Establishment and evaluation of a nomogram prediction model for the risk of vascular calcification in stage 5 chronic kidney disease patients.

Vascular calcification (VC) is a common complication of chronic kidney disease (CKD) that has a detrimental effect on patients' survival and prognosis. The aim of this study was to develop and validate a practical and reliable prediction model for VC in CKD5 patients. The medical records of 544 CKD5 patients were reviewed retrospectively. Multivariate logistic regression analysis was used to identify the independent risk factors for vascular calcification in patients with CKD5 and then created a nomogram prediction model. The area under the receiver operating characteristic curve (AUC), Hosmer-Lemeshow test, and decision curve analysis (DCA) were used to assess model performance. The patients were split into groups with normal and high serum uric acid levels, and the factors influencing these levels were investigated. Age, BUN, SUA, P and TG were independent risk factors for vascular calcification in CKD5 patients in the modeling group (P < 0.05). In the internal validation, the results of model showed that the AUC was 0.917. No significant divergence between the predicted probability of the nomogram and the actual incidence rate (x2 = 5.406, P = 0.753) was revealed by the calibration plot and HL test, thus confirming that the calibration was satisfactory. The external validation also showed good discrimination (AUC = 0.973). The calibration chart and HL test also demonstrated good consistency. Besides, the correlation analysis of serum uric acid levels in all CKD5 patients revealed that elevated uric acid levels may be related to gender, BUN, P, and TG.

MARCH1 promotes the growth and maintaining of stem cell-like characteristics of gastric cancer cells by activating the Wnt/ß-catenin signaling pathway.

Gastric carcinoma (GC) is a malignant tumor, which is an important cause of death in all tumor deaths. The role of MARCH1 in GC has not been studied, this study aims to investigate the function of MARCH1 in GC. The expression of MARCH1 in normal tissue and tumor tissue was analyzed by TCGA-based GEPIA platform and UALCAL website and verified by RT-qPCR, Western blotting (WB), and Immunohistochemistry (IHC); CCK8 assay and crystal violet assay were separately used to detect cell viability and cell cloning ability. Cell spheroidization assay and Fluorescence-activated cell sorting (FACS) were performed to determine CD44+, CD133+ cell numbers to study the stemness characteristics of GC cells. While, WB was used to study the specific signaling pathway regulated by MARCH1. Animal model of GC was established to study the regulation of MARCH1 on GC growth in vivo. It showed that the expression of MARCH1 in GC tissues was higher than that in normal tissues; CCK8 and crystal violet assay showed that MARCH1 could promote cell viability and cloning ability of GC cells; cell spheroidization experiments and FACS showed that MARCH1 promoted the cloning ability of GC cells; WB results revealed that MARCH1 mainly regulated GC through the Wnt/ß-catenin signaling pathway; In-vivo results showed that MARCH1 can promote the growth of GC. This study found that MARCH1 maintained the stemness characteristics and promoted the proliferation of GC cells by activating the Wnt/ß-catenin signaling pathway.

A novel subgenotype C6 Enterovirus A71 originating from the recombination between subgenotypes C4 and C2 strains in mainland China.

Recombination plays important roles in the genetic diversity and evolution of Enterovirus A71 (EV-A71). The phylogenetics of EV-A71 in mainland China found that one strain DL71 formed a new subgenotype C6 with unknown origin. This study investigated the detailed genetic characteristics of the new variant. DL71 formed a distinct cluster within genotype C based on the genome and individual genes (5'UTR, VP4, VP1, 2A, 2B, 2C, 3D, and 3'UTR). The average genetic distances of the genome and individual genes (VP3, 2A, 2B, 2C, 3A, 3C, and 3D) between DL71 and reference strains were greater than 0.1. Nine recombination events involving smaller fragments along DL71 genome were detected. The strains Fuyang-0805a (C4) and Tainan/5746/98 (C2) were identified as the parental strains of DL71. In the non-recombination regions, DL71 had higher identities with Fuyang-0805a than Tainan/5746/98, and located in the cluster with C4 strains. However, in the recombination regions, DL71 had higher identities with Tainan/5746/98 than Fuyang-0805a, and located in the cluster with C2 strains. Thus, DL71 was a novel multiple inter-subgenotype recombinant derived from the dominant subgenotype C4 and the sporadic subgenotype C2 strains. Monitoring the emergence of new variants by the whole-genome sequencing remains essential for preventing disease outbreaks and developing new vaccines.

The urinary exosomes derived from premature infants attenuate cisplatin-induced acute kidney injury in mice via microRNA-30a-5p/ mitogen-activated protein kinase 8 (MAPK8).

Acute kidney injury (AKI) is a susceptible factor for chronic kidney disease (CKD). There is still a lack of effective prevention methods in clinical practice. This study investigated the protective effect of the urinary exosomes from premature infants on cisplatin-induced acute kidney injury. Here we isolated exosomes from the fresh urine of premature infants. A C57BL/6 mice model of cisplatin-induced acute kidney injury was given 100 ug urinary exosomes 24 hours after model establishment. The kidneys were collected for pathological examination and the evaluation of renal tubular damage and apoptosis. In the in vitro experiment, human renal cortex/proximal tubular cells (HK-2) were induced by cisplatin to assess the effect of the urine exosomes from premature infants. Exosome microRNA (miRNA) sequencing technology was applied to investigate the miRNAs enriched in exosomes and the dual-luciferase gene reporter system to examine the targeting relationship of the miRNA with target genes. The results indicated that the urinary exosomes could decrease the serum creatinine level and the apoptosis of renal tubular cells, and reduce mice mortality. In addition, miR-30a-5p was the most abundant miRNA in the exosomes. It protected HK-2 cells from cisplatin-induced apoptosis by targeting and down-regulating the mitogen-activated protein kinase 8 (MAPK8). Together, our findings identified that the urinary exosomes derived from premature infants alleviated cisplatin-induced acute kidney injury and inhibited the apoptosis of HK-2 via miR-30a-5p, which could target MAPK8. These findings implied that urinary exosomes from premature infants riched in miR-30a-5p might become a potential treatment for AKI.

Characterization of the gut microbiota in hemodialysis patients with sarcopenia.

PURPOSE: Maintenance hemodialysis (MHD) patients are at high risk of sarcopenia. Gut microbiota affects host metabolic and may act in the occurrence of sarcopenia importantly. This study aimed to study the characterization of the gut microbiota in MHD patients with sarcopenia, and to further reveal the complex pathophysiology of sarcopenia in MHD patients. METHODS: Fecal samples and clinical data were collected from 30 MHD patients with sarcopenia, and 30 age-and-sex-matched MHD patients without sarcopenia in 1 general hospital of Jiangsu Province from December 2020 to March 2021. 16S rRNA sequencing technology was used to analyze the genetic sequence of the gut microbiota for evaluation of the diversity, species composition, and differential microbiota of the two groups. RESULTS: Compared to MHD patients without sarcopenia, the ACE index of patients with sarcopenia was lower (P = 0.014), and there was a structural difference in the ß-diversity between the two groups (P = 0.001). At the genus level, the relative abundance of Tyzzerella_4 in the sarcopenia group was significantly higher than in the non-sarcopenia group (P = 0.039), and the relative abundance of Megamonas (P = 0.004), Coprococcus_2 (P = 0.038), and uncultured_bacterium_f_Muribaculaceae (P = 0.040) decreased significantly. CONCLUSION: The diversity and structure of the gut microbiota of MHD patients with sarcopenia were altered. The occurrence of sarcopenia in MHD patients may be influenced by gut microbiota.

N-terminal prohormone B-type natriuretic peptide variability acts as a predictor of poor prognosis in patients with cardiorenal syndrome type 2.

This study aims to explore the effect of N-terminal pro-brain natriuretic peptide (NT-proBNP) variability (mean absolute difference of the log2 NT-proBNP level measured in hospital) on the prognosis of patients with cardiorenal syndrome (CRS) type 2. Patients with CRS type 2 were retrospectively included. The varied NT-proBNP indications were analyzed. They were NT-proBNP I(pre-treatment), NT-proBNP II(post-treatment), NT-proBNP II/I, ΔNT-proBNP, log2 (NT-proBNP) variability and mean log2 (NT-proBNP). A logistic regression model and survival curves (Kaplan-Meier analysis) were built to identify independent predictors associated with poor prognosis. The primary outcomes were major adverse renal and cardiac events. The secondary outcome was all-cause mortality. From 2012 to 2016, 136 patients were included in this study with 69 (50.7%) had high log2 (NT-proBNP) variability level. The optimal cutoff level for each NT-proBNP indication that predicts poor prognosis was calculated, and the area under curves ranged from 0.668 to 0.891 with different indications. Kaplan-Meier analysis revealed that there was significantly correlated with prevalence of primary outcomes and NT-proBNP variability. The hazard ratios (HRs) ranged from 1.67 to 6.61 with different indications. The multivariate regression analyses also identified the risk of the primary outcomes were associated with elevated NT-proBNP values, except NT-proBNP I. The odds ratio (ORs) ranged from 1.83 to 6.61 with different indications. When analyzing the relationship between NT-proBNP variability and all-cause mortality, the results were the same. NT-proBNP variability might serve as an independent predictor for poor prognosis and all-cause mortality in patients with CRS type 2.

Physiological analysis of the effect of altitudinal gradients on Leymus secalinus on the Qinghai-Tibetan Plateau.

On the Qinghai-Tibetan Plateau, the high-altitudinal gradients can negatively affect plant distribution and limit plant growth and reproduction. Leymus secalinus (Georgi) Tzvel. is an important forage crop on the Qinghai-Tibetan Plateau and has an excellent ability to fix sand and improve soil. To evaluate the effect of altitude on the physiological characteristics of L. secalinus on the Qinghai-Tibetan Plateau, we measured the lipid peroxidation; chlorophyll a (Chl a), chlorophyll b (Chl b), total carotenoid (Car), soluble protein, proline and soluble sugar contents; and the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in leaves from eight different altitudes in Minhe County and Huangzhong County. The leaves were collected at the initial bloom stage, and the average vertical distance between two adjacent collection sites was approximately 100 meters. The reduction in Chl a and Chl b contents and the increase in Car contents can allow plants to weaken their light absorption and avoid photodamage to the chloroplast. The decrease in malondialdehyde (MDA) content associated with lower lipid peroxidation, and the changes of CAT, SOD and POD activities reflect a higher reactive oxygen species (ROS) scavenging capacity in high-altitude plants. The increase in proline and soluble sugar contents with elevation suggests that proline and soluble sugar may play a key role in the osmotic adjustment of plants in alpine regions. The results suggested that altitudinal gradients negatively affect L. secalinus on the Qinghai-Tibetan Plateau and that the adaptation mechanism and survival strategies of L. secalinus were attributed to the combined effects of multiple protective strategies.

In vitro study on human umbilical cord mesenchymal stem cells transfected with lentivirus-mediated hNIS-EGFP dual reporter gene and co-labeled with superparamagnetic iron oxide.

The aim of the present study was to establish a stem cell line for multi-mode imaging (in vivo fluorescence imaging, magnetic resonance imaging and 99mTc single-photon emission computed tomography) and to study the biological activity, stemness, proliferative activity and differentiation ability of superparamagnetic iron oxide (SPIO), human sodium/iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) co-labeled human umbilical cord mesenchymal stem cells (hUCMSCs). The EGFP reporter gene was selected to indirectly reflect the expression of target gene hNIS, and hUCMSCs were re-transfected with the successfully constructed recombinant plasmid pCMV-NIS-EF1-GFP-PGK-puro. When a stem cell line stably expressing hNIS and EGFP was obtained, the cells were incubated with 30 µg/ml SPIO to obtain hNIS, EGFP and SPIO co-labeled stem cells. The protein expressions of hNIS and EGFP were identified using western blot analysis, and the protein function of hNIS was identified by 125I influx and 125I efflux experiments. hNIS-EGFP-hUCMSCs were labeled with SPIO under the mediation of poly-L-lysine, and SPIO, hNIS and EGFP co-labeled hUCMSCs were established successfully. Staining with Prussian blue confirmed that 98% of cells were successfully labeled with SPIO. Western blotting results demonstrated positive hNIS and EGFP protein expression levels, and 125I influx and 125I efflux experiments confirmed that the protein function of hUCMSCs after expressing hNIS was normal. The uptake of 125I was higher in cell lines hNIS-EGFP-hUCMSCs than in control hUCMSCs (fold change: 16.43±2.30 times; P<0.05). The stemness of hNIS-EGFP-hUCMSCs was found to be slightly decreased but not statistically significant; the overall characteristics of stem cells remained unchanged. The assessments of adipogenic and osteogenic differentiation suggest that hNIS-EGFP-hUCMSCs have no significantly different characteristics compared with primary hUCMSCs.

Functional and molecular features of the calmodulin-interacting protein IQCG required for haematopoiesis in zebrafish.

We previously reported a fusion protein NUP98-IQCG in an acute leukaemia, which functions as an aberrant regulator of transcriptional expression, yet the structure and function of IQCG have not been characterized. Here we use zebrafish to investigate the role of iqcg in haematopoietic development, and find that the numbers of haematopoietic stem cells and multilineage-differentiated cells are reduced in iqcg-deficient embryos. Mechanistically, IQCG binds to calmodulin (CaM) and acts as a molecule upstream of CaM-dependent kinase IV (CaMKIV). Crystal structures of complexes between CaM and IQ domain of IQCG reveal dual CaM-binding footprints in this motif, and provide a structural basis for a higher CaM-IQCG affinity when deprived of calcium. The results collectively allow us to understand IQCG-mediated calcium signalling in haematopoiesis, and propose a model in which IQCG stores CaM at low cytoplasmic calcium concentrations, and releases CaM to activate CaMKIV when calcium level rises.

Iqcg is essential for sperm flagellum formation in mice.

Mammalian spermatogenesis comprises three successive phases: mitosis phase, meiosis phase, and spermiogenesis. During spermiogenesis, round spermatid undergoes dramatic morphogenesis to give rise to mature spermatozoon, including the condensation and elongation of nucleus, development of acrosome, formation of flagellum, and removal of excessive cytoplasm. Although these transformations are well defined at the morphological level, the mechanisms underlying these intricate processes are largely unknown. Here, we report that Iqcg, which was previously characterized to be involved in a chromosome translocation of human leukemia, is highly expressed in the spermatogenesis of mice and localized to the manchette in developing spermatids. Iqcg knockout causes male infertility, due to severe defects of spermiogenesis and resultant total immobility of spermatozoa. The axoneme in the Iqcg knockout sperm flagellum is disorganized and hardly any typical ("9+2") pattern of microtubule arrangement could be found in Iqcg knockout spermatids. Iqcg interacts with calmodulin in a calcium dependent manner in the testis, suggesting that Iqcg may play a role through calcium signaling. Furthermore, cilia structures in the trachea and oviduct, as well as histological appearances of other major tissues, remain unchanged in the Iqcg knockout mice, suggesting that Iqcg is specifically required for spermiogenesis in mammals. These results might also provide new insights into the genetic causes of human infertility.

Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling.

LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem ß-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites.

Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia.

Abnormal epigenetic regulation has been implicated in oncogenesis. We report here the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). We discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOXB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed Arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases. Our work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases.

Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML.

Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARalpha (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARalpha degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARalpha and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)-conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug's mechanism of action and its specificity for APL.

Expression, crystallization and preliminary X-ray studies of the immunoglobulin-like domain 3 of human palladin.

Palladin is a member of the recently discovered palladin/myotilin/myopalladin family, the members of which associate with alpha-actinin. Palladin may play important roles in actin stress-fibre formation, cell adhesion and migration. The immunoglobulin-like domain 3 of human palladin has been overexpressed in Escherichia coli and crystallized suitable for X-ray crystallographic study. Crystals have been obtained using the vapour-diffusion method and belong to space group P2(1). X-ray diffraction data were collected in-house to 1.8 A resolution from a single crystal. The unit-cell parameters are a = 40.9, b = 33.3, c = 34.8 A, beta = 90.3 degrees . One molecule was predicted to be present in the asymmetric unit.

Design of wide-spectrum inhibitors targeting coronavirus main proteases.

The genus Coronavirus contains about 25 species of coronaviruses (CoVs), which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens), and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (M(pro)s), which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV M(pro)s, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.

AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec.

To explore the genetic abnormalities that cooperate with AML1-ETO (AE) fusion gene to cause acute myeloid leukemia (AML) with t(8;21), we screened a number of candidate genes and identified 11 types of mutations in C-KIT gene (mC-KIT), including 6 previously undescribed ones among 26 of 54 (48.1%) cases with t(8;21). To address a possible chronological order between AE and mC-KIT, we showed that, among patients with AE and mC-KIT, most leukemic cells at disease presentation harbored both genetic alteration, whereas in three such cases investigated during complete remission, only AE, but not mC-KIT, could be detected by allele-specific PCR. Therefore, mC-KIT should be a subsequent event on the basis of t(8;21). Furthermore, induced expression of AE in U937-A/E cells significantly up-regulated mRNA and protein levels of C-KIT. This may lead to an alternative way of C-KIT activation and may explain the significantly higher C-KIT expression in 81.3% of patients with t(8;21) than in patients with other leukemias. These data strongly suggest that t(8;21) AML follows a stepwise model in leukemogenesis, i.e., AE represents the first, fundamental genetic hit to initiate the disease, whereas activation of the C-KIT pathway may be a second but also crucial hit for the development of a full-blown leukemia. Additionally, Gleevec suppressed the C-KIT activity and induced proliferation inhibition and apoptosis in cells bearing C-KIT N822K mutation or overexpression, but not in cells with D816 mC-KIT. Gleevec also exerted a synergic effect in apoptosis induction with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

Cloning, expression, purification and crystallization of NHR3 domain from acute myelogenous leukemia-related protein AML1-ETO.

The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. This translocation creates a fusion between the acute myelogenous leukemia 1 (AML1, a transcription factor) gene on chromosome 21 and the eight-twenty-one (ETO, a zinc finger nuclear protein) gene on chromosome 8, leading to the repression of certain AML1 target genes. We cloned NHR3 domain coding fragment into vector pGEX-6p-1 using PCR and obtained recombinant plasmid pGEX-6p-1-NHR3, which can be induced to stably overexpress fusion protein in E. coli. Through the purification on GST affinity chromatography column and PreScission protease cleavage, a large amount of NHR3 protein with high purity was obtained. In order to avoid possible interference of some strong negative charged molecules, NHR3 protein was further purified by Mono Q anion exchange chromatography. The NHR3 crystals were grown with hanging drop/vapor diffusion method and the first crystals appeared after four weeks at 18 degrees in 0.2 M Tris-sodium citrate dihydrate, 0.1 M sodium cacodylate, pH 6.5, and 30% iso-propanol (V/V). ESI mass spectrum showed that the molecular weight of this domain was in agreement with its primary structure sequence prediction, and circular dichroism spectral data (190-250 nm) showed that NHR3 had a well-defined secondary structure of 25.9% alpha-helix, 23.2% random coil and 50.9% turn, which was consistent with GOV4 software prediction.