Network Pharmacology and Experimental Validation of Qingwen Baidu Decoction Therapeutic Potential in COVID-19-related Lung Injury.

BACKGROUND AND PURPOSE: Coronavirus disease 2019 (COVID-19) is a lifethreatening disease worldwide due to its high infection and serious outcomes resulting from acute lung injury. Qingwen Baidu decoction (QBD), a well-known herbal prescription, has shown significant efficacy in patients with Coronavirus disease 2019. Hence, this study aims to uncover the molecular mechanism of QBD in treating COVID-19-related lung injury. METHODS: Traditional Chinese Medicine Systems Pharmacology database (TCMSP), DrugBanks database, and Chinese Knowledge Infrastructure Project (CNKI) were used to retrieve the active ingredients of QBD. Drug and disease targets were collected using UniProt and Online Mendelian Inheritance in Man databases (OMIM). The core targets of QBD for pneumonia were analyzed by the Protein-Protein Interaction Network (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) to reveal the underlying molecular mechanisms. The analysis of key targets using molecular docking and animal experiments was also validated. RESULTS: A compound-direct-acting target network mainly containing 171 compounds and 110 corresponding direct targets was constructed. The key targets included STAT3, c-JUN, TNF-α, MAPK3, MAPK1, FOS, PPARG, MAPK8, IFNG, NFκB1, etc. Moreover, 117 signaling pathways mainly involved in cytokine storm, inflammatory response, immune stress, oxidative stress and glucose metabolism were found by KEGG. The molecular docking results showed that the quercetin, alanine, and kaempferol in QBD demonstrated the strongest affinity to STAT3, c- JUN, and TNF-α. Experimental results displayed that QBD could effectively reduce the pathological damage to lung tissue by LPS and significantly alleviate the expression levels of the three key targets, thus playing a potential therapeutic role in COVID-19. CONCLUSION: QBD might be a promising therapeutic agent for COVID-19 via ameliorating STAT3-related signals.

Genetic alterations and molecular mechanisms underlying hereditary intrahepatic cholestasis.

Hereditary cholestatic liver disease caused by a class of autosomal gene mutations results in jaundice, which involves the abnormality of the synthesis, secretion, and other disorders of bile acids metabolism. Due to the existence of a variety of gene mutations, the clinical manifestations of children are also diverse. There is no unified standard for diagnosis and single detection method, which seriously hinders the development of clinical treatment. Therefore, the mutated genes of hereditary intrahepatic cholestasis were systematically described in this review.

Study on the Mechanism of Action of Different Acupuncture Regimens on Premature Ovarian Failure Model Rats.

Objective: To study the mechanism of acupuncture on premature ovarian failure (POF) through the apoptosis pathway mediated by Bcl-2/Bax. Methods: POF rats were successfully obtained by cyclophosphamide. They were divided into five groups. After that, acupuncture was performed. The blank group and model group were not treated. The routine acupuncture group was acupuncture at Guanyuan, Sanyinjiao, Zhongji, and Guilai four points. The Neck-seven-acupuncture group was selected from Fengchi, Fengfu, Tianzhu, and Wangu four acupoints; the three-viscera simultaneous treatment group selected Guanyuan, Shenshu, Sanyinjiao, Taichong, and Baihui five points; and the data mining group selected Guanyuan and Sanyinjiao two points for 14 days of treatment. During the treatment, some rats were shed one after another due to the side effects of bone marrow suppression caused by mold-making. After treatment, serum estradiol (E2), follicle forming hormone (FSH), and luteinizing hormone (LH) were detected by radioimmunoassay, Bcl-2 and Bax proteins were analyzed by Western blot method, and Bcl-2 and Bax RNA were analyzed by PCR method. Results: Bcl-2 increased and Bax decreased in rats with premature ovarian failure treated with acupuncture. It shows that acupuncture can affect the secretion of ovarian-related hormones and the expression of apoptosis-related proteins, which is more significant in the conventional acupuncture point group. Conclusion: Acupuncture can inhibit the apoptosis of granulosa cells in ovarian tissue of rats with premature ovarian failure and improve ovarian function. The mechanism of its effect is to promote Bcl-2 gene expression and protein synthesis and inhibit Bax gene expression and protein synthesis. The conventional treatment group works best. This provides an experimental basis for the clinical use of acupuncture to intervene in the treatment of premature ovarian failure.

Spatial Analysis of Schistosomiasis in Hunan and Jiangxi Provinces in the People's Republic of China.

Understanding the spatial distribution of schistosome infection is critical for tailoring preventive measures to control and eliminate schistosomiasis. This study used spatial analysis to determine risk factors that may impact Schistosoma japonicum infection and predict risk in Hunan and Jiangxi Provinces in the People's Republic of China. The study employed survey data collected in Hunan and Jiangxi in 2016. Independent variable data were obtained from publicly available sources. Bayesian-based geostatistics was used to build models with covariate fixed effects and spatial random effects to identify factors associated with the spatial distribution of infection. Prevalence of schistosomiasis was higher in Hunan (12.8%) than Jiangxi (2.6%). Spatial distribution of schistosomiasis varied at pixel level (0.1 × 0.1 km), and was significantly associated with distance to nearest waterbody (km, ß = -1.158; 95% credible interval [CrI]: -2.104, -0.116) in Hunan and temperature (°C, ß = -4.359; 95% CrI: -9.641, -0.055) in Jiangxi. The spatial distribution of schistosomiasis in Hunan and Jiangxi varied substantially and was significantly associated with distance to nearest waterbody. Prevalence of schistosomiasis decreased with increasing distance to nearest waterbody in Hunan, indicating that schistosomiasis control should target individuals in close proximity to open water sources as they are at highest risk of infection.

Validation of MAPK signalling pathway as a key role of paeoniflorin in the treatment of intrahepatic cholestasis of pregnancy based on network pharmacology and metabolomics.

Numerous studies have clarified the effectiveness of paeoniflorin in the treatment of cholestasis. However, the therapeutic efficacy and mechanisms of action of paeoniflorin in intrahepatic cholestasis of pregnancy (ICP) were still unknown. This study aimed to investigate the molecular biological mechanisms of paeoniflorin against ICP by combining network pharmacology and metabolomics. The effects of paeoniflorin were investigated in the ICP rat model induced by 17α-ethinylestradiol, showing improvements in the liver indices, liver histopathological changes, bile flow rate, and serum levels of TBA and ALP. The underlying mechanisms and metabolic pathways of paeoniflorin were revealed by network pharmacology and untargeted metabolomics, showing that paeoniflorin exerted its curative effect against ICP-induced ferroptosis through PI3K/AKT and MAPK signalling pathways. In conclusion, paeoniflorin protected against ICP-induced liver injury through MAPK signaling pathways.

Predicting the Occurrence of Advanced Schistosomiasis Based on FISHER Discriminant Analysis of Hematological Biomarkers.

We established a model that predicts the possibility of chronic schistosomiasis (CS) patients developing into advanced schistosomiasis (AS) patients using special biomarkers that were detected in human peripheral blood. Blood biomarkers from two cohorts (132 CS cases and 139 AS cases) were examined and data were collected and analyzed by univariate and multivariate logistic regression analysis. Fisher discriminant analysis (FDA) for advanced schistosomiasis was established based on specific predictive diagnostic indicators and its accuracy was assessed using data of 109 CS. The results showed that seven indicators including HGB, MON, GLB, GGT, APTT, VIII, and Fbg match the model. The accuracy of the FDA was assessed by cross-validation, and 86.7% of the participants were correctly classified into AS and CS groups. Blood biomarker data from 109 CS patients were converted into the discriminant function to determine the possibility of occurrence of AS. The results demonstrated that the possibility of occurrence of AS and CS was 62.1% and 89.0%, respectively, and the accuracy of the established model was 81.4%. Evidence displayed that Fisher discriminant analysis is a reliable predictive model in the clinical field. It's an important guide to effectively control the occurrence of AS and lay a solid foundation for achieving the goal of schistosomiasis elimination.

Metabolomics Profiles Associated with the Treatment of Zuojin Pill on Patients with Chronic Nonatrophic Gastritis.

Objective: Chronic nonatrophic gastritis (CNG) is the most common digestive disease. In China, Zuojin pill (ZJP) is considered an effective medicine formula for CNG. However, its efficacy and mechanism have never been explored. In order to understand how and why ZJP demonstrates therapeutic effect on CNG, a clinical trial was conducted. Metabolomics was used to explore its deep mechanism. Methods: A total of 14 patients with CNG were recruited from October 2020 to March 2021 (ChiCTR2000040549). The endoscopy and histopathological changes were evaluated as efficacy. Serum samples were prepared and detected by performing widely targeted metabolome using UPLC. Multivariate statistical analysis was conducted to identify potential differential metabolites and signaling pathways. Last, the signal-related inflammatory factors containing COX-2, IL-4, and IL-17 were confirmed via immunohistochemical staining and enzyme-linked immunosorbent assay. Results: ZJP was able to alleviate several indexes of mucosal injury under endoscopy and histology. Erosion and bile reflux, but not red plaques and hemorrhage, were downregulated by ZJP. In addition, it could remarkably alleviate active chronic inflammation. A total of 14 potential metabolites, namely, hypoxanthine, adipic acid, D-ribono-1,4-lactone, L-sepiapterin, imidazoleacetic acid, sebacate, ADP-ribose, 4-hydroxybenzyl alcohol, 11,12-EET, 15-OxoETE, 12-OxoETE, (±)8-HETE, glycyrrhizinate, and DL-aminopimelic acid, were discriminated by metabolomics. Moreover, certain amino acid metabolism got significance during the disease progress and treatment. The related inflammatory factors including COX-2, IL-4, and IL-17 were inhibited by ZJP in both mucosa and serum. Conclusion: All these results indicated that ZJP partially acts as an inflammatory suppressor to regulate comprehensive metabolism disorders. This might be an important mechanism of ZJP in the treatment of CNG.

Schistosomiasis in the People's Republic of China - down but not out.

Schistosomiasis has been subjected to extensive control efforts in the People's Republic of China (China) which aims to eliminate the disease by 2030. We describe baseline results of a longitudinal cohort study undertaken in the Dongting and Poyang lakes areas of central China designed to determine the prevalence of Schistosoma japonicum in humans, animals (goats and bovines) and Oncomelania snails utilizing molecular diagnostics procedures. Data from the Chinese National Schistosomiasis Control Programme (CNSCP) were compared with the molecular results obtained.Sixteen villages from Hunan and Jiangxi provinces were surveyed; animals were only found in Hunan. The prevalence of schistosomiasis in humans was 1.8% in Jiangxi and 8.0% in Hunan determined by real-time polymerase chain reaction (PCR), while 18.3% of animals were positive by digital droplet PCR. The CNSCP data indicated that all villages harboured S. japonicum-infected individuals, detected serologically by indirect haemagglutination assay (IHA), but very few, if any, of these were subsequently positive by Kato-Katz (KK).Based on the outcome of the IHA and KK results, the CNSCP incorporates targeted human praziquantel chemotherapy but this approach can miss some infections as evidenced by the results reported here. Sensitive molecular diagnostics can play a key role in the elimination of schistosomiasis in China and inform control measures allowing for a more systematic approach to treatment.

Prevalence of Histamine-Forming Bacteria in Two Kinds of Salted Fish at Town Markets of Guangdong Province of South China.

ABSTRACT: Histamine-forming bacteria (HFB) were isolated from the 70 salted fish samples bought from town markets of Guangdong Province of south China. In addition, the histamine-forming ability of HFB was analyzed. There were 31 strains of HFB isolated from 36 salted fish pickled overnight. They were identified as six bacteria species: Vibrio alginolyticus, Vibrio rumoiensis, Staphylococcus saprophyticus, Staphylococcus xylosus, Lactococcus lactis, and Morganella morganii. The rate of confirmation of V. alginolyticus was highest (23 of 31), exceeding 200 mg/kg histamine. In particular, M. morganii produced a histamine amount that exceeded 2,000 mg/kg, although it was only one isolate in this study. In addition, five bacteria species of HFB were isolated from 34 dried salted fish. Among them, S. saprophyticus was dominant in the dried salted fish but produced histamines below 200 mg/kg. However, Enterobacter aerogenes from dried salted fish formed a histamine amount exceeding 200 mg/kg. The study showed that the dominant strain of HFB was different in two kinds of salted fish. Both kinds of salted fish contained HFB whose histamine-forming capacity exceeded 200 mg/kg. As a result, the safety of salted fish should be of concern, especially salted fish pickled overnight.

Interaction between bacterial diversity and biogenic amines production in a salted mackerel stored at soft frozen (-7℃-0℃) storage.

The bacterial diversity of salted mackerel "one-night courtyard" at soft frozen area (-7℃-0℃) storage was studied. The fish samples at 0, 14, 21, 28, and 35 days were analysis for bacterial structure using high-throughput sequencing technologies (HTS) and biogenic amines using high-performance liquid chromatography (HPLC). The analysis results of HTS showed that the dominant bacteria species was varied gradually following with storage time. On the 0th, 21st, and 28th days of storage, dominant Vibrionaceae was accounting for 71.70%, 59.16%, and 70.68% of the total sequences analyzed, respectively. On the 14th and 35th days, Shewanellaceae was the dominant bacterial, accounting for 87.53% and 70.95% of the total sequences analyzed, respectively. In addition, 21st and 28th days, an abundance of Operational Taxonomic Units (OTUs) was top. The dominant bacterial of Proteobacteria, Firmicutes, was producer of biogenic amines. Furthermore, the analysis results of HPLC shown the total biogenic amines of maximum amount 363.01 mg/kg in the sample of HY.14 lower than 1000 mg/kg of the FDA regulation. The range ability of cadaverine was obvious following with the storage time. Cadaverine was 87.36 mg/kg on the 0th day, and it was maximum amount of 276.89 mg/kg on the 14th days. Putrescine was 20 mg/kg on the 0th day and maximum amount of 55.04 mg/kg on the 28thdays of storage. The tyramine was smallest amount of production, and the largest amount was 38.99 mg/kg on 28th, and the smallest amount was 11.97 mg/kg on 35th. Nevertheless, the maximum amount of histamine was 55.04 mg/kg on the 0th day and about 23.14 mg/kg of histamine was little change from 14th to 35th days of storage. Dominant bacteria affect the change of biogenic amines. The study can help understand the interaction between microbial flora and biogenic amines in the salted mackerel of one-night courtyard.

The Dynamics of Hepatic Fibrosis Related to Schistosomiasis and Its Risk Factors in a Cohort of China.

China has had a long history against schistosomiasis japonica. The most serious prognosis of chronic schistosome infection is hepatic fibrosis, which develops into advanced schistosomiasis if the process is not effectively controlled. After a more than seven decades endeavor, China has gained remarkable achievements in schistosomiasis control and achieved transmission control nationwide (infection rate of schistosomes in residents and domestic animals both less than 1%) by 2015. However, new advanced schistosomiasis cases emerge annually in China, even in areas where the transmission of schistosomiasis had been interrupted. In the present study, the residents (>5 years old) in a schistosomiasis endemic village were examined for schistosomiasis every year during 1995-2019 by the modified Kato-Katz thick smear method and/or miracidium hatching technique. Residents who were identified to have an active infection method were treated with praziquantel at a dose of 40 mg/kg body weight. Ultrasonography was carried out to assess the liver morbidity related to schistosomiasis in 1995 and 2019, respectively. The prevalence of schistosomiasis among residents presented a downward trend annually, from 17.89% (175/978) in 1995 to 0 (0/475) in 2019. Among 292 residents who received ultrasound scan both in 1995 and 2019, 141 (48.29%) presented stable liver damage, while liver fibrosis was developed severely in 86 (29.45%) and reversed in 65 (22.26%) residents. Univariate and multivariate analysis showed that anti-fibrosis treatment was the protective factor against schistosomiasis hepatic fibrosis. Males, residents aged 38 and above, fishermen, and people who did not receive anti-fibrosis treatment were groups with higher risk of liver fibrosis development. Our results revealed that although the infection rate of schistosome dropped significantly in endemic areas, liver fibrosis was still developing among some residents, even though they had received deworming treatment. Liver protection/anti-fibrosis treatment should be administered in endemic regions and regions with historically uncontrolled transmission to slow down the deterioration of hepatic fibrosis among patients in schistosomiasis endemic areas.

Long-Term Survival After Salvage Thoracic Surgery on a Patient with ALK-Rearranged Metastatic Lung Adenocarcinoma After Progression on Targeted Therapy.

Targeted therapy for patients with advanced non-small cell lung cancer (NSCLC) is often challenged by the arising of drug resistance. After progression to targeted therapy, treatment options include continued targeted therapy, definitive local therapy, and the combination of both. While there is evidence that local ablative radiotherapy may prolong the disease control by targeted therapy, little is known regarding the relevance of salvage thoracic surgery in this setting. Herein, we presented a case of stage IV lung adenocarcinoma with concurrent EML4-ALK and TAC1-ALK fusion who had long-term survival after salvage thoracic surgery. The patient underwent a multidisciplinary treatment scheme that consisted of radiotherapy, ALK inhibitor crizotinib, and surgery, with blood-based genomic profiling for monitoring disease progression. Notably, salvage thoracic surgery was performed after progression on the crizotinib therapy and acquired ALK F1174C mutation was identified, which has been shown to be resistant to crizotinib and possibly sensitive to ceritinib. The patient benefited from salvage thoracic surgery with a remarkable progression-free survival of 31 months at last follow-up, and the patient maintained high-performance status throughout the course of management. To the best of our knowledge, this is the first case reporting on the long-term survival outcome from salvage thoracic surgery after crizotinib treatment in an NSCLC patient carrying double ALK fusion.

3D Freeform Printing of Nanocomposite Hydrogels through in situ Precipitation in Reactive Viscous Fluid.

Composite hydrogels have gained great attention as three-dimensional (3D) printing biomaterials because of their enhanced intrinsic mechanical strength and bioactivity compared to pure hydrogels. In most conventional printing methods for composite hydrogels, particles are preloaded in ink before printing, which often reduces the printability of composite ink with little mechanical improvement due to poor particle-hydrogel interaction of physical mixing. In contrast, the in situ incorporation of nanoparticles into a hydrogel during 3D printing achieves uniform distribution of particles with remarkable mechanical reinforcement, while precursors dissolved in inks do not influence the printing process. Herein, we introduced a "printing in liquid" technique coupled with a hybridization process, which allows 3D freeform printing of nanoparticle-reinforced composite hydrogels. A viscoplastic matrix for this printing system provides not only support for printed hydrogel filaments but also chemical reactants to induce various reactions in printed objects for in situ modification. Nanocomposite hydrogel scaffolds were successfully fabricated through this 3D freeform printing of hyaluronic acid (HAc)-alginate (Alg) hydrogel inks through a two-step crosslinking strategy. The first ionic crosslinking of Alg provided structural stability during printing, while the secondary crosslinking of photo-curable HAc improved the mechanical and physiological stability of the nanocomposite hydrogels. For in situ precipitation during 3D printing, phosphate ions were dissolved in the hydrogel ink and calcium ions were added to the viscoplastic matrix. The composite hydrogels demonstrated a significant improvement in mechanical strength, biostability, as well as biological performance compared to pure HAc. Moreover, the multi-material printing of composites with different calcium phosphate contents was achieved by adjusting the ionic concentration of inks. Our method greatly accelerates the 3D printing of various functional or hybridized materials with complex geometries through the design and modification of printing materials coupled with in situ post-printing functionalization and hybridization in reactive viscoplastic matrices.

Combination therapy of first and third generation EGFR-TKIs for an advanced lung adenocarcinoma patient harboring EGFR mutations and amplification: a case report.

The paradigm for the pharmacological management of advanced non-small cell lung cancer (NSCLC) has been revolutionized by the development of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Developing resistance to target therapy is unavoidable. Mostly, treatments for single molecular alteration after acquiring EGFR-TKI treatment resistance are well studied. However, there is limited evidence of treatment strategies for complex resistance mechanisms. Presented here is a case of an EGFR-mutated NSCLC patient who developed a complex resistance profile: T790M point mutation and EGFR amplification after first-line EGFR-TKI. This patient was safely treated with a combination of osimertinib and icotinib and achieved a significant clinical response and clear molecular response. Here we present the clinical evidence of the efficacy of osimertinib combined with icotinib in the treatment of EGFR classical mutation along with resistant mutation of T790M point mutation and EGFR amplification.

Discovering small molecules as Wnt inhibitors that promote heart regeneration and injury repair.

There are intense interests in discovering proregenerative medicine leads that can promote cardiac differentiation and regeneration, as well as repair damaged heart tissues. We have combined zebrafish embryo-based screens with cardiomyogenesis assays to discover selective small molecules that modulate heart development and regeneration with minimal adverse effects. Two related compounds with novel structures, named as Cardiomogen 1 and 2 (CDMG1 and CDMG2), were identified for their capacity to promote myocardial hyperplasia through expansion of the cardiac progenitor cell population. We find that Cardiomogen acts as a Wnt inhibitor by targeting ß-catenin and reducing Tcf/Lef-mediated transcription in cultured cells. CDMG treatment of amputated zebrafish hearts reduces nuclear ß-catenin in injured heart tissue, increases cardiomyocyte (CM) proliferation, and expedites wound healing, thus accelerating cardiac muscle regeneration. Importantly, Cardiomogen can alleviate the functional deterioration of mammalian hearts after myocardial infarction. Injured hearts exposed to CDMG1 display increased newly formed CMs and reduced fibrotic scar tissue, which are in part attributable to the ß-catenin reduction. Our findings indicate Cardiomogen as a Wnt inhibitor in enhancing injury-induced CM proliferation and heart regeneration, highlighting the values of embryo-based small molecule screens in discovery of effective and safe medicine leads.

Effect of integrated control intervention on soil-transmitted helminth infections in Jiangxi province in southeast China.

Soil transmitted helminths (STHs) burden was enormous in China several decades ago, however, rigorous control efforts have been successful with appreciable reduction in diseases burden. Here, we assessed provincial-level data derived from cross sectional surveys, executed in 1989, 2002 and 2014, on the prevalence of STHs among populations in Jiangxi province, China. This study, also, reported STHs integrated control intervention aimed at reducing STHs transmission and worm burden among population at county-level. The intervention strategies included mass drug administration (MDA), health education, improved water supply for drinking, improved sanitary facilities and environmental modification in Guixi municipality. The overall infection rate of STHs in Jiangxi province decreased from 77.7% (1989) to 6.3% (2014), while Ascaris lumbricoides, hookworm and Trichuris trichiura decreased from 71.1%, 17.6% and 17.0% (1989) to 0.9%, 4.7% and 1.0% (2014), respectively. STHs infection rates in female population were higher than male in the three surveys. Reduction in STHs prevalence was observed in all age groups, but the decline was less in higher age group. STHs prevalence in Guixi intervention region indicated remarkable reduction from 31.8% (2006) to 6.1% (2009) (χ2＝255.22, P＜0.01). A. lumbricoides, hookworm and T. trichiura infection rates decreased from 10.4%, 17.0% and 7.1% (2006) to 0.1%, 4.1% and 2.2%, respectively (2009) (X2A.l = 110.23, P<0.01; X2hk = 103.57, P < 0.01; X2T.t = 32.0, P < 0.01). A. lumbricoides infection rate declined the most of all STHs. Following control efforts with integrated control intervention strategies, STHs prevalence in Jiangxi province experienced remarkable trend in decline between 1989 and 2014. Consolidating control efforts with sustained integrated control strategies is, therefore, important to achieving STHs elimination in China.

[Study on immune status of patients with schistosomiasis japonica in Poyang Lake region III Humoral and cellular immune characteristics between Schistosoma japonicum high and low antibody responders].

OBJECTIVE: To explore the immune mechanism of negative results of immune tests of schistosomiasis japonica patients. METHODS: Totally 142 schistosomiasis patients (positive stool examinations) of Poyang Lake region were tested by ELISA method, and the ROC curve was applied to determine the high and low response of the patients. The levels of cellular immunity and cytokines of high and low responders were compared. RESULTS: Totally eight schistosomiasis patients were found as low responders. Besides SWAP-IgA (t = -1.588, P > 0.1), the levels of isotype antibodies were significantly lower in the low responders compared with those in the high responders (t = -14.517 to -2.866, all P < 0.05). In the low responders, the propor- tion of CD3⁺T was increased; and the proportions of CD4⁺T, CD8⁺T, CD4⁺CD25⁺Treg, and the ratio of CD4⁺/CD8⁺ were all de- creased, but all of them were not significant (t = -1.72 to 0.974, all P > 0.05) compared with those in the high responders. The differences of IFN-γ and IL-10 between the high and low responders were both not significant (t = -2.426 to 0.216, all P > 0.05). CONCLUSIONS: There is a significant difference between the high and low responders only in the levels of isotype antibodies. One of the reasons of low response in the immune tests is the much lower antibody level after the antigen-antibody compound is completely formulated.

Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.

Reference genes used in normalizing qRT-PCR data are critical for the accuracy of gene expression analysis. However, many traditional reference genes used in zebrafish early development are not appropriate because of their variable expression levels during embryogenesis. In the present study, we used our previous RNA-Seq dataset to identify novel reference genes suitable for gene expression analysis during zebrafish early developmental stages. We first selected 197 most stably expressed genes from an RNA-Seq dataset (29,291 genes in total), according to the ratio of their maximum to minimum RPKM values. Among the 197 genes, 4 genes with moderate expression levels and the least variation throughout 9 developmental stages were identified as candidate reference genes. Using four independent statistical algorithms (delta-CT, geNorm, BestKeeper and NormFinder), the stability of qRT-PCR expression of these candidates was then evaluated and compared to that of actb1 and actb2, two commonly used zebrafish reference genes. Stability rankings showed that two genes, namely mobk13 (mob4) and lsm12b, were more stable than actb1 and actb2 in most cases. To further test the suitability of mobk13 and lsm12b as novel reference genes, they were used to normalize three well-studied target genes. The results showed that mobk13 and lsm12b were more suitable than actb1 and actb2 with respect to zebrafish early development. We recommend mobk13 and lsm12b as new optimal reference genes for zebrafish qRT-PCR analysis during embryogenesis and early larval stages.

[Preservation of live eggs of Schistosoma japonicum].

OBJECTIVE: To observe the preservation time and activity of miracidium hatching from schistosome eggs preserved in different solutions, so as to obtain the optimal preservation conditions and then provide quality control products for field application. METHODS: The rectum stool of rabbits infected with Schistosoma japonicum was collected and the coarse fecal residue was removed with a series of sample sieves of 80, 100, 160 and 200 meshes respectively, and then the schistosome eggs were concentrated with the sample sieve of 260 meshes. The concentrated eggs were preserved in 0.9% sodium chloride solution, 1.2% sodium chloride solution, phosphate buffered saline solution (PBS, PH 7.2), 1.0% sucrose solution, and Mili-Q water, respectively, and then were conserved in a 4 °C refrigerator and 15 °C constant temperature incubator, respectively. The preserved eggs were hatched in different time (7-day interval) , the vitality and quantity of the miracidia were observed, and the hatching rates were calculated. RESULTS: Under the condition of 4 °C, the hatching rates of eggs dropped to 0 in 1.0% sucrose solution and 1.2% sodium chloride solution at the 49th and 126th day, respectively, and the hatching rates of eggs in the 0.9% sodium chloride solution and PBS solution dropped to 10% at the 112th day, and the activity of miracidium was weakened since 140th. In the Mili-Q water, the hatching rate dropped less than 10% at the 196th day and the activity of miracidia was weakened since the 280th day. Under the condition of 15 °C, the hatching rate of eggs in different solutions gradually dropped to 0 from the 49th day to 105th day. CONCLUSION: The eggs preserved in Mili-Q water at the temperature of 4 °C can be used as the positive reference for hatching tests within 196 days.

[Survey of epidemic status of principal human parasites in Jiangxi Province in 2014].

OBJECTIVE: To understand and analyze the epidemic status of principal human parasites in Jiangxi Province, so as to provide the evidence for formulating The 13rd Five-Year Project of Parasites Control. METHODS: A survey was performed according to the scheme of The 3rd National Survey of Principal Human Parasites as well as the specific situation of Jiangxi Province. The survey of the soil-transmitted nematodes and intestinal protozoa were performed based on the ecological regions and stratified by economic and geographic situation. The survey of Clonorchis sinensis was performed along with the soil-transmitted nematodes in rural, and was performed by the sample method of random cluster in cities and towns. RESULTS: Totally 23 606 residents of 92 survey sites from 32 counties were surveyed, all of them were tested for the infection of soil-transmitted nematodes, in which 21,569 residents were tested for intestinal protozoa infection, and 1 486 children were tested for eggs of Enterobius vermicularis. Twenty kinds of intestinal parasites were found, with the total infection rate of 9.64%, and 4.296 millions of patients were reckoned in the whole Jiangxi Province. The infection rate of children of E. vermicularis was 13.73%. The infection rates of C. sinensis and intestinal protozoa were 0.58% and 1.42% respectively. The infection rate of Toxoplasma gondii was 7.50%. CONCLUSION: In Jiangxi Province, the infection rates of soil-transmitted nematodes are decreasing but the infection rate of C. sinensis is increasing in local areas, and the control work of parasites still should be strengthened.