-
Vavilovskii Zhurnal Genetiki I Selektsii Oct 2021Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene...
Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identif ication of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact f iltration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene's f lanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two f lanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the f ield of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method's feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to eff iciently localize genomic transposon integrations, which was conf irmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.
PubMed: 34755021
DOI: 10.18699/VJ21.068 -
Molecular Metabolism Mar 2023Oligodendrocyte progenitor cell differentiation is regulated by nutritional signals in the adult median eminence (ME), but the consequences on local myelination are...
OBJECTIVE
Oligodendrocyte progenitor cell differentiation is regulated by nutritional signals in the adult median eminence (ME), but the consequences on local myelination are unknown. The aim of this study was to characterize myelin plasticity in the ME of adult mice in health or in response to chronic nutritional challenge and determine its relevance to the regulation of energy balance.
METHODS
We assessed new oligodendrocyte (OL) and myelin generation and stability in the ME of healthy adult male mice using bromodeoxyuridine labelling and genetic fate mapping tools. We evaluated the contribution of microglia to ME myelin plasticity in PLX5622-treated C57BL/6J mice and in Pdgfra-Cre/ER;R26R-eYFP;Myrf mice, where adult oligodendrogenesis is blunted. Next, we investigated how high-fat feeding or caloric restriction impact ME OL lineage progression and myelination. Finally, we characterized the functional relevance of adult oligodendrogenesis on energy balance regulation.
RESULTS
We show that myelinating OLs are continuously and rapidly generated in the adult ME. Paradoxically, OL number and myelin amounts remain remarkably stable in the adult ME. In fact, the high rate of new OL and myelin generation in the ME is offset by continuous turnover of both. We show that microglia are required for continuous OL and myelin production, and that ME myelin plasticity regulates the recruitment of local immune cells. Finally, we provide evidence that ME myelination is regulated by the body's energetic status and demonstrate that ME OL and myelin plasticity are required for the regulation of energy balance and hypothalamic leptin sensitivity.
CONCLUSIONS
This study identifies a new mechanism modulating leptin sensitivity and the central control of energy balance and uncovers a previously unappreciated form of structural plasticity in the ME.
Topics: Mice; Male; Animals; Myelin Sheath; Leptin; Mice, Transgenic; Median Eminence; Mice, Inbred C57BL
PubMed: 36739968
DOI: 10.1016/j.molmet.2023.101690 -
Methods in Molecular Biology (Clifton,... 20223D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture...
3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes. Such improvements successfully resolve the fine-scale level of chromatin folding, including enhancer-promoter or promoter-promoter interactions, genic and nucleosomal folding, and boost the signal-to-noise ratio in detecting loops and substructures underlying TADs. In this chapter, we will thoroughly discuss the details of the Micro-C protocol and critical parameters to consider for generating high-quality Micro-C maps.
Topics: Animals; Chromatin; Chromosome Mapping; Genome; Mammals; Micrococcal Nuclease; Nucleosomes
PubMed: 35867245
DOI: 10.1007/978-1-0716-2497-5_4 -
Globalization and Health Sep 2021Substandard and falsified (SF) medical products are a global public health threat. The presence and spread of SF drugs negatively affect (1) patients' safety and health...
OBJECTIVE
Substandard and falsified (SF) medical products are a global public health threat. The presence and spread of SF drugs negatively affect (1) patients' safety and health outcomes, (2) national economy, (3) public trust in the healthcare system, and (4) the international fight against serious health challenges such as malaria and antimicrobial resistance. The objective of the current study was to investigate and provide a snapshot analysis of the evolution and developmental patterns of global research publications on SF products.
METHODS
A bibliometric approach was adopted using terms such as fake, falsified, counterfeit, substandard, and others. No language restriction was made. The study period was from 1900 up to 2020. The search strategy was validated and implemented using Scopus database.
RESULTS
The search strategy retrieved 978 documents authored by 2861 researchers from 100 different countries and published in 421 different journals. The retrieved documents received 11,237 citations (11.5 citations per document) with an H-index of 53. The 978 documents retrieved from Scopus were published from 1961 to 2020, giving an average of 16.6 publications per year. The present study indicated that research on SF medical products: (a) has experienced a steep growth from 2001 to 2012 followed by a steady-state growth; (b) was disseminated in a wide range of journals, mainly in the fields of the pharmaceutical industry, analytical chemistry, public health, infectious diseases, and internal medicine; (c) was published by scholars with diverse and distant geographical backgrounds; (d) was mainly produced in the United States, United Kingdom, and Germany; (d) has fragmented research networks and a limited number of researchers per network; (e) has limited cross-country collaboration except for that between the US and the UK in one hand and countries in the Mekong region in the other hand; (f) emphasized on medications related to malaria and sexual stimulants; and (g) received relatively inadequate funding.
CONCLUSIONS
Research on SF medical products is important and should remain a priority to ensure good quality of medications. Research activity in the field needs to be encouraged in world regions such as Africa and the Middle East where drug regulations are unsatisfactory and cross-border trade of illegal medications is common.
Topics: Bibliometrics; Delivery of Health Care; Drug and Narcotic Control; Humans; Public Health; Publications
PubMed: 34556126
DOI: 10.1186/s12992-021-00766-5 -
Genes & Development Oct 2021Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA...
Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.
Topics: DNA Breaks, Double-Stranded; DNA Repair; DNA Replication; DNA, Single-Stranded; Homologous Recombination; Tumor Suppressor p53-Binding Protein 1
PubMed: 34503990
DOI: 10.1101/gad.348667.121 -
Nature Communications Jan 2024Although chromatin organizations in plants have been dissected at the scales of compartments and topologically associating domain (TAD)-like domains, there remains a gap...
Although chromatin organizations in plants have been dissected at the scales of compartments and topologically associating domain (TAD)-like domains, there remains a gap in resolving fine-scale structures. Here, we use Micro-C-XL, a high-throughput chromosome conformation capture (Hi-C)-based technology that involves micrococcal nuclease (instead of restriction enzymes) and long cross-linkers, to dissect single nucleosome-resolution chromatin organization in Arabidopsis. Insulation analysis reveals more than 14,000 boundaries, which mostly include chromatin accessibility, epigenetic modifications, and transcription factors. Micro-C-XL reveals associations between RNA Pols and local chromatin organizations, suggesting that gene transcription substantially contributes to the establishment of local chromatin domains. By perturbing Pol II both genetically and chemically at the gene level, we confirm its function in regulating chromatin organization. Visible loops and stripes are assigned to super-enhancers and their targeted genes, thus providing direct insights for the identification and mechanistic analysis of distal CREs and their working modes in plants. We further investigate possible factors regulating these chromatin loops. Subsequently, we expand Micro-C-XL to soybean and rice. In summary, we use Micro-C-XL for analyses of plants, which reveal fine-scale chromatin organization and enhancer-promoter loops and provide insights regarding three-dimensional genomes in plants.
Topics: Chromatin; Nucleosomes; Promoter Regions, Genetic; Transcription Factors; Genome
PubMed: 38167349
DOI: 10.1038/s41467-023-44347-z -
Analytical Chemistry Jul 2022As the key player of a new restriction modification system, DNA phosphorothioate (PT) modification, which swaps oxygen for sulfur on the DNA backbone, protects the...
As the key player of a new restriction modification system, DNA phosphorothioate (PT) modification, which swaps oxygen for sulfur on the DNA backbone, protects the bacterial host from foreign DNA invasion. The identification of PT sites helps us understand its physiological defense mechanisms, but accurately quantifying this dynamic modification remains a challenge. Herein, we report a simple quantitative analysis method for optical mapping of PT sites in the single bacterial genome. DNA molecules are fully stretched and immobilized in a microfluidic chip by capillary flow and electrostatic interactions, improving the labeling efficiency by maximizing exposure of PT sites on DNA while avoiding DNA loss and damage. After screening 116 candidates, we identified a bifunctional chemical compound, iodoacetyl-polyethylene glycol-biotin, that can noninvasively and selectively biotinylate PT sites, enabling further labeling with streptavidin fluorescent nanoprobes. With this method, PT sites in PT DNA can be easily detected by fluorescence, while almost no detectable ones were found in PT DNA, achieving real-time visualization of PT sites on a single DNA molecule. Collectively, this facile genome-wide PT site detection method directly characterizes the distribution and frequency of DNA modification, facilitating a better understanding of its modification mechanism that can be potentially extended to label DNAs in different species.
Topics: DNA; DNA, Bacterial; Genome, Bacterial; Microfluidics; Sulfur
PubMed: 35834188
DOI: 10.1021/acs.analchem.2c01752 -
Seminars in Immunology Aug 2020Epitopes, in the context of T cell recognition, are short peptides typically derived by antigen processing, and presented on the cell surface bound to MHC molecules (HLA... (Review)
Review
Epitopes, in the context of T cell recognition, are short peptides typically derived by antigen processing, and presented on the cell surface bound to MHC molecules (HLA molecules in humans) for TCR scrutiny. The identification of epitopes is a context-dependent process, with consideration given to, for example, the source pathogen and protein, the host organism, and state of the immune reaction (e.g., following natural infection, vaccination, etc.). In the following review, we consider the various approaches used to define T cell epitopes, including both bioinformatic and experimental approaches, and discuss the concepts of immunodominance and immunoprevalence. We also discuss HLA polymorphism and epitope restriction, and the resulting impact on the identification of, and potential population coverage afforded by, epitopes or epitope-based vaccines. Finally, some examples of the practical application of T cell epitope identification are provided, showing how epitopes have been valuable for deriving novel immunological insights in the context of the immune response to various pathogens and allergens.
Topics: Animals; Computational Biology; Epitope Mapping; Epitopes, T-Lymphocyte; HLA Antigens; Humans; Immunoassay; Immunodominant Epitopes; Polymorphism, Genetic; Protein Binding; T-Lymphocytes; Vaccines
PubMed: 33131981
DOI: 10.1016/j.smim.2020.101418 -
Frontiers in Nutrition 2021Healthcare systems worldwide are seriously challenged by a rising prevalence of neurodegenerative diseases (NDDs), which mostly, but not exclusively, affect the... (Review)
Review
Healthcare systems worldwide are seriously challenged by a rising prevalence of neurodegenerative diseases (NDDs), which mostly, but not exclusively, affect the ever-growing population of the elderly. The most known neurodegenerative diseases are Alzheimer's (AD) and Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis, but some viral infections of the brain and traumatic brain injury may also cause NDD. Typical for NDD are the malfunctioning of neurons and their irreversible loss, which often progress irreversibly to dementia and ultimately to death. Numerous factors are involved in the pathogenesis of NDD: genetic variability, epigenetic changes, extent of oxidative/nitrosative stress, mitochondrial dysfunction, and DNA damage. The complex interplay of all the above-mentioned factors may be a fingerprint of neurodegeneration, with different diseases being affected to different extents by particular factors. There is a voluminous body of evidence showing the benefits of regular exercise to brain health and cognitive functions. Moreover, the importance of a healthy diet, balanced in macro- and micro-nutrients, in preventing neurodegeneration and slowing down a progression to full-blown disease is evident. Individuals affected by NDD almost inevitably have low-grade inflammation and anomalies in lipid metabolism. Metabolic and lipid profiles in NDD can be improved by the Mediterranean diet. Many studies have associated the Mediterranean diet with a decreased risk of dementia and AD, but a cause-and-effect relationship has not been deduced. Studies with caloric restriction showed neuroprotective effects in animal models, but the results in humans are inconsistent. The pathologies of NDD are complex and there is a great inter-individual (epi)genetic variance within any population. Furthermore, the gut microbiome, being deeply involved in nutrient uptake and lipid metabolism, also represents a pillar of the gut microbiome-brain axis and is linked with the pathogenesis of NDD. Numerous studies on the role of different micronutrients (omega-3 fatty acids, bioactive polyphenols from fruit and medicinal plants) in the prevention, prediction, and treatment of NDD have been conducted, but we are still far away from a personalized diet plan for individual NDD patients. For this to be realized, large-scale cohorts that would include the precise monitoring of food intake, mapping of genetic variants, epigenetic data, microbiome studies, and metabolome, lipidome, and transcriptome data are needed.
PubMed: 34422879
DOI: 10.3389/fnut.2021.688086 -
JMIR MHealth and UHealth Nov 2022Digital health interventions are efficacious in health-promoting behaviors (eg, healthy eating and regular physical activity) that mitigate health risks and menopausal... (Review)
Review
BACKGROUND
Digital health interventions are efficacious in health-promoting behaviors (eg, healthy eating and regular physical activity) that mitigate health risks and menopausal symptoms in midlife. However, integrated evidence-based knowledge about the mechanisms of change in these interventions is unclear.
OBJECTIVE
This systematic review aimed to evaluate studies on behavior change techniques (BCTs) and mechanisms of change in digital health interventions aimed at promoting health-enhancing behaviors in midlife women (aged 40-65 years).
METHODS
A systematic literature search of the electronic databases PubMed, Web of Science, PsycINFO, and Cochrane Central Register of Controlled Trials in the Cochrane Library was conducted. In total, 2 independent reviewers selected the studies for inclusion, extracted data, and completed BCT mapping of eligible studies. The mechanism of action and intervention functions of eligible studies were evaluated using the behavior change wheel framework. Reporting of psychological theory use within these interventions was explored using the Theory Coding Scheme. Mode of delivery, psychological theory, and BCTs were presented as descriptive statistics.
RESULTS
In total, 13 interventions (including 1315 women) reviewed used 13 (SD 4.30, range 6-21) BCTs per intervention on average. The "Shaping knowledge" and "Repetition and substitution" behavior change categories were used most frequently, with 92% (12/13) of the interventions implementing at least one of the BCTs from these 2 categories. Only 13.98% (169/1209) of the 93 available BCTs were used, with "Instructions on behaviour" most frequently used (12/13, 92%). The behavior change wheel mapping suggests that half of the intervention content aimed to increase "Capability" (49/98, 50% of the intervention strategies), "Motivation" (41/98, 42%), and "Opportunity" (8/98, 8%). "Behavioural Regulation" was the most frequently used mechanism of action (15/98, 15%), followed by increasing "Knowledge" (13/98, 13%) and "Cognitive and Interpersonal skills" (10/98, 10%). A total of 78% (7/9) of the intervention functions were used in the studies to change behavior, primarily through "Enablement" (60/169, 35.5%), whereas no study used "Restriction" or "Modelling" functions. Although 69% (9/13) of the interventions mentioned a psychological theory or model, most (10/13, 77%) stated or suggested rather than demonstrated the use of a theoretical base, and none reported explicit links between all BCTs within the intervention and the targeted theoretical constructs. Technological components were primarily based on web-based (9/13, 69%) modes of delivery, followed by phone or SMS text message (8/13, 62%) and wearables (7/13, 54%).
CONCLUSIONS
The findings of this review indicate an overall weak use of theory, low levels of treatment fidelity, insignificant outcomes, and insufficient description of several interventions to support the assessment of how specific BCTs were activated. Thus, the identified limitations in the current literature provide an opportunity to improve the design of lifestyle health-enhancing interventions for women in midlife.
TRIAL REGISTRATION
PROSPERO CRD42021259246; https://tinyurl.com/4ph74a9u.
Topics: Humans; Female; Behavior Therapy; Text Messaging; Health Behavior; Motivation
PubMed: 36350694
DOI: 10.2196/37234