Sample Preparation and Phosphopeptide Enrichment for Plant Phosphoproteomics via Label-Free Mass Spectrometry.

Phosphopeptide enrichment is the main bottleneck of every phosphorylation study. Therefore, in this chapter, a general workflow tries to overbridge the hurdles of plant sample handling from sample collection to protein extraction, protein solubilization, enzymatic digestion, and enrichment step prior to mass spectrometry. The workflow provides information to perform global proteomics as well as phosphoproteomics enabling the researcher to use the protocol in both fields.

Capillarity-driven Enrichment and Hydrodynamic Trapping of Trace Nucleic Acids by Plasmonic Cavity Membrane for Rapid and Sensitive Detections.

Small reactor-based Polymerase chain reaction (PCR) has attracted considerable attention. A significant number of tiny reactors must be prepared in parallel to capture, amplify and accurately quantify few target genes in clinically relevant large volume, which, however, requires sophisticated microfabrication and longer sample-to-answer time. Here we report single plasmonic cavity membrane that not only enriches and captures few nucleic acids by taking advantage of both capillarity and hydrodynamic trapping but also quickly amplifies them for sensitive plasmonic detection. We fabricate the plasmonic cavity membrane with few nanoliters in a void volume by self-assembling gold nanorods with SiO2 tips. Simulations reveal that hydrodynamic stagnation between the SiO2 tips is mainly responsible for the trapping of the nucleic acid in the membrane. Finally, we show that our plasmonic cavity membrane is capable of enriching SARS-CoV-2 genes up to 20000-fold within 1 min, amplifying within 3 min, and detecting the trace genes as low as a single copy per microliter. We anticipate that our work not only expands the utility of PCR but also provide an innovative way of the enrichment and detection of trace biomolecules in a variety of point-of-care testing applications. This article is protected by copyright. All rights reserved.

Molecular mechanisms underlying structural plasticity of electroconvulsive therapy in major depressive disorder.

Although previous studies reported structural changes associated with electroconvulsive therapy (ECT) in major depressive disorder (MDD), the underlying molecular basis of ECT remains largely unknown. Here, we combined two independent structural MRI datasets of MDD patients receiving ECT and transcriptomic gene expression data from Allen Human Brain Atlas to reveal the molecular basis of ECT for MDD. We performed partial least square regression to explore whether/how gray matter volume (GMV) alterations were associated with gene expression level. Functional enrichment analysis was conducted using Metascape to explore ontological pathways of the associated genes. Finally, these genes were further assigned to seven cell types to determine which cell types contribute most to the structural changes in MDD patients after ECT. We found significantly increased GMV in bilateral hippocampus in MDD patients after ECT. Transcriptome-neuroimaging association analyses showed that expression levels of 726 genes were positively correlated with the increased GMV in MDD after ECT. These genes were mainly involved in synaptic signaling, calcium ion binding and cell-cell signaling, and mostly belonged to excitatory and inhibitory neurons. Moreover, we found that the MDD risk genes of CNR1, HTR1A, MAOA, PDE1A, and SST as well as ECT related genes of BDNF, DRD2, APOE, P2RX7, and TBC1D14 showed significantly positive associations with increased GMV. Overall, our findings provide biological and molecular mechanisms underlying structural plasticity induced by ECT in MDD and the identified genes may facilitate future therapy for MDD.

A PCR-independent approach for mtDNA enrichment and next-generation sequencing: comprehensive evaluation and clinical application.

BACKGROUND: Sequencing the mitochondrial genome has been increasingly important for the investigation of primary mitochondrial diseases (PMD) and mitochondrial genetics. To overcome the limitations originating from PCR-based mtDNA enrichment, we set out to develop and evaluate a PCR-independent approach in this study, named Pime-Seq (PCR-independent mtDNA enrichment and next generation Sequencing). RESULTS: By using the optimized mtDNA enrichment procedure, the mtDNA reads ratio reached 88.0 ± 7.9% in the sequencing library when applied on human PBMC samples. We found the variants called by Pime-Seq were highly consistent among technical repeats. To evaluate the accuracy and reliability of this method, we compared Pime-Seq with lrPCR based NGS by performing both methods simultaneously on 45 samples, yielding 1677 concordant variants, as well as 146 discordant variants with low-level heteroplasmic fraction, in which Pime-Seq showed higher reliability. Furthermore, we applied Pime-Seq on 4 samples of PMD patients retrospectively, and successfully detected all the pathogenic mtDNA variants. In addition, we performed a prospective study on 192 apparently healthy pregnant women during prenatal screening, in which Pime-Seq identified pathogenic mtDNA variants in 4 samples, providing extra information for better health monitoring in these cases. CONCLUSIONS: Pime-Seq can obtain highly enriched mtDNA in a PCR-independent manner for high quality and reliable mtDNA deep-sequencing, which provides us an effective and promising tool for detecting mtDNA variants for both clinical and research purposes.

Identification of key mRNAs and signaling pathways in obsessive compulsive disorder based on weighted gene co-expression network analysis and cytoHubba plugin.

PURPOSE: Obsessive-compulsive disorder (OCD) is a complex psychiatric disorder. Genetic and broad environmental factors are common risk factors for OCD. The purpose of this study is to explore the molecular mechanism of OCD and to find new molecular targets for the diagnosis and management of OCD. METHODS: All data were downloaded from public dataset. Key modules and candidate key mRNAs were identified based on weighted gene co-expression network analysis (WGCNA). The "limma" R package was used for differential expression analysis of mRNAs. Subsequently, functional enrichment analysis of differentially expressed mRNAs (DEmRNAs) was also carried out. In addition, a diagnostic model was constructed. Finally, the infiltration level of immune cells in OCD and its correlation with multicentric key DEmRNAs were analyzed. RESULTS: Green and red modules were selected as the hub modules. A total of 447 mRNAs were considered candidate key mRNAs according to GS > 0.2 and MM > 0.3. A total of 26 DEmRNAs in the same direction were identified in the GSE60190 and GSE78104 datasets. A total of 26 DEmRNAs were intersected with candidate key mRNAs in WGCNA to obtain 10 intersection DEmRNAs (HSPB1, ITPK1, CBX7, PPP1R10, TAOK1, PISD, MKNK2, RWDD1, PPA1, and RELN). However, only four DEmRNAs (HSPB1, TAOK1, MKNK2, and PPA1) predicted related drugs. Subsequently, receiver operating characteristic analysis shows that the diagnostic model has high diagnostic value. Moreover, six multicentric key DEmRNAs (SNRPF, SNRNP70, PRPF8, NOP56, EPRS, and CCT2) were screened by UpSet package. Finally, six multicentric key DEmRNAs were found to be associated with immune cells. CONCLUSION: The key molecules obtained in this study lay a foundation for further research on the molecular mechanism of OCD.

Next-generation sequencing survey of acute febrile illness in Senegal (2020-2022).

Introduction: Acute febrile illnesses (AFI) in developing tropical and sub-tropical nations are challenging to diagnose due to the numerous causes and non-specific symptoms. The proliferation of rapid diagnostic testing and successful control campaigns against malaria have revealed that non-Plasmodium pathogens still contribute significantly to AFI burden. Thus, a more complete understanding of local trends and potential causes is important for selecting the correct treatment course, which in turn will reduce morbidity and mortality. Next-generation sequencing (NGS) in a laboratory setting can be used to identify known and novel pathogens in individuals with AFI. Methods: In this study, plasma was collected from 228 febrile patients tested negative for malaria at clinics across Senegal from 2020-2022. Total nucleic acids were extracted and converted to metagenomic NGS libraries. To identify viral pathogens, especially those present at low concentration, an aliquot of each library was processed with a viral enrichment panel and sequenced. Corresponding metagenomic libraries were also sequenced to identify non-viral pathogens. Results and Discussion: Sequencing reads for pathogens with a possible link to febrile illness were identified in 51/228 specimens, including (but not limited to): Borrelia crocidurae (N = 7), West Nile virus (N = 3), Rickettsia felis (N = 2), Bartonella quintana (N = 1), human herpesvirus 8 (N = 1), and Saffold virus (N = 1). Reads corresponding to Plasmodium falciparum were detected in 19 specimens, though their presence in the cohort was likely due to user error of rapid diagnostic testing or incorrect specimen segregation at the clinics. Mosquito-borne pathogens were typically detected just after the conclusion of the rainy season, while tick-borne pathogens were mostly detected before the rainy season. The three West Nile virus strains were phylogenetically characterized and shown to be related to both European and North American clades. Surveys such as this will increase the understanding of the potential causes of non-malarial AFI, which may help inform diagnostic and treatment options for clinicians who provide care to patients in Senegal.

A proof of concept for a targeted enrichment approach to the simultaneous detection and characterization of rickettsial pathogens from clinical specimens.

Infection with either Rickettsia prowazekii or Orientia tsutsugamushi is common, yet diagnostic capabilities are limited due to the short window for positive identification. Until now, although targeted enrichment had been applied to increase sensitivity of sequencing-based detection for various microorganisms, it had not been applied to sequencing of R. prowazekii in clinical samples. Additionally, hybridization-based targeted enrichment strategies had only scarcely been applied to qPCR of any pathogens in clinical samples. Therefore, we tested a targeted enrichment technique as a proof of concept and found that it dramatically reduced the limits of detection of these organisms by both qPCR and high throughput sequencing. The enrichment methodology was first tested in contrived clinical samples with known spiked-in concentrations of R. prowazekii and O. tsutsugamushi DNA. This method was also evaluated using clinical samples, resulting in the simultaneous identification and characterization of O. tsutsugamushi directly from clinical specimens taken from sepsis patients. We demonstrated that the targeted enrichment technique is helpful by lowering the limit of detection, not only when applied to sequencing, but also when applied to qPCR, suggesting the technique could be applied more broadly to include other assays and/or microbes for which there are limited diagnostic or detection modalities.

Identification of potential key ferroptosis- and autophagy-related genes in myelomeningocele through bioinformatics analysis.

Myelomeningocele is a common congenital anomaly associated with polygenic disorders worldwide. However, the intricate molecular mechanisms underlying myelomeningocele remain elusive. To investigate whether ferroptosis and ferritinophagy contribute to the pathomechanism of myelomeningocele, differentially expressed genes (DEGs) were identified as novel biomarker and potential treatment agents. The GSE101141 dataset from Gene Expression Omnibus (GEO) was analyzed using GEO2R web tool to obtain DEGs based on |log2 fold change (FC)|≥1.5 and p < 0.05. Two datasets from the Ferroptosis Database (481 genes) and Autophagy Database (551 genes) were intersected with the DEGs from the GSE101141 dataset to identify ferroptosis- and autophagy-related DEGs using Venn diagrams. Functional and pathway enrichment, protein-protein interaction (PPI) network analyses were performed, and candidate genes were selected. Transcription factors (TFs), microRNAs (miRNAs), diseases and chemicals interacting with the candidate genes were identified. Receiver operating characteristic (ROC) curve analysis was performed to validate the diagnostic value of the candidate genes. Sixty ferroptosis-related and 74 autophagy-related DEGs were identified. These DEGs are involved in FoxO signaling pathway. Six candidate genes (EGFR, KRAS, IL1B, SIRT1, ATM, and MAPK8) were selected. miRNAs such as hsa-miR-27a-3p, hsa-miR-877-5p, and hsa-miR-892b, and TFs including P53, POU3F2, TATA are involved in regulation of candidate genes. Diseases such as schizophrenia, fibrosis, and neoplasms are the most relevant to the candidate genes. Chemicals, such as resveratrol, curcumin, and quercetin may have significant implications in the treatment of myelomeningocele. The candidate genes, especially MAPK8, also showed a high diagnostic value for myelomeningocele. These results help to shed light on the molecular mechanism of myelomeningocele and may provide new insights into diagnostic biomarker in the amniotic fluid and potential therapeutic agents of myelomeningocele.

Burlap and buddies: the effects of social enrichment (preweaning mixing) and object enrichment (burlap) on piglet performance, behavior, and welfare in the preweaning environment.

When weaned in commercial operations, piglets are not only separated from their sow but also mixed with unfamiliar pigs in an unfamiliar environment with a new diet. These abrupt changes can be stressful for piglets, often having negative welfare consequences. Our objective was to study the effects of early-life preweaning socialization and object enrichment in the preweaning environment. We compared piglet performance, behavior, and welfare across six treatments that combined multi-litter group size (1 vs. 2 vs. 4 litters) and burlap (yes vs. no). We recorded piglet behavior and lesion-scored sows and piglets. Normal conforming data, expressed per experimental unit (and behavior data were averaged over time), were analyzed by ANOVA. When given the opportunity in the sow barn, piglets in multi-litter groups socialized with other litters. Burlap use (P = 0.08) was observed in < 5% of the observations but tended to increase in mixed litter groups. Cross-sucking was observed in ~12% of the observations within mixed litter groups and tended to increase with mixed group size (P = 0.08). While there was no difference in the proportion of piglets nursing (P > 0.10), piglets were less active in the single crate groups and most active in the groups of two litters mixed (P = 0.03). Additionally, observed piglet/sow biting behaviors did not differ between treatments; however, piglet-piglet biting (P = 0.07), and pen object manipulation (P = 0.07) tended to be observed more frequently in non-enriched groups. Piglet displacements were observed more often in non-enriched groups around the pen (P = 0.03) but tended to be observed more often in enriched groups at the teat (P = 0.07). Preweaning socialization and object enrichment had no impact on the average number of piglets weaned per sow or total piglet mortality (P > 0.10). However, the proportion of laid-on piglets decreased as the number of mixed litters increased (P = 0.02). The average number of lesions per piglet did not differ between treatments. Although the final lesion scores of sow teat and udder condition did not differ between treatments (P > 0.10), sow udder scores tended to worsen more in the single litters than in the mixed litters (P = 0.08). Overall, social and object enrichment allows piglets to socialize at a younger age and to redirect their attention toward an object (burlap) which does not negatively impact piglet or sow performance, or behavior, and may improve piglet welfare around weaning.

Critical gene signature and immunological characterization in peripheral vascular atherosclerosis: novel insights from mendelian randomization and transcriptomics.

Introduction: Peripheral vascular atherosclerosis (PVA) is a chronic inflammatory disease characterized by lipid accumulation in blood vessel walls, leading to vessel narrowing and inadequate blood supply. However, the molecular mechanisms underlying PVA remain poorly understood. In this study, we employed a combination of Mendelian randomization (MR) analysis and integrated transcriptomics to identify the critical gene signature associated with PVA. Methods: This study utilized three public datasets (GSE43292, GSE100927 and GSE28829) related to peripheral vascular atherosclerosis obtained from the Gene Expression Omnibus database. Instrumental variables (IVs) were identified through expression quantitative trait loci (eQTL) analysis, and two-sample MR analysis was performed using publicly available summary statistics. Disease critical genes were identified based on odds ratios and intersected with differentially expressed genes in the disease dataset. GSE28829 dataset was used to validate the screened disease critical genes. Functional enrichment analysis, GSEA analysis, and immune cell infiltration analysis were performed to further characterize the role of these genes in peripheral vascular atherosclerosis. Results: A total of 26,152 gene-related SNPs were identified as IVs, and 242 disease-associated genes were identified through MR analysis. Ten disease critical genes (ARHGAP25, HCLS1, HVCN1, RBM47, LILRB1, PLAU, IFI44L, IL1B, IFI6, and CFL2) were significantly associated with peripheral vascular atherosclerosis. Functional enrichment analysis using KEGG pathways revealed enrichment in the NF-kappa B signaling pathway and osteoclast differentiation. Gene set enrichment analysis further demonstrated functional enrichment of these genes in processes related to vascular functions and immune system activation. Additionally, immune cell infiltration analysis showed differential ratios of B cells and mast cells between the disease and control groups. The correlations analysis highlights the intricate interplay between disease critical genes and immune cells associated with PVA. Conclusion: In conclusion, this study provides new insights into the molecular mechanisms underlying PVA by identifying ten disease critical genes associated with the disease. These findings, supported by differential expression, functional enrichment, and immune system involvement, emphasize the role of these genes in vascular function and immune cell interactions in the context of PVA. These findings contribute to a better understanding of PVA pathogenesis and offer potential targets for further mechanistic exploration and therapeutic interventions.

Gene ontology functional annotation datasets for the ITAG3.2 and ITAG4.0 tomato (Solanum lycopersicum) genome annotations.

Functional annotation based on Gene Ontology has provided a structured and comprehensive system to access the current knowledge about the function of genes. For model plants such as Arabidopsis thaliana, there is a constant updating and restructuring of the functional annotation that increases the reliability of the analyses that use it. For tomato (Solanum lycopersicum), a crop widely used as a model plant for the study of fleshy fruits, there is no functional annotation, at least not freely accessible, even though its genome has long been sequenced and annotated. In this work, we generated, using a simplified version of the maize GAMER pipeline, a tomato Gene Ontology functional annotation with 72.42% (ITAG3.2) and 74.2% (ITAG4.0) of protein-coding genes with at least one GO term association. With this dataset, we share a reliable and easy-to-use tool with the tomato community.

Rapidly improving ARDS differs clinically and biologically from persistent ARDS.

BACKGROUND: Rapidly improving acute respiratory distress syndrome (RIARDS) is an increasingly appreciated subgroup of ARDS in which hypoxemia improves within 24 h after initiation of mechanical ventilation. Detailed clinical and biological features of RIARDS have not been clearly defined, and it is unknown whether RIARDS is associated with the hypoinflammatory or hyperinflammatory phenotype of ARDS. The purpose of this study was to define the clinical and biological features of RIARDS and its association with inflammatory subphenotypes. METHODS: We analyzed data from 215 patients who met Berlin criteria for ARDS (endotracheally intubated) and were enrolled in a prospective observational cohort conducted at two sites, one tertiary care center and one urban safety net hospital. RIARDS was defined according to previous studies as improvement of hypoxemia defined as (i) PaO2:FiO2 > 300 or (ii) SpO2: FiO2 > 315 on the day following diagnosis of ARDS (day 2) or (iii) unassisted breathing by day 2 and for the next 48 h (defined as absence of endotracheal intubation on day 2 through day 4). Plasma biomarkers were measured on samples collected on the day of study enrollment, and ARDS phenotypes were allocated as previously described. RESULTS: RIARDS accounted for 21% of all ARDS participants. Patients with RIARDS had better clinical outcomes compared to those with persistent ARDS, with lower hospital mortality (13% vs. 57%; p value < 0.001) and more ICU-free days (median 24 vs. 0; p value < 0.001). Plasma levels of interleukin-6, interleukin-8, and plasminogen activator inhibitor-1 were significantly lower among patients with RIARDS. The hypoinflammatory phenotype of ARDS was more common among patients with RIARDS (78% vs. 51% in persistent ARDS; p value = 0.001). CONCLUSIONS: This study identifies a high prevalence of RIARDS in a multicenter observational cohort and confirms the more benign clinical course of these patients. We report the novel finding that RIARDS is characterized by lower concentrations of plasma biomarkers of inflammation compared to persistent ARDS, and that hypoinflammatory ARDS is more prevalent among patients with RIARDS. Identification and exclusion of RIARDS could potentially improve prognostic and predictive enrichment in clinical trials.

A System Biology Approach Reveals New Targets for Human Thyroid Gland Toxicity in Embryos and Adult Individuals.

Compounds of natural or synthetic origin present in personal care products, food additives, and packaging may interfere with hormonal regulation and are called endocrine-disrupting chemicals (EDCs). The thyroid gland is an important target of these compounds. The objective of this study was to analyze public data on the human thyroid transcriptome and investigate potential new targets of EDCs in the embryonic and adult thyroid glands. We compared the public transcriptome data of adult and embryonic human thyroid glands and selected 100 up- or downregulated genes that were subsequently subjected to functional enrichment analysis. In the embryonic thyroid, the most highly expressed gene was PRMT6, which methylates arginine-4 of histone H2A (86.21%), and the downregulated clusters included plasma lipoprotein particles (39.24%) and endopeptidase inhibitory activity (24.05%). For the adult thyroid gland, the most highly expressed genes were related to the following categories: metallothionein-binding metals (56.67%), steroid hormone biosynthetic process (16.67%), and cellular response to vascular endothelial growth factor stimulus (6.67%). Several compounds ranging from antihypertensive drugs to enzyme inhibitors were identified as potentially harmful to thyroid gland development and adult function.

Chemical characterization and source apportionment of PM2.5 in a Northeastern China city during the epidemic period.

To investigate the influence of COVID-19 lockdown measures on PM2.5 and its chemical components in Shenyang, PM2.5 samples were continuously collected from January 1 to May 31, 2020. The samples were then analyzed for water-soluble inorganic ions, metal elements, organic carbon, and elemental carbon. The findings indicated a significant decrease in PM2.5 and its various chemical components during the lockdown period, compared to pre-lockdown levels (p < 0.05), suggesting a substantial improvement in air quality. Water-soluble inorganic ions (WSIIs) were identified as the primary contributors to PM2.5, accounting for 47% before the lockdown, 46% during the lockdown, and 37% after the lockdown. Ionic balance analysis revealed that PM2.5 exhibited neutral, weakly alkaline, and alkaline characteristics before, during, and after the lockdown, respectively. NH4+ was identified as the main balancing cation and was predominantly present in the form of NH4NO3 in the absence of complete neutralization of SO42- and NO3-. Moreover, the higher sulfur oxidation ratio (SOR) and nitrogen oxidation ratio (NOR), along with the significant increase in PM2.5/EC, suggested intense secondary transformation during the lockdown period. The elevated OC/EC ratio during the lockdown period implied higher secondary organic carbon (SOC), and the notable increase in SOC/EC ratio indicated a significant secondary transformation of total carbon. The enrichment factor (EF) results revealed that during the lockdown, 9 metal elements (As, Sn, Pb, Zn, Cu, Sb, Ag, Cd, and Se) were substantially impacted by anthropogenic emissions. Source analysis of PMF was employed to identify the sources of PM2.5 in Shenyang during the study period, and the analysis identified six factors: secondary sulfate and vehicle emissions, catering fume sources, secondary nitrate and coal combustion emissions, dust sources, biomass combustion, and industrial emissions, with secondary sulfate and vehicle emissions and catering fume sources contributing the most to PM2.5.

Profiles of psychological flexibility and caregiving experience in dementia family caregivers: A latent profile analysis.

OBJECTIVES: To explore the profiles of psychological flexibility among dementia family caregivers and examine their associations with psychological well-being and caregiving factors. METHODS: Participants were 521 dementia family caregivers in Japan. Latent profile analysis was conducted to explore the profiles of psychological flexibility. The analyses examined differences in depression, anxiety, life satisfaction, and work-family conflict/enrichment between the profiles, and whether sociodemographic variables and caregiving stressors predict the profile. RESULTS: Four distinct profiles were identified: high psychological flexibility (14.2%), moderate psychological flexibility with high commitment (24.7%), moderate psychological flexibility with low commitment (48.0%), and low psychological flexibility (13.1%). The low psychological flexibility profile exhibited the highest scores of depression, anxiety and work-family conflict, followed by the moderate psychological flexibility with low/high commitment profiles, and the high psychological flexibility profile. The high psychological flexibility and moderate psychological flexibility with high commitment profiles exhibited higher life satisfaction than the moderate psychological flexibility with low commitment profile. Caregiving stressors, marital status, and caregiver status predicted the profile. CONCLUSION: Enhancing defusion and acceptance, rather than increasing commitment to personal values, may be beneficial in supporting distressed caregivers. Having more caregiving stressors, being single/divorced/bereaved, and being a primary caregiver may be useful indicators of decreased psychological flexibility among dementia family caregivers.

Phylogenomics of the leafhopper genus Neoaliturus Distant, 1918 (Hemiptera: Cicadellidae: Deltocephalinae) reveals genetically divergent lineages in the invasive beet leafhopper.

Phylogenomic analysis based on nucleotide sequences of 398 nuclear gene loci for 67 representatives of the leafhopper genus Neoaliturus yielded well-resolved estimates of relationships among species of the genus. Subgenus Neoaliturus (Neoaliturus) is consistently paraphyletic with respect to Neoaliturus (Circulifer). The analysis revealed the presence of at least ten genetically divergent clades among specimens consistent with the previous morphology-based definition of the leafhopper genus "Circulifer" which includes three previously recognized "species complexes." Specimens of the American beet leafhopper, N. tenellus (Baker), collected from the southwestern USA consistently group with one of these clades, comprising specimens from the eastern Mediterranean. Some of the remaining lineages are consistent with ecological differences previously observed among eastern Mediterranean populations and suggest that N. tenellus, as previously defined, comprises multiple monophyletic species, distinguishable by slight morphological differences.

Enricherator: A Bayesian Method for Inferring Regularized Genome-wide Enrichments from Sequencing Count Data.

A pervasive question in biological research studying gene regulation, chromatin structure, or genomics is where, and to what extent, does a signal of interest arise genome-wide? This question is addressed using a variety of methods relying on high-throughput sequencing data as their final output, including ChIP-seq for protein-DNA interactions,1 GapR-seq for measuring supercoiling,2 and HBD-seq or DRIP-seq for R-loop positioning.3,4 Current computational methods to calculate genome-wide enrichment of the signal of interest usually do not properly handle the count-based nature of sequencing data, they often do not make use of the local correlation structure of sequencing data, and they do not apply any regularization of enrichment estimates. This can result in unrealistic estimates of the true underlying biological enrichment of interest, unrealistically low estimates of confidence in point estimates of enrichment (or no estimates of confidence at all), unrealistic gyrations in enrichment estimates at very close (<10 bp) genomic loci due to noise inherent in sequencing data, and in a multiple-hypothesis testing problem during interpretation of genome-wide enrichment estimates. We developed a tool called Enricherator to infer genome-wide enrichments from sequencing count data. Enricherator uses the variational Bayes algorithm to fit a generalized linear model to sequencing count data and to sample from the approximate posterior distribution of enrichment estimates (https://github.com/jwschroeder3/enricherator). Enrichments inferred by Enricherator more precisely identify known binding sites in cases where low coverage between binding sites leads to false-positive peak calls in these noisy regions of the genome; these benefits extend to published datasets.

Integrated transcriptome sequencing and weighted gene co-expression network analysis reveals key genes of papillary thyroid carcinomas.

Objective: Papillary thyroid carcinoma (PTC) accounts for the majority of thyroid cancers and has a high recurrence rate. We aimed to screen key genes involved in PTC to provide novel insights into the mechanisms of PTC. Methods: Seven microarray datasets of PTC were downloaded from gene expression omnibus database. Differentially expressed genes (DEGs) between PTC and normal samples were screened in the merged dataset. Then, protein-protein interaction (PPIs) functional modules analysis and weighted gene co-expression network analysis (WGCNA) were utilized to identify PTC-associated key genes. The identified key genes were then characterized from various aspects, including gene set enrichment analysis (GSEA) and the associations with immune infiltration, methylation levels and prognosis. Results: A large numbers of DEGs were identified, and these DEGs are involved in several cancer pathways. Nine key genes (including down-regulated genes GNA14, AVPR1A, and WFS1, and up-regulated genes LAMB3, PLAU, MET, MFGE8, PRSS23, and SERPINA1) were identified. Patients in the AVPR1A and GNA14 high expression groups had better disease-free survival (DFS) than those in the low expression group. Key genes were mainly involved in P53 pathway, estrogen response, apoptosis, glycolysis, NOTCH signaling, epithelial mesenchymal transition, WNT_beta catenin signaling, and inflammatory response. The expression of key genes was associated with immune cell infiltration and corresponding methylation levels. The verification results of key gene proteins and mRNA expression levels using external validation datasets were consistent with our expectations, implying the involvements of key genes in PTC. Conclusion: The key genes may serve as potential therapeutic targets for PTC. This study provides novel insights into the mechanisms underlying PTC development.

Effects of the combination of chronic unpredictable stress and environmental enrichment on anxiety-like behavior assessed using the elevated plus maze in Swiss male mice: Hypothalamus-Pituitary-Adrenal Axis-mediated mechanisms.

Environmental enrichment (EE) is a paradigm that offers the animal a plethora of stimuli, including physical, cognitive, sensory, and social enrichment. Exposure to EE can modulate both anxiety responses and plasma corticosterone. In this study, our objective was to explore how chronic unpredictable stress (CUS) impacts anxiety-related behaviors in male Swiss mice raised in EE conditions. Additionally, we investigated corticosterone and adrenocorticotropic hormone (ACTH) levels to assess the involvement of the hypothalamic-pituitary-adrenal (HPA) axis in mediating these responses. Mice were housed under either EE or standard housing conditions for 21 days. Afterward, they were exposed to 11 days of CUS while still reared in their distinct housing conditions, with half of the mice receiving daily pretreatment with the vehicle and the other half receiving daily metyrapone (MET) injections, an inhibitor of steroid synthesis, 30 mins before CUS exposure. Blood samples were obtained to assess plasma corticosterone and ACTH levels. The 11-day CUS protocol induced anxiety-like phenotype and elevated ACTH levels in EE mice. Chronic MET pretreatment prevented anxiety-like behavior in the EE-CUS groups, by mechanisms involving increased plasma corticosterone levels and decreased ACTH. These results suggest a role of the HPA axis in the mechanism underlying the anxiogenic phenotype induced by CUS in EE mice and shed light on the complex interplay between environmental factors, stress, and the HPA axis in anxiety regulation.

RepEnTools: an automated repeat enrichment analysis package for ChIP-seq data reveals hUHRF1 Tandem-Tudor domain enrichment in young repeats.

BACKGROUND: Repeat elements (REs) play important roles for cell function in health and disease. However, RE enrichment analysis in short-read high-throughput sequencing (HTS) data, such as ChIP-seq, is a challenging task. RESULTS: Here, we present RepEnTools, a software package for genome-wide RE enrichment analysis of ChIP-seq and similar chromatin pulldown experiments. Our analysis package bundles together various software with carefully chosen and validated settings to provide a complete solution for RE analysis, starting from raw input files to tabular and graphical outputs. RepEnTools implementations are easily accessible even with minimal IT skills (Galaxy/UNIX). To demonstrate the performance of RepEnTools, we analysed chromatin pulldown data by the human UHRF1 TTD protein domain and discovered enrichment of TTD binding on young primate and hominid specific polymorphic repeats (SVA, L1PA1/L1HS) overlapping known enhancers and decorated with H3K4me1-K9me2/3 modifications. We corroborated these new bioinformatic findings with experimental data by qPCR assays using newly developed primate and hominid specific qPCR assays which complement similar research tools. Finally, we analysed mouse UHRF1 ChIP-seq data with RepEnTools and showed that the endogenous mUHRF1 protein colocalizes with H3K4me1-H3K9me3 on promoters of REs which were silenced by UHRF1. These new data suggest a functional role for UHRF1 in silencing of REs that is mediated by TTD binding to the H3K4me1-K9me3 double mark and conserved in two mammalian species. CONCLUSIONS: RepEnTools improves the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, enhancing accessibility and adding some key functions. RepEnTools can analyse RE enrichment rapidly, efficiently, and accurately, providing the community with an up-to-date, reliable and accessible tool for this important type of analysis.