Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures.

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

Current Trends and Challenges of Microbiome Research in Prostate Cancer.

PURPOSE OF REVIEW: The role of the gut microbiome in prostate cancer is an emerging area of research interest. However, no single causative organism has yet been identified. The goal of this paper is to examine the role of the microbiome in prostate cancer and summarize the challenges relating to methodology in specimen collection, sequencing technology, and interpretation of results. RECENT FINDINGS: Significant heterogeneity still exists in methodology for stool sampling/storage, preservative options, DNA extraction, and sequencing database selection/in silico processing. Debate persists over primer choice in amplicon sequencing as well as optimal methods for data normalization. Statistical methods for longitudinal microbiome analysis continue to undergo refinement. While standardization of methodology may help yield more consistent results for organism identification in prostate cancer, this is a difficult task due to considerable procedural variation at each step in the process. Further reproducibility and methodology research is required.

Evolutionary Couplings and Molecular Dynamic Simulations Highlight Details of GPCRs Heterodimers' Interfaces.

A growing body of evidence suggests that only a few amino acids ("hot-spots") at the interface contribute most of the binding energy in transient protein-protein interactions. However, experimental protocols to identify these hot-spots are highly labor-intensive and expensive. Computational methods, including evolutionary couplings, have been proposed to predict the hot-spots, but they generally fail to provide details of the interacting amino acids. Here we showed that unbiased evolutionary methods followed by biased molecular dynamic simulations could achieve this goal and reveal critical elements of protein complexes. We applied the methodology to selected G-protein coupled receptors (GPCRs), known for their therapeutic properties. We used the structure-prior-assisted direct coupling analysis (SP-DCA) to predict the binding interfaces of A2aR/D2R, CB1R/D2R, A2aR/CB1R, 5HT2AR/D2R, and 5-HT2AR/mGluR2 receptor heterodimers, which all agreed with published data. In order to highlight details of the interactions, we performed molecular dynamic (MD) simulations using the newly developed AWSEM energy model. We found that these receptors interact primarily through critical residues at the C and N terminal domains and the third intracellular loop (ICL3). The MD simulations showed that these residues are energetically necessary for dimerization and revealed their native conformational state. We subsequently applied the methodology to the 5-HT2AR/5-HTR4R heterodimer, given its implication in drug addiction and neurodegenerative pathologies such as Alzheimer's disease (AD). Further, the SP-DCA analysis showed that 5-HT2AR and 5-HTR4R heterodimerize through the C-terminal domain of 5-HT2AR and ICL3 of 5-HT4R. However, elucidating the details of GPCR interactions would accelerate the discovery of druggable sites and improve our knowledge of the etiology of common diseases, including AD.

Comparison of rectal swab, glove tip, and participant-collected stool techniques for gut microbiome sampling.

BACKGROUND: Studies of the gut microbiome are becoming increasingly important. Such studies require stool collections that can be processed or frozen in a timely manner so as not to alter the microbial content. Due to the logistical difficulties of home-based stool collection, there has been a challenge in selecting the appropriate sample collection technique and comparing results from different microbiome studies. Thus, we compared stool collection and two alternative clinic-based fecal microbiome collection techniques, including a newer glove-based collection method. RESULTS: We prospectively enrolled 22 adult men from our prostate cancer screening cohort SABOR (San Antonio Biomarkers of Risk for prostate cancer) in San Antonio, TX, from 8/2018 to 4/2019. A rectal swab and glove tip sample were collected from each participant during a one-time visit to our clinics. A single stool sample was collected at the participant's home. DNA was isolated from the fecal material and 16 s rRNA sequencing of the V1-V2 and V3-V4 regions was performed. We found the gut microbiome to be similar in richness and evenness, noting no differences in alpha diversity among the collection methods. The stool collection method, which remains the gold-standard method for the gut microbiome, proved to have different community composition compared to swab and glove tip techniques (p< 0.001) as measured by Bray-Curtis and unifrac distances. There were no significant differences in between the swab and glove tip samples with regard to beta diversity (p> 0.05). Despite differences between home-based stool and office-based fecal collection methods, we noted that the distance metrics for the three methods cluster by participant indicating within-person similarities. Additionally, no taxa differed among the methods in a Linear Discriminant Analysis Effect Size (LEfSe) analysis comparing all-against-all sampling methods. CONCLUSION: The glove tip method provides similar gut microbiome results as rectal swab and stool microbiome collection techniques. The addition of a new office-based collection technique could help easy and practical implementation of gut microbiome research studies and clinical practice.

Nanopore-based DNA long-read sequencing analysis of the aged human brain.

Aging disrupts cellular processes such as DNA repair and epigenetic control, leading to a gradual buildup of genomic alterations that can have detrimental effects in post-mitotic cells. Genomic alterations in regions of the genome that are rich in repetitive sequences, often termed "dark loci," are difficult to resolve using traditional sequencing approaches. New long-read technologies offer promising avenues for exploration of previously inaccessible regions of the genome. Using nanopore-based long-read whole-genome sequencing of DNA extracted from aged 18 human brains, we identify previously unreported structural variants and methylation patterns within repetitive DNA, focusing on transposable elements ("jumping genes") as crucial sources of variation, particularly in dark loci. Our analyses reveal potential somatic insertion variants and provides DNA methylation frequencies for many retrotransposon families. We further demonstrate the utility of this technology for the study of these challenging genomic regions in brains affected by Alzheimer's disease and identify significant differences in DNA methylation in pathologically normal brains versus those affected by Alzheimer's disease. Highlighting the power of this approach, we discover specific polymorphic retrotransposons with altered DNA methylation patterns. These retrotransposon loci have the potential to contribute to pathology, warranting further investigation in Alzheimer's disease research. Taken together, our study provides the first long-read DNA sequencing-based analysis of retrotransposon sequences, structural variants, and DNA methylation in the aging brain affected with Alzheimer's disease neuropathology.

Differential Patterns of Gut and Oral Microbiomes in Hispanic Individuals with Cognitive Impairment.

Alzheimer's disease and related dementias (ADRD) have been associated with alterations in both oral and gut microbiomes. While extensive research has focused on the role of gut dysbiosis in ADRD, the contribution of the oral microbiome remains relatively understudied. Furthermore, the potential synergistic interactions between oral and gut microbiomes in ADRD pathology are largely unexplored. This study aims to evaluate distinct patterns and potential synergistic effects of oral and gut microbiomes in a cohort of predominantly Hispanic individuals with cognitive impairment (CI) and without cognitive impairment (NC). We conducted 16S rRNA gene sequencing on stool and saliva samples from 32 participants (17 CI, 15 NC; 62.5% female, mean age = 70.4 ± 6.2 years) recruited in San Antonio, Texas, USA. Correlation analysis through MaAslin2 assessed the relationship between participants' clinical measurements (e.g., fasting glucose and blood cholesterol) and their gut and saliva microbial contents. Differential abundance analysis evaluated taxa with significant differences between CI and NC groups, and alpha and beta diversity metrics assessed within-sample and group compositional differences. Our analyses revealed no significant differences between NC and CI groups in fasting glucose or blood cholesterol levels. However, a clear association was observed between gut microbiome composition and levels of fasting glucose and blood cholesterol. While alpha and beta diversity metrics showed no significant differences between CI and NC groups, differential abundance analysis revealed an increased presence of oral genera such as Dialister , Fretibacterium , and Mycoplasma in CI participants. Conversely, CI individuals exhibited a decreased abundance of gut genera, including Shuttleworthia , Holdemania , and Subdoligranulum , which are known for their anti-inflammatory properties. No evidence was found for synergistic contributions between oral and gut microbiomes in the context of ADRD. Our findings suggest that similar to the gut microbiome, the oral microbiome undergoes significant modifications as individuals transition from NC to CI. Notably, the identified oral microbes have been previously associated with periodontal diseases and gingivitis. These results underscore the necessity for further investigations with larger sample sizes to validate our findings and elucidate the complex interplay between oral and gut microbiomes in ADRD pathogenesis.

Quinolinic acid links kidney injury to brain toxicity.

Kidney dysfunction often leads to neurological impairment, yet the complex kidney-brain relationship remains elusive. We employed spatial and bulk metabolomics to investigate a mouse model of rapid kidney failure induced by mouse double minute 2 ( Mdm2) conditional deletion in the kidney tubules to interrogate kidney and brain metabolism. Pathway enrichment analysis of focused plasma metabolomics panel pinpointed tryptophan metabolism as the most altered pathway with kidney failure. Spatial metabolomics showed toxic tryptophan metabolites in the kidneys and brains, revealing a novel connection between advanced kidney disease and accelerated kynurenine degradation. In particular, the excitotoxic metabolite quinolinic acid was localized in ependymal cells adjacent to the ventricle in the setting of kidney failure. These findings were associated with brain inflammation and cell death. A separate mouse model of acute kidney injury also had an increase in circulating toxic tryptophan metabolites along with altered brain inflammation. Patients with advanced CKD similarly demonstrated elevated plasma kynurenine metabolites and quinolinic acid was uniquely correlated with fatigue and reduced quality of life in humans. Overall, our study identifies the kynurenine pathway as a bridge between kidney decline, systemic inflammation, and brain toxicity, offering potential avenues for diagnosis and treatment of neurological issues in kidney disease.

Dementia Prevalence and Onchocerca volvulus Infection among Rural Elderly Persons in the Ntui Health District, Cameroon: A Population-Based Study.

Recent research suggests that infection with Onchocerca volvulus induces neurocognitive decline. This study sought to compare the cognitive outcomes of elderly persons based on onchocerciasis infection status and report the overall prevalence of dementia in the rural Ntui Health District in Cameroon. A community-based approach was used to recruit 103 participants aged ≥60 years. Dementia screening was done using the Community Screening Interview for Dementia (CSID) tool with a cut-off value of ≤29.5. O. volvulus infection was determined via microscopic examination of skin snips and serological testing of Ov16 antibodies using rapid diagnostic tests. Overall, the prevalence of dementia was 10.7%. Among the tested individuals, 17.9% (15/84) and 62.1% (41/66) were positive for O. volvulus and Ov16 antibodies, respectively. A multivariable linear regression model of CSID scores found a significant positive association with education level (8.654; 95% CI: 2.0870 to 15.222). However, having a positive skin snip for O. volvulus (-3.399; 95% CI: -6.805 to 0.007) and inhaling tobacco (-5.441; 95% CI: -9.137 to -1.744) tended to lower the CSID scores. Ongoing onchocerciasis transmission in the Ntui Health District may constitute a risk factor for dementia. Strengthening onchocerciasis elimination and adopting healthier lifestyles would contribute to dementia prevention among the elderly residing in endemic communities.

The precise timeline of transcriptional regulation reveals causation in mouse somitogenesis network.

BACKGROUND: In vertebrate development, the segmental pattern of the body axis is established as somites, masses of mesoderm distributed along the two sides of the neural tube, are formed sequentially in the anterior-posterior axis. This mechanism depends on waves of gene expression associated with the Notch, Fgf and Wnt pathways. The underlying transcriptional regulation has been studied by whole-transcriptome mRNA profiling; however, interpretation of the results is limited by poor resolution, noisy data, small sample size and by the absence of a wall clock to assign exact time for recorded points. RESULTS: We present a method of Maximum Entropy deconvolution in both space and time and apply it to extract, from microarray timecourse data, the full spatiotemporal expression profiles of genes involved in mouse somitogenesis. For regulated genes, we have reconstructed the temporal profiles and determined the timing of expression peaks along the somite cycle to a single-minute resolution. Our results also indicate the presence of a new class of genes (including Raf1 and Hes7) with two peaks of activity in two distinct phases of the somite cycle. We demonstrate that the timeline of gene expression precisely reflects their functions in the biochemical pathways and the direction of causation in the regulatory networks. CONCLUSIONS: By applying a novel framework for data analysis, we have shown a striking correspondence between gene expression times and their interactions and regulations during somitogenesis. These results prove the key role of finely tuned transcriptional regulation in the process. The presented method can be readily applied to studying somite formation in other datasets and species, and to other spatiotemporal processes.

Coevolution combined with molecular dynamics simulations provides structural and mechanistic insights into the interactions between the integrator complex subunits.

Finding the 3D structure of large, multi-subunit complexes is difficult, despite recent advances in cryo-EM technology, due to remaining challenges to expressing and purifying subunits. Computational approaches that predict protein-protein interactions, including Direct Coupling Analysis (DCA), represent an attractive alternative for dissecting interactions within protein complexes. However, they are readily applicable only to small proteins due to high computational complexity and a high number of false positives. To solve this problem, we proposed a modified DCA approach, a powerful tool to predict the most likely interfaces of protein complexes. Since our modified approach cannot provide structural and mechanistic details of interacting peptides, we combine it with Molecular Dynamics (MD) simulations. To illustrate this novel approach, we predict interacting domains and structural details of interactions of two Integrator complex subunits, INTS9 and INTS11. Our predictions of interacting residues of INTS9/INTS11 are highly consistent with crystallographic structure. We then expand our procedure to two complexes whose structures are not well-studied: 1) The heterodimer formed by the Cleavage and Polyadenylation Specificity Factor 100-kD (CPSF100) and 73-kD (CPSF73); 2) The heterotrimer formed by INTS4/INTS9/INTS11. Experimental data supports our predictions of interactions within these two complexes, demonstrating that combining DCA and MD simulations is a powerful approach to revealing structural insights of large protein complexes.

Cerebral small vessel disease burden is associated with decreased abundance of gut Barnesiella intestinihominis bacterium in the Framingham Heart Study.

A bidirectional communication exists between the brain and the gut, in which the gut microbiota influences cognitive function and vice-versa. Gut dysbiosis has been linked to several diseases, including Alzheimer's disease and related dementias (ADRD). However, the relationship between gut dysbiosis and markers of cerebral small vessel disease (cSVD), a major contributor to ADRD, is unknown. In this cross-sectional study, we examined the connection between the gut microbiome, cognitive, and neuroimaging markers of cSVD in the Framingham Heart Study (FHS). Markers of cSVD included white matter hyperintensities (WMH), peak width of skeletonized mean diffusivity (PSMD), and executive function (EF), estimated as the difference between the trail-making tests B and A. We included 972 FHS participants with MRI scans, neurocognitive measures, and stool samples and quantified the gut microbiota composition using 16S rRNA sequencing. We used multivariable association and differential abundance analyses adjusting for age, sex, BMI, and education level to estimate the association between gut microbiota and WMH, PSMD, and EF measures. Our results suggest an increased abundance of Pseudobutyrivibrio and Ruminococcus genera was associated with lower WMH and PSMD (p values < 0.001), as well as better executive function (p values < 0.01). In addition, in both differential and multivariable analyses, we found that the gram-negative bacterium Barnesiella intestinihominis was strongly associated with markers indicating a higher cSVD burden. Finally, functional analyses using PICRUSt implicated various KEGG pathways, including microbial quorum sensing, AMP/GMP-activated protein kinase, phenylpyruvate, and ß-hydroxybutyrate production previously associated with cognitive performance and dementia. Our study provides important insights into the association between the gut microbiome and cSVD, but further studies are needed to replicate the findings.

Cognitive screening with functional assessment improves diagnostic accuracy and attenuates bias.

INTRODUCTION: Cognitive screening measures often lack sensitivity and are hampered by inequities across ethnoracial groups. A multitrait multimethod (MTMM) classification may attenuate these shortcomings. METHODS: A sample of 7227 participants across the diagnostic spectrum were selected from the National Alzheimer's Coordinating Center cohort. Random forest ensemble methods were used to predict diagnosis across the sample and within Black American (n = 1025) and non-Hispanic White groups (n = 5263) based on: (1) a demographically corrected Montreal Cognitive Assessment (MoCA), (2) MoCA and Functional Assessment Questionnaire (FAQ), (3) MoCA and FAQ with demographic correction. RESULTS: The MTMM approach with demographic correction had the highest diagnostic accuracy for the cognitively unimpaired (area under curve [AUC] [95% confidence interval (CI)]): 0.906 [0.892, 0.920]) and mild cognitive impairment (AUC: 0.835 [0.810, 0.860]) groups and reduced racial disparities. DISCUSSION: With further validation, the MTMM approach combining cognitive screening and functional status assessment may serve to improve diagnostic accuracy and extend opportunities for early intervention with greater equity.

Regulation of local GTP availability controls RAC1 activity and cell invasion.

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.

Coevolution of Residues Provides Evidence of a Functional Heterodimer of 5-HT2AR and 5-HT2CR Involving Both Intracellular and Extracellular Domains.

Serotonin is a neurotransmitter that plays a role in regulating activities such as sleep, appetite, mood and substance abuse disorders; serotonin receptors 5-HT2AR and 5-HT2CR are active within pathways associated with substance abuse. It has been suggested that 5-HT2AR and 5-HT2CR may form a dimer that affects behavioral processes. Here we study the coevolution of residues in 5-HT2AR and 5-HT2CR to identify potential interactions between residues in both proteins. Coevolution studies can detect protein interactions, and since the thus uncovered interactions are subject to evolutionary pressure, they are likely functional. We assessed the significance of the 5-HT2AR/5-HT2CR interactions using randomized phylogenetic trees and found the coevolution significant (p-value = 0.01). We also discuss how co-expression of the receptors suggests the predicted interaction is functional. Finally, we analyze how several single nucleotide polymorphisms for the 5-HT2AR and 5-HT2CR genes affect their interaction. Our findings are the first to characterize the binding interface of 5-HT2AR/5-HT2CR and indicate a correlation between this interface and location of SNPs in both proteins.

qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing.

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

Comparison between Timelines of Transcriptional Regulation in Mammals, Birds, and Teleost Fish Somitogenesis.

Metameric segmentation of the vertebrate body is established during somitogenesis, when a cyclic spatial pattern of gene expression is created within the mesoderm of the developing embryo. The process involves transcriptional regulation of genes associated with the Wnt, Notch, and Fgf signaling pathways, each gene is expressed at a specific time during the somite cycle. Comparative genomics, including analysis of expression timelines may reveal the underlying regulatory modules and their causal relations, explaining the nature and origin of the segmentation mechanism. Using a deconvolution approach, we computationally reconstruct and compare the precise timelines of expression during somitogenesis in chicken and zebrafish. The result constitutes a resource that may be used for inferring possible causal relations between genes and subsequent pathways. While the sets of regulated genes and expression profiles vary between different species, notable similarities exist between the temporal organization of the pathways involved in the somite clock in chick and mouse, with certain aspects (as the phase of expression of Notch genes) conserved also in the zebrafish. The regulated genes have sequence motifs that are conserved in mouse and chicken but not zebrafish. Promoter sequence analysis suggests involvement of several transcription factors that may bind these regulatory elements, including E2F, EGR and PLAG, as well as a possible role of G-quadruplex DNA structure in regulation of the cyclic genes. Our research lays the groundwork for further studies that will probe the evolution of the regulatory mechanism of segmentation across all vertebrates.

A Conserved Structural Signature of the Homeobox Coding DNA in HOX genes.

The homeobox encodes a DNA-binding domain found in transcription factors regulating key developmental processes. The most notable examples of homeobox containing genes are the Hox genes, arranged on chromosomes in the same order as their expression domains along the body axis. The mechanisms responsible for the synchronous regulation of Hox genes and the molecular function of their colinearity remain unknown. Here we report the discovery of a conserved structural signature of the 180-base pair DNA fragment comprising the homeobox. We demonstrate that the homeobox DNA has a characteristic 3-base-pair periodicity in the hydroxyl radical cleavage pattern. This periodic pattern is significant in most of the 39 mammalian Hox genes and in other homeobox-containing transcription factors. The signature is present in segmented bilaterian animals as evolutionarily distant as humans and flies. It remains conserved despite the fact that it would be disrupted by synonymous mutations, which raises the possibility of evolutionary selective pressure acting on the structure of the coding DNA. The homeobox coding DNA may therefore have a secondary function, possibly as a regulatory element. The existence of such element may have important consequences for understanding how these genes are regulated.