Human steroid 5α-reductase 2 (SRD5α2) as a critical integral membrane enzyme in steroid metabolism catalyzes testosterone to dihydrotestosterone. Mutations on its gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride as SRD5α2 inhibitors are widely used anti-androgen drugs for benign prostate hyperplasia, which have recently been indicated in the treatment of COVID-19. The molecular mechanisms underlying enzyme catalysis and inhibition remained elusive for SRD5α2 and other eukaryotic integral membrane steroid reductases due to a lack of structural information. Here, we report a crystal structure of human SRD5α2 at 2.8 Å revealing a unique 7-TM structural topology and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride in a largely enclosed binding cavity inside the membrane. Structural analysis together with computational and mutagenesis studies reveals molecular mechanisms for the 5α-reduction of testosterone and the finasteride inhibition involving residues E57 and Y91. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region regulating the NADPH/NADP + exchange. Mapping disease-causing mutations of SRD5α2 to our structure suggests molecular mechanisms for their pathological effects. Our results offer critical structural insights into the function of integral membrane steroid reductases and will facilitate drug development.

Intramuscular fat (IMF) is one of the most critical parameters affecting meat quality and mainly affected by genetic factors. MicroRNA as an important regulatory factor, which is still a lack of research in the development of sheep IMF deposition. We used RNA sequencing (RNA-seq) and cell-level validation to explore the role of miRNA in IMF deposition. As for this purpose, longissimus thoracis et lumborum (LTL) samples of 2 month-old (Mth-2) and 12 months-old (Mth-12) Aohan fine-wool sheep (AFWS) were used to identified miRNAs expression. We found 59 differentially expressed miRNAs (DE-miRNA) between these age groups and predicted their 1,796 target genes. KEGG functional enrichment analysis revealed eight pathways involved in lipid metabolism-related processes, including fatty acid elongation and the AMPK signaling pathway. A highly expressed DE-miRNA, miR-193a-5p, was found to serve a function in 3T3-L1 preadipocyte differentiation. Luciferase assay demonstrated that miR-193a-5p directly binds to the 3'-UTR region of ACAA2. By constructing mimics and inhibitor vector transfecting into 3T3-L1 cells to explore the effect of miR-193a-5p on cell proliferation and differentiation, we demonstrated that overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte proliferation, as evidenced by decreased mRNA and protein expression of CDK4 and CyclinB. CCK-8 assay showed that miR-193a-5p significantly inhibited cell proliferation. Similarly, the overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte differentiation and adipocyte-specific molecular markers' expression, leading to a decrease in PPARγ and C/EBPα and ACAA2. Inhibition of miR-193a-5p had the opposite effects. Our study lists the miRNAs associated with intramuscular lipid deposition in sheep and their potential targets, striving to improve sheep meat quality.

Reproductive transition of grasses is characterized by switching the pattern of lateral branches, featuring the suppression of outgrowth of the subtending leaves (bracts) and rapid formation of higher-order branches in the inflorescence (panicle). However, the molecular mechanisms underlying such changes remain largely unknown. Here, we show that bract suppression is required for the reproductive branching in rice. We identified a pathway involving the intrinsic time ruler microRNA156/529, their targets SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, NECK LEAF1 (NL1), and PLASTOCHRON1 (PLA1), which regulates the bract outgrowth and thus affects the pattern switch between vegetative and reproductive branching. Suppression of the bract results in global reprogramming of transcriptome and chromatin accessibility following the reproductive transition, while these processes are largely dysregulated in the mutants of these genes. These discoveries contribute to our understanding of the dynamic plant architecture and provide novel insights for improving crop yields.

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.

FOXO3a (forkhead box transcription factor 3a) is involved in regulating multiple biological processes in cancer cells. BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a receptor accounting for priming damaged mitochondria for autophagic removal. In this study we investigated the role of FOXO3a in regulating the sensitivity of glioma cells to temozolomide (TMZ) and its relationship with BNIP3-mediated mitophagy. We showed that TMZ dosage-dependently inhibited the viability of human U87, U251, T98G, LN18 and rat C6 glioma cells with IC50 values of 135.75, 128.26, 142.65, 155.73 and 111.60 μM, respectively. In U87 and U251 cells, TMZ (200 μM) induced DNA double strand breaks (DSBs) and nuclear translocation of apoptosis inducing factor (AIF), which was accompanied by BNIP3-mediated mitophagy and FOXO3a accumulation in nucleus. TMZ treatment induced intracellular ROS accumulation in U87 and U251 cells via enhancing mitochondrial superoxide, which not only contributed to DNA DSBs and exacerbated mitochondrial dysfunction, but also upregulated FOXO3a expression. Knockdown of FOXO3a aggravated TMZ-induced DNA DSBs and mitochondrial damage, as well as glioma cell death. TMZ treatment not only upregulated BNIP3 and activated autophagy, but also triggered mitophagy by prompting BNIP3 translocation to mitochondria and reinforcing BNIP3 interaction with LC3BII. Inhibition of mitophagy by knocking down BNIP3 with SiRNA or blocking autophagy with 3MA or bafilomycin A1 exacerbated mitochondrial superoxide and intracellular ROS accumulation. Moreover, FOXO3a knockdown inhibited TMZ-induced BNIP3 upregulation and autophagy activation. In addition, we showed that treatment with TMZ (100 mg·kg-1·d-1, ip) for 12 days in C6 cell xenograft mice markedly inhibited tumor growth accompanied by inducing FOXO3a upregulation, oxidative stress and BNIP3-mediated mitophagy in tumor tissues. These results demonstrate that FOXO3a attenuates temozolomide-induced DNA double strand breaks in human glioma cells via promoting BNIP3-mediated mitophagy.

Accumulating evidence has demonstrated that circular RNAs (circRNAs) are involved in the pathogenesis of cancer, including that of esophageal squamous cell carcinoma (ESCC). The current study aimed to investigate the role of hsa_circ_0000700 in ESCC. hsa_circ_0000700, miR-1229, and related functional gene expression was measured by RT-qPCR. To characterize the functions of hsa_circ_0000700 and miR-1229, ESCC cells were infected with hsa_circ_0000700-specific siRNA, miR-1229 mimics, and an inhibitor alone or in combination. Cell Counting Kit-8 (CCK8), colony formation, EdU, flow cytometry, and Transwell assays were employed to evaluate cell proliferation, apoptosis, and migration. Luciferase reporter and RNA immunoprecipitation assays were used to confirm the targeting relationship between hsa_circ_0000700 and miR-1229. Finally, a competing endogenous RNAs (ceRNA) network was built for hsa_circ_0000700, and miR-1229 targets were analyzed by bioinformatics. circ_0000700 expression was significantly upregulated in ESCC cell lines. Actinomycin D and RNase R treatment confirmed that circ_0000700 was more stable than its linear CDH9 mRNA form. Moreover, a cytoplasmic and nuclear fractionation assay suggested that circ_0000700 was mainly distributed in the cytoplasm of ECA-109 and TE-1 cells. In vitro, the proliferative and migratory capacities of ECA-109 and TE-1 cells were inhibited by knocking down circ_0000700 expression. Additionally, miR-1229 silencing reversed the circ_0000700-specific siRNA-induced attenuation of malignant phenotypes. Mechanistically, circ_0000700 was identified as a sponge of miR-1229 and could activate PRRG4, REEP5, and PSMB5 indirectly to promote ESCC progression. In summary, our results suggest that hsa_circ_0000700 functions as an oncogenic factor by sponging miR-1229 in ESCC.

Promoting the differentiation of oligodendrocyte precursor cells (OPCs) is important for fostering remyelination in multiple sclerosis. Catalpol has the potential to promote remyelination and exert neuroprotective effects, but its specific mechanism is still unclear. Recent studies have shown that the NOTCH1 signaling pathway is involved in mediating OPC proliferation and differentiation. In this study, we elucidated that catalpol promoted OPC differentiation in vivo and vitro and explored the regulatory role of catalpol in specific biomolecular processes. Following catalpol administration, better and faster recovery of body weight and motor balance was observed in mice with cuprizone (CPZ)-induced demyelination. Luxol fast blue staining (LFB) and transmission electron microscopy (TEM) showed that catalpol increased the myelinated area and improved myelin ultrastructure in the corpus callosum in demyelinated mice. In addition, catalpol enhanced the expression of CNPase and MBP, indicating that it increased OPC differentiation. Additionally, catalpol downregulated the expression of NOTCH1 signaling pathway-related molecules, such as JAGGED1, NOTCH1, NICD1, RBPJ, HES5, and HES1. We further demonstrated that in vitro, catalpol enhanced the differentiation of OPCs into OLs and inhibited NOTCH1 signaling pathway activity. Our data suggested that catalpol may promote OPC differentiation and remyelination through modulation of the NOTCH1 pathway. This study provides new insight into the mechanism of action of catalpol in the treatment of multiple sclerosis.

Objective: This work proposes to predict target genes and pathways for uveal melanoma (UM) based on an ensemble method and pathway analyses.

Bloodstream infection is a major cause of morbidity and mortality worldwide. We explored whether MinION nanopore sequencing could accelerate diagnosis, resistance, and virulence profiling prediction in simulated blood samples and blood cultures. One milliliter of healthy blood samples each from direct spike (sample 1), anaerobic (sample 2), and aerobic (sample 3) blood cultures with initial inoculation of ∼30 CFU/ml of a clinically isolated Klebsiella pneumoniae strain was subjected to DNA extraction and nanopore sequencing. Hybrid assembly of Illumina and nanopore reads from pure colonies of the isolate (sample 4) was used as a reference for comparison. Hybrid assembly of the reference genome identified a total of 39 antibiotic resistance genes and 77 virulence genes through alignment with the CARD and VFDB databases. Nanopore correctly detected K. pneumoniae in all three blood samples. The fastest identification was achieved within 8 h from specimen to result in sample 1 without blood culture. However, direct sequencing in sample 1 only identified seven resistance genes (20.6%) but 28 genes in samples 2-4 (82.4%) compared to the reference within 2 h of sequencing time. Similarly, 11 (14.3%) and 74 (96.1%) of the virulence genes were detected in samples 1 and 2-4 within 2 h of sequencing time, respectively. Direct nanopore sequencing from positive blood cultures allowed comprehensive pathogen identification, resistance, and virulence genes prediction within 2 h, which shows its promising use in point-of-care clinical settings.

Oncolytic viruses are emerging as therapeutic agents in oncology. However, resistance of tumor cells to HSV oncolysis pose significant barriers to antitumor response. Thus, study on the mechanisms of therapeutic resistance to oHSV and finding strategies for overcoming these mechanisms are needed. In this study, Rab27a, a small GTPase involved in exosome biogenesis, was noticed to highly correlate with the susceptibility of tumor cells to oHSV. We found that i) lower abundance of Rab27a in oHSV resistant mouse tumor cells was shown when compared to that of sensitive tumor cells through deep-sequencing; ii) the resistance of human tumor cells to oHSV infection is associated with a downregulation of Rab27a expression and overexpression of Rab27a can promote the replication capacity of oHSV; iii) Interestingly, a stabilizer protein of Rab27a, KIBRA, highly accumulated in oHSV resistant tumor cells, which is in contrast with the expression pattern of Rab27a. Furthermore, knockdown of KIBRA expression reduced oHSV replication in oHSV resistant tumor cells. Consequently, Rab27a was found to be relevant with oHSV replication without cell type specificity, and low abundance of Rab27a contributes to oHSV resistance in both mouse and human tumor cells, which will give new insights in the identification of potential targets or biomarkers for oHSV cancer therapy.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

Molecular bases of eukaryotic circadian clocks mainly rely on transcriptional-translational feedback loops (TTFLs), while epigenetic codes also play critical roles in fine-tuning circadian rhythms. However, unlike histone modification codes that play extensive and well-known roles in the regulation of circadian clocks, whether DNA methylation (5mC) can affect the circadian clock, and the associated underlying molecular mechanisms, remains largely unexplored in many organisms. Here we demonstrate that global genome DNA hypomethylation can significantly lengthen the circadian period of Arabidopsis. Transcriptomic and genetic evidence demonstrate that SUPPRESSOR OF drm1 drm2 cmt3 (SDC), encoding an F-box containing protein, is required for the DNA hypomethylation-tuned circadian clock. Moreover, SDC can physically interact with another F-box containing protein ZEITLUPE (ZTL) to diminish its accumulation. Genetic analysis further revealed that ZTL and its substrate TIMING OF CAB EXPRESSION 1 (TOC1) likely act downstream of DNA methyltransferases to control circadian rhythm. Together, our findings support the notion that DNA methylation is important to maintain proper circadian pace in Arabidopsis, and further established that SDC links DNA hypomethylation with a proteolytic cascade to assist in tuning the circadian clock.

Retinoblastoma (RB) is an intraocular malignancy that mainly affects young children. Previous reports have demonstrated that mutations or the inactivation of the RB1 gene were the main cause of RB; however, disruption of the intracellular signaling pathways following deficiency of RB1 requires further investigation. Based on the Gene Expression Omnibus data and bioinformatics prediction, the present study aimed to investigate the microRNA (miR)‑338‑3p/neuro‑oncological ventral antigen 1 (NOVA1) axis in RB. Subsequently, overexpression and knockdown of miR‑338‑3p and NOVA1, respectively, were performed to study the role of miR‑338‑3p/NOVA1 in the progression of the RB cells. The results demonstrated that overexpression of miR‑338‑3p significantly inhibited cell proliferation, migration and invasion, and promoted apoptosis of the RB cells. Moreover, knockdown of NOVA1 showed similar results. A dual‑luciferase reporter assay and rescue experiments further confirmed the direct binding between miR‑338‑3p and NOVA1. Taken together, the results indicated that miR‑338‑3p acted as tumor suppressor by targeting the oncogene of NOVA1 in RB, which may serve as potential therapeutic targets in RB.

Sex chromosomes are a peculiar constituent of the genome because the evolutionary forces that fix the primary sex-determining gene cause genic degeneration and accumulation of junk DNA in the heterogametic partner. One of the most spectacular phenomena in sex chromosome evolution is the occurrence of neo-Y chromosomes, which lead to X1X2Y sex-determining systems. Such neo-sex chromosomes are critical for understanding the processes of sex chromosome evolution because they rejuvenate their total gene content. We assembled the male and female genomes at the chromosome level of the spotted knifejaw (Oplegnathus punctatus), which has a cytogenetically recognized neo-Y chromosome. The full assembly and annotation of all three sex chromosomes allowed us to reconstruct their evolutionary history. Contrary to other neo-Y chromosomes, the fusion to X2 is quite ancient, estimated at 48 Ma. Despite its old age and being even older in the X1 homologous region which carries a huge inversion that occurred as early as 55-48 Ma, genetic degeneration of the neo-Y appears to be only moderate. Transcriptomic analysis showed that sex chromosomes harbor 87 genes, which may serve important functions in the testis. The accumulation of such male-beneficial genes, a large inversion on the X1 homologous region and fusion to X2 appear to be the main drivers of neo-Y evolution in the spotted knifejaw. The availability of high-quality assemblies of the neo-Y and both X chromosomes make this fish an ideal model for a better understanding of the variability of sex determination mechanisms and of sex chromosome evolution.

Of all known small molecules targeting human immunodeficiency virus (HIV) capsid protein (CA), PF74 represents by far the best characterized chemotype, due to its ability to confer antiviral phenotypes in both early and late phases of viral replication. However, the prohibitively low metabolic stability renders PF74 a poor antiviral lead. We report herein our medicinal chemistry efforts toward identifying novel and metabolically stable small molecules targeting the PF74 binding site. Specifically, we replaced the inter-domain-interacting, electron-rich indole ring of PF74 with less electron-rich isosteres, including imidazolidine-2,4-dione, pyrimidine-2,4-dione, and benzamide, and identified four potent antiviral compounds (10, 19, 20 and 26) with markedly improved metabolic stability. Compared to PF74, analog 20 exhibited similar submicromolar potency, and much longer (51-fold) half-life in human liver microsomes (HLMs). Molecular docking corroborated that 20 binds to the PF74 binding site, and revealed distinct binding interactions conferred by the benzamide moiety. Collectively, our data support compound 20 as a promising antiviral lead.

Mesenchymal stem/stromal cells (MSCs) are multipotent cells with a promising application potential in regenerative medicine and immunomodulation. However, MSCs cultured in vitro exhibit functional heterogeneity. The underlying molecular mechanisms that define MSC heterogeneity remain unclear.

Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration.

BACKGROUND Mitophagy, a selective autophagy process, plays various roles in tumors. Prohibitin 2 (PHB2) is an inner-mitochondrial membrane protein that participates in parkin-induced mitophagy. However, the role of PHB2 in non-small cell lung carcinoma (NSCLC) has not been previously reported. MATERIAL AND METHODS PHB2 protein or PHB2-mRNA in NSCLC and paired normal tissues was determined by Western blot, qRT-PCR, and immunohistochemical staining. Cell proliferation was detected by CCK-8 assay. Cell migration was evaluated by wound healing and transwell migration assays. A 3D live-cell confocal system was used to monitor autophagic flux. Mitochondrial autolysosomes were observed by transmission electron microscopy (TEM). Finally, we performed JC-1 assay to measure mitochondrial membrane potential (MMP). RESULTS The level of PHB2 was significantly increased in human NSCLC specimens compared to paired adjacent specimens. Inhibition of PHB2 expression attenuated mitophagy in A549 and H1299 cells, as indicated by decreased levels of LC3 II/I and parkin markers and increased level of p62 protein. Furthermore, the inhibition caused reduction in mitochondrial autolysosomes and autophagic flux, as shown by TEM and live-cell imaging, respectively. In addition, PHB2 inhibition caused a remarkable increase in MMP and suppressed the proliferation and migration of A549 and H1299 cells. CONCLUSIONS Our results suggest that downregulation of PHB2 reduced parkin-mediated mitophagy, which suppressed proliferation and migration of A549 and H1299 cells.

RSL3 is a type of small molecular compound which can inactivate glutathione peroxidase 4 (GPX4) and induce ferroptosis, but its role in glioma cell death remains unclear. In this study, we found RSL3 inhibited the viabilities of glioma cells and induced glioma cell death in a dose-dependent manner. In vitro studies revealed that RSL3-induced cell death was accompanied with the changes of autophagy-associated protein levels and was alleviated by pretreatment of 3-Methyladenine, bafilomycin A1 and knockdown of ATG5 with siRNA. The ATP and pyruvate content as well as the protein levels of HKII, PFKP, PKM2 were decreased in cells treated by RSL3, indicating that RSL3 induced glycolysis dysfunction in glioma cells. Moreover, supplement of exterior sodium pyruvate, which was a final product of glycolysis, not only inhibited the changes of autophagy-associated protein levels caused by RSL3, but also prevented RSL3-induced cell death. In vivo data suggested that the inhibitory effect of RSL3 on the growth of glioma cells was associated with glycolysis dysfunction and autophagy activation. Taken together, RSL3 induced autophagic cell death in glioma cells via causing glycolysis dysfunction.

Endoscopic resection is increasingly performed for gastric gastrointestinal stromal tumors (GIST). However, the safety and outcomes remain elusive. We aimed in this retrospective study to compare operative complications and prognosis between endoscopically and surgically resected small (≤ 5 cm) GIST tumor groups.