Pleiotropy of positive selection in ancient ACE2 suggests an alternative hypothesis for bat-specific adaptations to host coronaviruses.

Angiotensin-convertingenzyme 2 (ACE2) has dual functions, regulating cardiovascular physiology and serving as the receptor for coronaviruses. Bats, the only true flying mammals and natural viral reservoirs, have evolved positive alterations in traits related to both functions of ACE2. This suggests significant evolutionary changes in ACE2 during bat evolution. To test this hypothesis, we examine the selection pressure in ACE2 along the ancestral branch of all bats (AncBat-ACE2), where powered flight and bat-coronavirus coevolution occurred, and detect a positive selection signature. To assess the functional effects of positive selection, we resurrect AncBat-ACE2 and its mutant (AncBat-ACE2-mut) created by replacing the positively selected sites. Compared to AncBat-ACE2-mut, AncBat-ACE2 exhibits stronger enzymatic activity, enhances mice's performance in exercise fatigue, and shows lower affinity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our findings indicate the functional pleiotropy of positive selection in the ancient ACE2 of bats, providing an alternative hypothesis for the evolutionary origin of bats' defense against coronaviruses.

The Development of a CRISPR-FnCpf1 System for Large-Fragment Deletion and Multiplex Gene Editing in Acinetobacter baumannii.

Acinetobacter baumannii is a low-GC-content Gram-negative opportunistic pathogen that poses a serious global public health threat. Convenient and rapid genetic manipulation is beneficial for elucidating its pathogenic mechanisms and developing novel therapeutic methods. In this study, we report a new CRISPR-FnCpf1-based two-plasmid system for versatile and precise genome editing in A. baumannii. After identification, this new system prefers to recognize the 5'-TTN-3' (N = A, T, C or G) and the 5'-CTV-3' (V = A, C or G) protospacer-adjacent motif (PAM) sequence and utilize the spacer with lengths ranging from 19 to 25 nt. In direct comparison with the existing CRISPR-Cas9 system, it exhibits approximately four times the targetable range in A. baumannii. Moreover, by employing a tandem dual crRNA expression cassette, the new system can perform large-fragment deletion and simultaneous multiple gene editing, which is difficult to achieve via CRISPR-Cas9. Therefore, the new system is valuable and can greatly expand the genome editing toolbox of A. baumannii.

SARS-CoV-2 NSP12 utilizes various host splicing factors for replication and splicing regulation.

The RNA-dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. In this study, the biochemical and cell biological results demonstrate the interactions between SARS-CoV-2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8). The entry efficacy of SARS-CoV-2 considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. Furthermore, the polymerase activity and stability of SARS-CoV-2 RdRp were affected by the three splicing factors to varying degrees. In addition, NSP12 and its homologues from SARS-CoV and MERS-CoV suppressed the alternative splicing of cellular genes, which were influenced by the three splicing factors. Overall, our research illustrates that SARS-CoV-2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies.

Pyroptosis-related gene signature elicits immune response in rosacea.

Rosacea is a complex chronic inflammatory skin disorder with high morbidity. Pyroptosis is known as a regulated inflammatory cell death. While its association with immune response to various inflammatory disorders is well established, little is known about its functional relevance of rosacea. So, we aimed to explore and enrich the pathogenesis involved in pyroptosis-related rosacea aggravations. In this study, we evaluated the pyroptosis-related patterns of rosacea by consensus clustering analysis of 45 ferroptosis-related genes (FRGs), with multiple immune cell infiltration analysis to identify the pyroptosis-mediated immune response in rosacea using GSE65914 dataset. The co-co-work between PRGs and WGCNA-revealed hub genes has established using PPI network. FRG signature was highlighted in rosacea using multi-transcriptomic and experiment analysis. Based on this, three distinct pyroptosis-related rosacea patterns (non/moderate/high) were identified, and the notably enriched pathways have revealed through GO, KEGG and GSEA analysis, especially immune-related pathways. Also, the XCell/MCPcount/ssGSEA/Cibersort underlined the immune-related signalling (NK cells, Monocyte, Neutrophil, Th2 cells, Macrophage), whose hub genes were identified through WGCNA (NOD2, MYD88, STAT1, HSPA4, CXCL8). Finally, we established a pyroptosis-immune co-work during the rosacea aggravations. FRGs may affect the progression of rosacea by regulating the immune cell infiltrations. In all, pyroptosis with its mediated immune cell infiltration is a critical factor during the development of rosacea.

Portal vein velocity predicts portal vein system thrombosis after splenectomy with esophagogastric devascularization.

BACKGROUND: Portal vein system thrombosis (PVST) is a potentially fatal complication after splenectomy with esophagogastric devascularization (SED) in cirrhotic patients with portal hypertension. However, the impact of portal vein velocity (PVV) on PVST after SED remains unclear. Therefore, this study aims to explore this issue. METHODS: Consecutive cirrhotic patients with portal hypertension who underwent SED at Tongji Hospital between January 2010 and June 2022 were enrolled. The patients were divided into two groups based on the presence or absence of PVST, which was assessed using ultrasound or computed tomography after the operation. PVV was measured by duplex Doppler ultrasound within one week before surgery. The independent risk factors for PVST were analyzed using univariate and multivariate logistic regression analysis. A nomogram based on these variables was developed and internally validated using 1000 bootstrap resamples. RESULTS: A total of 562 cirrhotic patients with portal hypertension who underwent SED were included, and PVST occurred in 185 patients (32.9%). Multivariate logistic regression analysis showed that PVV was the strongest independent risk factor for PVST. The incidence of PVST was significantly higher in patients with PVV ≤ 16.5 cm/s than in those with PVV > 16.5 cm/s (76.2% vs. 8.5%, p < 0.0001). The PVV-based nomogram was internally validated and showed good performance (optimism-corrected c-statistic = 0.907). Decision curve and clinical impact curve analyses indicated that the nomogram provided a high clinical benefit. CONCLUSION: A nomogram based on PVV provided an excellent preoperative prediction of PVST after splenectomy with esophagogastric devascularization.

Carbon dioxide production index (VCO2i) predicts hyperlactatemia during cardiopulmonary bypass in pediatric carDiac surGery (pGDP- VCO2i): Study protocol for a nested case-control trial.

BACKGROUND: Hyperlactatemia (HL) during cardiopulmonary bypass (CPB) is relatively frequent in infants and associates with increased morbidity and mortality. Studies on adults have shown that carbon dioxide production index (VCO2i) during CPB is linked to the occurrence of HL, with 'critical thresholds' for VCO2i reported to be 60 mL/min/m2. However, considering infants have a higher metabolic rate and lower tolerance to hypoxia, the critical threshold of VCO2i in infants cannot be replied to the existing adults' standards. The objective of this study is to investigate the association of VCO2i during CPB and HL, and explore the critical VCO2i threshold during CPB in infants. METHODS: VCO2i predicts hyperlactatemia during cardiopulmonary bypass in pediatric cardiac surgery (pGDP-VCO2i) is a nested case-control study. A cohort of consecutive pediatric patients of less than 3 years of age, undergoing congenital cardiac surgeries between May 2021 and December 2023 in West China Hospital will be enrolled. The VCO2i levels of each patient will be recorded every 5 min during CPB. The primary outcome is the rate of HL. The infants will be divided into two groups based on the presence or not of HL. Pre- and intraoperative factors will be tested for independent association with HL. Then, we will make an analysis, and the critical value of VCO2i will be obtained. The postoperative outcome of patients with or without HL will be compared. DISCUSSION: This will be the first trial to investigate the association of VCO2i during CPB and HL, and explore the critical VCO2i threshold during CPB in pediatrics. The results of this study are expected to lay a foundation for clinical application of goal-directed perfusion (GDP) management strategy, and optimize the perfusion strategy and improve the prognosis of pediatric patients undergoing cardiac surgery. TRIAL REGISTRATION: Chictr.org.cn, ChiCTR2100044296 on 16 March 2021.

[Microcirculatory visualization and metabolomics of Dalbergia cochinchinensis in ameliorating coronary microvascular dysfunction in rats].

This study employed microcirculation visualization and metabolomics methods to explore the effect and possible mechanism of Dalbergia cochinchinensis in ameliorating coronary microvascular dysfunction(CMD) induced by microsphere embolization in rats. Sixty SPF-grade male SD rats were randomized into sham, model, and low-, medium-, and high-dose [1.5, 3.0, and 6.0 g·kg~(-1)·d~(-1), respectively] D. cochinchinensis water extract groups. The rats in sham and model groups were administrated with equal volume of normal saline by gavage once a day for 7 consecutive days. The rat model of CMD was prepared by injecting polyethylene microspheres into the left ventricle, while the sham group was injected with an equal amount of normal saline. A blood flow meter was used to measure blood flow, and a blood rheometer to measure blood viscosity and fibrinogen content. An automatic biochemical analyzer and reagent kits were used to measure the serum levels of myocardial enzymes, glucose, and nitric oxide(NO). Hematoxylin-eosin(HE) staining was used to observe the pathological changes of myocardial tissue. DiI C12/C18 perfusion was used to infuse coronary microvessels, and the structural and morphological changes were observed using a confocal laser scanning microscope. AngioTool was used to analyze the vascular area, density, radius, and mean E lacunarity in the microsphere embolization area, and vascular blood flow resistance was calculated based on Poiseuille's law. Non-targeted metabolomics based on high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed screen potential biomarkers and differential metabolites regulated by D. cochinchinensis and the involved metabolic pathways were enriched. The pharmacodynamic results showed that compared with the model group, D. cochinchinensis significantly increased mean blood flow, reduced plasma fibrinogen content, lowered the levels of myocardial enzymes such as creatine kinase(CK), creatine kinase-MB(CK-MB), and lactate dehydrogenase(LDH), alleviate myocardial injury, and protect damaged myocardium. In addition, D. cochinchinensis significantly increased serum NO content, promoted vascular smooth muscle relaxation, dilated blood vessels, lowered serum glucose(GLU) level, improved myocardial energy metabolism, and alleviated pathological changes in myocardial fibrosis and inflammatory cell infiltration. The results of coronary microcirculation perfusion showed that D. cochinchinensis improved the vascular morphology, increased the vascular area, density, and radius, reduced vascular mean E lacunarity and blood flow resistance, and alleviated vascular endothelial damage in CMD rats. The results of metabolomics identified 45 differential metabolites between sham and model groups, and D. cochinchinensis recovered the levels 25 differential metabolites, which were involved in 8 pathways including arachidonic acid metabolism, arginine biosynthesis, and sphingolipids metabolism. D. cochinchinensis can ameliorate coronary microcirculation dysfunction caused by microsphere embolization in rats, and it may alleviate the pathological changes of CMD rats by regulating inflammatory reaction, endothelial damage, and phospholipid metabolism.

Teraryl Braces in Macrocycles: Synthesis and Conformational Landscape Remodeling of Peptides.

The three-dimensional structure of medium-sized cyclic peptides accounts for their biological activity and other important physiochemical properties. Despite significant advances in the past few decades, chemists' ability to fine-tune the structure, in particular, the backbone conformation, of short peptides made of canonical amino acids is still quite limited. Nature has shown that cross-linking the aromatic side chains of linear peptide precursors via enzyme catalysis can generate cyclophane-braced products with unusual structures and diverse activities. However, the biosynthetic path to these natural products is challenging to replicate in the synthetic laboratory using practical chemical modifications of peptides. Herein, we report a broadly applicable strategy to remodel the structure of homodetic peptides by cross-linking the aromatic side chains of Trp, His, and Tyr residues with various aryl linkers. The aryl linkers can be easily installed via copper-catalyzed double heteroatom-arylation reactions of peptides with aryl diiodides. These aromatic side chains and aryl linkers can be combined to form a large variety of assemblies of heteroatom-linked multi-aryl units. The assemblies can serve as tension-bearable multijoint braces to modulate the backbone conformation of peptides as an entry to previously inaccessible conformational space.

VPS37C facilitates erythroid differentiation by promoting EKLF stability.

The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.

A machine learning framework develops a DNA replication stress model for predicting clinical outcomes and therapeutic vulnerability in primary prostate cancer.

Recent studies have identified DNA replication stress as an important feature of advanced prostate cancer (PCa). The identification of biomarkers for DNA replication stress could therefore facilitate risk stratification and help inform treatment options for PCa. Here, we designed a robust machine learning-based framework to comprehensively explore the impact of DNA replication stress on prognosis and treatment in 5 PCa bulk transcriptomic cohorts with a total of 905 patients. Bootstrap resampling-based univariate Cox regression and Boruta algorithm were applied to select a subset of DNA replication stress genes that were more clinically relevant. Next, we benchmarked 7 survival-related machine-learning algorithms for PCa recurrence using nested cross-validation. Multi-omic and drug sensitivity data were also utilized to characterize PCa with various DNA replication stress. We found that the hyperparameter-tuned eXtreme Gradient Boosting model outperformed other tuned models and was therefore used to establish a robust replication stress signature (RSS). RSS demonstrated superior performance over most clinical features and other PCa signatures in predicting PCa recurrence across cohorts. Lower RSS was characterized by enriched metabolism pathways, high androgen activity, and a favorable prognosis. In contrast, higher RSS was significantly associated with TP53, RB1, and PTEN deletion, exhibited increased proliferation and DNA replication stress, and was more immune-suppressive with a higher chance of immunotherapy response. In silico screening identified 13 potential targets (e.g. TOP2A, CDK9, and RRM2) from 2249 druggable targets, and 2 therapeutic agents (irinotecan and topotecan) for RSS-high patients. Additionally, RSS-high patients were more responsive to taxane-based chemotherapy and Poly (ADP-ribose) polymerase inhibitors, whereas RSS-low patients were more sensitive to androgen deprivation therapy. In conclusion, a robust machine-learning framework was used to reveal the great potential of RSS for personalized risk stratification and therapeutic implications in PCa.

A retrospective assessment of real-world experience with venetoclax and azacitidine therapy in elderly acute myeloid leukemia.

This study aimed to examine the effect of venetoclax coupled with azacytidine in treating older adults with relapsed and refractory (R/R) acute myeloid leukemia (AML). The clinical data of 10 senior patients with AML over 65 years old who were treated with venetoclax and azacytidine, including six patients with R/R AML, were retrospectively evaluated. This study comprised seven males and three females with a median age of 71 years. Five patients had at least one relapse, and one patient did not achieve remission after four cycles of azacytidine monotherapy, considering it resistant. AML with myelodysplasia-related changes was found in four cases. One of the 10 patients died early after 1-13 cycles of venetoclax plus azacytidine treatment due to a protracted period of neutropenia and severe lung infection induced by medications. Six of the remaining nine patients, including six R/R patients, achieved a complete remission (CR) or a CR with incomplete hematologic recovery (CRi). After two cycles of therapy, one patient did not react. Neutropenia lasted an average of 10.5 (6-15) days in all patients, with the most severe cases occurring in the second and third weeks of therapy. Three patients who tested positive for the TP53 gene mutation had the following outcomes: One relapsed patient has been in progression-free remission (PFS) for the past 24 months, whereas another has been in full remission but relapsed 2 months later. Another patient experienced complete remission in myelology for 4 months, but the variable allele fraction (VAF) value steadily rose, suggesting that the illness was on the verge of progressing. IDH2 gene alterations were found in three of four patients who obtained maintained CR for more than 18 months following recurrence. Venetoclax in combination with azacytidine is a successful and well-tolerated therapy for R/R AML in the elderly. Venetoclax and azacytidine may help patients with TP53 mutations and reduce VAF. The IDH2 mutation might be a good predictor of veneclax sensitivity. A notable adverse response in the treatment phase of the regimen is severe infection induced by neutropenia.

HCC treated with immune checkpoint inhibitors: a hyper-enhanced rim on Sonazoid-CEUS Kupffer phase images is a predictor of tumor response.

OBJECTIVES: We aimed to evaluate the efficacy of anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody therapy by assessing the hyper-enhanced rim phenomenon of hepatocellular carcinoma (HCC) on Sonazoid-contrast-enhanced ultrasound (CEUS) Kupffer phase images. METHODS: This retrospective study included 61 patients with HCC who received anti-PD-1/PD-L1 antibody therapy from August 1, 2020, to January 31, 2022. We compared the progression-free survival (PFS) of patients with hyper-enhanced rim+ and hyper-enhanced rim-nodules and the time to nodule progression (TTnP) of hyper-enhanced rim+ and hyper-enhanced rim- nodules. RESULTS: Thirty-nine patients received postoperative therapy, and 22 patients had unresectable HCC. The mean PFS was 11.8 months (95% confidence interval [CI]: 8.7-14.9) for patients with hyper-enhanced rim+ HCC nodules and 16.5 months (95% CI: 14.9-18.1) for patients with hyper-enhanced rim- HCC nodules in the surgery group (p = 0.017). The mean PFS was 9.2 months (95% CI: 3.6-14.8) for patients with hyper-enhanced rim+ HCC nodules and 17.8 months (95% CI: 14.9-20.6) for patients with hyper-enhanced rim- HCC nodules in the non-surgery group (p = 0.015). For hyper-enhanced rim+ HCC nodules, TTnP for each nodule exceeding the specified threshold was 10.1 months, whereas that for hyper-enhanced rim- HCC nodules was 17.6 months (p = 0 .018). The disease control rate was 42.9% (3/7) for hyper-enhanced rim+ HCC nodules and 85.7% (21/24) for hyper-enhanced rim- HCC nodules (p = 0.013). CONCLUSIONS: The presence of hyper-enhanced rim on the Kupffer phase images obtained via the non-invasive Sonazoid-CEUS is a promising imaging biomarker for predicting unfavorable response with anti-PD-1/PD-L1 therapy in patients with HCC. KEY POINTS: • The mean progression-free survival was 11.8 months for patients with hyper-enhanced rim+ HCC nodules and 16.5 months for patients with hyper-enhanced rim- HCC nodules in the surgery group. • The mean progression-free survival was 9.2 months for patients with hyper-enhanced rim+ HCC nodules and 17.8 months for patients with hyper-enhanced rim- HCC nodules in the non-surgery group. • The disease control rate was 42.9% for hyper-enhanced rim+ HCC nodules and 85.7% for hyper-enhanced rim- HCC nodules (p = 0.013).

Activating α7nAChR helps post-myocardial infarction healing by regulating macrophage polarization via the STAT3 signaling pathway.

BACKGROUND: Monocytes/macrophages play critical roles in inflammation and cardiac remodeling following myocardial infarction (MI). The cholinergic anti-inflammatory pathway (CAP) modulates local and systemic inflammatory responses by activating α7 nicotinic acetylcholine receptors (α7nAChR) in monocytes/macrophages. We investigated the effect of α7nAChR on MI-induced monocyte/macrophage recruitment and polarization and its contribution to cardiac remodeling and dysfunction. METHODS: Adult male Sprague Dawley rats underwent coronary ligation and were intraperitoneally injected with the α7nAChR-selective agonist PNU282987 or the antagonist methyllycaconitine (MLA). RAW264.7 cells were stimulated with lipopolysaccharide (LPS) + interferon-gamma (IFN-γ) and treated with PNU282987, MLA, and S3I-201 (a STAT3 inhibitor). Cardiac function was evaluated by echocardiography. Masson's trichrome and immunofluorescence were used to detect cardiac fibrosis, myocardial capillary density, and M1/M2 macrophages. Western blotting was used to detect protein expression, and the proportion of monocytes was measured using flow cytometry. RESULTS: Activating the CAP with PNU282987 significantly improved cardiac function and reduced cardiac fibrosis and 28-day mortality after MI. On days 3 and 7 post-MI, PNU282987 reduced the percentage of peripheral CD172a + CD43low monocytes and the infiltration of M1 macrophages in the infarcted hearts, whereas it increased the recruitment of peripheral CD172a + CD43high monocytes and M2 macrophages. Conversely, MLA exerted the opposite effects. In vitro, PNU282987 inhibited M1 macrophage polarization and promoted M2 macrophage polarization in LPS + IFN-γ-stimulated RAW264.7 cells. These PNU282987-induced changes in LPS + IFN-γ-stimulated RAW264.7 cells were reversed by administering S3I-201. CONCLUSION: Activating α7nAChR inhibits the early recruitment of pro-inflammatory monocytes/macrophages during MI and improves cardiac function and remodeling. Our findings suggest a promising therapeutic target for regulating monocyte/macrophage phenotypes and promoting healing after MI.

Association between increased serum interleukin-8 levels and improved cognition in major depressive patients with SSRIs.

BACKGROUND: The effect of neuroinflammatory cytokines on cognitive deficits in patients with major depressive disorder (MDD) can be altered by selective serotonin reuptake inhibitors (SSRIs). This study aimed to examine serum interleukin-8 (IL-8) levels, cognitive function, and their associations in MDD patients with SSRIs. METHODS: Thirty SSRI-treated MDD patients and 101 healthy controls were recruited for this study. We examined cognitive performance using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and serum IL-8 levels using the Human Inflammatory Cytokine Cytometric Bead Array in both cases and controls. RESULTS: The RBANS test scores were significantly lower in MDD patients with SSRIs than in healthy controls after controlling for covariates (all p < 0.001). Serum levels of IL-8 were higher in MDD patients with SSRIs than in healthy controls after adjusting for covariates (F = 3.82, p = 0.05). Serum IL-8 levels were positively correlated with sub-scores of delayed memory (r = 0.37, p = 0.04) and visuospatial/constructional (r = 0.43, p = 0.02) in MDD patients with SSRIs but not in in healthy controls (delayed memory score: r = -0.12, p = 0.24; visuospatial/constructional score: r = 0.02, p = 0.81). CONCLUSIONS: Our findings suggested that increased serum IL-8 level might not only be involved in the MDD psychopathology or the use of SSRIs but also correspond to improving MDD delayed memory and visuospatial/constructional function.

Detection of aberrant DNA methylation patterns in sperm of male recurrent spontaneous abortion patients.

Aberrant DNA methylation patterns in sperm are a cause of embryonic failure and infertility, and could be a critical factor contributing to male recurrent spontaneous abortion (RSA). The purpose of this study was to reveal the potential effects of sperm DNA methylation levels in patients with male RSA. We compared sperm samples collected from fertile men and oligoasthenospermia patients. Differentially methylated sequences were identified by reduced representation bisulfite sequencing (RRBS) methods. The DNA methylation levels of the two groups were compared and qRT-PCR was used to validate the expression of genes showing differential methylation. The results indicated that no difference in base distribution was observed between the normal group and the patient group. However, the chromosome methylation in these two groups was markedly different. One site was located on chromosome 8 and measured 150 bp, while the other sites were on chromosomes 9, 10, and X and measured 135 bp, 68 bp, and 136 bp, respectively. In particular, two genes were found to be hypermethylated in these patients, one gene was DYDC2 (placed in the differential methylation region of chromosome 10), and the other gene was NXF3 (located on chromosome X). Expression levels of DYDC2 and NXF3 in the RSA group were significantly lower than those in the normal group (P < 0.05). Collectively, these results demonstrated that changes in DNA methylation might be related to male RSA. Our findings provide important information regarding the potential role of sperm DNA methylation in human development.

The possible impact of the universal two-child policy on pregnancy outcomes.

PURPOSE: The implementation of the universal two-child policy contributes to adverse pregnancy outcomes, but how the policy change leads to adverse pregnancy outcomes is not well elaborated. In this study, we aimed to compare maternal characteristics and complications, accessed the change in the proportion of maternal characteristics and maternal complications, and evaluated the mediation of maternal characteristics on maternal complications. METHODS: Demographic and clinical data of three-level sample facilities were extracted from China's National Maternity Near Miss Obstetrics Surveillance System from Jan 1, 2012 to May 31, 2021. The associations between the universal two-child policy and maternal risk factors, the universal two-child policy and maternal complications, and maternal risk factors and maternal complications were evaluated using multivariate logistic regression analyses, with odds ratios (ORs) and 95% confidence intervals (CIs). Mediation analysis was used to estimate the potential mediation effects on the associations between the policy and maternal complications. Population-attributable fractions (PAF) were conducted to quantify the maternal complications burden attributable to the implementation of the universal two-child policy. RESULTS: In the context of the universal two-child policy, the incidence of maternal near miss, antepartum or intrapartum complication, and post-partum complication increased at municipal- and county-level sample facilities. After adjusting for covariables, there were significant associations between the universal two-child policy and maternal risk factors (P < 0.001), the universal two-child policy and an increased risk of maternal complications (P < 0.001), and maternal risk factors and maternal complications(P < 0.001). The effects of the universal two-child policy on maternal near miss and medical disease were significantly mediated by maternal risk factors with mediation proportions of 19.77% and 4.07% at the municipal-level sample facility, and mediation proportions for 2.72% at the county-level sample facility on medical disease. The universal two-child policy contributed 19.34%, 5.82%, 8.29%, and 46.19% in the incidence of the maternal near miss, antepartum or intrapartum complication, post-partum complication, and medical disease at municipal-level sample facility, respectively. The corresponding PAF% at county-level sample facility was 40.49% for maternal near miss, 32.39% for the antepartum or intrapartum complication, 61.44% for post-partum complication, and 77.72% for medical disease. For provincial-level sample facility, the incidence of maternal near miss, antepartum or intrapartum complications, and medical diseases decreased (P < 0.05) and no statistically significant difference occurred in the incidence of post-partum complications. CONCLUSIONS: In the context of the universal two-child policy, the incidence of maternal near miss, antepartum or intrapartum complication, and post-partum complication increased at municipal- and county-level sample facility. Maternal risk factors may play a mediating role in the effect of policy change and maternal complications. Provincial hospitals have been able to improve the quality of perinatal health care and reduce adverse pregnancy outcomes by adjusting their obstetric service strategies in the context of the new birth policy.

Synthesis and anti-HIV activity of non-nucleoside reverse-transcriptase inhibitor DB02 phosphate derivatives based on water-soluble optimization.

To improve the water solubility of anti-human immunodeficiency virus (HIV) agent DB02, an excellent non-nucleoside reverse-transcriptase inhibitor (NNRTI) obtained in our previous efforts, we designed and synthesized four phosphate derivatives of DB02 based on the molecular model of DB02 with RT. Here, the antiviral activity of these four derivatives was detected, leading to the discovery of compound P-2, which possessed a superior potency to the lead compound DB02 against wild-type HIV-1 and a variety of HIV-resistant mutant viruses significantly. Furthermore, the water solubility of P-2 was nearly 17 times higher than that of DB02, and the pharmacokinetic test in rats showed that P-2 demonstrate significantly improved oral bioavailablity of 14.6%. Our study showed that the introduction of a phosphate ester group at the end of the C-2 side chain of DB02 was beneficial to the improvement of its antiviral activity and pharmacokinetic properties, which provided a promising lead for the further development of S-DACOs type of NNRTIs.

[ZEB2 Regulates the Migration and Invasion of PANC-1 Pancreatic Cancer Cells: An Experimental Study].

Objective: To investigate the effects and mechanisms of zinc finger E-box binding homeobox transcription factor-2 ( ZEB2) on the proliferation, colony formation, migration, and invasion abilities and the epithelial-mesenchymal transition (EMT) of PANC-1 cells, a human pancreatic cancer cell line. Methods: Data on the expression of ZEB2 in pancreatic cancer tissues and paracancerous tissues from The Cancer Genome Atlas (TCGA) database were analyzed. PANC-1 pancreatic cancer cells were divided into si-NC group, si- ZEB2 group, pcDNA3.1 group, and pcDNA3.1- ZEB2 group. qRT-PCR and Western blot were conducted to confirm the effectiveness of ZEB2 knockdown or overexpression. CCK-8, colony formation, wound healing, and Transwell assays were conducted to examine the effects of ZEB2 on the proliferation, colony formation, migration, and invasion of PANC-1 cells. qRT-PCR and immunofluorescence assays were performed to examine the expression of E-cadherin and vimentin, the EMT markers, in the cells. Prediction of proteins interacting with ZEB2 was made through the STRING database. Results: TCGA database analysis showed that the expression level of ZEB2 in pancreatic cancer tissues was significantly higher than that in adjacent tissues ( P<0.05). Compared with those of cells in the control group, the proliferation, colony formation, migration, and invasion of cells in the si- ZEB2 group were decreased ( P<0.05). Compared with those of cells in the pcDNA3.1 group, the proliferation, colony formation, migration and invasion of cells in the pcDNA3.1- ZEB2 group were increased (all P<0.05). According to the results of qRT-PCR and immunofluorescence assays, compared with those of the si-NC group, the expression of E-cadherin mRNA, an epithelial marker, in the si- ZEB2 group increased, while the expression of vimentin mRNA, an mesenchymal marker, and the protein decreased. Compared with those of the pcDNA3.1 group, the expression of E-cadherin mRNA in the PANC-1 cells of the pcDNA3.1- ZEB2 group decreased, while the expression of vimentin mRNA and the protein increased (all P<0.05). Analysis with the STRING database predicted that 10 proteins had close interaction with ZEB2. Conclusion: Overexpression of ZEB2 promotes the migration, invasion, and the EMT process of PANC-1 pancreatic cancer cells.

[Methyl ferulic acid ameliorates ethanol-induced L02 cell steatosis through microRNA-378b-mediated CaMKK2-AMPK pathway].

Alcoholic liver disease(ALD), with its increasing morbidity and mortality, has seriously and extensively affected the health of people worldwide. Methyl ferulic acid(MFA) has been proven to significantly inhibit alcohol-induced lipid production in L02 cells through the AMP-activated protein kinase(AMPK) pathway, but its in-depth mechanism remains unclear. This study aimed to further clarify the mechanism of MFA in improving lipid accumulation in L02 cells through the microRNA-378b(miR-378b)-mediated calcium/calmodulin-dependent protein kinase kinase 2(CaMKK2)-AMPK signaling pathway based on existing researches. L02 cells were induced by 100 mmol·L~(-1) ethanol for 48 h to establish the model of ALD in vitro, and 100, 50, and 25 µmol·L~(-1) concentration of MFA was treated. MiR-378b plasmids(containing the overexpression plasmid-miR-378b mimics, silence plasmid-miR-378b inhibitor, and their respective negative control-miR-378b NCs) were transfected into L02 cells by electroporation to up-regulate or down-regulate the levels of miR-378b in L02 cells. The levels of total cholesterol(TC) and triglyceride(TG) in cells were detected by commercial diagnostic kits and automatic biochemical analyzers. The expression levels of miR-378b in L02 cells were detected by real-time quantitative polymerase chain reaction(qRT-PCR). CaMKK2 mRNA levels were detected by PCR, and protein expressions of related factors involved in lipid synthesis, decomposition, and transport in lipid metabolism were detected by Western blot. The results displayed that ethanol significantly increased TG and TC levels in L02 cells, while MFA decreased TG and TC levels. Ethanol up-regulated the miR-378b level, while MFA effectively inhibited the miR-378b level. The overexpression of miR-378b led to lipid accumulation in ethanol-induced L02 cells, while the silence of miR-378b improved the lipid deposition induced by ethanol. MFA activated the CaMKK2-AMPK signaling pathway by lowering miR-378b, thus improving lipid synthesis, decomposition, and transport, which improved lipid deposition in L02 cells. This study shows that MFA improves lipid deposition in L02 cells by regulating the CaMKK2-AMPK pathway through miR-378b.

[UPLC-Q-TOF-MS metabolomic study on improvement of acute myocardial ischemia in rats by Dalbergia cochinchinensis heartwood].

This paper aimed to study the effect of Dalbergia cochinchinensis heartwood on plasma endogenous metabolites in rats with ligation of the left anterior descending coronary artery, and to analyze the mechanism of D. cochinchinensis heartwood in improving acute myocardial ischemic injury. The stability and consistency of the components in the D. cochinchinensis heartwood were verified by the establishment of fingerprint, and 30 male SD rats were randomly divided into a sham group, a model group, and a D. cochinchinensis heartwood(6 g·kg~(-1)) group, with 10 rats in each group. The sham group only opened the chest without ligation, while the other groups established the model of ligation. Ten days after administration, the hearts were taken for hematoxylin-eosin(HE) staining, and the content of heart injury indexes in the plasma creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH), energy metabolism-related index glucose(Glu) content, and vascular endothelial function index nitric oxide(NO) was determined. The endogenous metabolites were detected by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS). The results showed that the D. cochinchinensis heartwood reduced the content of CK-MB and LDH in the plasma of rats to relieve myocardial injury, reduced the content of Glu in the plasma, improved myocardial energy metabolism, increased the content of NO, cured the vascular endothelial injury, and promoted vasodilation. D. cochinchinensis heartwood improved the increase of intercellular space, myocardial inflammatory cell infiltration, and myofilament rupture caused by ligation of the left anterior descending coronary artery. The metabolomic study showed that the content of 26 metabolites in the plasma of rats in the model group increased significantly, while the content of 27 metabolites decreased significantly. Twenty metabolites were significantly adjusted after the administration of D. cochinchinensis heartwood. D. cochinchinensis heartwood can significantly adjust the metabolic abnormality in rats with ligation of the left anterior descending coronary artery, and its mechanism may be related to the regulation of cardiac energy metabolism, NO production, and inflammation. The results provide a corresponding basis for further explaining the effect of D. cochinchinensis on the acute myocardial injury.