Development of site-specific antibody-conjugated immunoliposomes for sensitive detection of disease biomarkers.

Liposome-based immunoassay (LIA) is an attractive protocol for amplifying the detection signals because of the excellent ability of liposomes to encapsulate signal marker compounds. The antigen-binding activity of the conjugated antibodies on the liposomal surface is crucial for the specificity and sensitivity of LIA. We present here a general platform to ensure that antibodies can conjugate onto the surface of liposomes in a site-specific and oriented manner. A His-handle-modified antibody with Fc region-specific and covalent conjugation was first fabricated using a photoactivatable ZBpa-His tag that was engineered using the aminoacyl-tRNA synthetase/suppressor tRNA technique. Based on the high affinity between the His tag and divalent metal ions, the novel His-modified antibody was oriented onto the surface of nickel ion-modified liposomes encapsulating horseradish peroxidase. With the prostate-specific antigen as a model, the detection efficiency of the new immunoliposomes was evaluated by chemiluminescence immunoassay. The immunoliposomes exhibited a limit of detection of 0.2 pg mL-1, which was a six time improvement compared with that of the chemical-coupled antibody-liposome conjugates. Thus, the proposed immunoliposomes are expected to hold potential applications for the sensitive detection of various biomarkers in complicated serum samples.

Exploration of severe Covid-19 associated risk factor in China: Meta-analysis of current evidence.

AIM: This meta-analysis aimed to explore potential risk factors for severe Covid-19. METHODS: We systemically and comprehensively retrieved the eligible study evaluating clinical differences between severe vs non-severe Covid-19. Main effect sizes were demographic characteristics, comorbidities, signs and symptoms, laboratory findings as well as radiological features of chest CT. RESULTS: A total of 2566 Covid-19 people (771 in the severe group and 1795 in the non-severe group) from 14 studies were eligible for this meta-analysis. It was demonstrated that older age and males were more likely to have severe Covid-19. Patients with underlying comorbidities, such as hypertension, diabetes, heart disease and COPD were significantly more susceptible to severe Covid-19. Patients with dyspnoea were more likely to be severely ill. Depressed total lymphocytes were observed in this article. Meanwhile, although reticulation (30.8%), intrathoracic lymph node enlargement (20.5%) and pleural effusions (30.8%) were relatively infrequent, meta-analysis revealed that patients with these presentations in chest CT were associated with increased risk of severe Covid-19. CONCLUSIONS: There are significant differences in clinical characteristics between the severe and non-severe Covid-19 patients. Many factors are related to the severity of the disease, which can help clinicians to differentiate severe patients from non-severe patients.

Losartan Protects Podocytes against High Glucose-induced Injury by Inhibiting B7-1 Expression.

The role of B7-1 in podocyte injury has received increasing attention. The aim of this study was to investigate whether losartan protects podocytes of patients with diabetic kidney disease (DKD) by regulating B7-1 and the underlying mechanisms. Rats with streptozotocin-induced DKD were treated with losartan for 8 weeks. Biochemical changes in blood and urine were analyzed. Kidneys were isolated for electron microscopy, immunofluorescence, real-time quantitative PCR (RT-PCR), and Western blot analysis. Immortalized mouse podocyte cells were cultured in normal or high glucose medium in the presence or absence of losartan for 48 h, and then the cells were collected for immunofluorescence, PCR, Western blotting and monolayer permeability detection. The phosphatidylinositol 3-kinase (PI3K) 110α subunit and angiotensin II type 1 receptor (AT1R) plasmids were transfected into podocytes, respectively, and then Western blotting was performed to assess the expression of B7-1 protein. The results showed that losartan ameliorated podocyte structure and function in the rat model of DKD, and reduced the expression of B7-1 protein. Overexpression of PI3K 110α subunit in podocytes attenuated the inhibitory effect of losartan on B7-1 expression in high glucose-stimulated podocytes. The expression of B7-1 was significantly increased by overexpression of AT1R and significantly reduced by blocking PI3K 110α subunit. We conclude that losartan protects podocytes against high glucose-induced injury by inhibiting AT1R-mediated B7-1 expression. This effect is dependent on the AT1R-PI3K 110α subunit pathway.

Suggestive Serological Evidence of Infection with Shrew-Borne Imjin Virus (Hantaviridae) in Humans.

The pathogenicity of the shrew-borne Imjin virus (MJNV) is unknown. The objective of our study was to find serological evidence of MJNV infection in humans. Partial MJNV nucleocapsid protein (NP) was cloned and expressed as an antigen for double-antigen sandwich ELISA, IgM capture ELISA, and dot blot to detect MJNV specific antibodies in hemorrhagic fever with renal syndrome (HFRS) patients' and healthy persons' sera from endemic areas in China. The purified recombinant NP reacted with neither the 90 healthy individuals' sera from non-endemic areas of MJNV nor the 100 antisera to HFRS-causing virus, indicating that the MJNV NP had no cross-reaction with normal human sera and HFRS-causing viral antibodies. As determined by screening ELISA and dot blot analysis, IgG antibodies against MJNV NP were detected in sera from two of 385 healthy individuals from MJNV-endemic areas, suggesting infection with MJNV or MJNV-like thottimvirus. Based on the suggestive evidence, healthcare workers should be alert to febrile diseases occurring among individuals with exposure to shrew-infested habitats.

Efficacy and safety of empagliflozin for type 2 diabetes mellitus: Meta-analysis of randomized controlled trials.

BACKGROUND: This study was designed to evaluate the efficiency and tolerability of empagliflozin (EMPA) as monotherapy or add-on to existing therapy in patients with type 2 diabetes mellitus (T2DM). METHODS: Randomized controlled trials (RCTs) comparing efficacy and safety of EMPA vs placebo or EMPA plus other antidiabetes drugs vs placebo plus other oral antidiabetes drugs (OADs) in T2DM were recruited from electronic database Pubmed, Web of Knowledge, and Cochrane Central Register of Controlled Trials (CENTRAL), supplemented by a hand search of the reference lists of selected articles. Main effect sizes were change from baseline on glycemia control, body weight, blood pressure, and complications (i.e., incidence of urinary and genital tract infections, and morbidity of hypoglycemia and hyperglycemia). Random-effects model was used to account for clinical or methodologic heterogeneity across studies. RESULTS: Fifteen RCTs with a total number of 7891 individuals (5374 in EMPA group and 2517 in control group) were suitable for this meta-analysis. The results demonstrated that significant improvements in glycemia control, body weight, and blood pressure were associated with EMPA application (i.e., monotherapy and add-on therapy) in patient with T2DM when compared with placebo. Meanwhile, EMPA 10 and 20 mg improved glycemia, body weight, and blood pressure control for patients with T2DM. There was no significant difference in incidence of hypoglycemia and urinary tract infections across EMPA and placebo group. Significant reduced risk of hyperglycemia was revealed in EMPA group vs placebo (risk ratio: 0.34, 95%confidence interval: 0.23-0.49, P < .00001), except in patients on background insulin therapy. However, increased risk of genital infection was noted across EMPA vs placebo (risk ratio: 2.59, 95% confidence interval: 1.80-3.71, P < .00001). CONCLUSION: Our evidence supports the application of EMPA in treatment of patients with T2DM who are obesity or at risk of weight gain.

Anaplasma species detected in Haemaphysalis longicornis tick from China.

Anaplasma are tick-borne obligatory intracellular bacteria, which infect humans and other animals. The Anaplasma species carried by ticks in China are not well studied. We collected 3145 questing Haemaphysalis longicornis ticks including 120 larvae, 2460 nymphs and 565 adults from vegetation in Jiaonan County, Shandong Province, China from 2013 to 2015. All ticks were examined for the presence of Anaplasma species by nested PCR amplification of the 16S rRNA gene. For further differentiation of A. capra from A. centrale, gltA and msp2 genes were sequenced for A. capra. Three Anaplasma species were detected in the nymph and/or adult ticks with the minimum infection rate of ticks as follows: 1.55% for A. bovis, 0.10% for A. phagocytophilum, and 0.03% for A. capra. These results indicated that the H. longicornis tick in Jiaonan County carried multiple Anaplasma species, which may be a challenge for public health in the studying area.

Global Prevalence of Asymptomatic Norovirus Infection: A Meta-analysis.

BACKGROUND: Studies about asymptomatic norovirus infections have been frequently reported. We aim to assess the global prevalence of asymptomatic infections. METHOD: We identified publications that included the proportion of asymptomatic norovirus infections by searching in PubMed, Ovid, Scopus, and Web of Science and by screening references from the articles reviewed. The principal summary data were the prevalence of asymptomatic norovirus infection. Random-effect models for meta-analysis were fitted to generate estimates of overall and subgroup prevalence. FINDINGS: Of 81 studies included, asymptomatic norovirus prevalence was estimated at 7% (95% CI: 6%-9%). Africa, Meso America and South America had higher prevalence (15%, 14%, 11%, respectively) while the prevalence in Europe and North America was lower (4%). Prevalence was similar between community and hospital (9%). Prevalence was higher in children (8%) than adults (4%). For food handlers, prevalence was estimated at 3%. In context of outbreaks, prevalence estimated from 15 studies was as high as 18% (95% CI: 10%-30%). INTERPRETATION: This knowledge could have an impact on the development of transmission prevention strategies in the future. The high prevalence indicated asymptomatic individuals must not be overlooked. OUTSTANDING QUESTIONS: The high prevalence indicated asymptomatic individuals must not be overlooked. Asymptomatic individuals may play an important role in norovirus transmission. This knowledge could have an impact on the development of transmission prevention strategies.

Detection of Imjin Virus and Seoul Virus in Crocidurine Shrews in Shandong Province, China.

INTRODUCTION: Recently, hantaviruses have been discovered in insectivores in Europe, Asia, Africa, and North America. Imjin virus (MJNV) was first isolated from the lung tissues of Ussuri white-toothed shrew (Crocidura lasiura) from South Korea in 2009. We aim to detect the species and prevalence of insectivore- and rodent-borne hantaviruses in shrews and rodents. MATERIALS AND METHODS: Shrews and rodents were captured in Jiaonan County of Shandong Province, China, in 2014. RT-PCR was used to amplify viral RNA of Hantavirus species, including insectivore-borne Imjin virus (MJNV), rodent-borne Hantaan virus (HTNV), and Seoul virus (SEOV) from shrews and rodents. RESULTS AND DISCUSSION: We found that MJNV infected 10.7% (19/178) of Crocidura shrews, but it infected none of rodents (0/475); we also found that 2 of 178 (1.1%) Crocidura shrews were PCR positive to SEOV. This study indicated that the major animal hosts of Imjin virus are shrews, and rodent-borne SEOV can infect shrews.

Well-oriented ZZ-PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies.

The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ-PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab')2 anti-goat IgG were used to detect goat IgG. The ZZ-PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ-PS-tag) surface is used.

Role of CD2-associated protein in albumin overload-induced apoptosis in podocytes.

Proteinuria is a well-established exacerbating factor of chronic kidney diseases. However, the harmful effects of protein overload on podocytes and the underlying mechanisms are still poorly understood. In the present study, we examined the effects of high concentrations of albumin on podocytes and investigated the role of CD2AP (CD2-associated protein) in albumin overload-induced podocyte apoptosis. Conditionally immortalized mouse podocytes were cultured in vitro and treated with different concentrations of BSA. In addition, CD2AP eukaryotic expression vector or siRNA (small interfering RNA) was transfected into podocytes before they were exposed to BSA. Podocyte apoptosis, expressions of active caspase-3 (p17) and CD2AP, and the distribution of F-actin cytoskeleton were detected by flow cytometry, Western-blot analysis and fluorescent staining respectively. It was found that exposure of podocytes to BSA induced podocyte apoptosis in a concentration-dependent manner that was accompanied by up-regulation of active caspase-3, the disruption of F-actin cytoskeleton, and decreased expression of CD2AP. Transfection of CD2AP eukaryotic expression vector into podocytes increased CD2AP expression, partially restored F-actin distribution, blocked active caspase-3 expression and inhibited podocyte apoptosis. In contrast, transfection of CD2AP siRNA deteriorated the above changes induced by BSA. It is concluded that protein overload induces podocyte apoptosis via the down-regulation of CD2AP and subsequent disruption of cytoskeleton of podocytes, and CD2AP may play an important role in protein overload-induced podocyte injury.

Primary syphilis cases in Guangdong Province 1995-2008: opportunities for linking syphilis control and regional development.

BACKGROUND: Syphilis cases have risen in many parts of China, with developed regions reporting the greatest share of cases. Since syphilis increases in these areas are likely driven by both increased screening and changes in sexual behaviours, distinguishing between these two factors is important. Examining municipal-level primary syphilis cases with spatial analysis allows a more direct understanding of changing sexual behaviours at a more policy-relevant level. METHODS: In this study we examined all reported primary syphilis cases from Guangdong Province, a southern province in China, since the disease was first incorporated into the mandatory reporting system in 1995. Spatial autocorrelation statistics were used to correlate municipal-level clustering of reported primary syphilis cases and gross domestic product (GDP). RESULTS: A total of 52,036 primary syphilis cases were reported over the period 1995-2008, and the primary syphilis cases increased from 0.88 per 100,000 population in 1995 to 7.61 per 100,000 in 2008. The Pearl River Delta region has a disproportionate share (44.7%) of syphilis cases compared to other regions. Syphilis cases were spatially clustered (p = 0.01) and Moran's I analysis found that syphilis cases were clustered in municipalities with higher GDP (p = 0.004). CONCLUSIONS: Primary syphilis cases continue to increase in Guangdong Province, especially in the Pearl River Delta region. Considering the economic impact of syphilis and its tendency to spatially cluster, expanded syphilis testing in specific municipalities and further investigating the costs and benefits of syphilis screening are critical next steps.

Downregulation of CD2-associated protein impaired the physiological functions of podocytes.

Emerging evidences show that CD2-associated protein (CD2AP) is involved in podocyte injury and the pathogenesis of proteinuria. However, the exact molecular mechanism by which CD2AP exerts its biological function is elusive. We knocked down CD2AP gene by target siRNA in conditionally immortalized mouse podocytes, which showed lowered cell adhesion and spreading ability (P<0.05). At the same time, cell cycle was arrested in G2/M phase (P<0.05), and pathologic nuclear division could easily be seen in CD2AP siRNA-transfected podocytes. The proliferation of podocytes were also inhibited significantly by CD2AP siRNA transfection (P<0.05). Further study revealed disordered distributions of F-actin, as well as lowered nephrin expression and phosphorylation in podocytes. These data suggest that CD2AP may play a crucial role in maintaining the normal function of podocytes and lowered CD2AP causes podocyte injury by disrupting the cytoskeleton and disturbing the nephrin-CD2AP signaling pathway.