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Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals.

Biomarkers for the progression of lung function in COPD are currently scarce. Plasma fetuin-B (FETUB) was identified by iTRAQ-based proteomics and was verified by ELISA in another group. Information regarding acute exacerbation (AE) was collected in a one-year follow-up programme. FETUB concentrations (1652 ± 427 ng/ml) were greater in COPD patients than in controls (1237 ± 77 ng/ml). The concentrations of FETUB in GOLD II (1762 ± 427 ng/ml), III (1650 ± 375 ng/ml) and IV (1800 ± 451 ng/ml) groups were greater than those in the controls (1257 ± 414 ng/ml) and the GOLD I (1345 ± 391 ng/ml) group. ROCs indicated that FETUB distinguished COPD patients from controls (AUC 0.747, 95% CI: 0.642–0.834) and also GOLD II, III and IV from GOLD I COPD patients (AUC: 0.770, 95% CI: 0.634–0.874). The combination of FETUB and fibrinogen performed better (AUC: 0.804, 95% CI: 0.705–0.881). FETUB also predicted the occurrence of AE (AUC: 0.707, 95% CI: 0.566–0.824) or frequent AE (AUC: 0.727, 95% CI: 0.587–0.840). FETUB concentrations were negatively correlated with FEV1%pred (r = −0.446, p = 0.000) and positively correlated with RV%pred (r = 0.317, p = 0.004), RV/TLC% (r = 0.360, p = 0.004), CT emphysema% (r = 0.322, p = 0.008) and grades of lung function (r = 0.437, p = 0.000). In conclusion, FETUB is likely to assist the diagnosis and management of COPD as a complement for other markers.

Maleic acid has been used as an etchant or non-rinse conditioner in adhesive dentistry. However, the inherent mechanisms of the interaction of maleic acid with hydroxyapatite/enamel have never been fully elucidated. The purpose of this study was to provide evidence for the chemisorption of maleic acid onto hydroxyapatite/enamel, and to identify the reaction products obtained following the interaction of maleic acid with hydroxyapatite. Hydroxyapatite particles were dissolved in a 15% (w/v) aqueous solution of maleic acid (pH = 0.98). Half of the solution was dried to obtain a desiccated mixture. This mixture, hydroxyapatite, maleic acid and self-prepared calcium maleate were analysed by X-ray diffraction (XRD). Acetone was added to the other half of the solution to obtain a precipitate. This precipitate, hydroxyapatite, maleic acid, unetched enamel and maleic acid-etched enamel were analysed by X-ray photoelectron spectroscopy (XPS). The precipitate was also analysed by (1)H NMR. A new binding energy, indicating carboxylate groups, was detected by XPS on the precipitate and maleic acid-etched enamel surface. XRD data indicated the formation of calcium maleate and calcium hydrogen phosphate after the reaction. NMR data revealed that one carboxylic group of maleic acid reacted with hydroxyapatite. Hence, maleic acid can chemisorb to hydroxyapatite and enamel via ionic interactions.

Background Ultrafiltration (UF) failure mostly contributes to technical failure in peritoneal dialysis (PD) patients, and one of its responsible factors is peritoneal angiogenesis. Resveratrol has been proposed to have an angiogenesis-ameliorating effect on tumor patients. We hypothesize transresveratrol has beneficial effects on angiogenesis-related markers in PD patients. Methods In this prospective, randomized, and double-blind trial, 72 patients were randomly assigned to 12-week treatment of low-dose or high-dose (150 or 450 mg/d) trans-resveratrol or a placebo. Visits were scheduled at 0, 4, 8, and 12 weeks after treatment. Clinical indices including 24-hour UF volume, UF rate, 24-hour urine volume, residual renal function, and dialysis adequacy (kt/v) were measured. Angiogenesis markers including vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), angiopoietin-2 (Ang-2), tyrosine kinase 2 (Tie-2), and thrombospondin-1 (Tsp-1) in peritoneal effluent were also assessed by enzyme-linked immunosorbent assay. Results Finally, 64 out of 72 patients were analyzed, 18 in the high-dose group, 22 in the low-dose group, and 24 in the placebo group. Over the 12-week period, patients in the high-dose group [mean change from baseline (95% CI): 171.4 (141.3-201.5) (mL), p ¼ 0.003 (Net UF); 11.3(10.5-12.1) (mL/h), p ¼ 0.02 (UF rate)] or the low-dose group [mean change from baseline (95% CI: 98.1 (49.5-146.7) (mL), p ¼ 0.007 (Net UF); 6.5 (4.4-8.6) (mL/h), p ¼ 0.04 (UF rate)] versus the placebo group had a significantly greater improvement in mean net UF volume and UF rate. The appearance rates of VEGF, Flk-1, and Ang-2 were more significantly reduced (appearance rates of Tie-2 and Tsp-1 increased) in the high-dose group versus the placebo group, but not in the low-dose group. Conclusion Supplementation with trans-resveratrol is beneficial to improve ultrafiltration in PD patients, and high-dose supplementation may improve ultrafiltration by ameliorating angiogenesis induced by conventional lactate-buffered PD solutions.

Purpose To investigate a novel mutation in the WEE2 gene in a female patient with primary infertility and fertilization failure. Methods Sanger sequencing was used to detect mutations in WEE2. The pathogenicity of the identified variant and its possible effects on the WEE2 protein were evaluated with in silico tools and molecular modeling. We used the calcium ionophore A23187 as a chemical activator of oocytes after intracytoplasmic sperm injection (ICSI). Results We identified a consanguineous family with a novel homozygous missense mutation in WEE2 (c.619C>T [p.R207C]). Based on preliminary bioinformatics analysis, we speculate that the novel homozygous missense mutation is pathogenic. ICSI combined with assisted oocyte activation (ICSI-AOA) did not overcome fertilization failure in this patient with WEE2 mutation. Conclusions We identified a novel mutation in WEE2 (c.619C>T [p.R207C]) in a female patient with fertilization failure after ICSI, and we provide evidence that this novel homozygous missense mutation can cause fertilization failure.

STUDY QUESTION What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility? SUMMARY ANSWER Novel compound heterozygous variants (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) in actin-like protein 7A (ACTL7A) were identified as a causative genetic factor for human TFF. WHAT IS KNOWN ALREADY ACTL7A, an actin-related protein, is essential for spermatogenesis. ACTL7A variants have been reported to cause early embryonic arrest in humans but have not been studied in human TFF. STUDY DESIGN, SIZE, DURATION We recruited a non-consanguineous family whose son was affected by infertility characterized by TFF after ICSI. Whole-exome sequencing was used to identify the potential pathogenic variants. Artificial oocyte activation (AOA) after ICSI was performed to overcome TFF and any resulting pregnancy was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS Sanger sequencing was performed to validate the variants. Pathogenicity of the identified variants was predicted by in silico tools. The ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM). Immunofluorescence staining and western blotting were used to investigate the mechanism of the variants on the affected spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE Novel compound heterozygous variants in ACTL7A (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) were identified in a family with TFF after ICSI. In silico analysis predicted that the variants lead to a disease-causing protein. TEM showed that the ACTL7A variants caused ultrastructural defects in the acrosome and perinuclear theca. Protein expression of ACTL7A and phospholipase C zeta, a key sperm-borne oocyte activation factor, was significantly reduced in the affected sperm compared to healthy controls, suggesting that the ACLT7A variants lead to an oocyte activation deficiency and TFF. AOA by calcium ionophore (A23187) after ICSI successfully rescued the TFF and achieved a live birth for the patient with ACTL7A variants. LIMITATIONS, REASONS FOR CAUTION Given the rarity of sperm-associated TFF, only one family with an only child carrying the ACTL7A variants was found. In addition, the TFF phenotype was not assessed in two or more ICSI cycles, due to the intervention in ICSI with AOA after one failed ICSI cycle. Further studies should validate the ACTL7A variants and its effect on male infertility in larger independent cohorts. WIDER IMPLICATIONS OF THE FINDINGS : Our findings revealed a critical role of ACTL7A in male fertility and identified bi-allelic variants in ACTL7A associated with human TFF, which expands the genetic spectrum of TFF and supports the genetic diagnosis of TFF patients. We also rescued TFF by AOA and obtained a healthy live birth, which provides a potentially effective intervention for patients with ACTL7A pathogenic variants. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared. TRIAL REGISTRATION NUMBER N/A.

BackgroundThe role of airway microbiota in COPD is highly debated. Symptomology assessment is vital for the management of clinically stable COPD patients; however, the link between symp toms and the airway microbiome is currently unknown.PurposeThe present study aimed to evaluate the relationship among stable COPD patients.Patients and methodsWe conducted pyrosequencing of bacterial 16S rRNA using induced sputum samples in a Han Chinese cohort that included 40 clinically stable COPD patients and 19 healthy controls.ResultsAlterations in community composition and core bacte rial taxa (Neisseria subflava, etc.) were observed in patients with severe symptoms compared with controls. The co-occurrence network indicated that the key microbiota enriched in COPD patients showed higher expression in patients with severe symptoms. The association pattern of symptoms with the sputum microbiome was obviously different from that of lung function in COPD patients.ConclusionThese findings broaden our insights into the relationship between the sputum microbiota and the symptom severity in COPD patients, emphasizing the role of symptoms in the airway microbiome, independent of lung function.