The structure of the archaeal nuclease RecJ2 implicates its catalytic mechanism and inability to interact with GINS.

Bacterial RecJ exhibits 5'→3' exonuclease activity that is specific to ssDNA; however, archaeal RecJs show 5' or 3' exonuclease activity. The hyperthermophilic archaea Methanocaldococcus jannaschii encodes the 5'-exonuclease MjRecJ1 and the 3'-exonuclease MjRecJ2. In addition to nuclease activity, archaeal RecJ interacts with GINS, a structural subcomplex of the replicative DNA helicase complex. However, MjRecJ1 and MjRecJ2 do not interact with MjGINS. Here, we report the structural basis for the inability of the MjRecJ2 homologous dimer to interact with MjGINS and its efficient 3' hydrolysis polarity for short dinucleotides. Based on the crystal structure of MjRecJ2, we propose that the interaction surface of the MjRecJ2 dimer overlaps the potential interaction surface for MjGINS and blocks the formation of the MjRecJ2-GINS complex. Exposing the interaction surface of the MjRecJ2 dimer restores its interaction with MjGINS. The cocrystal structures of MjRecJ2 with substrate dideoxynucleotides or product dCMP/CMP show that MjRecJ2 has a short substrate binding patch, which is perpendicular to the longer patch of bacterial RecJ. Our results provide new insights into the function and diversification of archaeal RecJ/Cdc45 proteins.

The biochemical characterization of a TatD nuclease from Thermus thermophilus.

Nucleases play pivotal roles in DNA repair and apoptosis. Moreover, they have various applications in biotechnology and industry. Among nucleases, TatD has been characterized as an exonuclease with various biological functions in different organisms. Here, we biochemically characterized the potential TatD nuclease from Thermus thermophilus. The tatD gene from T. thermophilus was cloned, then the recombinant TatD nuclease was expressed and purified. Our results revealed that the TthTatD nuclease could degrade both single-stranded and double-stranded DNA, and its activity is dependent on the divalent metal ions Mg2+ and Mn2+. Remarkably, the activity of TthTatD nuclease is highest at 37 °C and decreases with increasing temperature. TthTatD is not a thermostable enzyme, even though it is from a thermophilic bacterium. Based on the sequence similarity and molecular docking of the DNA substrate into the modeled TthTatD structure, several key conserved residues were identified and their roles were confirmed by analyzing the enzymatic activities of the site-directed mutants. The residues E86 and H149 play key roles in binding metal ions, residues R124/K126 and K211/R212 had a critical role in binding DNA substrate. Our results confirm the enzymatic properties of TthTatD and provide a primary basis for its possible application in biotechnology.

NEAT1 inhibits the angiogenic activity of cerebral arterial endothelial cells by inducing the M1 polarization of microglia through the AMPK signaling pathway.

BACKGROUND: Enhancing angiogenesis may be an effective strategy to promote functional recovery after ischemic stroke. Inflammation regulates angiogenesis. Microglia are crucial cells that initiate inflammatory responses after various brain injuries. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) plays a role in regulating brain injury. This study aimed to explore the effects of NEAT1-regulated microglial polarization on the neovascularization capacity of cerebrovascular endothelial cells and the underlying molecular regulatory mechanisms. METHODS: Mouse cerebral arterial endothelial cells (mCAECs) were co-cultured with BV-2 cells in different groups using a Transwell system. NEAT1 expression levels were measured by fluorescence quantitative reverse transcription PCR. Levels of IL-1ß, IL-6, TNF-α, Arg-1, IL-4, and IL-10 were determined using ELISA. Expression levels of CD86 and CD163 were detected by immunofluorescence. The neovascularization capacity of mCAECs was assessed using CCK-8, Transwell, Transwell-matrigel, and tube formation assays. Label-free quantification proteomics was carried out to identify differentially expressed proteins. Protein levels were measured by Western blotting. RESULTS: NEAT1 overexpression induced M1 polarization in BV-2 cells, whereas NEAT1 knockdown blocked lipopolysaccharide-induced M1 polarization in microglia. NEAT1-overexpressing BV-2 cells suppressed the angiogenic ability of mCAECs, and NEAT1-knocking BV-2 cells promoted the angiogenic ability of mCAECs under lipopolysaccharide treatment. Label-free quantitative proteomic analysis identified 144 upregulated and 131 downregulated proteins that were induced by NEAT1 overexpression. The AMP-activated protein kinase (AMPK) signaling pathway was enriched in the Kyoto Encyclopedia of Genes and Genomes analysis of the differentially expressed proteins. Further verification showed that NEAT1 inactivated the AMPK signaling pathway. Moreover, the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide reversed the effect of NEAT1 on BV-2 polarization and the regulatory effect of NEAT1-overexpressing BV-2 cells on the angiogenic ability of mCAECs. CONCLUSIONS: NEAT1 inhibits the angiogenic activity of mCAECs by inducing M1 polarization of BV-2 cells through the AMPK signaling pathway. This study further clarified the impact and mechanism of NEAT1 on microglia and the angiogenic ability of cerebrovascular endothelial cells.

The Association of Blood Urea Nitrogen to Creatinine Ratio and the Prognosis of Critically Ill Patients with Cerebral Infarction: A Cohort Study.

Background: To evaluate the association between blood urea nitrogen (BUN) to creatinine (Cr) (BUN/Cr) ratio and the in-hospital mortality of critically ill patients with cerebral infarction in intensive care unit (ICU). Methods: In this cohort study, the data of 3059 participants with cerebral infarction were collected from the Medical Information Mart for Intensive Care (MIMIC)-III and the MIMIC-IV database. After propensity score matching (PSM) on age and gender, 2085 people were involved in and divided into the alive group (n = 1390) and the dead group (n = 695) based on the results of follow-up. Multivariate logistic analyses were applied to identify the confounders and the association between BUN/Cr and mortality of cerebral infarction. Results: The median follow-up time was 10.5 days. Among 2778 participants, 695 were dead at the end of follow-up. Univariate analysis revealed that BUN/Cr [risk ratio (RR) = 1.01, 95% confidence interval (CI): 1.01-1.02] might be associated with the in-hospital mortality of cerebral infarction patients. After adjusting for respiratory failure, malignant cancer, anticoagulation, liver disease, white blood cell (WBC), red cell distribution width (RDW), glucose, bicarbonate, and temperature, BUN/Cr had week correlation with the increased risk of in-hospital mortality of cerebral infarction patients (RR = 1.01, 95% CI: 1.01-1.02). Conclusion: This study evaluated the association between BUN/Cr and the in-hospital mortality of cerebral infarction patients in ICU and found that BUN/Cr had weak correlation with the increased risk of in-hospital mortality of patients with cerebral infarction in ICU especially in males and those with respiratory failure, malignant cancer, and without liver disease, as well as those receiving anticoagulation.

Calcimycin Suppresses S100A4 Expression and Inhibits the Stimulatory Effect of Transforming Growth Factor ß1 on Keloid Fibroblasts.

Recent researches have indicated that S100A4 participates in tissue fibrosis, whereas calcimycin inhibits this process as a novel S100A4 transcription inhibitor. However, the relationship and mechanisms between calcimycin and S100A4 in keloid fibroblasts (KFs) remain unknown. The present research was aimed to evaluate the effect of calcimycin on S100A4 expression and pathogenesis in KFs. Keloid fibroblasts were cultured and exposed to different concentrations of calcimycin in the absence or presence of transforming growth factor ß1 (TGF-ß1). The results showed that the expression of S100A4 was significantly increased in keloid derived fibroblasts compared with normal skin fibroblasts. Calcimycin depressed S100A4 in a concentration- and time-dependent manner. Moreover, calcimycin suppressed TGF-ß1-induced collagen type I, fibronectin, and α-smooth muscle actin expression and cell viability in cultured KFs. Furthermore, calcimycin modulated expression of TGF-ß/Smad target genes Smad7 and phosphorylation of TGF-ß1-induced Smad2/3. This research for the first time confirmed the presence of S100A4 in KFs. Calcimycin inhibits the expression of S100A4, as well as KF proliferation and migration and extracellular matrix (ECM) synthesis. Taken together, these results indicate that calcimycin might be a therapeutic candidate to keloid or other related fibrotic disorders.

Role of Homeodomain-Interacting Protein Kinase 2 in the Pathogenesis of Tissue Fibrosis in Keloid-Derived Keratinocytes.

Epithelial-mesenchymal transition (EMT) plays a critical role in fibrotic keloid formation, which is characterized by excessive collagen and extracellular matrix synthesis and deposition. Growing evidence suggests that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) acts upstream of several major fibrosis signaling pathways; however, the role of HIPK2 in the keloid fibrogenesis remains unknown. In the current study, we investigated the roles of HIPK2 in the pathogenesis of keloids. Primary normal skin and keloid keratinocytes were cultured and pretreated with transforming growth factor (TGF)-ß1. Next, keratinocytes were transfected with scrambled small interfering RNA (siRNA) and anti-HIPK2 siRNA. The TGF-ß1-associated HIPK2 alterations were investigated by quantitative real-time polymerase chain reaction. Protein levels were analyzed by western blotting. The HIPK2 was markedly increased in the keloid-derived keratinocytes compared with normal skin keratinocytes. In addition, HIPK2 induced the expression of EMT markers in normal skin keratinocytes by TGF-ß1-SMAD family member 3 (SMAD3). The effect of TGF-ß1-related EMT markers and SMAD3 phosphorylation in response to added TGF-ß1 was significantly abrogated when the cells were transfected with HIPK2 siRNA. We conclude that HIPK2 is a crucial factor in the pathogenesis of keloids, suggesting that HIPK2 might be a novel potential drug target for antikeloid therapy.

Advances in highly active one-carbon metabolism in cancer diagnosis, treatment and drug resistance: A systematic review.

Study background objectives: Cancer poses a significant health concern as it is incurable. Every year, research on how to treat and eradicate this chronic condition is done. This systematic review will unmask the recent developments concerning highly active 1C metabolism with regard to cancer diagnosis, treatment, and drug resistance. The significance of this study is rolling out evidence-based evidence on the importance of one-carbon metabolism in cancer diagnosis and treatment. Methods: Eight randomized controlled trials (RCTs) were reviewed from five electronic databases - EMBASE, Scopus Review, Google Scholar, Web of Science, and PubMed. Outcomes from the eight studies were analyzed to paint a picture of the topic in question. While the Preferred Reporting Items for Systematic Reviews and Meta-Analysis' (PRISMA) protocol guided the initial literature search, The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach informed quality assessments of the eligible studies. Conclusion: Since its emergence in the 1980s, 1C metabolism has been investigated and broadened to capture essential aspects of cancer treatment, diagnosis, and drug resistance. The review found that metabolites like folic acid could be used to detect different types of cancer. The metabolic pathways could induce tumorigenesis and DNA methylation, hence drug resistance. Systematic review registration: https://inplasy.com/projects/, identifier INPLASY2022110099.

Effect of Si-Miao-Yong-An decoction on the differentiation of monocytes, macrophages, and regulatory T cells in ApoE-/- mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Si-Miao-Yong-An decoction (SMYAD) is a renowned traditional Chinese medicinal formula. SMYAD was originally recorded in the "Shi Shi Mi Lu", which was edited by medical scientist Chen Shi'duo during the Qing Dynasty. SMYAD has been traditionally used to treat thromboangiitis obliterans. At present, it is mainly used in clinical applications and research of cardiovascular diseases. AIM OF THE STUDY: To explore the effects of SMYAD on the pathological changes of atherosclerosis (AS) and the differentiation of monocytes, macrophages, and regulatory T (Treg) cells in apolipoprotein E knockout (ApoE-/-) mice. MATERIALS AND METHODS: Eight C57BL/6J mice, which were fed with normal diet for 16 weeks, were used as control group. Forty ApoE-/- mice were randomly divided into model group, atorvastatin group, SMYAD low-dose (SMYAD-LD) group, SMYAD medium-dose (SMYAD-MD) group, and SMYAD high-dose (SMYAD-HD) group. ApoE-/- mice were fed with western diet (WD) for 8 weeks, and the drugs were continuously administered for 8 weeks. The levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured by the esterase method. Morphological changes of the aortic sinus in mice were observed by hematoxylin-eosin (HE) staining, the lipid infiltration of the aorta and aortic sinus were observed by oil red O staining, and the spleen index was calculated. The proportion of Ly6Chigh and Ly6Clow monocyte subsets, macrophages, and their M1 phenotype, as well as Treg cells in spleen were measured by flow cytometry. The expressions of cluster of differentiation 36 (CD36), scavenger receptor A1 (SRA1), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), F4/80, and fork head frame protein 3 (FOXP3) in aortic sinus were assessed by immunohistochemical staining. The serum levels of oxidized low density lipoprotein (ox-LDL), interleukin-1ß (IL-1ß), IL-18, transforming growth factor-ß (TGF-ß), and IL-10 were measured by enzyme-linked immunosorbent assays (ELISA). RESULTS: Compared with the model group, the level of serum TC and LDL-C decreased in the SMYAD group, the pathological changes of aortic sinus decreased, and lipid infiltration of aorta and aortic sinus also decreased. These decreases were accompanied by a significant downregulation of CD36, SRA1, and LOX-1. Furthermore, the proportions of Ly6Chigh pro-inflammatory monocyte subsets, macrophages, and their M1 phenotypes in spleen decreased significantly, while the proportion of Treg cells increased. In addition, while the expression of F4/80 decreased, the expression of FOXP3 increased in the aorta sinus. The levels of serum pro-inflammatory factors IL-1ß and IL-18 decreased. CONCLUSIONS: SMYAD can improve the pathological changes associated with AS and can inhibit lipid deposition in ApoE-/- mice induced by WD diet. The likely mechanism is the inhibition of the differentiation and recruitment of monocytes and macrophages, the promotion of the differentiation and recruitment of Treg cells, as well as the reduction of the secretion of pro-inflammatory factors.

Serum CYR61 as a potential biomarker for the diagnosis of esophagogastric junction tumor.

BACKGROUND: Esophagogastric junction tumor (EGJ) is a rare but fatal disease with a rapid rising incidence worldwide in the late 20 years, and it lacks a convenient and safe method for diagnosis. The present study aimed to evaluate the potential of serum CYR61 as a biomarker for the diagnosis of EGJ tumor. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to estimate CYR61 levels in sera of 152 EGJ tumor patients and 137 normal controls. Receiver operating characteristics (ROC) was carried out to evaluate the diagnostic accuracy. The Mann-Whitney's U test was used to compare the difference of serum levels of CYR61 between groups. And chi-square tests were employed to estimate the correlation of the positive rate of serum CYR61 between/among subgroups. RESULTS: Serum CYR61 levels were statistically lower in EGJ tumor and early-stage EGJ tumor patients than those in normal controls (P<0.0001). The sensitivity, specificity and the area under the curve (AUC) of this biomarker in EGJ tumor were 88.2%, 43.8% and 0.691, respectively, and those for early stage of EGJ tumor were 80.0%, 66.4% and 0.722, respectively. Analyses showed that there was no correlation between the clinical data and the levels of CYR61 (P>0.05). CONCLUSION: The present study showed that CYR61 might be a potential biomarker to assist the diagnosis of EGJ tumor.

Diagnostic and prognostic values of contrast-enhanced ultrasound in breast cancer: a retrospective study.

This study aimed to explore the diagnostic and prognostic values of contrast-enhanced ultrasound (CEUS) in breast cancer. Between September 2009 and October 2011, a total of 143 breast cancer patients and 161 healthy people were selected as case group and control group, respectively. After the identification of lesions by conventional ultrasound, all patients underwent CEUS. The CEUS images were analyzed, and time-intensity curves (TICs) were obtained. Hematoxylin-eosin and immunohistochemistry staining was performed on tissue specimens, according to which the expressions of estrogen receptor (ER), c-erb-B2, p53, and Ki-67 were measured. Multivariate logistic regression analysis was used to compare CEUS and TIC parameters between the two groups. Compared with the control group, cancer patients showed high enhancement, heterogeneous enhancement or defects in the central region, expansion of lesion diameter after enhancement and crab-like blur lesion edges. The peak intensity (PI), relative start time of enhancement, relative PI, and relative area under the curve in the case group were significantly higher than those in the control group. Logistic analysis showed that the uniformity of enhancement, expansion of lesion diameter, and relative PI were significant diagnostic parameters of breast cancer, with area under the curve being 0.798, 0.776, and 0.919, respectively. There were strong associations between CEUS characteristics and expressions of prognostic factors in breast cancer: the heterogeneous enhancement was common in c-erb-B2-positive tumors; the centripetal enhancement occurred more in ER-negative tumors; perforator vessels were often seen in tumors at high histological grade; perfusion defects were common in ER-negative, c-erb-B2-positive, and Ki-67-positive tumors. CEUS is a useful tool for the early diagnosis and prognosis of breast cancer.

Subclavian steal syndrome like appearance resulting from a vertebral artery origin stenosis: a case report.

Partial steal has been regarded as a classic ultrasound appearance of subclavian steal syndrome. We report a case with the vertebral artery origin stenosis and intact subclavian artery, which showed the similar partial steal ultrasound features. The following computerized tomography angiography confirmed the stenosis. Therefore, when an alternating flow in the vertebral artery is detected, the investigation of its origin must be performed besides the ipsilateral subclavian artery.