Natural products and their derivatives alleviating cerebral white matter lesions.

White matter lesions (WMLs), characterized by focal demyelination or myelination disorders, are commonly present in cerebral small vessel disease and various neurological diseases. Multiple etiologies lead to WMLs. However, there is no specific therapy or effective drugs for relieving WMLs. Natural products and their derivatives originate from bacterial, fungal, plant, and marine animal sources, many of which have multiple therapeutic targets. Compared to single target compounds, natural products and their derivatives are promising to be developed as better drugs to attenuate WMLs. Thus, this review attempts to summarize the status of natural products and their derivatives (2010-to date) alleviating cerebral white matter lesions for the discovery of new drugs.

Effects of co-infection with Clonorchis sinensis on T cell exhaustion levels in patients with chronic hepatitis B.

To investigate the effects of co-infection with Clonorchis sinensis (C. sinensis) on T cell exhaustion levels in patients with chronic hepatitis B, we enrolled clinical cases in this study, including the patients with concomitant C. sinensis and HBV infection. In this study, we detected inhibitory receptors and cytokine expression in circulating CD4+ and CD8+ T cells by flow cytometry. PD-1 and TIM-3 expression levels were significantly higher on CD4+ T and CD8+ T cells from co-infected patients than on those from the HBV patients. In addition, CD4+ T cells and CD8+ T cells function were significantly inhibited by C. sinensis and HBV co-infection compared with HBV single infection, secreting lower levels of Interferon gamma (IFN-γ), Interleukin-2 (IL-2), and TNF-α. Our current results suggested that C. sinensis co-infection could exacerbate T cell exhaustion in patients with chronic hepatitis B. PD-1 and TIM-3 could be novel biomarkers for T cell exhaustion in patients with Clonorchis sinensis and chronic hepatitis B co-infection. Furthermore, it may be one possible reason for the weaker response to antiviral therapies and the chronicity of HBV infection in co-infected patients. We must realize the importance of C. sinensis treatment for HBV-infected patients. It might provide useful information for clinical doctors to choose the right treatment plans.

CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccßA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, ß-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccßA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

Effect of Lacking ZKSCAN3 on Autophagy, Lysosomal Biogenesis and Senescence.

Transcription factors can affect autophagy activity by promoting or inhibiting the expression of autophagic and lysosomal genes. As a member of the zinc finger family DNA-binding proteins, ZKSCAN3 has been reported to function as a transcriptional repressor of autophagy, silencing of which can induce autophagy and promote lysosomal biogenesis in cancer cells. However, studies in Zkscan3 knockout mice showed that the deficiency of ZKSCAN3 did not induce autophagy or increase lysosomal biogenesis. In order to further explore the role of ZKSCAN3 in the transcriptional regulation of autophagic genes in human cancer and non-cancer cells, we generated ZKSCAN3 knockout HK-2 (non-cancer) and Hela (cancer) cells via the CRISPR/Cas9 system and analyzed the differences in gene expression between ZKSCAN3 deleted cells and non-deleted cells through fluorescence quantitative PCR, western blot and transcriptome sequencing, with special attention to the differences in expression of autophagic and lysosomal genes. We found that ZKSCAN3 may be a cancer-related gene involved in cancer progression, but not an essential transcriptional repressor of autophagic or lysosomal genes, as the lacking of ZKSCAN3 cannot significantly promote the expression of autophagic and lysosomal genes.

Effects of co-infection with Clonorchis sinensis on the sex hormones levels in male patients with chronic hepatitis B.

OBJECTIVE: We evaluated the levels of sex hormones in male hepatitis B patients co-infected with Clonorchis sinensis (C. sinensis). METHODS: A total of 136 male individuals were enrolled in this study, including 27 healthy controls, 28 patients with C. sinensis mono-infection, 19 patients with only chronic hepatitis B, 18 patients with post-hepatitis B liver cirrhosis, 26 chronic hepatitis B patients co-infected with C. sinensis, and 18 post-hepatitis B liver cirrhosis patients coinfected with C. sinensis. Serum levels of progesterone (P), luteinizing hormone (LH), estradiol (E2), testosterone (T), prolactin (PRL), and follicle stimulating hormone (FSH) in these groups were measured. RESULTS: The results showed that compared with the LC group, the LC+ C. sinensis co-infected group had an increase in E2 but decrease in T and FSH. The levels of E2 in CHB+ C. sinensis co-infected patients were significantly higher than those in CHB mono-infected patients, but the significantly lower levels of T were observed. Compared with HCs group, the LC group showed significant increase in all terms of sex hormones, except PRL. By contrast, the CHB mono-infected group presented an apparent decrease in E2, T, and PRL than the HCs group. However, there were no significant differences in sex hormone levels between the C. sinensis mono-infected patients and HCs. CONCLUSION: This study suggests that C. sinensis co-infection aggravates the sex hormone disturbance in HBV patients at both chronic hepatitis and cirrhosis stages, providing evidences for potential strategies in disease prevention and treatment.

Temperature preference can bias parental genome retention during hybrid evolution.

Interspecific hybridization can introduce genetic variation that aids in adaptation to new or changing environments. Here, we investigate how hybrid adaptation to temperature and nutrient limitation may alter parental genome representation over time. We evolved Saccharomyces cerevisiae x Saccharomyces uvarum hybrids in nutrient-limited continuous culture at 15°C for 200 generations. In comparison to previous evolution experiments at 30°C, we identified a number of responses only observed in the colder temperature regime, including the loss of the S. cerevisiae allele in favor of the cryotolerant S. uvarum allele for several portions of the hybrid genome. In particular, we discovered a genotype by environment interaction in the form of a loss of heterozygosity event on chromosome XIII; which species' haplotype is lost or maintained is dependent on the parental species' temperature preference and the temperature at which the hybrid was evolved. We show that a large contribution to this directionality is due to a temperature dependent fitness benefit at a single locus, the high affinity phosphate transporter gene PHO84. This work helps shape our understanding of what forces impact genome evolution after hybridization, and how environmental conditions may promote or disfavor the persistence of hybrids over time.

The storage stability of Bacillus subtilis spore displaying cysteine protease of Clonorchis sinensis and its effect on improving the gut microbiota of mice.

Bacillus subtilis (B. subtilis) spore can serve as an ideal vehicle for expressing heterologous antigens, and elicit specific immune responses by oral administration. In previous studies, we successfully constructed the recombinant B. subtilis spores expressing cysteine protease of Clonorchis sinensis (C. sinensis, B.s-CsCP), and confirmed that oral administration of B.s-CsCP could elicit good protective immune responses in mice. In this study, Gram staining was used to observe the morphology of B.s-CsCP in different form, and the storage of liquid spores and lyophilized spores at different temperatures was compared. The mice were orally immunized with three different doses of spores (2×108, 1×109, and 5×109 CFU/day) for three times in total at biweekly interval. Then, antibody levels of mice were measured, the safety of spores was evaluated, and the changes of gut microbiota after oral gavage of spores (1×109 dose) were investigated. Results showed that B. subtilis was a typical Gram-positive bacterium, and its spore had good resistance to chemical dye. Liquid B. subtilis spores resuspended in sterile water could be stored for a long time at 4 °C or below, while lyophilized spores could be well stored even at RT and better at lower temperatures. Oral administration of B. subtilis spores to mice could stimulate both local mucosal and systemic immune responses in a dose-dependent manner without toxic side effects. Besides, beneficial bacteria producing butyrate such as Odoribacter were increased, while potential pathogens such as Escherichia-Shigella were decreased in mice intestine. Therefore, our work further confirmed that B. subtilis spores expressing CsCP could be a promising oral vaccine against C. sinensis with the advantages of stability, safety, easy storage, and promotion of intestinal health.Key Points• Recombinant CsCP B. subtilis spores could be easily preserved in either liquid or freeze-dried state.• Oral immunization of recombinant spores in mice could increase both local and system immune levels in a dose-dependent manner.• Oral administration of recombinant spores increased the number of beneficial bacteria and reduced the number of harmful bacteria in the intestinal tract of mice.

Innovative health risk assessment of heavy metals in Chinese herbal medicines based on extensive data.

In the present study, the concentrations of lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg), and copper (Cu) in 2245 batches of Chinese herbal medicines (CHMs) were measured using inductively coupled plasma-mass spectroscopy (ICP-MS). We developed a risk assessment strategy that assessed the heavy metal-associated health risk of CHMs based on our large dataset. Using a combination of the mean and 95th percentile (P95) values of the chronic daily intake (CDI), hazard quotient (HQ), hazard index (HI), and lifetime cancer risk (CR), the health risks of the average exposure population and the high exposure population were estimated, respectively. To obtain a precise and realistic risk assessment, the exposure frequency and exposure duration were determined using questionnaire data from 20,917 randomly selected volunteers. Additionally, given the specific ingestion characteristics of CHMs, the safety factor and the transfer rates of heavy metals were highlighted as well. The concentrations of Pb, Cd, As, Hg, and Cu in 2245 batches of CHMs were 1.566, 0.299, 0.391, 0.074, and 8.386 mg/kg, respectively. The mean HI values indicated that consumption of most CHMs would not pose an unacceptable health risk to the average exposure population, except for argy wormwood leaf (1.326), morinda root (2.095), plantain herb (1.540), chrysanthemum flower (1.146), and Indian madder root (2.826). In addition, CR assessment for Pb and As revealed that, for the average exposure population, the risk of developing cancers was lower than the acceptable levels (1 × 10-4) in the clinic. However, the P95 of the HI and CR values indicated that more attention should be paid to the systemic effects of CHMs in terms of both non-carcinogenic and carcinogenic health risks for the high exposure population. Furthermore, in order to serve population health better, national and international guidelines have now been established. The risk assessment strategy developed in this study is the first of its kind, and contributed to the risk assessment, guidelines, and safety standards for heavy metals in CHMs.

Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection.

Clonorchis sinensis (C. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. Effective strategies are still urgently expected to prevent against C. sinensis infection. In the present study, paramyosin of C. sinensis (CsPmy) was stably and abundantly expressed on the surface of Bacillus subtilis spores. The recombinant spores (B.s-CotC-CsPmy) were incorporated in the basal pellets diet in three different dosages (1 × 105, 1 × 108, 1 × 1011 CFU/g pellets) and orally administrated to grass carp (Ctenopharyngodon idella). The immune responses and intestinal microbiota in the treated grass carp were investigated. Results showed that specific anti-CsPmy IgM levels in sera, skin mucus, bile, and intestinal mucus, as well as mRNA levels of IgM and IgZ in the spleen and head kidney, were significantly increased in B.s-CotC-CsPmy-1011 group. Besides, transcripts levels of IL-8 and TNF-αin the spleen and head kidney were also significantly elevated than the control groups. Moreover, mRNA levels of tight junction proteins in the intestines of B.s-CotC-CsPmy-1011 group increased. Potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 1011 CFU/g B.s-CotC-CsPmy spores administrated fishes could be detected compared with control group. The amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 1011 CFU/g B.s-CotC-CsPmy spores orally immunized group. Our work demonstrated that B. subtilis spores presenting CsPmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against C. sinensis infection for grass carp.

Comparison of 2013 and 2009 versions of Caprini risk assessment models for predicting VTE in Chinese cancer patients: a retrospective study.

This study aimed to compare the predictive value of 2009 and 2013 version of Caprini risk assessment models (RAM) for venous thromboembolism (VTE) in cancer patients by receiver operating characteristic (ROC) analysis. This retrospective study reviewed a total of 1439 VTE and 1439 non-VTE Chinese cancer inpatients. The baseline demographic data of these patients were recorded. 2009 and 2013 versions Caprini RAMs were applied, and cumulative risk scores were obtained by adding the scores of each risk factor. The specificity, sensitivity, positive predictive value and negative predictive value of these two models were analyzed. ROC curve was drawn to calculate the area under the curve (AUC) and the Youden index. Significant differences were observed in the risk factors between VTE and non-VTE Group. The specificity and negative predictive value of 2013 version were higher than those of 2009 version (P < 0.05). No significant differences were found in the sensitivity or positive predictive value between 2009 and 2013 versions of the Caprini RAM (P > 0.05). The AUC and Youden index of 2013 Caprini RAM were significantly higher than those of 2009 Caprini RAM (P < 0.001), whereas the Youden index of the 2009 Caprini RAM at critical point 4 was higher than that at critical point 3 (0.362 vs 0.067, P < 0.05). Compared with 2009 version, 2013 version of the Caprini RAM provides a more accurate and efficacious method for the risk assessment of VTE in Chinese cancer patients.

Urine cell-free DNA as a promising biomarker for early detection of non-small cell lung cancer.

BACKGROUND: While blood-derived cell-free DNA has been shown to be a candidate biomarker able to provide diagnostic and prognostic insight in cancer patients, little is known regarding the potential application of urine cell-free DNA (ucfDNA) in diagnosis of cancer. Thus, the aim of this study was to investigate ucfDNA concentration and integrity index as potential biomarkers for early detection of non-small-cell lung cancer (NSCLC). METHODS: Urine samples were collected from 35 healthy controls and 55 NSCLC patients at various tumor node metastasis (TNM) stages. Two long interspersed nuclear element 1 (LINE1) fragments (LINE1-97 and 266 bp) were quantified via quantitative real-time PCR (qPCR). DNA integrity index was calculated as the ratio of LINE1-266/LINE-97. RESULTS: LINE1 fragments concentrations of ucfDNA (LINE1-97, 266 bp) were significantly higher in NSCLC patients with stage III/IV than in stage I/II and in healthy controls. The receiver operating characteristic (ROC) curves for discriminating patients with stage III/IV from healthy controls had areas under the curves (AUC) of 0.84 and 0.886, respectively. Moreover, ucfDNA integrity LINE1-266/97 was significantly higher in patients with stage III/IV than in stage I/II and in healthy controls. The AUC of ROC curve for discriminating patients with stage III/IV from healthy controls was 0.800. Furthermore, LINE1-266 fragment concentration was significantly higher in lymph node metastasis (LNM)-positive patients relative to LNM-negative patients. The ROC curve for discriminating LNM-positive from LNM-negative patients had an AUC of 0.822. CONCLUSION: UcfDNA could serve as a promising biomarker for early detection of NSCLC.

Crosstalk between PKC and MAPK pathway activation in cardiac fibroblasts in a rat model of atrial fibrillation.

OBJECTIVE: Atrial fibrillation (AF) is the most frequent form of cardiac arrhythmia and major cause of cardiac ischemia. Defective calcium homeostasis due to anomalous expression of ryanodine receptor type 2 (RyR2) or its hyperactivation by phosphorylation by serine threonine kinases has been implicated as a central mechanism of AF pathogenesis. Given the role of protein kinase C (PKC) isoforms in cardiac function we investigated role of PKC in AF using a rat model. RESULTS: PMA induced global increase in protein synthesis in cardiac fibroblasts isolated from AF rats, but not healthy controls, and the increase was inhibited by PKC inhibition. PMA mediated activation of both PKC and ERK and either inhibition of PKC by Go6983 or ERK by the MEK inhibitor Trametinib attenuated both P-ERK and P-PKC in both cardiac fibroblasts isolated from AF rats or from healthy rats but transduced with PKC-delta. The PKC and ERK mediated induction of global protein synthesis was found to be mediated by increased phosphorylation of the ribosomal protein S6. CONCLUSION: Our findings provide a foundation for future testing of PKC and MEK inhibitors to treat AF in pre-clinical models. It also needs to be determined if PKC and MAPK pathway activation is functioning via RyR2 or some yet undefined substrates.

In vivo and in vitro studies using Clonorchis sinensis adult-derived total protein (CsTP) on cellular function and inflammatory effect in mouse and cell model.

Clonorchis sinensis (C. sinensis) can induce a food-borne parasitic disease (clonorchiasis). Numerous studies have analyzed functional proteins, immunologic factors, pro-inflammatory cytokines, and cell signaling transduction that promote the development of clonorchiasis. In a previous study, it was shown that C. sinensis adult-derived total protein (CsTP) might be involved in the pathogenesis and development of liver fibrosis via bringing about Th2 immune response. In the present study, further investigation of CsTP on cellular function and inflammatory effect in vitro and in vivo has been elicited. CsTP induced inflammation and autophagy as evidenced by upregulation of TNF-α, IFN-γ, and autophagic markers LC3B and P62. Exposed to CsTP upregulated the antiapoptotic gene Bcl-2 expression, diminished the apoptosis induced by H2O2, but promoted the proliferation and migration of LX-2 cells in proper concentration range. Additionally, the protein levels of p-AKT and p-mTOR were repressed in response to CsTP, suggesting a correlation of blocking the activation of mTOR/AKT signaling pathway. These results revealed that CsTP might exacerbate hepatic pathological changes by regulating cell proliferation, apoptosis, autophagy, and inflammation in the liver and LX-2 cells. Some effects might be partially involved in the mTOR and AKT pathways.

[Ingredients changes in Rehmanniae Radix processing based by HPLC-ELSD specific chromatogram].

To establish the HPLC-ELSD specific chromatogram analysis method of Rehmanniae Radix and Rehmanniae Radix Prae-parata, and analyze and compare their chemical compositions, so as to reveal the change regularity of compositions during the proces-sing. By HPLC-ELSD method, the chromatographic column for Prevail Carbohydrate ES(4.6 mm ×250 mm, 5 µm) was adopted, with acetonitrile(A)-water(B) as mobile phase for gradient elution, and the evaporative light-scattering detector was used. A total of 23 batches of Rehmannia Radix samples, and 25 batches of Rehmanniae Radix Praeparata samples and processing dynamic samples were compared. The established method had a great repeatability, precision and stability. Eight common chromatographic peaks were extracted from 23 batches of Rehmanniae Radix samples, 8 common peaks were extracted from 25 Rehmanniae Radix Praeparata, and 7 chromatographic peaks were identified. The composition ratio of Rehmannia Radix was changed greatly during the processing. When the simila-rity≥0.95 and the fructose peak area was more than 2 times of stachyose tetrahydrate or more than 20 times of raffinose, the processing degree conformed to the requirements of empirical identification. The three main oligosaccharides of Rehmanniae Radix were sucrose that was heated to generate fructose and glucose, stachyose tetrahydrate that was heated to generate melibiose, sucrose and fructose, and stachyose tetrahydrate that was heated to generate manninotriose. The change in the index of proportion between monosaccharides and oligosaccharides can be used as the quantitative criterion for the processing quality of Rehmanniae Radix Praeparata.

[Herbalogical study on merit rating of Forsythiae Fructus based on near-mature fruit and hyper-mature fruit].

Forsythiae Fructus( Lianqiao) is classed from near-mature fruit and hyper-mature fruit,which are named as Qingqiao and Laoqiao,respectively. This article was based on the different views of which was better,Qingqiao or Laoqiao. Acorrding to the naming,varieties,habitat,harvesting and processing,used parts,medicinal properties and clinical efficacy,the herbalogical study was carried out. The results showed that Lianqiao had been sourced from the areial part of Hypericum ascyron and H. erectum of Clusiaceae before Tang Dynasty. Beside the former,and the fruit of Forsythia suspensa of Oleaceae was newly used as Lianqiao during the Southern and Northern Dynasties to the Tang Dynasty. The later had been the only origin of Lianqiao since the Song Dynasty. With the change of the medicinal varieties,the habitats of Lianqiao has also changed. The varieties of Clusiaceae were mainly produced in the Yellow River Basin from the Han Dynasty to the Tang Dynasty. After the Song Dynasty,they were produced in the south of the Yangtze River. The variety of Oleaceae was mainly produced in Shanxi,Henan,Shandong,Shaanxi,and northern Sichuan from the Tang and Song Dynasties. Currently,Shanxi and Henan have the largest output. Traditionally,there were two commercial varieties including Qingqiao and Laoqiao of Lianqiao based on the harvesting time. In traditional Chinese medicine( TCM) theory,Lianqiao removes evil heat and relieves toxicity,removes swelling and resolves enlarged nodes. Accroding to the effects of Lianqiao,Qingqiao was considered to be better than Laoqiao in TCM clinic. The modern research on main medicinal constituents and pharmacodynamic effects also confirmed the above mentioned facts. This paper can provide literature support for the rationalities of Qingqiao's mainstream medication and assay standard of Lianqiao in the Chinese Pharmacopoeia.

Elevated homocysteine increases the risk of left atrial/left atrial appendage thrombus in non-valvular atrial fibrillation with low CHA2DS2-VASc score.

Aims: To assess the association and the predictive value of plasma homocysteine (Hcy) levels with LA/LAA thrombus in non-valvular Atrial fibrillation (AF) patients with low CHA2DS2-VASc score. Methods and results: Eight hundred and eighty-eight consecutive patients in non-valvular AF with CHA2DS2-VASc score of 0 and 1 were enrolled. All patients routinely underwent transthoracic echocardiography and transoesophageal echocardiography. A total of thirty-two patients had LA/LAA thrombus. Compared with patients without LA/LAA thrombus, plasma Hcy levels were significantly higher in patients with LA/LAA thrombus (16.5 ± 4.8 mmol/L vs. 13.4 ± 4.1 mmol/L, P = 0.009). In multivariate analysis, Hcy was independently associated with LA/LAA thrombus (OR 1.048, 95% CI 1.007-1.090, P = 0.022). Hcy demonstrated a significant predictive value with area under the curve of 0.722 (95% CI 0.662-0.781, P < 0.001). The optimal cut-off point for Hcy predicting LA/LAA thrombus was 13.5 mmol/L (sensitivity 67%, specificity 65%). Patients with Hcy ≥13.5 mmol/L had higher prevalence of LA/LAA thrombus compared with those with Hcy <13.5 mmol/L (6.1% vs. 2.1%, P < 0.001). Elevated Hcy significantly increased the risk of LA/LAA thrombus in patients with CHA2DS2-VASc score of 0 and 1 (OR 11.789, 95% CI 1.437-96.746, P = 0.022; OR 2.256, 95% CI 1.007-5.155, P = 0.048, respectively). Conclusion: Elevated plasma Hcy increases the risk of LA/LAA thrombus in non-valvular AF patients with low CHA2DS2-VASc score, thus it should be taken into account in prediction of thromboembolism.

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 µm, diameter with distinct nucleus 7-8 µm diameter) and spermatogonia (12-15 µm, with distinct nucleus 6-7.5 µm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 µm) and type B spermatogonia (diameter 10-11 µm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.

[Residual status and risk assessment of forbidden and restricted pesticides of organophosphorus in Loincerae Japonicae Flos].

This paper reports the residual status of forbidden and restricted pesticides of organophosphorus in Loincerae Japonicae Flos to provide reference for the risk control and the formulation of maximum residue limits of the flower. A method for the determination of residues of 23 forbidden and restricted pesticides of organophosphorus was established, and 64 collected samples were tested. Then a risk assessment based on the maximum residue limit was carried out. Results showed that the detection rate of samples was 58%. 6 of 23 pesticides were detected and the ratio was chlorpyrifos 41%, omethoate 23%, triazophos 11%, isofenphos-methyl 6.3%, methamidophos 1.6%, isocarbophos 1.6%, respectively. And the median of pesticide residues in the positive samples was chlorpyrifos 0.037 mg·kg⁻¹, omethoate 0.043 mg·kg⁻¹, triazophos 0.030 mg·kg⁻¹, isofenphos-methyl 0.18 mg·kg⁻¹, methamidophos 0.041 mg·kg⁻¹, isocarbophos 0.041 mg·kg⁻¹, respectively. In the samples with pesticide residues, the residue amount of methamidophos and isocarbophos was lower than the theoretical maximum residue limit, and the residue amount of chlorpyrifos, isofenphos-methyl, triazophos were higher than it individually, while, all the residue amount of omethoate was higher than it. According to the assessment result of theoretical maximum residue limit, it is suggested that relevant departments should accelerate the formulation of the maximum residue limit standard, and strengthen the supervision of the use of forbidden and restricted pesticides of organophosphorus in Loincerae Japonicae Flos.

Identification, characterization and expression analysis of TLR5 in the mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

TLRs (Toll-like receptors) are very important pathogen pattern recognition receptors, which control the host immune responses against pathogens through recognition of molecular patterns specific to microorganisms. In this regard, investigation of the turbot TLRs could help to understand the immune responses for pathogen recognition. Here, transcripts of two TLR5 (TLR5a and TLR5b) were captured, and their protein structures were also predicted. Meanwhile, we characterized their expression patterns with emphasis on mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot TLR5 genes showed the closest relationship to Paralichthys olivaceus. These two TLR5 genes were ubiquitously expressed in healthy tissues although expression levels varied among the tested tissues. In addition, the two copies of turbot TLR5 showed different expression patterns after bacterial infections. After Vibrio anguillarum infection, TLR5a was generally up-regulated in intestine and skin while down-regulated in gill, while TLR5b showed a general down-regulation in mucosal tissues. After Streptococcus iniae infection, the TLR5a was down-regulated at 2 h while generally up-regulated after 4 h in mucosal tissues. Interestingly, the TLR5b was up-regulated in intestine while down-regulated in skin and gill after Streptococcus iniae infection. These findings suggested a possible irreplaceable role of TLR5 in the immune responses to the infections of a broad range of pathogens that include Gram-negative and Gram-positive bacteria. Future studies should apply the bacteriological and immune-histochemical techniques to study the main sites on the mucosal tissue for bacteria entry and identify the ligand specificity of the turbot TLRs after challenge.