Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus / 中国热带医学

@#Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

The protective role of raltegravir in experimental acute lung injury in vitro and in vivo

Disruption of pulmonary endothelial permeability and associated barrier integrity increase the severity of acute respiratory distress syndrome (ARDS). This study investigated the potential ability of the human immunodeficiency virus-1 (HIV-1) integrase inhibitor raltegravir to protect against acute lung injury (ALI) and the underlying mechanisms. Accordingly, the impact of raltegravir treatment on an in vitro lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMEC) model of ALI and an in vivo LPS-induced two-hit ALI rat model was examined. In the rat model system, raltegravir treatment alleviated ALI-associated histopathological changes, reduced microvascular permeability, decreased Evans blue dye extravasation, suppressed the expression of inflammatory proteins including HMGB1, TLR4, p-NF-κB, NLRP3, and MPO, and promoted the upregulation of protective proteins including claudin 18.1, VE-cadherin, and aquaporin 5 as measured via western blotting. Immunohistochemical staining further confirmed the ability of raltegravir treatment to reverse LPS-induced pulmonary changes in NLRP3, claudin 18.1, and aquaporin 5 expression. Furthermore, in vitro analyses of HPMECs reaffirmed the ability of raltegravir to attenuate LPS-induced declines in VE-cadherin and claudin 18.1 expression while simultaneously inhibiting NLRP3 activation and reducing the expression of HMGB1, TLR4, and NF-kB, thus decreasing overall vascular permeability. Overall, our findings suggested that raltegravir may represent a viable approach to treating experimental ALI that functions by maintaining pulmonary microvascular integrity.

Engeletin protects against cerebral ischemia/reperfusion injury by modulating the VEGF/vasohibin and Ang-1/Tie-2 pathways

Engeletin is a natural derivative of Smilax glabra rhizomilax that exhibits anti-inflammatory activity and suppresses lipid peroxidation. In the present study, we sought to elucidate the mechanistic basis for the neuroprotective and pro-angiogenic activity of engeltin in a human umbilical vein endothelial cells (HUVECs) oxygen-glucose deprivation and reoxygenation (OGD/R) model system and a middle cerebral artery occlusion (MCAO) rat model of cerebral ischemia and reperfusion injury. These analyses revealed that engeletin (10, 20, or 40 mg/kg) was able to reduce the infarct volume, increase cerebral blood flow, improve neurological function, and bolster the expression of vascular endothelial growth factor (VEGF), vasohibin-2 (Vash-2), angiopoietin-1 (Ang-1), phosphorylated human angiopoietin receptor tyrosine kinase 2 (p-Tie2), and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in MCAO rats. Similarly, engeletin (100, 200, or 400 nM) markedly enhanced the migration, tube formation, and VEGF expression of HUVECs in an OGD/R model system, while the VEGF receptor (R) inhibitor axitinib reversed the observed changes in HUVEC tube formation activity and Vash-2, VEGF, and CD31 expression. These data suggested that engeletin exhibited significant neuroprotective effects against cerebral ischemia and reperfusion injury in rats, and improved cerebrovascular angiogenesis by modulating the VEGF/vasohibin and Ang-1/Tie-2 pathways.

Association of Increased Urine Brain Derived Neurotrophic Factor with Lower Urinary Tract Symptoms in Men with Benign Prostatic Hyperplasia / 华中科技大学学报（医学）（英德文版）

Urinary brain-derived neurotrophic factor (BDNF),an ubiquitous neurotrophin,was found to rise in patients with benign prostatic hyperplasia (BPH).We hypothesized that the urinary level of BDNF could be a potential biomarker for lower urinary tract symptoms (LUTS) in patients with BPH.Totally,76 patients with BPH-caused LUTS and 32 male control subjects without BPH were enrolled.International Prostate Symptom Score (IPSS) was applied to assess the symptom severity of LUTS.Urodynamic tests were performed for the diagnosis of underlying detrusor overactivity (DO) in the patients with BPH.Urine samples were collected from all subjects.Urinary BDNF levels were measured using enzyme-linked immunosorbent assays and normalized by urinary creatinine (Cr) levels.Seventy-six BPH patients were divided into moderate LUTS group (n=51,7＜IPSS ≤ 20) and severe LUTS group (n=25,IPSS＞20) according to the IPSS.Of the 76 BPH patients,DO was present in 34 (44.7％)according to the urodynamic test.The urinary BDNF/Cr levels were significantly higher in BPH patients with moderate LUTS (8.29±3.635,P＜0.0001) and severe LUTS (11.8±6.44,P＜0.0001) than normal controls (1.71±0.555).Patients with severe LUTS tended to have higher urinary BDNF/Cr levels than patients with moderate LUTS (11.8±6.44 vs.8.29±3.635,P=0.000).The conditions of BPH with LUTS correlated with elevated urinary BDNF levels,and urinary BDNF levels were even higher in BPH-DO patients.The results of this study have provided evidence to suggest that urinary BDNF level test could evaluate the severity of LUTS in BPH patients,and BDNF level can be used as a biornarker for the diagnosis of DO in BPH patients.

Multi-center phase II clinical trial of humanized anti-epidermal factor receptor monoclonal antibody h-R3 combined with radiotherapy for locoregionally advanced nasopharyngeal carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safty of the humanized anti-epidermal factor receptor monoclonal antibody h-R3 in combination with radiotherapy for locoregionally advanced nasopharyngeal carcinoma.</p><p><b>METHODS</b>Totally, 137 patients from 7 medical center around China were randomly divided into combined therapy group or control group. There was no difference in Karnofsky performance score between two groups. All patients in both groups received radical conventionally fractionated radiotherapy to the total dose of D(T) 70-76 Gy. For the combined therapy group, h-R3 was added at a dose of 100 mg i.v. weekly for 8 weeks started at the beginning of radiotherapy.</p><p><b>RESULTS</b>Of the 137 eligilbe patients, 70 were in the combined therapy group treated by h-R3 plus radiotherapy and 67 in the control group by radiotherapy alone. The intent-to-treat (ITT) population consisted of 130 patients, while the per-protocol (PP) population was composed of 126 patients. The efficacy was assessed respectively at three point of time: the end of treatment, the 5th- and 17th-week after treatment. The complete response (CR) of the combined therapy group was significantly higher than that of the control group in both ITT and PP (ITT: 65.63%, 87.50%, 90.63% versus 27.27%, 42.42%, 51.52%; PP: 67.21%, 90.16%, 93.44% versus 27.69%, 43.08%, 52.31%; P < 0.05, respectively). The most common h-R3-related adverse reactions were fever (4.3%), hypotension (2.9%), nausea (1.4%), dizziness (2.9%) and rash (1.4%), which could be reversible if treated properly. Radiotherapy combined with 100 mg h-R3 i. v. weekly was tolerable and did not aggravate the side effects of radiation. The quality of life in the combined therapy group was comparable to that in the control group.</p><p><b>CONCLUSION</b>This phase 1 multicenter clinical trial shows that h-R3 in combination with radiotherapy is effective and well-tolerated for the treatment of locoregionally advanced nasopharyngeal carcinoma.</p>

Studyies on the Breeding and Cultivation of L-Lactic Acid Producing Strain / 微生物学通报

In order to obtain higher L-lactic acid yield industrial strain, the original strain Rhizopus oryzae PW352 was mutated by means of N+ ions implantation and a mutant strain Rhizopus oryzae RE3303 was obtained. Its lactic acid yield was increased by 75% than that of the original one. The acid producing condition was optimized by orthogonal design. The concentration of L-lactic acid reached to 131～136g/L and the conversion rate of glucose was as high as 86%～90% under the optimum condition.