Localization and expression of histone H2A variants during mouse oogenesis and preimplantation embryo development.

Epigenetic modifications of the genome, such as histone H2A variants, ensure appropriate gene activation or silencing during oogenesis and preimplantation embryo development. We examined global localization and expression of the histone H2A variants, including H2A.Bbd, H2A.Z and H2A.X, during mouse oogenesis and preimplantation embryo development. Immunocytochemistry with specific antibodies against various histone H2A variants showed their localization and changes during oogenesis and preimplantation development. H2A.Bbd and H2A.Z were almost absent from nuclei of growing oocytes (except 5-day oocyte), whereas H2A.X was deposited in nuclei throughout oogenesis and in preimplantation embryos. In germinal vesicle (GV) oocyte chromatin, H2A.Bbd was detected as a weak signal, whereas no fluorescent signal was detected in GV breakdown (GVBD) or metaphase II (MII) oocytes; H2A.Z showed intense signals in chromatin of GV, GVBD and MII oocytes. H2A. Bbd showed very weak signals in both pronucleus and 2-cell embryo nuclei, but intense signals were detected in nuclei from 4-cell embryo to blastula. The H2A.Z signal was absent from pronucleus to morula chromatin, whereas a fluorescent signal was detected in blastula nuclei. Our results suggest that histone H2A variants are probably involved in reprogramming of genomes during oocyte meiosis or after fertilization.

T cell protein tyrosine phosphatase (TCPTP) deficiency in muscle does not alter insulin signalling and glucose homeostasis in mice.

AIMS/HYPOTHESIS: Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle. METHODS: We generated muscle-specific TCPTP-deficient (Mck-Cre;Ptpn2(lox/lox)) mice (Mck, also known as Ckm) and assessed the impact on glucose homeostasis and muscle insulin receptor signalling in chow-fed versus high-fat-fed mice. RESULTS: Blood glucose and insulin levels, insulin and glucose tolerance, and insulin-induced muscle insulin receptor activation and downstream PI3K/Akt signalling remained unaltered in chow-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. In addition, body weight, adiposity, energy expenditure, insulin sensitivity and glucose homeostasis were not altered in high-fat-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. CONCLUSIONS/INTERPRETATION: These results indicate that TCPTP deficiency in muscle has no effect on insulin signalling and glucose homeostasis, and does not prevent high-fat diet-induced insulin resistance. Thus, despite their high degree of sequence identity, PTP1B and TCPTP contribute differentially to insulin receptor regulation in muscle. Our results are consistent with the notion that these two highly related PTPs make distinct contributions to insulin receptor regulation in different tissues.

Generation of induced pluripotent mouse stem cells in an indirect co-culture system.

Typically, production of induced pluripotent stem cells requires direct contact with feeder cells. However, once the stem cells have reached the appropriate maturation point, it is difficult to separate them from feeder cells, which must be irradiated with γ-rays or treated with the antibiotic mitomycin-C. We used a microporous poly-membrane-based indirect contact co-culture system with mouse embryonic fibroblasts to induce mouse pluripotent stem cells without radiation or antibiotics. We found that induced pluripotent stem cells induced by this co-culture method had a reprogramming efficiency and time similar to those induced using traditional methods. Furthermore, strongly expressed pluripotent markers showed a normal karyotype and formation and contained all three germ layers in a teratoma.

[Clinical application of a self-developed suction-irrigation device in endoscopic ear surgery for attic cholesteatoma].

Objective: To introduce a new self-developed irrigation device(SID) that does not employ a sheath or an irrigation-suction system and evaluate to its efficiency in transcanal endoscopic ear surgery (TEES) for attic cholesteatoma. Methods: 38 patients who were subjected to TEES for attic cholesteatoma between October 2019 to June 2021 were included in this study, including 17 males and 21 females with an average age of (38.6±11.9) years. SID and underwater continuous drilling were used during operation. Width of endoscope and irrigation speed were measured when SID was applied. The operating time, surgical view and complications were compared between two groups. Results: The width of the endoscope was 3.5-4.6 mm in diameter and the irrigation speed was 20-40 ml/min when SID was used. SID cleaned the lens at the tip of the endoscope and created a clear field of view during TEES. The operation time was (86.6±18.1) min. The skin of the external ear canal was found injured during operation in 3 patients, but there were no complications such as necrosis of the flap, stenosis of external ear canal, sensorineural hearing loss, facial paralysis and cerebrospinal fluid leakage. Conclusions: SID is simple and enhances the efficacy of TEES, providing a new irrigation choice in TEES for attic cholesteatoma.

The use of benzodiazepine monotherapy for major depression before and after implementation of guidelines for benzodiazepine use.

WHAT IS KNOWN AND OBJECTIVE: The Department of Health (DOH) in Taiwan issued the 'Guidelines for Benzodiazepine Use in Sedation and Hypnosis' in March 2004, which clearly stated that benzodiazepines (BZDs) should not be used alone for the treatment of depression. However, the extent to which clinicians comply with the BZD guidelines was not known. This study aimed to evaluate whether sole prescribing of BZDs for major depression decreased after the implementation of the BZD guidelines. METHODS: This was a retrospective longitudinal trend analysis by analyzing the Longitudinal Health Insurance Database (LHID) from September 2002 to September 2005. The LHID contains all claims data from a random sample of 1,000,000 beneficiaries of the universal National Health Insurance programme in Taiwan. The 3-year study period was divided equally into six periods, before and after the implementation of the guidelines respectively. For each period, the proportion of patients with major depression (ICD-9-CM code 296.2x, 296.3x) treated with BZDs without any concomitant antidepressant was calculated in order to conduct a trend analysis. RESULTS AND DISCUSSION: A total of 5463 prescriptions of BZDs solely used for major depression were observed in the entire study period. In more than 80% of the BZD prescriptions in which BZDs were used alone for major depression, they were prescribed at doses higher than one prescribed daily dose/defined daily dose and were supplied for more than 7 days. The number of outpatients with major depression ranged from 2137 to 3326 during the 12 periods. The proportion of depressed patients treated with BZDs alone per 3 months (i.e., the non-compliance rate) fluctuated from 6·7% to 9·4% before implementation of the guidelines, and from 8·0% to 9·4% after implementation, in outpatient settings. In addition, the guideline non-adherence rates in inpatient settings varied from 7·0% to 11·8% and from 7·8% to 12·6% before and after the implementation of the BZD guidelines respectively. Further trend analyses indicated that the implementation of the guidelines was not associated with a reduced rate of sole prescribing of BZDs for major depression in either inpatient (P = 0·083) or outpatient settings (P = 0·925). WHAT IS NEW AND CONCLUSION: The formulation and implementation of the BZD guidelines appear not to be associated with a reduced rate of sole prescribing of BZDs for major depression, and more comprehensive efforts are required.

A mutation in the Kit gene leads to novel gonadal phenotypes in both heterozygous and homozygous mice.

The direct sequencing of the Kit cDNA obtained from mutant mice was used to reveal the molecular nature of the W(-3Bao) ENU-induced mutation. There was a T to A transversion at the 441st nucleotide in the W(-3Bao) open reading frame (ORF), which introduced a pre-mature termination codon at residue 147. The gross embryonic development, hematopoiesis and spermatogenesis were examined in the mutant mice. There was no visible difference among the W(-3Bao/+), W(-3Bao/3Bao) and wild type embryos before embryonic day 12.5. W(-3Bao/3Bao) embryos appeared pale after E14.5 and dwarf after E16.5. An extremely low level of hematochrome and large red blood cells were found in W(-3Bao/3Bao) 18.5 days old embryos, leading to the stillbirth of the homozygotes. In 18.5 days old embryos the spermatogonia of W(-3Bao/3Bao) embryos did not migrate to the contorted seminiferous tubules properly, but instead were found in the interstitial tissue. The spermatogonia of W(-3Bao/+) or W(+/+) mice were present in both the interstitial tissue and contorted seminiferous tubules. In the adult male hetereozygotes, there are contorted seminiferous tubules with no spermatogonia, suggesting that the migration defect was dominant. In female W(-3Bao/3Bao) ovaries, primordial follicles were absent while primordial follicles appeared clearly in the ovaries of W(-3Bao/+) or W(+/+) mice. With a nonsense mutation in the Kit gene, W(-3Bao/+) mice show white spotting and an abnormal development of the contorted seminiferous tubules and W(-3Bao/3Bao) mice are stillborn due to severe macrocytic anemia, and have abnormal genital glands in both the male and female.

[Clinical study of butterfly cartilage myringoplasty for anterior quadrant tympanic perforation under endoscope].

Objective: To evaluate the results of butterfly cartilage myringoplasty for anterior quadrant tympanic perforation under endoscope. Methods: Thirty-eight patients with anterior quadrant tympanic perforations who were subjected to endoscopic butterfly cartilage myringoplasty from April 2016 to October 2018 were included in this study, including 16 males and 22 females, with an average age of (34.5±14.2) years. The patients were reviewed retrospectively, and the pre-and post-operative pure tone audiometry (PTA) thresholds, pre-and post-operative air-bone gaps (ABG), post-operative graft success rates and complications were evaluated. SPSS 23.0 was used to analyze data. Results: Mean post-operative follow-up duration was (9.4±3.1) months (range 6-18 months). The graft survival rate was 94.7% (36/38) . The preoperative and postoperative mean PTA was (30.9±8.9) dB HL and (21.4±7.7) dB HL respectively. Preoperative and postoperative mean ABG was (18.4±6.3) dB and (10.8±6.0) dB respectively. There was significant difference between pre-and postoperative PTA and ABG (t=5.353 and 4.162, P<0.05 for both). A postoperative ABG reduction of (8.3±1.5) dB was reached. Two (4.7%) patients had postoperative myringitis, two (4.7%) had recurrent perforation, and one (2.4%) had lateral healing of transplanted tympanic membrane in the postoperative follow-ups. No intratympanic cholesteatoma was observed. Conclusions: Endoscopic butterfly inlay myringoplasty is a reliable, minimally invasive alternative method to repair anterior tympanic membrane perforations, with high closure rate and low risk of complications.

Do faculty and resident physicians discuss their medical errors?

BACKGROUND: Discussions about medical errors facilitate professional learning for physicians and may provide emotional support after an error, but little is known about physicians' attitudes and practices regarding error discussions with colleagues. METHODS: Survey of faculty and resident physicians in generalist specialties in Midwest, Mid-Atlantic and Northeast regions of the US to investigate attitudes and practices regarding error discussions, likelihood of discussing hypothetical errors, experience role-modelling error discussions and demographic variables. RESULTS: Responses were received from 338 participants (response rate = 74%). In all, 73% of respondents indicated they usually discuss their mistakes with colleagues, 70% believed discussing mistakes strengthens professional relationships and 89% knew at least one colleague who would be a supportive listener. Motivations for error discussions included wanting to learn whether a colleague would have made the same decision (91%), wanting colleagues to learn from the mistake (80%) and wanting to receive support (79%). Given hypothetical scenarios, most respondents indicated they would likely discuss an error resulting in no harm (77%), minor harm (87%) or major harm (94%). Fifty-seven percent of physicians had tried to serve as a role model by discussing an error and role-modelling was more likely among those who had previously observed an error discussion (OR 4.17, CI 2.34 to 7.42). CONCLUSIONS: Most generalist physicians in teaching hospitals report that they usually discuss their errors with colleagues, and more than half have tried to role-model discussions. However, a significant number of these physicians report that they do not usually discuss their errors and some do not know colleagues who would be supportive listeners.

[Clinical application of a self-developed bone dust collector in mastoid cavity obliteration following mastoidectomy].

Objective: To introduce a self-developed bone dust collector designed by the authors and evaluate its efficiency in mastoid obliteration following mastoidectomy. Methods: Consecutive patients, from April 2017 to March 2018, who prepared to receive mastoidectomy were randomly divided into two groups, and in each group the bone dust was harvested by self-developed bone dust collector or by conventional used method respectively in mastoidectomy. The amount of the harvested bone dust and the time consumed in the collecting procedure were compared between two groups. The infection of the bone dust after mastoid obliteration was also evaluated during follow up. Results: 33 patients were recruited in bone dust collector group, and 31 patients in conventional method group.There is no significance of difference between two groups in sex ratio, age and pneumatization of mastoid cells (P>0.05 for all). The median amount of bone dust harvested by bone dust collector was significantly larger than that collected by conventional method (1.8 g vs 1.1 g, P<0.05). The median time spent in bone dust collector group was significantly shorter than that spent in conventional method group (4 minutes vs 6 minutes, P<0.05). No bone dust infection was found in the follow-up in all patients. Conclusion: The present self-developed bone dust collector is a easy and useful apparatus which can significantly improve the efficiency of collecting bone dust in mastoidectomy.

The E1A 13S product of adenovirus 5 activates transcription of the cellular human HSP70 gene.

Expression of the human gene encoding the major heat shock protein, HSP70, was induced during cell growth by serum stimulation and after infection with adenovirus 5. In this study we showed that HSP70 gene expression could be induced by adenovirus 5 infection, even in the absence of exogenous serum factors. Whereas serum stimulation induced the expression of the endogenous HSP70 gene, it had no effect on early adenovirus promoters. However, expression of both the cellular HSP70 gene and the adenovirus E3 promoter were activated during adenovirus infection. By using a collection of reconstructed mutant viruses, we identified the 13S product of the E1A region as the specific transcriptional trans-activator of the HSP70 gene.

A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter.

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

In vitro cytotoxicity evaluation of nano-carbon particles with different sp2/sp3 ratios.

Graphitization occurs during the long-term service of a diamond-like carbon (DLC) modified artificial joint. Then, DLC wear debris, which are carbon particles with different sp2/sp3 ratios and sizes ranging from the nano- to micro-meter scale produced. In this paper, to promote the application of DLC coating for artificial joint modification, the cytotoxicity of DLC debris (nano-carbon particles, NCs) with different sp2/sp3 ratios was studied. The microstructure and physical characteristics of NCs with different sp2/sp3 ratios were investigated by Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS). Meanwhile, osteoblasts and macrophages were applied to characterize the cytotoxicity of the NCs. In vitro cytotoxicity assay results indicated that cells incubated with NCs of different sp2/sp3 ratios had greater osteogenic capacity, and these particles caused a weaker immune response in comparison with CoCrMo particles. Taken together, the results indicated that NCs with different sp2/sp3 ratios presented a good cytocompatibility than CoCrMo particles. But no significant differences were observed among NCs with different sp2/sp3 ratios. The better cytocompatibility of NCs is mainly attributable to their surface charge.

Dose-dependent cytotoxicity evaluation of graphite nanoparticles for diamond-like carbon film application on artificial joints.

While a diamond-like carbon (DLC)-coated joint prosthesis represents the implant of choice for total hip replacement in patients, it also leads to concern due to the cytotoxicity of wear debris in the form of graphite nanoparticles (GNs), ultimately limiting its clinical use. In this study, the cytotoxicity of various GN doses was evaluated. Mouse macrophages and osteoblasts were incubated with GNs (<30 nm diameter), followed by evaluation of cytotoxicity by means of assessing inflammatory cytokines, results of alkaline phosphatase assays, and related signaling protein expression. Cytotoxicity evaluation showed that cell viability decreased in a dose-dependent manner (10-100 µg ml-1), and steeply declined at GNs concentrations greater than 30 µg ml-1. Noticeable cytotoxicity was observed as the GN dose exceeded this threshold due to upregulated receptor of activator of nuclear factor kB-ligand expression and downregulated osteoprotegerin expression. Meanwhile, activated macrophage morphology was observed as a result of the intense inflammatory response caused by the high doses of GNs (>30 µg ml-1), as observed by the increased release of TNF-α and IL-6. The results suggest that GNs had a significant dose-dependent cytotoxicity in vitro, with a lethal dose of 30 µg ml-1 leading to dramatic increases in cytotoxicity. Our GN cytotoxicity evaluation indicates a safe level for wear debris-related arthropathy and could propel the clinical application of DLC-coated total hip prostheses.

Biological responses of diamond-like carbon (DLC) films with different structures in biomedical application.

Diamond-like carbon (DLC) films are potential candidates for artificial joint surface modification in biomedical applications, and the influence of the structural features of DLC surfaces on cell functions has attracted attention in recent decades. Here, the biocompatibility of DLC films with different structures was investigated using macrophages, osteoblasts and fibroblasts. The results showed that DLC films with a low ratio of sp(2)/sp(3), which tend to have a structure similar to that of diamond, led to less inflammatory, excellent osteogenic and fibroblastic reactions, with higher cell viability, better morphology, lower release of TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6), and higher release of IL-10 (interleukin-10). The results also demonstrated that the high-density diamond structure (low ratio of sp(2)/sp(3)) of DLC films is beneficial for cell adhesion and growth because of better protein adsorption without electrostatic repulsion. These findings provide valuable insights into the mechanisms underlying inhibition of an inflammatory response and the promotion of osteoblastogenesis and fibrous propagation, and effectively build a system for evaluating the biocompatibility of DLC films.

I148M variant of PNPLA3 increases the susceptibility to non-alcoholic fatty liver disease caused by obesity and metabolic disorders.

BACKGROUND: The patatin-like phospholipase 3 (PNPLA3) rs738409 gene polymorphism is an important genetic determinant of non-alcoholic fatty liver disease (NAFLD). However, the associations between liver fat and metabolic traits in rs738409 G allele carriers and the allelic influence on this association have not been fully studied. AIM: To investigate the influence of the PNPLA3 gene polymorphism on the association of liver fat with serum metabolic factors and carotid atherosclerosis. METHODS: Liver fat was measured by quantitative ultrasound in 4300 subjects in the Shanghai Changfeng community and analysed for its association with obesity and metabolic factors in individuals with the PNPLA3 CC, CG and GG genotypes. RESULTS: Non-alcoholic fatty liver disease occurred in 37.9% and 28.8% of the subjects with the GG and CC genotypes respectively (P < 0.001). Liver fat was significantly associated with body mass index, waist circumference, serum triglycerides, high-density lipoprotein cholesterol, fasting blood glucose and insulin in the PNPLA3 rs738409 G allele carriers (P < 0.001). Compared with the CC homozygotes, the GG homozygotes presented higher liver fat and liver fibrosis scores despite their better metabolic status (comparison of regression line slopes, P < 0.05). An increase in liver fat was accompanied by a significant increase in the average and maximum carotid intima-media thickness in subjects with the PNPLA3 CC genotype but not in those with the GG genotype. CONCLUSIONS: PNPLA3 rs738409 G allele carriers were found to be more susceptible to the metabolic-related hepatic steatosis, and developed NAFLD and liver fibrosis despite presenting relatively better metabolic statuses and lower risks for carotid atherosclerosis.

Training affects muscle phospholipid fatty acid composition in humans.

Training improves insulin sensitivity, which in turn may affect performance by modulation of fuel availability. Insulin action, in turn, has been linked to specific patterns of muscle structural lipids in skeletal muscle. This study investigated whether regular exercise training exerts an effect on the muscle membrane phospholipid fatty acid composition in humans. Seven male subjects performed endurance training of the knee extensors of one leg for 4 wk. The other leg served as a control. Before, after 4 days, and after 4 wk, muscle biopsies were obtained from the vastus lateralis. After 4 wk, the phospholipid fatty acid contents of oleic acid 18:1(n-9) and docosahexaenoic acid 22:6(n-3) were significantly higher in the trained (10.9 +/- 0.5% and 3.2 +/- 0.4% of total fatty acids, respectively) than the untrained leg (8.8 +/- 0.5% and 2.6 +/- 0.4%, P < 0.05). The ratio between n-6 and n-3 fatty acids was significantly lower in the trained (11.1 +/- 0.9) than the untrained leg (13.1 +/- 1.2, P < 0.05). In contrast, training did not affect muscle triacylglycerol fatty acid composition. Citrate synthase activity was increased by 17% in the trained compared with the untrained leg (P < 0.05). In this model, diet plays a minimal role, as the influence of dietary intake is similar on both legs. Regular exercise training per se influences the phospholipid fatty acid composition of muscle membranes but has no effect on the composition of fatty acids stored in triacylglycerols within the muscle.

Relationships between the fatty acid composition of muscle and erythrocyte membrane phospholipid in young children and the effect of type of infant feeding.

Muscle membrane fatty acid (FA) composition is linked to insulin action. The aims of this study were to compare the FA composition of muscle and erythrocyte membrane phospholipid in young children; to investigate the effect of diet on these lipid compositions; and to investigate differential incorporation of FA into muscle, erythrocyte and adipose tissue membrane phospholipid, and adipose tissue triglyceride. Skeletal muscle biopsies and fasting blood samples were taken from 61 normally nourished children (45 males and 16 females), less than 2 yr old (means +/- SE, 0.80 +/- 0.06 yr), undergoing elective surgery. Adipose tissue samples were taken from 15 children. There were significant positive correlations between muscle and erythrocyte docosahexaenoic acid (DHA) (r = 0.44, P < 0.0001), total n-3 polyunsaturated fatty acids (PUFA) (r = 0.39, P = 0.002), and the n-6/n-3 PUFA ratio (r = 0.39, P = 0.002). Adipose tissue triglyceride had lower levels of long-chain PUFA, especially DHA, than muscle and erythrocytes (0.46 +/- 0.18% vs. 2.44 +/- 0.26% and 3.17 +/- 0.27%). Breast-fed infants had higher levels of DHA than an age-matched group of formula-fed infants in both muscle (3.91 +/- 0.21% vs. 1.94 +/- 0.18%) and erythrocytes (3.81 +/- 0.40% vs. 2.65 +/- 0.23%). The results of this study show that (i) erythrocyte FA composition is a reasonable index of muscle DHA, total n-3 PUFA, and the n-6/n-3 PUFA ratio; (ii) breast feeding has a potent effect on the FA composition of all these tissues; and (iii) there is a wide range in long-chain PUFA levels in muscle, erythrocytes, and adipose tissue.