Upregulation of TMEM40 is associated with the malignant behavior and promotes tumor progression in cervical cancer.

OBJECTIVE: Recent studies indicated that transmembrane protein 40 (TMEM40) is associated with several types of cancers but is not clear in cervical cancer (CC). The study aimed to examine the role of TMEM40 in CC and related mechanisms. METHODS: The expression of TMEM40 in CC tissues and cell lines was studied with western blot and real-time quantitative RT-PCR. The effect of TMEM40 on proliferation was evaluated by CCK-8, EdU and colony formation assay. The migration, invasion, cell cycle and apoptosis of CC cells were studied with wound healing, transwell assays and flow cytometry. Tumor growth was evaluated in vivo using a xenogenous subcutaneously implant model. RESULTS: The results revealed that the TMEM40 elevation in CC tissues and cell lines was closely correlated with tumor size and lymph node metastasis in clinical patients. Upregulation of TMEM40 with OE-TMEM40 vector promoted the invasion, migration and proliferation, inhibited the apoptosis and led to distinct S cell cycle arrest in CC cell lines. Silencing TMEM40 with shRNA inhibited the invasion, migration and proliferation, promoted apoptosis and led to a G0/G1 cell cycle arrest in CC cell lines. Silence of TMEM40 downregulated the expression of c-MYC, Cyclin D1, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-9 (MMP-9), but in contrast, activated p53 and several apoptosis related proteins such as p53, Caspase-3, Caspase-9 and PARP1. In addition, TMEM40 silencing dramatically decreased tumor growth in mice models. CONCLUSION: The present study demonstrates that TMEM40 upregulation can be a potential prognostic biomarker and contribute to CC development.

Enterovirus D68 in hospitalized children with respiratory symptoms in Guangdong from 2014 to 2018: Molecular epidemiology and clinical characteristics.

BACKGROUND: Enterovirus D68 (EV-D68) is an emerging pathogen in humans. EV-D68 causes a wide range of respiratory symptoms in children and has the propensity to cause severe complications. EV-D68 outbreaks are rarely investigated in mainland China. Therefore, in this study, we aimed to investigate the prevalence of EV-D68 in children and to describe the clinical manifestations as well as the phylogeny of EV-D68 in Guangdong Province from 2014 to 2018. METHODS: Nasopharyngeal swabs were collected from hospitalized children with respiratory symptoms and screened for respiratory pathogens by fluorescence quantitative PCR and culture. The EV-positive samples were subsequently typed by sequencing the 5'-untranslated region and EV-D68-specific VP1 capsid gene. A phylogenetic tree was constructed by the maximum-likelihood method based on the VP1 gene using ClustalW. RESULTS: A total of 1,498 (59.8%) out of 2,503 children were screened positive for ≥1 virus species. Among the 158 (6.31%) EV-positive samples, 17 (0.68%) were identified as EV-D68. Most EV-D68 cases (n = 14) were diagnosed with pneumonia and bronchial pneumonia. No deaths were found in EV-D68 cases. Wheezing occurred in EV-D68 cases more frequently (70.59% vs. 43.26%, P = 0.040) than that of other EVs. All the EV-D68 were of clade B3, which were highly similar to the strains circulating in China. CONCLUSION: EV-D68 was the predominant enterovirus type in hospitalized children with respiratory symptoms in Guangdong Province. All the EV-D68 strains belong to clade B3. The development of diagnostic tools is warranted in order to monitor EV-D68 infections in China.

Fatal Community-acquired Pneumonia in Children Caused by Re-emergent Human Adenovirus 7d Associated with Higher Severity of Illness and Fatality Rate.

Human adenoviruses (HAdVs) are highly contagious pathogens causing acute respiratory disease (ARD), such as community-acquired pneumonia. HAdV-7d, a re-emergent genomic variant, has been recently reported in Asia and the United States after a several-decade absence. However, whether HAdV-7d is associated with higher severity than other types is currently unclear. In this study, the clinical and epidemiological investigation showed that fever, cough, and sore throat were the three most common respiratory symptoms of HAdV infections. HAdV-7 caused longer duration of fever, higher morbidity of tachypnea/dyspnea, pleural effusion, diarrhea, hepatosplenomegaly, consciousness alteration, as well as higher rates of pneumonia, mechanical ventilation and higher fatality rate (28.6%) than other types, particularly HAdV-3 and HAdV-2. The genomes of seven HAdV-7d isolates from mild, severe, and fatal cases were sequenced and highly similar with each other. Surprisingly, two isolates (2011, 2012) had 100% identical genomes with an earlier strain from a fatal ARD outbreak in China (2009), which elucidates the virus origin and confirms the unexpected HAdV genomic conservation and stability. Phylogenetic analysis indicated that L1 52/55-kDa DNA packaging protein may be associated with the higher severity of illness and fatality rate of HAdV-7. Clinicians need to be aware of HAdVs in children with ARD.

Changes in enterovirus serotype constituent ratios altered the clinical features of infected children in Guangdong Province, China, from 2010 to 2013.

BACKGROUND: Enterovirus (EV)-related hand, foot, and mouth disease/herpangina (HFMD/HA) has been prevalent in Guangdong Province, China, since 2010. METHODS: Clinical data for EV-related HFMD/HA inpatients admitted to the Department of Paediatrics of Zhujiang Hospital from 2010 to 2013 were retrospectively reviewed. The corresponding EV serotypes were also determined by reverse transcription-polymerase chain reaction or BLAST analysis of the sequenced partial lengths of the viral protein1/5'-untranslated region. RESULTS: A total of 867 eligible inpatients admitted during 2010-2013 were included in the study. Of these, the serotype of the responsible EV was successfully identified in 824 cases. The incidence of enterovirus 71 (EV71) infection amongst pediatric HFMD/HA inpatients decreased dramatically from 55.5 % in 2010 to 8.1 % in 2013, with a similar decrease recorded for coxsackievirus A16 (CVA16). However, the incidence of non-EV71/CVA16 infection increased from 30.0 % in 2010 to 83.8 % in 2013. We noted that the types of infection caused by different EV serotypes varied: EV71 was responsible for 100 % of the paralysis cases (26/26), 84.6 % of the deaths (11/13), and 84.1 % of cases with severe central nervous system involvement (SCNSI) (74/88); echovirus contributed to 16.4 % of the deaths (2/13) and 4.4 % of the SCNSI cases; and coxsackievirus accounted for only 2.2 % of the SCNSI cases (2/90). The clinical features of HFMD/HA cases varied greatly during the time period examined, with drastic changes in the hospitalization rates (45.1, 63.7, 36.4, and 19.1 % for 2010, 2011, 2012, and 21013, respectively), mortality rates (2.3, 0.9, 2.5, and 0.0 %, respectively), paralysis (5.1, 1.2, 5.4, and 0.0 %, respectively), SCNSI (16.8, 7.1, 12.7, and 2.2 %, respectively), and acute respiratory infection (21.1, 22.0, 45.9, and 59.0 %, respectively). CONCLUSIONS: The incidences of infection caused by different EV serotypes, along with the clinical features of HFMD/HA cases, changed drastically in Guangdong Province, China, from 2010 to 2013, with the biggest changes observed in 2013. The changed constituent ratios of the different EV serotypes might therefore be responsible for the differences in the observed clinical features of HFMD/HA during this period.

Enterovirus-related diarrhoea in Guangdong, China: clinical features and implications in hand, foot and mouth disease and herpangina.

BACKGROUND: A series of complications caused by enteroviruses, including meningitis, encephalitis, acute flaccid paralysis, acute cardiopulmonary failure, respiratory infection, and myocardial injury have been reported in hand, foot and mouth disease/herpangina (HFMD/HA). However, the complication of diarrhoea caused by enteroviruses has been neglected, and a summary of its clinical features and impact on HFMD/HA is unavailable. METHODS: We included inpatients with HFMD/HA admitted to the Paediatric Department of Zhujiang Hospital during 2009-2012. We summarised and compared clinical data for cases with and without diarrhoea, and determined enterovirus serotypes by reverse transcriptase polymerase chain reaction and genotyping based on a partial-length fragment of viral protein 1 or the 5'-untranslated region. RESULTS: There were 804 inpatients with HFMD/HA and 28 (3.5%) presented with diarrhoea. Gastrointestinal symptoms were mild in most cases of diarrhoea (82.1%), with high prevalence of no dehydration (82.1%), short duration of diarrhoea (78.6%) and watery stools (75.0%). The prevalence of multi-organ dysfunction syndrome (10.7 vs 0.40%) (p = 0.001), hepatic injury (14.3 vs 3.4%) (p = 0.019), myocardial injury (21.4 vs 6.1%) (p = 0.002) and convulsion (21.4 vs 7.2%) (p = 0.016) was significantly higher in the diarrhoea than no diarrhoea group. There was no significant difference between the two groups regarding prevalence of death, altered consciousness, paralysis, central nervous system involvement, or acute respiratory infection. CONCLUSIONS: Most patients with diarrhoea caused by enteroviruses circulating in Guangdong Province in 2009-2012 had mild or moderate gastrointestinal symptoms. Although enterovirus-related diarrhoea caused additional multi-organ dysfunction syndrome, hepatic injury and myocardial injury in children with HFMD/HA, timely intervention efficiently reduced disease severity and improved outcome.

Certification of a new certified reference material of honokiol.

Honokiol is the most important active pharmaceutical ingredient in Magnolia officinalis, which is a famous traditional Chinese medicine and commonly used in clinical practice. In order to control the quality of honokiol and related pharmaceuticals, a new certified reference material (CRM) of honokiol was developed. The studies of sample preparation, homogeneity, stability, value assignment, and uncertainty evaluation were accomplished in this paper. Three different methods, including differential scanning calorimetry (DSC), mass balance method (MB), and coulometric titration (CT), were employed to determine the purity of honokiol. Specifically, the DSC and CT methods for purity determination of honokiol were established for the first time. The purity of honokiol CRM, after validation and evaluation, was found to be 99.3%, with an expanded uncertainty of 0.5% (k = 2).

[Study on identification of four kinds of Gentianaceae Mongolian medicine Digeda with spectroscopy techniques].

To study the identification of Gentianaceae Mongolian medicine Digeda with spectroscopy techniques, near infrared spectroscopy and differential scanning calorimetry techniques were applied to study on the identification of 4 kinds of Gentianaceae Mongolian medicine Digeda, and characteristic spectrums obtained were systematically analyzed. In NIR study, the four species of Digeda exist some differences in 4 250-4 400 cm(-1) and 5 650-5 800 cm(-1) of one-dimensional spectra, and show significant differences in 4 100- 4 400 cm(-1), 4 401-4 900 cm(-1) and 5 400-5 800 cm(-1) of the second derivative spectra. DSC curves of them present distinct topological pattern, characteristic peak and peak temperature. Using near infrared spectroscopy and differential scanning calorimetry analysis can realize efficient and accurate identification of four kinds of Mongolian medicine Digeda, and provide scientific basis for the efficient and accurate identification of other Gentianaceae Mongolian medicine Digeda.

[Mapping of the B Cell Neutralizing Epitopes on ED III of Envelope Protein from Dengue Virus].

Dengue virus (DENV) envelope [E] protein is the major surface protein of the virions that indued neutralizing antibodies. The domain III of envelope protein (EDIII) is an immunogenic region that holds potential for the development of vaccines; however, the epitopes of DENV EDIII, especially neutralizing B-cell linear epitopes, have not been comprehensively mapped. We mapped neutralizing B-cell linear epitopes on DENV-1 EDIII using 27 monoclonal antibodies against DENV-1 EDIII proteins from mice immunized with the DENV-1 EDIII. Epitope recognition analysis was performed using two set of sequential overlapping peptides (16m and 12m) that spanned the entire EDIII protein from DENV-1, respectively. This strategy identified a DENV-1 type- specific and a group-specific neutralizing epitope, which were highly conserved among isolates of DENV-1 and the four DENV serotypes and located at two regions from DENV-1 E, namely amino acid residues 309-320 and 381-392(aa 309-320 and 381-392), respectively. aa310 -319(310KEVAETQHGT319)was similar among the four DENV serotypes and contact residues on aa 309 -320 from E protein were defined and found that substitution of residues E309 , V312, A313 and V320 in DENV-2, -3, -4 isolates were antigenically silent. We also identified a DENV-1 type-specific strain-restricted neutralizing epitope, which was located at the region from DENV-1 E, namely amino acid residues 329-348 . These novel type- and group-specific B-cell epitopes of DENV EDIII may aid help us elucidate the dengue pathogenesis and accelerate vaccine design.

Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.

Development of a double antibody sandwich ELISA for West Nile virus detection using monoclonal antibodies against non-structural protein 1.

The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans.

The absence of exanthema is related with death and illness severity in acute enterovirus infection.

OBJECTIVE: To clarify whether exanthema is related to illness severity in acute enterovirus infection in children. METHODS: The data of pediatric inpatients at Zhujiang Hospital during 2009-2012 with an acute enterovirus infection were reviewed retrospectively. Enterovirus infection was determined by real-time reverse transcription PCR. Clinical data were summarized and compared between cases with and without exanthema. RESULTS: A total of 780 pediatric inpatients with an acute enterovirus infection were included in this study, of whom 83 (10.6%) presented no exanthema. The percentage of deaths in the group of patients without exanthema was significantly higher than that in the group with exanthema (7.2% vs. 1.1%; p = 0.002). Central nervous system involvement (41.0% vs. 30.0%; p = 0.041), severe central nervous system (CNS) involvement (21.7% vs. 11.0%; p = 0.005), severe CNS involvement with cardiopulmonary failure (9.6% vs. 2.3%; p = 0.002), an altered level of consciousness (15.7% vs. 7.6%; p = 0.013), and convulsions (14.4% vs. 6.3%; p = 0.007) occurred significantly more frequently in the group without exanthema. CONCLUSIONS: A considerable proportion of children with an acute enterovirus infection in Guangdong Province, China during 2009-2012 presented no exanthema, and the absence of exanthema was found to be related to death and illness severity for these acute enterovirus infections. Clinicians in China should consider enterovirus as the possible pathogen when treating children with an acute pathogen infection without exanthema.

[Establishment and evaluation of a double antibody sandwich ELISA to detect Csa2 protein of Candida albicans].

OBJECTIVE: To establish a double antibody sandwich ELISA for detecting Csa2 protein in Candida albicans infection and evaluate its specificity and sensitivity. METHODS: A recombinant expression vector pPIC9K-Csa2 was constructed and transformed into Pichia pastoris GS115. A large-scale expression of recombinant Csa2 protein (rCsa2) was optimized using methanol, and the protein was purified in P. pastoris expression system. New Zealand Rabbits and guinea pigs were respectively immunized with the purified rCsa2 to prepare polyclonal antisera. The double antibody sandwich ELISA was established by choosing the optimal dilution of coating antisera and detecting antisera. Different concentrations of rCsa2 and culture supernatants of C. albicans collected at different time points were used to evaluate the sensitivity of detection. The specificity of the sandwich ELISA was evaluated by detecting culture supernatants of other three Candida spp, five Aspergillus spp, Cryptococcus neoformans and Penicillium marneffei. RESULTS: The rCsa2 protein was successfully expressed and purified. SDS-PAGE showed that its Mr was 13 300. Western blotting demonstrated that the protein bound to specific antibody. The sensitivity of the sandwich ELISA we established using the high-titer antisera was about 240 pg/mL of rCsa2, and could detect Csa2 protein in the culture supernatant of C. albicans when cultured for as early as 18 hours. There was no cross-reactivity between the culture supernatants of other 10 clinically important fungi and C. albicans. CONCLUSION: The double antibody sandwich ELISA for detecting Csa2 protein has been established with good sensitivity and specificity. Csa2 protein could be used as a new diagnostic marker of C. albicans infection.

A unique and conserved neutralization epitope in H5N1 influenza viruses identified by an antibody against the A/Goose/Guangdong/1/96 hemagglutinin.

Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab' fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.

Dengue virus envelope domain III immunization elicits predominantly cross-reactive, poorly neutralizing antibodies localized to the AB loop: implications for dengue vaccine design.

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime-boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309-320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime-boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.

New 23-spirocholestane derivatives from Ypsilandra thibetica.

Three new unusual 23-spirocholestane derivatives, ypsilanogenin (1), ypsilanogenin 3-O-ß-D-glucopyranoside (2), and 4'-acetylypsilanogenin 3-O-ß-D-glucopyranoside (3), were isolated from the whole plants of Ypsilandra thibetica. The structures of compounds 1-3 were deduced by spectroscopic and chemical methods, and the structure of 1 was further confirmed by a single-crystal diffraction analysis. All isolates were evaluated for their inhibitory activities against HIV-1.

Three new triterpenoids containing four-membered ring from the fruiting body of Ganoderma sinense.

Methyl ganosinensate A (1), ganosinensic acid A (1a), and ganosinensic acid B (2), three new triterpenoids with an unusual four-membered ring skeleton produced by a bond across C-1 to C-11, were isolated from the fruiting body of Ganoderma sinense . Their structures were established on the basis of extensive spectroscopic methods, including 1D and 2D NMR techniques, and methyl ganosinensate A was confirmed by X-ray crystallographic analysis.

Comprehensive mapping of immunodominant and conserved serotype- and group-specific B-cell epitopes of nonstructural protein 1 from dengue virus type 1.

The dengue virus (DENV) nonstructural protein 1 (NS1) is an immunogenic protein that holds potential for the development of vaccines and diagnostic reagents; however, the epitopes of NS1 have not been comprehensively mapped. We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. This strategy identified three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15, 71-85, and 338-352. We also identified five group-specific B-cell epitopes that were highly conserved among isolates of the four DENV serotypes. These novel immunodominant serotype- and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.