CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation.

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.

Secondary Metabolites from Annulohypoxylon sp. and Structural Revision of Emericellins A and B.

Seven previously undescribed compounds were isolated from the endophytic fungus Annulohypoxylon sp. KYG-19 (family Xylariaceae), including three gymnomitrane-type sesquiterpenes xylariacinols A, B, and D (1, 2, and 4), one bisabolane-type sesquiterpene annulnol F (6), one phenol derivative lariacinol G (7), and two polyhydroxy compounds hypoxylonols H and I (8 and 9), together with two known gymnomitrane-type sesquiterpenes emericellin A (3) and 3-gymnomitren-15-ol (5). The assignments of their structures was determined by extensive spectroscopic and spectrometric analysis, acetonide analysis, Mosher's method, and X-ray crystallography. In addition, the structures of emericellins A and B, which were reported to possess an unprecedented tricyclo[4, 4, 2, 1]hendecane scaffold, were revised by comparing their spectroscopic data with those of 1 and 3. Compounds 1 and 4 exhibited antibacterial activity against Escherichia coli with minimum inhibitory concentrations of 4 and 2 µg/mL, respectively.

Arabidopsis SMALL ORGAN 4, a homolog of yeast NOP53, regulates cell proliferation rate during organ growth.

Cell proliferation is a fundamental event essential for plant organogenesis and contributes greatly to the final organ size. Although the control of cell proliferation in plants has been extensively studied, how the plant sets the cell number required for a single organ is largely elusive. Here, we describe the Arabidopsis SMALL ORGAN 4 (SMO4) that functions in the regulation of cell proliferation rate and thus final organ size. The smo4 mutant exhibits a reduced size of organs due to the decreased cell number, and further analysis reveals that such phenotype results from a retardation of the cell cycle progression during organ development. SMO4 encodes a homolog of NUCLEOLAR PROTEIN 53 (NOP53) in Saccharomyces cerevisiae and is expressed primarily in tissues undergoing cell proliferation. Nevertheless, further complementation tests show that SMO4 could not rescue the lethal defect of NOP53 mutant of S. cerevisiae. These results define SMO4 as an important regulator of cell proliferation during organ growth and suggest that SMO4 might have been evolutionarily divergent from NOP53.

Evolutionary Modes of wtf Meiotic Driver Genes in Schizosaccharomyces pombe.

Killer meiotic drivers are a class of selfish genetic elements that bias inheritance in their favor by destroying meiotic progeny that do not carry them. How killer meiotic drivers evolve is not well understood. In the fission yeast, Schizosaccharomyces pombe, the largest gene family, known as the wtf genes, is a killer meiotic driver family that causes intraspecific hybrid sterility. Here, we investigate how wtf genes evolve using long-read-based genome assemblies of 31 distinct S. pombe natural isolates, which encompass the known genetic diversity of S. pombe. Our analysis, involving nearly 1,000 wtf genes in these isolates, yields a comprehensive portrayal of the intraspecific diversity of wtf genes. Leveraging single-nucleotide polymorphisms in adjacent unique sequences, we pinpoint wtf gene-containing loci that have recently undergone gene conversion events and infer their ancestral state. These events include the revival of wtf pseudogenes, lending support to the notion that gene conversion plays a role in preserving this gene family from extinction. Moreover, our investigation reveals that solo long terminal repeats of retrotransposons, frequently found near wtf genes, can act as recombination arms, influencing the upstream regulatory sequences of wtf genes. Additionally, our exploration of the outer boundaries of wtf genes uncovers a previously unrecognized type of directly oriented repeats flanking wtf genes. These repeats may have facilitated the early expansion of the wtf gene family in S. pombe. Our findings enhance the understanding of the mechanisms influencing the evolution of this killer meiotic driver gene family.

Uncovering the key pharmacodynamic material basis and possible molecular mechanism of extract of Epimedium against liver cancer through a comprehensive investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver cancer is a worldwide malignant tumor, and currently lacks effective treatments. Clinical studies have shown that epimedium (YYH) has therapeutic effects on liver cancer, and some of its prenylflavonoids have demonstrated anti-liver cancer activity through multiple mechanisms. However, there is still a need for systematic research to uncover the key pharmacodynamic material basis and mechanism of YYH. AIM OF THE STUDY: This study aimed to screen the anti-cancer material basis of YYH via integrating spectrum-effect analysis with serum pharmacochemistry, and explore the multi-target mechanisms of YYH against liver cancer by combining network pharmacology with metabolomics. MATERIALS AND METHODS: The anti-cancer effect of the extract of YYH (E-YYH) was first evaluated in mice with xenotransplantation H22 tumor cells burden and cultured hepatic cells. Then, the interaction between E-YYH compounds and the cytotoxic effects was revealed through spectrum-effect relationship analysis. And the cytotoxic effects of screened compounds were verified in hepatic cells. Next, UHPLC-Q-TOF-MS/MS was employed to identify the absorbed components of E-YYH in rat plasma to distinguish anti-cancer components. Subsequently, network pharmacology based on anti-cancer materials and metabolomics were used to discover the potential anti-tumor mechanisms of YYH. Key targets and biomarkers were identified and pathway enrichment analysis was performed. RESULTS: The anti-cancer effect of E-YYH was verified through in vitro and in vivo experiments. Six anti-cancer compounds in plasma (icariin, baohuoside Ⅰ, epimedin C, 2″-O-rhamnosyl icariside Ⅱ, epimedin B and sagittatoside B) were screened out by spectrum-effect analysis. Forty-five liver-cancer-related targets were connected with these compounds. Among these targets, PTGS2, TNF, NOS3 and PPARG were considered to be the potential key targets preliminarily verified by molecular docking. Meanwhile, PI3K/AKT signaling pathway and arachidonic acid metabolism were found to be associated with E-YYH's efficacy in network pharmacology and metabolomics analysis. CONCLUSIONS: Our research revealed the characteristics of multi-component, multi-target and multi-pathway mechanism of E-YYH. This study also provided an experimental basis and scientific evidence for the clinical application and rational development of YYH.

A new cyclopentenone derivative from Trichoderma atroviride HH-01.

A new cyclopentenone derivative, atrovinol (1), together with ten known compounds (2-11) were isolated from Trichoderma atroviride HH-01, an endophytic fungus from Illigera rhodantha (Hernandiaceae). Their structures were identified by HRESIMS, 1 D/2D NMR, and electronic circular dichroism (ECD) spectra. Compound 1 exhibited moderate inhibitory activity against Staphylococcus aureus and Bacillus subtilis with MIC values of 8.0 µg/mL and 16.0 µg/mL, respectively.

The effects of hypothermia on glutamate and γ-aminobutyric acid metabolism during ischemia in monkeys: a repeated-measures ANOVA study.

During an ischemic stroke, the brain releases various factors, including glutamate and γ-aminobutyric acid. Glutamate can cause neurotoxic effects through certain receptors and exacerbate neurological damage, while γ-aminobutyric acid as an inhibitory neurotransmitter can antagonize the excitotoxic effects of glutamate and enhance the tolerance of neurons to ischemia. Therefore, in this study, the content of amino acid neurotransmitters in brain tissue before ischemia, after 10 min of ischemia, hypothermic perfusion, and rewarming were analyzed by high-performance liquid chromatography-UV in an animal model of ischemic stroke generated by blocking the bilateral common carotid arteries of rhesus monkeys. The changes in amino acid neurotransmitters in the rhesus monkey brain during post-ischemia hypothermic perfusion and rewarming were investigated by statistical methods of repeated measures ANOVA, showing that the concentration change of glutamate had not only a temporal factor but also was influenced by temperature, and there was an interaction effect between the two. Time but not temperature affected the change in γ-aminobutyric acid concentration, and there was an interaction effect between the two. Accordingly, hypoperfusion exerts a protective effect during ischemia by inhibiting the release of excitatory amino acid neurotransmitters, while the antagonistic effect of GABA on Glu is not significant.

An improved auxin-inducible degron system for fission yeast.

Conditional degron technologies, which allow a protein of interest to be degraded in an inducible manner, are important tools for biological research, and are especially useful for creating conditional loss-of-function mutants of essential genes. The auxin-inducible degron (AID) technology, which utilizes plant auxin signaling components to control protein degradation in nonplant species, is a widely used small-molecular-controlled degradation method in yeasts and animals. However, the currently available AID systems still have room for further optimization. Here, we have improved the AID system for the fission yeast Schizosaccharomyces pombe by optimizing all three components: the AID degron, the small-molecule inducer, and the inducer-responsive F-box protein. We chose a 36-amino-acid sequence of the Arabidopsis IAA17 protein as the degron and employed three tandem copies of it to enhance efficiency. To minimize undesirable side effects of the inducer, we adopted a bulky analog of auxin, 5-adamantyl-IAA, and paired it with the F-box protein OsTIR1 that harbors a mutation (F74A) at the auxin-binding pocket. 5-adamantyl-IAA, when utilized with OsTIR1-F74A, is effective at concentrations thousands of times lower than auxin used in combination with wild-type OsTIR1. We tested our improved AID system on 10 essential genes and achieved inducible lethality for all of them, including ones that could not be effectively inactivated using a previously published AID system. Our improved AID system should facilitate the construction of conditional loss-of-function mutants in fission yeast.

A new cyclohexenone derivative from phomopsis sp. XM-01.

A new cyclohexenone derivative, phomopine (1), together with five known compounds (2-6) were isolated from Phomopsis sp. XM-01. The structure of 1 was determined by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculation. In vitro bioassays, compounds 1 and 2 exhibited potent antimicrobial activities against Candida albicans and Staphylococcus aureus with their corresponding minimum inhibitory concentrations (MICs) of 64 µg/mL and 16 µg/mL, respectively.

Single-chromosome fission yeast models reveal the configuration robustness of a functional genome.

In eukaryotic organisms, genetic information is usually carried on multiple chromosomes. Whether and how the number and configuration of chromosomes affect organismal fitness and speciation remain unclear. Here, we have successfully established several single-chromosome fission yeast Schizosaccharomyces pombe strains, in which the three natural chromosomes have been fused into one giant chromosome in different orders. Chromosome fusions accompanied by the deletions of telomeres and centromeres result in the loss of chromosomal interactions and a drastic change of global chromosome organization, but alter gene expression marginally. The single-chromosome strains display little defects in cell morphology, mitosis, genotoxin sensitivity, and meiosis. Crosses between a wild-type strain and a single-chromosome strain or between two single-chromosome strains with different fusion orders suffer defective meiosis and poor spore viability. We conclude that eukaryotic genomes are robust against dramatic chromosomal reconfiguration, and stochastic changes in chromosome number and genome organization during evolution underlie reproductive isolation and speciation.

Nrf2 promotes esophageal squamous cell carcinoma (ESCC) resistance to radiotherapy through the CaMKIIα-associated activation of autophagy.

BACKGROUND: NF-E2-related factor 2 (Nrf2) is involved in the radiation resistance of esophageal squamous cell carcinoma (ESCC), but the underlying molecular mechanism is unclear. The purpose of our study was to explore the role of Nrf2 in the radiation resistance of ESCC and the potential molecular mechanism. RESULTS: Nrf2 expression was introduced into Ec109 and KYSE-30 ESCC cells with lentivirus. CCK-8 and colony formation assays were used to evaluate the effect of Nrf2 on radioresistance in culture. The autophagy level was assessed by western blotting, flow cytometry, and confocal fluorescence microscopy. The effect of Nrf2 on the transcription of Ca2 +/calmodulin-dependent protein kinase II α (CaMKIIα) was studied by chromatin immunoprecipitation. We found that the overexpression of Nrf2 increased the radiation resistance of ESCC cells. Mechanistically, Nrf2 triggered the radiation resistance of ESCC cells by targeting CaMKIIα and subsequently activating autophagy. In addition, we found that Nrf2 directly regulated the transcription of CaMKIIα by binding to its promoter region. The effect of Nrf2 on radiation resistance was also explored in both a xenograft mouse model and ESCC patient samples. Consistent with the results of the in vitro study, high Nrf2 expression level resulted in in vivo radioresistance in an Ec109-derived xenograft mouse model. Furthermore, we also demonstrated that upregulations of both Nrf2 and CaMKIIα was closely related to lower survival rates of ESCC patients. CONCLUSIONS: Our study reveals that Nrf2 promotes the radiation resistance of ESCC by targeting CaMKIIα and subsequently activating autophagy, which is characterized by the suppression of phosphorylated mTOR and p62, activation of Beclin 1, and transformation of LC3-I to LC3-II.

Biotransformation of α-terpineol by Alternaria alternata.

α-Terpineol (1), the main volatile constituent in some traditional Chinese medicines, has been reported to be metabolized to 4R-oleuropeic acid by the larvae of common cutworms. The present study verified that α-terpineol could be converted to 4R-oleuropeic acid (2) and (1S,2R,4R)-p-menthane-1,2,8-triol (3) by Alternaria alternata fermentation. Using shortened fermentation times, 7-hydroxy-α-terpineol (2a) was identified as an oxidative intermediate, which was consistent with the hypothesis put forward by previous studies. Cytochrome P450 enzymes were also confirmed to catalyze this biotransformation. This is the first study on the biotransformation of α-terpineol by microbial fermentation.

The role of Nrf2/PIWIL2/purine metabolism axis in controlling radiation-induced lung fibrosis.

NF-E2-related factor 2 (Nrf2) is a key transcription factor recently implicated in the control of radiation-induced lung fibrosis (RILF). However, the molecular mechanism of Nrf2 in the pathogenesis of RILF is still unclear. The purpose of this study was to evaluate the regulatory effect and mechanism of Nrf2 in the pathogenesis of RILF. The effects of different Nrf2 expression levels on RILF were explored in vitro and in vivo. The RILF model of Nrf2 knockout mice was established for in vivo study. In the study of the mechanism of action, ChIP-seq assay and metabolomics analysis were performed. The discovered mechanism of Nrf2-mediated RILF alleviation was further validated in vitro and in vivo. We found that overexpression of Nrf2 significantly alleviated the fibrosis caused by irradiation in vivo and in vitro. Conversely, Nrf2 silencing strongly aggravated the development of RILF. Mechanistically, Nrf2 signaling increased the expression of piwi-like RNA-mediated gene silencing 2 (PIWIL2), leading to the alteration of purine metabolism and contributing to the relief of RILF. These results suggest that Nrf2 promotes the attenuation of RILF in vivo and in vitro by directly targeting PIWIL2 and activating purine metabolism.

The complete mitochondrial genome of red-throated flycatcher Ficedula albicilla (Passeriformes: Ficedula).

We report the mitochondrial genome of Ficedula albicilla. The overall base composition of F. albicilla mitogenome is 29.49%A, 15.06%G, 32.98%C, and 22.47%T, with an A + T content of 51.96%. The total length of the sequence is 16,791 bp (13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control regions). Phylogenetic analysis was performed based on the concatenated nucleotide sequences of cytochrome c oxidase subunit I and cytochrome b using the neighbor-joining method and the Kimura 2-parameter model in MEGA 7.0 with 1000 bootstrap replicates.

Systematic analysis reveals the prevalence and principles of bypassable gene essentiality.

Gene essentiality is a variable phenotypic trait, but to what extent and how essential genes can become dispensable for viability remain unclear. Here, we investigate 'bypass of essentiality (BOE)' - an underexplored type of digenic genetic interaction that renders essential genes dispensable. Through analyzing essential genes on one of the six chromosome arms of the fission yeast Schizosaccharomyces pombe, we find that, remarkably, as many as 27% of them can be converted to non-essential genes by BOE interactions. Using this dataset we identify three principles of essentiality bypass: bypassable essential genes tend to have lower importance, tend to exhibit differential essentiality between species, and tend to act with other bypassable genes. In addition, we delineate mechanisms underlying bypassable essentiality, including the previously unappreciated mechanism of dormant redundancy between paralogs. The new insights gained on bypassable essentiality deepen our understanding of genotype-phenotype relationships and will facilitate drug development related to essential genes.

A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast.

The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.

Wharton's Jelly-derived mesenchymal stem cells alleviate memory deficits and reduce amyloid-ß deposition in an APP/PS1 transgenic mouse model.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly and is characterized by amyloid plaques, neurofibrillary tangles, and neuronal loss. Cumulative evidence supports that neuroinflammation is an important factor for the pathogenesis of AD and contributes to amyloid beta (Aß) generation. However, there has been no effective treatment for AD. Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) have a potential therapeutic effect in the treatment for neurological diseases. In the present study, we evaluated the therapeutic effect of WJ-MSC transplantation on the neuropathology and memory deficits in amyloid precursor protein (APP) and presenilin-1 (PS1) double-transgenic mice and discussed the mechanism. WJ-MSCs were intravenously transplanted into the APP/PS1 mice. Four weeks after treatment, WJ-MSCs significantly improved the spatial learning and alleviated the memory decline in the APP/PS1 mice. Aß deposition and soluble Aß levels were significantly reduced after WJ-MSC treatment. Furthermore, WJ-MSCs significantly increased the expression of the anti-inflammatory cytokine, IL-10. Meanwhile, pro-inflammatory microglial activation and the expressions of pro-inflammatory cytokines, IL-1ß and TNFα, were significantly down-regulated by WJ-MSC treatment. Thus, our findings suggest that WJ-MSCs might produce beneficial effects on the prevention and treatment for AD through modulation of neuroinflammation.

Percutaneous Repair Technique for Acute Achilles Tendon Rupture with Assistance of Kirschner Wire.

The aim of this study is to introduce a self-designed, minimally invasive technique for repairing an acute Achilles tendon rupture percutaneously. Comparing with the traditional open repair, the new technique provides obvious advantages of minimized operation-related lesions, fewer wound complications as well as a higher healing rate. However, a percutaneous technique without direct vision may be criticized by its insufficient anastomosis of Achilles tendon and may also lead to the lengthening of the Achilles tendon and a reduction in the strength of the gastrocnemius. To address the potential problems, we have improved our technique using a percutaneous Kirschner wire leverage process before suturing, which can effectively recover the length of the Achilles tendon and ensure the broken ends are in tight contact. With this improvement in technique, we have great confidence that it will become the treatment of choice for acute Achilles tendon ruptures.