RT-RPA-PfAgo detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/µL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.

The association between ambient air pollution exposure and connective tissue sarcoma risk: a nested case-control study using a nationwide population-based database.

A nationwide population-based database was utilized in a nested case-control study to explore the association between ambient air pollution exposure and the likelihood of developing connective tissue sarcoma. The study examined 280 cases of connective tissue sarcoma diagnosed between 2000 and 2012. A random sample of 1120 control subjects was selected from a subpopulation of claim records without a connective tissue sarcoma diagnosis in a 1:4 ratio. The control subjects were selected based on similar characteristics as the connective tissue sarcoma patients, including gender, birth year, and the year of diagnosis of the case group with medical records. Risk factors for connective tissue sarcoma were collected for analysis. Our data on exposure to air pollutants was collected from Taiwan's Air Quality Monitoring Network, which has been gathering air quality data from a growing network of sampling stations (now 76) throughout the country since 1997. It was discovered that the risk of connective tissue sarcoma was significantly increased by the Charlson comorbidity index (CCI), elevated levels of specific air pollution indices (e.g., total hydrocarbons (THC), fine particulate matter (PM2.5), and O3_8 (the annual mean of the daily maximum 8-h average concentration of O3), the High Pollutant Standards Index (hPSI) (the percentage of days in a given year in Taiwan where the PSI exceeds 100), and an insurable monthly wage over US$1100. Further investigation is needed to explore the involvement of these air pollutants in the formation of connective tissue sarcoma.

Proteomic analysis of exosome-like vesicles from Fasciola gigantica adult worm provides support for new vaccine targets against fascioliasis.

BACKGROUND: Extracellular vesicles (EVs) released by helminths play an important role in parasite-host communication. However, little is known about the characteristics and contents of the EVs of Fasciola gigantica, a parasitic flatworm that causes tropical fascioliasis. A better understanding of EVs released by F. gigantica will help elucidate the mechanism of F. gigantica-host interaction and facilitate the search for new vaccine candidates for the control and treatment of fascioliasis. METHODS: Two different populations of EVs (15k EVs and 100k EVs) were purified from adult F. gigantica culture media by ultracentrifugation. The morphology and size of the purified EVs were determined by transmission electron microscopy (TEM) and by the Zetasizer Nano ZSP high performance particle characterization system. With the aim of identifying diagnostic markers or potential vaccine candidates, proteins within the isolated 100k EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS). Mice were then vaccinated with excretory/secretory products (ESPs; depleted of EVs), 15k EVs, 100k EVs and recombinant F. gigantica heat shock protein 70 (rFg-HSP70) combined with alum adjuvant followed by challenge infection with F. gigantica metacercariae. Fluke recovery and antibody levels were used as measures of vaccine protection. RESULTS: TEM analysis and nanoparticle tracking analysis indicated the successful isolation of two subpopulations of EVs (15k EVs and 100k EVs) from adult F. gigantica culture supernatants using differential centrifugation. A total of 755 proteins were identified in the 100k EVs. Exosome biogenesis or vesicle trafficking proteins, ESCRT (endosomal sorting complex required for transport) pathway proteins and exosome markers, heat shock proteins and 14-3-3 proteins were identified in the 100k EVs. These results indicate that the isolated 100k EVs were exosome-like vesicles. The functions of the identified proteins may be associated with immune regulation, immune evasion and virulence. Mice immunized with F. gigantica ESPs, 15k EVs, 100k EVs and rFg-HSP70 exhibited a reduction in fluke burden of 67.90%, 60.38%, 37.73% and 56.6%, respectively, compared with the adjuvant control group. The vaccination of mice with F. gigantica 100k EVs, 15k EVs, ESP and rFg-HSP70 induced significant production of specific immunoglobulins in sera, namely IgG, IgG1 and IgG2a. CONCLUSION: The results of this study suggest that proteins within the exosome-like vesicles of F. gigantica have immunomodulatory, immune evasion and virulence functions. This knowledge may lead to new strategies for immunotherapy, vaccination and the diagnosis of fascioliasis.

Prevalence of Lower Urinary Tract Symptoms in Children with Attention-Deficit/Hyperactivity Disorder: Comparison of Hospital and Population-Based Cohorts of 13,000 Patients.

Background: This study investigates the prevalence of lower urinary tract symptoms (LUTS) in school-age children with Attention-Deficit/Hyperactivity Disorder (ADHD) based on hospital-based and population-based cohorts. Methods: The hospital-based sample comprised 42 children with ADHD and 65 without ADHD aged 6−12 years. Voiding dysfunction was assessed by the Dysfunctional Voiding Scoring System (DVSS) questionnaire. We compared the baseline data, DVSS score, and uroflowmetry between the two groups. For the population-based cohort in the national insurance database, we included 6526 children aged 6−12 years, whose claims record included the diagnosis of ADHD, and another 6526 control subjects matched by gender and age. We compared the presence of LUTS diagnosis codes between the two groups. Results: Our results showed that, for the hospital-based cohort, the mean total DVSS score and the proportion of significant LUTS in children in the ADHD group were significantly higher than in subjects in the non-ADHD group. The DVSS subscales showed that the item "I cannot wait when I have to pee" item was significantly higher in the ADHD group (1.62 ± 1.17 vs. 0.90 ± 1.09, p = 0.002). For the population-based cohort, children with ADHD had a significantly higher likelihood of storage symptoms (5.53% vs. 2.91%, p < 0.001) and enuresis (3.28% vs. 1.95%, p < 0.001) compared with those of the no ADHD group. Conclusions: Children with ADHD have a higher prevalence of significant LUTS, especially storage symptoms and enuresis, than children without ADHD. The observed correlations between ADHD and LUTS provided the supporting evidence to evaluate the concomitant voiding dysfunction in children with ADHD.

Genetic diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described. METHODS: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools. RESULTS: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure. CONCLUSIONS: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Estrogen receptor-associated receptor α and peroxisome proliferator-activated receptor γ in metabolism and disease (Review).

Estrogen receptor­associated receptor α (ERRα) is an orphan nuclear receptor that lacks corresponding ligands. ERRα recruits co­regulators to regulate gene transcription and plays an important role in human physiological functions. Peroxisome proliferator­activated receptor Î³ (PPARγ) is also a nuclear receptor that regulates the expression of target genes via a ligand­dependent mechanism, thereby participating in a series of physiological processes. Both ERRα and PPARγ are involved in the process of energy metabolism and tumorigenesis. In the present review, a concise overview of the important roles governed by ERRα and PPARγ in metabolism and their association with various disease are provided.

Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

The excretory-secretory products released by the liver fluke Fasciola gigantica (FgESPs) play important roles in regulating the host immune response during the infection. Identification of hepatic miRNAs altered by FgESPs may improve our understanding of the pathogenesis of F. gigantica infection. In this study, we investigated the alterations in the hepatic microRNAs (miRNAs) in mice treated with FgESPs using high-throughput small RNA (sRNA) sequencing and bioinformatics analysis. The expression of seven miRNAs was confirmed by quantitative stem-loop reverse transcription quantitative PCR (qRT-PCR). A total of 1,313 miRNAs were identified in the liver of mice, and the differentially expressed (DE) miRNAs varied across the time lapsed post exposure to FgESPs. We identified 67, 154 and 53 dysregulated miRNAs at 1, 4 and 12 weeks post-exposure, respectively. 5 miRNAs (miR-126a-3p, miR-150-5p, miR-155-5p, miR-181a-5p and miR-362-3p) were commonly dysregulated at the three time points. We also found that most of the DE miRNAs were induced by FgESPs in the mouse liver after 4 weeks of exposure. These were subjected to Gene Ontology (GO) enrichment analysis, which showed that the predicted targets of the hepatic DE miRNAs of mice 4 weeks of FgESPs injection were enriched in GO terms, including cell membrane, ion binding, cellular communication, organelle and DNA damage. KEGG analysis indicated that the predicted targets of the most downregulated miRNAs were involved in 15 neural activity-related pathways, 6 digestion-related pathways, 20 immune response-related pathways and 17 cancer-related pathways. These data provide new insights into how FgESPs can dysregulate hepatic miRNAs, which play important roles in modulating several aspects of F. gigantica pathogenesis.

Newly excysted juveniles of Fasciola gigantica trigger the release of water buffalo neutrophil extracellular traps in vitro.

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes and are among the first line of immune system defense. PMNs can form neutrophil extracellular traps (NETs) in response to some pathogens. The release of NETs plays an important role in trapping and killing invading parasites. However, the effects of NETs on parasitic trematode infections remain unclear. In the present study, water buffalo NET formation, triggered by the newly excysted juveniles (NEJs) of Fasciola gigantica, was visualized by scanning electron microscopy. The major components of the structure of NETs were characterized by immunofluorescence. Viability of flukes incubated with water buffalo PMNs were examined under light microscopy. The results revealed that F. gigantic juveniles triggered PMN-mediated NETs. These NETs were confirmed to comprise the classic characteristics of NETs: DNA, histones, myeloperoxidase and neutrophil elastase. Although NETs were formed in response to viable larvae, the larvae were not killed in vitro. These results suggest that NET formation may serve as a mechanism to hamper the migration of large larvae to facilitate immune cells to kill them. This study demonstrates, for the first time, that parasitic trematode juveniles can trigger NET formation.

Nasogastric tube decompression is unnecessary in patients undergoing laparoscopic nephroureterectomy for localized upper tract urothelial carcinoma.

BACKGROUND/PURPOSE: This study investigates the safety and feasibility to perform laparoscopic nephroureterectomy (LNU) for upper tract urothelial carcinoma (UTUC) without routine nasogastric tube (NGT) decompression. METHODS: The hospital-based samples comprised of 100 consecutive UTUC patients receiving elective LNU performed by two experienced surgeons. The nationwide data was based on LHID2005 composed of one million beneficiaries randomly selected from the Taiwan National Health Insurance Research Database to identify patients with the diagnoses of UTUCs receiving LNUs. We then compared baseline characteristics, peri-operative data, convalescence parameters and complications between two groups stratified by use of NGT tube. RESULTS: The hospital-based samples composed of 50 subjects with NGT and 50 without. There were no significant differences in baseline characteristics between two groups. Peri-operative and convalescence parameters were similar when comparing no NGT versus NGT: blood loss of 206 vs. 165 mL; operative time of 180.5 vs.181.1 min; days to intake was 2.1 vs.1.7 days; and hospital stay of 7.8 vs. 7.5 days (all p > 0.05). The nationwide study samples comprised 140 subjects, of which 72 were with NGT and 68 were with no NGT. The baseline data, complications and length of hospital stay were similar between two groups. CONCLUSION: Surgery-naïve patients with localized UTUC received LNU without peri-operative NGT is safe and feasible.

The role of lncRNA LSAT1 in the invasion and metastasis of non-small cell lung cancer under hypoxia.

BACKGROUND: To investigate the role of lncRNA LSAT1 in the hypoxia-induced invasion and metastasis of non-small cell lung cancer (NSCLC). METHODS: High-throughput microarrays were used to compare the lncRNA expression profile difference between normoxia-induced and hypoxia-induced NSCLC cell lines including A549, NCI-H1650 and NCI-H1299 in order to preliminarily screen key molecules related to hypoxia-induced invasion and metastasis of NSCLC. The different expression of lncRNA LSAT1 was measured in hypoxia-induced NSCLC cells (compared to normoxia-induced NSCLC cells) and 20 pairs of NSCLC tissues (compared to adjacent tissues) with RT-PCR. The expression of lncRNA LSAT1 in NSCLC cells A549 and NCI-H1299 was down-regulated by lentiviral transfection. Transwell migration and invasion assays and tail vein injection metastasis assay were employed for investigating the effect of lncRNA LSAT1 knockdown on the hypoxia-induced invasion and metastasis of NSCLC cells. RESULTS: RT-PCR showed that lncRNA LSAT1 was significantly increased in hypoxia-induced NSCLC cells A549, NCI-H1650 and NCI-H1299 and compared with adjacent tissues. The expression of lncRNA LSAT1 was also upregulated in NSCLC tissues. Transwell migration and invasion assays demonstrated that hypoxia could enhance the abilities of migration and invasion of NSCLC cells A549 and NCI-H1299. However, these two abilities were significantly inhibited after lncRNA LSAT1 knockdown. In addition, nude mice tail vein injection metastasis assay also verified that lncRNA LSAT1 knockdown inhibited liver metastasis and lung metastasis of NSCLC cell A549 in vivo. CONCLUSIONS: We found a series of hypoxia-related lncRNAs in NSCLC by means of high-throughput screening microarrays. Clinical specimen analysis and functional loss test confirmed that lncRNA LSAT1 is a key lncRNA for hypoxia-induced invasion and metastasis of NSCLC. In addition, STAT3 may be one of the target genes for lncRNA LSAT1 to play a regulatory role.

Trends in the use of antimuscarinics and alpha-adrenergic blockers in women with lower urinary tract symptoms in Taiwan: A nationwide, population-based study, 2007-2012.

BACKGROUND: We aim to examine the trend in the use of antimuscarinics and off-label alpha-adrenergic blockers for treatment of lower urinary tract symptoms (LUTS) in a Taiwanese Women Cohort between 2007 and 2012. METHODS: This population-based National Health Insurance Research Database (NHIRD) was used to examine the trends in the use of antimuscarinics or off-label alpha-adrenergic blockers in Taiwan. A sample of 1,000,000 individuals randomly drawn from the whole population of 23 million individuals who were registered in the NHI in 2005. From 2007 through 2012, women aged over 18 years whose claim record contained prescriptions of either of the two drugs for treatment of any of the LUTS-related diagnoses were identified and analyzed. The annual usage of the two drug classes were calculated by defined daily dose (DDD). RESULTS: From 2007-2012, there was a 0.80 fold (69676.8 to 125104.3) increase in DDD of antimuscarinics in our cohort. The overall healthcare seeking prevalence of LUTS was 7.33% in 2007 and 12.38% in 2012, in a rising trend. The prevalence of antimuscarinics-treated LUTS in our cohort increased from 2.53 in 2007 to 3.41 per 1000 women in 2012. The prevalence of LUTS treated by antimuscarinics increased especially for those older than 60 years during the study period. CONCLUSIONS: This 6-year observational study provided the epidemiologic information of clinically significant LUTS of Asian female population. Moreover, there was a rising trend in the use of antimuscarinics and off-label alpha-adrenergic blockers in the population-based cohort.

DNA methylation and hydroxymethylation profiles reveal possible role of highly methylated TLR signaling on Fasciola gigantica excretory/secretory products (FgESPs) modulation of buffalo dendritic cells.

BACKGROUND: Excretory/secretory products (ESPs) released by parasites influence the development and functions of host dendritic cells (DCs). However, little is known about changes of DNA (hydroxy)methylation on DC development during Fasciola gigantica infection. The present study aimed to investigate whether F. gigantica ESPs (FgESPs) affects the development and functions of buffalo DCs through altering the DNA (hydroxy)methylation of DCs. METHODS: Buffalo DCs were prepared from peripheral blood mononuclear cells (PBMCs) and characterized using scanning and transmission electron microscopy (SEM/TEM) and quantitative reverse transcriptional PCR (qRT-RCR). DCs were treated with 200 µg/ml of FgESPs in vitro, following DNA extraction. The DNA methylome and hydroxymethylome were profiled based on (hydroxy)methylated DNA immunoprecipitation sequencing [(h)MeDIP-Seq] and bioinformatics analyses. qRT-RCR was also performed to assess the gene transcription levels of interest. RESULTS: FgESPs markedly suppressed DC maturation evidenced by morphological changes and downregulated gene expression of CD1a and MHC II. Totals of 5432 and 360 genes with significant changes in the 5-methylcytosine (5-mC) and the 5-hydroxymethylcytosine (5-hmC) levels, respectively, were identified in buffalo DCs in response to FgESPs challenge. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these differentially expressed genes were highly enriched in pathways associated with immune response. Some cancer-related pathways were also indicated. There were 111 genes demonstrating changes in both 5-mC and 5-hmC levels, 12 of which were interconnected and enriched in 12 pathways. The transcription of hypermethylated genes TLR2, TLR4 and IL-12B were downregulated or in a decreasing trend, while the mRNA level of high-hydroxymethylated TNF gene was upregulated in buffalo DCs post-exposure to FgESPs in vitro. CONCLUSIONS: To our knowledge, the present study provides for the first time a unique genome-wide profile of DNA (hydroxy)methylation for DCs that interact with FgESPs, and suggests a possible mechanism of FgESPs in suppressing DC maturation and functions that are involved in TLR signaling.

miR-124 upregulates astrocytic glutamate transporter-1 via the Akt and mTOR signaling pathway post ischemic stroke.

High-concentration glutamic acid (Glu) induced by ischemic stroke can be inhibited by glutamate transporter-1 (GLT-1), which is the main mechanism for preventing excessive extracellular glutamate accumulation in the central nervous system. Upregulation of miR-124 could reduce the infarct area and promote the recovery of neurological function after ischemic stroke. A previous study investigated whether miR-124 could regulate GLT-1 expression in normal culture conditions. However, the role of miR-124 in the regulation of GLT-1 expression and further mechanisms after ischemic stroke remain unclear. In this study, the effects of miR-124 on GLT-1 expression in astrocytes after ischemic stroke were explored using an in vitro model of ischemic stroke (oxygen-glucose deprivation/reperfusion, OGD/reperfusion). The expression of GLT-1 was significantly decreased with lower expression of miR-124 in astrocytes injured by OGD/reperfusion. When miR-124 expression was improved, the expression of GLT-1 was notably increased in astrocytes injured by OGD/reperfusion. The results revealed that GLT-1 expression in astrocytes had a relationship with miR-124 after OGD/reperfusion. However, a direct interaction could not be confirmed with a luciferase reporter assay. Further results demonstrated that an inhibitor of Akt could decrease the increased protein expression of GLT-1 induced by miR-124 mimics, and an inhibitor of mTOR could increase the reduced protein expression of GLT-1 caused by a miR-124 inhibitor in astrocytes injured by different OGD/reperfusion conditions. These results indicated that miR-124 could regulate GLT-1 expression in astrocytes after OGD/reperfusion through the Akt and mTOR pathway.

Th2-related cytokines are associated with Fasciola gigantica infection and evasion in the natural host, swamp buffalo.

The infection of ruminants by Fasciola spp. always induces a non-protective Th2-type immune response. However, little is known about changes in the local and systemic immune environment during F. gigantica migration in buffalo. In this study, native swamp buffaloes were each infected with 500 viable F. gigantica metacercariae. Mesenteric lymph node (MLN), hepatic lymph node (HLN), spleen, and serum samples were collected from control and infected buffaloes at 3, 10, 28, 42, 70, and 98 days post-infection (DPI). The mRNA expression levels of the Th1- and Th2-related cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IFN-γ, TNF-α, and CD4 were measured during different infection stages in the MLNs, spleens, and HLNs using quantitative real-time PCR (qRT-PCR). Levels of the specific anti-ESP isotype antibodies IgG, IgG1, and IgG2 were used to reflect changes in humoral immunity. The results of this study indicated that swamp buffaloes were susceptible to F. gigantica infection, and that susceptibility to this infection was closely related to the cytokine environment associated with the Th2-type immune response. The MLNs showed a mixed Th1- and Th2-type immune response during the acute infection stages, after which the production of these cytokines returned to normal. Cytokine expression in the HLNs also expressed a mixed Th1- and Th2-type immune response during the early infection stages. When the infection became chronic, the typical Th2 immune response was induced in the HLNs. At the acute infection stages, the spleen exhibited a Th2 immune response. Nevertheless, cytokines associated with the Th1 and Th2 immune responses were upregulated at 98 DPI. In addition, the total IgG and IgG1 of the parasite-specific antibodies increased. This suggested that the Th2-related cytokines and IgG1 induced by F. gigantica infection might mediate successful F. gigantica infection in the natural host, swamp buffalo.

Expression profiles of genes involved in TLRs and NLRs signaling pathways of water buffaloes infected with Fasciola gigantica.

Infection of ruminants and humans with Fasciola gigantica is attracting increasing attention due to its economic impact and public health significance. However, little is known of innate immune responses during F. gigantica infection. Here, we investigated the expression profiles of genes involved in Toll-like receptors (TLRs) and NOD-like receptors (NLRs) signaling pathways in buffaloes infected with 500F. gigantica metacercariae. Serum, liver and peripheral blood mononuclear cell (PBMC) samples were collected from infected and control buffaloes at 3, 10, 28, and 70days post infection (dpi). Then, the levels of 12 cytokines in serum samples were evaluated by ELISA. Also, the levels of expression of 42 genes, related to TLRs and NLRs signaling, in liver and PBMCs were determined using custom RT2 Profiler PCR Arrays. At 3 dpi, modest activation of TLR4 and TLR8 and the adaptor protein (TICAM1) was detected. At 10 dpi, NF-κB1 and Interferon Regulatory Factor signaling pathways were upregulated along with activation of TLR1, TLR2, TLR6, TLR10, TRAF6, IRF3, TBK1, CASP1, CD80, and IFNA1 in the liver, and inflammatory response with activated TLR4, TLR9, TICAM1, NF-κB1, NLRP3, CD86, IL-1B, IL-6, and IL-8 in PBMCs. At 28 dpi, there was increase in the levels of cytokines along with induction of NLRP1 and NLRP3 inflammasomes-dependent immune responses in the liver and PBMCs. At 70 dpi, F. gigantica activated TLRs and NLRs, and their downstream interacting molecules. The activation of TLR7/9 signaling (perhaps due to increased B-cell maturation and activation) and upregulation of NLRP3 gene were also detected. These findings indicate that F. gigantica alters the expression of TLRs and NLRs genes to evade host immune defenses. Elucidation of the roles of the downstream effectors interacting with these genes may aid in the development of new interventions to control disease caused by F. gigantica infection.

Dynamic expression of cytokine and transcription factor genes during experimental Fasciola gigantica infection in buffaloes.

BACKGROUND: Determining the mechanisms involved in the immune-pathogenesis of the tropical liver fluke, Fasciola gigantica, is crucial to the development of any effective therapeutic intervention. Here, we examined the differential gene expression of cytokines and transcription factors in the liver of F. gigantica-infected buffaloes, over the course of infection. METHODS: Water buffaloes (swamp type) were infected orally with 500 F. gigantica encysted metacercariae. Liver tissue samples were collected 3, 10, 28, 42, 70 and 98 days post-infection (dpi). Levels of gene expression of nine cytokines (IFN-γ, TGF-ß, IL-1ß, IL-4, IL-6, IL-10, IL-12B, IL-13 and IL-17A) and four transcription factors (T-bet, GATA-3, Foxp3 and ROR-γτ) were determined using quantitative real-time PCR (qRT-PCR). We evaluated any correlation between gene expression of these immune-regulatory factors and the severity of liver pathology. RESULTS: Histopathological examination revealed that cellular infiltration, hemorrhage and fibrosis without calcification in the liver parenchyma of infected buffaloes, increased over the course of infection. This progressive pathology was attributed to dysregulated and excessive inflammatory responses induced by infection. The early infection phase (3-10 dpi) was marked by a generalized immunosuppression and elevated TGF-ß expression in order to facilitate parasite colonization. A mixed Th1/Th2 immune response was dominant from 28 to 70 dpi, to promote parasite survival while minimizing host tissue damage. During late infection (98 dpi), the response was biased towards Th1/Treg in order to inhibit the host's Th2 protective response and promote chronic infection. Both IL-10 and IL-17A and the Th17/Treg balance, played key roles in mediating the inflammatory and immunoregulatory mechanisms in the liver during chronic fasciolosis. CONCLUSIONS: Our data showed distinct CD4+ T helper (Th) polarization and cytokine dysregulation in response to F. gigantica infection in water buffaloes over the course of infection. Characterizing the temporal expression profiles for host immune genes during infection should provide important information for defining how F. gigantica adapts and survives in the liver of buffaloes and how host immune responses influence F. gigantica pathogenicity.

Retracted: siRNA Mediated Silencing of NIN1/RPN12 Binding Protein 1 Homolog Inhibits Proliferation and Growth of Breast Cancer Cells

Retraction: Retracted: siRNA mediated silencing of NIN1/RPN12 binding protein 1 homolog inhibits proliferation and growth of breast cancer cells Asian Pacific Journal of Cancer Prevention has retracted the article titled "siRNA mediated silencing of NIN1/RPN12 binding protein 1 homolog inhibits proliferation and growth of breast cancer cells"(1) for reason of similarity with a series of articles identified by Byrne and Labbé (2). 1. Huang WY1, Chen DH, Ning L, Wang LW. siRNA mediated silencing of NIN1/RPN12 binding protein 1 homolog inhibits proliferation and growth of breast cancer cells. Asian Pac J Cancer Prev. 2012;13(5):1823-7. 2. J. A. Byrne and C. Labbé, "Striking similarities between publications from China describing single gene knockdown experiments in human cancer cell lines," Scientometrics, vol. 110, no. 3, pp. 1471­1493, 2017. Authors did not respond to request for comment.

Serum levels of cytokines in water buffaloes experimentally infected with Fasciola gigantica.

Fasciola gigantica infection in water buffaloes causes significant economic losses especially in developing countries. Although modulation of the host immune response by cytokine neutralization or vaccination is a promising approach to control infection with this parasite, our understanding of cytokine's dynamic during F. gigantica infection is limited. To address this, we quantified the levels of serum cytokines produced in water buffaloes following experimental infection with F. gigantica. Five buffaloes were infected via oral gavage with 500 viable F. gigantica metacercariae and blood samples were collected from buffaloes one week before infection and for 13 consecutive weeks thereafter. The levels of 10 cytokines in serum samples were simultaneously determined using ELISA. F. gigantica failed to elicit the production of various pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), IL-2, IL-6, IL-12, and IFN-γ. On the other hand, evidence of a Th2 type response was detected, but only early in the course of parasite colonization and included modest increase in the levels of IL-10 and IL-13. The results also revealed suppression of the immune responses as a feature of chronic F. gigantica infection in buffaloes. Taken together, F. gigantica seems to elicit a modest Th2 response at early stage of infection in order to downregulate harmful Th1- and Th17-type inflammatory responses in experimentally infected buffaloes. The full extent of anti-F. gigantica immune response and its relation to pathogenesis requires further study.

Genomic comparison of virulent and non-virulent serotype V ST1 Streptococcus agalactiae in fish.

Streptococcus agalactiae or Group B Streptococcus (GBS) is the major pathogen causing pneumonia and meningitis in human, mastitis in dairy cows, and streptococcal disease in tilapia. Previous studies have shown that fish GBS strains are correlated with human GBS strains in evolution and might have cross-host infection ability. Although the invasive disease caused by ST1 GBS in non-pregnant adults and cows is increasing worldwide, infection of fish by ST1 GBS has not been reported. The aim of this study was to determine whether ST1 GBS was virulent in fish and to investigate the genomic characteristics of ST1 GBS strains with different pathogenicity in tilapia. The human-derived serotype V ST1 GBS strains NNA048 and NNA038 were used to intraperitoneally challenge Nile tilapia (Oreochromis niloticus) with doses of 1.0×109CFU/fish, 1.0×107CFU/fish, and 1.0×105CFU/fish, respectively. The cumulative mortality rates of NNA048 infection at three different doses were 100.00%, 83.33%, and 40.00%. In contrast, there were no any sick or dead fish in NNA038 infection group. Histopathological results indicated that challenge of tilapia with NNA048 caused different degree of degeneration and necrosis in brain, liver, spleen, head kidney, and gut, and a large number of blue-stained Streptococcus granules were observed in the tissues. In contrast, there were no any lesions in the tissues of tilapia that were challenged with NNA038. Genome comparison showed that the major genome differences between NNA048 and NNA038 were attributed to the different phage sequences, and there was a 49.8kb length, intact phage sequence encoding 68 proteins in NNA048 genome. SNV and Indels analysis between NNA038 and NNA048 genomes indicated that there were a total of 96 SNVs, 5 deletions and 1 insert. Taken together, serotype V ST1 GBS was comprised of virulent and nonvirulent strains to tilapia, and gene rearrangement might be the main reason of causing different levels of virulence between strains.

De novo transcriptome sequencing and analysis of the juvenile and adult stages of Fasciola gigantica.

Fasciola gigantica is regarded as the major liver fluke causing fasciolosis in livestock in tropical countries. Despite the significant economic and public health impacts of F. gigantica there are few studies on the pathogenesis of this parasite and our understanding is further limited by the lack of genome and transcriptome information. In this study, de novo Illumina RNA sequencing (RNA-seq) was performed to obtain a comprehensive transcriptome profile of the juvenile (42days post infection) and adult stages of F. gigantica. A total of 49,720 unigenes were produced from juvenile and adult stages of F. gigantica, with an average length of 1286 nucleotides (nt) and N50 of 2076nt. A total of 27,862 (56.03%) unigenes were annotated by BLAST similarity searches against the NCBI non-redundant protein database. Because F. gigantica needs to feed and/or digest host tissues, some proteases (including cysteine proteases and aspartic proteases), which play a role in the degradation of host tissues (protein), have been paid more attention in the present study. A total of 6511 distinct genes were found differentially expressed between juveniles and adults, of which 3993 genes were up-regulated and 2518 genes were down-regulated in adults versus juveniles, respectively. Moreover, stage-specific differentially expressed genes were identified in juvenile (17,009) and adult (6517) F. gigantica. The significantly divergent pathways of differentially expressed genes included cAMP signaling pathway (226; 4.12%), proteoglycans in cancer (256; 4.67%) and focal adhesion (199; 3.63%). The transcription pattern also revealed two egg-laying-associated pathways: cGMP-PKG signaling pathway and TGF-ß signaling pathway. This study provides the first comparative transcriptomic data concerning juvenile and adult stages of F. gigantica that will be of great value for future research efforts into understanding parasite pathogenesis and developing vaccines against this important parasite.