Principle and application of self-transcribing active regulatory region sequencing in enhancer discovery research.

Self-transcribing active regulatory region sequencing (STARR-seq) is a high-throughput sequencing method capable of simultaneously discovering and validating all enhancers within the genome. In this method, candidate sequences are inserted into plasmid vectors and electroporated into cells. Acting as both enhancers and target genes, the self-transcription of these sequences will also be enhanced by themselves. By sequencing the transcriptome and comparing the results with the non-inserted control, the locations and activity of enhancers can be determined. In traditional enhancer discovery strategies, the chromatin open regions and transcription active regions were sequenced and predicted as enhancers. However, the activity of these putative enhancers could only be validated one by one without a high-throughput method. STARR-seq solved this limitation, allowing simultaneous enhancers discovery and activity validation in a high-throughput manner. Since the introduction of STARR-seq, it has been widely used to discover enhancers and validate enhancer activity in a number of organisms and cells. In this review, we present the traditional enhancer prediction methods and the basic principles, development history, specific applications of STARR-seq, and its future prospects, aiming to provide a reference for researchers in related fields conducting enhancer studies.

Total flavonoids of litchi Seed alleviates schistosomiasis liver fibrosis in mice by suppressing hepatic stellate cells activation and modulating the gut microbiomes.

Infection with Schistosoma japonicum (S. japonicum) is an important zoonotic parasitic disease that causes liver fibrosis in both human and domestic animals. The activation of hepatic stellate cells (HSCs) is a crucial phase in the development of liver fibrosis, and inhibiting their activation can alleviate this progression. Total flavonoids of litchi seed (TFL) is a naturally extracted drug, and modern pharmacological studies have shown its anti-fibrotic and liver-protective effects. However, the role of TFL in schistosomiasis liver fibrosis is still unclear. This study investigated the therapeutic effects of TFL on liver fibrosis in S. japonicum infected mice and explored its potential mechanisms. Animal study results showed that TFL significantly reduced the levels of Interleukin-1ß (IL-1ß), Tumor Necrosis Factor-α (TNF-α), Interleukin-4 (IL-4), and Interleukin-6 (IL-6) in the serum of S. japonicum infected mice. TFL reduced the spleen index of mice and markedly improved the pathological changes in liver tissues induced by S. japonicum infection, decreasing the expression of alpha-smooth muscle actin (α-SMA), Collagen I and Collagen III protein in liver tissues. In vitro studies indicated that TFL also inhibited the activation of HCSs induced by Transforming Growth Factor-ß1 (TGF-ß1) and reduced the levels of α-SMA. Gut microbes metagenomics study revealed that the composition, abundance, and functions of the mice gut microbiomes changed significantly after S. japonicum infection, and TLF treatment reversed these changes. Therefore, our study indicated that TFL alleviated granulomatous lesions and improved S. japonicum induced liver fibrosis in mice by inhibiting the activation of HSCs and by improving the gut microbiomes.

Clonorchis sinensis infection amplifies hepatocellular carcinoma stemness, predicting unfavorable prognosis.

BACKGROUND: Extensive evidence links Clonorchis sinensis (C. sinensis) to cholangiocarcinoma; however, its association with hepatocellular carcinoma (HCC) is less acknowledged, and the underlying mechanism remains unclear. This study was designed to investigate the association between C. sinensis infection and HCC and reveal the relationship between C. sinensis infection and cancer stemness. METHODS: A comprehensive analysis of 839 HCC patients categorized into C. sinensis (-) HCC and C. sinensis (+) HCC groups was conducted. Chi-square and Mann-Whitney U tests were used to assess the association between C. sinensis infection and clinical factors. Kaplan-Meier and Cox regression analyses were used to evaluate survival outcomes. Immunohistochemistry was used to determine CK19 and EpCAM expression in HCC specimens. RESULTS: Compared to C. sinensis (-) HCC patients, C. sinensis (+) HCC patients exhibited advanced Barcelona Clinic Liver Cancer (BCLC) stage, higher male prevalence and more liver cirrhosis as well as elevated alpha-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), eosinophil, complement 3 (C3), and complement 4 (C4) values. C. sinensis infection correlated with shorter overall survival (OS) (p < 0.05) and recurrence-free survival (RFS) (p < 0.05). Furthermore, Cox multivariate analysis revealed that C. sinensis infection was an independent prognostic factor for OS in HCC patients. Importantly, C. sinensis infection upregulated the expression of HCC cancer stem cell markers CK19 and EpCAM. CONCLUSION: HCC patients with C. sinensis infection exhibit a poor prognosis following hepatectomy. Moreover, C. sinensis infection promotes the acquisition of cancer stem cell-like characteristics, consequently accelerating the malignant progression of HCC. AUTHOR SUMMARY: Clonorchis sinensis (C. sinensis) is a prominent food-borne parasite prevalent in regions such as China, particularly in Guangxi. C. sinensis has been associated with various hepatobiliary system injuries, encompassing inflammation, periductal fibrosis, cholangiocarcinoma and even hepatocellular carcinoma (HCC). A substantial body of evidence links C. sinensis to cholangiocarcinoma, However, the connection between C. sinensis and HCC and the intricate mechanisms underlying its contribution to HCC development remain incompletely elucidated. Our study demonstrates clear clinicopathological associations between C. sinensis and HCC, such as gender, BCLC stage, liver cirrhosis, MVI, AFP, CA19-9, circulating eosinophils and complements. Furthermore, we found that the co-occurrence of C. sinensis exhibited a significant association with shorter OS and RFS in patients diagnosed with HCC. A major finding was that C. sinensis infection promotes the acquisition of cancer stem cell-like characteristics, consequently accelerating the malignant progression of HCC. Our results provide a more comprehensive comprehension of the interplay between C. sinensis and HCC, shedding fresh light on the carcinogenic potential of C. sinensis.

Multi-omics approaches reveal the molecular mechanisms underlying the interaction between Clonorchis sinensis and mouse liver.

Introduction: Clonorchiasis remains a serious global public health problem, causing various hepatobiliary diseases. However, there is still a lack of overall understanding regarding the molecular events triggered by Clonorchis sinensis (C. sinensis) in the liver. Methods: BALB/c mouse models infected with C. sinensis for 5, 10, 15, and 20 weeks were constructed. Liver pathology staining and observation were conducted to evaluate histopathology. The levels of biochemical enzymes, blood routine indices, and cytokines in the blood were determined. Furthermore, alterations in the transcriptome, proteome, and metabolome of mouse livers infected for 5 weeks were analyzed using multi-omics techniques. Results: The results of this study indicated that adult C. sinensis can cause hepatosplenomegaly and liver damage, with the most severe symptoms observed at 5 weeks post-infection. However, as the infection persisted, the Th2 immune response increased and symptoms were relieved. Multi-omics analysis of liver infected for 5 weeks identified 191, 402 and 232 differentially expressed genes (DEGs), proteins (DEPs) and metabolites (DEMs), respectively. Both DEGs and DEPs were significantly enriched in liver fibrosis-related pathways such as ECM-receptor interaction and cell adhesion molecules. Key molecules associated with liver fibrosis and inflammation (Cd34, Epcam, S100a6, Fhl2, Itgax, and Retnlg) were up-regulated at both the gene and protein levels. The top three metabolic pathways, namely purine metabolism, arachidonic acid metabolism, and ABC transporters, were associated with liver cirrhosis, fibrosis, and cholestasis, respectively. Furthermore, metabolites that can promote liver inflammation and fibrosis, such as LysoPC(P-16:0/0:0), 20-COOH-leukotriene E4, and 14,15-DiHETrE, were significantly up-regulated. Conclusion: Our study revealed that the most severe symptoms in mice infected with C. sinensis occurred at 5 weeks post-infection. Moreover, multi-omics analysis uncovered predominant molecular events related to fibrosis changes in the liver. This study not only enhances our understanding of clonorchiasis progression but also provides valuable insights into the molecular-level interaction mechanism between C. sinensis and its host liver.

Magnetic resonance imaging and next-generation sequencing for the diagnosis of cystic echinococcosis in the intradural spine: a case report.

BACKGROUND: Cystic echinococcosis (CE) is a parasitic zoonotic disease caused by the larval stage of Echinococcus granulosus. The liver and lungs are the most common sites for infection. Infection of the intradural spine is rare. CASE PRESENTATION: A 45-year-old woman of Han ethnicity presented with a chronic history of recurrent lumbar pain. Magnetic resonance imaging of the lumbar spine revealed the classical characteristic of multiple cystic lesions of variable sizes, manifesting a "bunch of grapes" appearance, localized within the spinal canal at the L4-L5 vertebral level. In the meanwhile, metagenomic next-generation sequencing identified Echinococcosis granulosa. The patient underwent surgery to remove the cyst entirely and subsequently took albendazole 400 mg orally twice daily for 6 months. CONCLUSION: Spinal CE should be suspected in patients with multiple spinal cystic lesions and zoonotic exposure. metagenomic next-generation sequencing serves as a robust diagnostic tool for atypical pathogens, particularly when conventional tests are inconclusive. Prompt and aggressive treatment for spinal cystic echinococcosis is imperative, and further research is warranted for improved diagnostic and therapeutic strategies.

Multilayer omics reveals the molecular mechanism of early infection of Clonorchis sinensis juvenile.

BACKGROUND: Clonorchiasis remains a non-negligible global zoonosis, causing serious socioeconomic burdens in endemic areas. Clonorchis sinensis infection typically elicits Th1/Th2 mixed immune responses during the course of biliary injury and periductal fibrosis. However, the molecular mechanism by which C. sinensis juvenile initially infects the host remains poorly understood. METHODS: The BALB/c mouse model was established to study early infection (within 7 days) with C. sinensis juveniles. Liver pathology staining and observation as well as determination of biochemical enzymes, blood routine and cytokines in blood were conducted. Furthermore, analysis of liver transcriptome, proteome and metabolome changes was performed using multi-omics techniques. Statistical analyses were performed using Student's t-test. RESULTS: Histopathological analysis revealed that liver injury, characterized by collagen deposition and inflammatory cell infiltration, occurred as early as 24 h of infection. Blood indicators including ALT, AST, WBC, CRP and IL-6 indicated that both liver injury and systemic inflammation worsened as the infection progressed. Proteomic data showed that apoptosis and junction-related pathways were enriched within 3 days of infection, indicating the occurrence of liver injury. Furthermore, proteomic and transcriptomic analysis jointly verified that the detoxification and antioxidant defense system was activated by enrichment of glutathione metabolism and cytochrome P450-related pathways in response to acute liver injury. Proteomic-based GO analysis demonstrated that biological processes such as cell deformation, proliferation, migration and wound healing occurred in the liver during the early infection. Correspondingly, transcriptomic results showed significant enrichment of cell cycle pathway on day 3 and 7. In addition, the KEGG analysis of multi-omics data demonstrated that numerous pathways related to immunity, inflammation, tumorigenesis and metabolism were enriched in the liver. Besides, metabolomic screening identified several metabolites that could promote inflammation and hepatobiliary periductal fibrosis, such as CA7S. CONCLUSIONS: This study revealed that acute inflammatory injury was rapidly triggered by initial infection by C. sinensis juveniles in the host, accompanied by the enrichment of detoxification, inflammation, fibrosis, tumor and metabolism-related pathways in the liver, which provides a new perspective for the early intervention and therapy of clonorchiasis.

Activation of primary hepatic stellate cells and liver fibrosis induced by targeting TGF-ß1/Smad signaling in schistosomiasis in mice.

BACKGROUND: In mice, liver fibrosis is the most serious pathologic change during Schistosoma japonicum (S. japonicum) infection. Schistosomiasis is mainly characterized by schistosome egg-induced granulomatous fibrosis. Hepatic stellate cells (HSCs) are mainly responsible for the net accumulation of collagens and fibrosis formation in the liver. Activated HSCs regulated by transforming growth factor-ß1 (TGF-ß1)/Smad signaling have emerged as the critical regulatory pathway in hepatitis virus or carbon tetrachloride-induced liver fibrosis. However, the detailed mechanism of HSC activation in schistosome-induced liver fibrosis is poorly understood. METHODS: Schistosoma japonicum-induced murine models and a control group were generated by abdominal infection with 15 (± 1) cercariae. The purity of cultured primary HSCs was evaluated by immunocytochemistry. The histopathological changes in the livers of infected mice were estimated by hematoxylin-eosin and Masson staining. Dynamic expression of pro-fibrotic molecules and microRNAs was detected by real-time quantitative PCR (RT-qPCR). Mainly members involved in the TGF-ß1/Smad signaling pathway were examined via RT-qPCR and Western blot. RESULTS: The egg-induced granulomatous inflammation formed at 4 weeks post-infection (wpi) and developed progressively. Alpha-smooth muscle actin (α-SMA), collagen I, collagen III, TGF-ß1, Smad2, Smad3, and Smad4 showed a significant increase in mitochondrial RNA (mRNA) and protein expression compared with the control group at 7 and 9 weeks post-infection (wpi), while an opposite effect on Smad7 was observed. In addition, the mRNA expression of microRNA-21 (miRNA-21) was significantly increased at 7 wpi, and the mRNA expression of miRNA-454 was decreased starting from 4 wpi. CONCLUSION: Our present findings revealed that HSCs regulated by the TGF-ß1/Smad signaling pathway play an important role in liver fibrosis in S. japonicum-infected mice, which may provide proof of concept for liver fibrosis in schistosomiasis.

Cysteine protease of Clonorchis sinensis alleviates DSS-induced colitis in mice.

BACKGROUND: Currently, inflammatory bowel disease (IBD) has become a global chronic idiopathic disease with ever-rising morbidity and prevalence. Accumulating evidence supports the IBD-hygiene hypothesis that helminths and their derivatives have potential therapeutic value for IBD. Clonorchis sinensis (C. sinensis) mainly elicit Th2/Treg-dominated immune responses to maintain long-term parasitism in the host. This study aimed to evaluate the therapeutic effects of cysteine protease (CsCP) and adult crude antigen (CsCA) of C. sinensis, and C. sinensis (Cs) infection on DSS-induced colitis mice. METHODS: BALB/c mice were given 5% DSS daily for 7 days to induce colitis. During this period, mice were treated with rCsCP, CsCA or dexamethasone (DXM) every day, or Cs infection which was established in advance. Changes in body weight, disease activity index (DAI), colon lengths, macroscopic scores, histopathological findings, myeloperoxidase (MPO) activity levels, regulatory T cell (Treg) subset levels, colon gene expression levels, serum cytokine levels, and biochemical indexes were measured. RESULTS: Compared with Cs infection, rCsCP and CsCA alleviated the disease activity of acute colitis more significant without causing abnormal blood biochemical indexes. In comparison, rCsCP was superior to CsCA in attenuating colonic pathological symptoms, enhancing the proportion of Treg cells in spleens and mesenteric lymph nodes, and improving the secretion of inflammatory-related cytokines (e.g., IL-2, IL-4, IL-10 and IL-13) in serum. Combined with RNA-seq data, it was revealed that CsCA might up-regulate the genes related to C-type lectin receptor and intestinal mucosal repair related signal pathways (e.g., Cd209d, F13a1 and Cckbr) to reduce colon inflammation and benefit intestinal mucosal repair. Dissimilarly, rCsCP ameliorated colitis mainly through stimulating innate immunity, such as Toll like receptor (TLR) signaling pathway, down-regulating the expression of inflammatory cytokines (e.g., IL-12b, IL-23r and IL-7), thereby restraining the differentiation of Th1/Th17 cells. CONCLUSIONS: Both rCsCP and CsCA showed good therapeutic effects on the treatment of acute colitis, but rCsCP is a better choice. rCsCP is a safe, effective, readily available and promising therapeutic agent against IBD mainly by activating innate immunity and regulating the IL-12/IL-23r axis.

MiR-130a-3p Alleviates Liver Fibrosis by Suppressing HSCs Activation and Skewing Macrophage to Ly6Clo Phenotype.

Emerging evidences have highlighted the crucial role of microRNAs (miRNAs) in the liver cirrhosis, but the relationship between miR-130a-3p and liver cirrhosis is not entirely clear. As we all know, schistosomiasis, as one of the zoonoses, can lead to liver cirrhosis when it advances. In this study, we investigated the biological functions of miR-130a-3p on the liver fibrosis of schistosomiasis in vivo and in vitro. The mice infected with Schistosoma japonicum (S. japonicum) were treated with lentivirus vector (LV)-miR-130a-3p by hydrodynamic injection through the tail vein. Our findings showed significantly decreased expression of miR-130a-3p both in the serum of patients with cirrhosis and in the liver of mice infected with S. japonicum. The results showed that LV-miR-130a-3p could effectively enter into the liver and alleviate liver granulomatous inflammation and collagen deposition. Simultaneously, LV-miR-130a-3p-promoted macrophages presented the Ly6Clo phenotype, concomitant with the decreased expression of the tissue inhibitor of metalloproteinases (TIMP) 1, and increased the expression of matrix metalloproteinase (MMP) 2, which contributed to the dissolution of collagen. Furthermore, overexpression of miR-130a-3p not only inhibited the activation and proliferation of hepatic stellate cells (HSCs) but also induced the apoptosis of HSCs. In addition, we also confirmed that miR-130a-3p enables to bind with mitogen-activated protein kinase (MAPK) 1 and transforming growth factor-beta receptors (TGFBR) 1 and TGFBR2 genes and inhibit the expressions of these genes. Our findings suggested that miR-130a-3p might represent as the potential candidate biomarker and therapeutic target for the prognosis identification and treatment of schistosomiasis liver fibrosis.

Seasonal variations and public search interests in Toxoplasma: a 16-year retrospective analysis of big data on Google Trends.

BACKGROUND: This study aims to understand whether there is a seasonal change in the internet search interest for Toxoplasma by using the data derived from Google Trends (GT). METHODS: The present study searched for the relative search volume (RSV) for the search term 'Toxoplasma' in GT within six major English-speaking countries (Australia, New Zealand [Southern Hemisphere] and Canada, Ireland, the UK and the USA [Northern Hemisphere] from 1 January 2004 to 31 December 2019, utilizing the category of 'health'. Data regarding the RSV of Toxoplasma was obtained and further statistical analysis was performed in R software using the 'season' package. RESULTS: There were significantly seasonal patterns for the RSV of the search term 'Toxoplasma' in five countries (all p<0.05), except for the UK. A peak in December-March and a trough in July-September (Canada, Ireland, the UK and the USA) were observed, while a peak in June/August and a trough in December/February (Australia, New Zealand) were also found. Moreover, the presence of seasonal patterns regarding RSV for 'Toxoplasma' between the Southern and Northern Hemispheres was also found (both p<0.05), with a reversed meteorological month. CONCLUSIONS: Overall, our study revealed the seasonal variation for Toxoplasma in using internet search data from GT, providing additional evidence on seasonal patterns in Toxoplasma.

Multiplex PCR for sexing Schistosoma japonicum cercariae and its utility.

Accurate discrimination of the Schistosoma japonicum cercariae gender is very important for establishing monosexual infection animal models and for standardizing the real intensity of infection. In this study, a multiplex PCR technique consisting of two pairs of primers, of which one amplifies a 185-bp band specific for the W chromosome and the other amplifies a 420-bp band for the Z chromosome, was established to sex the S. japonicum cercariae. For male cercariae (ZZ), a single 420-bp band is expected, and for female cercariea (ZW), two distinct 185-bp and 420-bp bands can be observed. There was no cross-reaction with S. mansoni, S. haematobium, Clonorchis sinensis, Paragonimus westermani, and Trichinella spiralis. After sexing the cercariae escaped from a single snail, mice in group A were infected with 60 male cercariae and mice of group B were infected with 40 female cercariae. Meanwhile, mice in group C were infected with 10 male and 10 female cercariae that were sexed by multiplex PCR. At 45 days postinfection, male and female adult worms were recovered to verify the accuracy of multiplex PCR for sexing S. japonicum cercariae and to calculate the male and female survival rate and paired worm ratio. Our results showed that the multiplex PCR technique could distinguish male cercariae with 100% accuracy. However, sometimes the discrimination results of multiplex PCR mis-scored mixed sexual cercariae as female cercariae. The mean male adult worm burden in mice of group C was 10.7 ± 2.4, and the mean female adult worm burden was 7.7 ± 2.5. There was a significant difference between the male worm burden and female worm burden in group C. The P value was 0.013. The real paired worm ratio of group C was 74.2% (95%CI 56.6~91.8%). These results demonstrated a male-biased sex ratio in the mice model with equilibrated sex ratio cercariae infection, as predicted by our multiplex PCR technique. In conclusion, our multiplex PCR technique is an effective tool for sexing S. japonicum cercariae, especially for distinguishing male cercariae, which is of great value for establishing monosexual cercariae infection mice models to harvest male adult worms for anti-schistosomal drug screening.

Interleukin-9 blockage reduces early hepatic granuloma formation and fibrosis during Schistosoma japonicum infection in mice.

Hepatic fibrosis induced by schistosomes is regulated by a complex network of cytokines. T helper type 9 (Th9) cells are a new type of effector T helper cells, which mainly secrete the specific cytokine interleukin-9 (IL-9). Interleukin-9 has been shown to contribute to liver fibrosis in patients with chronic hepatitis B and in a mouse model due to carbon tetrachloride. However, the role of IL-9 in schistosomiasis fibrosis remains unknown. In this study, we investigated the roles of IL-9 in schistosomiasis through in vivo and in vitro studies. The in vivo studies found that neutralization of IL-9 reduced liver granulomatous inflammation and collagen deposition around parasite eggs. The in vitro studies found that the treatment of primary hepatic stellate cells with IL-9 induced a significant increase of collagen and α-smooth-muscle actin. Moreover, we also described the dynamics and relevance of IL-9 and IL-4 in mice infected with Schistosoma japonicum. We found that IL-9 might appear more quickly and at higher levels than IL-4. Hence, our findings indicated that IL-9 might play a role in regulating hepatic fibrosis in early-stage schistosomiasis and become a promising approach for regulating hepatic fibrosis caused by S. japonicum.

In vitro and in vivo activities of DW-3-15, a commercial praziquantel derivative, against Schistosoma japonicum.

BACKGROUND: Schistosomiasis is a debilitating neglected tropical disease that affects approximately 190 million people around the world. Praziquantel (PZQ) is the only drug available for use against all Schistosoma species. Although PZQ has a high efficacy, recognized concerns have prompted the development of new, alternative drugs for repeated use in endemic areas where PZQ efficacy against strains of Schistosoma is reduced. A hybrid drug containing different pharmacophores within a single molecule is a promising strategy. Our earlier in vivo studies showed the significant antiparasitic activity of a praziquantel derivative, DW-3-15, against Schistosoma japonicum. In the present study, DW-3-15 was synthesized in large amounts by a pharmaceutical company and its schistosomicidal efficacy and stability were further confirmed. Parameters such as parasite viability, pairing and oviposition were evaluated in vitro. An in vivo study was conducted to assess the effect of commercial DW-3-15 on worm burden, egg production and diameter of granulomas. Additionally, to gain insight into the mechanism of action for DW-3-15, morphological changes in the tegument of S. japonicum were also examined. RESULTS: The in vitro study showed the antiparasitic activity of DW-3-15 against S. japonicum, with significant reductions in viability of adult and juvenile worms, worm pairings and egg output. Compared to PZQ, DW-3-15 induced similar ultrastructural changes and evident destruction of the tegument surface in male worms. In vivo, the oral administration of DW-3-15 at a dose of 400 mg/kg per day for five consecutive days in mice significantly reduced the total worm burden and number of eggs in the liver. Histological analysis of the livers showed a marked reduction in the average diameter of the egg granuloma. CONCLUSIONS: Our findings suggest that DW-3-15, a PZQ derivative with the prospect of commercial production, can be developed as a potential promising schistosomicide.

Characterization of a non-sexual population of Strongyloides stercoralis with hybrid 18S rDNA haplotypes in Guangxi, Southern China.

Strongyloidiasis is a much-neglected but sometimes fatal soil born helminthiasis. The causing agent, the small intestinal parasitic nematode Strongyloides stercoralis can reproduce sexually through the indirect/heterogonic life cycle, or asexually through the auto-infective or the direct/homogonic life cycles. Usually, among the progeny of the parasitic females both, parthenogenetic parasitic (females only) and sexual free-living (females and males) individuals, are present simultaneously. We isolated S. stercoralis from people living in a village with a high incidence of parasitic helminths, in particular liver flukes (Clonorchis sinensis) and hookworms, in the southern Chinese province Guangxi. We determined nuclear and mitochondrial DNA sequences of individual S. stercoralis isolated from this village and from close by hospitals and we compared these S. stercoralis among themselves and with selected published S. stercoralis from other geographic locations. For comparison, we also analyzed the hookworms present in the same location. We found that, compared to earlier studies of S. stercoralis populations in South East Asia, all S. stercoralis sampled in our study area were very closely related, suggesting a recent common source of infection for all patients. In contrast, the hookworms from the same location, while all belonging to the species Necator americanus, showed rather extensive genetic diversity even within host individuals. Different from earlier studies conducted in other geographic locations, almost all S. stercoralis in this study appeared heterozygous for different sequence variants of the 18S rDNA hypervariable regions (HVR) I and IV. In contrast to earlier investigations, except for three males, all S. stercoralis we isolated in this study were infective larvae, suggesting that the sampled population reproduces predominantly, if not exclusively through the clonal life cycles. Consistently, whole genome sequencing of individual worms revealed higher heterozygosity than reported earlier for likely sexual populations of S. stercoralis. Elevated heterozygosity is frequently associated with asexual clonal reproduction.

Dynamics of Th9 cells and their potential role in immunopathogenesis of murine schistosomiasis.

BACKGROUND: Th1, Th2, Th17, Treg and Tfh cells play important roles in schistosomiasis. Th9 cells secrete IL-9 as a signature cytokine and contribute to several classes of inflammatory disease. However, the effects of Th9 cells in schistosomiasis are unknown. We aimed to explore the dynamic changes and potential roles of Th9 cells in the pathogenesis of hepatic egg granulomatous inflammation in mice infected with Schistosoma japonicum. METHODS: Twenty mice with S. japonicum infection and five normal controls (NC) were used as models. The average areas of egg granulomas were estimated by hematoxylin-eosin (H & E) staining. Hepatic IL-9 and transcription factor PU.1 levels were detected by immunohistochemistry. Flow cytometry techniques were used to analyze the proportions of Th9 cells. With the help of ELISA, serum levels of IL-9 were examined. RESULTS: The egg granulomas began to form from four weeks after infection and continued to develop. In parallel with the development of egg granulomas, the hepatic levels of IL-9 and PU.1 increased very slowly during the first four weeks post-infection and increased rapidly thereafter. Moreover, the proportions of splenic Th9 cells and levels of serum IL-9 had similar developmental trends with the egg granulomas. CONCLUSION: The proliferation of Th9 cells and levels of IL-9 were significantly higher in S. japonicum-infected mice compared to NC. In addition, dynamic changes of Th9 and IL-9 were synchronous with the developmental trend of hepatic egg granulomatous inflammation, suggesting that Th9 cells might be a new subset in the pathogenesis of schistosomiasis.

A novel genotype screening and phylogenetic analysis of Blastocystis hominis based on EF-1α.

Blastocystis hominis (B. h) is a kind of intestinal parasitic protozoa with the characteristic of worldwide distribution, morphology diversity, and diarrhea induced, etc. The traditional morphological classify was difficult to distinguish the genetic difference of B. h in different population and different geological strains. Recently, based on the small subunit ribosomal DNA sequence of B. h, the sequenced-tagged site (STS) primers was design, and successfully and widely applied to the distinguish the genotype of B. h, and however several B. h strains did not distinguish. To address it, the elongation factor-1 alpha (EF-1α) gene of B. h was screened due to its conservation here, and its specific primers were designed to distinguish the genotype of B. h. After epidemiological survey, the infection rate of B. h in boys was 14.74%, and that of girls was 15.05%, and the total infection rate of B. h was 14.93%. In total of 53 infection students, with the using of 7 pairs STS primers, 31 strains was validated by polymerase chain reaction (PCR), including 4 strains of Type 1, 17 strains of Type 3, 4 strains of Type 4, 1 strains of Type 6, and 5 strains of Type 7, and did not found the Type 2, Type 5 and mixture genotype. In the 23 unknown genotype strains of B. h, 15 strains were identified by PCR using EF-1α primers, and had a higher homology in the DNA sequence (70%), and was evolutionarily closer to the EF-1α sequence of S and H strains of B. h. This study indicated that STS primers could identify the genotype of B. h, and EF-1α primers as a novel diagnosis primers could auxiliary identify the unknown genotype strain of B. h, and exhibited a wide application on the identification of the genotype strain of B. h, and provided a significant reference on the study of B. h in clinic.

Comparative analysis of intestinal parasitic infections of outpatients in Guangxi medical university affiliated hospital in 2005 and 2013.

OBJECTIVE: To understand the hospital's status and trends of intestinal parasitic infections and to provide a reference for prevention. METHODS: Stool samples were treated by acid-ether centrifugation; iodine staining and direct-smearing were performed; intestinal parasites were examined under a microscope; characteristics of parasitic infections in population were analyzed using the descriptive epidemiological method. RESULTS: 10 kinds of parasites were detected; the infection rate of clonorchissinensis was the highest, followed by B. hominis, hookworm, whipworm and roundworm in order (x(2) = 131.188, 1261.928, 129.386, P < 0.01); The overall infection rates in 2013 and 2005 were 37.08% and 41.07% respectively, and the infection rate in 2013 was lower than that in 2005 (x(2) = 20.5003, P < 0.01); All the infection rates of clonorchissinensis, hookworm, whipworm and roundworm in 2013 were lower than those in 2005 (x(2) = 18.275, 45.449, 34.855, 12.435, P < 0.01); Both in 2005 and 2013, the male infection rate was higher than that in female (x(2) = 12.859, 24.924, P < 0.01); For male, the infection rate of clonorchissinensis was the highest, followed by B. hominis (x(2) = 313.621, 104.409, P < 0.01); for female, the infection rate of B. hominis was the highest, followed by clonorchissinensis (x(2) = 95.293, 43.357, P < 0.01). For male, the age group of 41~ had the highest infection rate of clonorchissinensis in 2005 (x(2) = 5.734, P < 0.05), and the age groups of 31~ and 41~ had the highest infection rate of clonorchissinensis in 2013 (x(2) = 8.908, P < 0.01); for female, both in 2005 and 2013, the age group of 21~, 31~, 41~ and 51~ had the highest infection rate of clonorchissinensis (x(2) = 6.508, 5.145, P < 0.05). There was no difference in male infection rate of B. hominis in 2005 (x(2) = 10.134, P > 0.05); in 2013, the age group of 0~ had the highest infection rate (x(2) = 3.825, P < 0.05); for women, it was the highest in the age groups of 11~, 21~ and 31~ in 2005 (x(2) = 10.459, P < 0.01), 0~ and 11~ in 2013 (x(2) = 53.669, P < 0.01). For Hookworm infection in male, the highest infection rate was found in the age group of 11~ 21~ and 61~ in 2005 (x(2) = 4.547, P < 0.05), 61~ and ≥ 71~ in 2013 (x(2) = 4.843, P < 0.05); for female, the highest infection rate was found in the age groups of 51~ and 61~ both in 2005 and 2013 (x(2) = 5.709, 5.958, P < 0.05). CONCLUSION: In Nanning city, although there was a decline in the infection rate of intestinal parasites of attenders compared with 8 years ago, the infection rate was still high and intestinal parasites were various; The infection rate of geohelminthes had been reduced to a low level; Clonorchissinensis and B. hominis were still the insect species with the highest infection rate.

Cloning, expression, and partial characterization of FBPA from Schistosoma japonicum, a molecule on that the fluke may develop nutrition competition and immune evasion from human.

Carbohydrate metabolism is the most important physiological process for Schistosoma japonicum which resides in host. However, as a key glycolytic enzyme in carbohydrate metabolism, fructose-1,6-bisphosphate aldolase (FBPA), there is no study on its enzymatic kinetics and antigenic peptides. Here, we report the gene cloning, expression, purification, and kinetics of the FBPA from S. japonicum (sjFBPA). After cloning, sjFBPA gene was introduced into pET-28a and transformed BL21, and a soluble His6-sjFBPA was expressed and purified successfully at the expected molecular mass of ~45 kDa. We first reported that the diversities in IGS regions and the features of residues position 346 and 357-362 of sjFBPA may be conferred either through conformational changes influencing easily the active site from a distance and/or causing the C-terminal region to interact directly with the active site, which lead His6-sjFBPA to exhibit a higher specific activity of 197.43 units/mg and degrades FBP with a typical substrate inhibition model and a higher efficiency of k cat = 6261.3/s and K m = 0.061 µM than human aldolases, which might be the strategy that S. japonicum gaining energy and surviving in its environment with low concentration of carbohydrate, and benefitting to get more metabolic substances for parasites in nutrition competition with their host. sjFBPA exhibits a high similarity of 81.46 % with that of hosts, especially in antigenic peptide regions, and 14 of 15 antigenic peptides of sjFBPA were conserved to those of human aldolase A, B, and/or C with high identity (17, 16, or 16 antigenic peptides, respectively), which may result in a molecular mimicry of FBPA with that of host, and an immune evasion from their hosts. This work would supply an experimental base for using FBPA to prevent the schistosomiasis in the future.

[PCR-based genotype classification of Blastocystis hominis isolates from college students of Guangxi].

Fifty-three Blastocystis hominis isolates were separated from the fecal specimens of carriers in college students from Guangxi and cultivated in vitro, and the genetic DNA was extracted. All the isolates were genotyped by PCR using seven pairs of known sequence-tagged site (STS) primers. The results showed there were five subtypes in the 53 isolates. Subtype 3 was the most popular one (32.1%, 17/53), followed by subtype 7 (9.4%, 5/53). Subtypes 1 (7.6%, 4/53), 4 (7.6%, 4/53), and 6 (1.9%, 1/53) were detected, while subtypes 2 and 5 were not detected. The genotypes of the other 22 isolates were unknown which were negative to all the STS primers.

[Genotype analysis and isoenzyme patterns of ten isolates of Blastocystis hominis from Guangxi].

OBJECTIVE: To analyze genotypes and lactate dehydrogenase (LDH) and esterase (EST) patterns in 10 isolates of Blastocystis hominis collected from Guangxi. METHODS: Ten B. hominis isolates (BhGX1-BhGX10) were obtained from the fecal specimens of patients and cultivated in vitro, and then the genomic DNA was extracted. The isolates were genotyped by PCR using seven pairs of known sequenced-tagged site (STS) primers. Isoenzyme patterns of LDH and EST were investigated by SDS-PAGE. RESULTS: Out of the 10 isolates, 8 were identified as genotype I and the genotypes of the other two (BhGX4 and BhGX7) were unknown which were negative to all the STS primers. Among the ten isolates, 10 LDH bands were found, more with Rm37, Rm49, Rm57, Rm68 and Rm92. 12 bands showed in EST patterns: Rm14, Rm18, Rm23, Rm27, Rm35, Rm41, Rm45, Rm50, Rm55, Rm68, Rm77 and Rm82. Difference existed with the LDH and EST patterns among the isolates. CONCLUSION: Genotype I is the major one in the 10 B. hominis isolates from Guangxi, and the isolates show different isoenzyme patterns for LDH and EST.