Disruption of a DNA G-quadruplex causes a gain-of-function SCL45A1 variant relevant to developmental disorders.

SLC45A1 encodes a glucose transporter protein highly expressed in the brain. Mutations in SLC45A1 may lead to neurological diseases and developmental disorders, but its exact role is poorly understood. DNA G-quadruplexes (DNA G4s) are stable structures formed by four guanine bases and play a role in gene regulation and genomic stability. Changes in DNA G4s may affect brain development and function. The mechanism linking alterations in DNA G-quadruplex structures to SLC45A1 pathogenicity remains unknown. In this study, we identify a functional DNA G-quadruplex and its key binding site on SLC45A1 (NM_001080397.3: exon 2: c.449 G>A: p.R150K). This variant results in the upregulation of mRNA and protein expression, which may lead to intellectual developmental disorder with neuropsychiatric features. Mechanistically, the mutation is found to disrupt DNA G-quadruplex structures on SLC45A1, leading to transcriptional enhancement and a gain-of-function mutation, which further causes increased expression and function of the SLC45A1 protein. The identification of the functional DNA G-quadruplex and its effects on DNA G4s may provide new insights into the genetic basis of SLC45A1 pathogenicity and highlight the importance of DNA G4s of SLC45A1 in regulating gene expression and brain development.

Clinical and molecular characteristics of RNF43 mutations as promising prognostic biomarkers in colorectal cancer.

Background: Transmembrane E3 ubiquitin ligase (RNF43) mutations are present in approximately 6-18% of colorectal cancers (CRC) and could enhance Wnt/ß-catenin signaling, which is emerging as a promising therapeutic target. This study aims to investigate the clinical and molecular characteristics and potential heterogeneity of RNF43-mutant CRC. Methods: A total of 78 patients with RNF43-mutant CRC were enrolled from July 2013 to November 2022. Demographic data, clinical characteristics, treatment regimens used, and survival outcomes were collected and analyzed. Results: Our study uncovered that patients with RNF43 mutations in the N-terminal domain (NTD; n = 50) exhibited shorter overall survival (OS; median months, 50.80 versus not reached; p = 0.043) compared to those in the C-terminal domain (CTD; n = 17). Most RNF43 mutations in NTD had positive primary lymph node status, low tumor mutation burden (TMB-L), and correlated with proficient mismatch repair (pMMR)/microsatellite stable (MSS) status. By contrast, RNF43 mutations in CTD were significantly enriched in deficient MMR (dMMR)/microsatellite instability (MSI-H) tumors with high TMB (TMB-H). N-terminal RNF43-mutated tumors harbored a hotspot variant (RNF43 R117fs), which independently predicted a significantly worse outcome in pMMR/MSS CRC with a median OS of 18.9 months. Patients with RNF43 mutations and the BRAF V600E alterations demonstrated sensitivity to BRAF/EGFR inhibitors. Moreover, we observed that pMMR/MSS patients with RNF43 R117fs mutation had a higher incidence of stage IV, ⩾2 metastatic sites, low TMB, and none of them received PD-1/PD-L1 inhibitor therapy. Conclusion: Our findings provide the first evidence that RNF43 mutations in NTD and the R117fs variant correlate with a poorer prognosis in CRC patients, providing strategies for Wnt-targeted therapy to improve clinical efficacy.

Research of Vertical via Based on Silicon, Ceramic and Glass.

With the increasing demand for high-density integration, low power consumption and high bandwidth, creating more sophisticated interconnection technologies is becoming increasingly crucial. Three-dimensional (3D) integration technology is known as the fourth-generation packaging technology beyond Moore's Law because of its advantages of low energy consumption, lightweight and high performance. Through-silicon via (TSV) is considered to be at the core of 3D integration because of its excellent electrical performance, lower power consumption, wider bandwidth, higher density, smaller overall size and lighter weight. Therefore, the particular emphasis of this review is the process flow of TSV technology. Among them, the research status of TSV hole etching, deep hole electroplating filling and chemical mechanical planarization (CMP) in TSV preparation process are introduced in detail. There are a multitude of inevitable defects in the process of TSV processing; thus, the stress problems and electrical characteristics that affect the reliability of TSV are summarized in this review. In addition, the process flow and process optimization status of through ceramic via (TCV) and through glass via (TGV) are discussed.

Dissecting Sperm Mitochondrial G-Quadruplex Structures Using a Fluorescent Probe Biomarker to Monitor and Regulate Fertilization Capability.

To monitor the levels of mitochondrial DNA G-quadruplexes (mtDNA G4s) in spermatozoa and to explore the possibility using mtDNA G4s as a reliable marker in patients with multiple clinical insemination failures, a novel chemical TPE-mTO probe engineered in our previous work was used on both samples from the mice sperm and from patients with fertilization failure. Expression of valosin-containing protein and the zona-free hamster egg assay were used to evaluate mitophagy and human sperm penetration. RNA-sequencing was used to explore expression changes of key genes affected by mtDNA G4s. Results showed that the probe can track mtDNA G4s in spermatozoa easily and quickly with fewer backgrounds. Significantly increased mtDNA G4s were also found in patients with fertilization failure, using the flow-cytometry-based TPE-mTO probe detection method. A sperm-hamster egg penetration experiment showed that abnormal fertilization caused by increased mtDNA G4s can be effectively restored by a mitophagy inducer. This study provides a novel method for monitoring etiological biomarkers in patients with clinical infertility and treatment for patients with abnormal fertilization caused by mtDNA G4 dysfunction.

A novel missense mutation in GREB1L identified in a three-generation family with renal hypodysplasia/aplasia-3.

BACKGROUND: Renal hypodysplasia/aplasia-3 (RHDA3), as the most severe end of the spectrum of congenital anomalies of the kidney and urinary tract, is mainly caused by mutations in GREB1L. However, the mutations in GREB1L identified to date only explain a limited proportion of RHDA3 cases, and the mechanism of GREB1L mutations causing RHDA3 is unclear. RESULTS: According to whole-exome sequencing, a three-generation family suffering from RHDA3 was investigated with a novel missense mutation in GREB1L, c.4507C>T. All three-generation patients suffered from unilateral absent kidney. This missense mutation resulted in sharp downregulation of mRNA and protein expression, which might lead to RHDA3. Mechanistically, through RNA-sequencing, it was found that the mRNA levels of PAX2 and PTH1R, which are key molecules involved in the development of the kidney, were significantly downregulated by knocking out GREB1L in vitro. CONCLUSIONS: This novel missense mutation in GREB1L can be helpful in the genetic diagnosis of RHDA3, and the discovery of the potential mechanism that GREB1L mutations involved in RHDA3 pathogenesis can promote the adoption of optimal treatment measures and the development of personalized medicine directly targeting these effects.

Monitoring tumour resistance to the BRAF inhibitor combination regimen in colorectal cancer patients via circulating tumour DNA.

AIMS: This study aimed to identify mechanisms of drug resistance to the combination of vemurafenib, irinotecan, and cetuximab (VIC) in BRAFV600E metastatic colorectal cancer (mCRC). METHODS: Forty-one patients with BRAFV600E mCRC from July 2018 and June 2020 were evaluated, with tissue and/or plasma samples collected. We profiled tissue and plasma samples using whole-exome sequencing and targeted sequencing of 425 cancer-relevant genes. Clinical cohort analysis from published studies was performed to consolidate our findings. RESULTS: BRAF mutant in baseline plasma and its dynamics are significantly associated with VIC-related response, and concurrent RNF43 mutation significantly sensitises tumour to VIC treatment. VIC resistance frequently involves genes in PI3K, MAPK pathway, and several novel resistance mechanisms such as TGFBR2 and SMAD4 mutations, and copy-number gains in PTK2, MYC, and GATA6 have been identified. We also firstly describe acquired altered genes in DNA damaging repair pathway, occurring in 33 % of patients after VIC treatment, and particularly, patients with this pre-treatment resistance subclones developed inferior responses, along with higher tumour mutation burden both at baseline and progression plasma. CONCLUSION: Analysis of ctDNA can provide novel insights into molecular resistance mechanisms to VIC in BRAFV600E mCRC patients, allowing accurate guidance for clinicians in personalised treatment strategies.

The novel BRDT inhibitor NHWD870 shows potential as a male contraceptive in mice.

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) family proteins have emerged as promising options not only for the treatment of multiple cancers but also for disturbing the process of sperm maturation with potential for use as viable contraceptive targets. In this study, we find that the BET family inhibitor NHWD870 and BRDT can bind well in vitro through bioinformatics software prediction and protein binding inhibition experiments. NHWD870 can produce a good contraceptive effect through animal experiments in vivo, and the fertility can be restored to normal after drug withdrawal. Transcriptomics and proteomics results suggest that NHWD870 affects pathways related to spermatogenesis and maturation, further contributing to the male infertility phenotype. Our results show that NHWD870 can induce a complete and reversible contraceptive effect in mice, which is stronger than that of JQ1 and its synthesized derivatives. This study is expected to eventually lead to clinical trials.

A loss-of-function variant in SSFA2 causes male infertility with globozoospermia and failed oocyte activation.

Globozoospermia (OMIM: 102530) is a rare type of teratozoospermia (< 0.1%). The etiology of globozoospermia is complicated and has not been fully revealed. Here, we report an infertile patient with globozoospermia. Variational analysis revealed a homozygous missense variant in the SSFA2 gene (NM_001130445.3: c.3671G > A; p.R1224Q) in the patient. This variant significantly reduced the protein expression of SSFA2. Immunofluorescence staining showed positive SSFA2 expression in the acrosome of human sperm. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and Coimmunoprecipitation (Co-IP) analyses identified that GSTM3 and Actin interact with SSFA2. Further investigation revealed that for the patient, regular intracytoplasmic sperm injection (ICSI) treatment had a poor prognosis. However, Artificial oocyte activation (AOA) by a calcium ionophore (A23187) after ICSI successfully rescued the oocyte activation failure for the patient with the SSFA2 variant, and the couple achieved a live birth. This study revealed that SSFA2 plays an important role in acrosome formation, and the homozygous c.3671G > A loss-of-function variant in SSFA2 caused globozoospermia. SSFA2 may represent a new gene in the genetic diagnosis of globozoospermia, especially the successful outcome of AOA-ICSI treatment for couples, which has potential value for clinicians in their treatment regimen selections.

Diagnostic Value of Combined Detection via Protein Induced by Vitamin K Absence or Antagonist II, Alpha-Fetoprotein, and D-Dimer in Hepatitis B Virus-Related Hepatocellular Carcinoma.

Purpose: We aimed to explore the clinical diagnostic value of combined detection via protein induced by vitamin K absence or antagonist II (PIVKA-II), alpha-fetoprotein (AFP), and D-dimer (D-D) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Materials and Methods: We analyzed PIVKA-II, AFP, and D-D levels in 291 subjects comprising liver cirrhosis (LC) patients (n = 143) and HCC patients (n = 148). Receiver operating characteristic (ROC) curves were used to analyze and compare the clinical diagnostic value of the three biomarkers for HBV-related HCC alone and in combination. Results: The levels of PIVKA-II, AFP, and D-D were positively correlated with tumor size in HCC patients. The levels of PIVKA-II and AFP in early-stage HCC, advanced HCC, HBV DNA+ HCC, and HBV DNA- HCC patients were higher than those in LC patients, while the levels of D-D were lower. The area under the curve for combined detection was greater than that for single-index detection in early-stage HCC, advanced HCC, HBV DNA+ HCC, and HBV DNA- HCC patients. Conclusion: D-D may be a useful biomarker for the diagnosis of HBV-related HCC. The combined detection of PIVKA-II, AFP, and D-D had better diagnostic value for different types of HCC than the detection of individual biomarkers.

A novel missense variant in the CASK gene causes intellectual developmental disorder and microcephaly with pontine and cerebellar hypoplasia.

BACKGROUND: Variants in the CASK gene result in a wide range of observed phenotypes in humans, such as FG Syndrome 4 and intellectual disabilities. Intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH) is an X-linked disorder that affects females and is characterized by severely impaired intellectual development and variable degrees of pontocerebellar hypoplasia. Variants in CASK are the main genetic cause of MICPCH. Variants in CASK can explain most patients with MICPCH, but there are still some patients whose disease aetiology cannot be explained. CASE PRESENTATION: An 11-month-old female diagnosed with MICPCH exhibited general developmental delays, microcephaly, and cerebellar hypoplasia. Whole-exome sequencing (WES) was used to find a novel heterozygous missense variant (NM_003688.3: c.638T>G) of CASK in this patient. Strikingly, this variant reduced the expression of CASK at the protein level but not at the mRNA level. By using protein structure prediction analysis, this study found that the amino acid change caused by the variant resulted in further changes in the stability of the protein structure, and these changes caused the downregulation of protein expression and loss of protein function. CONCLUSION: In this study, we first reported a novel heterozygous pathogenic variant and a causative mechanism of MICPCH. The amino acid change cause by this variant led to changes in the protein structure and a decrease in its stability, which caused a loss of protein function. This study could be helpful to the genetic diagnosis of this disease.

An Update on the Relationship of SARS-CoV-2 and Male Reproduction.

Since the outbreak of the COVID-19, up to now, infection cases have been continuously rising to over 200 million around the world. Male bias in morbidity and mortality has emerged in the COVID-19 pandemic. The infection of SARS-CoV-2 has been reported to cause the impairment of multiple organs that highly express the viral receptor angiotensin-converting enzyme 2 (ACE2), including lung, kidney, and testis. Adverse effects on the male reproductive system, such as infertility and sexual dysfunction, have been associated with COVID-19. This causes a rising concern among couples intending to have a conception or who need assisted reproduction. To date, a body of studies explored the impact of SARS-CoV-2 on male reproduction from different aspects. This review aims to provide a panoramic view to understand the effect of the virus on male reproduction and a new perspective of further research for reproductive clinicians and scientists.

Recent Advances on Drugs and Vaccines for COVID-19.

The current situation of Coronavirus Disease 2019 (COVID-19) worldwide is still very severe. Presently, many breakthroughs have been accomplished in the research and development of drugs for the treatment of COVID-19, especially vaccines; however, some of the so-called COVID-19-specific drugs highlighted in the early stage failed to achieve the expected curative effect. There is no antiviral therapy available, by stimulating protective immunity vaccine is the best choice for the future management of infection. Therefore, we aimed to identify the latest developments in the research and development of these drugs and vaccines and provide a reference for the prevention and treatment of COVID-19.

Expression and Correlation of IgG4 and IL-21 in Collagen-Induced Arthritis Rats.

PURPOSE: We explored the expression levels of IgG4 and interleukin (IL)-21 in the serum and ankle joints of collagen-induced arthritis (CIA) rats at different disease stages. MATERIALS AND METHODS: Wistar rats were randomly divided into normal and model groups, and the latter group was administered bovine type II collagen to induce arthritis. Enzyme-linked immunosorbent assay was performed at 21, 28, 35, and 42 days to detect IgG4 and IL-21 in the serum, followed by histological and immunohistochemical analyses of IgG4 and IL-21r in the ankle joint of rats. RESULTS: The contents of IgG4 and IL-21 in the serum of the CIA model group were positively correlated and increased with disease progression. The expression of IgG4 and IL-21 receptors in the ankle joint of the CIA model group was significantly higher than that in the control group. These proteins were closely related to the pathological score. The serum IL-21 level in the model group was closely related to the level of IL-21 receptor in the ankle joint. CONCLUSION: IL-21 may promote the occurrence and development of rheumatoid arthritis by combining with IL-21r to regulate the content of IgG4.

Spermidine Inhibits Joints Inflammation and Macrophage Activation in Mice with Collagen-Induced Arthritis.

PURPOSE: Spermidine (SPD) is a naturally occurring polyamine. In this study, we examined the role and possible mechanism of SPD in collagen-induced arthritis (CIA) mice. MATERIALS AND METHODS: CIA mice were intraperitoneally injected with SPD (2 and 50 mg/kg), dexamethasone (0.5 mg/kg), or saline daily for 21 days. The severity of the disease and inflammatory responses in the serum and joint tissue were assessed through macroscopic, immunohistochemical, and histological analyses. RESULTS: Macroscopic and histological results indicated that SPD protected against the development of CIA. SPD suppressed the levels of the pro-inflammatory cytokines IL-6 and IL-1ß and increased the levels of the anti-inflammatory factor IL-10 in the serum. Immunohistochemical staining showed that 50 mg/kg SPD inhibited iNOS expression in synovial macrophages in the ankle joints of CIA mice. CONCLUSION: These results suggest that SPD may protect CIA mice by inhibiting the polarization of M1 macrophages in the synovial tissue, reducing pro-inflammatory cytokines, and promoting anti-inflammatory factor release.

Clinical characteristics and laboratory features of COVID-19 in high altitude areas: A retrospective cohort study.

Coronavirus disease 2019 (COVID-19) is highly contagious and has affected the whole world. We seek to investigate the clinical and laboratory characteristics of COVID-19 patients in the high altitude areas of Sichuan, China. In this retrospective cohort study, a total of 67 patients with laboratory-confirmed SARS-CoV-2 infections in Sichuan's Ngawa Tibetan and Qiang Autonomous Prefecture were included from February 1, 2020, to March 2, 2020. Their clinical characteristics, as well as radiological and laboratory features, were extracted. Four (6.0%) patients were categorized as severe cases; 39 (58.2%) were non-severe cases, and 24 (35.8%) were asymptomatic cases. A total of 46 (68.7%) patients were associated with cluster infection events in this study. The most common symptoms were cough, sputum production, dyspnea, fatigue or myalgia, and headache. Seven (10.4%) patients showed leucopenia, and 20 (29.9%) patients showed lymphopenia. Lymphocyte counts and neutrophil-to-lymphocyte ratios (NPR) were different between the three groups. In total, 14 (20.9%) patients had thrombocytopenia, and prothrombin times (PT) and fibrinogen levels differed between groups. We also found significant differences in sodium, chloride and calcium levels between the three groups. Antiviral therapy did not lead to obvious adverse events or shortened durations from initial positive to subsequent negative nuclei acid tests. Advanced age, hypertension, high neutrophil count, the neutrophil-to-lymphocyte ratio, fibrinogen and lactate dehydrogenase levels were identified as independent risk factors for symptomatic cases of COVID-19. In conclusion, the symptoms of patients in high altitude areas were mild, and about one third were asymptomatic. We also identified several independent risk factors for symptomatic cases of COVID-19.

Flavonoid fisetin reverses impaired hippocampal synaptic plasticity and cognitive function by regulating the function of AMPARs in a male rat model of schizophrenia.

Cognitive deficits are the core feature of schizophrenia and effective treatment strategies are still missing. Previous studies have reported that fisetin promotes long-term potentiation (LTP) and cognitive function in normal rodents and other model animals of neurological diseases. The aim of this study was to assess the effect of fisetin on synaptic plasticity and cognitive deficits caused by a brief disruption of N-methyl-D-aspartate receptors (NMDARs) with dizocilpine (MK-801) during early development in rats. The cognitive performance was examined by the Morris water maze task and a fear conditioning test. Hippocampal synaptic plasticity was investigated by field potential recording. The expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) and cognition-related proteins was measured by western blotting. We found that intraperitoneal administration of fisetin rescued hippocampus-dependent spatial and contextual fear memory in MK-801 rats. In parallel with these behavioral results, fisetin treatment in MK-801 rats reversed the impairment of hippocampal LTP. At the molecular level, fisetin treatment selectively increased the phosphorylation and surface expression of AMPA receptor subunit 1 (GluA1) in MK-801-treated rats. Moreover, fisetin restored the phosphorylation levels of calcium-calmodulin-dependent kinaseII (CaMKII), cAMP response element-binding protein (CREB), and the extracellular signal-regulated kinase (ERK1/2) in MK-801-treated rats. Collectively, our findings demonstrate that fisetin treatment can reverse the deficits of hippocampal synaptic plasticity and memory in a male rat model of schizophrenia by restoring the phosphorylation and surface expression of AMPAR GluA1 subunit, suggesting fisetin as a promising therapeutic candidate for schizophrenia-associated cognitive deficits.

Serum hepatitis B virus ribonucleic acid and its influencing factors in chronic hepatitis B.

BACKGROUND: Hepatitis B virus (HBV) remains one of the most serious and prevalent health problems in the world. OBJECTIVES: To determine the serum hepatitis B virus (HBV) RNA levels in patients with chronic hepatitis B (CHB) with low HBV DNA levels and analyze the influencing factors. MATERIAL AND METHODS: Seventy-two CHB patients with low HBV DNA levels were enrolled and divided into 2 groups according to hepatitis B e antigen (HBeAg) status; their age, sex, the incidence of HBV RNA level < lower limit of detection (LLD), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), quantitative determination of HBsAg (qHBsAg), HBV DNA, and HBV RNA levels were compared. The factors influencing serum HBV RNA levels < LLD and the correlation between serum HBV RNA levels, and serum ALT, AST, qHBsAg and HBV DNA levels were analyzed. RESULTS: In HBeAg-positive patients, serum AST, qHBsAg and HBV RNA levels were higher, and serum HBV DNA levels and incidence of HBV RNA < LLD were lower than those in HBeAg-negative patients (p < 0.05). Multivariate linear regression analysis revealed that HBeAg is a factor that significantly influences serum HBV RNA levels in patients with CHB (p < 0.05). Multivariate logistic regression analysis indicated that HBeAg and qHBsAg are factors that influence serum HBV RNA levels < LLD in patients with CHB. In HBeAg-positive patients, serum HBV RNA levels were positively correlated with qHBsAg and HBeAg. CONCLUSIONS: The serum HBV RNA levels in CHB patients with low HBV DNA levels varied according to HBeAg status. The HBeAg is a factor that significantly influences serum HBV RNA levels in patients with CHB, while HBeAg and qHBsAg are factors that significantly influence serum HBV RNA levels < LLD in patients with CHB.