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Background: Understanding the molecular pathogenesis of inflammatory bowel disease (IBD) has led to the discovery of new therapeutic targets that are more specific and effective. Our aim was to explore the molecular pathways and genes involved in IBD pathogenesis and to identify new therapeutic targets and novel biomarkers that can aid in the diagnosis of the disease. Methods: To obtain the largest possible number of samples and analyze them comprehensively, we used a mega-analysis approach. This involved reprocessing raw data from multiple studies and analyzing them using bioinformatic and machine learning techniques. Results: We analyzed a total of 697 intestinal biopsies of Ulcerative Colitis (n = 386), Crohn’s disease (n = 183) and non-IBD controls (n = 128). A machine learning analysis detected 34 genes whose collective expression effectively distinguishes inflamed biopsies of IBD patients from non-IBD control samples. Most of these genes were upregulated in IBD. Notably, among these genes, three novel lncRNAs have emerged as potential contributors to IBD development: ENSG00000285744, ENSG00000287626, and MIR4435-2HG. Furthermore, by examining the expression of 29 genes, among the 34, in blood samples from IBD patients, we detected a significant upregulation of 12 genes (p-value < 0.01), underscoring their potential utility as non-invasive diagnostic biomarkers. Finally, by utilizing the CMap library, we discovered potential compounds that should be explored in future studies for their therapeutic efficacy in IBD treatment. Conclusion: Our findings contribute to the understanding of IBD pathogenesis, suggest novel biomarkers for IBD diagnosis and offer new prospects for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Purpose: One-half of hormone receptor-positive (HR +) breast cancer (BC) patients have low expression of HER2 (HER2-low) and may benefit from trastuzumab deruxtecan (TDXd). This study aimed to identify parameters associated with HER2-low levels in primary and metastatic tumors. We specifically sought to determine whether OncotypeDX and HER2 mRNA levels could identify patients who would otherwise be considered HER2-negative by immunohistochemistry (IHC). Methods: This retrospective analysis of all consecutive HR + patients who underwent OncotypeDX from January 2004 to December 2020 was conducted in a single medical center (n = 1429). We divided HER2-negative cases into HER2-low (IHC = 1 + or 2 + and non-amplified fluorescent situ hybridization) and HER2-0 (IHC = 0). HER2 RT-PCR was evaluated from the OncotypeDX results. Results: HER2-low cases exhibited significantly higher HER2 RT-PCR scores (p = 2.1e-9), elevated estrogen receptor (ER) levels (p = 0.0114), and larger tumor sizes compared to HER2-0 cases (> 2 cm; 36.6% vs. 22.1%, respectively, p < 0.00001). Primary tumors > 2 cm were more likely to be HER2-low (OR = 2.07, 95% CI: 1.6317 to 2.6475, p < 0.0001). Metastatic BCs expressed higher HER2 IHC scores compared with primary BCs (Wilcoxon signed-rank, p = 0.046). HER2 IHC scores were higher for low-risk vs. medium-risk OncotypeDX (p = 0.0067). No other clinical or pathological parameters were associated with the increase in HER2 levels in the metastatic samples. Conclusion: It might be beneficial to use clinical data from the primary tumor, including the HER2 RT-PCR score, to determine a HER2-low status. [ABSTRACT FROM AUTHOR]

Resistance to chemotherapy is a leading cause of treatment failure. Drug resistance mechanisms involve mutations in specific proteins or changes in their expression levels. It is commonly understood that resistance mutations happen randomly prior to treatment and are selected during the treatment. However, the selection of drug-resistant mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells and thus cannot result from the selection of pre-existent mutations. Accordingly, adaptation must involve the generation of mutations de novo upon drug treatment. Here we explored the origin of resistance mutations to a widely used Top1 inhibitor, irinotecan, which triggers DNA breaks, causing cytotoxicity. The resistance mechanism involved the gradual accumulation of recurrent mutations in non-coding regions of DNA at Top1-cleavage sites. Surprisingly, cancer cells had a higher number of such sites than the reference genome, which may define their increased sensitivity to irinotecan. Homologous recombination repairs of DNA double-strand breaks at these sites following initial drug exposures gradually reverted cleavage-sensitive "cancer" sequences back to cleavage-resistant "normal" sequences. These mutations reduced the generation of DNA breaks upon subsequent exposures, thus gradually increasing drug resistance. Together, large target sizes for mutations and their Top1-guided generation lead to their gradual and rapid accumulation, synergistically accelerating the development of resistance. [ABSTRACT FROM AUTHOR]

Background: Noncoding RNAs such as microRNAs (miRNAs) have attracted attention as biological pathway regulators, which differ from chromosomal translocations and gene point mutations. Their involvement in the molecular mechanisms underlying light chain (AL) amyloidosis pathogenesis is yet to be elucidated. Aims: To decipher specific miRNA expression profile in AL‐amyloidosis and to examine how miRNAs are involved in AL pathogenesis. Methods: The expression profile of miRNAs and mRNA from bone marrow (BM)‐derived CD138+ cells were determined using the NanoString nCounter assay and RNA‐Seq, respectively. The effect of aberrantly expressed miRNAs on potential molecular targets was analyzed by qRT‐PCR, Western blot, Mito‐potential assay, and Annexin‐PI staining. Results: Genes which were significantly differentially expressed between AL‐amyloidosis and MM, were found to be involved in cell growth and apoptotic mechanisms. Specifically, BCL2L1, MCL1, and BCL2 were upregulated in AL‐amyloidosis compared with MM and controls. The levels of miR‐181a‐5p and miR‐9‐5p, which regulate the above‐mentioned genes, were lower in BM samples from AL‐amyloidosis compared with controls, providing a mechanism for BCL2 family gene upregulation. When miR‐9‐5p and miR‐181a‐5p were overexpressed in ALMC1 cells, BCL2L1, MCL1, and BCL2 were downregulated and induced apoptosis. Treatment of ALMC‐1 cells with venetoclax, (BCL‐2 inhibitor), resulted in the upregulation of those miRNAs, the downregulation of BCL2, MCL1, and BCL2L1 mRNA and protein levels, and subsequent apoptosis. Conclusion: Our findings suggest that miR‐9‐5p and miR‐181a‐5p act as tumor‐suppressors whose downregulation induces anti‐apoptotic mechanisms underlying the pathogenesis of AL‐amyloidosis. The study highlights the post‐transcriptional regulation in AL‐amyloidosis and provides pathogenetic evidence for the potential use of BCL‐2 inhibitors in this disease. [ABSTRACT FROM AUTHOR]

This real-world cohort analysis assessed the efficacy of alpelisib and endocrine treatment (ET) combinations in a post-everolimus setting. Thirteen women who started alpelisib and ET at standard doses between 2018 and 2022 for advanced breast cancer (ABC), after undergoing CDK4/6i and everolimus treatment, were eligible for the study entry. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were the objective response rate (ORR) and clinical benefit rate (CBR), with different molecular profiling. The patients had previously received a median of four (range 3–8) systemic treatments, including CDK4/6i and everolimus. The median PFS on alpelisib was 5.5 months (range 0.5–10), and four women each had an ORR and three (23%) had a stable disease. The 6-month CBR was 46.1%, similar to the BYLeive study cohort C (47.8%). Notably, our cohort included patients with a long CBR under everolimus treatment (median 6 months, range 1–18); however, the responses to alpelisib and everolimus were not correlated (Pearson r = −0.23, p = 0.44). The PIK3CA, P53, ARID, GATA3, and ESR1 mutations were not associated with the 6-month CBR. Despite heavy pre-treatments, including everolimus, alpelisib was clinically relevant in our cohort, even among patients with an ESR1 mutation. The best treatment sequence for PIK3CA/mTOR inhibitors warrants examination in future trials on PIK3CA-mutant inpatients with luminal ABC. [ABSTRACT FROM AUTHOR]

Medicinal properties of plants are usually identified based on knowledge of traditional medicine or using low-throughput screens for specific pharmacological activities. The former is very biased since it requires prior knowledge of plants' properties, while the latter depends on a specific screening system and will miss medicinal activities not covered by the screen. We sought to enrich our understanding of the biological activities of Sarcopoterium spinosum L. root extract based on transcriptome changes to uncover a plurality of possible pharmacological effects without the need for prior knowledge or functional screening. We integrated Gene Set Enrichment Analysis of the RNAseq data to identify pathways affected by the treatment of cells with the extract and perturbational signatures in the CMAP database to enhance the validity of the results. Activities of signaling pathways were measured using immunoblotting with phospho-specific antibodies. Mitochondrial membrane potential was assessed using JC-1 staining. SARS-CoV-2-induced cell killing was assessed in Vero E6 and A549 cells using an MTT assay. Here, we identified transcriptome changes following exposure of cultured cells to the medicinal plant Sarcopoterium spinosum L. root extract. By integrating algorithms of GSEA and CMAP, we confirmed known anti-cancer activities of the extract and predicted novel biological effects on oxidative phosphorylation and interferon pathways. Experimental validation of these pathways uncovered strong activation of autophagy, including mitophagy, and excellent protection from SARS-CoV-2 infection. Our study shows that gene expression analysis alone is insufficient for predicting biological effects since some of the changes reflect compensatory effects, and additional biochemical tests provide necessary corrections. This study defines the advantages and limitations of transcriptome analysis in predicting the biological and medicinal effects of the Sarcopoterium spinosum L. extract. Such analysis could be used as a general approach for predicting the medicinal properties of plants. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to examine how various types of cancer, classified histologically, affect semen quality. Methods: The study group included 313 patients who were diagnosed with cancer and reached for a sperm cryopreservation before a gonadotoxic treatment (PG-Tx group). Their semen parameters were compared to those of two control groups: (a) individuals who attended a fertility investigation and were found to be above the limit of the lower reference value of the WHO 2010 manual (ARL group), and (b) fertile men, whose semen parameters were obtained from the dataset of the WHO 2020 manual. Results: Semen quality was significantly poorer in the PG-Tx group than in the ARL group. Differences included a 65.6% decrease in concentration, a 12.1% decrease in volume, a 72.7% decrease in total count, and a 33.0%, 22.2%, and 24.7% decrease in total motility, rapid motility, and progressive motility, respectively. Linear regression models comparing the PG-Tx and ARL groups revealed that the maximum reduction in total motility and concentration was in men with germ-cell tumors, whereas the minimum reduction was in hematological tumors. Similarly, all sperm quality parameters were significantly lower in the PG-Tx group than in the fertile-men group (p < 0.0001). Conclusions: While the effect of malignancy on semen parameters is debatable, we found that all examined types of cancer significantly impaired sperm quality parameters. Although the median of most semen parameters of patients with cancer were still in the normal WHO range, their fifth percentile, represents men with a delayed time to pregnancy. [ABSTRACT FROM AUTHOR]

Background: Previous meta‐analyses concluded that there is a decline in sperm parameters over time. This conclusion might be incorrect due to inherent biases or focusing only on a single parameter – sperm concentration. Objective: To study trends in sperm parameters over the past 20 years using data from the trials that defined the reference ranges of the World Health Organization manual. Materials and methods: Retrospective evaluation of the data used to define the World Health Organization reference ranges. The data from 11 studies, including 3589 participants between 1996 and 2016, were divided into three period groups based on the decade of study. Differences in semen parameters' distribution were presented in boxplot. p‐values were calculated by the Kruskal–Wallis rank‐sum test followed by Dunn post hoc test. Analyses were conducted using the R programming language. Results: A small decrease was noted in mean sperm concentrations (88.1 million/ml, 87.6 million/ml, and 77.2 million/ml for the first, second, and third decades, respectively) (p < 0.01). However, the 5th percentile of sperm concentration for the third decade was higher than the first or second decades (18 million/ml versus 14.9 million/ml and 15 million/ml, respectively). No significant differences were noted in progressive motility over the years (p = 0.32). The percent of morphologically normal sperm decreased between the first (24.2%) and the second (12.6%) periods of the study (p < 0.001) and then increased in the third decade (14.2%) (p < 0.01). Total motile sperm count (TMC) declined between the second and third decades (189 million and 153.9 million, respectively, p < 0.001), at levels unlikely to decrease fertility. However, the 5th percentile of the TMC remained stable at 24.9, 20.8, and 20.6 million, for the first, second, and third decades respectively (p = 0.36). Discussion and conclusion and relevance: Trends in sperm parameters over the last three decades do not seem to be clinically significant. [ABSTRACT FROM AUTHOR]

Marine plastic debris serve as substrates for the colonization of a variety of prokaryote and eukaryote organisms. Of particular interest are the microorganisms that have adapted to thrive on plastic as they may contain genes, enzymes or pathways involved in the adhesion or metabolism of plastics. We implemented DNA metabarcoding with nanopore MinION sequencing to compare the 1-month-old biomes of hydrolyzable (polyethylene terephthalate) and non-hydrolyzable (polyethylene) plastics surfaces vs. those of glass and the surrounding water in a Mediterranean Sea marina. We sequenced longer 16S rRNA, 18S rRNA, and ITS barcode loci for a more comprehensive taxonomic profiling of the bacterial, protist, and fungal communities, respectively. Long read sequencing enabled high-resolution mapping to genera and species. Using previously established methods we performed differential abundance screening and identified 30 bacteria and five eukaryotic species, that were differentially abundant on plastic compared to glass. This approach will allow future studies to characterize the plastisphere communities and to screen for microorganisms with a plastic-metabolism potential. [ABSTRACT FROM AUTHOR]

More than one third of the mammalian genome is in a close association with the nuclear lamina, thus these genomic regions were termed lamina-associated domains (LADs). This association is fundamental for many aspects of chromatin biology including transcription, replication, and DNA damage repair. LADs association with the nuclear envelope is thought to be dependent on two major mechanisms: The first mechanism is the interaction between nuclear membrane proteins such as LBR with heterochromatin modifications that are enriched in LADs chromatin. The second mechanism is based on proteins that bind the borders of the LADs and support the association of the LADs with the nuclear envelope. Two factors were suggested to support the second mechanism: CCCTC-binding factor (CTCF) and YY1 based on their enriched binding to LADs borders. However, this mechanism has not been proven yet at a whole genome level. Here, to test if CTCF supports the LADs landscape, we generated melanoma cells with a partial loss of function (pLoF) of CTCF by the CRISPR-Cas9 system and determined the LADs landscape by lamin B ChIP-seq analysis. We found that under regular growth conditions, CTCF pLoF led to modest changes in the LADs landscape that included an increase in the signal of 2% of the LADs and a decrease in the signal of 8% of the LADs. However, CTCF importance for the LADs landscape was much higher upon induction of a chromatin stress. We induced chromatin stress by inhibiting RNA polymerase II, an intervention that is known to alter chromatin compaction and supercoiling. Notably, only in CTCF pLoF cells, the chromatin stress led to the dissociation of 7% of the LADs from the lamina. The CTCF-dependent LADs had almost three times shorter median length than the non-affected LADs, were enriched in CTCF binding at their borders, and were higher in their facultative-status (cell-type specific). Thus, it appears that CTCF is a key factor in facilitating the association of short facultative LADs with the nuclear lamina upon chromatin stress. [ABSTRACT FROM AUTHOR]

Purpose: Patients with the autosomal recessive disorder of familial dysautonomia typically exhibit exacerbated adverse side effects to many common drugs. We aimed to catalog these adverse effects – with a focus on common drugs that are frequently administered to FD patients and compare their incidences to those within the general population. Methods: We used data of 595 FD patients from an international database with information on drugs received and adverse effects. To investigate the molecular causes of reported differences in drug responses in FD patients, we used expression microarrays to compare the mRNA expression profiles in peripheral blood leukocytes of FD patients (n = 12) and healthy individuals (n = 10). Results: Several drug classes, including cholinergics, anti‐cholinergics, anti‐convulsants, methylxanthines, SSRIs, and antibiotics caused either unreported symptoms or elevated rates of adverse events in FD patients. FD patients experienced different or more frequent adverse side effects than the general population in 31/123 drugs. These side effects included blood cell dyscrasias, amenorrhea, gastrointestinal bleeding, and bronchospasm. New findings include enhanced reaction of FD patients to H2 antagonist agents and to serotonin receptor agonists. We also detected eight genes differentially expressed between FD patients and healthy individuals that may underlie the differential drug responses of FD patients. Conclusion: We provide evidence that suggests the use of several common drugs should be discontinued or reduced in FD patients. [ABSTRACT FROM AUTHOR]

This study aimed to study whether IVF stimulation that results in one or two mature follicles should proceed to oocyte retrieval. This is a retrospective cohort study conducted at McGill University Health Center on 459 patients who underwent IVF treatment between 2011 and 2014, undergoing hormonal stimulation and monitoring of their ovarian response. The primary outcomes were pregnancy and live birth rates. Statistical modeling was used to determine individual roles of patient age and ovarian reserve on outcomes, while controlling for the other factors. Of the 459 cycles included in the study, 360 cycles (78.4%) ended in embryo transfer. Live birth rates per cycle were 15.6%, for the ≤ 34-year-olds; 6.5%, for the 35–39-year-olds; and 2.7%, for the ≥ 40-year-olds (p < 0.01). Twenty-five percent of the cycles in the ≥ 40-year-old group were canceled versus 17% and 15% in the 35–39-year-old and ≤ 34-year-old groups, respectively (p < 0.05). Testing likelihood of live birth as a function of age and antral follicular count (AFC) revealed that a 1-year increase in age reduces the likelihood of live birth by 11% (p < 0.05) and one-unit increase in AFC count leads to a 9% increase in the odds of a live birth (p < 0.05). For the youngest age group, the AFC had a most significant effect, and those with AFC > 11 had 56% live birth rate, while those with AFC ≤ 11 had only 6% of live birth rate. This study supports a shift in reasoning from age being the predictor of outcomes in women with a low response at IVF to both age and ovarian reserve needing to be taken into consideration. [ABSTRACT FROM AUTHOR]

Purpose: What is the trend in sperm parameters in a group of men attending a single reproductive center, over a 10-year period? Methods: A retrospective study was conducted on 12,188 semen samples obtained from unique individuals who attended a university reproductive clinic from 2009 to 2018, inclusively. Semen analysis was done using computer-assisted sperm analysis and verified by an andrologist. Analysis was done after dividing the dataset into two groups: above WHO 2010 lower reference limits (ARL) (N = 6325) and below the reference limits (BRL) (N = 5521). Results: Volume increased slightly (ARL, p = 0.049) before returning to baseline or was stable (BRL, p = 0.59). Sperm concentration and total count of the BRL and ARL group declined initially and then recovered slightly (p < 0.0001, in all cases). Although these changes were statistically significant, this was due to the large study population; clinically, these changes were quite mild and would not have been significant for fertility. Sperm total motility and progressive motility of both the BRL group and the ARL group increased slightly from 2009 until 2015 and then decreased back to baseline (p < 0.0001). This change offset the decrease in count seen in those years. A spurious change was observed with sperm morphology that declined after the first 2 years and remained stable thereafter (p < 0.0001, in both groups). However, this change was attributed to a contemporaneous change in the method of analyzing strict morphology which happened when the change occurred. Conclusion: While statistically significant changes were found, clinically, these changes were quite mild and would not have been significant for fertility. [ABSTRACT FROM AUTHOR]

Societal Impact Statement: Grapevine is a major horticultural crop that has been intertwined with human culture for Millennia. The Levant was an important center of the ancient winemaking industry until the collapse of the Byzantine Empire. Consequently, the identity of the ancient grapevine varieties of the Levant was considered lost. Here, we study the domestication history of Levantine grapevine samples using whole‐genome sequencing data and show that some of the samples are authentic descendants of ancient Levantine varieties and that they conform to an independent lineage. This precious germplasm paves a route to bridge the ancient culture of the Levant and modern grapevine varieties. However, accelerated anthropogenic development in this region poses a threat to sampling locations; thus, additional efforts are urged to explore and conserve grapevine and other crop germplasm collections in the Levant. Summary: The ancient wine varieties of the Levant have inspired beliefs and rituals in human societies. Despite their importance, the original Levantine wine‐grape varieties were considered lost due to cultural turnovers commencing in the 7th century CE, leading to the collapse of a flourishing winemaking industry in this region.We generated whole‐genome sequence data for a diversity panel of wild and domesticated grapevine accessions. Demographic models, pedigree analysis, and genome scans were conducted to estimate divergence among four distinct groups.Demographic models designated that the domestication in the Caucasus region predated the emergence of the Levantine varieties and that authentic descendants of ancient varieties are represented among the Levantine accessions. We further explored the pedigree relationship among domesticated grapevines and identified intriguing relationship between the "Chardonnay" variety and Levantine accessions. Genomic scans and genetic load analyses further supported the distinct lineage history of the Levantine and Eurasian accessions.Tracking the domestication history of crops is challenging especially in a perennial species like grapevine. Although the Levantine varieties represent a distinct genomic background, it remains unclear whether ancient extensive gene‐flow or direct domestication of Levantine wild grapevines has sparked the emergence of the ancient Levantine varieties. [ABSTRACT FROM AUTHOR]

As treatment protocols for medulloblastoma (MB) are becoming subgroup-specific, means for reliably distinguishing between its subgroups are a timely need. Currently available methods include immunohistochemical stains, which are subjective and often inconclusive, and molecular techniques—e.g., NanoString, microarrays, or DNA methylation assays—which are time-consuming, expensive and not widely available. Quantitative PCR (qPCR) provides a good alternative for these methods, but the current NanoString panel which includes 22 genes is impractical for qPCR. Here, we applied machine-learning–based classifiers to extract reliable, concise gene sets for distinguishing between the four MB subgroups, and we compared the accuracy of these gene sets to that of the known NanoString 22-gene set. We validated our results using an independent microarray-based dataset of 92 samples of all four subgroups. In addition, we performed a qPCR validation on a cohort of 18 patients diagnosed with SHH, Group 3 and Group 4 MB. We found that the 22-gene set can be reduced to only six genes (IMPG2 , NPR3 , KHDRBS2 , RBM24 , WIF1 , and EMX2) without compromising accuracy. The identified gene set is sufficiently small to make a qPCR-based MB subgroup classification easily accessible to clinicians, even in developing, poorly equipped countries. [ABSTRACT FROM AUTHOR]

Background/Aim: Germline mutations in PTCH1 or SUFU in the sonic hedgehog (SHH) pathway cause Gorlin's syndrome with increased risk of developing SHHsubgroup medulloblastoma. Gorlin's syndrome precludes the use of radiotherapy (a standard component of treatment) due to the development of multiple basal cell carcinomas. Also, current SHH inhibitors are ineffective against SUFU-mutated medulloblastoma, as they inhibit upstream genes. In this study, we aimed to detect differences in the expression of genes and microRNAs between SUFU- and PTCH1-mutated SHH medulloblastomas which may hint at new treatment directions. Patients and Methods: We sequenced RNA and microRNA from tumors of two patients with germline Gorlin's syndrome - one having PTCH1 mutation and one with SUFU mutation - followed by bioinformatics analysis to detect changes in genes and miRNAs expression in these two tumors. Expression changes were validated using qRT-PCR. Ingenuity pathway analysis was performed in search for targetable pathways. Results: Compared to the PTCH1 tumor, the SUFU tumor demonstrated lower expression of miR-301a-3p and miR-181c-5p, matrix metallopeptidase 11 (MMP11) and OTX2, higher expression of miR-7-5p and corresponding lower expression of its targeted gene, connexin 30 (GJB6). We propose mechanisms to explain the phenotypic differences between the two types of tumors, and understand why PTCH1 and SUFU tumors tend to relapse locally (rather than metastatically as in other medulloblastoma subgroups). Conclusion: Our results help towards finding new treatable molecular targets for these types of medulloblastomas. [ABSTRACT FROM AUTHOR]

The population of Israel is ethnically diverse, and individuals from different ethnic groups share specific genetic variations. These variations, which have been passed on from common ancestors, are usually reported in public databases as rare variants. Here, we aimed to identify ethnicity-based benign copy number variants (CNVs) and generate the first Israeli CNV database. We applied a data-mining approach to the results of 10,193 chromosomal microarray tests, of which 2150 tests were from individuals of 13 common ethnic backgrounds (n ≥ 10). We found 165 CNV regions (> 50 kbp) that are unique to specific ethnic groups (uCNVRs). The frequency of more than 19% of these uCNVRs is between 1 and 20% of the common ethnic origin, while their frequency in the overall cohort is between 0.5 and 1.6%. Of these 165 uCNVRs, 98 are reported as variants of unknown significance or as not available in dbVar; we postulate that these uCNVRs should be annotated as either "likely benign" or "benign". The ethnic-specific CNVs extracted in this study will allow geneticists to distinguish between relevant pathogenic genomic aberrations and benign ethnicity-related variations, thus preventing variant misinterpretation that may lead to unnecessary pregnancy terminations. [ABSTRACT FROM AUTHOR]

Study Question: How does age affect various semen parameters?Summary Answer: For most semen parameters, the nomogram of the entire population was biphasic, peaking around the fourth decade of life.What Is Known Already: In clinical practice, semen quality is examined by using the WHO 2010 reference limits but these limits do not account for male age. A percentile-based, large-scale nomogram describing how different semen parameters change throughout reproductive life has been lacking.Study Design, Size, Duration: A retrospective study was conducted with 12 188 sperm samples, obtained from individuals who attended the McGill University Health Centre reproductive clinic between 2009 and 2018.Participants/materials, Setting, Methods: One sample from each individual who attended the clinic during the study period was analysed by using computer-assisted sperm analysis (CASA). The analysed parameters were human-verified and included sperm concentration, motility, progressive motility, total count, morphology and semen volume. Based on this analysis, the entire dataset (n = 12 188) was further divided into two groups of samples: samples that surpassed the WHO 2010 lower reference limits ('above reference limits' group, ARL; n = 6305), and samples that did not ('below reference limit' group, BRL; n = 5883). Regression quantiles were fitted as a function of age to generate age-dependent nomograms, and these quantiles were divided into 5th, 25th, 50th, 75th and 95th percentiles.Main Results and the Role Of Chance: In the entire dataset, age had a significant influence (P < 0.001) on all parameters (except morphology) which demonstrated a biphasic trend peaking in the fourth decade of life. In the ARL group, age had a significant influence (P < 0.01) on all semen parameters except sperm concentration and morphology. However, unlike in the entire dataset, only semen volume demonstrated a biphasic trend in the ARL group (peaking in the fourth decade of life), whereas other parameters either remained unchanged (concentration and morphology) or consistently declined with age (sperm motility, progressive motility and total sperm count). Percentile-based nomograms were generated for individuals between the ages of 20 and 60 years in the entire dataset and in the ARL group.Limitations, Reasons For Caution: First, the semen samples were obtained from individuals who were referred to a fertility clinic, such that the entire dataset does not necessarily represent the general population. Second, the cross-sectional sampling design increases variance, and the nomograms are less accurate in the 5th and 95th percentiles and at the extremes of the age distributions. Third, the observed age-dependent changes in semen parameters do not necessarily indicate changes in fertility, as not all factors that affect male fertility were analysed. Fourth, some of our semen analyses employed CASA, which can have variability issues. Finally, our models did not incorporate possible secular trends.Wider Implications Of the Findings: We provide the first nomogram that correlates age with semen quality parameters in different population percentiles, thus complementing the current reference limits set by the WHO in 2010. Most examined semen parameters in our study changed non-linearly with age; therefore, age should be regularly employed as a factor in the clinical analysis of semen samples.Study Funding/competing Interest(s): The authors have not received any funding to support this study. There are no conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2‐positive breast cancer receiving adjuvant therapy. The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2‐positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease‐free survival. Gene signatures associated with reactive stroma in HER2‐positive early breast cancer (N = 209) were significantly associated with trastuzumab resistance in estrogen receptor (ER)‐negative tumors (hazard ratio [HR] = 1.27 p interaction = 0.014 [DCN], HR = 1.58, p interaction = 0.027 [PLAU], HR = 1.71, p interaction = 0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER‐positive tumors (HR = 0.73 p interaction = 0.47 [DCN], HR = 0.71, p interaction = 0.73 [PLAU], HR = 0.84; p interaction = 0.36 [HER2STROMA]). Pathological evaluation of HER2‐positive/ER‐negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor‐infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC <50%). In conclusion, reactive stroma in HER2‐positive/ER‐negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy. What's new Tumor cells can interact with the surrounding microenvironment, to alter gene expression, suppress immune function, and enhance their own growth. While trastuzumab has dramatically improved clinical outcomes for patients with HER2‐positive breast cancer, not all tumors respond to treatment. In this study, the authors found that one mechanism of trastuzumab resistance may be due to altered gene expression representing "reactive" stromal cells surrounding the tumor, even when high levels of tumor‐infiltrating lymphocytes (TILs) are present. These "reactive" genetic signatures may provide valuable biomarkers for predicting treatment response. [ABSTRACT FROM AUTHOR]

Extraintestinal multidrug resistant Escherichia coli sequence type (ST) 131 is a worldwide pandemic pathogen and a major cause of urinary tract and bloodstream infections. The role of this pandemic lineage in multidrug resistance plasmid dissemination is still scarce. We herein performed a meta-analysis on E. coli ST131 whole-genome sequence (WGS) databases to unravel ST131 plasmidome and specifically to decipher CTX-M encoding plasmids-clade associations. We mined 880 ST131 WGS data and proved that CTX-M-27-encoding IncF[F1:A2:B20] (Group1) plasmids are strictly found in clade C1, whereas CTX-M-15-encoding IncF[F2:A1:B-] (Group2) plasmids exist only in clade C2 suggesting strong plasmid-clade adaptations. Specific Col-like replicons (Col156, Col(MG828), and Col8282) were also found to be clade C1-associated. BLAST-based search revealed that Group1 and Group2 plasmids are narrow-host-range and restricted to E.coli. Among a collection of 20 newly sequenced Israeli ST131 CTX-M-encoding plasmids (2003–2016), Group1 and Group2 plasmids were dominant and associated with the expected clades. We found, for the first time in ST131, a CTX-M-15-encoding phage-like plasmid group (Group3) and followed its spread in the WGS data. This study offers a comprehensive way to decipher plasmid-bacterium associations and demonstrates that the CTX-M-encoding ST131 Group1 and Group2 plasmids are clade-restricted and presumably less transmissible, potentially contributing to ST131 clonal superiority. [ABSTRACT FROM AUTHOR]

The live attenuated Brucella melitensis Rev.1 (Elberg-originated) vaccine strain is widely used to control the zoonotic infection brucellosis in small ruminants, but the molecular mechanisms underlying the attenuation of this strain have not been fully characterized. Following their uptake by the host cell, Brucella replicate inside a membrane-bound compartment—the Brucella -containing vacuole—whose acidification is essential for the survival of the pathogen. Therefore, identifying the genes that contribute to the survival of Brucella in acidic environments will greatly assist our understanding of its molecular pathogenic mechanisms and of the attenuated virulence of the Rev.1 strain. Here, we conducted a comprehensive comparative transcriptome analysis of the Rev.1 vaccine strain against the virulent reference strain 16M in cultures grown under either normal or acidic conditions. We found 403 genes that respond differently to acidic conditions in the two strains (FDR < 0.05, fold change ≥ 2). These genes are involved in crucial cellular processes, including metabolic, biosynthetic, and transport processes. Among the highly enriched genes that were downregulated in Rev.1 under acidic conditions were acetyl-CoA synthetase, aldehyde dehydrogenase, cell division proteins, a cold-shock protein, GroEL, and VirB3. The downregulation of these genes may explain the attenuated virulence of Rev.1 and provide new insights into the virulence mechanisms of Brucella. [ABSTRACT FROM AUTHOR]

Objective:Inflammation of the meninges is a source of severe morbidity and therefore is an important health concerns worldwide. The conventional clinical microbiology approaches used today to identify pathogens suffer from several drawbacks and frequently provide false results. This research describes a fast method to detect the presence of pathogens using the frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) system.Methods:The study included 43 individuals divided into 4 groups: 9 diagnosed with different types of bacteria; 16 diagnosed with different types of viruses; 5 healthy samples served as a control; and 12 samples were negative to any pathogen, although presenting related symptoms. All samples contained leukocytes that were extracted from the cerebrospinal fluid (CSF) and were subjected to nuclear staining by 4′, 6-diamidino-2-phenylindole (DAPI) and FLT analyses based on phase and amplitude crossing point (CRPO).Results:Using notched boxplots, we found differences in 95% probability between the first three groups through different notch ranges (NR). Pathogen samples presented a longer median FLT (3.28 ns with NR of 3.24–3.32 ns in bacteria and 3.18 ns with NR of 3.16–3.21 ns in viruses) compared to the control median FLT (2.65 ns with NR of 2.63–2.67 ns). Furthermore, we found that the undetected forth group was divided into two types: a relatively normal median FLT (2.72 ns with NR of 2.68–2.76 ns) and a prolonged FLT (3.22 ns with NR of 3.17–3.27 ns).Conclusion:FLT measurements can differentiate between control and pathogen by the CRPO method.Significance:The FD-FLIM system can provide a high throughput diagnostic technique that does not require a physician. [ABSTRACT FROM AUTHOR]

Background: About 5%-10% of breast cancer and 10%-15% of ovarian cancer are hereditary. BRCA1 and BRCA2 are the most common germline mutations found in both inherited breast and ovarian cancers. Once these mutations are identified and classified, a course of action to reduce the risk of developing either ovarian or breast cancer - including surveillance and surgery - is carried out. Purpose: The purpose of the current research is to characterize the gene expression differences between healthy cells harboring a mutation in BRCA1/2 genes and normal cells. This will allow detection of candidate genes and help identify women who carry functional BRCA1/2 mutations, which cannot always be detected by the available sequencing methods, for example, carriers of mutations found in regulatory sequences of the genes. Materials and methods: Our cohort consisted of 50 healthy women, of whom 24 were individuals with BRCA1 or BRCA2 heterozygous mutations and 26 were non-carrier controls. RNA purified from non-irradiated lymphocytes of nine BRCA1/2 mutation carriers versus four control mutation-negative individuals was utilized for RNA-Seq analysis. The selected RNA-Seq transcripts were validated, and the levels of spleen tyrosine kinase (SYK) mRNA were measured by using real-time quantitative polymerase chain reaction. Results: Differences in gene expression were found when comparing untreated lymphocytes of BRCA1/2 mutation carriers and controls. Among others, the SYK gene was identified as being differently expressed for BRCAH2 mutation carriers. The expression level of SYK was significantly higher in untreated healthy lymphocytes of BRCA1 heterozygote carriers compared with controls, regardless of irradiation. In contrast to normal tissues, in cancerous breast tissues, the expression levels of the BRCA1 and SYK genes were not intercorrelated. Conclusion: Collectively, our observations demonstrate that SYK may prove to be a good candidate for better diagnosis, treatment, and prevention of BRCA1 mutation-associated breast cancer. [ABSTRACT FROM AUTHOR]

Purpose: Medulloblastoma (MB), the most common malignant brain tumor in children, is divided into four tumor subgroups: wingless-type (WNT), sonic hedgehog (SHH), Group 3, and Group 4. Ideally, clinical practice and treatment design should be subgroup specific. While WNT and SHH subgroups have well-defined biomarkers, distinguishing Group 3 from Group 4 is not straightforward. MicroRNAs (miRNAs), which regulate posttranscriptional gene expression, are involved in MB tumorigenesis. However, the miRNA-messenger RNA (mRNA) regulatory network in MB is far from being fully understood. Our aims were to investigate miRNA expression regulation in MB subgroups, to assess miRNA target relationships, and to identify miRNAs that can distinguish Group 3 from Group 4. Patients and methods: With these aims, integrated transcriptome mRNA and miRNA expression analysis was performed on primary tumor samples collected from 18 children with MB, using miRNA sequencing (miRNA-seq), RNA sequencing (RNA-seq), and quantitative PCR. Results: Of all the expressed miRNAs, 19 appeared to be significantly differentially expressed (DE) between Group 4 and non-Group 4 subgroups (false discovery rate [FDR] <0.05), including 10 miRNAs, which, for the first time, are reported to be in conjunction with MB. RNA-seq analysis identified 165 genes that were DE between Group 4 and the other subgroups (FDR <0.05), among which seven are predicted targets of five DE miRNAs and exhibit inverse expression pattern. Conclusion: This study identified miRNA molecules that may be involved in Group 4 etiology, in general, and can distinguish between Group 3 and Group 4, in particular. In addition, understanding the involvement of miRNAs and their targets in MB may improve diagnosis and advance the development of targeted treatment for MB. [ABSTRACT FROM AUTHOR]

The live attenuated Brucella melitensis Rev.1 Elberg-originated vaccine strain has been widely used to control brucellosis in small ruminants. However, despite extensive research, the molecular mechanisms underlying the attenuation of this strain are still unknown. In the current study, we conducted a comprehensive comparative analysis of the whole-genome sequence of Rev.1 against that of the virulent reference strain, B. melitensis 16M. This analysis revealed five regions of insertion and three regions of deletion within the Rev.1 genome, among which, one large region of insertion, comprising 3,951 bp, was detected in the Rev.1 genome. In addition, we found several missense mutations within important virulence-related genes, which may be used to determine the mechanism underlying virulence attenuation. Collectively, our findings provide new insights into the Brucella virulence mechanisms and, therefore, may serve as a basis for the rational design of new Brucella vaccines. [ABSTRACT FROM AUTHOR]

Exposure to dibutyl phthalate (DBP) is ubiquitous among women of reproductive age. Previous studies in animal models and in human cells in vitro have shown that exposure to DBP disrupts ovarian function. Here, we examined the effect of DBP on global gene expression in mural granulosa cells (MGCs) in vitro. Primary cultures of MGC obtained from 48 patients undergoing IVF were treated with increasing concentrations of DBP (0, 0.01, 0.1, 1, 10, or 100 μg/ml) for 48 h. Microarray analysis was used to identify genes exhibiting expression changes following DBP exposure. When compared with untreated cells, exposure to 100 mg/ml DBP resulted in significant differences in expression of 346 annotated genes (> 2-fold; q value<.05). Of them, 151 were upregulated and 195 downregulated. The main functional annotations affected by DBP were associated with cell cycle, mitosis, Rho GTPases, PLK1, Aurora B signaling pathways, and E2F-mediated regulation of DNA replication. No significant differences in gene expression were observed for the lower concentrations of DBP (0.01, 0.1, 1, and 10 mg/ml) compared with controls for both the microarray analysis and genes validated by quantitative real-time (qRT)-PCR. This study provides important molecular inputs on the effect of short-term DBP exposure on human MGCs in vitro. Our results indicate that acute treatment with high concentrations of DBP alters gene expression pathways mainly associated with the cell cycle. [ABSTRACT FROM AUTHOR]

Background: The RNA-seq technique is applied for the investigation of transcriptional behaviour. The reduction in sequencing costs has led to an unprecedented trove of gene expression data from diverse biological systems. Subsequently, principles from other disciplines such as the Benford law, which can be properly judged only in data-rich systems, can now be examined on this high-throughput transcriptomic information. The Benford law, states that in many count-rich datasets the distribution of the first significant digit is not uniform but rather logarithmic. Results: All tested digital gene expression datasets showed a Benford-like distribution when observing an entire gene set. This phenomenon was conserved in development and does not demonstrate tissue specificity. However, when obedience to the Benford law is calculated for individual expressed genes across thousands of cells, genes that best and least adhere to the Benford law are enriched with tissue specific or cell maintenance descriptors, respectively. Surprisingly, a positive correlation was found between the obedience a gene exhibits to the Benford law and its expression level, despite the former being calculated solely according to first digit frequency while totally ignoring the expression value itself. Nevertheless, genes with low expression that exhibit Benford behavior demonstrate tissue specific associations. These observations were extended to predict the likelihood of tissue specificity based on Benford behaviour in a supervised learning approach. Conclusions: These results demonstrate the applicability and potential predictability of the Benford law for gleaning biological insight from simple count data. [ABSTRACT FROM AUTHOR]

Genetic syndromes involving both brain and eye abnormalities are numerous and include syndromes such as Warburg micro syndrome, Kaufman oculocerebrofacial syndrome, Cerebro-oculo-facio-skeletal syndrome, Kahrizi syndrome and others. Using exome sequencing, we have been able to identify homozygous mutation p.(Tyr39Cys) in MED25 as the cause of a syndrome characterized by eye, brain, cardiac and palatal abnormalities as well as growth retardation, microcephaly and severe intellectual disability in seven patients from four unrelated families, all originating from the same village. The protein encoded by MED25 belongs to Mediator complex or MED complex, which is an evolutionary conserved multi-subunit RNA polymerase II transcriptional regulator complex. The MED25 point mutation is located in the von Willebrand factor type A ( MED25 VWA) domain which is responsible for MED25 recruitment into the Mediator complex; co-immunoprecipitation experiment demonstrated that this mutation dramatically impairs MED25 interaction with the Mediator complex in mammalian cells. [ABSTRACT FROM AUTHOR]

Of all known S almonella enterica serovars, S. Infantis is one of the most commonly isolated and has been recently emerging worldwide. To understand the recent emergence of S. Infantis in Israel, we performed extensive comparative analyses between pre-emergent and the clonal emergent S. Infantis populations. We demonstrate the fixation of adaptive mutations in the DNA gyrase ( gyrA) and nitroreductase ( nfsA) genes, conferring resistance to quinolones and nitrofurans, respectively, and the carriage of an emergent-specific plasmid, designated pESI. This self-transferred episome is a mosaic megaplasmid (∼280 kb), which increases bacterial tolerance to environmental mercury ( mer operon) and oxidative stress, and provides further resistance to tetracycline, sulfamethoxazole and trimethoprim, most likely due to the presence of tetRA, sulI and dfrA genes respectively. Moreover, pESI carries the yersiniabactin siderophore system and two novel chaperone-usher fimbriae. In vitro studies established that pESI conjugation into a plasmidless S. Infantis strain results in superior biofilm formation, adhesion and invasion into avian and mammalian host cells. In vivo mouse infections demonstrated higher pathogenicity and increased intestinal inflammation caused by an S. Infantis strain harboring pESI compared with the plasmidless parental strain. Our results indicate that the presence of pESI that was found only in the emergent population of S. Infantis in Israel contributes significantly to antimicrobials tolerance and pathogenicity of its carrier. It is highly likely that pESI plays a key role in the successful spread of the emergent clone that replaced the local S. Infantis community in the short time of only 2-3 years. [ABSTRACT FROM AUTHOR]

The principal architects of coral reefs are the scleractinian corals; these species are divided in two major clades referred to as “robust” and “complex” corals. Although the molecular diversity of the “complex” clade has received considerable attention, with several expressed sequence tag (EST) libraries and a complete genome sequence having been constructed, the “robust” corals have received far less attention, despite the fact that robust corals have been prominent focal points for ecological and physiological studies. Filling this gap affords important opportunities to extend these studies and to improve our understanding of the differences between the two major clades. Here, we present an EST library from Stylophora pistillata (Esper 1797) and systematically analyze the assembled transcripts compared to putative homologs from the complete proteomes of six well-characterized metazoans: Nematostella vectensis, Hydra magnipapillata, Caenorhabditis elegans, Drosophila melanogaster, Strongylocentrotus purpuratus, Ciona intestinalis and Homo sapiens. Furthermore, comparative analyses of the Stylophora pistillata ESTs were performed against several Cnidaria from the Scleractinia, Actiniaria and Hydrozoa, as well as against other stony corals separately. Functional characterization of S. pistillata transcripts into KOG/COG categories and further description of Wnt and bone morphogenetic protein (BMP) signaling pathways showed that the assembled EST library provides sufficient data and coverage. These features of this new library suggest considerable opportunities for extending our understanding of the molecular and physiological behavior of “robust” corals. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA-mRNA interactions rather than as on-off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer. [ABSTRACT FROM AUTHOR]

The transcription factors Foxa1 and Foxa2 promote the specification of midbrain dopaminergic (mDA) neurons and the floor plate. Whether their role is direct has remained unclear as they also regulate the expression of Shh, which has similar roles. We characterized the Foxa2 cis-regulatory network by chromatin immunoprecipitation followed by high-throughput sequencing of mDA progenitors. This identified 9160 high-quality Foxa2 binding sites associated with 5409 genes, providing mechanistic insights into Foxa2-mediated positive and negative regulatory events. Foxa2 regulates directly and positively key determinants of mDA neurons, including Lmx1a, Lmx1b, Msx1 and Ferd3l, while negatively inhibiting transcription factors expressed in ventrolateral midbrain such as Helt, Tle4, Otx1, Sox1 and Tal2. Furthermore, Foxa2 negatively regulates extrinsic and intrinsic components of the Shh signaling pathway, possibly by binding to the same enhancer regions of co-regulated genes as Gli1. Foxa2 also regulates the expression of floor plate factors that control axon trajectories around the midline of the embryo, thereby contributing to the axon guidance function of the floor plate. Finally, this study identified multiple Foxa2-regulated enhancers that are active in the floor plate of the midbrain or along the length of the embryo in mouse and chick. This work represents the first comprehensive characterization of Foxa2 targets in mDA progenitors and provides a framework for elaborating gene regulatory networks in a functionally important progenitor population. [ABSTRACT FROM AUTHOR]

An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N6-methyladenosine (m6A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m6A modification landscape in a transcriptome-wide manner, using a novel approach, m6A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m6A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks-around stop codons and within long internal exons-and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m6A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m6A has a fundamental role in regulation of gene expression. [ABSTRACT FROM AUTHOR]

Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage-specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD-mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene-specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development. [ABSTRACT FROM AUTHOR]

Background: A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8+ T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known. Methodology/Principal Findings: In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached. Conclusion: Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance. [ABSTRACT FROM AUTHOR]

Background: Functional genomic studies involving high-throughput sequencing and tiling array applications, such as ChIP-seq and ChIP-chip, generate large numbers of experimentally-derived signal peaks across the genome under study. In analyzing these loci to determine their potential regulatory functions, areas of signal enrichment must be considered relative to proximal genes and regulatory elements annotated throughout the target genome Regions of chromatin association by transcriptional regulators should be distinguished as individual binding sites in order to enhance downstream analyses, such as the identification of known and novel consensus motifs. Results: PeakAnalyzer is a set of high-performance utilities for the automated processing of experimentally-derived peak regions and annotation of genomic loci. The programs can accurately subdivide multimodal regions of signal enrichment into distinct subpeaks corresponding to binding sites or chromatin modifications, retrieve genomic sequences encompassing the computed subpeak summits, and identify positional features of interest such as intersection with exon/intron gene components, proximity to up- or downstream transcriptional start sites and cisregulatory elements. The software can be configured to run either as a pipeline component for high-throughput analyses, or as a cross-platform desktop application with an intuitive user interface. Conclusions: PeakAnalyzer comprises a number of utilities essential for ChIP-seq and ChIP-chip data analysis. Highperformance implementations are provided for Unix pipeline integration along with a GUI version for interactive use. Source code in C++ and Java is provided, as are native binaries for Linux, Mac OS X and Windows systems. [ABSTRACT FROM AUTHOR]

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for ''missing self'' recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an ''educating impact'' on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors. [ABSTRACT FROM AUTHOR]

Nucleosome core particles in eukaryotes are linked by a stretch of DNA that is usually associated with a linker histone. Here, we show in yeast, that the presence of yeast linker histone Hho1p represses expression of a pol II transcribed gene (MET15) embedded in the rDNA. In vivo deletions of Hho1p sequences showed that the second globular domain is sufficient for that repression, whereas the presence of the N terminus is required for its derepression. In contrast, a run-on assay confirmed by a ChlP experiment showed that Hho1p is required for maximal poll processivity during rDNA transcription. Psoralen accessibility experiments indicated that Hho1p is necessary for normal rDNA compaction. DNA array expression analysis comparing RNA transcripts in wild-type and hho1 strains before and after a heat-shock showed that Hho1p is necessary to achieve wild-type mRNA levels of transcripts that encode ribosomal components. Taken together, our results suggest that Hho1p is involved in rDNA compaction, and like core histones, is required for efficient rDNA transcription by pol I. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells lyse only cells that do not express sufficient levels of self class I MHC molecules. Inhibition of lysis is mediated by inhibitory receptors expressed by NK cells, such as the murine Ly49 receptors, that bind to MHC class I molecules. Since inhibitory receptor genes and MHC class I genes are located on different chromosomes, and are hence not automatically co-inherited, NK cells apparently adapt to the MHC environment during their development. Two models have been proposed to account for this "education" process of NK cells. The two-step selection model postulates that developing NK cells initiate the stable expression of a random set of Ly49 genes, and then undergo two selection steps, one for cells that express a sufficient number of self-MHC receptors, and one against cells that express too many inhibitory receptors. The sequential model postulates that a cell keeps initiating the stable expression of additional inhibitory receptors until a sufficient expression level of self-MHC specific receptors is reached, and the cell matures. In this study we implement both models in computer simulations, and compare simulation results to experimental data, in order to evaluate the relative plausibility of the two models. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are lymphocytes which can kill tumor and virus-infected cells, and mediate acute rejection of bone marrow grafts. NK-cell killing is directed primarily at target cells which do not express sufficient levels of self-major histocompatibility complex (MHC) class-I molecules. Inhibition of lysis of self-MHC-expressing target cells is mediated via inhibitory receptors on the NK cell, which bind to MHC molecules. Each NK cell expresses only a subset of all its inhibitory receptor gene repertoire, which may bind to only a subset of the self-MHC molecules expressed by other cells of the organism. Two conceptual models have been proposed to explain the process of NK cell ‘education’ in which these cells adapt, during their development, to the self-MHC environment: the two-step selection and the sequential models. In this manuscript we develop mathematical and computational models of the process of NK cell development and education, which implement the two conceptual models. We use this theoretical framework to examine the available data on NK cell repertoire compositions, and evaluate the degree these data support either of the two conceptual models. We show that the data published so far on NK cell receptor expression patterns is insufficient to refute either model, since data on NK cell receptor binding affinities to MHC is also needed. However, the models allow us to make predictions on these binding affinities, which can later be tested experimentally. [Copyright &y& Elsevier]

The original article can be found online at https://doi.org/10.1007/s10815-021-02298-8. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. [Extracted from the article]

B-lymphocytes are essential for an efficient immune response against a variety of pathogens. A large fraction of hematologic malignancies are of B-cell origin, suggesting that the development and activation of B cells must be tightly regulated. In recent years, differentially expressed non-coding RNAs have been identified in mantle cell lymphoma (MCL) tumor samples as opposed to their naive, normal B-cell compartment. These aberrantly expressed molecules, specifically microRNAs (miRNAs), circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs), have a role in cellular growth and survival pathways in various biological models. Here, we provide an overview of current knowledge on the role of non-coding RNAs and their relevant targets in B-cell development, activation and malignant transformation, summarizing the current understanding of the role of aberrant expression of non-coding RNAs in MCL pathobiology with perspectives for clinical use. [ABSTRACT FROM AUTHOR]

Brucella species are facultative intracellular bacteria that cause brucellosis, a zoonotic world-wide disease. The live attenuated B. melitensis Rev.1 vaccine strain is widely used for the control of brucellosis in the small ruminant population. However, Rev.1 induces antibodies against the O-polysaccharide (O-PS) of the smooth lipopolysaccharide thus, it is difficult to differentiate between infected and vaccinated animals. Hence, rough Brucella strains lacking the O-PS have been introduced. In the current study, we conducted a comprehensive comparative analysis of the genome sequence of two natural Rev.1 rough strains, isolated from sheep, against that of 24 Rev.1 smooth strains and the virulent reference strain B. melitensis 16M. We identified and characterized eight vital mutations within highly important genes associated with Brucella lipopolysaccharide (LPS) biosynthesis and virulence, which may explain the mechanisms underlying the formation of the Rev.1 rough phenotype and may be used to determine the mechanism underlying virulence attenuation. Further complementation studies aimed to estimate the specific role of these mutations in affecting Brucella morphology and virulence will serve as a basis for the design of new attenuated vaccines for animal immunization against brucellosis. [ABSTRACT FROM AUTHOR]

Cell migration requires reposition and reshaping of the cell nucleus. The nuclear lamina is highly important for migration of both primary and cancer cells. B-type lamins are important for proper migration of epicardial cells and neurons and increased lamin B to lamin A ratio accelerates cancer cell migration through confined spaces. Moreover, a positive association between lamin B1 levels and tumor formation and progression is found in various cancer types. Still, the molecular mechanism by which B-type lamins promote cell migration is not fully understood. To better understand this mechanism, we tested the effects of lamin B1 on perinuclear actin organization. Here we show that induction of melanoma cell migration leads to the formation of a cytosolic Linker of Nucleoskeleton and Cytoskeleton (LINC) complex-independent perinuclear actin rim, which has not been detected in migrating cells, yet. Significantly, increasing the levels of lamin B1 but not the levels of lamin A prevented perinuclear actin rim formation while accelerated the cellular migration rate. To interfere with the perinuclear actin rim, we generated a chimeric protein that is localized to the outer nuclear membrane and cleaves perinuclear actin filaments in a specific manner without disrupting other cytosolic actin filaments. Using this tool, we found that disruption of the perinuclear actin rim accelerated the cellular migration rate in a similar manner to lamin B1 over-expression. Taken together, our results suggest that increased lamin B1 levels can accelerate cell migration by inhibiting the association of the nuclear envelope with actin filaments that may reduce nuclear movement and deformability. [ABSTRACT FROM AUTHOR]