Nitric oxide synthase regulates coelomocytes apoptosis through the NF-κB signaling pathway in the sea cucumber Apostichopus japonicus.

Nitric oxide synthase (NOS) was initially discovered to participate in the generation of nitric oxide as a defense mechanism against pathogenic infections. In recent years, it has been found that NOS plays a pivotal role in regulating apoptosis and inflammation in mammals. However, the mechanisms underlying NOS-mediated apoptosis in invertebrates remain largely unclear. In this study, we found that the Apostichopus japonicus NOS (AjNOS) expression levels were upregulated by 2.20-fold and 3.46-fold after being challenged with Vibrio splendidus at concentrations of 107 CFU mL-1 and 108 CFU mL-1 for 12 h compared to the control group, respectively. Under these conditions, the rates of coelomocytes apoptosis were increased from 14.7% to 32.7% and 45.4%, respectively. Treatment with NOS inhibitor (l-NAME) resulted in a reduction of coelomocytes apoptosis rates from 32.6% to 26.5% in V. splendidus (107 CFU mL-1) groups and from 42.3% to 33.3% in V. splendidus (108 CFU mL-1) groups, respectively. NOS has been reported to regulate apoptosis through IκBα phosphorylation. Simultaneously, exposure to V. splendidus in conjunction with l-NAME resulted in down-regulation of AjIκBα phosphorylation levels compared to the group infected solely with V. splendidus. Furthermore, immunofluorescence analysis revealed that treatment with l-NAME or interference of AjNOS using siRNA inhibited translocation of AjNF-κB/p65 (RelA) into the nucleus. Previous studies have shown that NF-κB can down-regulate expression levels of Bcl-2 family members, which is an important pathway for regulating apoptosis. In the present study, treatment with l-NAME was found to promote anti-apoptotic AjBcl-2 mRNA increase to 1.41-fold and protein expression increase to 1.86-fold at 12 h post V. splendidus challenge. However, these effects were suppressed by PMA (an NF-κB activator). Overall, our findings demonstrate that AjNOS regulates coelomocytes apoptosis induced by V. splendidus through activation of the AjNF-κB signaling pathway and down-regulation of AjBcl-2 in A. japonicus.

AjTGFß alleviates V. splendidus-induced inflammation through SMADs pathway in Apostichopus japonicus.

The inhibition of inflammatory response is an essential process to control the development of inflammation and is an important step to protect the organism from excessive inflammatory damage. As a pleiotropic cytokine, transforming growth factor beta (TGF-ß) plays a regulatory role in inhibiting inflammation in vertebrates. To investigate the role of TGF-ß in the regulation of inflammation in invertebrates, we cloned and characterized the TGF-ß gene from Apostichopus japonicus via rapid amplification of cDNA ends, and the sample was designated as AjTGF-ß. For Vibrio splendidus-challenged sea cucumbers, the expression of AjTGF-ß mRNAs in coelomocytes decreased at 96 h (0.27-fold), which was contrary to the trend of inflammation. AjTGF-ß was expressed in all tissues with the highest expression in the body wall. When AjTGF-ß was knocked down by using small interfering RNA (siRNA-KD) to 0.45-fold, AjSMAD 2/3 and AjSMAD6 were downregulated to 0.32- and 0.05-fold compared with the control group, respectively. Furthermore, when the damaged sea cucumber was challenged by V. splendidus co-incubated with rAjTGF-ß, the damage area had no extensive inflammation, and damaged repair appeared at 72 h compared with the Vs + BSA group, in which the expression of AjSMAD 2/3 was upregulated by 1.35-fold. Under this condition, AjSMAD 2/3 silencing alleviated rAjTGF-ß-induced damage recovery. Moreover, rAjTGF-ß slightly induced the collagen I expression from 6.13 ng/mL to 7.84 ng/mL, and collagen III was upregulated from 6.23 ng/mL to 6.89 ng/mL compared with the Vs + BSA group. This finding indicates that AjTGF-ß negatively regulated the inflammatory progress and accelerated the repair of damage by AjSMADs to regulate the collagens expression.

Investigation and management of an anaphylactic shock case after inoculation with COVID-19 vaccine / 上海预防医学

Anaphylactic shock is a serious and rare adverse reaction， which can be life-threatening if not treated in time. COVID-19 vaccine is a newly marketed vaccine， and people pay high attention to its adverse reactions. This report summarized the investigation and management process of a case of anaphylactic shock after inoculation with COVID-19 vaccine， in order to provide reference for standardizing the diagnosis and management of anaphylactic shock after vaccination.

Target of rapamycin signaling inhibits autophagy in sea cucumber Apostichopus japonicus.

Autophagy mediated by mTOR pathway is a particularly important immune defense mechanism in the pathogens infected mammals. However, the role of TOR in echinoderm autophagy is largely unknown. Here, a cDNA encoding TOR protein was cloned and characterized from sea cucumber Apostichopus japonicus (designated as AjTOR) and its biological functions were also investigated. The AjTOR gene encoded a peptide of 2499 amino acids with the representative domains of DUF3385, FAT, FRB, PI3Kc, and FATC, which exhibited highly conservation with vertebrate orthologs. Phylogenetic analysis supported that AjTOR belonged to a new member of TOR family. Moreover, tissues distribution analysis indicated that AjTOR was ubiquitously expressed in all the tested tissues, with the highest transcription in muscle. Vibrio splendidus infection in vivo and LPS challenge in vitro could both significantly down-regulate the mRNA expression of AjTOR. What's more, transmission electron microscopy observations showed that rapamycin treatment resulted in rapid formation of autophagosomes in coelomocytes both at 3 and 6 h, however, injection with mTOR activator of MHY1485 showed an inhibitory effect on autophagosomes formation compared to the control, suggesting blocking the expression of AjTOR could accelerates autophagy signals. Our findings supported that AjTOR served as a negative regulator in sea cucumber authophay.

Analysis of overseas patent registration of the classical prescription of traditional Chinese medicine / 国际中医中药杂志

Through the analysis of patent jurisprudence, it is proved that the Classical Prescription of Traditional Chinese Medicine (CPTCM) belongs to the scope of "existing technology" in the Law of Patent, and has lost the foundation of obtaining patent rights. Taking Japan's CPTCM as an example, based on the analysis of the administration of CPTCM and patent applications related to CPTCM in Japan, it is proved that CPTCM can not obtain patent authorization in Japan. Through the comparison of patent in China, the United States, Europe and worldwide region, it is proved that China is still the main source of patent applications for Traditional Chinese Medicine. At the end of the article, the author puts forward the suggestion of "active protection". It is believed that we should abandon the concept of patent-only protection and improve the influence of Traditional Chinese Medicine in worldwide from the aspects of industrial development, promotion ofits application, and intellectual property protection.

Analysis of overseas patent registration of the classical prescription of traditional Chinese medicine / 国际中医中药杂志

Through the analysis of patent jurisprudence, it is proved that the Classical Prescription of Traditional Chinese Medicine (CPTCM) belongs to the scope of "existing technology" in the Law of Patent, and has lost the foundation of obtaining patent rights. Taking Japan's CPTCM as an example, based on the analysis of the administration of CPTCM and patent applications related to CPTCM in Japan, it is proved that CPTCM can not obtain patent authorization in Japan. Through the comparison of patent in China, the United States, Europe and worldwide region, it is proved that China is still the main source of patent applications for Traditional Chinese Medicine. At the end of the article, the author puts forward the suggestion of "active protection". It is believed that we should abandon the concept of patent-only protection and improve the influence of Traditional Chinese Medicine in worldwide from the aspects of industrial development, promotion ofits application, and intellectual property protection.

Pathogen spectrum and epidemic status of major human parasites in Chen-zhou City,Hunan Province / 中国血吸虫病防治杂志

Objective To understand the pathogen spectrum and epidemic status of major human parasites in Chenzhou City,Hunan Province,so as to provide the evidence for parasitic diseases control. Methods The survey sites were selected by the stratified cluster sampling method. The intestinal helminthic eggs were detected by Kato?Katz technique. The trophozoites or cysts of intestinal protozoa were detected by saline smear and iodine staining methods. The eggs of Enterubius vermicularis of chil?dren from 3 to 6 years old were detected by the cellophane anal swab method. The species of hookworm were identified by the fil?ter paper strip culture method. Results A total of 7 031 people were detected with the intestinal helminthic infective rate of 1.83%(129 cases). The major parasite was hookworm and there was a statistically significant difference of the infection rates among various parasites(c2=107.77,P<0.01). All the hookworm larvae were Necator americanus. No intestinal protozoon was detected. There were statistically significant differences of the infection rates among the counties(c2=25.77,P<0.01). The age of the patients was mainly focused on 30 and above years old and the infection rate was increased with the growth of age(c2=26.21,P<0.01). Farmers were the main population of the patients and there was a statistically significant difference of the in?fection rates between farmer and others(c2=29.67,P<0.01). Conclusions The infection rates of parasites are low and hook?worm is the main parasite in the pathogen spectrum in Chenzhou City. However,the infection factors still exist,therefore,effec?tive and scientific measures should be taken to consolidate the achievement.

Investigation on the iodine nutritional status among the key population in Chenzhou City, Hunan Province,2015 / 中华地方病学杂志

Objective To study the iodine nutritional status among the key population in Chenzhou City of Hunan Province,and to provide scientific basis for prevention and control of iodine deficiency disorders (IDD).Methods According to the "Hunan Iodized Salt Monitoring Pilot Scheme",total of 11 counties (cities,districts) were selected in Chenzhou City in 2015.One township was selected by east,west,south,north and center in each county (city,district).Four villages were drawn from each township,and 15 households were selected from each village to collect salt samples.Forty children aged 8 to 10 years old (half male half female) were selected from each primary school in each township.Urine samples were taken and the goiter was examined.Totally 20 pregnant women were selected from each township and urine samples were collected.The content of iodine in salt samples and urine samples were detected by the method of direct titration and As-Ce catalytic spectrophotometry,respectively.The status of goiter was detected using palpation.Results Totally 3 300 salt samples were detected in 2015,the median of salt iodine content was 25.2 mg/kg.The coverage and qualified iodized salt consumption rates were 99.61％ (3 287/3 300) and 95.30％ (3 145/3 300),respectively,which achieved the standard for the elimination of IDD.Totally 2 200 urine samples of children aged 8 to 10 years old were tested,the median of urinary iodine was 238.98 μg/L.There was variations of urinary iodine in children aged 8 to 10 years old in each county (H =32.25,P ＜ 0.01).Totally 2 200 children aged 8 to 10 years old were checked,and no goiter was found.Totally 1 100 urine samples of pregnant women were detected,the median of urinary iodine was 204.25 μg/L.There was variations of urinary iodine in pregnant women in each county (H =117.47,P ＜ 0.01).Conclusions Totally,the iodine nutritional status among the key population is appropriate in Chenzhou City.However,surveillance and health education should be continuously strengthened in the future.

Investigation on the iodine nutritional status among the key population in Chenzhou City, Hunan Province,2015 / 中华地方病学杂志

Objective To study the iodine nutritional status among the key population in Chenzhou City of Hunan Province,and to provide scientific basis for prevention and control of iodine deficiency disorders (IDD).Methods According to the "Hunan Iodized Salt Monitoring Pilot Scheme",total of 11 counties (cities,districts) were selected in Chenzhou City in 2015.One township was selected by east,west,south,north and center in each county (city,district).Four villages were drawn from each township,and 15 households were selected from each village to collect salt samples.Forty children aged 8 to 10 years old (half male half female) were selected from each primary school in each township.Urine samples were taken and the goiter was examined.Totally 20 pregnant women were selected from each township and urine samples were collected.The content of iodine in salt samples and urine samples were detected by the method of direct titration and As-Ce catalytic spectrophotometry,respectively.The status of goiter was detected using palpation.Results Totally 3 300 salt samples were detected in 2015,the median of salt iodine content was 25.2 mg/kg.The coverage and qualified iodized salt consumption rates were 99.61％ (3 287/3 300) and 95.30％ (3 145/3 300),respectively,which achieved the standard for the elimination of IDD.Totally 2 200 urine samples of children aged 8 to 10 years old were tested,the median of urinary iodine was 238.98 μg/L.There was variations of urinary iodine in children aged 8 to 10 years old in each county (H =32.25,P ＜ 0.01).Totally 2 200 children aged 8 to 10 years old were checked,and no goiter was found.Totally 1 100 urine samples of pregnant women were detected,the median of urinary iodine was 204.25 μg/L.There was variations of urinary iodine in pregnant women in each county (H =117.47,P ＜ 0.01).Conclusions Totally,the iodine nutritional status among the key population is appropriate in Chenzhou City.However,surveillance and health education should be continuously strengthened in the future.

The first description of complete invertebrate arginine metabolism pathways implies dose-dependent pathogen regulation in Apostichopus japonicus.

In this study, three typical members representative of different arginine metabolic pathways were firstly identified from Apostichopus japonicus, including nitric oxide synthase (NOS), arginase, and agmatinase. Spatial expression analysis revealed that the AjNOS transcript presented negative expression patterns relative to those of Ajarginase or Ajagmatinase in most detected tissues. Furthermore, Vibrio splendidus-challenged coelomocytes and intestine, and LPS-exposed primary coelomocytes could significantly induce AjNOS expression, followed by obviously inhibited Arginase and AjAgmatinase transcripts at the most detected time points. Silencing the three members with two specific siRNAs in vivo and in vitro collectively indicated that AjNOS not only compete with Ajarginase but also with Ajagmatinase in arginine metabolism. Interestingly, Ajarginase and Ajagmatinase displayed cooperative expression profiles in arginine utilization. More importantly, live pathogens of V. splendidus and Vibrio parahaemolyticus co-incubated with primary cells also induced NO production and suppressed arginase activity in a time-dependent at an appropriate multiplicity of infection (MOI) of 10, without non-pathogen Escherichia coli. When increasing the pathogen dose (MOI = 100), arginase activity was significantly elevated, and NO production was depressed, with a larger magnitude in V. splendidus co-incubation. The present study expands our understanding of the connection between arginine's metabolic and immune responses in non-model invertebrates.

Identification and characterization of miR-31 potential targets by RNA-seq.

In our previous work, miR-31 displayed differential significant expression in Apostichopus japonicus sea cucumber with skin ulcer syndrome and modulated coelomocytes ROS production by targeting p105. To identify other promising targets ofmiR-31, 4 transcriptome libraries of coelomocytes, as well as 2 control libraries, were constructed frommiR-31 mimics (31 M) or AMO-miR-31 (31I) and injected into a sea cucumber at 12 and 24 h. A total of207,977 unigenes with an average length of 363 bp were assembled, in which17,204 distinct sequences (8.27% of the unigenes) were successfully matched with annotated protein sequences. Fragments per kilobase of transcript per million fragments mapped analysis indicated that 1325 unigenes displayed up-regulated expression profiles in the 31I-12 group and were depressed in the 31M - 12 group compared with the control group. A total of 1470 unigenes showed down-regulated expressions in 31I-12 and were induced in 31 M-12. Similarly, 2079 and 2098 unigenes were detected at 24 h post-injection. Among these unigenes, 36 unigenes (depressed expression in the 31 M group and induced in the 31I group) showed consistent expression patterns at 2 examined time points and were considered promising targets of miR-31. qPCR analysis confirmed that all 4 unigenes showed opposite expression profiles to miR-31 in cultured coelomocytes. Our present work provided a fast and feasible method of identifying miR-31 targets by transcriptome analysis. The results of this study would enhance our present understanding ofmiR-31 function insea cucumber immune regulation.

Study of the RNA interference silencing of survivin gene and chemosensitivity to docetaxel in MCF-7 cells / 中国药理学通报

Aim Through interfering the expression of survivin with short hairpin RNA(shRNA) technology,to investigate the effect of downregulation of survivin on cell apoptosis and chemosensitivity to docetaxel in breast cancer MCF-7 cells.Methods(1) Using MCF-7 cells as a model system,three groups were set up transfected with lipofectamine,RNAi control plasmid and survivin RNAi plasmid,respectively.The expression of survivin in MCF-7 cells was measured at transcriptional and translational level by using RT-PCR and Western blot methods.(2) The effect on the cell cycle and apoptosis was analyzed with flow cytometry.(3) The viability of cells applied with different doses of ducetaxel was determined by using the method of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction(MTT method).Results(1) RT-PCR and Western blot demonstrated that survivin expression was significantly decreased by transfection with RNAi targeting plasmid;the expression proportion was reduced by nearly 75.4% and 79.8%(P