Inflammation of the gastrointestinal tract and liver can be caused by a variety of factors including genetic diseases, immune mediated conditions, environmentally induced reactions, toxic exposure, tissue injury, abnormalities in the gut-liver axis, and general infectious diseases. The consequence of inflammation is wound healing following fibrous tissue repair and regeneration. Although it occurs in all organs, each organ shows a specific pattern of reactivity.In this Research Topic, leading experts from the field of Gastroenterology and Hepatology share new findings and current concepts in the pathogenesis and management of inflammation and fibrosis in the gastrointestinal tract and liver. In total about 200 basic scientists and clinicians from eight countries  contributed 28 originals or review articles about current perspectives and findings in corresponding diseases. These articles cover a wide range of aspects in the pathogenesis of liver inflammation and fibrosis, gastrointestinal disease, and inflammatory bowel disease (IBD). The contributions show that these fascinating fields have made conceptual advances that in the near future will hopefully lead to novel treatment options.An et al. discuss the main roles of interleukins (ILs) in the context of hepatic inflammation and how this knowledge can be used for the development of therapeutic drugs. In particular, they highlight the dynamics by which interleukins mediate the cross-talk between hepatic cells during liver inflammation and injury. It becomes clear that abrogating IL signaling might prevent the progression of hepatic fibrogenesis in the early stages of liver injury. However, as the authors discuss there are some practical problems that need to be addressed to move IL-targeted therapies from the experimental stage to the clinical setting.Han et al. investigated the impact of a traditional medicinal herbal extract derived from Chicory (Cichorium pumilum Jacq) in a rat model of liver fibrosis induced by chronic colitis. The authors demonstrate that this herb can promote probiotic growth and prevent liver fibrosis. In addition, the authors demonstrate that Lactucin that is one of the bitter tasting sesquiterpene lactone of Chicory is able to inhibit lipopolysaccharide-induced inflammatory responses in vitro and activation of the Mitogen-activated protein kinase and Akt signaling. Therefore, the authors proposed Lactucin as one of the most significant anti-inflammatory compounds in Chicory extracts.In an experimental animal study, Rahman et al. In their study, the authors provide evidence that carveol improves liver detriments and liver metabolic deficits in mice subjected to APAP, while the nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor all-trans retinoic acid exaggerated APAP toxicity. Mechanistically, the authors found that carveol treatment increases Nrf2 expression that combats various reactive oxygen species and other stress kinases. Additional, comprehensible in-silico docking studies showed that carveol might have an affinity to the active catalytic pockets of COX-2, HO-1, IL-1, NF-κB, iNOS, Nrf2 and TNF-α that all impact inflammation.Another compound boosting endogenous antioxidant mechanisms in the context of acetaminophen (APAP)-induced liver damage was identified by Nrf2 pathway was also investigated in an experiment study by Zhao et al., who analyzed the effect of bicyclol in cholestatic mice caused by bile duct ligation. Interestingly, it was found that bicyclol acts as a significant hepatoprotective agent that attenuates liver damage by increasing the levels of hydropholic bile acids such as α-muricholic acid (MCA) and β-MCA. Moreover, bicyclol promoted autophagy and activated Nrf2 expression and antioxidant downstream genes.The Pu et al. provide evidence that the 5-lipoxygenase (5-LO) that catalyzes the oxidation of essential fatty acid substrates into inflammatory leukotrienes is a key enzyme involved in mediating hepatic fibrosis. The authors could show that genetic ablation or pharmacological inhibition of 5-LO is therapeutically beneficial to block hepatic fibrosis in vitro and in vivo. In line, patients suffering from non-alcoholic steatohepatitis and liver fibrosis showed elevated levels of 5-LO suggesting that strategies to target 5-LO may offer new therapeutic avenues.Shouman et al. They evaluated antisense oligonucleotides directed against the tissue factor in rats in which liver fibrosis were induced by a single administration of Diethylnitrosamine followed by repeated doses of carbon tetrachloride once weekly for 6 weeks. The authors could demonstrate that the blockage of tissue factor expression resulted in a significant downregulation of the protease-activated receptor 1 (PAR1) and toll-like receptor 4 (TLR4) that are both critical drivers in hepatic inflammation and fibrosis.A new network in the pathogenesis of hepatic fibrosis was introduced by Roeb and Weiskirchen summarized findings from a selective literature search on topics related to non-alcoholic fatty liver disease (NAFLD), fructose and fibrosis. The study showed that the rate of overweight and obesity is significantly higher in adult and pediatric patients suffering from non-alcoholic steatohepatitis (NASH). Although it is presently not known whether this is due to an excess of energy or the particular metabolism of fructose, the authors suggest that reduction in sugar consumption in conjunction with avoidance of foods enriched in saturated fats and weight loss is recommended for NAFLD and NASH patients.Xiang et al. analyzed the effects of kaempferol on the liver X receptor α (LXRα)/lysophosphatidylcholine acyltransferase 3 (LPCAT3) pathway in livers of mice fed a high fat diet inducing NASH. They could show that this natural flavonol reduces endoplasmic reticulum stress and inflammation as assessed in vitro and in vivo by reduced expression of LXRα, LPCAT3 and genes involved in the initiation or execution of endoplasmic reticulum stress. Based on their results the authors conclude that the LXRα/LPCAT network offers new potential targets for NASH treatment.Acharya et al. summarized recent aspects in the understanding of hepatic fibrosis including relevant disease-associated pathways, cellular and molecular drivers of fibrogenesis, and how this knowledge can be translated into therapy of respective disease. The review shows that hepatic fibrogenesis is a highly complex and orchestrated process that offers many therapeutic opportunities for impeding or reversing this disease process.Li et al. ameliorates hepatic steatosis by decreasing the de novo lipogenesis enzyme acetyl-CoA carboxylase 1 (ACC1), increasing the fatty oxidation enzyme acyl-CoA oxidase 1 (ACOX1), and inducing autophagy via the AMPK-mTOR pathway. This may be in future used as therapeutic approach to treat NAFLD by a functional mechanism.The sodium-glucose cotransporter two inhibitor dapaglifloxin was shown by Ye et al. showed that Salidroside inhibits carbon tetrachloride-induced liver fibrosis in mice by suppression of JNK activation and modulating the sphingosine kinase 1/sphingosine-1-phosphate signaling pathway for inhibition of Akt. This reduces activation and migration of hepatic stellate cells (HSC) as the executive players in fibrosis. Salidroside alleviates by this mechanism liver injury, hepatocyte apoptosis and consequently liver fibrosis.The group of Drescher et al. discusses developmental differences between individual immune cells and their role in health and disease, with a focus on liver diseases. They summarized findings showing there exists a fragile balance between T helper 17 (TH17) and regulatory T cells (TReg) that is critical in maintaining immune homeostasis during acute and chronic inflammation.A review article by Zhu et al. focused on Hengshun aromatic vinegar as a food additive to improve NAFLD. It improved cell viability and attenuated cell damage. Serum levels of triglycerides, transaminases  and malondialdehyde (MDA) dropped. Inflammation and lipogenesis were reduced by interference with metabolic key regulators, i.e. by enhancing silent information regulator of transcription 1 (Sirt1). Thus, it represents a new assistant strategy to fight NAFLD.Kong et al. investigated the role of the AMP analog 5-aminoimidazole-4-carboxamide (AICAR) which acts as an activator of the AMP-kinase (AMPK) on the pathogenesis of acute pancreatitis-associated liver injury (PALI). In a rat model of sodium taurocholate-induced acute pancreatitis, they found that AICAR attenuated PALI and restored liver function. Moreover, the compound activated the AMPK/Nrf2 signaling pathway, while the AMPK inhibitor Compound C aggravated PALI. In Nrf2 null mice, the effect of AICAR on alleviation of liver injury was significantly weakened suggesting that AICAR protects against PALI by interfering with Nrf2-triggered processes.Lamas-Paz et al. identified a novel mechanism that triggers acute alcoholic hepatic injury in the context of the gut-liver axis. The authors could demonstrate mice subjected to acute alcohol injury develop significant alterations in the gut microbiota and in intestinal epithelial barrier function. Interestingly, these alterations are triggered by yet unknown mediators released from extracellular vesicles produced by intestinal epithelial cells provoking deleterious effects on hepatocyte viability and steatosis.Ren et al. demonstrated that the COX-2 inhibitor rutaecaprine has gastroprotective effects on ethanol-induced acute gastric mucosal injury in mice. In the respective model, rutaecaprine augmented cellular antioxidant capacity, most likely by triggering antioxidant and anti-apoptosis defense capacities that results in inhibition of the inflammatory acting NF-κB, pathway.A thematically related study by Chen et al. performed an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based study to investigate the ameliorative effects of palmatine on Heliobacter pylori-induced chronic atrophic gastritis in rats using serum and urine metabolomics. Interestingly, this alkaloid attenuated pathological damage and inflammation and improved integrity of the gastric mucosal epithelial barrier. Mechanistically, the authors suggested that the therapeutic effects of palmatine are majorly attributable to its capacity to impact the metabolism of taurine, subtaurine, glycerol phospholipid and the mutual transformation of pentose and glucoronide.Yang et al. found that Gremlin 1 (GREM1) is significantly increased in mouse fibrotic colon. In their original study, the authors demonstrated that GREM1 promoted the proliferations and activation of intestinal fibroblasts by activating the VEGFR2 receptor and triggering fatty acid oxidation. In line with these findings, the authors could block intestinal fibrosis progression in vivo by blocking the GREM1-VEGFR2 axis.Enck and Klosterhalfen addressed the interesting question why in trials of irritable bowel syndrome (IBS) and IBD the placebo rates are remarkably high. Based on a meta-analysis, they report that the spontaneous improvement accounts for 50% of the placebo effect. Moreover, nocebo effects are easily detectable in randomized control trials (RCTs), whereas adverse events are difficult to see in respective reports.Chen Y et al. developed a novel prediction model useful to estimate the risk of primary non-response rate to infliximab therapy and select the optimal treatment for individual patients suffering from Crohn’s disease. In their retrospective study including 322 Crohn’s disease patients and data from the Gene Expression Omnibus (GEO) database, the authors performed and intensive bioinformatics analysis and provide evidence that IL-6 is a good predictive factor for risk assessment.Schmidt et al. evaluated the clinical efficacy of tumor necrosis factor antibodies on chronic IBD outcome. In total one third of the patients do not respond and in many cases drug efficacy is lost over time. Contraindications, adverse events and intolerance have to be considered when these biologicals are administered. This overview of new therapeutic approaches with novel biologicals is helpful for clinicians treating IBD.Zhong et al. showed that the serine-threonine kinase inhibitor rapamycin is effective in the treatment of upper gastrointestinal Crohn’s disease-related stricture formation, but has no effect in lower gastrointestinal Crohn’s disease. The drug was recently shown to reduce intestinal fibrosis by inhibiting CX3Cr1-mTOR-induced autophagy in mononuclear phagocytes and upregulating the IL-23/IL-22 axis. This was now evaluated by a Gastrointestinal obstruction symptoms score and diet score, which both showed improvement in upper gastrointestinal Crohn’s disease, but not in lower gut stricture disease. Adverse events were recorded in 40% of the patients, mostly mouth ulcers. However, no death or serious opportunistic infections were observed. This therapy may avoid surgical or endoscopic interventions. These promising results should now be confirmed in randomized trials with larger numbers of patients.The group of Al-Bawardy et al. highlighted the relevance of continued development of new therapeutic strategies and modifications of existing therapies to improve the outcome of IBD. In particular, they suggested small molecule inhibitors targeting the Janus kinase or the sphingosine-1-phosphate receptor that both critically contribute to the pathogenesis of IBD as promising therapeutic targets. Most important is that most of these agents can be administered orally and have a favorable safety profile compared to established anti-TNF agents.Ayyar and Moss summarized the actual knowledge on extracellular vesicles including exosomes in intestinal inflammation. The compiled information illustrates that exosomes in particular and extracellular vesicles in general are capable of modulating gene expression and cellular functions, thereby critically impacting inflammation, immune responses, and composition of gut microbiota. As such these vesicles might be suitable biomarkers of IBD and molecular structures for therapeutic targeting specific cargos to the inflamed gastrointestinal tract.Ferretti et al. provide an update on current sonographic methods to discriminate inflammation and fibrosis in Crohn’s disease, mainly focusing on studies investigating pathological features or the response to treatment as direct and indirect reference parameters. In their contribution, the authors highlight strengths and weaknesses of individual ultrasound methods and conclude that despite the promising results obtained with novel sonographic techniques such as contrast-enhanced ultrasound (CEUS) and sonoelastrography, several limitations must be addressed before using these techniques in routine clinical practice and trials.Matsumoto et al. investigated potential therapeutic effects of the RXR agonist Net-3IB that was previously developed in their team in an experimental model of colitis induced through the adoptive transfer of CD45RBhighCD4+ cells. The authors showed that Net-3IB ameliorates colitis by inhibiting both the expansion of Th1 cells and the activation of inflammatory macrophages locally in the colon. Thus, the small molecule inhibitor appears to be a promising candidate for the treatment of IBD.Lactobacillus and Akkermansia species. With antibiotic exposure the anti-inflammatory and mucus-protecting effect of metformin was abolished. It is concluded that metformin-induced changes of the microbiota are therapeutically effective in ulcerative colitis.Ke and colleagues addressed the question whether the anti-inflammatory effect of metformin in the intestine is due to a change of the microbiota. They examined this in the experimental colitis model of dextran sulfate sodium (DSS)-induced ulcerative colitis. Indeed the inflammation was attenuated by metformin. An increased expression of mucin 2 was noted. The gut microbiota changed under metformin by increasing protective Nguyen et al. demonstrates the importance of the noncanonical NF-κB signaling pathway in IBD patients. In their study, the authors analyzed the expression of 88 target genes known to be associated with noncanonical NF-κB signaling in biopsy specimens that were collected during colonoscopy. Importantly, the expression of a number of genes was associated with increased gastrointestinal inflammation and in particular in patients unresponsive to anti-TNF agents. Although the study is based on a relatively small sample size, the study suggests that the noncanonical NF-κB signaling is an understudied pathway that critically impacts the pathogenesis of IBD.The study by All these 28 articles show that inflammation and fibrosis of the gastrointestinal tract and liver are complex problems. Currently, there are a number of real breakthroughs in this research area, but of course scientists and clinicians are still at the beginning in the identification of suitable therapeutic targets. Since the frequency of these malignancies and their complications currently increase dramatically, new strategies anti-inflammatory and anti-fibrotic therapies are urgently needed.


Alzheimer’s disease (AD) is the most prevalent form of dementia, and the numbers of AD patients are expected to increase as human life expectancy improves. Deposition of β-amyloid protein (Aβ) in the extracellular matrix and intracellular neurofibrillary tangles are molecular hallmarks of the disease. Since the precise pathophysiology of AD has not been elucidated yet, effective treatment is not available. Thus, understanding the disease pathology, as well as identification and development of valid biomarkers, is imperative for early diagnosis as well as for monitoring disease progression and therapeutic responses. Keeping this goal in mind several studies using quantitative proteomics platform have been carried out on both clinical specimens including the brain, cerebrospinal fluid (CSF), plasma and on animal models of AD. In this review, we summarize the mass spectrometry (MS)-based proteomics studies on AD and discuss the discovery as well as validation stages in brief to identify candidate biomarkers. Alzheimer’s disease (AD) is the most common progressive neurodegenerative disorder with memory loss, cognitive impairment, disorientation, and psychiatric symptoms. These characteristics of AD render patients incapable of performing day-to-day activities independently. With an annual global increase in patients with cognitive impairment, the cost of caring for these patients is increasing. As the number of AD-affected people increases due to the aging population, it is becoming one of the greatest healthcare challenges of the 21st century . DespiteThe current clinical diagnosis of AD heavily relies on neuropsychological testing, clinical assessment, imaging, and exclusion of other neurological disorders . PresentIn this review, we will describe the different biological samples and proteomics technologies that have been used for furthering our understanding of disease pathogenesis and the discovery of candidate biomarkers.Over the past few decades, several studies on both patient-derived biological samples as well as in vivo and in vitro Alzheimer’s disease models utilizing proteomics technologies have been carried out. These studies have contributed greatly toward the detection of a biomarker as well as understanding the disease pathogenesis .Understanding the function of the brain and the related disease mechanisms requires comprehensive knowledge of the changes in the neuronal proteome and post-translational modifications (PTMs). The past decade has seen remarkable advances in high-resolution mass-spectrometry-based proteomics that have enabled precise analysis of thousands of proteins from brain tissue samples in a relatively shorter amount of time. Quantitative proteome analysis of the human hippocampus at different Braak stages identified alteration in 372 proteins. These proteins were components of the extracellular matrix and calcium-dependent signaling . SimilarApart from brain tissues, protein aggregates such as extracellular amyloid-beta plaques and tau neurofibrillary tangles have also been extensively studied using proteomics techniques providing insights into their role in AD pathology and progression . Liao etSw,Ind J20 mouse along with control. They reported altered mTOR signaling and β-spectrin, Rab3-mediated APP trafficking and proteolysis as early molecular events associated with AD pathogenesis [The elucidation of molecular mechanisms and cellular signaling pathways, that are responsible for neuronal cell death and advancement of the disease, is critical for discovering novel therapeutic targets. There are several limitations to use human postmortem brain tissue for research purposes: major limitations being the availability of tissue, quality of the tissue, genetic heterogeneity, pre-terminal medication or concomitant disease, cause of death, and postmortem interval. This large variability in sample procurement and other confounding factors produce inconsistent results in proteomics analyses. In vivo animal model systems serve as a better alternative to postmortem brain tissue in understanding disease mechanism as many of these issues related to consistency can be controlled. Such models can also be used for studying the molecular progression of the disease as well as different region-specific changes in the brain. Multiple mouse models have been employed for studying AD-related neurodegeneration. Tauopathy mouse model with human P301L mutation along with non-transgenic littermate controls has been used by Vega et al. for two-dimensional gel electrophoresis (2-DiGE)-based proteomics. This proteome analysis identified alteration in Ezrin expression in the early stages of neurodegeneration in tauopathy models . Mazi etogenesis . Systemaogenesis . AlthougStudies on animal models of AD have greatly improved our understanding of disease pathogenesis. However, translational clinical research is still heavily reliant on human tissue specimens. One of the other alternatives to fresh human tissue is formalin-fixed and paraffin-embedded (FFPE) tissue sample. These FFPE tissues can be stored for the long term and also have a plethora of associated clinical data like survival time and therapy response. Although such archives are immensely valuable, the use of formalin for fixation induces cross-linkages in proteins and makes it technically challenging for proteomic analyses. Several researchers have successfully adopted proteomics protocols for analyses of FFPE samples, and recent technological advancements have yielded reliable recovery and reproducible quantification of proteins comparable to fresh frozen tissues. Drummond et al. isolated neuronal cells from temporal cortex FFPE samples with severe Alzheimer’s using the LCM-LC-MS/MS technique and identified more than 400 proteins of which 78% were of neuronal origin and about 200 proteins were associated with Alzheimer’s disease . SimilarThe goal of the protein marker discovery is to detect a protein or panel of proteins that differentiates affected patients with a specific disease from healthy individuals. For biomarker analysis, it is preferred that it would be accessible through minimal invasion. There are multiple studies on identifying markers for AD using biofluids such as cerebrospinal fluid , plasma Cerebrospinal fluid (CSF) is a clear liquid produced in the ventricles of the brain that surrounds and protects the central nervous system, making it an important source of biomarkers for AD. As CSF surrounds the brain tissue, it would reflect overall brain physiology and processes like synapses loss and neuronal damage . It has Serum or plasma represents the largest version of the human proteome present in any sample as it also contains proteins from all tissue as leakage markers apart from classical “plasma proteins” ,57. Seru669–711/Aβ1–42 and Aβ1–40/Aβ1–42 ratios [Several discovery proteomics ,64,65 asUrine is formed through the filtration of plasma by glomeruli in the kidneys. Urine contains water, glucose, salt, and other metabolites and proteins derived from the serum proteins. Thus, urine can provide readout of the systemic physiology . IncreasSaliva is a physiological fluid that contains mucous and serous secretions containing mucin, alpha-amylase, and other proteins. For biomarker analysis, CSF is a relatively invasive procedure that requires the participation of specially trained medical professionals. This makes CSF a non-ideal sample for screening and early diagnosis. Saliva is noninvasive and comparatively very easily accessible compared to both blood and CSF. Expression of tau mRNA and amyloid-β precursor protein (AβPP) has been reported in the salivary gland and human salivary epithelial cells, respectively ,76. Min Currently, an autopsy is the only method that gives a confirmed AD diagnosis. The eye shares a neural and vascular resemblance to the brain, thus providing an opportunity to access the cerebral pathology . Visual Evolution of the proteomics technologies started from the development of the separation of proteins by two-dimensional polyacrylamide gel electrophoresis . PreviouData-dependent acquisition or information-dependent acquisition has been the cornerstone of shotgun-discovery-based proteomics technology. In the data-dependent acquisition (DDA) in each duty cycle, the instrument cycles through first a short high-resolution MS1 survey scan of the peptides and based on the peptide intensity it selects potential precursor ions for fragmentation followed by series of quick low-resolution MS2 scan for detection of the fragment ions (product ions). The resolution setting and automatic-gain control for MS1 and MS2 scans are optimized to get fast scan speed and lower duty cycle while providing high accuracy for precursor identification. Extensive pre-fractionation strategies or enrichment strategies are employed to either increase the proteome coverage or to access low abundant post-translationally modified proteome. Data from DDA is analyzed by comparing the experimental mass spectrum to a theoretical mass-spectrum generated from a protein reference database or a spectral library matching using different vendor-specific or open-source algorithms. Further, in discovery-based analysis, two often-used proteomics approaches are label-free and isobaric multiplex labeling strategies for relative quantitative proteomicsLabel-free quantitative (LFQ) proteomics approaches take advantage of the correlation between high-resolution LC/MS extracted ion currents (XIC) and peptide abundances for the identification of differentially abundant proteins between the different sample groups . SignalsIsobaric multiplex labeling strategies allows for relative quantitation of proteins across multiple samples in a single LC-MS run, thereby greatly increasing the throughput. Digested peptides from each sample are N-terminally tagged by one of the isobaric labels either with isobaric tag for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT). With iTRAQ reagent, eight samples can be multiplexed, while TMT reagent allows multiplexing up to 16 samples. After complete labeling of the peptides, samples are pooled followed by fractionation and LC-MS/MS analysis which drastically improves the proteome coverage. High-resolution tandem mass spectrometry can easily distinguish these tags where the relative intensity of these tags corresponds to the relative abundance of the peptides across the samples.Several studies have carried out proteomics analysis for the discovery of biomarkers using either iTRAQ or TMT. Sathe et al. used TMT-based multiplexing for the identification of CSF-based biomarker for Alzheimer’s disease. In this study, they identified several known and novel protein markers for AD. In this study, several previously known AD markers including MAPT, NPTX2, SCG2, VGF, GFAP, SST, and NCAM1 as well as novel biomarkers such as GSN, PKM, and YWHAG were identified . In anotPost-translational modifications (PTMs) are key regulators of cell signaling. There are more than 200 types of PTMs that are known to control cellular functions. Phosphorylation of proteins, at serine, threonine, or tyrosine residues, are major post-translational modifications that control cellular signaling. Protein phosphorylation and dephosphorylation is a reversible process that is regulated by kinases and phosphatases. Global characterization of changes in protein phosphorylation is needed for the understanding of the complex regulatory circuits needed for cellular response to a stimulus.Quantitative phosphoproteomics analysis of the hippocampus of the Alzheimer’s disease subjects by Domenico et al. identified significant alteration in the phosphorylation of the proteins involved in important neuronal processes such as formation, outgrowth, and guidance of neurites . In anotOnce the discovery data are acquired, rigorous statistical methods are applied to identify significantly differential proteins that can be used as potential markers for the disease. Multiple tools including Perseus , PANDA , and DAnIn a targeted proteomic analysis, proteotypic peptides are selected as a proxy for quantifying proteins of interest. Single-reaction monitoring (SRM) or multiple-reaction monitoring (MRM) analyses are routinely used for quantitation of proteins and require a tandem mass spectrometer with more than one mass analyzer. In both SRM and MRM experiments, the first mass analyzer, quadrupole (Q1), selects the precursor ion and the third quadrupole (Q3) selects the desired fragment ion using narrow mass windows. In the second quadrupole (Q2) or collision cell, precursor ions are fragmented via collisional induced disassociation (CID). Therefore, SRM requires a signal from both precursor and fragments ion pair to generate a positive result. This allows SRM to achieve high specificity along with very low background noise thereby enhancing the sensitivity of detection. Monitoring multiple precursor-fragment ion transitions for the same or different analytes in a single mass-spectrometer analysis is referred to as multiple-reaction monitoring (MRM). Several studies have employed an MRM-based targeted proteomics approach for biomarker analysis for Alzheimer’s disease.viz. creatine kinase B and fructose-bisphosphate aldolase C in AD hippocampus tissue [Multiple-reaction monitoring used for the quantification of Alpha-, Beta-, and Gamma-synuclein identified an increase in concentrations in the cerebrospinal fluid of Alzheimer’s patients . Similars tissue . Althougs tissue ,114,115.s tissue ,116. SucParallel-reaction monitoring is a rapidly emerging application of high-resolution mass spectrometry for targeted proteomics analysis. In parallel-reaction monitoring, the third quadrupole of a triple quadrupole mass spectrometer is replaced with a high resolution and accurate mass analyzer to allow parallel detection of all target product ions in one concerted high-resolution mass spectrum . PRM anaData-independent analysis (DIA) or sequential window acquisition of all theoretical mass spectra  is a recHowever, the major challenges of DIA-MS lie in data analysis and interpretation of the mixed tandem spectra originating from multiple precursors. For the analysis of the DIA data, several tools were available including Skyline , SpectroChang et al. used DIA for quantifying the synaptic proteome of synaptosomes isolated from the hippocampus and motor cortex tissue from Alzheimer’s disease cases and subjects with normal aging. They identified a total of 2077 unique proteins and reported differential expression of 30 protein including 17 novel proteins in AD . Bader eRecent developments in sample preparation methodologies along with high-throughput mass spectrometry have improved the proteome coverage. Broadly, the proteomic analysis methods can be divided into discovery and targeted mass-spectrometry-based methods. Several quantitative proteomics methods including label-free, isobaric labelling based in the discovery platform and MRM, PRM and DIA in the targeted platform have been employed for AD biomarker discover and understanding AD pathogenesis. These advancements in technologies have rapidly generated several datasets on various biofluids, tissues, and animal models related to Alzheimer’s disease. In this review, we have discussed these AD-related studies on the various specimens as well as on different mass-spectrometry platforms in detail. Although these studies have dramatically improved our understanding of disease pathology, as the complexity of the disease is high, studies at a large scale in diverse populations will be required in future for the identification of the potential biomarkers. One of the other limitations in the identification of robust and reliable biomarker candidates is the current “triangular strategy” of discovering candidates from a smaller subset of samples and validation in a larger cohort. Where many of the identified candidates turn out to be specific to the discovery cohort and fail at validation stages in larger cohorts. Such limitations are now being addressed by a shift in the study design of clinical discovery proteomics with a “rectangular strategy”. Such strategies employ high proteome depth workflows for simultaneous discovery and validation on hundreds of samples in a population-wide setting. In the future, it is also necessary to have a stringent reporting system for study and patient selection criteria, clinical metadata, and parameter used for analysis. This information will be useful for comparison of the datasets as well as to build a knowledge-base that will help to further our understanding of the disease and associated neurodegeneration.


To adapt ‘fishplots’ to describe real-time evolution of SARS-CoV-2 genomic clusters.This novel analysis adapted the fishplot to depict the size and duration of circulating genomic clusters over time in New South Wales, Australia. It illuminated the effectiveness of interventions on the emergence, spread and eventual elimination of clusters and distilled genomic data into clear information to inform public health action.The online version contains supplementary material available at 10.1186/s13104-021-05827-x. Since the arrival of the first COVID-19 case in Australia, the Pathogen Genomics Team at New South Wales (NSW) Health Pathology’s Institute of Clinical Pathology and Medical Research (ICPMR) and the University of Sydney have implemented prospective and responsive whole genome sequencing (WGS) of confirmed SARS-CoV-2 infections. A strong positive correlation between genomics-informed clusters and epidemiologically linked cases was rapidly established, and prospective SARS-CoV-2 WGS transitioned from a novel tool with unproven relevance to an essential element of NSW’s COVID-19 public health response [In this correspondence we describe the adaptation and application of ‘fishplots’ to display COVID-19 genomic cluster architecture and evolution over time. The fishplot is a variant of a streamgraph, originally developed to enable visualisation of clonal tumour evolution . We haveth and Australians returning from overseas have been required to complete a mandatory 14-day quarantine in designated hotels since March 28th, NSW has accommodated over half of the travellers returning to Australia by air. Despite this and at the time of writing , NSW accounted for just 17% (n = 5094) of Australia’s confirmed COVID-19 cases with no extensive local transmission of any strains linked to foreign travellers returning after March 15th. In total, overseas acquired cases restricted to hotel quarantine outweighed locally acquired cases, accounting for 59% and 41%, respectively. The multidisciplinary public health response to COVID-19 implemented in NSW is co-ordinated by the Public Health Emergency Operations Centre within the NSW Ministry of Health  [The state of NSW is Australia’s most populous with 8 million residents accounting for 31% of the Australian population, 65% of whom live in Greater Sydney. Since the Australian border was closed to all non-Australian travellers on March 15The integration of SARS-CoV-2 genomics into routine public health response has addressed key limitations of conventional epidemiological methods including poor or incorrect case recall, and confirmation of contentious or tenuous links. Furthermore, genomics has been instrumental in the timely identification of links between cases for which epidemiological links were not immediately apparent, supplementing conventional contact tracing methods and informing targeted public health resource allocation . In NSW,th. Sustained local transmission of introduced strains was the exception and the median duration in weeks for the 17 first wave clusters identified (≥ 5 cases) was 4  was high compared to that reported by most countries around the world  and moreAdditional file 1. SARS-CoV-2 genome sequences included in the visualisation of COVID-19 activity in New South Wales, Australia.


To report the rate of fetal anomalies detected on anatomy ultrasound in pregnant patients who underwent IVF with preimplantation genetic testing for aneuploidy (PGT-A) compared to patients who conceived following IVF with unscreened embryos and age-matched patients with natural conceptions.Retrospective cohort study at a single maternal-fetal medicine practice. Patients with singleton pregnancies who had a mid-trimester anatomy ultrasound between January 2017 and December 2018 were screened for inclusion. A total of 712 patients who conceived after IVF with or without PGT-A were age-matched with natural conception controls. The primary outcome was the rate of fetal and placental anomalies detected on mid-trimester anatomical survey. Secondary outcomes included the rates of abnormal nuchal translucency (NT), second trimester serum analytes, non-invasive prenatal testing (NIPT), and invasive diagnostic testing.There were no differences in the rate of fetal anomalies in patients who underwent IVF with PGT-A compared to patients who conceived following IVF with unscreened embryos and age-matched patients with natural conceptions. Rate of abnormal NT, high-risk NIPT, and abnormal invasive diagnostic testing were also similar. Patients who conceived after IVF with or without PGT-A had higher rates of abnormal placental ultrasound findings and abnormal second trimester serum analytes compared to natural conception controls.The use of PGT-A was not associated with a difference in risk of fetal anomaly detection on a mid-trimester anatomical survey. The results of this study highlight the importance of improved patient counseling regarding the limitations of PGT-A, and of providing standard prenatal care for pregnancies conceived through ART, regardless of whether PGT-A was performed.The online version contains supplementary material available at 10.1007/s10815-021-02069-5. Preimplantation genetic testing for aneuploidy (PGT-A) has been increasingly adopted by in vitro fertilization (IVF) clinics across the USA, with reported use in over 40% of autologous IVF cycles in 2018 . HoweverEthical concerns pertaining to the rapid uptake of the PGT-A technology include safety, cost, and a misunderstanding of the predictive value of PGT-A results. A survey of 300 patients undergoing IVF at a high-volume academic IVF center found that patients often hold misconceptions about the utility of PGT-A, with the majority of patients indicating that their primary reason for pursuing PGT-A was to increase the chance of having a “healthy baby,” and nearly one-fifth of patients indicating that it was to reduce the risk of congenital anomalies or “birth defects” .Abnormalities of fetal anatomy detected on prenatal ultrasound are reported in approximately 3–4% of pregnancies . It is eFew studies have investigated the rate of congenital anomalies in pregnancies resulting from IVF with PGT-A. Zhang et al. found no overall difference in the rate of congenital anomalies between patients who underwent IVF with or without PGT-A followed by frozen or fresh embryo transfer (ET) . ImportaThe goal of the present study was to report the rates and specific types of fetal and placental anomalies detected on mid-trimester anatomy ultrasound in patients who underwent IVF with PGT-A compared to patients who conceived following IVF with unscreened embryos and age-matched patients with natural conceptions.This was a retrospective cohort study at a single high-volume metropolitan maternal-fetal medicine referral center. All patients with singleton pregnancies who had a mid-trimester anatomy ultrasound between January 1, 2017, and December 31, 2018, were screened for inclusion. The electronic medical record was queried to identify patients with singleton pregnancies conceived with IVF with and without PGT-A during the study period. Multifetal gestations and patients without a mid-trimester anatomy ultrasound were excluded. All anatomical surveys at the center were performed by board-certified maternal-fetal medicine physicians using standardized protocols. One-to-one matching with naturally conceived pregnancies during the same period was subsequently performed based on patient age at the time of anatomy ultrasound. For patients who conceived with IVF using donor egg, the age of the egg donor at the time of retrieval was used for age matching.The charts were systematically reviewed and the data extracted by three members of the research team  using a shared protocol. Accurate categorization of pregnancies  was confirmed by a chart review. Fetal anomaly data was obtained from the targeted anatomy ultrasound report and categorized as major or minor and by organ system according to the Center for Disease Control and Prevention (CDC) guidelines . AbnormaTo detect a reduction in ultrasound-detected anomalies from 3 to 1% (OR 0.33), 768 patients would be needed per group. Our convenience sample of 2 years of ultrasounds performed on patients who had undergone IVF yielded 712 patients. A power calculation indicated that a sample size of 710 patients per group would afford 80% power to detect an OR of 0.27, or a difference from a baseline rate of ultrasound-detected anomalies of 3 to 0.8%.t test. Categorical variables were compared using the chi-square or fisher’s exact test where applicable. Logistic regression was used for multivariate analysis. Statistical significance was set at a p value of < 0.05 with two-tailed testing. Statistical analyses were performed using STATA version 14 .Comparison of continuous variables was performed using Student’s This study was approved by the Institutional Review Board of the University of California, Los Angeles (IRB# 19-000222).p < 0.001) and a higher rate of maternal comorbidities . There were no differences in BMI (Table p < 0.001). The average age of the egg donor was also younger in IVF with the unscreened embryo group . BMI, parity, rate of comorbidities, and use of gestational carrier did not differ between the two groups  (Table p < 0.001). The breakdown in frequency of various placental anomalies is available in Supplemental Table There were no significant differences in the rate of abnormal anatomy ultrasound, detection of major or minor fetal anomalies, or rate of high-risk NT in patients who underwent IVF with PGT-A compared to patients who conceived following IVF with unscreened embryos and age-matched patients with natural conceptions. The characterization of major and minor anomalies by group is shown in Table 1) Table . This fip = 0.003). This finding of a difference in rate of abnormal second trimester screen between treatment groups persisted in a multivariate logistic regression adjusting for the presence of medical comorbidities in the mother (p = 0.002). A total of 131 patients elected to undergo invasive diagnostic testing via amniocentesis/CVS. The rate of abnormal invasive diagnostic testing was significantly higher in patients with natural conceptions compared to patients who conceived after IVF with unscreened embryos or IVF with PGT-A  (Table The rate of high-risk NIPT did not differ among patients who underwent IVF with PGT-A compared to patients who conceived following IVF with unscreened embryos and age-matched patients with natural conceptions. Compared to patients who conceived after IVF with unscreened embryos and IVF with PGT-A, patients with natural conceptions had a lower rate of abnormal second trimester serum analytes (0.9% vs 3.5% vs 4.5%, In this large cohort, the rate of fetal anomalies detected on mid-trimester ultrasound did not differ in patients who conceived after IVF with PGT-A compared to patients who conceived following IVF with unscreened embryos and age-matched patients with natural conceptions. Rates of abnormal NT and NIPT were also similar between groups. These findings are consistent with prior studies that have reported similar rates of congenital anomalies in neonates conceived through IVF with PGT compared to those conceived with unscreened embryos , 20–23. A significantly higher rate of abnormal placental ultrasound findings and abnormal second trimester serum analytes were seen in patients who conceived following IVF with or without PGT-A compared to natural conception controls. Placental complications such as placental insufficiency and preeclampsia have been associated with alterations in second trimester maternal serum analytes, and patients with subfertility who conceive with and without IVF have been demonstrated to have higher rates of abnormal placentation in several large studies –30. The p value that just met significance, p = 0.049. This difference was driven by a 0% abnormal invasive testing rate in the IVF with the PGT-A group, who accounted 39 of the 56 IVF patients who underwent invasive testing. This result was not surprising given that PGT-A would be expected to reduce the number of chromosomal abnormalities detectable on CVS/amniocentesis karyotype. While discordance between PGT-A and invasive diagnostic testing may occur in rare cases, we would not expect to see it in our cohort given the sample size.The rate of abnormal invasive diagnostic testing was significantly higher among patients with natural conceptions compared to patients who conceived following IVF with or without PGT-A, with a Previous studies have shown that patients have misconceptions about the utility of PGT-A and that this impacts decision-making regarding prenatal care. In a prior publication by one of the authors, the majority of patients undergoing IVF believed PGT-A would improve the chance of having a “healthy baby” and reduce the risk of birth defects . A studyLimitations of this study include its retrospective design and the potential for selection bias given that the study setting is a high-risk referral center. The rates of anomalies in this patient population, therefore, may not reflect those in the general population. We aimed to mitigate this by inclusion of a natural conception control group in which we found a baseline incidence of anomalies similar to what has previously reported in the general population. We did not include birth outcome, newborn exam data, or placental pathology data and therefore could not corroborate the anomalies detected on mid-trimester ultrasound with clinical outcomes. While this is a limitation, it is worth noting that many pregnancies complicated by major anomalies detected on mid-trimester ultrasound are terminated and, thus, would not necessarily be captured by inclusion of newborn examination data. Finally, because only women who underwent mid-trimester ultrasound were included, this would not capture early miscarriages or terminations.Strengths of our study include the large sample size, inclusion of a natural conception control group, and that all analyzed data originated from 2017 to 2018, which increases the likelihood that IVF and PGT-A technologies used were more homogenous. Additionally, to our knowledge, this is the first study to report the rate of ultrasound anomalies, fetal and placental, in IVF patients with and without PGT-A.In conclusion, the use of PGT-A was not associated with a difference in the rate of fetal anomalies detected on mid-trimester ultrasound in this large cohort. While the available evidence does not support the use of PGT-A for the purpose of reducing the risk of birth defects, patients consistently report this as a primary reason for pursuing this technology . Improve


Toxoplasma gondii modifies the levels of microRNAs and mRNAs in infected host cells by modulating the innate and adaptive immune responses, facilitating its survival within the host. Some studies have shown that microRNAs are promising molecular markers for developing diagnostic tools for human toxoplasmosis. MicroRNAs can be detected in human specimens collected using non-invasive procedures. changes in the circulating host microRNAs have been associated with T. gondii infection in mice and ocular toxoplasmosis in humans. Besides, microRNAs can be amplified from samples using sensitive and molecular-specific approaches such as real-time PCR. This review presents recent findings of the role that microRNAs play during T. gondii infection and discuss their potential use of these small nuclei acid molecules to different approaches such as laboratory diagnosis, modulation of cell and tissue infected as other potential applications in human toxoplasmosis.MicroRNAs are molecules belonging to an evolutionarily conserved family of small non-coding RNAs, which act on post-transcriptional gene regulation, causing messenger RNA (mRNA) degradation or inhibiting mRNA translation into proteins. These molecules represent potential biomarkers for diagnosis, non-invasive prognosis, and monitoring the development of the disease. Moreover, they may provide additional information on the pathophysiology of parasitic infections and guide strategies for treatment. The Apicomplexan parasite  MicroRNAs are small non-coding RNAs acting on post-transcriptional regulation of gene expression, causing messenger RNA (mRNA) degradation or blocking mRNA translation . Since mPlasmodium falciparum, Plasmodium vivax, Cryptosporidium parvum, and Toxoplasma gondii  or inhibiting its translation into proteins . MicroRNCaenorhabditis elegans (http://www.mirbase.org/)   of whichse.org/) . DetailsThe study and processing of microRNAs as biomarkers requires attention since it’s fragile structure and stability may be compromised depending on the methods and the biological material used  . NeverthMicroRNA expression is complex and begins in the cell nucleus. The miRNA gene transcription by the action of RNA polymerase II results in the formation of a double-stranded primary miRNA (pri-miRNA) with a tail at its 5’ end and a poly-A tail at the 3’ end . Pri-miRToxoplasma gondii (T. gondii), the etiological agent for toxoplasmosis, is an intracellular parasite that infects nucleated cells from all warm-blooded animals . Types I and III  cause direct and prolonged phosphorylation of host transcription factors STAT3 and STAT6  activate the host transcription factor NF-kB, which leads to the production of pro-inflammatory cytokines in the host cell  the inhibitor of STAT1-dependent transcription (TgIST), which is a protein, binds to activated STAT1 dimers in the nucleus of IFN-γ-dependent cells, and (ii) the Mi2/NuRD complex, which can modify the chromatin of cells, blocking IFN-γ-dependent transcriptional mechanisms  and by transplacental transmission, the foetus may develop severe stages of the pulmonary disease disseminated and cerebral toxoplasmosis  for the diagnosis of human ocular toxoplasmosis using blood samples. Our study may contribute to a better understanding of microRNAs’ role during n humans Table 1.The data presented in this revision concerning microRNAs attract the attention for the potential use of these small nucleic acid molecules to explore at least four different aspects of human toxoplasmosis. One of them refers to laboratory diagnosis. Diagnosis methods based on early detection of microRNAs could be an essential tool, especially for detecting microRNA in peripheral blood before IgM antibodies appear. In this context, microRNAs could be explored as biological markers of infection, especially in the acute phase of the disease, allowing the early treatment of human acute toxoplasmosis. This strategy could reduce tissue inflammation and, consequently, the tissue damage in target organs such as the eye and brain.T. gondii. Investigations aiming to check if one or more specific microRNA might be expressed in some particular infected tissues would establish a correlation with the clinical form of human toxoplasmosis. Despite the parasite infecting any nucleated cell, it remains in a latent phase, and the majority of individuals remains asymptomatic. However, the human toxoplasmosis clinical manifestation occurs in some target organs and would be appropriate to understand the potential relation between the T. gondii and the infected tissues. This strategy could favour the comprehension of new aspects of the host-parasite interaction in human toxoplasmosis.Another potential application of the detection of microRNA in human toxoplasmosis refers to the tissue targeted by T. gondii strains modulate the host cells affecting cell mobility through blood, neurological, and ocular barriers. Besides, it would be possible to verify microRNAs also correlates the different ways of T. gondii acquisition of the infection by placenta or acquired after birth.The observations that some types of microRNA are up-regulated in ocular toxoplasmosis and cerebral toxoplasmosis in HIV patients open opportunity to investigate if this up- or down-regulation correlates specific strains. This strategy could be adding new information on how different T. gondii interacts with human hosts.Finally, exploring mi-RNA in the different clinical forms of human toxoplasmosis would clarify how host microRNAs modulate this parasite infection and how GMFJr, CA, FM, and HL wrote the manuscript. LA and BC drew the table and figure in the manuscript. CB and LM revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grants: 2018/09448-8 to GMFJr) and NIH NIAID award number U19AI110819 (to HL). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) CMA and LCPA Finance Code 001 and by CNPq 303281/2020-0 (to CCB).The opinions, assumptions, and conclusions or recommendations expressed in this material are strictly those of the authors and do not necessarily reflect the views of FAPESP.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.


This may explain the differences in μTBS values and morphological appearance of the ABRZ. NaF is effective in enhancing the enamel/dentin bond durability and also helps create a high quality of ARBZ to improve the clinical success of restorations.A fluoride-releasing adhesive system is expected to promote mineralization of demineralized dentin/enamel around a composite restoration, thereby contributing to the longevity of the restoration. Scanning electron microscopic (SEM) and transmission electron microscopic (TEM) observations revealed the formation of an “acid–base resistant zone” (ABRZ) beneath the hybrid layer when dentin was treated with a self-etch adhesive system. A thicker ABRZ was formed from the upper slope to the end of the outer lesion, indicating greater resistance against an acid-base challenge, when a fluoride-releasing self-etch adhesive system was used. The slope formation of a fluoride-releasing adhesive is believed to be due to fluoride-release from the adhesive. Quantitative assessment of the acid resistance was performed at the interface using the region of interest (ROI) mode of a digital image analysis software package. The area of the ABRZ is influenced by the concentration of fluoride release from the adhesive resin. The threshold of fluoride concentration in the adhesive may exist to influence the morphology of the ABRZ. X-ray absorption fine structure (XAFS) analysis of the dentin treated with different concentrations of NaF-mouth rinses suggested that different fluoride concentrations result in the formation of different chemical compounds, such as fluorapatite and CaF Fluoride has been shown to exhibit anticariogenic activity by increasing enamel and dentin resistance to acid attack as well as inhibit carbohydrate metabolism in dental plaque , 2. AlsoA fluoride-releasing adhesive system is expected to promote the mineralization of demineralized dentin/enamel around a composite restoration, thereby contributing to the longevity of the restoration –8. In coUltrastructural examinations of the adhesive–dentin interface after an acid–base challenge have been carried out to investigate the mechanism of recurrent caries at the margins of restorations , 11. ThiScanning electron microscopic (SEM) and transmission electron microscopic (TEM) observations have revealed the formation of an “acid–base resistant zone” (ABRZ) found beneath the hybrid layer when dentin was treated with a self-etch adhesive system , 13. Theμm. Apatite crystals were sparse at the top of the ABRZ  is composed of a self-etching primer and a fluoride-containing adhesive . The bonthe ABRZ  and werethe ABRZ . The thi3+), boron (BO33-), fluoride (F-), sodium (Na+), silicon (SiO32-), and strontium (Sr2+), in both neutral and acidic conditions [A two-step self-etching primer system, FL-Bond II  contains a multi-ion releasing component made up of surface pre-reacted glass ionomer (S-PRG) fillers. The S-PRG filler-containing adhesive of FL II is able to release various ions, such as aluminum  analysis  of dentiAbbassy et al.  reportedA fluoride-releasing adhesive has the potential to create a thick ABRZ at the adhesive interface, protecting restoration margins from recurrent caries initiation. However, the area of the ABRZ is influenced by the concentration of fluoride ion release from the adhesive resin. NaF is effective in enhancing the enamel/dentin bond durability and also creating a high quality of ARBZ to improve the clinical success of restorations.


Changes in cellular chromatin states fine-tune transcriptional output and ultimately lead to phenotypic changes. Here we propose a novel application of our reproducibility-optimized test statistics (ROTS) to detect differential chromatin states (ATAC-seq) or differential chromatin modification states (ChIP-seq) between conditions. We compare the performance of ROTS to existing and widely used methods for ATAC-seq and ChIP-seq data using both synthetic and real datasets. Our results show that ROTS outperformed other commonly used methods when analyzing ATAC-seq data. ROTS also displayed the most accurate detection of small differences when modeling with synthetic data. We observed that two-step methods that require the use of a separate peak caller often more accurately called enrichment borders, whereas one-step methods without a separate peak calling step were more versatile in calling sub-peaks. The top ranked differential regions detected by the methods had marked correlation with transcriptional differences of the closest genes. Overall, our study provides evidence that ROTS is a useful addition to the available differential peak detection methods to study chromatin and performs especially well when applied to study differential chromatin states in ATAC-seq data. Chromatin states can be seen as the collection of proteins or histone modifications that regulate the openness and activity of a given chromatin region ,2. DynamThe output of both ChIP-seq and ATAC-seq analysis consists of reads enriched in genomic locations  representing the presence of a protein in the case of ChIP-seq or open chromatin state in the case of ATAC-seq. Thus, methods first developed for ChIP-seq data analysis, such as MACS2  and HOMEIn addition to peak calling, it is of specific importance to find significant differences in chromatin states between the biological conditions or groups of interest from the ChIP-seq and ATAC-seq data. A major challenge in such differential ChIP-seq and ATAC-seq data analysis is the large search space, as it is not limited only to, e.g. protein coding genes as in gene expression analysis, but in theory, open chromatin and protein binding events take place across most of the genome. Additionally, the range of the signal does not have intrinsic boundaries like the 0 to 100% in DNA methylation, as there is no theoretical upper limit for the read enrichments. An additional challenge in detecting the differential states in the context of ChIP-seq also lies in the amount of noise generated by the immunoprecipitation step, which makes it difficult to detect subtle changes between conditions ,16. SeveThe existing methods for differential peak calling can be classified into one-step methods that inherently include the initial peak calling, and two-step methods that require the prior use of a separate peak caller to produce the peaks for differential analysis. The one-step methods can be further separated into sliding window methods , but the differential peak calling methods have been previously compared only using ChIP-seq data , constitHere, we introduce the application of reproducibility optimized test statistic (ROTS)  for robuz-statistic k by considering the reproducibility of the k top-ranked features t-type statistics The ROTS is an approach that is based on investigating the inherent characteristics of the data and thus is able to free itself from any distributional assumptions . Specifik top-ranked features over B pairs of bootstrap datasets. The reproducibility for each pair b of bootstrap data matrices    and usesWe selected five datasets for our study: two biological datasets were based on ATAC-seq [Interferon response (IFN) and Yellow Fever vaccine (YF)], two were from ChIP-seq studies (Rheumatoid Arthritis H3K4me3 and H3K36me3), while the fifth was a synthetic dataset. The four biological datasets Table  were selhttps://www.encodeproject.org/chip-seq/histone). For the comparative purpose of this study, the ChIP-seq datasets were compared without subtracting the input chromatin sample, because as such the detection of differential binding between two conditions does not require an input ChIP-seq control  and converted to fastq format with fastq-dump tool from the SRA Toolkit . Reads w control ,41. The We used the GenomicRanges R/Bioconductor package  to detecEach of the biological ATAC-seq/ChIP-seq datasets included in our study contained matching gene expression data. For ATAC-seq datasets, we used the normalized gene expression data available from the original studies; in the YF dataset the available read counts had been corrected for batch effect with ComBat , and in Evaluation of differential peak calling in real biological data is challenging, as there is no existing biological gold standard. To circumvent this, we used the correlation to gene expression data as suggested previously ,45–48. TWe compared the performance of ROTS with five popular differential peak detection methods; DiffBind, diffReps, MAnorm2, PePr and THOR, on a synthetic dataset adapted from a previous study , containWe also tested the speed and the memory consumption of the methods when run on a modern computer cluster managed by the open-source Simple Linux Utility for Resource Management. In our inspection, ROTS was by far the fastest and the most memory efficient method Figure . DiffBinThe number of significant peaks (FDR < 0.05) reported varied considerably between the different methods within each dataset Table . THOR anNext, we compared the overlap of the most significant differential peaks between the methods across each of the biological datasets, with focus on top 2000 peaks Figure .In order to compare the shape of the peaks detected by the different methods, we assessed the intensity and width of the most significant differential peaks called by each method. We used heatmaps, average read counts across the peak regions and average peak widths to evaluate the 2000 most significant differential peaks Figure . AdditioIn the ATAC-seq datasets, all methods detected peaks with clear differences in read count values between the conditions Figure  and B, wP < 2.2×10–16 for the top 2000 peaks)  overall detected narrower peaks than the one-step methods  (0 peaks) . In the 0 peaks)  and B. I0 peaks) .Visual inspection of the example loci further illustrates that results of diffReps, PePr and THOR, based on inbuilt candidate peak identification, included many lower intensity segments on both sides of the peak summit Figure . THOR anIn order to have experiment-specific external evaluation criteria, we used the closest phenotypic data, the transcriptomic data, available for each of the studied chromatin datasets as conceived previously . The undDifferential peaks detected by ROTS consistently had a high overall correlation with differential transcription across the four datasets while the behavior of the other methods was more variable between the datasets Figure . Among tAccurate differential peak calling remains a challenge in the study of chromatin and histone modification states despite recent methodological advancements. ROTS is a computational tool that has been shown to work well in the statistical analysis of several types of high-throughput omics data by bootstrapping over the data to improve the reproducibility of the results and adapting the statistical test according to the intrinsic properties of the data . In thisThe differential peak detection software compared in this study can be defined as one-step methods that include the peak calling step as part of the method, and two-step methods that use a separate peak caller to produce the initial peaks for the differential analysis. By visual inspection of the mean signals Figure  and examBased on our results, the two-steps methods relying on MACS2 input peaks were more consistent in finding the peaks with high-intensity enrichment in the middle of the peak than the one-step methods considered in this study. For instance, many of the THOR and diffReps (one-step methods) ATAC-seq peak regions showed a pattern where the highest signal of the broadest peaks was not in the middle of the peak but on the two sides of the peak centre Figure , suggestper se. However, we observed that ROTS consistently appeared among the methods reporting highest correlations across the datasets and especially in ATAC-seq data the differential peaks detected by ROTS had stronger correlation with differential transcription compared to the other methods  data where cell type or condition specific clusters may be pooled and analyzed in a similar manner as the bulk ATAC-seq data.https://bioconductor.org/packages/release/bioc/html/ROTS.html).ROTS (1.16.0) is a Bioconductor R package for differential testing in omics data THOR is part of the regulatory genomic toolbox (0.11.4) software for differential peak calling (https://github.com/shenlab-sinai/diffreps)diffReps (1.55.6) is a software for differential peak calling (https://github.com/shawnzhangyx/PePr)PePr (1.1.10) is a software for differential peak calling (https://bioconductor.org/packages/release/bioc/html/DiffBind.html)DiffBind (3.0.15) is a software for differential peak calling (https://github.com/tushiqi/MAnorm2)MAnorm2 (1.0.0) is a software for differential peak calling (https://github/elolab/Faux_et_al_NARGAB2021The code used to produce the figures in this study and to perform the differential peak calling with the different methods is available at: The ATAC-seq Yellow-Fever dataset is available on the GEO accession number GSE101609; updated 15 May 2019.The ATAC-seq IFN dataset is available on the GEO accession number GSE100383; updated 15 May 2019.The ChIP-seq RA dataset for H3K4me3 and H3K36me3 is available on the GEO accession number GSE112655; updated 19 March 2019.lqab059_Supplemental_FileClick here for additional data file.


Large scale viral genome sequencing and analysis have uncovered the functional impact of numerous genetic variants in disease pathogenesis and transmission. Emerging evidence of mutations in spike protein domains escaping antibody neutralization is reported. We have built a database with precise collation of manually curated variants in SARS-CoV-2 from literature with potential escape mechanisms from a range of neutralizing antibodies. This comprehensive repository encompasses a total of 5258 variants accounting for 2068 unique variants tested against 230 antibodies, patient convalescent plasma and vaccine breakthrough events. This resource enables the user to gain access to an extensive annotation of SARS-CoV-2 escape variants which would contribute to exploring and understanding the underlying mechanisms of immune response against the pathogen. The resource is available at   Genomic approaches have been instrumental in understanding the origin and evolution of SARS-CoV-2, the causative agent for the COVID-19 pandemic  and GlobThere has been a significant interest in recent times in understanding the functional impact of genetic variants in SARS-CoV-2 apart from exploring the genetic epidemiology. The variant D614G present in spike protein has been one the earliest and prominent examples with potential implications associated with the infectivity of the virus . StudiesWith the advent of therapies including monoclonal antibodies, convalescent plasma as well as the recent availability of vaccines, interest in genetic variants which could affect the efficacy of such modalities of therapy has accelerated. The targeting of spike proteins by broad-neutralizing antibodies against SARS-CoV-2 offers a potential means of treating and preventing further infections of COVID-19 . Evidenchttp://clingen.igib.res.in/esc/.The lack of a systematic effort to compile genetic variants in SARS-CoV-2 associated with immune escape motivated us to compile the information in a relevant, searchable and accessible format. Towards this goal, we systematically evaluated publications for evidence on immune escape associated with genetic variants in SARS-CoV-2 and created a database named as ESC. User-friendly web interface is made available to retrieve information on immune escape variants as well as their extensive functional annotations. To the best of our knowledge, this is the first most comprehensive resource for immune escape variants for SARS-CoV-2. The resource can be accessed online at Genetic variants in the SARS-CoV-2 genome and evidence suggesting association with immune escape were systematically catalogued. A significant number of variants were associated with escape or resistance to a range of neutralizing and monoclonal antibodies, while a subset was associated with resistance to convalescent plasma. The data was compiled by manual curation of literature available from peer-reviewed publications and preprints. Literature reports with relevant information on antibody escape variants were retrieved from sources including PubMed, LitCovid, Google Scholar and preprint servers. The reports were systematically checked for details pertaining to the variation, antibodies tested and experimental methods followed in the study. In addition, the variants were systematically categorized based on experimental validation and computational prediction. Collated data was organized in a pre-formatted template based on their protein positions. This comprehensive compendium was used for further functional annotations.The variant information and annotations were retrieved from annotation tables for individual features using ANNOVAR . VariantDetails on B-cell and T-cell epitopes spanning the protein residues of SARS-CoV-2 were retrieved from Immune Epitope Database and Analysis Resource (IEDB) . All epiInformation pertaining to the list of antibodies associated with escape mechanisms was retrieved from available public sources. Compiled antibodies were systematically mapped back to the AntiBodies Chemically Defined (ABCD) database which provides integrated information regarding the antibodies along with its corresponding antigens and protein cross links to fetch unique antibody identifiers ,19.HTTPS for information transfer. The Beacon API has one endpoint: /beacon?Variant; query interface is provided by the beacon endpoint. Contents of the database gets updated every month by systematic literature curations and also made available to users for bulk download. The back-end of the web interface was implemented using Apache web server and MongoDB v3.4.10 in order to provide a user-friendly interface for variant search. The JavaScript Object Notation (JSON) file format was used to systematically store the data. PHP 7.0, AngularJS, HTML, Bootstrap 4 and CSS were used to code the web interface for querying. Highcharts javascript library was also used for improved presentation and interactivity. A Beacon API has been created using the PHP programming language. The ESC Beacon API v1.0.0 is a read-only API with specifications written in OpenAPI. It uses JSON in requests and responses and standard We compiled a total of 5258 variant entries from over 60 articles which studied SARS-CoV-2 variants and their effect on immune escape. This included a total of 2068 unique variants mapping to spike protein, ORF1ab and ORF3a. Out of the total unique variants, 2060 variants mapped to the gene coding for spike protein with potential immune escape mechanisms elucidated through experimental evidence as well as computational predictions. The remaining eight variants were found in ORF1ab and ORF3a genes, out of which, three were reported to confer potential epitope loss. The compiled list of variants was found associated with 230 unique SARS-CoV-2 antibodies and patient polyclonal sera. A handful of SARS-CoV-2 variations associated with vaccine breakthrough events have also been documented. A brief comparison of the curations in the ESC database with other publicly available resources is summarized in By scanning through the spike protein residues and their associations with SARS-CoV-2 neutralizing and monoclonal antibodies, we were able to compile the exact count of antibodies reported to have potential associations with the residues. From our analysis we observed that spike protein residues ranging from 350 to 500 amino acid positions exhibited potential antibody associations with the possibility of immune escape against at least one antibody. A total of 22 hotspot residues  were found to possess immune evasion capability against > 10 monoclonal antibodies. A schematic representation of the number of antibodies associated with spike protein residues along with their domain annotations is shown in Figure With the aim of mapping back the known B and T cell epitopes encompassing the variant compendium, SARS-CoV-2 epitope details were extracted. There were a total of 310 and 472 experimentally validated B cell and T cell epitopes respectively. This precisely included 263 linear and 47 discontinuous B cell epitopes in spike protein. Reported B cell and T cell epitope information was mapped back to residues possessing antibody escape mutations, which provided a brief insight on the potential impact of these variations in immune recognition and responses.The database offers a user-friendly interface enabling the users to search for variants based on their amino acid change, gene name or the antibody name as per the specified format. The search query returns a list of matching results, whose complete functional annotations can be viewed by clicking on the displayed elements. The resource provides a list of annotation features for each variant precisely organized into eight major sections namely Variant details, Antibody details, Variants of Concern/Interest, Protein domain details, Epitope details, Functional annotation, Literature Evidence and Variant frequency. Figure https://virological.org/t/issues-with-sars-cov-2-sequencing-data/473) and diagnostic primer/probe sites are also mapped. Extensive evidence from literature curation including the methods of the study, neutralization quantification profiles and details of antibody/mutant generation are summarized in the literature evidence section. Variant frequency section exclusively summarizes the estimated frequencies of the variant on a global scale as well as by its geography. In addition, characteristic mutations of VoCs and VoIs have also been annotated with brief descriptions in the Variants of Concern/Interest section.Basic details pertaining to the variant like the amino acid change, genomic coordinates and the variant type have been enlisted in the Variant details section. Information on the associated neutralizing antibodies and their identifiers are provided in the Antibody details section. Domain and epitope details section exclusively comprises details on the protein domain, epitopes reported to span the protein residue through experimental validations. Computationally predicted functional annotations on deleteriousness from SIFT , evolutiin-vitro assays, unique insights into the structural and functional mechanisms whereby the pathogen could evolve and evade antibodies have become possible. These insights could have enormous implications in efficacy of vaccines currently being used as well as under trials. One of the recent studies has reported the impact of a few immune escape variants on the efficacy of vaccines (With evidence emerging on genetic variants in SARS-CoV-2 associated with resistance to monoclonal antibodies and convalescent plasma using vaccines . It is ehttps://github.com/mercywilliams160896/ESC_COVID19.The completed data curated from various literature sources are collated and made available for access and bulk download at https://clingen.igib.res.in/esc/api/beacon?Variant=A475V. A detailed overview of The ESC Beacon API v1.0.0 has been documented and linked to the webpage duly for user interests.An API for ESC is also made available for ease of access to data. Example search: gkab895_Supplemental_FileClick here for additional data file.


The hepatitis C virus (HCV) infection is a candidate disease for micro-elimination. Accurate baseline HCV prevalence estimation is essential to monitor progress to micro-elimination but can be methodologically challenging in low-endemic regions like the Netherlands due to lack of disaggregated data by age or risk-groups on the number of chronic HCV patients (i.e. HCV RNA positive). This study estimates the number of patients that has had a chronic HCV infection (ever-chronic) in the Utrecht region of the Netherlands.In the Utrecht province in the Netherlands, positive HCV tests from the period 2001–2015 from one diagnostic center and four hospital laboratories were collected. A two-source capture-recapture method was used to analyze the overlap between the two registries (with 92% HCV RNA and 8% HCV immunoblot confirmed infections) to obtain the number of ever-chronic HCV infections in the Utrecht region. The Utrecht region was defined as an area with a 25 km radius from the Utrecht city center. The current viremic HCV prevalence was calculated by taking into account the proportion of cured and deceased HCV patients from a local HCV retrieval (REACH) project.The estimated number of ever-chronic HCV patients was 1245 (95% CI 1164–1326) and would indicate a prevalence of 0.10 (95% CI 0.09–0.10) in the Utrecht region. This is 30% (95% CI 21–38%) more than the number of known HCV patients in the records. The ever-chronic HCV prevalence was highest in the 1960–1969 age cohort . Since 50% of the HCV patients were cured or deceased in the REACH-project, the number of current viremic HCV patients was estimated at 623 individuals in the Utrecht region .The results of this study suggest a low ever-chronic and current HCV prevalence in the Utrecht area in the Netherlands, but other studies need to confirm this.The online version contains supplementary material available at 10.1186/s12879-021-06324-z. Estimates based on modelling suggest that 71 million individuals are chronically infected with the hepatitis C virus (HCV) worldwide and consequently are at risk for developing the associated long-term complications such as cirrhosis and hepatocellular carcinoma . The micIn the Utrecht province in the Netherlands, positive HCV test results (i.e. either an anti-HCV or a HCV-RNA PCR) from the period 2001–2015 were extracted from the Laboratory Information System (LIS) of all four regional hospitals and one diagnostic center for general practitioners (GPs) as part of a regional micro-elimination project (REACH) that aimed to identify and trace untreated HCV patients . The curThe REACH-project aimed to trace previously diagnosed but untreated HCV patients by extracting positive HCV test-results (i.e. either an anti-HCV test or a HCV-RNA PCR test) from the LIS from all four regional hospitals and one diagnostic center for GPs in the Utrecht region in the Netherlands as published before . PositivThe capture-recapture method originates from the field of ecology where it was often adopted to estimate an animal population size. First, a portion of a population is captured, marked and released. Later on, another sample is extracted from the population and the number of marked individuals in this second sample is counted. If the population mixes homogeneously and every individual is captured with the same probability, the proportion of marked individuals in the second sample is an estimate for the proportion of marked individuals in the entire population. Estimation of the population size can then be obtained by dividing the number of marked individuals by the fraction of marked individuals in the second sample. This approach is often referred to as the Lincoln-Petersen method, named after those who popularized its use , 8. The ever-chronic) in the entire study period (2001–2015). The HCV prevalence in Utrecht was calculated by dividing the result of the capture-recapture analysis by the total population size of the Utrecht province on the 1st of January 2016 (Statistics Netherlands - CBS). Finally, the REACH-project generated information about the proportion of cured and deceased HCV patients (=50%) [viremic population size.The modified Lincoln-Petersen estimator with Chapman adjustment was used in this study to estimate the prevalence of chronic HCV in Utrecht with a 95% confidence interval (CI) by means of a two-source capture-recapture analysis Fig. . The twos (=50%) . This prAll cleared or cured acute HCV infections and false positive test results were excluded from the analysis through screening of the patient clinical records during the REACH project. The majority of the identified HCV infections (95%) were HCV RNA confirmed and the remaining 5% only had a positive anti-HCV which can indicate both past as well as ongoing HCV infection. Since the latter was a relatively small proportion and progression from acute to chronic HCV occurs in ~ 75% , 13 all 2, but also one of the most densely populated with approximately 1274 million inhabitants in 2016. The home residencies of the HCV patients in our study sample were located throughout the Netherlands, but only patients living in the Utrecht region were included. For the purpose of the capture-recapture analysis, the Utrecht region was set as the area within a 25-km radius from the Utrecht city center as this most approximated the actual Utrecht province borders, excluding any large cities from neighboring provinces but still including the most important cities from hospitals participating in REACH (including the most distant city Amersfoort of which the outer border is at a linear distance of 25 km from Utrecht)  residency location was based on their six digit postal code. Patients for whom no information about their current or last available residency was available were excluded from the capture-recapture analysis. The postal codes were translated into geographic coordinates in order to select all HCV patients living within 25-km radius from Utrecht.The Utrecht province is the smallest province of the Netherlands as its borders demarcate approximately 1400 kmht) Fig. b. The 25province on the 1st of January 2016 (Statistics Netherlands - CBS).The capture-recapture analysis was performed on different HCV patient groups based on the distance of their residency from the Utrecht city center with incremental steps of 10 km (i.e. 10 to a maximum of > 120 km distance from Utrecht). In this manner, the patient subgroups gradually increased in size until the entire study sample was included. For each distance, the estimated/recorded population size ratio with 95% CI was also calculated. In addition, we estimated the number of ever-chronic HCV infected in five specific age cohorts  and risk-groups (migrants vs. non-migrants) in the Utrecht region with stratified capture-recapture analyses in these groups. The ever-chronic HCV prevalence in each age cohort in Utrecht was calculated by dividing the result of the stratified capture-recapture analysis by the total population size of each age cohort in the Utrecht The geographical patient selection was done in R studio version 1.1.463 and the “FSA” and “FSAdata” packages were used for the capture-recapture analysis.p, < 0.001) compared to those with a registered address. There were no significant differences in the decade of birth between those with and without a known residency location.From the generated laboratory list of positive HCV tests in the Utrecht province, 1853 individual HCV patients could be identified and for 1738 of them, their (last) postal codes were known. Basic patient characteristics are described in Table The geographical location of home addresses of all 1738 known HCV patients was mapped for all recorded HCV patients  are depicted in Supplementary Figure The estimated-to-recorded population size ratio for five different age cohorts varied between 1.1–1.6 with overlapping confidence intervals. The estimated ever-chronic HCV prevalence in each age cohort is depicted in Fig. Population size estimation of the population of chronically HCV infected persons in a low prevalence country, where HCV prevalence is concentrated in risk groups, can be methodologically challenging. This study aimed to estimate the ever-chronic HCV prevalence in the Utrecht region by means of a two-source capture-recapture analysis on two extensive lists of positive HCV tests performed between 2001 and 2015 in one diagnostic center for GPs and four hospital laboratories. The number of estimated ever-chronic HCV infected patients in the Utrecht region was 1245 (95% CI 1164–1326), indicating a local prevalence of 0.10 (95% CI 0.09–0.10).The most recent benchmark study on HCV prevalence in the Netherlands, adopting the Workbook method, reported a slightly higher nationwide ever-chronic HCV prevalence of 0.16%  compared to our study, but the confidence intervals overlap .The current capture-recapture approach might, however, have underestimated the total number of HCV infected individuals, since the undiagnosed HCV patients in the population were not sampled. In contrast, between 0 and 8% of the included HCV infections may actually have been cleared or cured HCV infections since they were only HCV immunoblot confirmed. It is probable that this has lead to overestimation of the ever-chronic HCV prevalence. In addition, a closed population was assumed although birth, death and migration did occur. This assumption would probably not have greatly affected the number of ever-chronic HCV infected, which was the main aim of our study. Moreover, the death rate was accounted for by taking the proportion of deceased HCV patients in the REACH-project as a proxy. Also, birth is presumed to have a low contribution to the current chronic infections due to the limited perinatal transmission of HCV 4–10%) in women and because the predominantly male HCV population does not contribute to perinatal transmission . With re0% in womTo further increase our knowledge on the viremic fHCV population and to monitor micro-elimination progress, the optimal solution would be mandatory registration of both acute and chronic HCV infections. In the Netherlands, mandatory notification of HCV infections to Public Health Services for the purpose of contact tracing is only required for acute HCV infections and chronic HCV infections of unknown duration at the moment . PrivacyThe results of the capture-recapture analysis suggest that the number of ever-chronic HCV infections in the Utrecht region is lower than previously assumed. Further studies our needed to confirm these results. To monitor the HCV population with ongoing viremia and the progress to micro-elimination, mandatory notification of all HCV infections is essential. A capture-recapture analysis will remain of additional value to assess completeness of HCV registries in the future.Additional file 1: Supplementary Figure 1. Capture-recapture analysis: the estimated/recorded population size ratio and the HCV population size estimation. A: The ratio between the estimated and baseline HCV population size was calculated for each distance including for the 95% confidence intervals. B: A capture-recapture analysis was performed on different HCV patients selections based on their residency distance from the Utrecht city center with incremental steps of 10-kilometer. HCV population size estimates with 95% confidence intervals are depicted for each distance.


METHODS/STUDY POPULATION: This study will use a cohort of 106 patients undergoing treatment for head and neck cancers at a single institution to temporally validate a model for post-treatment ototoxicity. We are interested in understanding if this model will be able to predict ototoxic risk  and if this model can differentiate high- and low-risk patients (discrimination). Observed and predicted values for audiometric hearing loss will be calculated and then compared using a calibration curve available in SAS v9.4, while the c-index (area under the receiver-operator curve) will be used to assess discrimination. The implementation of this model will be assessed in a clinical setting. RESULTS/ANTICIPATED RESULTS: The validation cohort is generally similar in age (61 years) and sex-mix  to the original cohort. However, there seems to be a different case-mix the types of treatments with more patients receiving cisplatin overall (59% vs. 43%), but fewer getting induction and high-dose cisplatin (1% vs. 13%). The original model showed good calibration and fair discrimination in the validation cohort with and area under the curve of 0.700. This concordance statistic suggests possibly-useful discrimination and the calibration curve suggests the model is well-calibrated. DISCUSSION/SIGNIFICANCE OF IMPACT: This project can improve clinical treatment paradigms, enhance patient education, and reduce healthcare costs. Our model allows oncologists to weigh the risks of hearing loss with the benefits of treatment on an individualized level