Predicting the risk of pulmonary infection in patients with chronic kidney failure: A-C2GH2S risk score-a retrospective study.

PURPOSE: The objective of this study is to investigate the associated risk factors of pulmonary infection in individuals diagnosed with chronic kidney disease (CKD). The primary goal is to develop a predictive model that can anticipate the likelihood of pulmonary infection during hospitalization among CKD patients. METHODS: This retrospective cohort study was conducted at two prominent tertiary teaching hospitals. Three distinct models were formulated employing three different approaches: (1) the statistics-driven model, (2) the clinical knowledge-driven model, and (3) the decision tree model. The simplest and most efficient model was obtained by comparing their predictive power, stability, and practicability. RESULTS: This study involved a total of 971 patients, with 388 individuals comprising the modeling group and 583 individuals comprising the validation group. Three different models, namely Models A, B, and C, were utilized, resulting in the identification of seven, four, and eleven predictors, respectively. Ultimately, a statistical knowledge-driven model was selected, which exhibited a C-statistic of 0.891 (0.855-0.927) and a Brier score of 0.012. Furthermore, the Hosmer-Lemeshow test indicated that the model demonstrated good calibration. Additionally, Model A displayed a satisfactory C-statistic of 0.883 (0.856-0.911) during external validation. The statistical-driven model, known as the A-C2GH2S risk score (which incorporates factors such as albumin, C2 [previous COPD history, blood calcium], random venous blood glucose, H2 [hemoglobin, high-density lipoprotein], and smoking), was utilized to determine the risk score for the incidence rate of lung infection in patients with CKD. The findings revealed a gradual increase in the occurrence of pulmonary infections, ranging from 1.84% for individuals with an A-C2GH2S Risk Score ≤ 6, to 93.96% for those with an A-C2GH2S Risk Score ≥ 18.5. CONCLUSION: A predictive model comprising seven predictors was developed to forecast pulmonary infection in patients with CKD. This model is characterized by its simplicity, practicality, and it also has good specificity and sensitivity after verification.

Pharmacological inhibition of dynamin-related protein 1 attenuates skeletal muscle insulin resistance in obesity.

Dynamin-related protein-1 (Drp1) is a key regulator in mitochondrial fission. Excessive Drp1-mediated mitochondrial fission in skeletal muscle under the obese condition is associated with impaired insulin action. However, it remains unknown whether pharmacological inhibition of Drp1, using the Drp1-specific inhibitor Mitochondrial Division Inhibitor 1 (Mdivi-1), is effective in alleviating skeletal muscle insulin resistance and improving whole-body metabolic health under the obese and insulin-resistant condition. We subjected C57BL/6J mice to a high-fat diet (HFD) or low-fat diet (LFD) for 5-weeks. HFD-fed mice received Mdivi-1 or saline injections for the last week of the diet intervention. Additionally, myotubes derived from obese insulin-resistant humans were treated with Mdivi-1 or saline for 12 h. We measured glucose area under the curve (AUC) from a glucose tolerance test (GTT), skeletal muscle insulin action, mitochondrial dynamics, respiration, and H2 O2 content. We found that Mdivi-1 attenuated impairments in skeletal muscle insulin signaling and blood glucose AUC from a GTT induced by HFD feeding (p < 0.05). H2 O2 content was elevated in skeletal muscle from the HFD group (vs. LFD, p < 0.05), but was reduced with Mdivi-1 treatment, which may partially explain the improvement in skeletal muscle insulin action. Similarly, Mdivi-1 enhanced the mitochondrial network structure, reduced reactive oxygen species, and improved insulin action in myotubes from obese humans (vs. saline, p < 0.05). In conclusion, inhibiting Drp1 with short-term Mdivi-1 administration attenuates the impairment in skeletal muscle insulin signaling and improves whole-body glucose tolerance in the setting of obesity-induced insulin resistance. Targeting Drp1 may be a viable approach to treat obesity-induced insulin resistance.

Distinct Adaptations of Mitochondrial Dynamics to Electrical Pulse Stimulation in Lean and Severely Obese Primary Myotubes.

BACKGROUND: Skeletal muscle from lean and obese subjects elicits differential adaptations in response to exercise/muscle contractions. In order to determine whether obesity alters the adaptations in mitochondrial dynamics in response to exercise/muscle contractions and whether any of these distinct adaptations are linked to alterations in insulin sensitivity, we compared the effects of electrical pulse stimulation (EPS) on mitochondrial network structure and regulatory proteins in mitochondrial dynamics in myotubes from lean humans and humans with severe obesity and evaluated the correlations between these regulatory proteins and insulin signaling. METHODS: Myotubes from human skeletal muscle cells obtained from lean humans (body mass index, 23.8 ± 1.67 kg·m-2) and humans with severer obesity (45.5 ± 2.26 kg·m-2; n = 8 per group) were electrically stimulated for 24 h. Four hours after EPS, mitochondrial network structure, protein markers of insulin signaling, and mitochondrial dynamics were assessed. RESULTS: EPS enhanced insulin-stimulated AktSer473 phosphorylation, reduced the number of nonnetworked individual mitochondria, and increased the mitochondrial network size in both groups (P < 0.05). Mitochondrial fusion marker mitofusin 2 was significantly increased in myotubes from the lean subjects (P < 0.05) but reduced in subjects with severe obesity (P < 0.05). In contrast, fission marker dynamin-related protein 1 (Drp1Ser616) was reduced in myotubes from subjects with severe obesity (P < 0.05) but remained unchanged in lean subjects. Reductions in DrpSer616 phosphorylation were correlated with improvements in insulin-stimulated AktSer473 phosphorylation after EPS (r = -0.679, P = 0.004). CONCLUSIONS: Our data demonstrated that EPS induces more fused mitochondrial networks, which are associated with differential adaptations in mitochondrial dynamic processes in myotubes from lean humans and human with severe obesity. It also suggests that improved insulin signaling after muscle contractions may be linked to the reduction in Drp1 activity.

Roux-en-Y gastric bypass surgery restores insulin-mediated glucose partitioning and mitochondrial dynamics in primary myotubes from severely obese humans.

BACKGROUND/OBJECTIVES: Impaired insulin-mediated glucose partitioning is an intrinsic metabolic defect in skeletal muscle from severely obese humans (BMI ≥ 40 kg/m2). Roux-en-Y gastric bypass (RYGB) surgery has been shown to improve glucose metabolism in severely obese humans. The purpose of the study was to determine the effects of RYGB surgery on glucose partitioning, mitochondrial network morphology, and the markers of mitochondrial dynamics skeletal muscle from severely obese humans. SUBJECT/METHODS: Human skeletal muscle cells were isolated from muscle biopsies obtained from RYGB patients (BMI = 48.0 ± 2.1, n = 7) prior to, 1 month and 7 months following surgery and lean control subjects (BMI = 22.4 ± 1.1, n = 7). Complete glucose oxidation, non-oxidized glycolysis rates, mitochondrial respiratory capacity, mitochondrial network morphology, and the regulatory proteins of mitochondrial dynamics were determined in differentiated human myotubes. RESULTS: Myotubes derived from severely obese humans exhibited enhanced glucose oxidation (13.5%; 95% CI [7.6, 19.4], P = 0.043) and reduced non-oxidized glycolysis (-1.3%; 95% CI [-11.1, 8.6]) in response to insulin stimulation at 7 months after RYGB when compared with the presurgery state (-0.6%; 95% CI [-5.2, 4.0] and 19.5%; 95% CI [4.0, 35.0], P = 0.006), and were not different from the lean controls (16.7%; 95% CI [11.8, 21.5] and 1.9%; 95% CI [-1.6, 5.4], respectively). Further, the number of fragmented mitochondria and Drp1(Ser616) phosphorylation were trended to reduce/reduced (0.0104, 95% CI [0.0085, 0.0126], P = 0.091 and 0.0085, 95% CI [0.0068, 0.0102], P = 0.05) in myotubes derived from severely obese humans at 7 months after RYGB surgery in comparison with the presurgery state. Finally, Drp1(Ser616) phosphorylation was negatively correlated with insulin-stimulated glucose oxidation (r = -0.49, P = 0.037). CONCLUSION/INTERPRETATION: These data indicate that an intrinsic metabolic defect of glucose partitioning in skeletal muscle from severely obese humans is restored by RYGB surgery. The restoration of glucose partitioning may be regulated through reduced mitochondrial fission protein Drp1 phosphorylation.

Exercise preconditioning reduces ischemia reperfusion-induced focal cerebral infarct volume through up-regulating the expression of HIF-1α.

To study the effect and mechanism of exercise preconditioning on focal cerebral ischemia reperfusion induced cerebral infarction via rat model; Sixty Sprague Dawley rats were divided into three groups at random: ischemia reperfusion group (IR, n=24), sham group (sham, n=12) and exercise preconditioning group (EP, n=24). Group EP carried out moderate exercise preconditioning for 4 weeks (swimming with non-weight bearing, 60 minutes/day, 6 days/week), Rats in Group EP and IR were established cerebral ischemia reperfusion injury model by Zea Longa's thread method. The cerebral infarct volume in rat of different group was evaluated after 2%TTC staining, expression of HIF-1α in rats' brain was detected by real-time RT-PCR, immunohistochmeistry method and western blot. No cerebral infarction and significant expression of HIF-1α in Group sham. Compared with Group IR, there was smaller infarct volume and stronger HIF-1α expression in Group EP (P<0.05). Moderate exercise preconditioning reduces ischemia reperfusion induced focal cerebral infarct volume through up-regulating the expression of HIF-1α.

Zili antagonizes Bmp signaling to regulate dorsal-ventral patterning during zebrafish early embryogenesis.

Bone morphogenetic protein (Bmp) signaling plays a pivotal role in dorsal-ventral (DV) patterning in vertebrate embryos. Piwi proteins are required for germline and stem cell development. Our previous study demonstrated that Zili, zebrafish Piwil2, inhibits transforming growth factor (TGF)-ßsignaling by interacting with Smad4, suggesting a role for zili in Bmp signaling. In the present study, zili-MO or zili mRNA was microinjected into one-cell embryos to knock down or elevate the expression of zili to study the role of zili during early zebrafish embryogenesis. Knockdown of zili inhibited the expression of dorsal marker genes, and enhanced that of ventral marker genes. In contrast, overexpression of zili promoted expression of dorsal marker genes, while it inhibited ventral marker genes. These results suggest that zili regulates DV patterning. The influence of zili on the Bmp pathway was further explored. Knockdown of zili resulted in higher expression levels of bmp2b, and bmp4, the Bmp signaling ligands, and reduced expression of chordin (chd), noggin (nog1), and follistatin (fst), which encode BMP antagonists. Meanwhile, overexpression of zili produced opposite effects. In conclusion, our results indicate that zili regulates dorsal-ventral patterning by antagonizing Bmp signaling during early embryogenesis in zebrafish.

Piwi-like 2 mediates fibroblast growth factor signaling during gastrulation of zebrafish embryo.

Piwi (P-element-induced wimpy testis) proteins have been shown to play important roles in maintenance of germ line stem cells, germ cell proliferation and differentiation, and control of Piwi-interacting RNAs (PiRNAs). PiRNAs comprise a broad class of small noncoding RNAs that function as an endogenous defense system against transposable elements. Fibroblast growth factor (Fgf) signals, mediated partly by no tail gene (ntl), are responsible for patterning embryo and mesoderm formation. To understand the function of Piwi proteins, we used zebrafish as a model system. In zebrafish, piwi-like 2 gene (piwil2) is also required for germ cell differentiation and meiosis. Here we report that piwil2 knockdown is able to inhibit the expression of fibroblast growth factor 8a (fgf8a). In contrast, injection with piwil2 mRNA enhances fgf8a expression. Knockdown of piwil2 reduces the inductive effect of fgf8a on dorsalized phenotype, in which embryos extend to an oval shape at the end of epiboly stage. Coinjection with fgf8a and piwil2 mRNAs led to more seriously dorsalized phenotype than coinjection with fgf8a mRNA and piwil2-cMO. In addition, knockdown of piwil2 inhibits the inductive effect of fgf8a on ntl, whereas overexpression of piwil2 enhances the inductive effect of fgf8a on ntl. We also demonstrate that piwil2 positively regulates ntl expression at bud stage, while piwil2 negatively regulates ntl expression at 24 hours post-fertilization. Thus, the functional consequences of piwil2 expression vary during early development of zebrafish embryo. Taken together, we suggest that zebrafish piwil2 is a mediator of Fgf signals in gastrula period.

Zebrafish Dnd protein binds to 3'UTR of geminin mRNA and regulates its expression.

Dnd (dead end) gene encodes an RNA binding protein and is specifically expressed in primordial germ cells (PGCs) as a vertebrate-specific component of the germ plasma throughout embryogenesis. By utilizing a technique of specific nucleic acids associated with proteins (SNAAP), 13 potential target mRNAs of zebrafish Dnd (ZDnd) protein were identified from 8-cell embryo, and 8 target mRNAs have been confirmed using an RT-PCR analysis. Of the target mRNAs, the present study is focused on the regulation of geminin, which is an inhibitor of DNA replication. Using electrophoretic mobility shift assay (EMSA), we demonstrated that ZDND protein bound the 67-nucleotide region from 864 to 931 in the 3'UTR of geminin mRNA, a sequence containing 60.29% of uridine. Results from a dual-luciferase assay in HEK293 cells showed that ZDND increases the translation of geminin. Taken together, the identification of target mRNA for ZDnd will be helpful to further explore the biological function of Dnd in zebrafish germ-line development as well as in cancer cells.

Sp1 plays an important role in regulating the transcription of ZNF313.

The ZNF313 gene has the highest transcription level in fertile male testes and may be related to human spermatogenesis. The deletion-mutated plasmids of ZNF313 promoter were constructed and transfected into HEK293 cells. The result showed that the fragment from nt -157 to +8 has a basal transcriptional activity. A functional Sp1 binding site was identified by site-directed mutation test and mithramycin A treatment. A 447-bp based at +233 to -213 exhibits a characteristic CpG island, which overlaps with the promoter region. Our work suggests that ZNF313 is controlled at the transcriptional level, and a common mechanism controlling the basal transcription of ZNF313 gene exists.

[Initial study on zili functions to the development of the germ layers during zebrafish early embryogenesis].

OBJECTIVE: To study the role for piwil2 gene (zili) in the development of the ectoderm, mesoderm and endoderm during early embryogenesis of zebrafish. METHODS: zili morpholino antisense oligonucleotide and 5 mis-pair control morpholino were used in this study. zili was cloned into expression vector. zili mRNA was synthesized in vitro. The antisense RNA probes of gsc, evel and sox17 were synthesized. zili-MO, zili-cMO and zili mRNA was microinjected into one-cell embryos, respectively. Whole-mount in situ hybridization was used to monitor the expressions of marker genes. RESULTS: Microinjection of zili-MO, which knocked down the expression of zili, downregulated the expression of the ectodermal and mesodermal marker gene gsc, promoting the expression of the ectodermal marker gene evel and resulting in the decrease of endodermal cell expressed sox17. The overexpression of zili, promoting the expression of gsc, inhibiting the expression of eve1 and resulting in the decrease of endodermal cell expressed sox17 were observed after microinjection of zili-mRNA. CONCLUSION: zili might have some effect on the formation of the ectoderm, mesoderm and endoderm during early embryogenesis and might be important for normal embryonic development.

Zili inhibits transforming growth factor-beta signaling by interacting with Smad4.

Piwi proteins are required for germ cell proliferation, differentiation, and germ line stem cell maintenance. In normal tissues, human and mouse Piwil2 are primarily expressed in testis but widely expressed in tumors. However, the underlying mechanism remains largely unknown. In vertebrates, transforming growth factor (TGF)-beta signaling plays an important role in patterning embryo and control of cell growth and differentiation. A previous study has shown a role for Zili, a Piwil2 gene in zebrafish, in germ cells in zebrafish. Here we report that zili functions in patterning the early embryo and inhibits TGF-beta signaling. Whole mount expression analysis shows that zili expresses not only in PGCs but also in axis. Ectopic expression of zili causes fusion of the eyes and reduction of mesodermal marker genes expression, suggesting that zili functions to inhibit Nodal signaling and mesoderm formation. Genetic interaction shows that zili inhibits Nodal and bone morphogenetic protein signaling. The results of protein interaction assays identify that Zili binds to Smad4 via its N-terminal domain and prevents the formation of Smad2/3/4 and Smad1/5/9/4 complexes to antagonize TGF-beta signaling. This work shows that zili plays a role in early embryogenesis beyond germ line as a novel negative regulator of TGF-beta signaling, extending the function of Piwi proteins in vertebrates.

Molecular cloning and expression analysis of a zebrafish novel zinc finger protein gene rnf141.

ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE). The open reading frame (ORF) encodes a polypeptide of 222 amino acids which shares 75.65% identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates.

Molecular cloning and expression analysis of a zebrafish novel zinc finger protein gene rnf141

ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE). The open reading frame (ORF) encodes a polypeptide of 222 amino acids which shares 75.65 percent identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates.

DAZL binds to the transcripts of several Tssk genes in germ cells.

The Dazl gene encodes a germ-cell-specific RNA-binding protein which is essential for spermatogenesis. It has been proposed that this protein (DAZL) binds to RNA in the cytoplasm of germ cells and controls spermatogenesis. Using the specific nucleic acids associated with proteins (SNAAP) technique, we identified 17 target mRNAs bound by mDAZL. Among these transcripts, we focused on TSSK2, which encodes a testis-specific serine/threonine kinase. To date, five TSSK family members have been cloned, and all are exclusively expressed in the testis. We demonstrated that in addition to the TSSK1 3'UTR , the 3'UTRs of TSSKs 2 and 4 were bound by human and mouse DAZL, and that human DAZL (hDAZL) bound to the 3'UTR of human TSSK5 (hTSSK5). Our results suggest that the Dazl gene may play different roles in human and mouse spermatogenesis by regulating different members of the downstream gene family.