Lentivirus-mediated overexpression of angiotensin-(1-7) attenuated ischaemia-induced cardiac pathophysiology

Myocardial infarction (MI) results in cell death, development of interstitial fibrosis, ventricular wall thinning and ultimately, heart failure. Angiotensin-(1-7) [Ang-(1-7)] has been shown to provide cardioprotective effects. We hypothesize that lentivirus-mediated overexpression of Ang-(1-7) would protect the myocardium from ischaemic injury. A single bolus of 3.5 × 10(8) transducing units of lenti-Ang-(1-7) was injected into the left ventricle of 5-day-old male Sprague-Dawley rats. At 6 weeks of age, MI was induced by ligation of the left anterior descending coronary artery. Four weeks after the MI, echocardiography and haemodynamic parameters were measured to assess cardiac function. Postmyocardial infarction, rats showed significant decreases in fractional shortening and dP/dt (rate of rise of left ventricular pressure), increases in left ventricular end-diastolic pressure, and ventricular hypertrophy. Also, considerable upregulation of cardiac angiotensin-converting enzyme (ACE) mRNA was observed in these rats. Lentivirus-mediated cardiac overexpression of Ang-(1-7) not only prevented all these MI-induced impairments but also resulted in decreased myocardial wall thinning and an increased cardiac gene expression of ACE2 and bradykinin B2 receptor (BKR2). Furthermore, in vitro experiments using rat neonatal cardiac myocytes demonstrated protective effects of Ang-(1-7) against hypoxia-induced cell death. This beneficial effect was associated with decreased expression of inflammatory cytokines (tumour necrosis factor-α and interleukin-6) and increased gene expression of ACE2, BKR2 and interleukin-10. Our findings indicate that overexpression of Ang-(1-7) improves cardiac function and attenuates left ventricular remodelling post-MI. The protective effects of Ang-(1-7) appear to be mediated, at least in part, through modulation of the cardiac renin-angiotensin system and cytokine production.     

Dengue virus PrM/M proteins fail to show pH-dependent ion channel activity in Xenopus oocytes

The transmembrane domains (TMDs) of dengue virus type-1 M protein (DENV-1M) were reported to form cation-selective channels in artificial lipid bilayers. We further explored this observation using the two-electrode voltage clamp (TEVC) method on the Xenopus laevis oocytes expressing DENV PrM and M proteins. Using myc epitope tagged M proteins, M was first shown to adopt its predicted native topology in mammalian cells when expressed on its own. The recombinant proteins were then successfully expressed on the surface of Xenopus oocytes. Using influenza A M2 (Inf A/M2) protein as a control, we measured the conductance of oocytes expressing DENV proteins under hyperpolarized or low-pH conditions. Inf A/M2 showed pH-dependent, amantadine-sensitive channel activity that was consistent with previously published reports. However, no activity was detected for DENV proteins. We conclude that DENV PrM and M proteins do not show pH-activated ion channel activity.     

Phenotyping interindividual variability in obstructive sleep apnoea response to temazepam using ventilatory chemoreflexes during wakefulness

Centrally active agents have a variable impact in patients with obstructive sleep apnoea (OSA) that is unexplained. How to phenotype the individual OSA response is clinically important, as it may help to identify who will be at risk of respiratory depression and who will benefit from a centrally active agent. Based on loop gain theory, we hypothesized that OSA patients with higher central chemosensitivity have higher breathing instability following the use of a hypnosedative, temazepam. In 20 men with OSA in a double‐blind, placebo‐controlled cross‐over trial we tested the polysomnographically (PSG) measured effects of temazepam 10 mg versus placebo on sleep apnoea. Treatment nights were at least 1 week apart. Ventilatory chemoreflexes were also measured during wakefulness in each subject. The patients (mean ± standard deviation; 44 ± 12 years) had predominantly mild‐to‐moderate OSA [baseline apnoea–hypopnoea index (AHI) = 16.8 ± 14.1]. Patients’ baseline awake central chemosensitivity correlated significantly with both the change of SpO₂ nadir between temazepam and placebo (r = ?0.468, P = 0.038) and oxygen desaturation index (ODI; r = 0.485, P = 0.03), but not with the change of AHI (r = 0.18, P = 0.44). Peripheral chemosensitivity and ventilatory recruitment threshold were not correlated with the change of SpO₂ nadir, ODI or AHI (all P > 0.05). Mild–moderate OSA patients with higher awake central chemosensitivity had greater respiratory impairment during sleep with temazepam. Relatively simple daytime tests of respiratory control may provide a method of determining the effect of sedative–hypnotic medication on breathing during sleep in OSA patients.     

Toll-like receptor 7 deficiency suppresses type 1 diabetes development by modulating B-cell differentiation and function

Innate immunity mediated by Toll-like receptors (TLRs), which can recognize pathogen molecular patterns, plays a critical role in type 1 diabetes development. TLR7 is a pattern recognition receptor that senses single-stranded RNAs from viruses and host tissue cells; however, its role in type 1 diabetes development remains unclear. In our study, we discovered that Tlr7-deficient (Tlr7^−/−) nonobese diabetic (NOD) mice, a model of human type 1 diabetes, exhibited a significantly delayed onset and reduced incidence of type 1 diabetes compared with Tlr7-sufficient (Tlr7^+/+) NOD mice. Mechanistic investigations showed that Tlr7 deficiency significantly altered B-cell differentiation and immunoglobulin production. Moreover, Tlr7^−/− NOD B cells were found to suppress diabetogenic CD4⁺ T-cell responses and protect immunodeficient NOD mice from developing diabetes induced by diabetogenic T cells. In addition, we found that Tlr7 deficiency suppressed the antigen-presenting functions of B cells and inhibited cytotoxic CD8⁺ T-cell activation by downregulating the expression of both nonclassical and classical MHC class I (MHC-I) molecules on B cells. Our data suggest that TLR7 contributes to type 1 diabetes development by regulating B-cell functions and subsequent interactions with T cells. Therefore, therapeutically targeting TLR7 may prove beneficial for disease protection.     

Genetic polymorphisms of cytochrome p4502E1 and susceptibility to alcoholic liver disease and hepatocellular carcinoma in a white population: a study and literature review, including meta-analysis.

AIMS: To investigate the associations between the Rsa I, Dra I, and Taq I genetic polymorphisms of cytochrome p4502E1 and susceptibility to alcoholic liver disease or to hepatocellular carcinoma. METHODS: DNA samples isolated from 61 patients with alcoholic liver disease, 46 patients with hepatocellular carcinoma, and 375 healthy controls were subjected to polymerase chain reaction amplification followed by digestion with the endonucleases Rsa I, Dra I, or Taq I. Meta-analysis was performed using data from previous studies of Rsa I polymorphism and the risk of alcoholic liver disease. RESULTS: No association was found between any of the three polymorphisms and susceptibility to hepatocellular carcinoma. The distributions of Rsa I and Dra I alleles among the patients with alcoholic liver disease were not significantly different from those among the control group. Meta-analysis of this data and previous data concerning Rsa I polymorphism and alcoholic liver disease risk failed to demonstrate any significant association between the two. However, the alcoholic liver disease group in this study showed a significantly lower frequency of the less common Taq I allele compared with the healthy control group (odds ratio, 0.33; 95% confidence interval, 0.12 to 0.78). CONCLUSIONS: Possession of the less common Taq I cytochrome p4502E1 allele is associated with reduced susceptibility to alcoholic liver disease. There is no existing evidence that the Taq I polymorphism is directly associated with altered alcohol metabolism, but it might be in linkage disequilibrium with as yet unidentified protective factors.     

Early direct and transneuronal effects in mice with targeted expression of a toxin gene to D1 dopamine receptor neurons

The neurochemical profile was examined at postnatal day 3-4 in mutant mice generated by in vivo Cre mediated activation of an attenuated diphtheria toxin gene inserted into the D1 dopamine receptor gene locus. An earlier study of this model had shown that D1 dopamine receptor, substance P and dynorphin were not expressed in the striatum. Quantitative in situ hybridization analysis showed an increase in D2 dopamine receptor and enkephalin messenger RNA expression. The nigrostriatal pathway in the mutant pups was intact with a normal number of dopaminergic neurons in the substantia nigra and the ventral tegmental area in addition to a normal pattern of striatal dopamine transporter and tyrosine hydroxylase immunoreactivity. Quantitative analysis of striatal dopamine transporter density using [3H]mazindol showed a reduction of 26% suggesting a degree of transneuronal down-regulation. There was also a 49% reduction of striatal GABA receptor binding and a 36% reduction of striatal muscarinic receptor binding in mutant pups. The number of healthy striatal neuropeptide Y-containing interneurons was also substantially down-regulated in the mutant striatum. In contrast, there was an increase in the number of striatal cholinergic interneurons. Down-regulated cortical GABA receptor and muscarinic receptor binding was also observed in addition to subtle morphological changes in the neuropeptide Y-expressing population of cortical neurons. The changes reflect the early cascade of events which follows the ablation of D1 dopamine receptor-positive cells. Although extensive changes in a number of striatal and cortical neurons were demonstrated, only subtle transneuronal effects were seen in the nigrostriatal pathway.     

Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival environment

Proteins secreted or exported by Treponema denticola have been implicated as mediators of specific interactions between the spirochete and subgingival tissues in periodontal diseases. However, limited information is available on the ability of this peptidolytic organism to bind or transport soluble peptides present in the subgingival environment. A prominent 70-kDa protein was isolated from surface extracts of T. denticola ATCC 35405. A clone expressing a portion of the protein was identified in an Escherichia coli expression library of T. denticola DNA. DNA sequence analysis showed that the cloned gene encoded a peptide homologous to OppA, the solute binding protein of an ATP-binding cassette-type peptide transporter involved in peptide uptake and environmental signaling in a wide range of bacteria. Genes encoding OppB, -C, -D, and -F were identified directly downstream of oppA in T. denticola. OppA was present in representative strains of T. denticola and in Treponema vincentii but was not detected in Treponema pectinovorum or Treponema socranskii. Immunogold electron microscopy suggested that OppA was accessible to proteins at the surface of the spirochete. Native OppA bound soluble plasminogen and fibronectin but did not bind to immobilized substrates or epithelial cells. A T. denticola oppA mutant bound reduced amounts of soluble plasminogen, and plasminogen binding to the parent strain was inhibited by the lysine analog epsilon-aminocaproic acid. Binding of soluble host proteins by OppA may be important both for spirochete-host interactions in the subgingival environment and for uptake of peptide nutrients.     

COVID-19 instructional approaches (in-person,online, hybrid), school start times,and sleep in over 5,000 U.S. adolescents

Study ObjectivesTo examine associations among instructional approaches, school start times, and sleep during the COVID-19 pandemic in a large, nationwide sample of U.S. adolescents.MethodsCross-sectional, anonymous self-report survey study of a community-dwelling sample of adolescents (grades 6–12), recruited through social media outlets in October/November 2020. Participants reported on instructional approach (in-person, online/synchronous, online/asynchronous) for each weekday (past week), school start times (in-person or online/synchronous days), and bedtimes (BT) and wake times (WT) for each identified school type and weekends/no school days. Sleep opportunity was calculated as BT-to-WT interval. Night-to-night sleep variability was calculated with mean square successive differences.ResultsRespondents included 5,245 racially and geographically diverse students (~50% female). BT and WT were earliest for in-person instruction; followed by online/synchronous days. Sleep opportunity was longer on individual nights students did not have scheduled instruction (>1.5 h longer for online/asynchronous than in-person). More students obtained sufficient sleep with later school start times. However, even with the same start times, more students with online/synchronous instruction obtained sufficient sleep than in-person instruction. Significantly greater night-to-night variability in sleep-wake patterns was observed for students with in-person hybrid schedules versus students with online/synchronous + asynchronous schedules.ConclusionsThese findings provide important insights regarding the association between instructional approach and school start times on the timing, amount, and variability of sleep in U.S. adolescents. Given the public health consequences of short and variable sleep in adolescents, results may be useful for education and health policy decision-making for post-pandemic secondary schools.