Background: 2019-Novel coronavirus (2019-nCoV) outbreaks create challenges for hospital laboratories because thousands of samples must be evaluated each day. Sample types, interpretation methods, and corresponding laboratory standards must be established. The possibility of other infections should be assessed to provide a basis for clinical classification, isolation, and treatment. Accordingly, in the present study, we evaluated the testing methods for 2019-nCoV and co-infections. Methods: We used a fluorescence-based quantitative PCR kit urgently distributed by the Chinese CDC to detect 8274 close contacts in the Wuhan region against two loci on the 2019-nCoV genome. We also analyzed 613 patients with fever who underwent multiple tests for 13 respiratory pathogens; 316 subjects were also tested for 2019-nCoV. Findings: Among the 8274 subjects, 2745 (33.2%) had 2019-nCoV infection; 5277 (63.8%) subjects showed negative results in the 2019-nCoV nucleic acid test (non-019-nCoV); and 252 cases (3.0%) because only one target was positive, the diagnosis was not definitive. Sixteen patients who originally had only one positive target were re-examined a few days later; 14 patients (87.5%) were finally defined as 2019-nCoV-positive, and 2 (12.5%) were finally defined as negative. The positive rates of nCoV-NP and nCovORF1ab were 34.7% and 34.7%, respectively. nCoV-NP-positive only and nCovORF1ab-positive cases accounted for 1.5% and 1.5%, respectively. In the 316 patients with multiple respiratory pathogens, 104 were positive for 2019-nCov and 6/104 had co-infection with coronavirus (3/104), influenza A virus (2/104), rhinovirus (2/104), and influenza A H3N2 (1/104); the remaining 212 patients had influenza A virus (11/202), influenza A H3N2 (11/202), rhinovirus (10/202), respiratory syncytial virus (7/202), influenza B virus (6/202), metapneumovirus (4/202), and coronavirus (2/202). Interpretation: Clinical testing methods for 2019-nCoV require improvement. Importantly, 5.8% of 2019-nCoV infected and 18.4% of non-2019-nCoV-infected patients had other pathogen infections. It is important to treat combined infections and perform rapid screening to avoid cross-contamination of patients. A test that quickly and simultaneously screens as many pathogens as possible is needed.

Protein phosphatase 2A (PP2A) is indispensable in development, and deficits of PP2A and deterioration of neuronal axons have been observed in several neurodegenerative disorders, but the direct link between PP2A and the neuronal axon development is still missing. Here, we show that PP2A is essential for axon development in transfected rat brain and the dissociated hippocampal neurons. Upregulation of PP2A catalytic subunit (PP2Ac) not only promotes formation and elongation of the functional axons but also rescues axon retardation induced by PP2A inhibition. PP2A can dephosphorylate collapsin response mediator protein-2 (CRMP2) that implements the axon polarization, whereas constitutive expression of phosphomimic-CRMP2 abrogates the effect of PP2A upregulation. We also demonstrate that PP2Ac is enriched in the distal axon of the hippocampal neurons. Our results reveal a mechanistic link between PP2A and axonogenesis/axonopathy, suggesting that upregulation of PP2A may be a promising therapeutic for some neurodegenerative disorders.

Background and objective Acute respiratory infection is the major cause of disease and death in children, particularly in developing countries. However, the spectrum of pathogenic viruses and atypical bacteria that exist in many of these countries remains incompletely characterized. The aim of this study was to examine the spectrum of pathogenic viruses and atypical bacteria associated with acute respiratory infection in children under the age of 16. Methods A total of 10 435 serum sera specimens were collected from hospitalized children presenting with acute respiratory infection symptoms. Indirect immunofluorescence assays were performed to detect immunoglobulin M antibodies against nine common pathogens: mycoplasma pneumonia, influenza virus B, respiratory syncytial virus, parainfluenza virus, adenovirus, influenza virus A, legionella pneumophila, coxiella burnetii and chamydophila pneumonia. Results Of the 10 435 specimens examined, 7046 tested positive for at least one pathogen. Among all of the tested pathogens, mycoplasma pneumonia had the highest detection rate (56.9%). Influenza virus A and influenza virus B epidemics occurred during both winter and summer. The detection rate of respiratory syncytial virus and adenovirus was higher in spring. Cases of mixed infection were more complex: 4136 specimens (39.6%) tested positive for ≥2 pathogens. There were statistically significant difference in detection rates of mycoplasma pneumonia, influenza virus B, respiratory syncytial virus, parainfluenza virus, adenovirus, influenza virus A, legionella pneumophila and chamydophila pneumonia among different age groups (P < 0.05). Conclusions The most common pathogens causing acute respiratory infection among children in Hubei of China were mycoplasma pneumonia, influenza virus B and respiratory syncytial virus. The detection rates for each pathogen displayed specific seasonal and age group variations.

Mangrove is a rich and underexploited ecosystem with great microbial diversity for discovery of novel and chemically diverse antimicrobial compounds. The goal of the study was to explore the pharmaceutical actinobacterial resources from mangrove soil and gain insight into the diversity and novelty of cultivable actinobacteria. Consequently, 10 mangrove soil samples were collected from Futian and Maoweihai of China, and the culture-dependent method was employed to obtain actinobacteria. A total of 539 cultivable actinobacteria were isolated and distributed in 39 genera affiliated to 18 families of 8 orders by comparison analysis of partial 16S rRNA gene sequences. The dominant genus was Streptomyces (16.0 %), followed by Microbacterium (14.5 %), Agromyces (14.3 %), and Rhodococcus (11.9 %). Other 35 rare actinobacterial genera accounted for minor proportions. Notably, 11 strains showed relatively low 16S rRNA gene sequence similarities (< 98.65 %) with validly described species. Based on genotypic analyses and phenotypic characteristics, 115 out of the 539 actinobacterial strains were chosen as representative strains to test their antibacterial activities against “ESKAPE” bacteria by agar well diffusion method and antibacterial mechanism by the double fluorescent protein reporter system. Fifty-four strains in 23 genera, including 2 potential new species, displayed antagonistic activity in antibacterial assay. Meanwhile, 5 strains in 3 genera exhibited inhibitory activity on protein biosynthesis due to ribosome stalling. These results demonstrate that cultivable actinobacteria from mangrove soil are potentially rich sources for discovery of new antibacterial metabolites and new actinobacterial taxa.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] plays an anticancer role in multiple types of cancer and potentiates the cytotoxic effects of several common chemotherapeutic agents. The hypercalcemia caused by 1,25(OH)2D3 alone or resistance to cisplatin weaken the anticancer effects of vitamin D. Thus, in this study, we aimed to investigate the synergistic effects of 1,25(OH)2D3 and cisplatin on the apoptosis and cell cycle progression of gastric cancer cells. BGC-823 human gastric cancer cells were treated with 1,25(OH)2D3 or cisplatin alone, or a combination of both agents. Cell apoptosis was assessed by TUNEL assay and flow cytometry. The expression of the apoptosis-related proteins, poly(ADP-ribose) polymerase (PARP), Bax, Bcl-2, caspase-3 and caspase-8, was examined using immunoblot analysis. ERK and AKT phosphorylation were examined by immunoblot analysis. The cell cycle distribution was determined by propidium iodide staining and flow cytometric analysis. p21 and p27 protein expression was also examined using immunoblot analysis. Our results revealed that co-treatment with 1,25(OH)2D3 enhanced cisplatin-induced apoptosis and upregulated the expression of Bax, and promoted the cleavage of PARP and caspase-3. The phosphorylation levels of ERK and AKT were reduced following combined treatment with 1,25(OH)2D3 and cisplatin. The percentage of cells in the G0/G1 phase was greater in the cells treated with the combined treatment than in those treated with either 1,25(OH)2D3 or cisplatin alone. p21 and p27 expression was upregulated following co-treatment with both agents. The results of this study suggest that 1,25(OH)2D3 potentiates cisplatin-mediated cell growth inhibition and cell apoptosis, which involves the upregulation of Bax, a decrease in ERK and AKT phosphorylation levels, and increased p21 and p27 levels.

The high expression of BMI-1 in cervical cancer is related to tumor progression, lymph node metastasis and HPV infection, suggesting that cervical cancer with excessive BMI-1 expression possesses high metastases potential and that BMI-1 may be a promising biomarker for predicting metastasis in cervical cancer.

Cardiac myocytes undergo apoptosis under conditions of high free fatty acid concentrations, including palmitate, which is implicated in lipotoxic cardiomyopathy. However, the underlying mechanisms remain unknown. The aim of the present study was to understand the role of reactive oxygen species (ROS) production and the extracellular signal‑regulated kinase 1/2 (ERK1/2) signaling pathway in palmitate‑induced apoptosis in H9c2 cells. H9c2 cells were exposed to palmitate for 12 h. The effect on the cell viability of H9c2 cells was evaluated using the 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assay and cell apoptosis was determined by Hoechst 33342 staining. Levels of intracellular ROS were determined using a peroxide‑sensitive fluorescent probe, 2',7'‑dichlorofluorescein diacetate. Protein expression was measured by western blot analysis. Following treatment with palmitate for 12 h, H9c2 cells apoptosis was demonstrated as increased brightly condensed chromatin or unclear fragments by staining with Hoechst 33342, which was associated with increasing levels of active caspase‑3 and cleaved poly (ADP-ribose) polymerase (PARP). In this model of treatment with palmitate, H9c2 cell apoptosis correlated with increased levels of p53 and Bax expression and reduced levels of Bcl-2 expression. Palmitate‑induced apoptosis was observed to increase levels of intracellular ROS production and p‑ERK1/2 and decrease p‑Akt significantly. Consistent with these results, palmitate‑induced apoptosis was attenuated by the ERK1/2 inhibitor, U0126, through partial reduction of intracellular ROS generation. Collectively, these results indicate that palmitate‑induced apoptosis in H9c2 cells is mediated by activation of the ERK1/2 signaling pathway and increased ROS generation.

BackgroundDetecting an ALK fusion gene in patients with non-small cell lung cancer (NSCLC) could provide evidence to guide individualized therapy.MethodsThe 5′/3′ imbalance strategy for quantitative reverse transcription-PCR (RT-qPCR) was developed to detect ALK fusion genes in circulating tumor RNA (ctRNA) of NSCLC patients.ResultsThis method was validated in patients with the ALK fusion gene confirmed by next generation sequencing (NGS). The amount of the ALK fusion gene detected by the new method ranged from 33.2 to 987.4, (mean 315.2), in the patients confirmed to have the ALK fusion gene (+). This is much higher than the amount of fusion gene detected in the patients who are negative for the ALK fusion gene (−). The amount detected in the ALK fusion gene (−) samples ranged from 0.36 to 13.04, (mean 4.58). In 188 NSCLC patients, the specificity and sensitivity of the method was compared to that of the FISH method. About 10.64% of the patients showed higher ALK fusion gene expression, and were classified as ALK fusion gene (+). This is identical to the percentage of patients detected by the FISH method to be ALK fusion gene (+). The cutoff value for diagnosis of ALK fusion (+) is 32.9 as determined by this method.ConclusionsA new RT-PCR method using a 5′/3′ imbalance strategy was developed, with high specificity and sensitivity, for detection of the ALK fusion gene in ctRNA of NSCLC patients. This method can rapidly detect ALK fusion genes in patients, which will be helpful for guiding targeted therapy, particularly the individualized usage of TKIs in these patients.