or conditions other than arthritisInability to answer the survey questions in English.Participants were recruited through poster advertisements at The Roth McFarlane Hand and Upper Limb Centre (HULC) at St. Joseph’s Health Care Hospital in London, Ontario and through The Arthritis Society main website. The patients that expressed interest to participate in the study received a letter of information about the survey. Both electronic and paper versions of the survey were available for participants. An email with the link of the online survey was sent out to the participants that were interested to complete the electronic version. The electronic version was hosted on Qualtrics from May 2019 till February 2020 which is a secure data collection platform . ParticiThe Australian/Canadian Osteoarthritis Hand Index (AUSCAN) is a 15-item self-reported disease specific questionnaire measuring pain (5-items), function (9-items) and stiffness (1-item) in the hand on a scale from 0 – none to 4 – extreme for all items , 20. TheN with N being the number of raters (patients with arthritis) and A is the number of patients that agree that the item was clear or relevant  *0.5relevant . Then Karelevant . Kappa vrelevant . We perfrelevant . We condOverall, 64 individuals with hand arthritis  participated in the study. Four individuals were excluded from the analysis because their arthritis was not affecting their hand. The majority of the participants (66%) were taking pain medication on a daily basis Table . All indThe I-CVI and the S-CVI supported the content validity of the hand pain, stiffness and function items and subscales of the AUSCANs Table . Five itThe I-CVI and the S-CVI of the PRWHE for pain subscale and function subscales all supported the content validity of the PRWHE Table . Five itThe I-CVI and the S-CVI supported the content validity of the TDX for hand function, pain and satisfaction subscales Table . Eleven This study established a high level of content validity for AUSCAN, PRWHE and TDX for patients with hand arthritis. The content validity index was very high for all the individual items for each questionnaire (I-CVI > 0.77) and for the overall score (S-CVI > 0.85) in terms of relevancy and clarity, exceeding the recommended benchmarks of 0.78 respectively . The KapFor the AUSCAN the content validity was established during development using a formal clinimetric process where patients in a tertiary care centre rated items by importance and frequency to establish relevance . This stContent validity of PRWHE was established during the development of the PRWHE by using semi-structured interviews in patients with distal radius fracture and expert opinion . Later tIt might have been expected that the AUSCAN would have more relevance to our sample, than the PRWHE since it a disease-specific PROM. Both point estimate and CI comparisons indicate that AUSCAN had slightly higher overall scores in terms of relevancy  and clarity  than the PRWHE . Although the CIs of the respective S-CVIs indicate that there was a small statistically significant difference   that wasn > 60) generated similar scores between the I-CVI and K scores. This has been previously described in the literature when the number of raters increasing and the probability of chance (Pc) decreases the K agreement and I-CVI values tend to converge [Our kappa statistics indicated excellent agreement between patient raters after correcting for chance agreement. (K > 0.77). The assessment from a large pool of patients (converge .This study provided novel data on the content validity index in 3 different PROMs in patients with hand arthritis. Since few studies address content validity, this is important to support the conceptual foundations of these measures and support their use in clinical practice. While the computation of CVI is relatively easy, its major weakness is the failure to adjust for chance agreement. However, the authors tried to mitigate this problem by calculating a modified kappa agreement , 36. A pThis study demonstrated evidence of very high content validity index for all the individual items and for the overall scale of AUSCAN, PRWHE and TDX for patients with hand arthritis, with high agreement across raters. This augments prior statistical evidence supporting statistical measurement properties, to provide support for the content validity.


Hepatitis B virus (HBV) infection is an important cause of morbidity and mortality with a very high burden in Africa. The risk of developing chronic infection is marked if the infection is acquired perinatally, which is largely preventable through a birth dose of HBV vaccine. We examined the cost-effectiveness of a birth dose of HBV vaccine in a medical setting in Ethiopia.We constructed a decision analytic model with a Markov process to estimate the costs and effects of a birth dose of HBV vaccine (the intervention), compared with current practices in Ethiopia. Current practice is pentavalent vaccination (DPT-HiB-HepB) administered at 6, 10 and 14 weeks after birth. We used disability-adjusted life years (DALYs) averted to quantify the health benefits while the costs of the intervention were expressed in 2018 USD. Analyses were based on Ethiopian epidemiological, demographic and cost data when available; otherwise we used a thorough literature review, in particular for assigning transition probabilities.In Ethiopia, where the prevalence of HBV among pregnant women is 5%, adding a birth dose of HBV vaccine would present an incremental cost-effectiveness ratio (ICER) of USD 110 per DALY averted. The estimated ICER compares very favorably with a willingness-to-pay level of 0.31 times gross domestic product per capita (about USD 240 in 2018) in Ethiopia. Our ICER estimates were robust over a wide range of epidemiologic, vaccine effectiveness, vaccine coverage and cost parameter inputs.Based on our cost-effectiveness findings, introducing a birth dose of HBV vaccine in Ethiopia would likely be highly cost-effective. Such evidence could help guide policymakers in considering including HBV vaccine into Ethiopia’s essential health services package. Hepatitis B virus (HBV) infection remains an important cause of morbidity and mortality globally. According to 2015 estimates by the World Health Organization (WHO), about 260 million individuals (3.5% of the world’s population) were living with chronic HBV infection in the world [The prevalence of HBV infection is estimated at 7.4% among the general population of Ethiopia, which is at the highest end of intermediate endemicityMeasures to control viral hepatitis have progressively increased globally . Most coThe fight against viral hepatitis is gathering momentum globally. Combating hepatitis is included in the Sustainable Development Goals (SDG 3.3) . The WorEvidence on the cost-effectiveness of HepB-BD vaccine is scarce in sub-Saharan Africa. A cost-effectiveness analysis (CEA) of adding HepB-BD vaccine in Mozambique has shown that it could be highly cost-effective within the Mozambique health system . Yet, CEWe constructed a decision analytic model with a Markov process , otherwise the two strategies would have similar efficacy on infections that occur later in life . We used data on HBV prevalence among pregnant women, vaccine effectiveness, and the risk of perinatal transmission to calculate the percentage of children born with HBV infection. Infected infants either develop acute symptomatic infection with a risk of fulminant hepatitis or remain asymptomatic  were not available locally and from other comparable sub-Saharan African countries. Therefore we used data from other settings that were based on a systematic review of the efficacy of hepatitis B immunization for newborn infants of HBsAg-positive mothers where the protective efficacy of the vaccine was 72% 95% CI 60 to 80%) in preventing perinatal HBV transmission . Vaccine0 to 80% Costs were estimated from a health provider perspective and only included direct medical costs. We estimated the incremental cost of introducing HepB-BD vaccine in its monovalent form. We preferred the monovalent form of the vaccine in order to minimize wastage rates. Both recurrent and capital costs were included using an ingredients-based approach following WHO guidelines . RecurreThe medical care costs included initial assessment and diagnosis costs, antiviral drug costs, costs associated with monitoring those on treatment and not on treatment, and cost of managing decompensated cirrhosis (DCC) and hepatocellular carcinoma (HCC) Table . In ordeWe conducted a series of one-way sensitivity analyses where we varied key input parameters one at a time over plausible ranges to test the robustness of our findings Table . Based oCompared to the current strategy, an additional HepB-BD vaccine would have an incremental cost-effectiveness ratio of 110 USD per DALY averted Table . LeavingThe one-way sensitivity analyses showed that the ICERs would range from cost savings to maxima of USD625 per DALY averted. The highest ICER was observed for a 10% vaccine utilization, followed by a 2% risk of perinatal transmission when the mother was HBeAg negative and a 2.5% prevalence of HBV infection among mothers with an ICER of USD373 to 328 per DALY averted  .We conducted a cost-effectiveness analysis of HepB-BD vaccine in its monovalent form (compared with current practices) in Ethiopia and found incremental cost-effectiveness ratio of USD110 per DALY averted. The ICER for HepB-BD is lower than the willingness-to-pay thresholds suggested by Ochalek J et al. < 0.31 times the GDP per capita in Ethiopia . This maDespite demonstrating that HepB-BD vaccine is highly cost-effective in an Ethiopian setting, our analysis presents a number of important limitations. First, local data on vaccine effectiveness, health state transition probabilities, and the cost of different health states were not readily available which may decrease the accuracy behind our ICER estimates. Second, we only assessed the cost of tenofovir as the treatment of choice in our analysis but using other drugs such as entecavir might be an alternative cost-effective therapy . TreatmeSuccessful implementation of HepB-BD vaccine is largely dependent on the timing of its administration. Timely administration (within 24 h of birth) of the vaccine may require integration with maternal and child health (MCH) programs rather than through the Expanded Program on Immunization (EPI). The suggested change in the delivery of HepB-BD vaccine requires redesigning the MCH (immediate post-partum services) and EPI programs and could include other birth dose vaccines such as Bacille Calmette-Guérin (BCG) and the birth dose of oral polio vaccine (OPV-0). This will entail not only additional work on midwives  but also logistic implications for vaccine storage and delivery within the maternity setting. Further, this might require training of health personnel and awareness-raising campaigns nationally in Ethiopia. With additional efforts on social mobilization, the introduction of HepB-BD vaccine may positively contribute to the improvement in the coverage of skilled birth attendance and other birth dose vaccines in Ethiopia.Besides, resources for health care are scarce in Ethiopia. The total health expenditure per capita in Ethiopia was about US$33 for the years 2016/2017 . The annBirth dose of HB vaccine is one of the most important preventive strategies recommended by the WHO and the national viral hepatitis control and prevention guidelines in Ethiopia. Given the formidable resource constraints in low-income countries such as Ethiopia, evidence on cost-effectiveness of interventions is vital towards setting priorities. Currently, the Ethiopian government is revising its essential health services package (EHSP). Given the substantial health dividend in investing in the birth dose of HB vaccine in Ethiopia and the fact that it is highly cost-effective, it should be considered for inclusion in the Ethiopia’s EHSP.Additional file 1. Decision tree structure.


Aureobasidium pullulans AFO-202 which has been proven to bring under control blood sugar levels in human subjects and also has potential in enhancing & regulating the immune parameters in relevance to COVID-19. We further recommend that this BRMG be tried in clinical studies of COVID-19 to provide a prophylactic effect for validation.With the COVID-19 pandemic causing huge threat to public health and definite treatment modalities and preventive vaccines yet to be arrived at, some of the key indicators of relevance to its prognosis have started emerging. One such independent predictor of outcome has been fasting plasma glucose (FPG) at the time of admission. Earlier, co-morbidities such as diabetes also have been reported to have a risk of relatively increased mortality due to COVID-19. In this background, we herein report on the beneficial effects of Biological response modifier glucan (BRMG) secreted by the black yeast  Fasting plasma glucose (FPG) level at admission has been indicated as a strong independent predictor of mortality in COVID-19 affected patients 1.2]. Wang et al. published their report of 605 COVID-19 patients in whom fasting plasma glucose (FPG) level ≥ 7.00 mmol/L at admission was a strong independent predictor of 28-day mortality [. Wang etBeta-D-glucans are highly conserved structural components of cell walls in yeast, fungi, or seaweed which are naturally occurring physiologically active compounds termed as biological response modifier glucans (BRMGs) . BRMGs fAureobasidium pullulans. This BRMG is secreted as an exo-polysacchride by the AFO-202 black yeast and hence does not involve additional purification or extraction processes, thus giving a highly pure BRMG [Given this background on BRMGs, we wish to refer to a BRMG derived from the AFO-202 strain of ure BRMG . The purure BRMG . Dectin-ure BRMG , it is nure BRMG .We wish to highlight three clinical studies of this AFO-202 BRMG in relevance to its action of bringing down high FPG levels to normal values in patients with diabetes while maintaining the levels at a normal standard in healthy volunteers. The first study involved 22 healthy volunteers for whom regular consumption of this AFO-202 BRMG for three months helped to maintain FPG levels in subjects whose levels were within the normal standard while decreasing the high levels to normal values in 95% of the subjects who had high FPG at the start of the study . In anotThe possible role of AFO-202 BRMG’s anti-glycation effect results from its anti-inflammatory effect in diabetes. Patients with diabetes generally have a weakened immune system , 16, andEspecially at this time, when there is no definite treatment for COVID-19-infected patients or a preventative vaccine available, safe measures such as BRMGs that may provide a prophylactic effect should undergo clinical studies for further validation so that their consumption can help high-risk individuals to keep their FPG levels under control, thereby providing them with a safety net. Further research on the specific mechanisms of BRMGs on metabolic and immune system pathways are necessary to validate their beneficial effects in those with co-morbidities that make them prone to COVID-19 & complications.


The rate of Cesarean sections (C-sections) in Poland increased from 21.7% in 2001 to 43.85% in 2017 even though the Polish Society of Gynecologists and Obstetricians highlights the negative consequences of C-section for both mother and child and recommends to make every possible effort to reduce its percentage, following the World Health Organization recommendations. There is a long list of possible complications related to the uterine scar after C-section, including uterine scar dehiscence, uterine rupture, abdominal and pelvic adhesions, uterine synechiae, ectopic pregnancy, anomalous location of the placenta, placental invasion, and—rarely—vesicouterine or uterocutaneous fistulas. Ultrasound (US) remains the first-line modality; however, its strong operator- and equipment dependence and other limitations require further investigations in some cases. Magnetic resonance imaging (MRI) is the second-line tool which is supposed to confirm, correct, or complete the sonographic diagnosis thanks to its higher tissue resolution and bigger field of view. This article will discuss the spectrum of C-section complications in the MR image-rich form and will provide a systematic discussion of the possible pathology that can occur, showing comprehensive anatomical insight into the pelvis after C-section thanks to MRI that facilitates clinical decisions. Cesarean section (C-section) is a popular delivery technique which—being necessary for certain conditions—can lead to a considerable percentage of complications.Ultrasound (US) is the first-line imaging tool in the detection of these complications.Acute intraoperative and postoperative complications are addressed with the use of computed tomography (CT) if diagnostic imaging is necessary.Magnetic resonance imaging (MRI) reveals consequences and complications of C-section in a targeted pelvic examination but also incidentally in the L-S spine examination.The rate of Cesarean sections (C-sections) in Poland increased from 21.7% in 2001 to 43.85% in 2017 even though the Polish Society of Gynecologists and Obstetricians highlights the negative consequences of C-section for both mother and child; recommends to make every possible effort to reduce its percentage, among healthy primiparous women with uncomplicated pregnancies in particular; and determines the optimum indications for C-sections . Di. DiAbnorAll the authors in the literature stress the necessity of complementary use of both US and MRI in these clinical situations –29.Magnetic resonance imaging provides comprehensive anatomical insight into the pelvis and its pathology facilitating clinical decisions thanks to its higher tissue resolution and a bigger field of view than ultrasound. The sequelae of Cesarean section can be depicted on lumbar spine MRI in a certain percentage of cases as an incidental finding and corroborated in detail on targeted pelvic MRI in order to cope with complications.