Ionic liquid-based transparent membrane-coupled human lung epithelium-on-a-chip demonstrating PM0.5 pollution effect under breathing mechanostress

The plausibility of human exposure to particulate matter(PM)has witnessed an increase within the last several years.PM of different sizes has been discovered in the atmosphere given the role of dust transport in weather and climate composition.As a regulator,the lung epithelium orchestrates the innate response to local damage.Herein,we developed a lung epithelium-ona-chip platform consisting of easily moldable polydimethylsiloxane layers along with a thin,flexible,and transparent ionic liquid-based poly(hydroxyethyl)methacrylate gel membrane.The epithelium was formed through the culture of human lung epithelial cells(Calu-3)on this membrane.The mechanical stress at the air–liquid interface during inhalation/exhalation was recapitulated using an Arduino-based servo motor system,which applied a uniaxial tensile strength from the two sides of the chip with 10%strain and a frequency of 0.2 Hz.Subsequently,the administration of silica nanoparticles(PM0.5)with an average size of 463 nm to the on-chip platform under static,dynamic,and dynamic+mechanical stress(DMS)conditions demonstrated the effect of environmental pollutants on lung epithelium.The viability and release of lactate dehydrogenase were determined along with proinflammatory response through the quantification of tumor necrosis factor-α,which indicated alterations in the epithelium.

人肝细胞癌中抑癌基因PTEN/MMAC1/TEP1的突变分析

PTEN/MMAC1/TEP1(PTEN)是新近分离到的抑癌基因,在多种肿瘤中存在突变。我们检测了34例人肝细胞癌中PTEN基因第5外显子和第8外显子的突变。采用聚合酶链方法以内含子引物扩增第5和第8外显子,继之以单链构象多态性和测序技术分析PTEN基因突变。有4例肝细胞癌SSCP显示异常条带并经测序证实存在突变。2例发生于第4内含子,突变位点相同;另两例发生于第8外显子,其中1例碱基颠换导致PTEN蛋白产物304位半胱氨酸突变为甘氨酸。

蛋白质酪氨酸磷酸化在抗失巢凋亡的癌细胞中的失调变化

失巢凋亡是细胞与细胞外基质脱离发生的一种特定的凋亡方式.癌细胞抗失巢凋亡或失巢生存能力可以使之在转移过程中生存.业已发现癌细胞失巢生存与PI3K-PKB/Akt、MAPK这两条重要信号途径有关,但是PI3K-PKB/Akt、MAPK通路的上游酪氨酸激酶途径还不甚清楚.为此设计了一种基于SH2-pTyr特异性结合特性的功能性筛选方法,以期发现癌细胞失巢生存相关的酪氨酸磷酸化蛋白质,为最终明确酪氨酸激酶途径提供有力的实验依据.实验发现,MDCK细胞悬浮培养后失巢凋亡,但癌细胞可以失巢生存.与这一现象相一致的是,悬浮培养后,MDCK细胞中一系列SH2结合的酪氨酸磷酸化蛋白质水平急剧下降,而癌细胞中蛋白质酪氨酸磷酸化水平并不呈锚着依赖性.细胞悬浮培养后,随着培养时间的延长,MDCK细胞中AblSSH2结合的靶蛋白酪氨酸磷酸化水平逐渐降低,在H460肺癌细胞中经过短暂下降后升高,H1792肺癌细胞随着培养时间的延长,AblSH2结合的靶蛋白酪氨酸磷酸化水平逐渐增加.FynSH2和CrkSH2结合的蛋白质分别为FAK和p130Cas,后者是重要的失巢生存信号.这些结果提示,酪氨酸磷酸化蛋白质可能赋予肺癌细胞失巢生存能力.结果也表明,功能性SH2筛查方法可以有效地发现肿瘤细胞中失巢生存相关的酪氨酸磷酸化蛋白质.

神经肌肉疾病患儿肺内振荡通气与激发性肺量测定的比较

Pulmonary infections can be life threatening for children with neuromuscular diseases who have impaired ability to clear secretions. Intrapulmonary percussive ventilation (IPV) is a pneumatic device that delivers air and aerosol to the lungs at frequencies of 200 to 300 cycles per minute at peak pressures from 20 to 40 cm H2O. Anecdotal reports and pilot studies show its safety and effectiveness in mobilizing secretions in patients with cystic fibrosis. Objective: To test the hypothesis that IPV used in a pulmonary program for adolescents with neuromuscular disease would reduce the number of days of antibiotic use for pulmonary infection. Methods: A randomized, controlled study was conducted to compare efficacy of IPV with incentive spirometry (IS) in reducing number of days of antibiotic use in adolescents with neuromuscular disease. The secondary endpoints were the number of respiratory infections, hospitalizations, and school days missed. Results: A total of 18 patients were enrolled (9 IPV, 9 IS). Antibiotic use was significantly higher with IS (24/1000 patient-days) compared with IPV(0/1000 patient-days), (incidence rate ratio, 43; 95% confidence interval, 6- 333). The IS group spent more days hospitalized (4.4/1000 patient-days vs 0/1000 patient- days) than the IPV group (incidence rate ratio, 8.5; 95% confidence interval, 1.1- 67). The IPV group had 0 episodes of pneumonia or bacterial bronchitis compared with 3 events in the IS group, although this did not meet statistical significance. Conclusion: Intrapulmonary percussive ventilation as part of a preventive pulmonary regimen reduced days of antibiotic use and hospitalization for respiratory illness in adolescents with neuromuscular disease.

Preventive fraction of physical fitness on risk factors in cardiac patients:Retrospective epidemiological study

AIM To quantify the preventive fraction of physical fitness on the risk factors in patients with cardiovascular diseases(CVDs).METHODS A total of 249 subjects(205 men and 44 women) suffering from CVD were categorized into four groups, according to their percentage of physical fitness. We calculated the odds ratio to obtain the preventive fraction in order to evaluate the impact of the physical fitness level on the risk factors(i.e., abdominal obesity, depression, diabetes, dyslipidemia, hypertension, obesity, overweight and smoking).RESULTS It is observed that a normal physical fitness level is sufficient to induce a preventive action on abdominal obesity(38%), diabetes(12%), hypertension(33%), obesity(12%) and overweight(11%). Also, the preventive fraction increases with the level of physical fitness, in particular for hypertension(36%) and overweight(16%). A high physical fitness level does not necessarily induce a preventive action in most risk factors, excluding depression. CONCLUSION This is the first study which demonstrates that reaching a normal physical fitness level is enough to induce a protection for some risk factors, despite having a CVD.

Effectiveness and Safety of 9-Month Treatment Regimen for Multidrug-Resistant Tuberculosis in the Philippines

Background: The Philippines has a burden of drug-resistant tuberculosis (DR-TB). One of the key challenges in the programmatic management of DR-TB (PMDT) is the high rate of loss to follow-up (38% in the 2010 cohort). An urgent need for a shorter, more tolerable, less expensive treatment regimen exists. The aim of the operational study is to determine the efficacy and safety of the short treatment regimen among drug resistant TB. Methods: This is a prospective single-arm cohort study evaluating the effectiveness and safety of a shorter 9 - 11-month treatment regimen (9MTR) for rifampicin-resistant/multi-drug resistant TB (RR/MDR-TB) in 10 PMDT facilities. All eligible consenting adult patients with rifampicin-resistant TB were enrolled and received the standardized 9-month treatment regimen (9MTR), including injectables, with a follow-up after 12 months of treatment completion. Results: A total of 329 patients were enrolled from July 2015 to December 2016. At the 6th month post-enrollment, 256 (77.8%) of them had culture-negative test results. The end-of-treatment success rate was 74.1% (224 [68.0%] were cured and 20 [6.1%] completed the treatment). On the other hand, 10 (3.0%) died, 41 (12.5%) lost to follow-up, 33 (10.0%) withdrawn, 1 (0.3%) treatment failure. In the 12th month after 9MTR completion, among the 244 patients with successful treatment, 198 (81.1%) had culture-negative results, while there were 46 patients whose culture tests were not done. One patient developed TB relapse with fluoroquinolone resistance. The majority of the adverse events were mild that occurred mostly during the first 6 months of treatment. Conclusion: The 9-month treatment regimen had a high treatment success rate with a favorable safety profile. The loss to follow-up was reduced;however, it was still a challenge. The introduction of the 9MTR via operational research had a major impact on building national capacity and infrastructure for the programmatic adoption of a new regimen. Ten PMDT centers received training and experience, created diagnostic pathways, and active drug safety monitoring and management were built.

Impact of HIV Status,CD4 Count and Antiretroviral Treatment on Tuberculosis Treatment Outcomes in a Low-Burden Country

Background: Few data is available in low-burden TB-HIV countries on TB treatment outcomes. We evaluated TB patients (pts) characteristics by HIV status and compared treatment outcomes in relation to HIV status, CD4 count and antiretroviral therapy (ART). Methods: We performed a retrospective analysis of 447 adult TB pts who registered and initiated treatment in our center from 2014 to 2015. Results: Pts were categorized as HIV-negative (n = 399, 89.3%), HIV-positive on ART (n = 42, 9.4%) and HIV-positive not on ART (n = 6, 1.3%). The proportion of pts with extended TB therapy was higher among HIV-positive pts (p = 0.03). Increased age was associated with higher death rates regarding treatment success (OR 1.08;95% CI 1.01 - 1.17, p = 0.03). Statistically significant higher mortality was found among HIV-positive pts on ART (OR 9.93;95% CI 1.36 - 72.37, p = 0.03) and HIV-positive pts not on ART (OR 397.00;95% CI 44.27 to 3559.91, p ≤ 0.0001) compared to HIV-negative pts. When multivariate analyses were restricted to HIV-positive pts, being not on ART was associated with higher mortality (OR 40.0;95% CI 4.37 - 365.78, p = 0.001). Conclusions: There was significant difference in death rates between HIV-positive and HIV-negative TB pts. HIV-positive pts not on ART had a significantly higher mortality.

Association of latitude and altitude with adverse outcomes in patients with COVID-19: The VIRUS registry

BACKGROUND The coronavirus disease 2019(COVID-19)course may be affected by environmental factors.Ecological studies previously suggested a link between climatological factors and COVID-19 fatality rates.However,individual-level impact of these factors has not been thoroughly evaluated yet.AIM To study the association of climatological factors related to patient location with unfavorable outcomes in patients.METHODS In this observational analysis of the Society of Critical Care Medicine Discovery Viral Infection and Respiratory Illness Universal Study:COVID-19 Registry cohort,the latitudes and altitudes of hospitals were examined as a covariate for mortality within 28 d of admission and the length of hospital stay.Adjusting for baseline parameters and admission date,multivariable regression modeling was utilized.Generalized estimating equations were used to fit the models.RESULTS Twenty-two thousand one hundred eight patients from over 20 countries were evaluated.The median age was 62(interquartile range:49-74)years,and 54%of the included patients were males.The median age increased with increasing latitude as well as the frequency of comorbidities.Contrarily,the percentage of comorbidities was lower in elevated altitudes.Mortality within 28 d of hospital admission was found to be 25%.The median hospital-free days among all included patients was 20 d.Despite the significant linear relationship between mortality and hospital-free days(adjusted odds ratio(aOR)=1.39(1.04,1.86),P=0.025 for mortality within 28 d of admission;aOR=-1.47(-2.60,-0.33),P=0.011 for hospital-free days),suggesting that adverse patient outcomes were more common in locations further away from the Equator;the results were no longer significant when adjusted for baseline differences(aOR=1.32(1.00,1.74),P=0.051 for 28-day mortality;aOR=-1.07(-2.13,-0.01),P=0.050 for hospital-free days).When we looked at the altitude’s effect,we discovered that it demonstrated a non-linear association with mortality within 28 d of hospital admission(aOR=0.96(0.62,1.47),1.04(0.92,1.19),0.49(0.22,0.90),and 0.51(0.27,0.98),for the altitude points of 75 MASL,125 MASL,400 MASL,and 600 MASL,in comparison to the reference altitude of 148 m.a.s.l,respectively.P=0.001).We detected an association between latitude and 28-day mortality as well as hospital-free days in this worldwide study.When the baseline features were taken into account,however,this did not stay significant.CONCLUSION Our findings suggest that differences observed in previous epidemiological studies may be due to ecological fallacy rather than implying a causal relationship at the patient level.

Membrane and Subcellular Muscle Injury Are Induced by Single or Multiple Exposure to Cigarette Smoke

Cigarette smoking is the main cause of chronic obstructive pulmonary disease (COPD). Diaphragm injury is observed in patients with COPD. However, the potential role of smoking in triggering or perpetuating muscle injury is unknown. The present study was aimed at evaluating the potential role of commercial tobacco smoke as a direct cause of skeletal muscle injury in experimental conditions. Seventy Wistar rats (170 - 250 g) were assigned to smoking (n = 49) or non-smoking (n = 21) groups. The smoking groups were submitted to a single or multiple (i.e., five or thirty) daily sessions of cigarette smoking in an inhalatory chamber (time length: 2 h each session). The level of exposure was constant and assessed by CO concentrations (50 ppm) and serum cotinine analysis. Animals submitted to a single smoke exposure and the corresponding controls were euthanized in groups at 0 h, 2 h, 4 h, 24 h or 48 h after completing the exposure. Animals submitted to multiple exposures were euthanized at 0 h after smoking. Samples from vastus lateralis muscle were obtained and processed for assessing cell injury and selected protein expression. Monoclonal anti-albumin antibodies were used to identify muscle fibers with sarcolemmal (membrane) injury. Subcellular muscle injury was assessed using transmission electron microscopy (EM). MyoD, myogenin and α-tubulin were immunodetected using western blot techniques. Exposure to cigarette smoke associated with significant membrane damage (mean relative difference (MRD) with controls: +181%, p = 0.004) and sarcomere disruptions (MRD: +226%, p = 0.001). Expression of MyoD and myogenin (normalized to α-tubulin) were significantly increased at 4 h and remained increased at 48 h post-exposure. We conclude that not only a single but also consecutive exposure to tobacco smoke have acute deleterious effects on peripheral muscle structure. A rapid induction of subrogate markers of skeletal muscle stress and repair processes associates to sarcolemmal and sarcomere damage.

小鼠眼角膜上皮细胞中IL-1β对Fas配体的表达调控研究

Fas配体(Fas Ligand,FasL)在多种不同的上皮细胞中均见表达,在炎症反应中诱导Fas阳性免疫细胞凋亡以保护组织.为了研究眼角膜炎症反应过程与Fas配体之间的关系,本研究检测了在眼角膜上皮细胞中IL-1β对FasL的表达翻译水平以及细胞毒性的影响,明确了在眼角膜上皮细胞中IL-1β抑制FasL的表达以及蛋白翻译,FasL的启动子活性受到IL-1β作用浓度的影响,两者呈相关性表现,发现IL-1β阻断由FasL诱导Fas阳性细胞对于眼角膜上皮细胞的细胞毒性作用.这些结果表明,眼角膜上皮细胞FasL受IL-1β的调控.

IL-1β regulates the mouse Fas ligand expression in corneal endothelial cells

Constitutively expressed Fas ligand (FasL) in several distinct epithelial cell types appears to protect tissues by inducing apoptosis of Fas+ immune cells during inflammatory reactions. To study the rela-tionship of FasL and inflammation process in cornea,we examined the effects of inflammatory cytokine IL-1β on the FasL production,expression and cytotoxic function in corneal endothelial cells. In this paper,we demonstrate that IL-1β inhibits the FasL production and expression in corneal endothelial cells. The promoter activities of FasL in these cells are reduced by IL-1β in a dose-dependent manner. Finally,we also find that IL-1β block the cytotoxic effects of FasL derived from corneal endothelial cells to the Fas+ target cells. These data support the view that FasL derived from corneal endothelial cells modulate inflammation within cornea.

Effects of Combinatorial Expression of selA, selB and selC Genes on the Efficiency of Selenocysteine Incorporation in Escherichia coli

In Escherichia coil four gene products（selA, selB, selC and selD） and a selenocysteine（Sec） insertion sequence（SECIS） are required for the correct translation of UGA codons encoding Sec. Previous studies have shown that the stoichiometry of selenoproteine mRNA and elongation factor SelB affect the efficiency of Sec incorporation. Herein lies a detailed analysis of the effects of co-expressing selA, selB and selC genes under inducible promoters on the incorporation efficiency of Sec. Over-expression of either selA or selB reduced the efficiency of Sec incorporation by 61.1% or 11.6%, respectively, compared to the over-expression of the reporter vector alone did. Concomitant over-expression ofselC with selA or selB completely reversed the reduce of the efficiency of Sec but still reduced the efficiency of the incorporation relative to that observed for expression of selC alone. Over-expression of selC gene alone under L-arabinose induction reduced the efficiency of the incorporation relative to that observed for co-expressing selC with selA and selB under the control of its endogenous promoter in the absence of L-arabinose. Co-expression of selA, selB and selC with selA or selB under the control of inducible promoters increased the effi- ciency of Sec incorporation by 69.7%. Moreover, inducing selenoprotein-related gene expression during the late exponential phase increased the efficiency of Sec incorporation by a factor of 5.4 relative to that observed for the reporter vector alone. These results suggest that the co-expression of selA, selB and selC in Escherichia coli under the control of inducible promoters is a viable and promising strategy for increasing the yields of selenoproteins.

Seedy CD8+ T_(RM) cells in aging lungs drive susceptibility to pneumonia and sequelae

Aging is a leading risk factor for pneumonia and subsequent comorbidities,including chronic pulmonary diseases.1 The underlying causes of age-related susceptibility to pneumonia-related morbidity and mortality are complex and poorly understood.While aging has been associated with many changes in diverse immune compartments,if and how aging affects resident immune cells in the lung have not been examined.