Structural basis of the human negative elongation factor NELF-B/C/E ternary complex.

Negative elongation factor (NELF) is a four-subunit transcription elongation factor that mainly functions in maintaining the paused state of RNA polymerase II in eukaryotes. Upon binding to Pol II, NELF works synergistically with DRB sensitivity-inducing factor (DSIF) and inhibits transcription elongation of Pol II, which subsequently retains a stably paused state 20-60 base pairs downstream of the promoter. The promoter-proximal pausing of Pol II caused by NELF is a general mechanism of transcriptional regulation for most signal-responsive genes. To date, structural studies have significantly advanced our understanding of the molecular mechanisms of NELF. However, a high quality structural model clarifying the interaction details of this complex is still lacking. In this study, we solved the high resolution crystal structure of the NELF-B/C/E ternary complex. We observed detailed interactions between subunits and identified residues important for the association between NELF-B and NELF-E. Our work presents a precise model of the NELF complex, which will facilitate our understanding of its in vivo function.

Comprehensive analysis of differences in N6-methyladenosine RNA methylomes in the rat adenohypophysis after GnRH treatment.

N6-methyladenosine is considered to be the most common and abundant internal chemical modification among the more than 150 identified chemical RNA modifications. It is involved in most biological processes and actively participates in the regulation of animal reproduction. However, the potential function of m6 A in the pituitaries of mammals is not yet clear. It is also unknown whether m6 A is involved in the secretion and regulation of FSH by GnRH, which in turn affects mammalian reproduction. In this study, rats were treated with gonadorelin to simulate physiological GnRH-mediated regulation of FSH synthesis and secretion, and m6 A-seq was used to analyze the differential m6 A modification of the rat pituitary after gonadorelin treatment. A whole-transcriptome map of m6 A in the rat pituitary gland before and after gonadorelin treatment was successfully created. A total of 6413 differential peaks were identified, of which 3764 m6 A peaks were upregulated and 2649 m6 A peaks were downregulated. Among the 709 differentially expressed genes, 250 genes were discovered with differential methylation modifications. Intriguingly, the altered m6 A peaks within mRNAs were enriched in steroid biosynthetic processes and responses to cAMP. The results of the study will lay a foundation for further exploration of the potential role of m6 A modification in the regulation of reproductive hormone secretion and provide a theoretical basis for the application of GnRH analogs in mammalian artificial reproduction.

Polycomb-like proteins link the PRC2 complex to CpG islands.

The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.

Wnt8B, transcriptionally regulated by ZNF191, promotes cell proliferation of hepatocellular carcinoma via Wnt signaling.

Dysregulation of wingless-type (Wnt) signaling is implicated in hepatocellular carcinoma (HCC). Wnt family member 8B (Wnt8B), one of the canonical Wnt ligands, is implicated in oncogenesis. However, the role of Wnt8B in human HCCs and its transcriptional regulation mechanism are presently unknown . Here, we report that Wnt8B expression was frequently increased in HCCs and was significantly associated with poorer patient prognosis. Wnt8B knockdown suppresses HCC cell growth both in vitro and in vivo via inhibiting the canonical Wnt signaling. Zinc finger transcription factor 191 (ZNF191) can positively regulate Wnt8B mRNA and protein expression, and promoter luciferase assay indicated that ZNF191 can increase the transcription activity of the 2-Kbps WNT8B promoter. Chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assay showed that ZNF191 protein directly binds to the WNT8B promoter, and the binding sites are at nt-1491(ATTAATT) and nt-1178(ATTCATT). Moreover, Wnt8B contributes to the effect of ZNF191 on cell proliferation, and Wnt8B expression correlates positively with ZNF191 in human HCCs. Our findings suggested that Wnt8B, directly transcriptionally regulated by ZNF191, plays a pivotal role in HCC proliferation via the canonical Wnt pathway and may serve as a new prognostic biomarker and a potential therapeutic target for HCC patients.

Cyclin Y binds and activates CDK4 to promote the G1/S phase transition in hepatocellular carcinoma cells via Rb signaling.

Inactivation of Rb is a major event in the development of hepatocellular carcinoma (HCC). The activity of CDK4, determined by T172 phosphorylation, correlates with the onset of RB phosphorylation and G1/S cell cycle transition. However, the regulation of CDK4 activation and of the Rb pathway in HCC remain unclear. Here, we report that cyclin Y, a novel member of the cyclin family, is a potential regulator of the Rb pathway. We demonstrate that the Cyclin Y protein was overexpressed in human HCC tissues and that it was associated with poor patient prognosis. Cyclin Y could regulate the G1/S phase transition in human HCC cell lines. We found that CDK4 can bind to Cyclin Y in vitro. Furthermore, the accumulation of Cyclin Y could activate CDK4 through T172 phosphorylation of CDK4, inactivate Rb with increasing Rb phosphorylation, and enable the expression of E2F target genes such as CDK2 and Cyclin A. Thus, our findings suggest that Cyclin Y plays a role in the G1/S phase transition of HCC cells via Cyclin Y/CDK4/Rb signaling and that Cyclin Y could be used as a potential prognostic biomarker in HCC.

Research on the evaluation system of "vegetation-atmosphere-water" environmental coordination development under ecological management.

As environmental issues on a global scale continue to worsen, all regions are pursuing ecological management and sustainable development strategies. The coordinated development of the "vegetation-air-water" environment is one of the most essential research topics in ecological governance. The purpose of this paper is to develop an evaluation system for the development of environmental governance in Shaanxi Province, as well as to evaluate the benefits of environmental governance in Shaanxi Province from 2012 to 2021 and its influencing factors. An index system for the coordinated development of "vegetation-atmosphere-water" is constructed, and the benefits and influencing factors of environmental governance are comprehensively analysed by using comprehensive analysis methods such as the coupled coordination model of the system and entropy weight TOPSIS. The results indicate that the development trend of the coupling coordination degree has evolved through the stages of "uncoordinated development at the early stage of governance, transformed development at the middle stage of governance, and coordinated development at the first success stage of governance." In addition, we identify the obstacles to the coordinated development of the environment and suggest appropriate countermeasures. The efficacy of environmental policy governance provides recommendations for future enhancements. It is important to note that ecological governance is influenced by both policy and nature; political influences, such as the switch from "returning farmland to forests" to "returning forests to farmland," will result in the destruction of the original good vegetation growth, which will significantly reduce the coordinated benefits of ecological governance. The original coordinated system will also be fractured, which is a problem worth contemplating. And policymakers, researchers, and practitioners can use the evaluation system and analysis method proposed in this paper as an effective tool to promote sustainable development and ecological governance.

Protocol for vision transformer-based evaluation of drug potency using images processed by an optimized Sobel operator.

Conventional approaches for screening anticancer drugs rely on chemical reactions, which are time consuming, labor intensive, and costly. Here, we present a protocol for label-free and high-throughput assessment of drug efficacy using a vision transformer and a Conv2D. We describe the steps for cell culture, drug treatment, data collection, and preprocessing. We then detail the building of deep learning models and their use to predict drug potency. This protocol can be adapted for screening chemicals that affect the density or morphological features of cells. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

SIC50: Determining drug inhibitory concentrations using a vision transformer and an optimized Sobel operator.

As a measure of cytotoxic potency, half-maximal inhibitory concentration (IC50) is the concentration at which a drug exerts half of its maximal inhibitory effect against target cells. It can be determined by various methods that require applying additional reagents or lysing the cells. Here, we describe a label-free Sobel-edge-based method, which we name SIC50, for the evaluation of IC50. SIC50 classifies preprocessed phase-contrast images with a state-of-the-art vision transformer and allows for the continuous assessment of IC50 in a faster and more cost-efficient manner. We have validated this method using four drugs and 1,536-well plates and also built a web application. We anticipate that this method will assist in the high-throughput screening of chemical libraries (e.g., small-molecule drugs, small interfering RNA [siRNA], and microRNA and drug discovery).

A Novel Acyl-AcpM-Binding Protein Confers Intrinsic Sensitivity to Fatty Acid Synthase Type II Inhibitors in Mycobacterium smegmatis.

The fatty acid synthase type II (FAS-II) multienzyme system is the main target of drugs to inhibit mycolic acid synthesis in mycobacterium. Meromycolate extension acyl carrier protein (AcpM) serves as the carrier of fatty acyl chain shuttling among the individual FAS-II components during the progression of fatty acid elongation. In this paper, MSMEG_5634 in Mycobacterium smegmatis was determined to be a helix-grip structure protein with a deep hydrophobic pocket, preferring to form a complex with acyl-AcpM containing a fatty acyl chain at the C36-52 length, which is the medium product of FAS-II. MSMEG_5634 interacted with FAS-II components and presented relative accumulation at the cellular pole. By forming the MSMEG_5634/acyl-AcpM complex, which is free from FAS-II, MSMEG_5634 could transport acyl-AcpM away from FAS-II. Deletion of the MSMEG_5634 gene in M. smegmatis resulted in a mutant with decreased sensitivity to isoniazid and triclosan, two inhibitors of the FAS-II system. The isoniazid and triclosan sensitivity of this mutant could be restored by the ectopic expression of MSMEG_5634 or Rv0910, the MSMEG_5634 homologous protein in Mycobacterium tuberculosis H37Rv. These results suggest that MSMEG_5634 and its homologous proteins, forming a novel acyl-AcpM-binding protein family in mycobacterium, confer intrinsic sensitivity to FAS-II inhibitors.

LncRNA-m18as1 competitively binds with miR-18a-5p to regulate follicle-stimulating hormone secretion through the Smad2/3 pathway in rat primary pituitary cells.

Long noncoding RNAs (lncRNAs) are expressed in different species and different tissues, and perform different functions, but little is known about their involvement in the synthesis or secretion of follicle-stimulating hormone (FSH). In general, we have revealed lncRNA|‒|microRNA (miRNA)|‒||messenger RNA (mRNA) interactions that may play important roles in rat primary pituitary cells. In this study, a new lncRNA was identified for the first time. First, we analyzed the gene expression of lncRNA-m18as1 in different tissues and different stages by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and observed the localization of lncRNA-m18as1 with fluorescence in situ hybridization, which indicated that this lncRNA was distributed mainly in the cytoplasm. Next, we used RT-qPCR and enzyme-linked immunosorbent assay (ELISA) to analyze the regulation of FSH synthesis and secretion after overexpression or knockdown of lncRNA-m18as1 and found that lncRNA-m18as1 was positively correlated with FSH synthesis and secretion. In addition, mothers against decapentaplegic homolog 2 (Smad2) was highly expressed in our sequencing results. We also screened miR-18a-5p from our sequencing results as a miRNA that may bind to lncRNA-m18as1 and Smad2. We used RNA immunoprecipitation-qPCR (RIP-qPCR) and/or dual luciferase assays to confirm that lncRNA-m18as1 interacted with miR-18a-5p and miR-18a-5p interacted with Smad2. Fluorescence in situ hybridization (FISH) showed that lncRNA-m18as1 and miR-18a-5p were localized mainly in the cytoplasm. Finally, we determined the relationship among lncRNA-m18as1, miR-18a-5p, and the Smad2/3 pathway. Overall, we found that lncRNA-m18as1 acts as a molecular sponge of miR-18a-5p to regulate the synthesis and secretion of FSH through the Smad2/3 pathway.

CircRNA-WNK2 Acts as a ceRNA for miR-328a-3p to Promote AANAT Expression in the Male Rat Pineal Gland.

Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and circular RNAs (circRNAs), which are expressed with a daily rhythm in the rat pineal gland, are associated with the regulation of melatonin secretion and other biological functions. However, the mechanisms of these molecules in the rat pineal gland are not yet fully understood. In this study, we found that circR-WNK2 was highly expressed at night, which may be involved in the regulation of melatonin secretion through the competitive endogenous RNA (ceRNA) mechanism. By dual luciferase reporter, RNA pull-down, and fluorescence in situ hybridization (FISH) assays, we found that miR-328a-3p can target circR-WNK2 and the Aa-nat mRNA 3'UTR. Transfection experiments indicated that circR-WNK2 could competitively bind to miR-328a-3p, reduce miR-328a-3p expression, and promote Aa-nat gene expression and melatonin secretion. And by constructing a superior cervical ganglionectomy (SCGx) rat model, we found that ncRNAs expression in the pineal gland was regulated by signals from the suprachiasmatic nucleus. This finding supports the hypothesis that these noncoding RNAs may interact to shape the circadian rhythm through transcriptional processing in melatonin synthesis.

Advances in the Regulation of Mammalian Follicle-Stimulating Hormone Secretion.

Mammalian reproduction is mainly driven and regulated by the hypothalamic-pituitary-gonadal (HPG) axis. Follicle-stimulating hormone (FSH), which is synthesized and secreted by the anterior pituitary gland, is a key regulator that ultimately affects animal fertility. As a dimeric glycoprotein hormone, the biological specificity of FSH is mainly determined by the ß subunit. As research techniques are being continuously innovated, studies are exploring the underlying molecular mechanism regulating the secretion of mammalian FSH. This article will review the current knowledge on the molecular mechanisms and signaling pathways systematically regulating FSH synthesis and will present the latest hypothesis about the nuclear cross-talk among the various endocrine-induced pathways for transcriptional regulation of the FSH ß subunit. This article will provide novel ideas and potential targets for the improved use of FSH in livestock breeding and therapeutic development.

CircAgtpbp1 Acts as a Molecular Sponge of miR-543-5p to Regulate the Secretion of GH in Rat Pituitary Cells.

CircRNAs have been identified to be expressed differently and stably in numerous species and tissues, but their functions in growth hormone (GH) secretion are still largely unknown. In summary, we have revealed a circRNA-miRNA-mRNA network that may play a biological role in the rat pituitary gland. First, we verified the chromosome location information of circAgtpbp1 according to sequencing analysis. The circAgtpbp1 characteristics were authenticated through PCR, qRT-PCR, treating with RNase and fluorescent in situ hybridization (FISH). Second, we detected the expression pattern of circAgtpbp1 in the rat anterior pituitary by qRT-PCR. We also designed circAgtpbp1 siRNA and constructed overexpression plasmid to evaluate the effect of circAgtpbp1 function on GH secretion by qRT-PCR, ELISA and Western blot. CircAgtpbp1 is a stable, truly circular molecule. We found that circAgtpbp1 interacted with miR-543-5p and can regulate GH secretion in pituitary cells through a circAgtpbp1-miR-543-5p-GH axis. Overall, the evidence generated by our study suggests that circAgtpbp1 can act as a sponge of miR-543-5p to reduce the inhibitory effect of miR-543-5p on Gh1 and further promote GH secretion. These findings expand our existing knowledge on the mechanisms of hormone regulation in the pituitary gland.

Clinical significance of tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma.

Circulating tumor cells (CTCs) are suspected of predicting the prognosis of malignant tumor, but there are few relevant reports specific to esophageal squamous cell carcinoma (ESCC). This study investigated the clinical significance of CTCs in patients with ESCC.Sixty patients with ESCC were enrolled, from whom CTCs had been tested by our team previously. Peripheral blood samples were obtained from these patients before treatment; and CTCs were assayed by isolation by size of epithelial tumor cells (ISET). Associations between the presence of CTCs and patients' clinicopathological parameters and clinical outcomes were analyzed.CTCs were detected in 20 patients (33.3%), who experienced significantly shorter progression-free survival (PFS) than did the CTC-negative patients. Overall, PFS was negatively associated with the number of CTCs. Multivariate analyses showed that a CTC count >2 was a strong independent prognostic indicator of tumor recurrence (hazard ratio [HR] 5.63; 95% confidence interval [CI] 1.77-17.89; P = .003). In the subgroup of 50 patients who underwent R0 resection and postoperative adjuvant radiotherapy or chemotherapy, CTC was a strong, independent, and prognostic indicator of tumor recurrence (HR 10.70; 95% CI, 1.40-81.91; P = .022). The number of CTCs correlated with the T stage (r = 0.26, P = .043) but not with the N or M stage. For subgroups in stages II or I-IIIB or T3 or T3 + T4, the PFS of patients with CTCs > 1 or > 2 was significantly shorter than that of the patients with CTCs ≤ 1 or CTCs ≤ 2. In the stage III or T3 + T4 groups, the PFS of patients with CTCs > 0 was significantly shorter than that of patients with CTC = 0.This is the first study to report that the CTC detected by ISET is an independent and prognostic indicator of patients' outcome in ESCC. Consideration of CTCs may improve the accuracy of preoperative staging in ESCC.

Autopsy interrogation of emergency medicine dispute cases: how often are clinical diagnoses incorrect?

AIMS: Emergency medicine is a 'high risk' specialty. Some diseases develop suddenly and progress rapidly, and sudden unexpected deaths in the emergency department (ED) may cause medical disputes. We aimed to assess discrepancies between antemortem clinical diagnoses and postmortem autopsy findings concerning emergency medicine dispute cases and to figure out the most common major missed diagnoses. METHODS: Clinical files and autopsy reports were retrospectively analysed and interpreted. Discrepancies between clinical diagnoses and autopsy diagnoses were evaluated using modified Goldman classification as major and minor discrepancy. The difference between diagnosis groups was compared with Pearson χ2 test. RESULTS: Of the 117 cases included in this study, 71 of cases (58 class I and 13 class II diagnostic errors) were revealed as major discrepancies (60.7%). The most common major diagnoses were cardiovascular diseases (54 cases), followed by pulmonary diseases, infectious diseases and so on. The difference of major discrepancy between the diagnoses groups was significant (p<0.001). Aortic dissection and myocardial infarction were the most common cause of death (15 cases for each disease) and the most common missed class I diagnoses (80% and 66.7% for each), higher than the average 49.6% of all class I errors of the study patients. CONCLUSIONS: High major disparities between clinical diagnoses and postmortem examinations exist in emergency medical disputes cases; acute aortic dissection and myocardial infarction are the most frequently major missed diagnoses that ED clinicians should pay special attention to in practice. This study reaffirmed the necessity and usefulness of autopsy in auditing death in EDs.

Effects of different sequences of pulmonary artery and vein ligations during pulmonary lobectomy on blood micrometastasis of non-small cell lung cancer.

The aim of this study was to investigate the effects of different sequences of pulmonary artery and vein ligations during lobectomy on blood micrometastasis of non-small cell lung cancer (NSCLC). Cytokeratin 19 (CK19)/adhesion molecule CD44v6 mRNA were used as markers. A total of 30 NSCLC patients undergoing pulmonary lobectomy were randomly divided into pulmonary artery (PA)-first and pulmonary vein (PV)-first groups according to the order of artery or vein ligation (15 cases in each). Fluorescent quantitative-RT-PCR (FQ-RT-PCR) was used to detect the mRNA expression of CK19 and CD44v6 in pulmonary venous blood at the early and late periods during surgery, and ΔCt values were calculated. Meanwhile, the peripheral blood samples from 10 healthy volunteers were selected as the control. ΔCt values of CD44v6 and CK19 of NSCLC groups at the early period during surgery were 7.83±1.70 and 10.76±2.74, while those of the control group were 9.17±1.04 and 12.76±2.36. The expression of CD44v6 and CK19 genes in venous blood of NSCLC groups was significantly higher than that of the control group (P<0.05). In addition, the ΔCt values of CD44v6 and CK19 in the early and late periods during surgery in the PA-first group were 7.92±1.97 vs. 5.67±2.11 (P= 0.008) and 11.21±3.14 vs. 8.60±4.02 (P= 0.05), respectively. The expression of CD44v6 and CK19 in the late period were both significantly higher than those in the early period, while neither the ΔCt value of CD44v6 nor that of CK19 in the early vs. late periods in the PV-first group exhibited statistically significant differences (7.95±1.91 vs. 7.74±2.10 and 10.60±3.15 vs. 10.30±2.98) (P<0.05). Surgical manipulation itself may stimulate the occurrence of blood micrometastasis and the ligation of the PV first during surgery may help prevent blood micrometastasis.

Using 18F-fluorodeoxyglucose positron emission tomography to estimate the length of gross tumor in patients with squamous cell carcinoma of the esophagus.

PURPOSE: To determine the optimal method of using (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) to estimate gross tumor length in esophageal carcinoma. METHODS AND MATERIALS: Thirty-six patients with esophageal squamous cell carcinoma treated with radical surgery were enrolled. Gross tumor volumes (GTVs) were delineated using three different methods: visual interpretation, standardized uptake value (SUV) 2.5, and 40% of maximum standard uptake value (SUV(max)) on FDG-PET imaging. The length of tumors on PET scan were measured and recorded as Length(vis), Length(2.5), and Length(40), respectively, and compared with the length of gross tumor in the resected specimen (Length(gross)). All PET data were reviewed again postoperatively, and the GTV was delineated using various percentages of SUV(max). The optimal-threshold SUV was generated when the length of PET matched the Length(gross). RESULTS: The mean (+/-SD) Length(gross) was 5.48 +/- 1.98 cm. The mean Length(vis), Length(2.5), and Length(40) were 5.18 +/- 1.93 cm, 5.49 +/- 1.79 cm, and 4.34 +/- 1.54 cm, respectively. The mean Length(vis) (p = 0.123) and Length(2.5) (p = 0.957) were not significantly different from Length(gross), and Length(2.5) seems more approximate to Length(gross.) The mean Length(40) was significantly shorter than Length(gross) (p < 0.001). The mean optimal threshold was 23.81% +/- 11.29% for all tumors, and it was 19.78% +/- 8.59%, 30.92% +/- 12.28% for tumors >/=5 cm, and <5 cm, respectively (p = 0.009). The correlation coefficients of the optimal threshold were -0.802 and -0.561 with SUV(max) and Length(gross), respectively. CONCLUSIONS: The optimal PET method to estimate the length of gross tumor varies with tumor length and SUV(max); an SUV cutoff of 2.5 provided the closest estimation in this study.