Distinct clinical features of transplanted children with Parvovirus B19 infection.

BACKGROUND: The immature and suppressed immune response makes transplanted children a special susceptible group to Parvovirus B19 (PVB19). However, the clinical features of transplanted children with PVB19 infection haven't been comprehensively described. METHODS: We searched the medical records of all the transplant recipients who attended the Children's Hospital of Fudan University from 1 Oct 2020 to 31 May 2023, and reviewed the medical literature for PVB19 infection cases among transplanted children. RESULTS: A total of 10 cases of PVB19 infection were identified in 201 transplanted children at our hospital, and the medical records of each of these cases were shown. Also, we retrieved 40 cases of PVB19 infection among transplanted children from the literature, thus summarizing a total of 50 unique cases of PVB19 infection. The median time to the first positive PVB19 DNA detection was 14 weeks post-transplantation. PVB19 IgM and IgG were detected in merely 26% and 24% of the children, respectively. The incidence of graft loss/dysfunction was as high as 36%. Hematopoietic stem cell transplant (HSCT) recipients showed higher PVB19 load, lower HGB level, greater platelet damage, lower PVB19 IgM/IgG positive rates, and more graft dysfunction than solid-organ transplant (SOT) recipients, indicating a more incompetent immune system. CONCLUSIONS: Compared with the published data of transplanted adults, transplanted children displayed distinct clinical features upon PVB19 infection, including lower PVB19 IgM/IgG positive rates, more graft dysfunction, and broader damage on hematopoietic cell lines, which was even more prominent in HSCT recipients, thus should be of greater concern.

Nano-flow cytometry unveils mitochondrial permeability transition process and multi-pathway cell death induction for cancer therapy.

Mitochondrial permeability transition (mPT)-mediated mitochondrial dysfunction plays a pivotal role in various human diseases. However, the intricate details of its mechanisms and the sequence of events remain elusive, primarily due to the interference caused by Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP). To address these, we have developed a methodology that utilizes nano-flow cytometry (nFCM) to quantitatively analyze the opening of mitochondrial permeability transition pore (mPTP), dissipation of mitochondrial membrane potential ( Δ Ψm), release of cytochrome c (Cyt c), and other molecular alternations of isolated mitochondria in response to mPT induction at the single-mitochondrion level. It was identified that betulinic acid (BetA) and antimycin A can directly induce mitochondrial dysfunction through mPT-mediated mechanisms, while cisplatin and staurosporine cannot. In addition, the nFCM analysis also revealed that BetA primarily induces mPTP opening through a reduction in Bcl-2 and Bcl-xL protein levels, along with an elevation in ROS content. Employing dose and time-dependent strategies of BetA, for the first time, we experimentally verified the sequential occurrence of mPTP opening and Δ Ψm depolarization prior to the release of Cyt c during mPT-mediated mitochondrial dysfunction. Notably, our study uncovers a simultaneous release of cell-death-associated factors, including Cyt c, AIF, PNPT1, and mtDNA during mPT, implying the initiation of multiple cell death pathways. Intriguingly, BetA induces caspase-independent cell death, even in the absence of Bax/Bak, thereby overcoming drug resistance. The presented findings offer new insights into mPT-mediated mitochondrial dysfunction using nFCM, emphasizing the potential for targeting such dysfunction in innovative cancer therapies and interventions.

Surveillance and epidemiological characterization of human adenovirus infections among outpatient children with acute gastroenteritis during the COVID-19 epidemic in Shanghai, China.

BACKGROUND: Human adenovirus (HAdV) has been recognized as one of the common enteric viruses associated with acute gastroenteritis (AGE) in children. The aim of this study was carried out to illustrate the epidemiological characterization of HAdV Infections among children younger than 15 years in Shanghai during COVID-19. METHODS: During May 2020 and April 2022, 1048 fecal samples were collected from children ≤ 15 years diagnosed with AGE in the Children's Hospital of Fudan University. HAdV was identified by PCR and sequenced with specific primers. All the obtained sequences were analyzed by MEGA (version 6.0). Demographic information and clinical features data were also collected and analyzed. RESULTS: In total, 97 (9.3%, 97/1048) samples were detected to be HAdV during May 2020 and April 2022. We found an atypical upsurge in HAdV infection in the year 2021 after a major suppression in the year 2020. Approximately 84.5% (82/97) of HAdV-infected children were aged 0-60 months. Among the 97 HAdV-positive samples, only two species and five genotypes were detected. HAdV-F (88.7%, 86/97) was the most prevalent species and HAdV-F41 (87.6%, 85/97) was the most common genotype. Diarrhea, vomiting, and fever were the main clinical manifestations in children infected with HAdV. The children aged from 0 to 12 months showed simpler patterns of clinical presentation than those of children older than 13 months. CONCLUSIONS: Our findings described the epidemiological changes of HAdV infection in children with AGE during the COVID-19, which further underscored the importance of continuous surveillance of HAdV at both local and global scales.

Comparison of Respiratory Pathogens in Children With Lower Respiratory Tract Infections Before and During the COVID-19 Pandemic in Shanghai, China.

Objectives: This study aimed to assess the impact of COVID-19 on the prevalence of respiratory pathogens among hospitalized children with lower respiratory tract infections (LRTIs) in Shanghai. Methods: Respiratory specimens were collected from children with LRTIs in Children's Hospital of Fudan University from February 2019 to January 2021 and common respiratory pathogens were detected using multiplex PCR. The data of 13 respiratory pathogens were analyzed and compared between the year of 2020 (from February 2020 to January 2021) and 2019 (from February 2019 to January 2020). Results: A total of 1,049 patients were enrolled, including 417 patients in 2019 and 632 patients in 2020. In 2020, 27.53% of patients were tested positive for at least one pathogen, which was significantly lower than that in 2019 (78.66%). The top three pathogens were Mycoplasma pneumoniae (Mp), human adenovirus (ADV) and human rhinovirus (RV) in 2019, whereas RV, human respiratory syncytial virus (RSV) and human parainfluenza virus (PIV) were the predominant ones in 2020. The positive rates of Mp, ADV, RV, PIV, Influenza virus B (InfB), H3N2, and H1N1 were significantly decreased in 2020. RV was the most detectable respiratory pathogen in 2020, and become the most frequent pathogen in all five age groups. PIV had a high prevalence from October to December 2020 which was even higher than that in 2019. Influenza virus A (InfA) was not detected in 2020. Co-infection was significantly less frequent in 2020. Conclusion: The public health interventions aiming to eliminate COVID-19 have great impact on the prevalence of common respiratory pathogens. The prevalence of RV and PIV reminds us a possible resurgence of some pathogens.

The changing pattern of common respiratory and enteric viruses among outpatient children in Shanghai, China: Two years of the COVID-19 pandemic.

Nonpharmaceutical interventions (NPIs) taken to combat the coronavirus disease 2019 (COVID-19) pandemic have not only decreased the spread of severe acute respiratory syndrome coronavirus 2 but also have had an impact on the prevalence of other common viruses. This study aimed to investigate the long-term impact of NPIs on common respiratory and enteric viruses among children in Shanghai, China, as NPIs were relaxed after June 2020. The laboratory results and clinical data of outpatient children with acute respiratory tract infections (ARTI) and acute gastroenteritis (AGE) were analyzed and compared between the post-COVID-19 period (from June 2020 to January 2022) and pre-COVID-19 period (from June 2018 to January 2020). A total of 107 453 patients were enrolled from June 2018 to January 2022, including 43 190 patients with ARTI and 64 263 patients with AGE. The positive rates of most viruses decreased during the post-COVID-19 period, with the greatest decrease for influenza A (-0.94%), followed by adenoviruses (AdV) (-61.54%), rotaviruses (-48.17%), and influenza B (-40%). However, the positive rates of respiratory syncytial virus (RSV) and enteric AdV increased during the post-COVID-19 period as the NPIs were relaxed. Besides this, in the summer of 2021, an unexpected out-of-season resurgence of RSV activity was observed, and the resurgence was more prominent among children older than 5 years. The effectiveness of the current relaxed NPIs in control of common respiratory and enteric viruses was variable. Relaxation of NPIs might lead to the resurgence of common viruses.

Mycoplasma pneumoniae and Adenovirus Coinfection Cause Pediatric Severe Community-Acquired Pneumonia.

Consolidation is one complication of pediatric severe community-acquired pneumonia (SCAP) that can respond poorly to conservative medical treatment. We investigated the pathogens that cause pediatric SCAP including cases with persistent consolidation that need bronchoscopy intervention. Alveolar lavage fluid (ALF) samples collected from cases admitted to Children's Hospital of Fudan University with SCAP during January 2019 to March in 2019 were retrospectively tested by the RespiFinder 2SMART multiplex PCR (multi-PCR) assay targeting 22 respiratory pathogens. A total of 90 cases and 91 samples were enrolled; 80.0% (72/90) of the cases had pulmonary consolidation and/or atelectasis. All samples were positive with targeted pathogens tested by multi-PCR, and 92.3% (84/91) of the samples were co-detected with pathogens. Mycoplasma pneumoniae (MP) and adenovirus (ADV) as the two dominant pathogens, with the positive rates of 96.7% (88/91) and 79.1% (72/91), respectively. Most of the samples were positive with MP and ADV simultaneously. As a control, 78.0% (71/91) of the samples were positive by conventional tests (CT), in which MP had the detection rate of 63.9% (55/86) by a traditional real-time PCR assay, while ADV were positive in 13.1% (12/91) of the samples by a direct immunofluorescence assay (DFA). In cases with persistent pulmonary consolidation, the positive rates of pathogens by multi-PCR and CT were 100% (72/72) and 81.9% (59/72), respectively. There were no significant differences of MP or ADV positive rates between cases with and without pulmonary consolidation. MP and ADV most prevalent in pediatric SCAP cases required fiberscope intervention, and presented with coinfections dominantly. IMPORTANCE Pathogens that cause pediatric severe community-acquired pneumonia (SCAP) requiring bronchoscopy intervention are understudied. Through this study, we explore the etiology of SCAP form alveolar lavage fluid (ALF) samples by the RespiFinder 2SMART multi-PCR assay. It is observed that high mixed detection rates of Mycoplasma pneumoniae and adenovirus in ALF samples collected from hospitalized SCAP children experienced bronchoscopy intervention. Eighty percent of the cases had pulmonary consolidation and/or atelectasis. The presence of possible coinfection of these two pathogens might contribute to poor clinical anti-infection response. The results of this study might be helpful for the selection of clinical strategies for the empirical treatment of such pediatric SCAP cases.

Epidemiology of Viruses Causing Pediatric Community Acquired Pneumonia in Shanghai During 2010-2020: What Happened Before and After the COVID-19 Outbreak?

INTRODUCTION: Since the global outbreak of COVID-19, there has been a significant reduction in pediatric outpatient and emergency visits for infectious diseases. The purpose of this study was to analyze the changes in respiratory viruses in children with community-acquired pneumonia (CAP) in Shanghai in the past 10 years, especially in the first year after COVID-19. METHODS: We conducted a retrospective, observational study; the results for eight common respiratory viruses (respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1-3 (PIV), adenovirus (ADV) and human metapneumovirus) tested by direct fluorescent antibody assays in hospitalized CAP cases in Children's Hospital of Fudan University during 2010-2020 were analyzed. RESULTS: Of the 5544 hospitalized CAP patients included in this study, 20.2% (1125/5544) were positive for the eight respiratory viruses. The top three pathogens were RSV, PIV3 and ADV, detected from 9.8% (543/5544), 5.3% (294/5544) and 2.0% (111/5544) of the samples, respectively. RSV had the highest positive rates among children < 2 years old. In 2020, the detection rate of all viruses showed a sharp decline from February to August compared with the previous 9 years. When the Shanghai community reopened in August 2020, the detection rate of eight viruses rebounded significantly in September. CONCLUSIONS: These eight respiratory viruses, especially RSV and PIV, were important pathogens of CAP in Shanghai children in the past 10 years. The COVID-19 pandemic had a significant impact on the detection rates for eight respiratory viruses in children with CAP in Shanghai.

Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour.

Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named "Sonication and Extrusion-assisted Active Loading" (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin-loaded milk-derived EVs (Dox-mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10-fold enhancement of drug encapsulation efficiency compared with passive loading. Single-particle analysis by nano-flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox-mEVs which would otherwise be overlooked by bulk-based approaches. Correlation analysis between doxorubicin auto-fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid-enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid-probe- and immuno-mediated magnetic isolation techniques to selectively remove the contaminants of non-lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL-prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as-developed cargo-loading approach and nano-flow cytometry-based characterization method will provide an instructive insight in the development of EV-based delivery systems.

Impact of COVID-19 pandemic on the prevalence of respiratory viruses in children with lower respiratory tract infections in China.

BACKGROUND: The multifaceted non-pharmaceutical interventions (NPIs) taken during the COVID-19 pandemic not only decrease the spreading of the SARS-CoV-2, but have impact on the prevalence of other viruses. This study aimed to explore the prevalence of common respiratory viruses among hospitalized children with lower respiratory tract infections (LRTI) in China during the COVID-19 pandemic. METHODS: Respiratory specimens were obtained from children with LRTI at Children's Hospital of Fudan University for detection of respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV) 1 to 3, influenza virus A (FluA), influenza virus B (FluB), human metapneumovirus (MPV) and rhinovirus (RV). The data were analyzed and compared between the year of 2020 (COVID-19 pandemic) and 2019 (before COVID-19 pandemic). RESULTS: A total of 7107 patients were enrolled, including 4600 patients in 2019 and 2507 patients in 2020. Compared with 2019, we observed an unprecedented reduction of RSV, ADV, FluA, FluB, and MPV infections in 2020, despite of reopening of schools in June, 2020. However, the RV infection was significantly increased in 2020 and a sharp increase was observed especially after reopening of schools. Besides, the PIV infection showed resurgent characteristic after September of 2020. The mixed infections were significantly less frequent in 2020 compared with the year of 2019. CONCLUSIONS: The NPIs during the COVID-19 pandemic have great impact on the prevalence of common respiratory viruses in China. Meanwhile, we do need to be cautious of a possible resurgence of some respiratory viruses as the COVID-19 restrictions are relaxed.

High-Throughput Human Telomere Length Analysis at the Single-Chromosome Level by FISH Coupled with Nano-Flow Cytometry.

Telomere length (TL) is a highly relevant biomarker for age-associated diseases and cancer, yet its clinical applications have been hindered by the inability of existing methods to rapidly measure the TL distribution and the percentage of chromosomes with critically short telomeres (CSTs, < 3 kb). Herein, we report the development of a high-throughput method to measure TL at the single-chromosome level. Metaphase chromosomes are isolated, hybridized with the Alexa Fluor 488-labeled telomeric peptide nucleic acid probe, and analyzed using a laboratory-built ultrasensitive nano-flow cytometer. The fluorescence intensity of individual chromosomes is converted to TL in kilobases upon external calibration. With an analysis rate of several thousand chromosomes per minute, a statistically robust TL distribution histogram is acquired in minutes, and the percentage of chromosomes with CSTs can be quickly assessed. By analyzing peripheral blood lymphocytes of 158 healthy donors, TL is found to shorten with age at a rate of 64 ± 3 bp/year and the percentage of chromosomes with CSTs increases with age at a rate of 0.32 ± 0.02%/year. Moreover, the data of 28 patients with chronic myeloid leukemia (CML) indicate that telomeres are significantly shorter at the time of diagnosis and the clinical phases of CML are closely associated with TL and the percentage of chromosomes with CSTs. This powerful tool could greatly deepen our understanding of telomere biology and improve the clinical utility of telomere biomarkers.

Etiology of Severe Pneumonia in Children in Alveolar Lavage Fluid Using a High-Throughput Gene Targeted Amplicon Sequencing Assay.

Objective: To evaluate the diagnostic value of a high-throughput gene targeted amplicon sequencing (TAS) assay for detecting pathogenic microorganisms in alveolar lavage fluid (ALF) from children with severe community-acquired pneumonia (SCAP). Methods: A retrospective study was performed on 48 frozen ALF samples from 47 severe pneumonia cases admitted to Children's Hospital of Fudan University from January 1, 2019, to March 31, 2019. All samples were tested by a multiplex PCR (Multi-PCR) assay and a TAS assay. The results of the TAS panels were parallel compared with Multi-PCR and Conventional Tests (CT) including culture, direct fluorescent antibody method (DFA), and singleplex polymerase chain reaction (PCR). Results: The proportion of pathogens detection by CT was 81.2% (39/48). The 8 common respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), influenza A virus (FLUA), influenza B virus (FLUB), parainfluenza virus 1-3 (PIV1-3), and human Metapneumovirus (hMPV) were found in 31.2% (15/48) of the 48 samples by DFA. With the criteria of CT results used as "Golden Standard" for determing of TAS results, the proportion of pathogens detection by TAS was 70.8% (34/48). The difference of proportion of pathogens detection between TAS and CT was not statistically significant (p = 0.232). The sensitivity and specificity of TAS for pathogens detection based on CT were 87.1% (95% CI, 71.77-95.18%) and 100.0% (95% CI, 62.88-100%), the positive predictive value (PPV) and negative predictive value (NPV) were 100.0% (95% CI, 87.35-100%) and 64.2% (95% CI, 35.62-86.02%), respectively. While Multi-PCR results were used as "Golden Standard," the total pathogens detection rate of TAS was 83.3% (40/48), which had a significant difference with that of Multi-PCR (p = 0.003). The sensitivity and PPV of TAS compared with Multi-PCR were 83.3% (95% CI, 69.23-92.03%) and 100.0% (95% CI, 89.08-100%), respectively. High rates of co-infection were proved by CT, Multi-PCR, and TAS. Mycoplasma pneumoniae (MP) and ADV were the two most frequently detected pathogens in all three assays. Conclusion: Compared with the CT and Multi-PCR methods, this TAS assay had a good performance in detecting bacteriological and viral pathogens from ALF. More research is needed to establish interpretation criteria based on TAS reads or analysis platforms.

Molecular and epidemiological characterization of human adenovirus and classic human astrovirus in children with acute diarrhea in Shanghai, 2017-2018.

BACKGROUND: In addition to rotavirus and norovirus, human adenovirus (HAdV) and classic human astrovirus (classic HAstV) are important pathogens of acute diarrhea in infants and young children. Here, we present the molecular epidemiology of HAdV and classic HAstV in children with acute diarrhea in Shanghai. METHODS: Fecal specimens were collected from 804 outpatient infants and young children diagnosed with acute diarrhea in Shanghai from January 2017 to December 2018. All of the samples were screened for the presence of HAdV and classic HAstV. HAdV and classic HAstV were detected using traditional PCR and reverse-transcription PCR, respectively. All of the HAdV and classic HAstV positive samples were genotyped by phylogenetic analysis. RESULTS: Among the 804 fecal samples, 8.58% (69/804) of samples were infected with either HAdV or classic HAstV, and five were co-infected with two diarrhea viruses. The overall detection rates of HAdV and classic HAstV were 3.47% (28/804) and 5.22% (42/804), respectively. Four subgroups (A, B, C, and F) and seven genotypes (HAdV-C1, -C2, -B3, -C5, -A31, -F40, and -F41) of HAdV were detected. Subgroup F had the highest constituent ratio at 64.29% (18/28), followed by non-enteric HAdV of subgroup C (21.43%, 6/28) and subgroup B 10.71% (3/28). HAdV-F41 (60.71%, 17/28) was the dominant genotype, followed by HAdV-C2 (14.29%, 4/28) and HAdV-B3 (10.71%, 3/28). Two genotypes of classic HAstV (HAstV-1 and HAstV-5) were identified in 42 samples during the study period; HAstV-1 (95.24%, 40/42) was the predominant genotype, and the other two strains were genotyped as HAstV-5. No significant differences were found between boys and girls in the detection rates of HAdV (P = 0.604) and classic HAstV (P = 0.275). Over half of the HAdV infections (82.14%, 23/28) and classic HAstV infections (66.67%, 28/42) occurred in children less than 36 months. Seasonal preferences of HAdV and classic HAstV infections were summer and winter, respectively. In this study, the common clinical symptoms of children with acute diarrhea were diarrhea, vomiting, fever and abdominal pain. CONCLUSIONS: Our findings indicate that HAdV and classic HAstV play important roles in the pathogenesis of acute diarrhea in children in Shanghai. Systematic and long-term surveillance of HAdV and classic HAstV are needed to monitor their prevalence in children and prevent major outbreak.

General and mild modification of food-derived extracellular vesicles for enhanced cell targeting.

Food-derived extracellular vesicles (FDEVs) have attracted increasing attention as potential delivery vehicles for therapeutic agents due to their desirable features such as excellent biocompatibility, easy accessibility and cost effectiveness. However, the intrinsic targeting capability of FDEVs is unsatisfactory compared to artificial nanoparticles or other source-derived EVs, which calls for efficient surface engineering strategies to equip them with specific ligands. Here we report a general and mild modification method via reduction of disulfide groups to maleimide reactive thiols. Taking milk-derived EVs (mEVs) as a model system, we demonstrated the feasibility for tethering various ligands on the surface without compromising the vesicular structures. Building an ultra-sensitive nano-flow cytometer (nFCM), the heterogeneous nature of the functionalized samples was revealed, and a magnetic separation approach was proposed accordingly to remove the as-observed non-EV particles. The cellular uptake and cytotoxicity experiments provided direct evidence showing an enhanced cell targeting and cargo delivery capability of the ligand conjugated mEVs. In addition, the in vivo imaging further proved the applicability of transferrin conjugation for increased tumor enrichment of mEVs. Collectively, this general and mild ligand conjugation method enables an efficient surface functionalization of FDEVs, which is of vital importance for enhanced targeting delivery.

Activatable Mitochondria-Targeting Organoarsenic Prodrugs for Bioenergetic Cancer Therapy.

Despite widespread applications for cancer treatment, chemotherapy is restricted by several limitations, including low targeting specificity, acquired drug resistance, and concomitant adverse side effects. It remains challenging to overcome these drawbacks. Herein, we report a new bioenergetic approach for treating cancer efficiently. As a proof-of-concept, we construct activatable mitochondria-targeting organoarsenic prodrugs from organoarsenic compounds and traditional chemotherapeutics. These prodrugs could accomplish selective delivery and controlled release of both therapeutic agents to mitochondria, which synergistically promote mitochondrial ROS production and induce mitochondrial DNA damage, finally leading to mitochondria-mediated apoptosis of cancer cells. Our in vitro and in vivo experiments reveal the excellent anticancer efficacy of these prodrugs, underscoring the encouraging outlook of this strategy for effective cancer therapy.

Rapid and quantitative in vitro analysis of mitochondrial fusion and its interplay with apoptosis.

Mitochondrial fusion is essential to maintain genomic stability and physiological functions of mitochondria. Since mitochondrial fusion and fission work in concert to regulate mitochondrial morphology and functions, it has been challenging to quantitatively measure the direct roles of mitochondrial fusion in apoptosis and cancer progression. Here, we report the development of a high-throughput in vitro method to quantify mitochondrial fusion through single mitochondria analysis by a laboratory-built nano-flow cytometer (nFCM). Isolated mitochondria expressing green fluorescent protein (GFP-mito) or discosoma red fluorescent protein (DsRed-mito) were mixed together, induced to fuse, and analyzed by nFCM. A particle exhibiting both green and red fluorescence was identified as an event of heterotypic fusion, and the efficiency of heterotypic fusion was used as a surrogate of overall fusion efficiency. The as-developed method was applied to reveal the interplay between mitochondrial fusion and apoptosis without the interference of fission. We show that cytosolic components promoted mitochondrial fusion, and this upregulation was diminished during apoptosis. Combined with the translocation of Bid and Bax from cytosol to mitochondria, these findings suggest that cytosolic pro-apoptotic Bcl-2 family proteins could be the positive mediators of mitochondrial fusion. On the other hand, fusion also renders mitochondria more resistant to membrane potential collapse upon apoptosis induction. Our data suggest that disruption of mitochondrial fusion could be a potent strategy for cancer therapy. Furthermore, the as-developed method offers an effective approach to identify fusion inhibitors, including betulinic acid and antimycin A, giving reasons for their powerful utility in cancer treatment.

Whole transcriptome profiling reveals major cell types in the cellular immune response against acute and chronic active Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a common human pathogen that infects over 95% of the population worldwide. In the present study, the whole transcriptome microarray data were generated from peripheral blood mononuclear cells from Chinese children with acute infectious mononucleosis (AIM) and chronic active EBV infection (CAEBV) that were also compared with a publicly available microarray dataset from a study of American college students with AIM. Our study characterized for the first time a broad spectrum of molecular signatures in AIM and CAEBV. The key findings from the transcriptome profiling were validated with qPCR and flow cytometry assays. The most important finding in our study is the discovery of predominant γδ TCR expression and γδ T cell expansion in AIM. This finding, in combination with the striking up-regulation of CD3, CD8 and CD94, suggests that CD8+ T cells and CD94+ NK cells may play a major role in AIM. Moreover, the unique up-regulation of CD64A/B and its significant correlation with the monocyte marker CD14 was observed in CAEBV and that implies an important role of monocytes in CAEBV. In conclusion, our study reveals major cell types (particularly γδ T cells) in the host cellular immune response against AIM and CAEBV.

Detection and Molecular Characterization of Human Adenovirus Infections among Hospitalized Children with Acute Diarrhea in Shanghai, China, 2006-2011.

Background: Human adenovirus (HAdV) is considered a significant enteropathogen associated with sporadic diarrhea in children. However, limited data are available regarding the epidemiology of HAdV in hospitalized children with viral diarrhea in Shanghai. The aim of this study was to characterize the epidemiology of HAdVs and describe their association with acute diarrhea in hospitalized children. Methods: A total of 674 fecal samples were subjected to PCR or RT-PCR to detect RVA, HuCV, HAstV, and HAdV. Results: HAdV infections were detected in 4.7% (32/674) of specimens, with detection rates of 13.4% (11/82), 4.6% (8/174), 3.2% (4/124), 4.1% (3/74), 2.0% (2/100), and 3.3% (4/120) from 2006 to 2011, respectively. Comprehensive detection of the four viruses revealed the presence of a high percentage (90.6%) of coinfections among HAdV-positive samples, where HAdV+RVA was the most prevalent coinfection. Of the 32 HAdV-positive samples, 50.0% (16/32) were classified as HAdV-41, and 18.8% (6/32) were classified as HAdV-3. Almost 94.0% of children infected with HAdV were less than 24 months of age. Conclusions: These results clearly indicated diversity across the HAdV genotypes detected in inpatient children with acute diarrhea in Shanghai and suggested that HAdVs play a role in children with acute diarrhea.

Molecular characterization and multiple infections of rotavirus, norovirus, sapovirus, astrovirus and adenovirus in outpatients with sporadic gastroenteritis in Shanghai, China, 2010-2011.

Rotavirus (RV), norovirus (NoV), sapovirus (SaV), human astrovirus (HAstV) and human adenovirus (HAdV) are significant because they are the most common pathogens that cause diarrhea in young children. The aim of this study was to investigate the genetic characteristics and compare the roles of these five viruses in outpatient children with diarrhea in Shanghai. A total of 436 fecal samples were collected from pediatric patients with acute gastroenteritis from January 2010 to December 2011. The selected samples were subjected to reverse transcription PCR (RT-PCR) or PCR to detect and genotype RV, NoV, SaV, HAstV and HAdV. RV (43.3 %, 189/436) was the most prevalent virus, followed by NoV (28.9 %, 126/436), HAdV (7.1 %, 31/436). HAstV (1.8 %, 8/436) and SaV (0.5 %, 2/436). The percentage of multiple infection cases was 14.9 % (65/436), and RV + NoV was the predominant mixed infection. The RV genotype combinations of P[8]G3 (52/189, 27.5 %), P[8]G1 (51/189, 26.9 %) and P[8]G9 (48/189, 25.4 %) occurred most frequently. The predominant NoV genotype was GII.4 (73.0 %, 92/126), and the majority of GII.4 clustered as GII.4-2006b (65.2 %, 60/92). Two of the SaV cases were identified as GI.2 and GII.1. All HAstV-positive samples belonged to HAstV-1. The predominant HAdV type was HAdV-41 (45.2 %, 14/31). This study clearly shows the diversity of the viral causative agents of acute gastroenteritis in outpatient children in Shanghai, which will provide baseline information for future vaccination strategies and development in this area.

A new accurate assay for Coxsackievirus A 16 by fluorescence detection of isothermal RNA amplification.

Coxsackievirus A 16 (CA16) is one of the most common causes of hand, foot, and mouth disease (HFMD) worldwide. Without a vaccine or antiviral drug early, rapid, and accurate detection is critical for preventing and controlling HFMD. A simultaneous amplification and testing (SAT) assay was developed for detecting CA16 based on isothermal RNA amplification with fluorescence using standard, real-time PCR equipment. Primers and probes were designed to target the VP1 region of CA16. Virus strains and clinical specimens were used to evaluate the diagnostic performance characteristics of the assay. The assay detected as few as 10 copies of CA16 RNA transcripts. Using real-time PCR plus sequencing as the reference standard, the sensitivity and specificity of the SAT-CA16 assay were 100% and 99.2%, respectively. These findings indicate that SAT-CA16 is a rapid and reliable method for detecting CA16.