Establishing the program of origin firing during S phase in fission Yeast.

Initiation of eukaryotic DNA synthesis occurs at origins of replication that are utilized with characteristic times and frequencies during S phase. We have investigated origin usage by evaluating the kinetics of replication factor binding in fission yeast and show that similar to metazoa, ORC binding is periodic during the cell cycle, increasing during mitosis and peaking at M/G1. At an origin, the timing of ORC binding in M and pre-RC assembly in G1 correlates with the timing of firing during S, and the level of pre-IC formation reflects origin efficiency. Extending mitosis allows ORC to become more equally associated with origins and leads to genome-wide changes in origin usage, while overproduction of pre-IC factors increases replication of both efficient and inefficient origins. We propose that differential recruitment of ORC to origins during mitosis followed by competition among origins for limiting replication factors establishes the timing and efficiency of origin firing.

The effects of mindfulness-based interventions on alleviating academic burnout in medical students: a systematic review and meta-analysis.

BACKGROUND: Mindfulness-based interventions have been tested to be the effective approach for preventing/reducing burnout in medical students. Therefore, this systematic review and meta-analysis aimed to synthesize the scientific evidence and quantify the pooled effect of MBIs on the burnout syndrome in medical students. METHODS: A comprehensive literature search was conducted in the databases, including PubMed, Embase, ERIC, PsycINFO, Scopus, Cochrane Central Register of Controlled Trials (CENTRAL), China National knowledge Information Database (CNKI) and WanFang Database from database inception to February 2023 using the terms of "mindfulness", "burnout" and "medical students". Two reviewers independently reviewed the studies, and extracted the data of the eligible studies, as well as assessed the risk of bias. A random-effects model was employed to calculate the standardized mean differences (SMD) with 95% confidence intervals (CI) of overall burnout and its sub-domains of burnout (i.e., emotional exhaustion, cynicism, and academic efficacy). RESULTS: Of 316 records in total, nine studies (with 810 medical students) were ultimately included. The four RCT studies demonstrated an overall judgment of some concerns risk of bias, and the overall risk of biases of the five qRCT studies were judged as serious. In term of the SORT, the RCT and qRCT studies were evaluated as level 2 evidence, and the overall strength of recommendation was classified as B (limited-quality patient-oriented evidence). The pooled analysis showed that MBIs were associated with significant small to moderate improvements for medical students' overall burnout (SMD=-0.64; 95% CI [-1.12, -0.16]; P = 0.009) in the included four RCTs, emotional exhaustion (SMD=-0.27; 95% CI [-0.50, -0.03]; P = 0.03) and academic efficacy (SMD = 0.43; 95% CI [0.20, 0.66]; P<0.001) in the four qRCTs. CONCLUSIONS: MBIs can serve as an effective approach for reducing burnout symptoms in medical students. Future high-quality studies with a larger sample size and robust randomized controlled trial methodologies should be obtained to reinforce the effectiveness of MBIs for reducing academic burnout in medical students.

Replication origin selection regulates the distribution of meiotic recombination.

The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome-wide modifications in replication program can modulate cellular physiology.

The organization of genome duplication is a critical determinant of the landscape of genome maintenance.

Genome duplication is essential for cell proliferation, and the mechanisms regulating its execution are highly conserved. These processes give rise to a spatiotemporal organization of replication initiation across the genome, referred to as the replication program. Despite the identification of such programs in diverse eukaryotic organisms, their biological importance for cellular physiology remains largely unexplored. We address this fundamental question in the context of genome maintenance, taking advantage of the inappropriate origin firing that occurs when fission yeast cells lacking the Rad3/ATR checkpoint kinase are subjected to replication stress. Using this model, we demonstrate that the replication program quantitatively dictates the extent of origin de-regulation and the clustered localization of these events. Furthermore, our results uncover an accumulation of abnormal levels of single-stranded DNA (ssDNA) and the Rad52 repair protein at de-regulated origins. We show that these loci constitute a defining source of the overall ssDNA and Rad52 hotspots in the genome, generating a signature pattern of instability along the chromosomes. We then induce a genome-wide reprogramming of origin usage and evaluate its consequences in our experimental system. This leads to a complete redistribution of the sites of both inappropriate initiation and associated Rad52 recruitment. We therefore conclude that the organization of genome duplication governs the checkpoint control of origin-associated hotspots of instability and plays an integral role in shaping the landscape of genome maintenance.

CDK activity provides temporal and quantitative cues for organizing genome duplication.

In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome.

Regulation of the program of DNA replication by CDK: new findings and perspectives.

Progression through the cell cycle is driven by the activities of the cyclin-dependent kinase (CDK) family of enzymes, which establish an ordered passage through the cell cycle phases. CDK activity is crucial for the cellular transitions from G1 to S and G2 to M, which are highly controlled to promote the faithful duplication of the genetic material and the transmission of the genome into daughter cells, respectively. While oscillations in CDK activity are essential for cell division, how its specific dynamics may shape cellular processes remains an open question. Recently, we have investigated the potential role of CDK in establishing the profile of replication initiation along the chromosomes, also referred to as the replication program. Our results demonstrated that the timing and level of CDK activity at G1/S provide two critical and independent inputs that modulate the pattern of origin usage. In this review, we will present the conclusions of our study and discuss the implications of our findings for cellular function and physiology.

Linking the organization of DNA replication with genome maintenance.

The spatial and temporal organization of genome duplication, also referred to as the replication program, is defined by the distribution and the activities of the sites of replication initiation across the genome. Alterations to the replication profile are associated with cell fate changes during development and in pathologies, but the importance of undergoing S phase with distinct and specific programs remains largely unexplored. We have recently addressed this question, focusing on the interplay between the replication program and genome maintenance. In particular, we demonstrated that when cells encounter challenges to DNA synthesis, the organization of DNA replication drives the response to replication stress that is mediated by the ATR/Rad3 checkpoint pathway, thus shaping the pattern of genome instability along the chromosomes. In this review, we present the major findings of our study and discuss how they may bring new perspectives to our understanding of the biological importance of the replication program.

Quantitative control of protein S-palmitoylation regulates meiotic entry in fission yeast.

Protein S-palmitoylation, a lipid modification mediated by members of the palmitoyltransferase family, serves as an important membrane-targeting mechanism in eukaryotes. Although changes in palmitoyltransferase expression are associated with various physiological and disease states, how these changes affect global protein palmitoylation and cellular function remains unknown. Using a bioorthogonal chemical reporter and labeling strategy to identify and analyze multiple cognate substrates of a single Erf2 palmitoyltransferase, we demonstrate that control of Erf2 activity levels underlies the differential modification of key substrates such as the Rho3 GTPase in vegetative and meiotic cells. We show further that modulation of Erf2 activity levels drives changes in the palmitoylome as cells enter meiosis and affects meiotic entry. Disruption of Erf2 function delays meiotic entry, while increasing Erf2 palmitoyltransferase activity triggers aberrant meiosis in sensitized cells. Erf2-induced meiosis requires the function of the Rho3 GTPase, which is regulated by its palmitoylation state. We propose that control of palmitoyltransferase activity levels provides a fundamental mechanism for modulating palmitoylomes and cellular functions.

[HPLC-ELSD for determining contents of three triterpenoidal saponins in fruits of Buddleja lindleyana from different habitats].

To establish an HPLC-ELSD method for the quantification of triterpenoids in the fruits of Buddleja lindleyana. The RP-HPLC-ELSD method was used for the determination of triterpenoids in B. lindleyana fruits, which were collected from different habitats. The column used was a packed with 5 µm stationary phase Waters SunFireTM C18 (4.6 mm×150 mm, 5 µm). The mobile phase consisted of Methanol-water(82∶18) at a flow rate of 1 mL•min⁻¹. Column temperature： 30 ℃. ELSD conditions： drift tube temperature： 106 ℃; carrier gas (nitrogen) flow rate： 1.5 L•min⁻¹; amplification factor： 1. The calibration curves showed good linear relationship on a range from 0.702 to 28.08 µg(r=0.999 2) for Clinoposaponin III, 0.390 to 15.60 µg(r=0.998 9) for Desrhamnoverbascosaponin and 0.192 to 7.68µg(r=0.999 0) for Mimengoside I. The average recovery rate(n=6) were 99.41%, 99.08% and 98.67% and it's RSD were 0.86%, 1.56% and 1.80%. This method can be used to determine the contents of triterpenoids in the fruits of Buddleja lindleyana for its simplicity, accurateness and reliability.

Artificial Modulation and Rewiring of Cell Cycle Progression Using Synthetic Circuits in Fission Yeast.

Cell cycle control is a central aspect of the biology of proliferating eukaryotic cells. However, progression through the cell cycle relies on a highly complex network, making it difficult to unravel the core design principles underlying the mechanisms that sustain cell proliferation and the ways in which they interact with other cellular pathways. In this context, the use of a synthetic approach to simplify the cell cycle network in unicellular genetic models such as fission yeast has opened the door to studying the biology of proliferating cells from unique perspectives. Here, we provide a series of methods based on a minimal cell cycle module in the fission yeast Schizosaccharomyces pombe that allows for an unprecedented artificial control of cell cycle events, enabling the rewiring and remodeling of cell cycle progression.

Serum FT3/FT4, but not TSH is associated with handgrip strength in euthyroid U.S. population: evidence from NHANES.

Objective: Although several studies have examined the relationship between thyroid function and muscle strength, their population primarily derived from Asian areas, and their results were controversial. Thus, this study aimed to explore the association between thyroid function and handgrip strength (HGS) in the U.S. population. Methods: A total of 1,067 participants from NHANES were categorized into three different age groups including young (<45 years), middle (45~64 years), and old (≥65 years) age groups. Thyroid function was measured by the competitive binding immune-enzymatic assays, while HGS was examined by a trained evaluator using a dynamometer. The weighted multiple linear regression models were used to examine the association between thyroid function and handgrip strength. The restricted cubic splines were employed to explore the non-linear relationship between these two variables. All statistical analyses were performed using the SPSS version 20.0 and R software. Results: After adjustment for potential covariates, FT3/FT4, but not TSH was positively associated with HGS in middle age group (ß=0.091, t=2.428, P=0.016). The subgroup analysis by sex revealed that the positive association between FT3/FT4 and HGS was observed in the middle age group for both male and female participants (ß=0.163, t=2.121, P=0.035; ß=0.157, t=2.180, P=0.031). The RCS analysis showed a statistically significant non-linear association between FT3/FT4 and HGS in overall population (P for non-linear=0.026). After adjustment for covariates, men with low HGS had a significant lower FT3/FT4 than those without low HGS in old age group (P=0.013). There was a significant increase in TSH level for female participants with low HGS in old age group compared to those with normal HGS (P=0.048). Conclusions: This study demonstrated FT3/FT4, but not TSH, was positively associated with HGS in middle age group, and the different association was observed in men in middle age group when participants were stratified by sex. Future longitudinal cohort study should be conducted to reveal the causal relationship between thyroid function and muscle strength.

The programme of DNA replication: beyond genome duplication.

The accurate duplication and transmission of genetic information is critical for cell growth and proliferation, and this is ensured in part by the multi-layered regulation of DNA synthesis. One of the key steps in this process is the selection and activation of the sites of replication initiation, or origins, across the genome. Interestingly, origin usage changes during development and in different pathologies, suggesting an integral interplay between the establishment of replication initiation along the chromosomes and cellular function. The present review discusses how the spatiotemporal organization of replication origin activation may play crucial roles in the control of biological events.

Bleeding risk after acupuncture in patients taking anticoagulant drugs: A case control study based on real-world data.

OBJECTIVES: Patients on anticoagulant medications may be at a higher risk of bleeding after acupuncture. This study aimed to assess the association between anticoagulant drug use and bleeding after acupuncture. DESIGN: Case control study SETTING: We analysed the diagnosis and treatment records (2000-2018) of a random sample of two million patients from the National Health Insurance Research Database in Taiwan. INTERVENTIONS: anticoagulant and antiplatelet drugs MAIN OUTCOME MEASURES: The incidence rates of major (visceral bleeding or ruptured blood vessels requiring transfusion) and minor (skin bleeding or contusion) bleeding after acupuncture RESULTS: We included the records of 13,447,563 acupuncture sessions in 821,946 participants and followed up the patients for 14 days after each session. The incidence of minor bleeding was 8.31 per 10,000 needles, whereas that of major bleeding was 4.26 per 100,000 needles. Anticoagulants significantly increased the risk of minor bleeding (adjusted OR = 1.15 (1.03-1.28)), but the risk of major bleeding did not reach statistical significance (adjusted OR = 1.18 (0.8 0-1.75)). Anticoagulants, such as warfarin (adjusted OR = 4.95 (2.55-7.64)), direct oral anticoagulants (adjusted OR = 3.07 (1.23-5.47)), and heparin (adjusted OR = 3.72 (2.18-6.34)) significantly increased the risk of bleeding. However, antiplatelet drug was not significantly associated with post-acupuncture bleeding. Comorbidities including liver cirrhosis, diabetes, and coagulation defects, were the risk factors for bleeding after acupuncture. CONCLUSIONS: Anticoagulant drugs may increase the risk of bleeding after acupuncture. We encourage physicians to ask patients in detail about their medical history and drug use prior to acupuncture treatment.

Glycyrrhizic acid modified Poria cocos polyscaccharide carbon dots dissolving microneedles for methotrexate delivery to treat rheumatoid arthritis.

Introduction: Rheumatoid arthritis is an autoimmune disease characterized by chronic joint inflammation. Methotrexate is one of the most effective drugs for rheumatoid arthritis, but the adverse reactions caused by oral methotrexate greatly limit its clinical application. Transdermal drug delivery system is an ideal alternative to oral methotrexate by absorbing drugs into the human body through the skin. However, methotrexate in the existing methotrexate microneedles is mostly used alone, and there are few reports of combined use with other anti-inflammatory drugs. Methods: In this study, glycyrrhizic acid was first modified onto carbon dots, and then methotrexate was loaded to construct a nano-drug delivery system with fluorescence and dual anti-inflammatory effects. Then hyaluronic acid was combined with nano-drug delivery system to prepare biodegradable soluble microneedles for transdermal drug delivery of rheumatoid arthritis. The prepared nano-drug delivery system was characterized by transmission electron microscopy, fluorescence spectroscopy, laser nanoparticle size analyzer, ultraviolet-visible absorption spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimeter and nuclear magnetic resonance spectrometer. The results showed that glycyrrhizic acid and methotrexate were successfully loaded on carbon dots, and the drug loading of methotrexate was 49.09%. The inflammatory cell model was constructed by lipopolysaccharide-induced RAW264.7 cells. In vitro cell experiments were used to explore the inhibitory effect of the constructed nano-drug delivery system on the secretion of inflammatory factors by macrophages and the cell imaging ability. The drug loading, skin penetration ability, in vitro transdermal delivery and in vivo dissolution characteristics of the prepared microneedles were investigated. The rat model of rheumatoid arthritis was induced by Freund's complete adjuvant. Results: The results of in vivo animal experiments showed that the soluble microneedles of the nano drug delivery system designed and prepared in this study could significantly inhibit the secretion of pro-inflammatory cytokines and had a significant therapeutic effect on arthritis. Discussion: The prepared glycyrrhizic acid-carbon dots-methotrexate soluble microneedle provides a feasible solution for the treatment of Rheumatoid arthritis.

Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair.

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

Impact of Chromosomal Context on Origin Selection and the Replication Program.

Eukaryotic DNA replication is regulated by conserved mechanisms that bring about a spatial and temporal organization in which distinct genomic domains are copied at characteristic times during S phase. Although this replication program has been closely linked with genome architecture, we still do not understand key aspects of how chromosomal context modulates the activity of replication origins. To address this question, we have exploited models that combine engineered genomic rearrangements with the unique replication programs of post-quiescence and pre-meiotic S phases. Our results demonstrate that large-scale inversions surprisingly do not affect cell proliferation and meiotic progression, despite inducing a restructuring of replication domains on each rearranged chromosome. Remarkably, these alterations in the organization of DNA replication are entirely due to changes in the positions of existing origins along the chromosome, as their efficiencies remain virtually unaffected genome wide. However, we identified striking alterations in origin firing proximal to the fusion points of each inversion, suggesting that the immediate chromosomal neighborhood of an origin is a crucial determinant of its activity. Interestingly, the impact of genome reorganization on replication initiation is highly comparable in the post-quiescent and pre-meiotic S phases, despite the differences in DNA metabolism in these two physiological states. Our findings therefore shed new light on how origin selection and the replication program are governed by chromosomal architecture.

Insights into the Link between the Organization of DNA Replication and the Mutational Landscape.

The generation of a complete and accurate copy of the genetic material during each cell cycle is integral to cell growth and proliferation. However, genetic diversity is essential for adaptation and evolution, and the process of DNA replication is a fundamental source of mutations. Genome alterations do not accumulate randomly, with variations in the types and frequencies of mutations that arise in different genomic regions. Intriguingly, recent studies revealed a striking link between the mutational landscape of a genome and the spatial and temporal organization of DNA replication, referred to as the replication program. In our review, we discuss how this program may contribute to shaping the profile and spectrum of genetic alterations, with implications for genome dynamics and organismal evolution in natural and pathological contexts.

Aspergilolide, a steroid lactone produced by an endophytic fungus Aspergillus sp. MBL1612 isolated from Paeonia ostii.

A new steroid lactone aspergilolide (1), and nine known compounds helvolic acid (2), verruculogen (3), tryprostatin B (4), 13-oxofumitremorgin B (5), fumitremorgin C (6), demethoxy fumitremorgin C (7), terezine D (8), aszonalenin (9), 12, 13-dihydroxy-fumitremorgin C (10) from cultures of the endophytic fungus Aspergillus sp. MBL1612. Their chemical structures were determined by a series of extensive spectroscopic methods. All of the compounds were isolated from this genus for the first time. The cytotoxicity against five human cancer cell lines of new compound were detected.

Analysis of Spt7 function in the Saccharomyces cerevisiae SAGA coactivator complex.

The Saccharomyces cerevisiae SAGA complex is required for the normal transcription of a large number of genes. Complex integrity depends on three core subunits, Spt7, Spt20, and Ada1. We have investigated the role of Spt7 in the assembly and function of SAGA. Our results show that Spt7 is important in controlling the levels of the other core subunits and therefore of SAGA. In addition, partial SAGA complexes containing Spt7 can be assembled in the absence of both Spt20 and Ada1. Through biochemical and genetic analyses of a series of spt7 deletion mutants, we have identified a region of Spt7 required for interaction with the SAGA component Spt8. An adjacent Spt7 domain was found to be required for a processed form of Spt7 that is present in a previously identified altered form of SAGA called SLIK, SAGA(alt), or SALSA. Analysis of an spt7 mutant with greatly reduced levels of SLIK/SAGA(alt)/SALSA suggests a subtle role for this complex in transcription that may be redundant with a subset of SAGA functions.

Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities.

Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism.