Replication Patterns Research Articles

Spotlight on the Gut Microbiome in Menopause: Current Insights.

The gut microbiome is an important contributor to human health, shaped by many endogenous and exogenous factors. The gut microbiome displays sexual dimorphism, suggesting influence of sex hormones, and also has been shown to change with aging. Yet, little is known regarding the influence of menopause – a pivotal event of reproductive aging in women – on the gut microbiome. Here, we summarize what is known regarding the interrelationships of female sex hormones and the gut microbiome, and review the available literature on menopause, female sex hormones, and the gut microbiome in humans. Taken together, research suggests that menopause is associated with lower gut microbiome diversity and a shift toward greater similarity to the male gut microbiome, however more research is needed in large study populations to identify replicable patterns in taxa impacted by menopause. Many gaps in knowledge remain, including the role the gut microbiome may play in menopause-related disease risks, and whether menopausal hormone therapy modifies menopause-related change in the gut microbiome. Given the modifiable nature of the gut microbiome, better understanding of its role in menopause-related health will be critical to identify novel opportunities for improvement of peri- and post-menopausal health and well-being.

Serotype I and II Feline Coronavirus Replication and Gene Expression Patterns of Feline Cells-Building a Better Understanding of Serotype I FIPV Biology.

Feline infectious peritonitis (FIP) is a disease of domestic cats caused by the genetic variant of the feline coronavirus (FCoV) and feline infectious peritonitis virus (FIPV), currently grouped into two serotypes, I and II. Although serotype I FIPV is more prevalent in cats with FIP, serotype II has been more extensively studied in vitro due to the relative ease in propagating this viral serotype in culture systems. As a result, more is known about serotype II FIPV than the more biologically prevalent serotype I. The primary cell receptor for serotype II has been determined, while it remains unknown for serotype I. The recent development of a culture-adapted feline cell line that more effectively propagates serotype I FIPV, FCWF-4 CU, derived from FCWF-4 cells available through the ATCC, offers the potential for an improved understanding of serotype I FIPV biology. To learn more about FIPV receptor biology, we determined targeted gene expression patterns in feline cells variably permissive to replication of serotype I or II FIPV. We utilized normal feline tissues to determine the immunohistochemical expression patterns of two known coronavirus receptors, ACE2 and DC-SIGN. Lastly, we compared the global transcriptomes of the two closely related FCWF-4 cell lines and identified viral transcripts with potential importance for the differential replication kinetics of serotype I FIPV.

Perception of major life events and personality trait change

Major life events can trigger personality trait change. However, a clear, replicable pattern of event-related personality trait change has yet to be identified. We examined whether the perception of major life events is associated with personality trait change. Therefore, we assessed young adults’ personality traits at five measurement occasions within 1 year. At the second measurement occasion, we also assessed their perception of a recently experienced major life event using the Event Characteristics Questionnaire. Contrary to our expectations, perceived impact of the event was not associated with the amount of personality trait change, but perceived valence was associated with changes in agreeableness and neuroticism. Exploratory analyses revealed some weak associations between other perceived event characteristics and the amount of personality trait change as well as interactions between perceived event characteristics and event categories in predicting changes in neuroticism. In general, effect sizes were small, and associations depended on the time interval between pre-event and post-event personality assessment. Results indicate that perceived event characteristics should be considered when examining event-related personality trait change.

Abstract 5123: Establishing circulating tumor cell-derived xenografts following a period of in vitro culture

Abstract Cancer metastasis—the spread of cancer cells from a primary node to a distant site—is responsible for as much as 90% of all cancer-associated deaths due to treatment resistance and increased tumor burden. Once established, metastatic nodules oftentimes exhibit distinct molecular phenotypes from their original primary tumor, undermining gold-standard methods for determining treatments for patients, which typically rely on molecular characterization of the primary tumor. Unfortunately, a lack of robust, scalable, and appropriate models for studying differences in patient-derived primary and metastatic tumors has hindered progress in identifying metastasis-specific therapies. Previously, we reported the ability to establish in vitro cultures of circulating tumor cells (CTCs), a unique precursor population of cells that exhibits an ability to escape the primary tumor but not yet fully complete metastasis. We have since expanded the application of this GU-CTC platform to culture CTCs from the blood of patients diagnosed with a variety of metastatic cancers, resulting in the successful culture of 31 unique patient-derived CTC cell lines. Furthermore, following an initial short-term culture period, we also performed subcutaneous injection of mixed CTC cultures into immunocompromised mice, resulting in not only primary tumor formation, but also systemic metastatic nodule formation in as short as 3 months. These resulting CTC-derived xenografts (CDXs) have been demonstrated using metastatic pancreatic, colorectal, and lung adenocarcinoma-derived CTCs. Encouragingly, all CDX models also exhibited replicable metastatic patterns upon reimplantation, reinforcing the important role of CTCs as precursors to metastasis. Bulk RNA-sequencing of patient-matched whole blood, cultured CTCs, and CDX-derived primary tumor and metastatic tissues further identified enrichment of an epithelial-mesenchymal transition as well as several cancer-specific gene signatures consistent with an invasive phenotype. This extended GU-CTC platform therefore enables not only the robust and rapid expansion of CTCs through both in vitro and in vivo methods, but also offers an avenue for personalized drug screens in the effort to target cancer metastasis. Citation Format: Jerry Xiao, Richard Schlegel, Seema Agarwal. Establishing circulating tumor cell-derived xenografts following a period of in vitro culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5123.

A simple pipeline for cell cycle kinetic studies in the root apical meristem.

Root system architecture ultimately depends on precise signaling between different cells and tissues in the root apical meristem (RAM) and integration with environmental cues. This study describes a simple pipeline to simultaneously determine cellular parameters, nucleus geometry, and cell cycle kinetics in the RAM. The method uses marker-free techniques for nucleus and cell boundary detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining for DNA replication quantification. Based on this approach, we characterized differences in cell volume, nucleus volume, and nucleus shape across different domains of the Arabidopsis RAM. We found that DNA replication patterns were cell layer and region dependent. G2 phase duration, which varied from 3.5 h in the pericycle to more than 4.5 h in the epidermis, was found to be associated with some features of nucleus geometry. Endocycle duration was determined as the time required to achieve 100% EdU-positive cells in the elongation zone and, as such, it was estimated to be in the region of 5 h for the epidermis and cortex. This experimental pipeline could be used to precisely map cell cycle duration in the RAM of mutants and in response to environmental stress in several plant species without the need for introgressing molecular cell cycle markers.

Replicable patterns of causal information flow between hippocampus and prefrontal cortex during spatial navigation and spatial-verbal memory formation.

Interactions between the hippocampus and prefrontal cortex (PFC) play an essential role in both human spatial navigation and episodic memory, but the underlying causal flow of information between these regions across task domains is poorly understood. Here we use intracranial EEG recordings and spectrally resolved phase transfer entropy to investigate information flow during two different virtual spatial navigation and memory encoding/recall tasks and examine replicability of information flow patterns across spatial and verbal memory domains. Information theoretic analysis revealed a higher causal information flow from hippocampus to lateral PFC than in the reverse direction. Crucially, an asymmetric pattern of information flow was observed during memory encoding and recall periods of both spatial navigation tasks. Further analyses revealed frequency specificity of interactions characterized by greater bottom-up information flow from hippocampus to PFC in delta-theta band (0.5-8Hz); in contrast, top-down information flow from PFC to hippocampus was stronger in beta band (12-30Hz). Bayesian analysis revealed a high degree of replicability between the two spatial navigation tasks (Bayes factor > 5.46e+3) and across tasks spanning the spatial and verbal memory domains (Bayes factor > 7.32e+8). Our findings identify a domain-independent and replicable frequency-dependent feedback loop engaged during memory formation in the human brain.

Genomic and neuroimaging approaches to bipolar disorder.

To date, besides genome-wide association studies, a variety of other genetic analyses (e.g. polygenic risk scores, whole-exome sequencing and whole-genome sequencing) have been conducted, and a large amount of data has been gathered for investigating the involvement of common, rare and very rare types of DNA sequence variants in bipolar disorder. Also, non-invasive neuroimaging methods can be used to quantify changes in brain structure and function in patients with bipolar disorder. To provide a comprehensive assessment of genetic findings associated with bipolar disorder, based on the evaluation of different genomic approaches and neuroimaging studies. We conducted a PubMed search of all relevant literatures from the beginning to the present, by querying related search strings. ANK3, CACNA1C, SYNE1, ODZ4 and TRANK1 are five genes that have been replicated as key gene candidates in bipolar disorder pathophysiology, through the investigated studies. The percentage of phenotypic variance explained by the identified variants is small (approximately 4.7%). Bipolar disorder polygenic risk scores are associated with other psychiatric phenotypes. The ENIGMA-BD studies show a replicable pattern of lower cortical thickness, altered white matter integrity and smaller subcortical volumes in bipolar disorder. The low amount of explained phenotypic variance highlights the need for further large-scale investigations, especially among non-European populations, to achieve a more complete understanding of the genetic architecture of bipolar disorder and the missing heritability. Combining neuroimaging data with genetic data in large-scale studies might help researchers acquire a better knowledge of the engaged brain regions in bipolar disorder.

A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing DsRed Protein Based on Bacterial Artificial Chromosome System.

Recombinant viruses possessing reporter proteins as tools are widely applied in investigating viral biology because of the convenience for observation. Previously, we generated a recombinant pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with enhanced green fluorescent protein (EGFP) reporter for monitoring virus spread and screening of neutralizing antibodies. PRRSV with different kinds of reporters can support more application scenarios. Here, we described a new genetically stable infectious clones of a highly pathogenic PRRSV (HP-PRRSV) harboring the DsRed (a red fluorescent protein isolated from the coral Discosoma) gene. In the recombinant infectious clone, the transcription regulatory sequence 2 (TRS2) of PRRSV was inserted between the open reading frame 7 (ORF7) and 3′UTR to drive the transcription of DsRed gene, which makes it a separate transcription unit in the viral genome. Using the bacterial artificial chromosome (BAC) system and cytomegalovirus (CMV) promoter, the recombinant HP-PRRSV with the DsRed insertion was successfully rescued and showed similar growth and replication patterns compared with the wild-type virus in the MARC-145 cells. In addition, the DsRed protein was stably expressed in the recombinant virus for at least 10 passages with consistent fluorescence intensity and density. Using the recombinant HP-PRRSV with DsRed protein, the virus tracking in MARC-145 was observed by live-cell imaging. Meanwhile, quantification of the DsRed fluorescence positive cells by flow cytometry provides an alternative to standard methods for testing the level of PRRSV infection. This recombinant PRRSV with DsRed fluorescence protein expression could be a useful tool for fundamental research on the viral biology and shows the new design for stable expression of foreign genes in PRRSV.

Organizational Replication and Spending Patterns among FBS Football and Division I Basketball Programs

Organizational Replication and Spending Patterns among FBS Football and Division I Basketball Programs

Emerging SARS-CoV-2 Genotypes Show Different Replication Patterns in Human Pulmonary and Intestinal Epithelial Cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) quickly spread worldwide following its emergence in Wuhan, China, and hit pandemic levels. Its tremendous incidence favoured the emergence of viral variants. The current genome diversity of SARS-CoV-2 has a clear impact on epidemiology and clinical practice, especially regarding transmission rates and the effectiveness of vaccines. In this study, we evaluated the replication of different SARS-CoV-2 isolates representing different virus genotypes which have been isolated throughout the pandemic. We used three distinct cell lines, including Vero E6 cells originating from monkeys; Caco-2 cells, an intestinal epithelium cell line originating from humans; and Calu-3 cells, a pulmonary epithelium cell line also originating from humans. We used RT-qPCR to replicate different SARS-CoV-2 genotypes by quantifying the virus released in the culture supernatant of infected cells. We found that the different viral isolates replicate similarly in Caco-2 cells, but show very different replicative capacities in Calu-3 cells. This was especially highlighted for the lineages B.1.1.7, B.1.351 and P.1, which are considered to be variants of concern. These results underscore the importance of the evaluation and characterisation of each SARS-CoV-2 isolate in order to establish the replication patterns before performing tests, and of the consideration of the ideal SARS-CoV-2 genotype–cell type pair for each assay.

Bayesian multistudy factor analysis for high-throughput biological data

This paper analyzes breast cancer gene expression across seven studies to identify genuine and thus replicable gene patterns shared among these studies. Our premise is that genuine biological signal is more likely to be reproducibly present in multiple studies than spurious signal. Our analysis uses a new modeling strategy for the joint analysis of high-throughput biological studies which simultaneously identifies shared as well as study-specific signal. To this end, we generalize the multi-study factor analysis model to handle high-dimensional data and generalize the sparse Bayesian infinite factor model to this context. We provide strategies for the identification of the loading matrices, common and study-specific. Through extensive simulation analysis, we characterize the performance of the proposed approach in various scenarios and show that it outperforms standard factor analysis in identifying replicable signal in all scenarios considered. The analysis of breast cancer gene expression studies identifies clear replicable gene patterns. These patterns are related to well-known biological pathways involved in breast cancer, such as the ER, cell cycle, immune system, collagen, and metabolic pathways. Some of these patterns are also associated with existing breast cancer subtypes, such as LumA, Her2, and basal subtypes, while other patterns identify novel pathways active across subtypes and missed by hierarchical clustering approaches. The R package MSFA implementing the method is available on GitHub.

SARS-CoV-2 Evolution among Oncological Population: In-Depth Virological Analysis of a Clinical Cohort.

Background: Oncological patients have a higher risk of prolonged SARS-CoV-2 shedding, which, in turn, can lead to evolutionary mutations and emergence of novel viral variants. The aim of this study was to analyze biological samples of a cohort of oncological patients by deep sequencing to detect any significant viral mutations. Methods: High-throughput sequencing was performed on selected samples from a SARS-CoV-2-positive oncological patient cohort. Analysis of variants and minority variants was performed using a validated bioinformatics pipeline. Results: Among 54 oncological patients, we analyzed 12 samples of 6 patients, either serial nasopharyngeal swab samples or samples from the upper and lower respiratory tracts, by high-throughput sequencing. We identified amino acid changes D614G and P4715L as well as mutations at nucleotide positions 241 and 3037 in all samples. There were no other significant mutations, but we observed intra-host evolution in some minority variants, mainly in the ORF1ab gene. There was no significant mutation identified in the spike region and no minority variants common to several hosts. Conclusions: There was no major and rapid evolution of viral strains in this oncological patient cohort, but there was minority variant evolution, reflecting a dynamic pattern of quasi-species replication.

Genome-wide investigation and expression profiling of polyphenol oxidase (PPO) family genes uncover likely functions in organ development and stress responses in Populus trichocarpa

BackgroundTrees such as Populus are planted extensively for reforestation and afforestation. However, their successful establishment greatly depends upon ambient environmental conditions and their relative resistance to abiotic and biotic stresses. Polyphenol oxidase (PPO) is a ubiquitous metalloproteinase in plants, which plays crucial roles in mediating plant resistance against biotic and abiotic stresses. Although the whole genome sequence of Populus trichocarpa has long been published, little is known about the PPO genes in Populus, especially those related to drought stress, mechanical damage, and insect feeding. Additionally, there is a paucity of information regarding hormonal responses at the whole genome level.ResultsA genome-wide analysis of the poplar PPO family was performed in the present study, and 18 PtrPPO genes were identified. Bioinformatics and qRT-PCR were then used to analyze the gene structure, phylogeny, chromosomal localization, gene replication, cis-elements, and expression patterns of PtrPPOs. Sequence analysis revealed that two-thirds of the PtrPPO genes lacked intronic sequences. Phylogenetic analysis showed that all PPO genes were categorized into 11 groups, and woody plants harbored many PPO genes. Eighteen PtrPPO genes were disproportionally localized on 19 chromosomes, and 3 pairs of segmented replication genes and 4 tandem repeat genomes were detected in poplars. Cis-acting element analysis identified numerous growth and developmental elements, secondary metabolism processes, and stress-related elements in the promoters of different PPO members. Furthermore, PtrPPO genes were expressed preferentially in the tissues and fruits of young plants. In addition, the expression of some PtrPPOs could be significantly induced by polyethylene glycol, abscisic acid, and methyl jasmonate, thereby revealing their potential role in regulating the stress response. Currently, we identified potential upstream TFs of PtrPPOs using bioinformatics.ConclusionsComprehensive analysis is helpful for selecting candidate PPO genes for follow-up studies on biological function, and progress in understanding the molecular genetic basis of stress resistance in forest trees might lead to the development of genetic resources.

Lesion covariance networks reveal proposed origins and pathways of diffuse gliomas.

Diffuse gliomas have been hypothesized to originate from neural stem cells in the subventricular zone and develop along previously healthy brain networks. Here, we evaluated these hypotheses by mapping independent sources of glioma localization and determining their relationships with neurogenic niches, genetic markers and large-scale connectivity networks. By applying independent component analysis to lesion data from 242 adult patients with high- and low-grade glioma, we identified three lesion covariance networks, which reflect clusters of frequent glioma localization. Replicability of the lesion covariance networks was assessed in an independent sample of 168 glioma patients. We related the lesion covariance networks to important clinical variables, including tumour grade and patient survival, as well as genomic information such as molecular genetic subtype and bulk transcriptomic profiles. Finally, we systematically cross-correlated the lesion covariance networks with structural and functional connectivity networks derived from neuroimaging data of over 4000 healthy UK BioBank participants to uncover intrinsic brain networks that may that underlie tumour development. The three lesion covariance networks overlapped with the anterior, posterior and inferior horns of the lateral ventricles respectively, extending into the frontal, parietal and temporal cortices. These locations were independently replicated. The first lesion covariance network, which overlapped with the anterior horn, was associated with low-grade, isocitrate dehydrogenase -mutated/1p19q-codeleted tumours, as well as a neural transcriptomic signature and improved overall survival. Each lesion covariance network significantly coincided with multiple structural and functional connectivity networks, with the first bearing an especially strong relationship with brain connectivity, consistent with its neural transcriptomic profile. Finally, we identified subcortical, periventricular structures with functional connectivity patterns to the cortex that significantly matched each lesion covariance network. In conclusion, we demonstrated replicable patterns of glioma localization with clinical relevance and spatial correspondence with large-scale functional and structural connectivity networks. These results are consistent with prior reports of glioma growth along white matter pathways, as well as evidence for the coordination of glioma stem cell proliferation by neuronal activity. Our findings describe how the locations of gliomas relate to their proposed subventricular origins, suggesting a model wherein periventricular brain connectivity guides tumour development.

Collective regulation of chromatin modifications predicts replication timing during cell cycle.

SUMMARYReplication timing (RT) associates with genome architecture, while having a mixed relationship to histone marks. By profiling replication at high resolution and assessing broad histone marks across the cell cycle at the resolution of RT with and without genetic perturbation, we address the causal relationship between histone marks and RT. Four primary chromatin states, including an uncharacterized H3K36me2 state, emerge and define 97% of the mappable genome. RT and local replication patterns (e.g., initiation zones) quantitatively associate with chromatin states, histone mark dynamics, and spatial chromatin structure. Manipulation of broad histone marks and enhancer elements by overexpressing the histone H3 lysine 9/36 tri-demethylase KDM4A impacts RT across 11% of the genome. Broad histone modification changes were strong predictors of the observed RT alterations. Lastly, replication within H3K36me2-enriched neighborhoods is sensitive to KDM4A overexpression and is controlled at a megabase scale. These studies establish a role for collective chromatin mark regulation in modulating RT.

Fingerprint of gait applied to Parkinson's disease

Many Neurological diseases provoke motor impairment. In order to obtain early diagnosis and effective assessment of therapeutic outcomes, it is desirable to quantify gait features and alterations. In this study, we showed that human gait is characterized by replicable patterns of consensual accelerations/decelerations of body segments, that are highly regulated. Hence, we analysed motion analysis data through a graph theoretical approach and applied this framework in Parkinson's disease (PD), showing the utility of our newly proposed technique in clinical settings.

Chromosomal Mcm2-7 distribution and the genome replication program in species from yeast to humans.

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.

Organization of DNA Replication Origin Firing in Xenopus Egg Extracts: The Role of Intra-S Checkpoint.

During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: (1) a segmentation of the genome into regions of low and high probability of origin firing; (2) that regions with high probability of origin firing escape intra-S checkpoint regulation and (3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering.

Meiotic recombination mirrors patterns of germline replication in mice and humans

Genetic recombination generates novel trait combinations, and understanding how recombination is distributed across the genome is key to modern genetics. The PRDM9 protein defines recombination hotspots; however, megabase-scale recombination patterning is independent of PRDM9. The single round of DNA replication, which precedes recombination in meiosis, may establish these patterns; therefore, we devised an approach to study meiotic replication that includes robust and sensitive mapping of replication origins. We find that meiotic DNA replication is distinct; reduced origin firing slows replication in meiosis, and a distinctive replication pattern in human males underlies the subtelomeric increase in recombination. We detected a robust correlation between replication and both contemporary and historical recombination and found that replication origin density coupled with chromosome size determines the recombination potential of individual chromosomes. Our findings and methods have implications for understanding the mechanisms underlying DNA replication, genetic recombination, and the landscape of mammalian germline variation.

Evidence of Transcriptional Shutoff by Pathogenic Viral Haemorrhagic Septicaemia Virus in Rainbow Trout.

The basis of pathogenicity of viral haemorrhagic septicaemia virus (VHSV) was analysed in the transcriptome of a rainbow trout cell line inoculated with pathogenic and non-pathogenic VHSV isolates. Although both VHSV isolates showed similar viral replication patterns, the number of differentially expressed genes was 42-fold higher in cells inoculated with the non-pathogenic VHSV at 3 h post inoculation (hpi). Infection with the non-pathogenic isolate resulted in Gene Ontologies (GO) enrichment of terms such as immune response, cytokine-mediated signalling pathway, regulation of translational initiation, unfolded protein binding, and protein folding, and induced an over-representation of the p53, PPAR, and TGF-β signalling pathways. Inoculation with the pathogenic isolate resulted in the GO enrichment of terms related to lipid metabolism and the salmonella infection KEGG pathway involved in the rearrangement of the cytoskeleton. Antiviral response was evident at 12hpi in cells infected with the pathogenic isolate. Overall, the data showed a delay in the response of genes involved in immune responses and viral sensing in cells inoculated with the pathogenic isolate and suggest transcriptional shutoff and immune avoidance as a critical mechanism of pathogenicity in VHSV. These pathways offer opportunities to further understand and manage VHSV pathogenicity in rainbow trout.