Leaf samples were collected after cold stress of 24 h (S1 stage) and 48 h (S2 stage). The results indicated that exogenous α-oxoglutarate significantly enhanced leaf GS activity, NADP-GDH activity, glutamate content, proline content and photosynthesis of soybean seedling exposed to cold stress at S1 and S2 stages. The ammonium content in leaf was significantly decreased by exogenous α-oxoglutarate at both stages. 5.0 mmol/L of exogenous α-oxoglutarate is the optimum concentration in this study. Leaf proline content for Henong60 and Heinong48 at A5.0 was 37.53% and 17.96% higher than that at A0 at S1 stage, respectively. Proline content for Henong60 and Heinong48 increased by 28.82% and 12.41% at A5.0 and A0, respectively, at S2 stage. Those results suggested that exogenous α-oxoglutarate could alleviate the adverse effects of cold stress.The objective of this study was to examine the effects of exogenous α-oxoglutarate on leaf proline accumulation, ammonium assimilation and photosynthesis of soybean when exposed to cold stress. To achieve this objective, exogenous α-oxoglutarate was sprayed to potted seedlings of Henong60 and Heinong48 at 0, 2.5, 5.0 and 7.5 mmol/L, identified as A Osmoregulation is an important mechanism that plants use to acclimate to different abiotic stresses. Proline is proposed to act as a main osmolyte in enhancing plant stress tolerance2.Low temperature often occurs during soybean seedling stage in Northeast China, which will pose great damage to soybean growth and development. Cold stress severely limits plant growth and leads to substantial productivity loss. Several plants species have evolved different mechanisms to minimize the adverse impact of cold stress3. Among the beneficial compounds, proline is greatly important for plants to cope with cold stress; the correlation between the acquisition of stress tolerance and proline accumulation has been verified4. In fact, proline is eventually synthesized from Glu in glutamate (Glu) pathway and ornithine (Orn) pathway5. Glutamate plays a significant role in amino acid metabolism in plants6. Glu biosynthesis is greatly linked with ammonium assimilation6. A mass of protein in plant can decompose into ammonium during osmotic stress2. Excessive ammonium can cause toxic effects on plants and must be rapidly transformed into non-toxic organic nitrogen compounds7. It has been demonstrated that GS/GOGAT cycle is the major route of ammonium assimilation in plants. Alternatively, NADH-dependent glutamate dehydrogenase  might also catalyze ammonium incorporation into glutamate8. The function of the alternative GDH pathway is still obscure and is believed to play a complementary role under conditions of environmental stress or excessive ammonia supply11.In plants, proline accumulation has been reported to occur after cold stress13 participates in a range of reactions in distinct plant cell compartments14, also being a key metabolite at the crossroads of carbon/nitrogen metabolism as it is required for ammonia assimilation16. Some information is available on the effects of exogenous α-oxoglutarate on nitrogen assimilation in plants, such as rice17, tobacco19, wheat20, and tomato21. Magalhaes et al.21 reported that NH4+ concentrations in tomato roots and shoots with exogenous α-oxoglutarate sharply decreased compared to control and the increase of NH4+ assimilation due to exogenous α-oxoglutarate clearly indicated that the availability of carbon skeletons is a key limiting factor for NH4+ assimilation in tomato plants. Yuan et al.17 investigated the role of α-oxoglutarate in regulation of carbon and nitrogen metabolisms in non-photosynthetic tissues of rice and found that exogenous α-oxoglutarate enhanced the activity of GOGAT (EC 1.4.1.14) and GS (EC 6.3.1.2) enzymes. And the increase in ammonium uptake and glutamate (Glu) was also observed in their study17. However, no information is available on the impacts of exogenous α-oxoglutarate on soybean seedling leaf ammonium assimilation and proline accumulation.2-oxoglutarate (2-OG), a key organic acid of the tricarboxylic acid (TCA) cycleThe objective of this investigation was to examine the impacts of exogenous α-oxoglutarate on ammonium assimilation proline accumulation and photosynthesis of soybean seedling exposed to cold stress (6 °C) in pot experiment. Through this study, we expected to understand the optimum dose of exogenous α-oxoglutarate applied to soybean seedling when exposed to cold stress. Our results could provide theoretical reference for taking measures against chilling in soybean production in Northeast China.A pot experiment was conducted at Experimental Station of Jiamusi Branch of Heilongjiang Academy of Agricultural Sciences, China. Experimental pot diameter is 14 cm and the depth is 16 cm. Each pot was filled with 500 g of soil. The soil used in the present study is the typical black soil with silty clay loam texture. Two spring soybean varieties Henong60 and Heinong48 were used in this experiment. Henong60, released in 2009, is cold-tolerant variety with 100-seed weight 18 g and plant height is 60 cm. The content of its protein and oil is 38.47% and 22.25%, respectively. The growth habit is determinate with white flowers. The effective accumulative temperature for maturity (≥ 10 °C) is about 2290 °C. Heinong48, released in 2004, is cold-sensitive variety with 100-seed weight 22 g and plant height 90 cm. The content of its protein and oil is 45.23% and 18.43%, respectively. The growth habit is semi-determinate with purple flowers. The effective accumulative temperature for maturity (≥ 10 °C) is about 2380 °C.21 with minor modifications. Soybean was not inoculated before seeding. Ten seeds of each cultivar were sown by hand in each pot. There are four soybean seedlings left in the pot after emergence. The seedlings of the two soybean varieties were grown in pots in field. Upon the appearance of the first true fully expanded leaves (V1 stage), 4 ml of exogenous α-oxoglutarate was sprayed to each pot of Henong60 and Heinong48. There were four exogenous α-oxoglutarate treatments including 0, 2.5, 5.0 and 7.5 mmol/L, identified as A0, A2.5, A5.0 and A7.5. Treated seedlings were transferred into controlled environment growth chambers at 14-h-light/10-h-dark. The light intensity was 120 µmol m−2 s−1 and the relative humidity was 65%. The cold stress degree is 6 °C. There were three replicates for each treatment and there were four pots for each replicates. Leaf samples for each treatment were collected after cold stress of 24 h (S1 stage) and 48 h (S2 stage).The simulation of cold stress was conducted according to the method by Tambussi et al.22. One unit of NADH-GDH activity is defined as the reduction of 1 µmol of coenzyme (NADH) per min at 30 °C. One unit of GS activity is the amount of enzyme that catalyzes the formation of 1 µmol γ-glutamylhydroxamate per min at 37 °C.The activities of NADH-GDH and GS were determined according to Lu et al.23. Photosynthetic rate was determined by using the CI-340 portable photosynthesis measuring system .Photosynthetic rate was measured according to Gai et al.SPAD) was conducted by using SPAD-502 Chlorophyll Meter Model .Measurement of chlorophyll content  according to the method by Lu et al.P < 0.05) between treatments were determined by using LSD test.All the data were statistically analyzed in SPSS 22 software. Analysis of variance (ANOVA) was used to test for significance, and significant differences  can also catalyze ammonium incorporation into glutamatet Tables , 5, 6. S15. The physiological mechanism by which α-oxoglutarate alleviates the adverse effect of cold on soybean seedlings in the present study is that foliar application of α-oxoglutarate increased GS activity, GDH activity, assimilated more ammonium and led to decreased ammonium content and synthesized more glutamate which is necessary for proline biosynthesis. In the end, an increase in proline content could explain the reason why α-oxoglutarate alleviates the adverse effect of cold on soybean seedlings.2-oxoglutarate (2-OG) is a key metabolite at the crossroads of carbon/nitrogen metabolism as it is required for ammonia assimilation2.5, A5.0, and A7.5 was significantly higher than that at A0 at S1 and S2 stages. In the present study, A5.0 gave the highest chlorophyll content for two soybean varieties at S1 and S2 stages. There was no significant difference in chlorophyll content for two soybean varieties between A2.5 and A7.5 at S1 stage.Results from this study indicated that leaf chlorophyll content was significantly affected by exogenous α-oxoglutarate, variety and their interaction at S1 stage and S2 stage under cold stress Table . The lea2.5, A5.0, and A7.5 than at A0 at both S1 and S2 stages. In our study, A5.0 produced the highest photosynthetic rate for two soybean cultivars at S1 and S2 stages. There was no significant difference in photosynthetic rate for two soybean varieties between A2.5 and A7.5 at S1 stage.The photosynthetic rate is an important parameter of photosynthesis. The data in Table 13. The chlorophyll content and photosynthetic rate are important parameters of photosynthesis. Many studies indicated that exogenous application of α-ketoglutarate had positive effect on photosynthesis38. Our results supported the work conducted by Ge37 who reported that α-oxoglutarate significantly increased leaf chlorophyll content and photosynthetic rate in winter-wheat seedling exposed to drought stress. Foliar application of α-oxoglutarate increased GS activity, GDH activity, assimilated more ammonium and led to increase in proline content for both varieties at S1 and S2 stages. Because there is a great need of NADPH when proline is synthesized, NADP+ will be produced39. As known to all, NADP+ is necessary to photosynthesis. Therefore, we infer that exogenous α-oxoglutarate increased photosynthetic rate by enhancing proline content.α-ketoglutarate plays an important role in nitrogen and carbon metabolism in plantsExogenous α-oxoglutarate significantly increased leaf GS activity, NADP-GDH activity, glutamate content, proline content and photosynthesis of soybean seedling exposed to cold stress at S1 and S2 stages. Accordlingly, the ammonium content was significantly decreased by exogenous α-oxoglutarate at S1 and S2 stages. 5.0 mmol/L of exogenous α-oxoglutarate is the optimum concentration in our study. There was a significant difference in leaf GS activity, NADP-GDH activity, glutamate content, proline content and photosynthesis between Henong60 and Heinong48 under cold stress at S1 and S2 stages. Those results suggested that exogenous α-oxoglutarate could alleviate the adverse impact of cold stress. In our region, cold stress usually not existed more than two days. Therefore, it is noted that foliar application of α-oxoglutarate is a feasible way to prevent soybean against cold stress in Northeast China.


In clinical cohort studies, high expression of long-chain acyl-coenzyme A synthetases 1 (ACSL1 gene) in peripheral white blood cells of patients with acute myocardial infarction (AMI) has been utilized as molecular markers of myocardial infarction diagnosis. The plasma triglyceride level of AMI patients is significantly higher than that of healthy individuals. We hypothesized that the high expression of ACSL1 increases the level of triglyceride, which is one of the pathogenesis of AMI promoted by ACSL1. In this report, cell culture based methods were adopted to test the hypothesis and further investigate the effect and mechanism of ACSL1 on lipid metabolism. In this study, liver cells of healthy individuals were cultured, the overexpression and the knockdown vectors of ACSL1 were constructed and transfected into liver cells. The transfection was verified at the mRNA and protein level. Intracellular triglyceride content was quantitatively analyzed using ELISA. Changes of genes related to lipid metabolism were subsequently measured through PCR array. Overexpression of ACSL1 led to higher gene expression and protein levels compared to control and the triglyceride content was significantly increased in overexpressing cells. The expression level of fatty acid oxidation pathway PPARγ was significantly down-regulated compared with the control group, as were genes associated with fatty acid synthesis pathways: SREBP1, ACC, FAS, and SCD1. ACSL1 knockdown decreased the content of triglyceride whereas PPARγ was up-regulated and SREBP1, ACC, FAS, and SCD1 were down-regulated compared with the control group. In summary, high expression of ACSL1 reduced fatty acid β-oxidation through the PPARγ pathway, thereby increasing triglyceride levels. Acute myocardial infarction (AMI) refers to myocardial necrosis caused by acute, persistent ischemia and hypoxia due to coronary atherosclerosis. AMI is a complex disease caused by the interaction of multiple factors, such as genetics, immunity and environment ACSL1 exists in cells of the liver, heart and fat Previous cohort studies indicated that the expression of ACSL1 in peripheral white blood cells of AMI patients increased compared with that of the healthy control group. The high expression of ACSL1 was an independent risk factor for AMI. The triglycerides content in peripheral white blood cells of patients with myocardial infarction was shown to increase Human liver cells and 1640 medium were purchased from Zhongqiao Xinzhou Biotechnology Co. Ltd . Australian fetal bovine serum and dual antibody were purchased from Invitrogen, Carlsbad, CA, USA and transfection reagents were purchased from Promega (Beijing) Biotech Co., Ltd. ACSL1 monoclonal antibody  was purchased from Abcam, Cambridge, MA, USA.Vectors which over express or under express ACSL1 were synthesized by GenePharma Co. Ltd.,  and were designed according to the ACSL1 mRNA sequence of human cells (NM_001995) published by the NCBI website. The empty vectors encoding for green fluorescent protein (GFP) were used as the negative control.2 incubator. The 1640 medium was used . When the confluency of cells reached 70% to 80%, the cells were harvested with trypsin and cultured for another 24 hours.Human hepatocytes were cultured at a constant temperature of 37 °C in a 5%COCells were transfected when the confluency reached over 70%. 3μg pBI-CMV3-ACSL1, pBI-CMV3, 3 μg pGPU6/GFP/Neo-ACSL1, or pGPU6/GFP/Neo-shNC vectors were added to 150μl serum-free RPMI 1640 media. 7.5 μl FuGENE®HD Transfection Reagent was added to the solution before being incubated for 15 minutes. The mixture was evenly dripped clockwise into a six-well plate and placed in the incubator. After 24 hours of culture, the cell state and distribution of green fluorescent protein could be observed under a fluorescence microscope.Protein lysates were prepared 48 hours after transfection, and the protein concentration was measured using the BCA protein detection kit. 30 μg of protein samples were denatured at 95 °C for 5 minutes. After SDS-PAGE electrophoresis the proteins were transferred onto a PVDF membrane at 200 mA for 60 min. The membrane was blocked at room temperature with a blocking buffer (5% skimmed milk powder) for 1 hour. Subsequently, the membranes were incubated with the primary antibody overnight at 4 °C. After washing, the membrane was then incubated with the secondary antibody at room temperature for 1 hour and the signal was developed using a chemiluminescent imaging system.® Premix Ex Taq. The primer sequences are shown in Table 24 hours after the cells were transfected, total RNA was extracted using the innuPREP RNA Mini Kit . The RNA quality of the collected samples was tested by agarose gel electrophoresis. The concentration and purity of RNA were tested using a nanodrop spectrophotometer . cDNA was obtained using reverse transcription kit . Quantitative real-time PCR was conducted using SYBRThe tissue cell triglyceride enzyme assay kit (Applygen Technologies Inc.) was used to measure triglyceride content. After cell lysis, 20 μl supernatant was mixed with the working solution. The elisa plates were shaken gently and incubated at 37 °C for 10 minutes. The OD value of the samples was tested .t-test was used to compare differences between groups, using P <0.05 as the level of statistical significance.Data were analyzed using SPSS 22.0 . Statistical analysis of the results and mapping were performed through GraphPad Prism 7.0. The results were expressed as mean ± standard deviation (SD). Independent sample After transfection of human hepatocytes with ACSL1 RNAi and overexpression vector for 24h, cell morphology, and fluorescence expression levels were observed under a fluorescence microscope. Green fluorescence could be observed in cells transfected with the pBI-CMV3-ACSL1 overexpression vector and the empty vector of pBI-CMV3, as well as the pGPU6/GFP/Neo-ACSL1 knockdown vector and the pGPU6/GFP/Neo-shNC negative control vector. GFP was evenly distributed in the Petri dish   . The relative expression levels of fatty acid synthesis pathway related genes were as follows: SREBP1 0.13 ± 0.02 vs 1.01 ± 0.20, (P=0.001), SCD 0.20 ± 0.01 vs 1.02 ± 0.28, (P=0.007), ACC 0.14 ± 0.12 vs 1.00 ± 0.14, (P=0.001), FAS 0.15 ± 0.03 vs 1.00 ± 0.09, (P<0.001)   Fig. .P=0.024) . The relative expression levels of genes related to fatty acid synthesis pathway were as follows: SREBP1 0.85 ± 0.03 vs 1.00 ± 0.01 (P<0.001), FAS 0.21 ± 0.02 vs 1.00 ± 0.01 (P<0.001), SCD 0.70 ± 0.02 vs 1.06 ± 0.44 (P=0.226), ACC 0.60 ± 0.06 vs 1.04 ± 0.36 (P=0.107) , which indicated overexpression of ACSL1 increased intracellular triglyceride levels by increasing intracellular fatty acid synthesis rather than by altering fatty acid oxidation Lipid accumulation in the liver was due to a combination of reduced fatty acid β-oxidation and increased fat synthesis Fatty acids, an important source of energy for mammals, are chemically inert and need to be activated by ACSL1 in cells outside of the mitochondria to form acyl-coenzyme A before they enter the metabolic pathway This study indicates that overexpression of ACSL1 in human liver cells results in decreased expression of the fatty acid oxidation-related gene PPARγ, thereby reducing the fatty acids β-oxidation and increasing triglyceride levels. PPARγ is upregulated after ACSL1 downregulation, thereby reducing triglyceride levels.It was shown that Peroxisome proliferators which activate receptor-α (PPAR α) and PPARγ are associated with inducing ACSL1 expression in the heart and adipose tissue, and have an essential role in β-oxidation PPARγ is a member of the peroxisome proliferation-activated receptor (PPAR) subfamily. Three types of PPAR subtypes are known: ppar-α, ppar-δ, and ppar-γ, with different distributions and specific functions In this study, it was also found that pathway genes related to fatty acid synthesis, such as SREBP1, ACC, FAS and SCD1, are significantly down-regulated after overexpression of ACSL1. No consistent results are found after ACSL1 downregulation. Therefore, whether ACSL1 gene affects triglyceride metabolism through the fatty acid synthesis pathway is still uncertain. However, our study confirms that ACSL1 gene affects plasma triglyceride levels through PPARγ.de novo fatty acids produced in the tissue Sterol regulatory element-binding proteins (SREBPs) consist of three closely related transcription factors SREBP-1a, SREBP-1c and SREBP-2 SREBPs are conserved from fission yeast to humans, which regulate the expression of genes that control cell lipid homeostasis, depending on species-specific requirements. Therefore, these proteins and their target genes perform complex feedback regulation on SREBP activity at multiple levels The liver is an important organ involved in lipid metabolism. Primary human hepatocytes are considered as the gold standard for investigating the molecular mechanisms involved in the development of nonalcoholic fatty liver disease (NAFLD) The ACSL1 gene affects triglyceride levels.The high expression of ACSL1 gene decreases fatty acid β-oxidation by PPARγ pathway, which increases the level of triglycerides.Therefore, this research confirms that the high expression of ACSL1 gene, which increases the level of plasma triglyceride, is one of the mechanisms that promote the incidence of AMI.


Drosophila melanogaster. Two proteins with DM9 repeats have been recently identified from Pacific oyster Crassostrea gigas as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as CgDM9CP-4) was identified from C. gigas. CgDM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of CgDM9CP-4 in circulating hemocytes were decreased after LPS, PGN and Vibrio splendidus stimulations. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs)  and microorganisms . CgDM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the CgDM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. CgDM9CP-4 was highly expressed in non-phagocytes . The reactive oxygen species (ROS) and the expression levels of cytokines in CgDM9CP-4-positive hemocytes were much lower than that in CgDM9CP-4-negative hemocytes. The mRNA expression level of CgDM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors . In addition, CgDM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that CgDM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens.DM9 refers to an uncharacterized protein domain that is originally discovered in  Innate immunity is the first line for organism to defense against infections , 2. PattDrosophila melanogaster, different marker proteins have been identified from subpopulations of hemocytes  with two tandem DM9 domains was identified from C. gigas with high sequence similarity with CgDM9CP-1 and CgDM9CP-2  determine its pathogen recognition activity and specific function in hemocytes, and (3) further cognize the involvement of DM9CPs in maturation and differentiation of mollusk hemocytes.The Pacific oyster gDM9CP-2 . ObjectiC. gigas, about two years old (average shell length of 13.0 cm), were collected from a commercial farm in Qingdao, Shandong Province, China, and cultured in filtered aerated seawater at 18°C for a week before processing.Adult pacific oysters g, 4°C for 10 min to harvest the hemocytes. All these samples were stored at -80°C using Trizol reagent (Invitrogen) for subsequent RNA extraction.Different tissues, including gonad, muscle, mantle, gill, and hepatopancreas, were obtained from six adult oysters as parallel samples. Hemolymph from the oysters was collected and immediately centrifuged at 800 × V. splendidus in sterile sea water suspension (1×109 CFU mL-1), 100 μL LPS from E. coli 0111:B4 , 100 μL of PGN from S. aureus , sterile sea water, respectively. The untreated oysters were employed as blank group. After treatment, the oysters were returned to water tanks and six individuals from each group were randomly sampled at 3, 6, 9, 12, 24, and 96 h post-injection, respectively. The hemolymph was collected from the oyster and centrifuged at 800 × g, 4°C for 10 min to harvest the hemocytes. All these samples were stored at -80°C using Trizol reagent for subsequent RNA extraction. The gills were also collected from the V. splendidus and sterile sea water stimulated oysters at 0, 3, 6, 9, 12, and 24 h for subsequent RNA extraction.After the acclimatization, two hundred oysters were randomly divided into five groups with forty individuals in each group and kept in aerated tanks. Each oyster in the four treated groups received an injection of 100 μL live Total RNA was extracted from samples using Trizol reagent. First-strand cDNA synthesis was carried out based on Promega M-MLV RT Usage information using the DNase I (Promega)-treated total RNA as template and oligo (dT)-adaptor  was retrieved from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). A pair of gene specific primers, P1 and P2 , and sequenced by sequencing primers (Sequence information of CgDM9CP-4. The protein motif feature of CgDM9CP-4 was predicted by Simple Modular Architecture Research Tool (SMART) 7.0 (http://smart.embl-heidelberg.de/). ClustalW multiple alignment program 1.81 and multiple alignment show program 2.0 (http://www.bioinformatics.org/sms2/) were used to perform the multiple sequence alignment of DM9 domain containing proteins.The EditSeq module of DNAStar Laser gene software suite 11.0.0 was used to analyze the deduced amino acid sequence of CgDM9CP-4 mRNA transcripts were detected by SYBR Green fluorescent qRT-PCR. Two gene specific primers for CgDM9CP-4, P3 and P4  and given in terms of relative mRNA expressed as mean ± SD (N = 4).The SYBR Green qRT-PCR assay was carried out in an ABI PRISM 7500 Sequence Detection System (Applied Biosystems) according to the manual. Dissociation curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The relative expression of CgDM9CP-4 was amplified with the primers P7 and P8 (NdeI and XholI (NEB) cleavage site sequences added to the 5’ end, respectively. The PCR fragment was digested by restriction enzymes NdeI and XholI, and ligated into the same restriction enzymes sites of expression vector pET-30a (Novagen). The recombinant plasmid (pET-30a-CgDM9CP-4) was transformed into E. coli Transetta (DE3) (TransGen). The positive transformants were incubated in LB medium containing 50 μg mL-1 kanamycin at 37°C with shaking at 220 rpm for 4 h. When the culture mediums reached OD600 of 0.5–0.7, IPTG was added to the LB medium at a final concentration of 1 mmol L-1, and incubated at 16°C with shaking at 180 rpm for 20 h. The bacterial culture was sonicated and centrifuged to get the supernatant containing soluble target protein. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) was purified by Ni2+ chelating Sepharose column (Roche), and the purified protein was dialyzed out of imidazole against ddH2O at 4°C for 24 h. The protein was resolved by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized with Coomassie Bright Blue R250. The concentration of purified soluble protein was quantified by BCA method. The obtained protein was stored at -80°C for subsequent experiment.The completed cDNA fragment of CgDM9CP-4 was immuned to 6 weeks old rats with a weight of approximately 120 g to acquire polyclonal antibody. Briefly, rCgDM9CP-4 of 1mg mL-1 was mixed with freund’s adjuvant to immunize the female rats for four times.The purified rCgDM9CP-4 were electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked with PBST containing 5% milk at room temperature for 1 h, and incubated with antibodies (diluted at 1:4000) for 1 h at room temperature with rocking, followed by three times of washes with PBST. Membranes were incubated with HRP-labeled Goat Anti-Rat IgG(H+L)  for 1 h at room temperature with rocking, followed by three times of washes with PBST. Membranes were incubated briefly in Western lighting ECL substrate system  before exposure to KODAK X-OMAT AR X-ray film .After SDS-PAGE, the samples of r-1, pH = 9.6) were used to coat 96-well microliter plates (Costar) and incubated at 37°C for 3 h. The wells were washed three times with PBST for 5 min per time and then blocked with 200 μL of 5% BSA in PBS at 37°C for 1 h. The plate was washed three times and 100 μL of rCgDM9CP-4 solution in different concentration was added to the wells with the presence of 5 mmol L-1 CaCl2 and 0.1 mg mL-1 BSA. After incubation at 37°C for 1 h, the plate was washed three times with PBST and then the bound protein was detected immunochemically. Rat anti-His taq monoclonal antibody  was added as primary antibody and incubated at 37°C for 1 h, and then incubated with 100 μL of goat-anti-rat Ig-alkaline phosphatase conjugate  as the second antibody. The wells were washed four times with TBST for 15 min after each incubation and then incubated with 100 μL of 0.1% (w/v) nitrophenyl phosphate  in 50 mmol L-1 carbonate buffer (pH= 9.8) containing 0.5 mmol L-1 MgCl2 at room temperature in dark for 15 min. The reaction was stopped by adding 50 μL of 2 mol L-1 H2SO4 per well and the absorbance was measured with an ELISA reader at 450 nm (Molecular Devices). The wells filled with 100 μL of TBS were used as negative control (blank). The apparent dissociation constant (Kd) values were calculated using Prism 5.00 software (GraphPad software) with a one-site binding model and nonlinear regression analysis where A is the absorbance at 450 nm.The PAMP binding assay was performed according to previously description with some modification . BrieflyS. aureus and M. luteus), gram-negative bacteria  and fungi (P. pastoris and Y. lipolytica) were used to exam the microbial binding activity of rCgDM9CP-4. The microbes were suspended in TBS (OD600 = 2), and incubated with rCgDM9CP-4 (100 μg mL-1) under rotation slightly at 4°C overnight. After three times washing, the bound proteins were dissociated from the microorganisms by loading buffer and analyzed by Western blot as described above. rTrx was employed as negative control, and the purified protein was set as positive control.Microbial binding activity was measured according to previous report with some modification . Gram-poS. aureus and M. luteus), Gram-negative bacteria (E. coli) and fungi (P. pastoris) were suspended in TBS buffer at 2×109 cells mL-1. An aliquot of 50 mL microbial suspension was incubated with 50 μL purified protein (1 mg mL-1) at room temperature under rotation for 30 min. rTrx protein was set as control, and 50 μL TBS was used as blank control. After three times of washing with PBS, 50 μL FITC-labeled rat anti-his taq antibody (diluted 1:1000) was used to suspend the microbial suspension, and incubated in dark for 20 min. The agglutination was observed under fluorescence microscopy (Olympus).The overnight cultured Gram-positive bacteria  according to the previous description were used to suspend the hemocytes, and the suspension was added into cell culture dishes (CgDM9CP-4 (diluted 1:500 in blocking buffer) as the primary antibody at 37°C for 1 h. After washing three times with PBST, the samples were incubated with Alexa Fluor 488-labeled goat-anti-rat antibody (diluted 1:1000 in PBST) as the second antibody at 37°C for 1 h. After another three times of washing with PBST, DAPI  was added to stain the nucleus and DIL  to stain the membrane. After the last three times of wash, samples were observed under fluorescence microscopy (Olympus) and Laser Scan Confocal Microscope (ZEISS).Immunohistochemistry (IHC) and immunocytochemistry (ICC) assays were conducted as previous description with some modification , 37. Fore dishes . After ig, 4°C for 10 min immediately. After three times of wash in M-L15, same volume of 4% PFA was added to fix the hemocytes for 5 min. After three times of wash in TBST, the hemocytes were then blocked with 500 μL of 3% BSA in PBS at room temperature for 1 h, and then the supernatant was removed and the hemocytes were incubated with 500 μL antibody of CgDM9CP-4  as the primary antibody at room temperature for 1 h. The hemocytes were then incubated with Alexa Fluor 488-labeled goat-anti-rat antibody  as the second antibody at 37°C for 1 h after three times washing. The expression pattern of CgDM9CP-4 was analyzed on an FACS Arial II flow cytometer .For flow cytometry analysis, hemocytes from six oysters were collected and centrifuged at 800 × To analyze hemocytes form the gills, gills from three oysters were collected and cut into small pieces, then soaked in M-L15 for 10 min to extract the hemocytes from gill. Flow cytometry analysis and immunocytochemistry were then performed as described above.CgDM9CP-4 antibody. For intracellular cytokine expression, antibody of CgTNF-α (CGI_10008787 or CGI_10008788), CgIFNLP or CgIL17-5  were added together with CgDM9CP-4 antibody as the primary antibodies. The hemocytes were then incubated with Alexa Fluor 488-labeled goat-anti-rat antibody and Alexa Fluor 594-labeled goat-anti-mouse antibodies (diluted 1:1000 in 3% BSA) as the second antibody at 37°C for 1 h after three times washing. The phagocytic rate and ROS level, as well as the expression levels of cytokines in CgDM9CP-4-positive hemocytes were analyzed on an FACS Arial II flow cytometer .Phagocytosis assay was performed following the previous description . Brieflyg, 4°C for 10 min to harvest the hemocytes. The hemocytes were suspended in resuspension and incubated with Latex beads  at room temperature for 1 h with rotation. The viability of hemocytes was measured by propidium iodide positivity under fluorescence microscopy (Olympus). The single-cell suspensions were sorted by flow cytometer at the sorting efficiency of 90%. The separated phagocytes and non-phagocytes were rechecked for the validity of the cell sorting. The total RNA was extracted from the hemocytes using Trizol reagent. cDNA synthesized and SYBR Green fluorescent qRT-PCR as described above.Phagocytes and non-phagocytes were separated following the previous description . In brieCgGATA, CgRunt, CgSCL and CgNotch cDNA fragments from C. gigas following the previous description  of CgGATA, CgRunt, CgSCL, CgNotch and EGFP were injected into the adductor of each oyster, respectively. The oysters received an injection of 100 μL sterile seawater were set as blank control. Hemolymph samples from six oysters of each group were collected at 24 h post-dsRNA injection for total RNA extraction. The efficiency of gene knock-down was checked using qRT-PCR. The expression level of CgDM9CP-4 was detected by qRT-PCR as described above.T7 promoter linked primers (cription . EGFP DN reports . The RNA-1) in an aerated tank for 48 h. Cross sections of gill were made as described above. The slides were then fixed with 4% PFA at room temperature for 15 min. After three times washing with 3% BSA in TBS, 0.1% Triton® X-100 in TBS was used to treat the samples at room temperature for 10 min. CgDM9CP-4 positive cells were stained as mentioned above and after the final three times washing with TBS (containing 3% BSA), 0.1% Triton® X-100 in TBS was used to treat the samples at room temperature for 10 min. Samples were then incubated with the 1 × Click-iT™ Reaction Buffer (provided with the kit) for 30 min, and washed thoroughly with PBS (3 × 15 min). EdU were analyzed on an FACS Arial II flow cytometer . For the new generated hemocytes in gill, slices were incubated with DAPI  for 5 min, washed extensively with PBS, mounted with 80% glycerin, and monitored under a Laser Scan Confocal Microscope (ZEISS).EdU labeling assay was performed as previously described . In briep < 0.05 and extremely significant at p < 0.01.All the data were expressed as mean ± standard deviation (N = 3 or 4), and analyzed by Statistical Package for Social Sciences (SPSS) 18.0. The significant differences among groups were tested by one-way analysis of variance (ANOVA) and multiple comparisons. Statistically significant differences were designated at CgDM9CP-4 was retrieved from NCBI (GenBank Accession No. XP_011421715). A cDNA fragment of 438 bp was amplified by one pair of gene specific primers. The open reading frame (ORF) of CgDM9CP-4 encodes a polypeptide of 145 amino acids with a predicted molecular mass of 16 kDa. There were only two tandem DM9 domains in CgDM9CP-4 identified by SMART analysis (The sequence of CgDM9CP-4 shared high similarities with other known DM9CPs. CgDM9CP-4 shared 63% similarity with Natterin-4 from C. gigas (XP_011426076), 38% with GL11485 from Drosophila persimilis, 42% with Natterin-3 from Zootermopsis nevadensis (KDR19911), 35% with Natterin-3 from Thalassophryne nattereri (AAU11824), 40% with Natterin-4 from Stegodyphus mimosarum (KFM79713) and 37% with GK20873 from Drosophila willistoni (XP_002061352)  and 30.78-fold (p < 0.01) higher than that of adductor, respectively.CgDM9CP-4 mRNA was then examined in oyster hemocytes at 0, 3, 6, 9, 12, 24 and 48 h after V. splendidus stimulation , 9  and 12 h  compared to blank group, respectively (p < 0.01) and 48 h , respectively , 9 , 12  and 24 h , and raised up to the original level at 48 h compared with 0 h after LPS stimulation , 9 , 12  and 24 h , and recovered to normal level at 48 h  while relative lower affinities to LPS and β-glucan (Kd=6.081×10-7M and 12.17×10-7M). As a control, no binding affinity was found in the group of rTrx (data not shown).The PAMPs binding activity of rS. aureus and M. luteus) and gram-negative (E. coli) bacteria, as well as fungi (Y. lipolytica) were used to test the agglutination activity of rCgDM9CP-4. rCgDM9CP-4 exhibited no agglutinating activities towards any of the microorganisms. Instead, rCgDM9CP-4 showed binding activity to all the tested microorganisms as clear signals of rCgDM9CP-4 were detected on the membranes , gram-negative  and fungi (P. pastoris and Y. lipolytica)] groups  , CgIFNLP and CgIL17-5, in CgDM9CP-4-positive and -negative hemocytes. The relative protein levels of CGI_10008787, CGI_10008788, IFNLP, IL-17 indicated by mean fluorescence intensity were 2566 ± 472, 2195 ± 231, 2895 ± 316 and 1523 ± 265 in CgDM9CP-4-positive hemocytes respectively   was 1190.3-fold (p < 0.01) higher than that in phagocytes , CgRunt , CgSCL  and CgNotch  knockdown groups were all significantly down-regulated in comparison with that in dsEGFP group, respectively , the National Natural Science Foundation of China , the Outstanding Talents and Innovative teams of Agricultural Scientific Research and Earmarked Fund (CARS-49) from Modern Agro-industry Technology Research System, the Fund for Outstanding Talents and Innovative Team of Agricultural Scientific Research in Ministry of Agriculture, the Research Foundation for Distinguished Professor in Liaoning (XLYC1902012 to LW) and the Climbing Scholar in Liaoning (to LS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The usefulness of a selected selenol compound is illustrated by the total synthesis of a biologically active human chemotactic protein, which plays an important role in innate and adaptive immunity.While thiol-based catalysts are widely employed for chemical protein synthesis relying on peptide thioester chemistry, this is less true for selenol-based catalysts whose development is in its infancy. In this study, we compared different selenols derived from the selenocysteamine scaffold for their capacity to promote thiol–thioester exchanges in water at mildly acidic pH and the production of peptide thioesters from  Chemical protein synthesis is now established as an alternative mode of production of tailored proteins for use in diverse fields of research, primarily biological and medicinal research . ThioestN-(2-mercaptoethyl)amides, i.e., N,S-acyl shift systems 21Step 1. Synthesis of peptide thioester off peptide 26  and AcA-MTG  were then successively dissolved in the MPAA solution (1.3 mL) and the pH of the mixture was adjusted to 7.2 by addition of 6 M NaOH. The mixture was stirred at 37 °C for 8 h. 3.0 additional equivalents of AcA-MTG were added and the mixture was stirred an additional 16 h at 37 °C under nitrogen atmosphere. Completion of the reaction was ascertained by UPLC-MS analysis.To a solution of Gn·HCl (1.1 g) in 0.1 M pH 7.4 phosphate buffer (1.2 mL) was added MPAA . SEA17  and MPA  were added to the previous mixture. The pH was adjusted to 4.0 by addition of 6 M NaOH and the reaction mixture was stirred at 37 °C for 72 h. After completion of the reaction, the mixture was diluted in 10 mL of water containing 0.1% TFA. The crude was purified by HPLC  to give peptide thioester 21 as a white solid after lyophilization .After protection of the N-terminal Cys residue (24 h), the subsequent SEA/MPA exchange reaction was carried out in one pot. TCEP∙HCl , diselenide catalyst 21 was analyzed by UPLC-MS : 4562.40, found: 4562.87.MALDI-TOF  and peptide 22  were then successively dissolved in the MPAA solution (0.12 mL) and the pH of the mixture was adjusted to 7.2 by addition of 6 M NaOH. The mixture was stirred at 37 °C for 24 h under nitrogen atmosphere. Completion of the reaction was insured by UPLC-MS analysis.To a solution of Gn·HCl (0.57 g) in 0.1 M, pH 7.4 phosphate buffer (0.60 mL) was added MPAA  and the pH of the mixture was adjusted to 7.2 by addition of 6 M NaOH. Peptide 2OH. The reaction mixture was first acidified to pH 3.0 with 6 M HCl and extracted 3 times with 1.0 mL of Et2O to remove MPAA. A 0.5 M solution of NH2OH·HCl in H2O  was then added and the pH was adjusted to 5.5. After 4 h stirring at 37 °C, pH was adjusted to 3.0 with 0.25 g mL−1 aqueous solution of TCEP∙HCl. The crude was further diluted in 4.0 mL of water containing 0.25% TFA and purified by HPLC  to afford peptide 23 as a white solid after lyophilization .After completion of the NCL ligation (24 h), the AcA protecting group was removed by reaction with NH23 was analyzed by UPLC-MS : 7900.22, found: 7901.49.MALDI-TOF  and peptide thioester 24  were then successively dissolved in the MPAA solution (91 µL) The mixture was stirred at 37 °C for 24 h under nitrogen atmosphere. Completion of the reaction was insured by UPLC-MS analysis.To a solution of Gn·HCl (0.57 g) in 0.1 M pH 7.4 phosphate buffer (0.60 mL) was added MPAA  and the pH of the mixture was adjusted to 7.7 by addition of 6 M NaOH. Peptide v/v containing 0.1% TFA, 50 °C, detection at 215 nm, 6 mL min−1, 0–38% eluent B in 10 min, then 38–58% eluent B in 60 min, C18XBridge column) to give 9-GN-l as a white solid after lyophilization .After completion of the NCL ligation (24 h), TCEP∙HCl  was added to the reaction medium to reduce any mixed disulfide formed during ligation. The resulting mixture was diluted in 3.0 mL of water containing 0.1% TFA. The crude was purified by HPLC : 8436.79, found: 8436.65.MALDI-TOF  was dissolved in PBS containing 10% by vol of glycerol, 2 M of Gn·HCl, 1 mM reduced glutathione, 0.2 mM oxidized glutathione . The reaction mixture was let 1 h at 0 °C, then 8 days at 20 °C.The linear polypeptide The protein was dialyzed twice (respectively 2 h and 24 h) at 20 °C against 200 mL PBS containing 10% in vol of glycerol. 0.71 mL of the dialyzed solution were recovered and the concentration of the folded protein was determined using UV quantification at 280 nm 9-GN was analyzed by UPLC-MS  as previously described .4 cells/well in 50 μL of RPMI 1640 medium with 0.1% FCS) was performed in a 48-well Boyden microchamber (Neuroprobe) through a standard 5-μm pore filter (Neuroprobe), at 37 °C for 1 h. Migration was evaluated in response to synthetic 9-GN (1 to 100 nM in RPMI 1640 medium containing 0.1% FCS), CCL2 used as positive control , and medium alone used as negative control. The filter was stained using Diff–Quik reagent. Migrated cells were counted on the lower side of the filter in three randomly selected high power fields (magnification × 100). Each assay was performed in triplicate. Results are expressed as the difference between mean numbers of cells per high power fields minus the negative control .Monocyte migration (5 × 10bis(2-sulfanylethyl)amido (SEA)/thiol exchange process by selenols derived from selenocysteamine. These data provide important clues regarding the role played by the basic amino group of the selenocysteamine scaffold in catalysis and how to evolve the catalyst to achieve useful catalytic potencies while minimizing synthetic cost. Catalysis by bisdiselenide proved particularly useful and involves presumably an intramolecular general acid catalysis by the proton of the dimethylammonium group. Bisdiselenide can be easily produced in multigram scale from cheap starting materials. Its usefulness is illustrated by the chemical synthesis of a biologically actice 9 kDa granulysin.We conducted detailed kinetic studies of the catalysis of the


Vitamin D, a secosteroid, plays an important role in several physiological processes, and its deficiency can lead to numerous pathophysiological conditions in humans. The primary objective of this study was to develop and validate the robustness of a mass spectrometry-based method capable of quantifying 25(OH)D3 for an upcoming epidemiological survey in India and to pilot test it on healthy volunteers. We first describe the development and validation of various experimental parameters that ascertain the robustness and reliability of 25-hydroxy-vitamin D3 (25(OH)D3) extractions and quantitative measurements from Dried Blood Spot (DBS) samples, where we used eight disks of 3 mm each, punched from the circular spot covering the entire circumference of the spot. Next, we conducted a pilot study, comparing 25(OH)D3 levels from serum and DBS samples from 45 participants using a protocol developed for specifically this purpose. We found that the mean 25(OH)D3 concentrations in DBS samples were comparable to the serum levels (P > 0.05). In summary, our extraction and LC–MS protocol for quantitative 25(OH)D3 measurements are robust and reproducible, and will serve as an invaluable tool for upcoming epidemiological surveys in India and perhaps around the world. Recent reports suggest that it also plays an important role in several other vital physiological functions such as the protection against respiratory tract infections, muscle contractions and nerve conduction2. Humans typically obtain vitamin D from two sources: exposure to sunlight which converts 7-dehydrocholesterol (7DHC) present in epidermal layer of skin into cholecalciferol (vitamin D3) and consumption of vitamin D rich foods such as certain fatty fish, shitake mushrooms and yeast which provide ergocalciferol (vitamin D2)3. In humans, both ergocalciferol and cholecalciferol are hydroxylated by hepatic hydroxylases to 25 hydroxy-vitamin D [25(OH)D2 and 25(OH)D3)]4. Being the primary storage form with longer half-life, the concentration of 25-hydroxy-vitamin D is measured to determine the physiological vitamin D status5.Vitamin D is a secosteroid that plays a significant role in bone mineralization and remodelling by regulating the calcium and phosphorus homeostatic balance4.Vitamin D bioavailability from commonly consumed foods is very low in most populations, particularly when the foods are not fortified. Therefore, the primary source for acquiring vitamin D in most populations is through sunlight exposure, and hence understandably, populations with limited sunlight availability  are more prone to its deficiency. Interestingly, vitamin D deficiency has also been reported in countries located in lower latitudes, where there is no scarcity of sunlight, with India being one of these nations. The majority of the Indian population lives in the region of ample sunshine throughout the year (8.4∘ to 37.6∘ north latitude), and despite this, studies have reported vitamin D deficiency in different age groups and geographical locations of India6. While there are several studies that have consistently reported high prevalence of vitamin D deficiency globally, comparable population scale studies in an Indian population, are to the best of our knowledge, not performed, and hence unavailable.Despite the lack of consensus within the scientific community on desired physiological concentrations of vitamin D, the most commonly accepted definition for vitamin D deficiency indicates a blood concentration less than 20 ng/mL (50 nmol/l) in adults7. The specific objectives our study were (1) to develop and validate the robustness of a mass spectrometry-based method capable of quantifying 25(OH)D3 that we intend using for subsequent epidemiological studies, (2) to assess 25(OH)D3 concentrations in serum and DBS (using the newly developed method) in a pilot sample of healthy adult volunteers.Thus, the primary purpose of the current study was to develop an efficient and robust epidemiological protocol for assessment of vitamin D in various representative groups of the Indian population using dried blood spots (DBS). Dried blood spotting is minimally invasive and a resource friendly alternative to serum/plasma samples, especially in large epidemiological studies. Being minimally invasive it is also the method of choice for paediatric and geriatric populations2H6]-25(OH)D3 were prepared at a concentration of 1 mg/mL in ethanol and stored at − 80 °C until use.Analytical standards for 25(OH)D3 were purchased from Sigma-Aldrich. LC–MS grade acetonitrile, methanol, and hexane were purchased from J.T. Baker (Thermo Fisher Scientific). Double deionized water was purchased from HiMedia Laboratories. Stock solutions of 25(OH)D3, and -25(OH)D3 on each spot. The blood spots were then allowed to dry at room temperature for 2 h and were subsequently transferred to light protective storage polythene bags along with a desiccant. The bags were stored at − 20 °C until extraction. 500 µL of whole blood was separately aliquoted and used for Haematocrit analysis using Yumizen H 500 . Approximately 30 min after the addition of [2H6]-25(OH)D3, the plain and heparin tubes were centrifuged for 10 min at 2500 rpm and aliquots of 500 µL of serum/plasma were prepared. All the aliquots were flash frozen in liquid nitrogen and stored at − 80 °C freezer until extraction. Samples were neither subjected to prolonged light exposure nor multiple freeze/thaw cycles.From each of the 45 study participants, intravenous samples were collected (approximately 3–4 mL) using plain and Heparin tubes. Immediately after collection, 3 mL whole blood from both tubes was aliquoted in an appropriate tube and Calcifediol-dThe charcoal treated serum was used for matrix calibration. Each tube containing 0.5 mL charcoal treated serum was spiked with 25(OH)D3 spanning a concentration range of 2.25, 11.25, 22.5, 67.5, 112.5, 225 ng/mL.The blank DBS cards (Whatman filter paper number 903) were prepared by spotting 55 µL of artificial blood in each spot. Eight discs of 3 mm diameter were punched into a 2 mL Eppendorf tube from the DBS. Each tube was spiked with 10 µL of 25(OH)D3 spanning a concentration range of 6.4, 9.6, 25.6, 51.2, 102.4,153.6 and 204.8 ng/mL.9. To the 500 µL serum sample a mixture of 200 µL of Acetonitrile: Methanol (70:30) was added. The tube was briefly vortexed and then sonicated for 30 min . After sonication, 1 mL hexane was added followed by brief vortexing and centrifugation at 10,000 rpm for 10 min. 900 µL of hexane was carefully transferred to a fresh tube without disturbing the phase separation. The hexane was evaporated to dryness using nitrogen concentrator . The residue was reconstituted in 200 µL of methanol followed by vortexing to ensure uniform mixing.The serum extraction method was a modification of a previously described method10. A circular disc of 3 mm diameter (corresponding to 3.2 µL of whole blood) was punched from a spot on the DBS card using a semi-automated puncher . For the assay, 8 discs from each participant were used (corresponding to 25.6 µL of whole blood). The punch was cleaned with 70% ethanol prior to and after taking sample from each study participant. The discs were placed into 2 mL Eppendorf tubes along with 250 µL of water. Each tube was vortexed for 30 s and then sonicated for 1 h using a sonicator . 250 µL of methanol was added to each tube and the tubes were sonicated for another 30 min. Subsequently, 500 µL of hexane was added to each tube followed by centrifugation at 10,000 rpm for 10 min. Approximately 400 µL of hexane was carefully aspirated in a fresh tube without disturbing the phase separation and evaporated to dryness using nitrogen concentrator . The residue was reconstituted in 25 µL of methanol followed by vortexing to ensure uniform mixing.DBS extraction method was a modification of a previously described method2H6]-25(OH)D3 = 407.5 → 389.5, 407.5 → 371.0, 407.5 → 107.09. Isolation and separation of analytes by chromatography was achieved by using a Shimadzu –Premier C18 150 mm × 2.1 × 3 µm column without derivatization. A typical LC–MS consisted of 10 min, with mobile phase A = water + 0.1% formic acid, and B = methanol + 0.1% formic acid, with a flow rate of 400 μL/min, and had the following gradient sequence: (i) equilibration with solvent B at 90% from 0.0 to 1.4 min, (ii) linear increase in solvent B from 90 to 100% from 1.5 to 4 min, and (iii) equilibration and washing with solvent B at 90% from 4.1 to 10 min. MS acquisition was stopped after 5 min of the LC gradient, and the remaining eluent was directed to waste collection system.The LC–MS system used was Shimadzu 8045 triple quadrupole mass spectrometer (MS) with the electrospray ionization (ESI) source in positive ion polarity mode fitted with Nexera X2 LC-30A UHPLC system. Data were collected in the multiple reaction monitoring (MRM) mode monitored for various vitamin D components in the extract using the following MS parameters: oven temperature 30 ∘C, interface temperature 298 ∘C, desolvation line temperature 196 ∘C, nebulizing gas flow 3.0 L/min, heating gas flow 10.0L/min, drying gas flow 18.0 L/min, IG vacuum 1.7e − 003 Pa, PG vacuum 6.9e + 001 Pa, CID Gas 230 KPa. Each vitamin D3 species and internal standard was identified and quantified using three MRM transitions reported previously, that for the various species were: (i) 25(OH)D3 = 401.2 → 383.3, 401.2 → 365.4, 401.2 → 159.1,; (ii) vitamin D3 = 385.2 → 259.2, 385.2 → 241.0, 385.2 → 107.3; and (iii) the internal standard -25(OH)D3 or 25(OH)D3. As the samples contained endogenous 25(OH)D3, area ratios of qualifier MRM transition 401.2 → 383.3 to quantifier MRM transition 401.2 → 365.4 were calculated and used for the assessment of selectivity for the detection of 25(OH)D3. Representative spectra for the qualifier and quantifier MRM for both the analyte 25(OH)D3, and the internal standard [2H6]-25(OH)D3 are presented in Fig. The method was selectivity tested by comparing chromatograms from five independent samples of vitamin D free serum and artificial DBS with and without spiking H6-25(OH)12. Matrix effects (ion suppression/ion enhancement) are recognized as one of the major potential sources of errors in serum LC–MS/MS analysis of 25(OH)D3, and hence it has been recommended that the matrix effects for any LC–MS/MS measurement of 25(OH)D3 should be evaluated thoroughly12. Also, the ionization efficiency of 25(OH)D3 is known to be low and in the absence of derivatization relatively larger volume of serum/plasma (0.1–0.5 mL) need to be used. Similarly, for DBS samples it has been reported that the composition of blood and its volume dramatically affects metabolite measurements from DSB samples. Hence, to overcome any matrix effects, matrix matched calibration using artificially prepared 25(OH)D3 free blood is recommended5.Although accepted as the gold standard, LC–MS/MS analysis of 25(OH)D3 from serum is not a universal assay and variability in sample preparation, chromatographic separation and ionization/fragmentation have been reported2H6]-25(OH)D3) was spiked in to establish an extraction recovery factor. Addition of internal standard was followed by separation as serum. The peak areas of the internal standard ([2H6]-25(OH)D3) were obtained at the end of extraction and were used to calculate the % recovery of [2H6]-25(OH)D3 in each sample. To ensure a uniform recovery calculation, same procedure was used to calculate recovery from corresponding DBS samples. The mean recovery for serum sample was found to be 79.03% while that for the corresponding DBS sample was found to be 69.72%. The % mean recovery was used to calculate the recovery factor (100/mean % recovery), and these were set at 1.27 and 1.43 for serum and DBS samples respectively.For the current study, we performed matrix matched calibration using charcoal treated serum for serum samples and artificially prepared blood using same charcoal treated serum for DBS samples. Considering the fact that 25(OH)D3 is hydrophobic in nature and is tightly bound to vitamin D binding protein(s), its extraction efficiency from serum may be affected. To ensure the maximum extraction efficiency in serum samples, we used a two-pronged strategy. Firstly, after separation of serum, the samples were flash frozen using liquid nitrogen. The goal of flash freezing was to minimize any potential biochemical reactions in serum that may result in metabolism/degradation of serum 25(OH)D3. Secondly, in a subset of the experimental samples (n = 31), a known concentration of internal standard D3 concentration needed to be normalized to its % haematocrit. The % haematocrit for the artificial blood used for calibration was 37%, and the following formula Eq.  was usedNext, for assessing accuracy and inter-day variance of this method, three samples representing low, medium and high concentrations with five aliquots of each were analysed on 3 different days for both serum and DBS samples. The artificial blood with 37% haematocrit using charcoal treated serum was used as a blank for both serum and DBS samples. To estimate inter-day precision, triplicates of each sample were analysed on three different days, and these were found to be identical in this method Fig. .Figure 42 . Mean vitamin 25(OH)D3 concentrations in serum and DBS samples from these human subjects were 47 ± 19 and 44 ± 19 ng/mL respectively. As the primary aim of our study was to develop an efficient epidemiological protocol for assessment of 25(OH)D3 concentrations in a representative Indian population, we classified the samples as vitamin D deficient (vitamin 25(OH)D3 levels < 20 ng/mL), insufficient (vitamin 25(OH)D3 < 30 ng/mL) and vitamin D sufficient (vitamin 25(OH)D3 > 30 ng/mL) by both the serum and DBS methods , thus validating the robustness of our method. In accordance with a previously reported analysis for such data, we also assessed these findings statistically using a Bland–Altman plot, and found the serum and DBS data to be in good accordance, and within experimental error limits9.Finally, to validate the robustness of our LC–MS/MS method in measuring 25(OH)D3 from human samples, we decided to perform a small pilot study in healthy human volunteers. In this pilot study, serum and DBS samples were extracted and analysed from 45 healthy volunteers, with a mean age ~ 37 years, mean body mass index = 24.5 kg/m13. The B-ring of 7DHC absorbs UVB radiation in the range of 280–320 nm which breaks the bond between C9 and C10 resulting in formation of pre-vitamin D3. Thermal isomerization converts pre-vitamin D3 to D314. The vitamin D3 is hydroxylated at 25th position by hepatic CYP27A1 or CYP2R1 to produce 25 (OH)D3). The 25(OH)D3 is subsequently hydroxylated at C1α in kidneys and other peripheral tissues by CYP27B1 to form 1,25(OH)2D3 which is the biologically active form of vitamin D14. Another cytochrome P450 enzyme (CYP11A1) catalyses sequential hydroxylation of D3 side chain to form several vitamin D metabolites such as 20(OH)D3, 22(OH)D3, ,  and 15. Among these, 20(OH)D3 is the predominant metabolite of this pathway, that can be detected in serum at an approximately 20 times lower concentration compared to D313.In humans approximately 95% of vitamin D3 is synthesized in epidermal keratinocytes from 7DHC17. The CYP11A1 derivatives can be further hydroxylated at C1α by CYP27A1, CYP24A1, CYP11A1, CYP2R1 and/or CYP2A4. It has also been reported that CYP11A1 derived 20,23(OH)2D3 and 1,25(OH)2D3 induce some overlapping gene expression patterns in keratinocytes18. CYP11A1 derived compounds i.e. both the D3 and L3 derivatives display anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties17.Prolonged exposure to UVB can cause photo-isomerization of pre-vitamin D3 to tachysterol3 (T3) which undergo UVB driven conversion to sterol3 (L3). Formation of T3 and L3 prevents excessive formation of pre-vitamin D3 and thus protects from D3 intoxication. Recently it has been reported that L3 undergoes CYP11A1 mediated hydroxylation and the hydroxylated lumisterol derivatives provide protection against UVB damage. The hydroxylumisterol derivatives are reported to be present in serum suggesting additional biological activity19. For the past decade, quantitative vitamin D analysis from serum/plasma using LC–MS has been considered the gold standard given its sensitivity, precision and lowered susceptibility to matrix effect compared to cell-based assays10. Previously published literature has reported that measurement of 25(OH)D3 from DBS using LC–MS/MS is a valid alternative to conventional methods of quantifying vitamin D metabolites from plasma/serum samples. However, being a relatively new and less explored method of analysis, vitamin D analysis from DBS samples shows considerable variability and optimization studies regarding this method are lacking in literature20. The primary goal of our study was to optimize and standardize a method for analysis of 25(OH)D3 using DBS for an upcoming epidemiological study in representative Indian population. For any clinical  assay, the most important performance parameters to be considered are sensitivity, specificity, throughput and labour for sample preparation21. Derivatization i.e. modification of original chemical structure of analyte with diverse tags22 is commonly used to improve the sensitivity of the vitamin D; however, it makes the assay laborious and may hamper accuracy by forming multiple stereoisomers10. Hence, here, we focused on developing an efficient, high throughput method for screening vitamin D deficiency without using chemical derivatization.Physiologically and clinically, vitamin D status refers to the concentration of hydroxylated D3 and D2 together, and the most widely accepted biochemical marker for assessing systemic vitamin D status is the total serum 25-hydroxyvitamin D [25(OH)D] concentration23. It has also been reported that, in a population that consumes food items not being fortified with vitamin D, 25(OH)D2 levels were below the detectable limit and hence its assessment of agreement between DBS and serum samples cannot be implemented in an Indian population24. Therefore here, we decided to assess the relative concentration of only 25(OH)D3 in DBS and serum, and validate a method for the same. There is a common agreement in the scientific community that 25(OH)D3 is a difficult analyte to quantitate and thus methods for the same are sparse in literature25. As a result, the implementation of novel LC–MS/MS based methods in a clinical lab is challenging due to the lack of agreement in serum and DBS samples. However, for an epidemiological study, with the primary aim of reporting prevalence of vitamin D deficiency, the goal should be to accurately report the vitamin D deficiency/sufficiency status compared to a diagnostic protocol where the aim is to derive the method for analysis of vitamin D in DBS that can closely reflect the physiological vitamin D status as closely possible to serum/plasma samples. The LC–MS/MS we report here, does all the above, and fits the bill for us to implement this for our upcoming epidemiological survey.It has been estimated that in the Indian population, sunlight exposure leading to indigenous synthesis of D3 is the primary determinant of vitamin D status as it contributes to > 90% of the total 25(OH)D concentration7. As a result, our method may sometimes lead to the overestimation of 25(OH)D concentrations. The accuracy of estimation of vitamin D requires selection of columns that offer high chromatographic selectivity prior to MS identification. However, this strategy sacrifices high throughput due to additional time requirement. Pentafluorophenyl (PFP) column which requires comparatively shorter run time can perhaps be used7 but will be substantially more expensive, additionally, it may not be economically feasible for a population scale study. Lastly, a 2-step derivatization i.e. a Diels–Alder reaction with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by acetylation, may be used7. However, derivatization can compromise accuracy because of its own limitations as discussed in the previous section. Selection of C18 column was a decision in interest of high throughput and cost-effective method. We acknowledge that because of these limitations, the protocol may be used for a population based epidemiological study rather than for diagnostic purposes.Despite its robustness, our method still has a few limitations. Here, we have used a C18 stationary phase and this cannot separate isomeric and isobaric vitamin D metabolites. For example, a C18 column can achieve only partial separation of 25(OH)D3 and 25(OH)D2 as well as between 25(OH)D3 and its C3-epimer


Edwardsiella tarda is a facultative intracellular pathogen in humans and animals. There is no effective way except vaccine candidates to eradicate intracellular E. tarda. In this study, four derivatives of marine peptide-N6NH2 were designed by an introduction of unnatural residues or substitution of natural ones, and their intracellular activities against E. tarda were evaluated in macrophages and in mice, respectively. The minimum inhibitory concentration (MIC) value of N6NH2 and GUON6NH2 against E. tarda was 8 μg/mL. GUON6NH2 showed higher stability to trypsin, lower toxicity (<1%) and longer post-antibiotic effect (PAE) than N6NH2 and other derivatives. Antibacterial mechanism results showed that GUON6NH2 could bind to LPS and destroyed outer/inner cell membranes of E. tarda, superior to N6NH2 and norfloxacin. Both N6NH2 and GUON6NH2 were internalized into macrophages mainly via lipid rafts, micropinocytosis, and microtubule polymerization, respectively, and distributed in the cytoplasm. The intracellular inhibition rate of GUON6NH2 against E. tarda was 97.05–100%, higher than that in case of N6NH2 (96.82–100%). In the E. tarda-induced peritonitis mouse model, after treatment with of 1 μmol/kg N6NH2 and GUON6NH2, intracellular bacterial numbers were reduced by 1.54- and 1.97-Log10 CFU, respectively, higher than norfloxacin (0.35-Log10 CFU). These results suggest that GUON6NH2 may be an excellent candidate for novel antimicrobial agents to treat infectious diseases caused by intracellular E. tarda. Edwardsiella tarda is a Gram-negative bacterium that can infect a wide range of hosts, including humans and animals  is a variant of arenicin-3  isolated from marine lugworm Arenicola marina and has strong intracellular antibacterial activity against Gram-negative bacteria  and fungi  open a new way for the development of antimicrobial agents . It has nd fungi . However than N1 . However2 were designed by using natural residues , unnatural residue  and D-amino acids to improve the stability of N6NH2 toward protease and the antibacterial activity  and other peptides were synthesized by Mimotopes . The purity of peptides was over 90%.The derivatives of N6NH2 were designed based on the replacement of L-type amino acids, Gly, and Arg by D-type ones, Pro, and Val or the addition of N,N,N’,N’-tetramethylguanidine and L-ornithine (Orn) at the N-terminus . The phy2O, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) solutions by CD spectroscopy  using a 1.0 mm path-length cuvette. The spectra of peptides were recorded from 180 to 260 nm at 25°C at a scanning speed of 100 nm/min with a step resolution of 2.0 nm and an integration time of 2 s. Data were analyzed using CDNN software.The secondary structure of peptides was investigated in ddHtroscopy . Peptide5 CFU/mL) were added into 96-well plates, followed by the addition of serial dilutions of peptides (from 0.0625 to 128 μg/mL). The plates were incubated at 37°C for 18–24 h. The minimal inhibitory concentration (MIC) value was determined as the lowest peptide concentration at which no bacterial growth was observed.The antimicrobial activity of peptides was determined by the microbroth dilution method . The bacE. tarda was cultured to logarithmic phase, diluted in Mueller Hinton broth (MHB) (1 × 105 CFU/mL) containing 1×, 2×, or 4× MIC of peptides, and cultured at 28°C (200 rpm). PBS was added as a control. Bacterial samples (100 μL) were removed at different time intervals  and were counted on MH solid plates. The time-killing curves of peptides were plotted. Bacterial cells treated with 2× MIC norfloxacin and without treatment were used as the positive and blank controls, respectively.Time-killing curve of peptides was performed according to the previous method . E. tard3 cells/well) were added into 96-well microtiter plates and incubated at 37°C for 24 h (5% CO2). A series of peptide solutions were then added into plates and incubated for 24 h. The cells without treatment were used as controls. The 3--2,5-diphenyl tetrazolium bromide 3--dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) solution was added and incubated for 4 h. Finally, dimethyl sulfoxide (DMSO) was added into the plates and the absorbance was measured at 570 nm with a spectrophotometer. The cell survival was calculated using the following formula: Survival (%) = (Abs control – Abs treated sample)/Abs control × 100.The cytotoxicity of N6NH2 and its derivatives to RAW 264.7 cells was determined by MTT . Brieflytreated sample – Abs negative control)/(Abs positive control – Abs negative control)] × 100.The hemolysis of peptides was evaluated by determining the amount of hemoglobin that was released from fresh mouse erythrocytes . The bloE. tarda cells by an inhibition zone method , sodium acetate buffer (pH 4.0), sodium phosphate buffer (pH 6.0), Tris-HCl buffer (pH 8.0), or glycine-NaOH buffer (pH 10.0). In addition, the peptides were incubated for 4 h at 37°C in pepsin  and trypsin   solutions, respectively. The antibacterial activity of peptides was estimated against e method .All tests were conducted in triplicate.E. tarda were determined as the MIC assay described above. Different concentrations of antibiotics and antimicrobial peptides were added to each row and column of the 96-well cell culture plates to make the final concentration of 1/16–8 × MIC. After adding 180 μL of test bacteria solution to give a total volume in each well of 200 μL, the plates were incubated at 28°C for 18–24 h to observe the bacterial growth during the combination of drugs. Briefly, E. tarda  was incubated with 10 μL of single peptides or antibiotics or their combination in 96-well plates. PBS was used as a negative control. After incubation at 37°C overnight, the OD600 nm value of bacterial cultures was measured with a microplate plate reader. The fractional inhibitory concentration index (FICI) is calculated as follows: FICI = FICA + FICB, where FICA and FICB are the MICs of drug A in the combination divided by the MICs of drug A alone and the MICs of drug B in the combination divided by the MICs of drug B alone, respectively. FICI ≤ 0.5, 0.5 < FICI ≤ 1.0, 1.0 < FICI ≤ 4.0, and FICI > 4.0 were defined as a synergy, addition, indifference, and antagonism, respectively. The results were performed from at least three independent experiments.Combination of N6NH2, GUON6NH2 and antibiotics were tested by using the checkerboard titration method as described previously . MIC valE. tarda cells were incubated with peptides  for 2 h, diluted, and cultured at 28°C and 200 rpm. An aliquot of samples (50 μL) were taken for CFU counts at different time intervals until bacterial cultures became turbid. Bacteria treated with the N6NH2, GUON6NH2 or vancomycin were used as the positive control; bacteria without treatment were used as the blank control. The PAE was calculated using the following formula: PAE = T–C, where T is the time for the CFU counts to increase by 10-fold above the count observed immediately after drug removal, and C is the corresponding time for the untreated control ( control .The BODIPY’-TR-cadaverine (BC) probe was used to determine the ability of peptides to bind to LPS . 0.5 μM E. tarda cells were collected by centrifugation and washed twice and then suspended in HEPES buffer (pH 7.4) to an OD600nm of 0.4. Cell suspensions and NPN solutions (10 μM) were added into 96-well black plates, followed by the addition of peptide solutions . Fluorescence intensity was recorded with a microplate reader . The cells treated PBS and without treatment were used as negative and blank controls, respectively.The outer membrane permeabilization ability of the peptides was determined using the fluorescent dye 1-N-Phenylnapthylamine (NPN) assay . Mid-logE. tarda cells (1 × 108 CFU/mL) were incubated with or without 1× MIC peptides solutions at 37°C for 5, 15, and 30 min, respectively. After washing twice with PBS, bacterial cells were then incubated with 50 μg/mL propidium iodide (PI) for 15 min and analyzed using a FACS Calibur Flow Cytometer  (Mid-log phase  States) .E. tarda was cultured to logarithmic growth stage (108 CFU/mL) and diluted with 0.01 M PBS (pH 7.4) to OD600 nm = 1. E. tarda cells were treated with 4× MIC peptides at 37°C for 2 h and the untreated cells were used as the control. Briefly, after centrifugation, the bacterial cells were washed three times with 0.1 M PBS (pH 7.2) and fixed overnight at 4°C with 2.5% glutaraldehyde. After washing again, the cells were fixed with 1% osmium tetroxide (OsO4) for 2 h and dehydrated in a graded ethanol series  for 15 min, respectively. The samples were then dried by CO2, sputtered with gold-palladium, and observed on a QUANTA200 SEM .To further characterize the bactericidal effects of the peptides, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to visualize morphological changes . E. tardE. tarda cells were treated peptides before fixation as described above. The bacteria were washed with 0.01 M PBS (pH 7.4) for five times, 7 min each time, and fixed with 1% osmium acid buffer (4°C) for 1 h. After dehydration with a graded acetone series (30%–50%–70%–85%–95%–100% × 3), the cells were immersed in mixtures of acetone and resin  for 40 min and in a pure epoxy resin overnight. The samples were then embedded in embedding medium and polymerized at 45°C for 3 h and at 65°C for 24 h, respectively. The sections were prepared using an ultramicrotome, stained with 1% uranyl acetate, and visualized by a JEM1400 .E. tarda cells in the exponential phase (108 CFU/mL) were treated with 2× MIC N6NH2 or GUON6NH2 at 37°C for 2 h or untreated as control. The cells were fixed with 4% formaldehyde at room temperature for 1 h and at 4°C overnight and then immersed in 1% formaldehyde. The samples were dehydrated in a graded ethanol series , infiltrated with LR Gold resin , and polymerized by at 52°C for 48 h. 70 nm sections were cut using Leica EM UC7 and collected on nickel grids with Formvar/carbon film. The sections were blocked at 2% BSA (Jackson ImmunoResearch) and incubated with murine anti-biotin antibodies at 1:40 dilution at room temperature for 1 h. After washing with PBS (containing 0.1% cold fish gelatin), the sections were incubated with 12 nm gold-labeled anti-mouse secondary antibody (Jackson ImmunoResearch) at room temperature for 1 h, washed with PBS, and fixed by 1% glutaraldehyde. After washing with ddH2O, the samples were stained with 2% uranyl acetate at room temperature for 30 min and imaged using a TEM H-7650 at 80 kV , centrifuged at 5,000 rpm for 5 min and diluted with 0.01 M PBS (pH 7.4) to OD600 nm = 1. In brief, E. tarda was treated with FITC-labeled peptides (1× or 4× MIC) at 37°C for 30 min. Then, the bacterial cells were washed three times with PBS, incubated with PI (10 μg/mL) at 4°C for 15 min, and washed again; 5 μL of bacterial suspension was coated on a slide and sealed with a cover slide. The samples were observed using a DeltaVision OMX SR system  at excitation wavelengths of 488 and 535 nm for PI and FITC, respectively. Cells without peptides served as controls.The site of action of the peptides on escribed . E. tardE. tarda and extracellular E. tarda was killed by adding gentamicin (1000 μg/ml) to the plate, followed by incubation at 37°C for 30 min. The cells were washed three times with PBS, and cultured in DMEM for 0, 6, 12, 18, and 24 h, respectively. At each time point, 500 μL 0.1% triton X-100 was added to the plate, and the samples were collected and diluted; the bacteria were counted as previously described (RAW 264.7 cells were infected with escribed . The exp4 cells) were cultured for 24 h and incubated with FITC-labeled N6NH2 and GUON6NH2 (5× and 25× MIC) for 18 h at 37°C. After incubation, the cells were then washed with PBS and stained with wheat germ agglutinin (WGA)-conjugated Alexa Fluor 555  and Hoechst 33342  for membrane and nuclear staining, respectively, for 10 min before confocal microscopy  for 24 h. Then, the cells were treated with FITC-N6NH2 and FITC-GUON6NH2  for 18 h and washed with PBS. To avoid the influence of membrane-bound FITC-labeled peptides, the cells were incubated with 0.04% trypan blue in PBS for 15 min and the fluorescence intensity was analyzed using a FACS Calibur Flow Cytometer   .To determine the effects of endocytosis inhibitors on peptide uptake, RAW 264.7 macrophages were pretreated with 3 mM amiloride, 5 mM methyl-β-cyclodextrin (MβCD), 20 μM nocodazole, and 6 μg/mL chlorpromazine for 1 h at 37°C. The cells were then treated with FITC-N6NH2 and FITC-GUON6NH2 at 37°C for 4 h. The cells treated with the FITC-labeled peptides were used as a positive control. Finally, the cells were mixed with 0.04% trypan blue and florescence within cells were measured using flow cytometry .5 cells/mL) were cultured for 24 h in DMEM with 10% FBS (without antibiotics) , and incubated with RAW 264.7 macrophages for 0.5 h. Gentamicin  was then added into the cells and incubated for 30 min to remove extracellular bacteria. After washing twice with PBS, RAW 264.7 macrophages were treated with different concentrations of N6NH2 and GUON6NH2  for 18 h, washed again, and lysed with Hanks buffered saline solution . The numbers of intracellular bacteria were counted at 0 and 18 h, respectively.For intracellular activity, RAW 264.7 macrophages (2.5 × 10biotics) . MeanwhiE. tarda . The infected mice were followed by the intraperitoneal injection of N6NH2, GUON6NH2 or norfloxacin  at 1 h postinfection, respectively. Mice injected with only bacteria or PBS were used as negative or blank controls. The survival of mice was recorded daily for 5 days.To establish a peritonitis mouse model, six-week-old female ICR mice (five mice/group) were intraperitoneally injected with E. tarda  and then injected with N6NH2 and GUON6NH2 (1 μmol/kg) or PBS (the blank control) in the same way at 1 h after infection. The mice were then killed at 24 h after treatment, and peritoneal fluids were obtained by washing with 5 mL of ice-cold PBS. The total number of E. tarda in the fluids was determined before any further procedures. For intracellular bacteria quantification, the other fraction was centrifuged for 5 min at 4°C . The cells were collected, treated with 1,000 μg/mL gentamicin for 30 min at 37°C to kill extracellular bacteria, and washed with ice-cold PBS twice to remove extracellular gentamicin. Subsequently, the cells were lysed and the bacteria were counted as described above for the in vitro experiment  with D-type ones; N6PNH2 was obtained by substitution of Arg in position 19 with Pro; Gly substitution with Val in position 1 and 12 led to V112N6NH2; GUON6NH2 was generated by addition of N,N,N’,N’-tetramethylguanidine and L-Orn to N-terminus of N6NH2. As shown in 2O, 20 mM SDS and 50% TFE, respectively. As shown in 2O was characterized by random coils (34.57–42.76%), antiparallel strands (15.17–30.36%) or β-turns (22.21–42.76%), with a negative minimum at 200 nm. However, GUON6NH2 showed a significant decrease in the proportion of anti-parallel strands (1.04%). DN6NH2 mainly contained anti-parallel chains and random coils, with a positive maximum at 200 nm. In SDS solution, N6NH2 had higher antiparallel chain (63.52%) than N6PNH2 (17.78%) and V112N6NH2 (14.77%); DN6NH2 changed from antiparallel strands to β-turns (38%) and random coils (52.22%). The secondary structures of N6PNH2, V112N6NH2 and GUON6NH2 in 50% TFE were characterized predominantly by β-turns, random coils, anti-parallel chains or β-turns, with a negative dichroic band at approximately 205 nm, and a positive maximum at 180 nm (strong) and 230 nm (weak).The anionic surfactant SDS and TFE are often used to mimic the cell membrane environment. To analyze the structural features of N6NH2 and its derivatives, the CD spectra of peptides were measured in ddHEscherichia coli, Salmonella and E. tarda than Gram-positive bacteria. N6NH2, DN6NH2 and GUON6NH2 exhibited the strongest antimicrobial activity against Gram-negative and Gram-positive bacteria with MIC values ranging from 2 to 32 μg/mL. The MIC values of N6PNH2 and V112N6NH2 against E. coli, Salmonella and E. tarda were 4–8, 2–32, and 8–32 μg/mL, respectively. The MIC value of N6NH2, DN6NH2 and GUON6NH2 against E. tarda was 8 μg/mL, indicating their similar antibacterial activity.As shown in The geometric mean (GM) of MICs of peptides against Gram-negative and Gram-positive bacteria was calculated, and the result was shown in E. tarda within 6 h, which were more rapid than norfloxacin, N6PNH2, and DN6NH2 , ofloxacin , enrofloxacin , norfloxacin , chloramphenicol , and kanamycin , respectively . GUON6NHE. tarda was 2.65 and 1.27 times longer than that of N6NH2 and norfloxacin, respectively , nocodazole (inhibiting tubulin polymerization into microtubules), chlorpromazine (inhibiting clathrin-mediated endocytosis), and MβCD (inhibiting caveolin-mediated endocytosis) were used to treat RAW 264.7 cells. As shown in E. tarda was evaluated in RAW 264.7 cells. As shown in E. tarda cells and the inhibition rates of intracellular bacteria were over 99% in the peptide-treated groups. The killing efficacy of 1× MIC GUON6NH2 against E. tarda was over 97.07%, which was slightly higher than that of N6NH2 (96.82%). 5–50× MIC N6NH2 and GUON6NH2 killed all intracellular bacteria. This result suggested that GUON6NH2 has more potent bacteriostatic effects than N6NH2 against intracellular E. tarda.The ability of N6NH2 and GUON6NH2 to eliminate intracellular in vivo antibacterial activity of peptides, the efficacy of N6NH2 and GUON6NH2 was determined in a mouse peritonitis model. The results showed the untreated mice began to die at 48 h after inoculation, and 80% were dead within 120 h. After treatment with 1 μmoL/kg N6NH2, the survival rate of mice was 80%, which was higher than that with 1 μmoL/kg GUON6NH2 (60%) and 1 μmoL/kg norfloxacin (40%)   . Moreoveg10 CFU) . ComparaEdwardsiella tarda can invade, survive, and proliferate in host cells (such as macrophages), leading to a high infection recurrence , within mammalian cells (E. tarda and macrophages (E. tarda a multiplicity of infection (MOI) of 1000:1 survived and replicated in macrophages with an increase of 7.36 Log10 CFU within 24 h  and antibacterial activity against The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Care and Use Committee of the Feed Research Institute of Chinese Academy of Agricultural Sciences (CAAS), and protocols were approved by the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of CAAS (AEC-CAAS-20090609).HH, DT, XW, and JW conceived and designed the experiments. HH carried out all the experiments. RM, NY, and ZW guided the methods. ZW, TL, and NY prepared partial materials in the laboratory. YH contributed materials and reagents. HH and XW contributed to writing. JW contributed to funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information on Vip3A and a clear understanding of its insecticidal mechanism at the molecular level limits its further development and broader application. Here we present the first crystal structure of the C-terminal fragment of Vip3A toxin (Vip3Aa11200–789). Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3A family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two potential glycan binding domains of Vip3A. In addition, cytotoxicity assays and insect bioassays show that the purified C-terminal fragment of Vip3Aa toxin alone have no insecticidal activity. Taken together, these findings provide insights into the mode of action of the Vip3A family of insecticidal toxins and will boost the development of Vip3A into more efficient bio-insecticides. The vegetative insecticidal proteins (Vips) secreted by  Bacillus thuringiensis (Bt), is the most widely used microbial insecticide in the world [The entomopathogenic bacteria he world ,2. It ishe world ,4,5. Howhe world ,6,7. TheVegetative insecticidal proteins (Vips), which are produced by Bt during its vegetative stages, have a wide spectrum of insecticidal activity, especially against lepidopteran pests . To dateVip3A toxins are large proteins (~789 amino acids) consisting of a conserved N-terminus and a variable C-terminal region. The ~88kDa Vip3A protoxin could be digested by insect midgut juices into two fragments: a ~20 kDa fragment corresponding to the N-terminal 198 amino acids, and a ~65 kDa fragment corresponding to the C-terminal fragment of Vip3A protein, which is regarded as an essential step for its activation and toxicity ,22,23. S200–789). The structure shows a four-domain organization which is likely to be conserved for all insecticidal Vip3A family toxins. We identify conserved hydrophobic α-helices in domain II, which we predict to be involved in the membrane insertion process. Structure-guided cell binding assays reveal that domain III may have a central role in host cell targeting and binding of Vip3A toxins. Structural analysis indicates that Vip3A toxins have potential for glycan binding through domains IV and V. Together, our structural and functional studies provide new insights into the molecular mechanisms underlying the mode of action of insecticidal Vip3A toxins.Here, we report the crystal structure of the C-terminal fragment of Vip3A toxin  from the Vip3Aa11199–end, we obtained the crystal of Vip3Aa11200–end, which diffracted to ~6 Å. Through the addition of an N-terminal MBP  tag, we were able to isolate crystals with improved diffraction. The structure was solved using a combination of anomalous phasing with a selenomethionine derivative crystal of Vip3Aa200–end and molecular replacement using MBP as a model in native crystals. The final structure of Vip3Aa11200–end was refined to a 3.2 Å resolution with R and Rfree values of 0.1980 and 0.2389, respectively  in this study. Full-length Vip3Aa11 consists of 789 amino acids, which have been demonstrated to be digested between residues K198 and D199 by insect midgut juice ,21. We iectively . 1–198 is a separate domain. Then the protoxin can be divided into five domains, starting from N-terminus: domain I, 1–198; domain II, 199–327; domain III, 328–518; domain IV, 537–667; and domain V, 679–789 in Vip3Aa11  consists of five helices, which form two layers A. The ou200–end structure  consists of twelve β strands and one short α-helix at the C-terminal end A. Twelve8–518 conSpodoptera frugiperda cells (Sf9 cells), which have previously been shown to be specifically targeted by Vip3 toxins [To explore this hypothesis, we used 3 toxins ,33. To dBoth domains IV and V are all β-sheets folds A,B. UnliThermoanaerobacterium polysaccharolyticum with high specificity toward β-1,4-glucose or β-1,4-mannose polymers [Domains IV and V are both built from two anti-parallel sheets of β sandwich, forming the “jelly-roll” topology. Despite showing only 17% sequence identity C, domainpolymers . Analysi200–end still have insecticidal activity, cytotoxicity assays and insect bioassays were carried out. As shown in 199–end, Vip3Aa200–end and MBP-Vip3Aa200–end have no toxicity to Sf9 cells. In addition, bioassay results showed that wild-type Vip3Aa was highly toxic against S. exigua at the concentration of 200 ng/cm2. However, the purified Vip3Aa199–end, Vip3Aa200–end and MBP-Vip3Aa200–end have no obvious insecticidal activity to S. exigua larvae even at the concentration of 2000 ng/cm2  is surrounded by other helices from domains I and II  for plasmid constructions and protein purification were cultured at 37 °C in lysogeny broth (LB) or agar. Methionine auxotrophic E. coli strain B834 (DE3)  were used for selenomethionine-substituted (SeMet) Vip3Aa200–end expressing. The S. frugiperda Sf9 cells  were maintained and propagated in Sf-900 II SFM  culture medium at 27 °C.200–end was amplified from the Vip3Aa11 gene (GenBank accession No. AY489126.1) using oligonucleotide primer Vip200-F and Vip200-R and cloned into the pET28a vector with an N-terminal 6×His-MBP tag. Plasmids used for RFP (red fluorescent protein) and C-terminal RFP tagged Vip3Aa (Vip3Aa-RFP) expression were constructed as described by Jiang et al. [The DNA of Vip3Aag et al. . The nuc200–end (Vip3Aa200–end) protein was expressed in E. coli B21(DE3) at 25 °C for 48 h in autoinduction Terrific broth (TB) medium. The cells were harvested by centrifugation at 5000× g at 4 °C for 15 min and the pellet was resuspended in lysis buffer (20 mM Tris–HCl pH 8 and 150 mM NaCl). After the cells were lysed by high pressure cell crusher , the supernatant was collected after centrifuged at 12000× g at 4 °C for 60 min. The proteins were purified using Ni-NTA agarose resin, washed with 20 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8.0, and then eluted with 300 mM imidazole. The Vip3Aa200–end proteins were further purified by HiTrap Q HP ion-exchange chromatography and Superdex 200 gel filtration chromatography . Fractions containing the Vip3Aa200–end protein were concentrated to ~7 mg/mL for crystallization. The expression and purification steps of other Vip3Aa truncations were the same as those of Vip3Aa200–end.Native His-MBP-Vip3Aa200–end was expressed in E. coli B834(DE3) strain. Briefly, the cells were cultured in the LB medium at 37 °C along with shaking until the OD600 of the bacterial culture reached 1.0. The cells were harvested by centrifugation at 4000× g at 4 °C for 15 min and the pellet was washed once with PBS. The pellet was resuspended in 1 L Medium A  and incubated for 3 h at 37 °C. We added 50 mg seleno-methionine in the medium and incubated for a further 30 min. The protein was incubated to express for a further 10 h by adding 200 mM IPTG . The SeMet-Vip3Aa200–end was purified by the same procedure as for the native Vip3Aa200–end protein.SeMet-substituted Vip3Aa6-tagged MBP-Vip3Aa200–end used for crystallization is described above. MBP-Vip3Aa200–end (5 mg/mL) was used to perform initial spare matrix crystal screening with a crystallization robot. After crystal optimization trials, MBP-Vip3Aa200–end (7 mg/mL) crystals grew in 3 days at 18 °C using the hanging-drop vapor-diffusion method in a mix of 1 μL of protein with 1 μL of reservoir solution consisting of 0.1 M sodium acetate pH 4.2, 0.5 M potassium formate, 0.1 M ammonium sulfate and 11% PEG4000. SeMet MBP-Vip3Aa200–end crystals grew in a similar condition. The purification of His12121 was collected at 3.62 Å (native I). A weak selenomethionine (SeMet) derivative data set was collected at 3.9 Å with the same symmetry as the native I crystal for the amino-acid assignment using the difference Fourier map of the SeMet derivative. After further crystallization optimization, another native crystal (native II) was obtained with the space group of P21 that could diffract to around 3.2 Å. Diffraction data were collected on BL17U1 and BL18U beamlines at Shanghai Synchrotron Radiation Facility  and processed by HKL2000 [A native data set with the space group of P2 HKL2000 .https://pymol.org/, Schrödinger, New York, NY, USA). Structure refinement was done by PHENIX [Molecular replacement was carried out to identity the MBP positions in the native crystals  by PHASEy PHENIX . The dat4 cells per ml were seeded into 6-well culture plates separately. After overnight culture, the cells were respectively treated with RFP tagged Vip3Aa or its truncations (0.15 μM) for 6 h. After treatment, the cells were washed three times with PBS to remove unbound proteins, and fixed with 4% paraformaldehyde at 37 °C for 15 min. The cell nuclei were labeled with DAPI (0.2 μg/mL) for 30 min. Cell images were captured using a Nikon TI-E inverted fluorescence Microscope .Sf9 cells with a density of 5 × 104 cells per ml were seeded into 96-well culture plates separately. After overnight incubation, the cells were treated with different Vip3Aa toxins for 72 h. WST-8 reagent was then added to each well. After incubating at 27 °C for 2 h, the absorbance was measured in microplate reader at 450 nm. Treatment with protein buffer was used as a control. All tests were performed in triplicate and were repeated at least three times. Cell viability (%) = average absorbance of treated group/average absorbance of control group × 100%.Cell viability assays were performed as described by Jiang et al. . BrieflyS. exigua first instar larvae and maintained in a rearing chamber at 27 °C, with 50% relative humidity, and 16:8 h light:dark photoperiod. The artificial diet was poured in a 1.8-cm2 24 well plate (about 5 mm thick per hole). 200 and 2000 ng/cm2 concentrations of Vip3Aa proteins  were spread on the diet. A tris buffer  was used as a blank control. Three independent replicates and 16 first instar larvae of S. exigua were used for each concentration. Mortality was recorded after 5 days. Bioassays were assessed using surface contamination method with p < 0.01; ***, p < 0.001. A p value of < 0.05 was considered to be statistically significant.All functional assays were performed at least three times independently. Data were shown as means ± SD. All statistical data were calculated using GraphPad Prism version 8.0 . One-way ANOVA followed by Dunnett’s test were used to identify statistically significant differences between treatments. Significance of mean comparison is annotated as follow: ns, nonsignificant; **, Coordinate for the atomic structure has been deposited in the RCSB Protein Data Bank under RCSB ID: 6VLS. The data that support the findings of this study are available from the corresponding author upon reasonable request.