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Genetic assessment of rice landraces is important for germplasm evaluation and genetic resource utilization. Rice landraces in peninsular Thailand have adapted to unique environmental stresses over time and have great significance as a genetic resource for crop improvement. In this study, rice landraces derived from rice research centers and farmers from different areas of peninsular Thailand were genetically assessed using 16 polymorphic InDel markers from putative stress-related genes. A total of 36 alleles were obtained. The average PIC value was 0.27/marker. The F(ST) varied from 0.46 to 1.00. Genetic diversity was observed both within and between populations. AMOVA indicated that genetic variations occurred mainly between populations (70%) rather than within populations (30%). The dendrogram, population structure, and PCoA scatter plot clearly demonstrated the differentiation of the two major groups, i.e., landraces from upland and lowland rice ecosystems. The unique alleles of Indel1922, -2543, -6746, -7447 and -8538, which lie in genes encoding putative WAX2, heavy metal-associated domain-containing protein, GA20ox2, PTF1, and PLETHORA2, respectively, were only found in rice from upland ecosystems. Putative WAX2, GA20ox2, and PLETHORA2 are likely related to drought and salt stress. Our findings demonstrate the diversity of landraces in peninsular Thailand. The preservation of these landraces should be facilitated with effective markers to maintain all variant alleles and to protect the genetic diversity. Indel1922, -2543, -6746, -7447 and -8538 have the potential to differentiate upland rice from lowland rice. Furthermore, Indel1922, -6746 and -8538 might be effective markers for drought and salt tolerance. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-020-00816-z) contains supplementary material, which is available to authorized users.

The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion.

Hyperglycemia-induced oxidative stress and inflammation are hallmarks of liver damage in diabetes mellitus. Accumulating evidence has demonstrated that Pluchea indica leaf ethanol extract (PILE) possesses strong antioxidant and anti-inflammatory properties. However, studies of its effects on liver damage in streptozotocin (STZ)-induced diabetic animals remain insufficient. To the best of our knowledge, the present study was the first to illustrate that PILE mitigated liver injury in STZ animals. Mice were first pretreated with PILE at either 50 mg/kg (PILE 50) or 100 mg/kg (PILE 100) 2 weeks prior to the induction of hyperglycemia by multiple low doses of STZ. The mice were then fed with PILE 50 or PILE 100 for 4 or 8 weeks, following which liver weight, pathological changes, oxidative stress parameters, inflammation-related markers and caspase-mediated apoptosis were measured at each time point. Untreated STZ mice exhibited abnormal increases in liver weight and severe pathological changes. However, PILE 100 reduced the severity of the STZ-induced diabetic phenotype at both time points. A significant decrease in the levels of superoxide dismutase and catalase, in addition to an increase in malondialdehyde, were observed in the livers of untreated STZ mice, all of which were significantly reversed by treatment with PILE 100 for 8 weeks. Western blot analysis revealed reduced levels of liver inflammatory markers, including interleukin-6, tumor necrosis factor-α, NF-κB p65, transforming growth factor-β1 and protein kinase C following PILE 100 treatment. Additionally, changes in the levels of apoptotic markers indicated that PILE 100 significantly attenuated caspase-9 and -3 expression, whilst preserving that of the Bcl-2 protein. In conclusion, the present study revealed that PILE alleviates hyperglycemia-induced liver injury by normalizing the various mediators of oxidative stress, inflammation and apoptosis.

Mangroves are susceptible to contamination due to their proximity to shores and human activities. Exposure to excessive trace metals can disturb their physiological functions and may eventually lead to death. Rhizophora mucronata is a common species growing in the mangrove forests of Thailand. Previous studies have shown that seedlings of R. mucronata are tolerant of trace metal and that they accumulate a large metal content in their root tissue. However, knowledge of their tolerance mechanisms is still lacking. To elicit the role of metal detoxification and sequestration by phytochelatins (PC) in the roots of R. mucronata seedlings, the impacts of Cu and Zn exposure were assessed on 1) physiological characteristics 2) the concentration of glutathione (GSH), a precursor of PC and 3) the level of the transcripts encoding phytochelatin synthase (PCS), the key enzyme for PC biosynthesis. Seedlings of R. mucronata were exposed to Cu and Zn in a hydroponic experiment (200 mg Cu or Zn/L in 1/4× Hoagland solution containing 8‰ NaCl, single addition). We found that both trace metals were largely accumulated in the roots. Only Cu-treated seedlings showed a decrease in the photosynthetic efficiency, in line with observed toxicity symptoms (i.e. bent stems and slight wilting of leaves). Metal accumulation, however, did not induce oxidative stress in the roots as indicated by similar level of total reactive species and lipid peroxidation across treatments. The GSH content in the roots exposed to Cu was significantly reduced while no change was observed in Zn-exposed roots. Coordinated semi-quantitative PCR and RT-qPCR revealed pcs down-regulation in Cu-treated roots, whereas Zn-treated roots showed a down-regulation on day 1 and a subsequent recovery on day 5. Failure of detoxification and sequestration of excess Cu due to GSH limitation and down-regulation of pcs may lead to the phytotoxic effects observed in Cu-treated plants. Our results suggest that both GSH and PC play an important role in trace metal tolerance in R. mucronata seedlings.

Metal oxide affinity chromatography (MOAC) is one of the most commonly used techniques for selective isolation phosphoproteins and phosphopeptides. This technique is capable of capturing the phosphorylated biomolecules through the affinity of the phosphoryl group for metal oxides/hydroxides. Fly-ash (FA), a by-product of coal-combustion power plants, is primarily composed of oxides of silicon and metals, among which iron and titanium. A number of studies have demonstrated the potential of these metal oxides for phosphoprotein and phosphopeptide enrichment. FA is annually produced over hundred million tons worldwide and generally considered as hazardous waste. It is thus of great importance to enhance its utilization. Here we present the first demonstration of the utility of FA as a low-cost MOAC material for the enrichment of phosphoproteins. With an FA-microcolumn, phosphoproteins can be successfully sequestered from other proteins. FA-microcolumns are shown to be simple, cheap and selective devices for phosphoprotein enrichment from a small volume of mixtures.

Isoflavonoids are the main compound in White Kwao Krua (Pueraria mirifica), which is an effective folk medicinal plant endemic to Thailand. It has been widely used for improving human physical and treating diseases. There are substances with estrogenic activities have been isolated from P. mirifica, such as puerarin, daidzein and genistein. Isoflavone synthase (IFS) is one of the key enzymes in Leguminous plants to convert liquiritigenin, liquiritigenin C-glucoside and naringenin chalcone to isoflavonoids. The aim of this research was to enhance the production of isoflavonoids by metabolic engineering. Transgenic plants were constructed by introducing P450 gene (EgP450) which is similar to IFS from oil palm (Elaeis guineensis), into P. mirifica by a biolistic method. After the transgenic plants had proved successfully, isoflavonoids of each group plants were determined by HPLC. The contents of daidzein and genistein in transgenic plants were higher than the control plants.

In a previous study, the gene EgP450 that encodes the proteins of 505 amino acids was isolated from oil palm. The recombinant protein EgP450 is bound to phenylurea-like herbicides which detoxify the substance. Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus sp., is another toxic compound that is known to cause acute toxic effects and act as a hepatocarcinogenic agent. This study aimed to examine the role of EgP450 enzyme in mycotoxin bioactivation in human mesenchymal stem cells (hMSCs). Docking analysis showed that EgP450 is bound to the group of carcinogens, which includes AFB1, n-(2-fluorenyl) acetamide, n-n-butyl-n-butan-4-ol-nitrosamine, n-nitrosodiethylamine, n-nitrosodiethylamine and n-nitrosodimethylamine. An in vivo aflatoxin toxicity test on hMSCs and AFB1 induces the expression of Bmi-1 which is one of the markers for the development of cancer. The presence of EgP450 at 0.15 μg/mL could reduced the Bmi-1 expression in AFB1 induced cells. Moreover, this protein also showed some antioxidant activity. These results exhibited the enormous potential of EgP450 in the detoxification processes.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is involved in the adaptive immune response via its effects on major histocompatibility complex (MHC)-restricted antigen presentation. In addition to antigen presentation, GILT exerts its antiviral activity by reducing disulfide bonds in proteins involved in viral infection and assembly, thereby inhibiting viral envelope-mediated infection and viral progeny production. In black tiger shrimp, Penaeus monodon GILT (PmGILT) was cloned and characterized, and found to be involved in the shrimp innate immune response and to exert neutralizing activity against white spot syndrome virus (WSSV) infection. However, the anti-WSSV mechanism of PmGILT in the shrimp innate immune response has not been defined. To explore the anti-WSSV activity of PmGILT, a yeast 2-hybrid (Y2H) assay was performed to identify WSSV proteins targeted by PmGILT. The assay revealed 4 potential PmGILT-interacting WSSV proteins: WSSV002, WSSV164, WSSV189, and WSSV471. Three of these 4 WSSV proteins (WSSV002, WSSV164 and WSSV189) were successfully produced and confirmed to interact with PmGILT in in vitro pull-down assays. WSSV189 and WSSV471 were previously identified as structural proteins, whereas WSSV164 is an immediate-early protein which has anti-melanization activity, and WSSV002 is an unknown. Because of the thiol reductase activity of PmGILT, WSSV164 and WSSV189, both of which are cysteine-containing WSSV proteins, were chosen for disulfide bond reduction assays. PmGILT reduced intrachain disulfide bonds in both WSSV proteins, suggesting that PmGILT exerts its anti-WSSV activity via its thiol reductase activity to disrupt the WSSV protein complex and restore the melanization activity of PmproPO1 and PmproPO2.

Rationale Since the last decade, mass spectrometry (MS) has become an essential technique for phosphoprotein analysis. Formidable analytical challenges of MS for phosphoprotein study are both the low abundance of phosphopeptides and the lack of an unambiguous diagnostic fragment ion for identification of phospho residues. These challenges can be met by a charge-based isolation of β-elimination products after tryptic digestion using diagonal strong cation-exchange chromatography. Methods β-Elimination combined with diagonal strong cation-exchange chromatography (BE/2SCX) was used for the enrichment of phosphorylated peptides prior to a mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (MS/MS). Bovine α-casein (≥70% purity) was used as a model protein. Results Conditions for β-elimination were optimized to maximize the efficiency of the reaction. With a β-elimination, all four model phosphopeptides from enolase (yeast) were correctly identified. The application of the BE/2SCX enrichment strategy for the analysis of β-elimination products of α-casein (bovine) allowed the identification of 11 phosphorylated products. Conclusions The introduction of a BE/2SCX-based enrichment step prior to LC/MS/MS analysis of β-elimination products facilitates the identification of phosphopeptides. Copyright © 2016 John Wiley & Sons, Ltd.

Translationally controlled tumor protein (TCTP) is a highly conserved protein found in various organisms and eukaryotes and is broadly expressed in several tissues. TCTP plays a key role in preventing apoptosis and promoting cell survival. The conserved tumor suppressor p53 is a transcription factor which responds to stress signals. p53 controls apoptosis through a series of quantitative and qualitative events which require several binding partners including TCTP. However, the exact coordination of these two molecules in the apoptosis pathway is not clearly understood. In this study, docking simulation successfully identified the binding sites of “human TCTP and human p53”, “Crustacean, Penaeus monodon TCTP and human p53”, and “plant, Elaeis guineensis TCTP and human p53” with binding energies of -843.10 Kcal/mol, -747.80Kcal/mol, and - 786.80 Kcal/mol, respectively. The results implied that non-human TCTPs have less binding activities with human p53 and they are not effective partners compared with human TCTP.

Rhizophora mucronata is a common mangrove growing in habitats subjected to heavy metal (HM) contamination. Understanding their physiological responses to copper (Cu) and zinc (Zn) excess and underlying tolerance mechanisms is crucial to assess impacts of metal pollution on mangrove community. Seedlings were treated with Cu or Zn (0, 50 or 100 mg per plant) by means of a single addition. At day 3 and 7, Cu and Zn accumulation, photosynthetic efficiency, superoxide dismutase and peroxidase activity, non-protein thiols, reactive oxygen species and lipid peroxidation in roots and leaves were measured. R. mucronata restricted Cu and Zn translocation, thus accumulated HM mainly in roots while kept the leaves unaffected. However, high root HM did not induce oxidative stress nor anti-oxidative defense as HM were largely deposited in cell wall. We concluded that HM tolerance strategies of R. mucronata seedlings are exclusion and restriction of translocation to the vital photosynthetic tissue.

Pm-fortilin is a recombinant protein of Penaeus monodon expressed in Escherichia coli. Activities regarding antioxidation and wound healing of the purified Pm-fortilin were investigated in vitro using normal fibroblast cell line L929. Relative viabilities of the cells were determined after exposure to either hydrogen peroxide (0.5 mmol/l for 4 h) or UV radiation (366 nm for 20 min). Significantly decreased viability was observed for the UV exposed cells compared with those incubated with the peroxide. Interestingly, proliferation of the stress-induced cells was improved by Pm-fortilin treatment. The protein’s curing effect was dose dependent. The scratch method was used in the wound healing test on L929 cells. The injured cells distinctively migrated if pre-incubation with Pm-fortilin was carried out. However, such migration ability was compromised if the injured cells were continually in contact with Pm-fortilin. The transcriptions of sydecan-2, dkk1, β-catenin, lrp6, and bmi1 genes in MG63 and HOS cancerous cells were down-regulated following incubation with Pm-fortilin for 5 days compared to those transcribed by L929 cells. The results suggested that Pm-fortilin might be non-oncogenic and is premised for use as a protective or repair agent in topical pharmaceutical products.

Pm-fortilin/TCTP is a highly expressed protein in white spot syndrome virus (WSSV) infected shrimp, Penaeus monodon. It was demonstrated in insect cell line that Pm-fortilin could down regulate the expression of WSSV early genes and inhibit viral replication. To confirm this function in shrimp, dsRNA was designed to target endogenous fortilin. The dsRNA significantly reduced the expression of Pm-fortilin mRNA within 12 days after introducing them in P. monodon. The mortality rates after Pm-fortilin knockdown in WSSV-challenged P. monodon significantly increased compared with those of the controls. The silencing of Pm-fortilin also lowers the expression levels of prophenoloxidase (proPO) and fortilin binding protein (FBP1). This implies the interconnection of these three molecules in immune responsive pathways. To investigate the practical approach of Pm-fortilin in aquaculture, the sonicated yeast Pichia pastoris that harbored recombinant Pm-fortilin protein was added to the food that was ingested by another commercially important species, Litopenaeus vannamei. We revealed that protection against WSSV but not YHV can be conferred in L. vannamei. The reduced levels of WSSV in surviving animals that were fed a diet containing 5% yeast harboring recombinant Pm-fortilin was confirmed. The purified recombinant protein was stable at pH values ranging from 6–9, salinities of 15–30 ppt and temperatures of 50–90 °C. These results imply that the Pm-fortilin confers a specific clearance effect on WSSV and can act as a useful feed additive. Pm-fortilin and some WSSV proteins may be involved in the process of latency or prevention of WSSV replication.

In Thailand, there are currently five recognized species members of the bubble-nesting Betta genus, namely Betta splendens, B. smaragdina, B. imbellis, B. mahachaiensis and B. siamorientalis. In 2010, we indicated the possibility, based on COI barcoding evidence, that there might be two additional species, albeit cryptic, related to the type-locality B. smaragdina in some provinces in the northeast of Thailand. In the present study, after a more extensive survey of the northeast, and phylogenetic analyses based on COI and ITS1 sequences, the B. smaragdina group may be composed of at least 3 cryptic species members. The phylogenetic positions of these B. smaragdina group members in the bubble-nesting bettas' tree together with those of their congeners have been consolidated by better DNA sequence quality and phylogenetic analyses. With a better supported tree, the species statuses of B. siamorientalis and the Cambodian B. smaragdina-like fish, B. stiktos, are also confirmed.

White spot syndrome virus proteins WSSV134 and WSSV322 have been shown to bind with the p20 domain (residues 55–214) of Penaeus monodon caspase ( Pm Casp) protein through yeast two-hybrid screening. Binding was confirmed for the p20 domain and the full-length caspase by co-immunoprecipitation. WSSV134 is also known as the WSSV structural protein VP36A, but no function or conserved domains have been ascribed to WSSV322. Discovery of the caspase binding activity of these two proteins led to an investigation of their possible anti-apoptotic roles. Full-length Pm Casp was confirmed to be an effector caspase by inducing apoptosis in transfected Sf-9 cells as assessed by DAPI staining. Using the same cell model, comparison of cells co-transfected with Pm Casp and either WSSV134 or WSSV322 revealed that both of the binding proteins had anti-apoptotic activity. However, using the same Sf-9 protocol with anti-apoptosis protein-1 (AAP-1; also called WSSV449) previously shown to bind and inactivate a different effector caspase from P. monodon ( Pm caspase) did not block apoptosis induced by Pm Casp. The results revealed diversity in effector caspases and their viral protein inhibitors in P. monodon .

The northeastern region of Thailand is well known to have a high incidence and mortality of cholangiocarcinoma (CCA). Protein phosphorylation status has been reported to reflect a key determinant of cellular physiology, but identification of phosphoproteins can be a problem due to the presence of phosphatase. Exosomes are stable toward circulating proteases and other enzymes in human blood and can be recognized before the onset of cancer progression. Here an in vitro metastatic model of isogenic CCA cells is used to provide insight into the phosphorylation levels of exosomal proteins derived from highly invasive cells. Gel-based and gel-free proteomics approaches are used to reveal the proteins differentially phosphorylated in relation to tumor cell phenotypes. Forty-three phosphoproteins are identified with a significant change in phosphorylation level. Phos-tag western blotting and immunohistochemistry staining are then employed to validate the candidate phosphoproteins. Heat shock protein 90 is successfully confirmed as being differentially phosphorylated in relation to tumor malignancy. Importantly, the aberrant phosphorylation of exosomal proteins might serve as a promising tool for the development of a biomarker for metastatic CCA.

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.

Plant 14-3-3 proteins are involved in signal transduction pathways of nitrogen and carbohydrate metabolism. An Eg14-3-3 ω gene was isolated from the mesocarp of oil palm. The 1055-bp cDNA had an open reading frame of 774 bp that encoded for 258 amino acids, and the cDNA had 113-bp and 195-bp 5′- and 3′-untranslated regions, respectively. The calculated molecular weight was 28.06 kDa, with a pI of 5.04. The palm 14-3-3 showed closest identity to 14-3-3 proteins of the omega group. The entire sequence of Eg14-3-3 ω showed 83% identity with 14-3-3 protein isoform 16R from Solanum tuberosum. Phylogenetic analysis showed that the Eg14-3-3 isoform was within the omega (ω) subgroup and, thus, was designated Eg14-3-3 ω. The Eg14-3-3 ω expression patterns were strong in the mesocarp as compared to the root. When Eg14-3-3 ω cDNA was overexpressed in transgenic calli, there was higher accumulation of oil in the transgenic calli than in the controls. Therefore, Eg14-3-3 ω has potential for applications in the breeding of oil palms in the future.

In our previous study, we reported that the nucleotide sequence of a 14-3-3ε cDNA clone from Litopenaeus vannamei encodes two transcripts, designated as 14-3-3EL and 14-3-3ES. Based on the retained introns we observed in 14-3-3EL by alternative splicing, we aimed to investigate the genomic structure of this gene. The 14-3-3ε gene, encoded by 2,311 bp of DNA sequence, is interrupted by three introns of 48, 1,124 and 365 bp in size. In addition, using the SWISS-MODEL, the predicted 3D model of 14-3-3EL has twelve α-helices and two β-sheets, whereas 14-3-3ES has only eleven helices in its tertiary structure. Additionally, we generated the 14-3-3EL and 14-3-3ES proteins and measured their thermal stabilities. The results demonstrated that the quantity of the 14-3-3EL protein decreased significantly at all temperatures tested. In contrast, the quantity of the 14-3-3ES protein remained unchanged, suggesting that 143-3-3EL is less stable than 14-3-3ES.

The aim of this study was to identify suitable artificial neural network (ANN) models for the EC-based and flow rate-based prediction of the nitrate nitrogen (NO3-N) concentration in the U-Tapao canal, located in the southern part of Thailand. Two types of four layer ANNs of the feed-forward back propagation (FFBP) and cascade-forward back propagation (CFBP) types were evaluated for this prediction. The selected inputs of the ANNs were EC and flow rate, which were collected daily from December 2014 to March 2015. Overall, the study found that the four layer FFBP with 2 neurons in the input layer, 20 neurons in the first hidden layer, 30 neurons in the second hidden layer, and a single neuron in the output layer with a tan-sigmoid transfer function was the optimal model. The FFBP model produced slightly more accurate results than the CFBP model. Linear regression analysis was used to predict NO3-N, which was compared with the results of the ANNs and the performance of the ANNs was better than that of the linear regression analysis. Therefore, the ANN approach proved to be suitable as an alternative to laboratory-based analysis for the prediction of NO3-N values in the U-Tapao canal.

Sequencing of cDNA clones from plant tissue to generate expressed sequence tags (ESTs) is an effective tool for gene discovery. Together with powerful bioinformatics tools, EST sequences allow the prediction of functions of putative bioactive compounds that can later be confirmed.To isolate a detoxification enzyme from an EST library from the oil palm (Elaeis guineensis Jacq. Arecaceae).In total, 750 clones from an oil palm cDNA library were randomly sequenced and analyzed. A clone homologous to cytochrome P450 monooxygenases (P450) was selected from the list of highly expressed genes. The full-length cDNA of P450 from E. guineensis (EgP450) was generated and transformed into a bacterial host to produce recombinant protein. A 3D model of EgP450 was generated and used in a molecular docking analysis to screen for target herbicide substrates. Finally, the detoxification activity of EgP450 was confirmed by an herbicide tolerance test with rice seedlings.The full-length EgP450 has an open reading frame (ORF) of 1515 bp that encodes a protein of 505 amino acids. Docking analysis showed that EgP450 bound to phenylurea-like herbicides such as isoproturon, chlortoluron and fluometuron. The herbicide tolerance test demonstrated that the presence of EgP450 protected the rice seedlings from the killing action of the phytotoxic agent isoproturon.The gene EgP450 was detected in the roots and stems of oil palm tissues, and its recombinant product was shown to protect rice seedlings from exogenous herbicides of the phenylurea family.

The 14-3-3 epsilon (14-3-3ε) is a member of the 14-3-3-protein family claimed to play important roles in many biological processes. In this study, two alternative 14-3-3 epsilon mRNAs, designated as 14-3-3EL and 14-3-3ES were identified from the shrimp L. vannamei. The 14-3-3EL isoform contains an insertion of 48 nucleotides by intron retention in the pre-mRNA of 14-3-3ε. While the 14-3-3ES occurred after being fully spliced. Using the yeast two hybrid method, the pattern of dimer formation by the two alternative 14-3-3ε isoforms revealed that the shrimp 14-3-3ε formed both homodimers and heterodimers. Both 14-3-3ε transcript variants were constitutively expressed in all shrimp tissues tested but the level of the 14-3-3ES isoform was always lower. However, after white spot syndrome virus (WSSV) infection, the expression level of the two transcript variants changed. At 48 h after infection, expression of 14-3-3EL mRNA increased significantly in the gill and muscle tissue whereas the expression 14-3-3ES increased only in muscle. It was of interest that in the lymphoid organ, there was a significant down-expression of both transcript variants. From these results we suggest that 14-3-3EL and 14-3-3ES might be related to different cellular processes that are modulated during virus infection.

Loss of β-cell mass and function is a fundamental feature of pathogenesis for type 1 and type 2 diabetes. Increasing evidence indicates that apoptosis is one of the main mechanisms of β-cell death in both types. Ethanolic extracts of Pluchea indica leaf (PILE) have been reported to possess blood glucose lowering actions in vivo. Nevertheless, further study is required to determine the underlying mechanisms. In this report, we have investigated the preventive effects of PILE on multiple low doses of streptozotocin (MLDS)-induced β-cell apoptosis. Mice were pre-treated with PILE at 50 mg/kg (PILE 50) or 100 mg/kg (PILE 100) for 2 weeks before streptozotocin (STZ) stimulation, and the treatment continued for 4 or 8 weeks. Results revealed that PILE 100 mice exhibited improved blood biochemistry, maintained a higher body weight, had decreased hyperglycemia, and restored islet architectures compared to non-treated STZ mice. Significantly, PILE 100 decreased levels of inflammatory response markers interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interlukin1-β (IL-1β), concomitant with the inhibition of caspase-3, caspase-8, capsepase-9, phosphorylation of signal transducer and activator of transcription 1 (pSTAT1), nuclear factor-κBp65 (NF-κBp65), and inducible nitric oxide synthase (iNOS). Additionally, survival and proliferative ability of β-cells was mediated by up-regulated Bcl-2 and Ki67, respectively. These results provide strong evidence that pretreatment with PILE 100 effectively attenuated STZ-induced diabetes-related symptoms and these effects could be associated with the inhibition of cytokine-induced β-cell apoptosis.

Ovarian maturation is a crucial step for shrimp brood stock. A suppressive subtractive hybridization was used to identify differentially expressed genes in the ovaries during vitellogenesis of Fenneropenaeus merguiensis. Three- to sevenfold up-regulated genes were selected. A blast search identified nine unique genes. The genes that may be involved in ovarian maturation, namely translationally controlled tumour protein (TCTP), heat shock protein 70 (HSP70), H-L(3)MBT-LIKE, shrimp ovarian peritrophin (SOP), vitellin (Vn), thrombospondin (TSP) and ribosomal protein L10a (RPL10a), were further studied. The transcripts of HSP70, TCTP, SOP and RPL10a in the ovary showed their highest expression in the early stage and declined in the later stages. In contrast, the transcripts of the H-L(3)MBT-LIKE, TSP and Vn genes increased from the early stage to be significantly up-regulated during the late stage. A comparison of gene expression among organs during the vitellogenesis showed that the transcripts of HSP70, SOP, H-L(3)MBT-LIKE and TSP were down-regulated in the brain, intestine, hepatopancreas and lymphoid (except for TSP) when compared with their expression in shrimp with non-developed ovaries. The mRNA of TCTP and RPL10a was significantly over-expressed in the lymphoid and heart, whereas TCTP transcripts were significantly down-regulated in the brain during the vitellogenesis. The molecular behaviour of the transcripts in this study may, in the future, lead to an ability to stimulate the ovarian development in shrimp.

Palm oil is the second largest traded oil or fat in the world market and palm is the most important crop grown mainly for its oil. Identified varieties of Elaeis guineensis Jacq., with a high oil content and produced through a selective breeding program, are desirable for improving the yield of oil, subsequently enhancing the economic feasibility of using oil palm in various applications, including bio-diesel. We have cloned the gene of biotin carboxylase ( accD ) from E. guineensis Jacq. This gene encodes a plastid-coded subunit of heteromeric acetyl-CoA carboxylase (ACCase). The cDNA of accD gene (accession number DQ004687 ) has an open reading frame of 1479 bp that encodes a putative protein of 492 amino acid residues ( AAY86362 ) with a predicted molecular mass of 55.47 kDa. The heteromeric form of ACCase is important as it catalyzes the first committed step of fatty acid synthesis. There is evidence that collectively suggests that the expression of accD in plastids is crucial to the levels of heteromeric ACCase and in turn, to the amount of seed oil in plant. Here we support the hypothesis that the expression level of accD is correlated with the oil palm production by using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (real-time PCR) analysis. Moreover, we observed the similar expression profile in nuclear-encoded subunit, biotin carboxylase ( accC ). This finding represents the genetic background of the expressed genes that correlate to high yield in plant and that ACCase can be used as a marker in the breeding program.

Since the last decade, mass spectrometry (MS) has become an essential technique for phosphoprotein analysis. Formidable analytical challenges of MS for phosphoprotein study are both the low abundance of phosphopeptides and the lack of an unambiguous diagnostic fragment ion for identification of phospho residues. These challenges can be met by a charge-based isolation of β-elimination products after tryptic digestion using diagonal strong cation-exchange chromatography.β-Elimination combined with diagonal strong cation-exchange chromatography (BE/2SCX) was used for the enrichment of phosphorylated peptides prior to a mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (MS/MS). Bovine α-casein (≥70% purity) was used as a model protein.Conditions for β-elimination were optimized to maximize the efficiency of the reaction. With a β-elimination, all four model phosphopeptides from enolase (yeast) were correctly identified. The application of the BE/2SCX enrichment strategy for the analysis of β-elimination products of α-casein (bovine) allowed the identification of 11 phosphorylated products.The introduction of a BE/2SCX-based enrichment step prior to LC/MS/MS analysis of β-elimination products facilitates the identification of phosphopeptides. Copyright © 2016 John WileySons, Ltd.

1, 38, Songklanakarin Journal of Science and Technology (SJST)

Fishmeal quality is of great importance for production of aquatic animals particularly shrimp; however it varies widely depending on raw materials and processing methods. A seven week feeding trial was conducted using five diets to examine effects of fishmeal quality on growth performance, protein digestibility and expression of gene regulating trypsin in Litopenaeus vannamei. Each test diet was fed four times daily to four groups of shrimp with an average initial weight of 2.2 g shrimp-1. The shrimp fed S1-premium grade fish meal (S1-PGFM) diet attained the highest weight gain, specific growth rate with the best feed conversion ratio (FCR) and protein utilization efficiency. Growth of shrimp fed S2-PGFM, grade 1 FM and grade 2 FM containing diets was inferior to those of S1-PGFM fed group. The imported FM diet fed group showed the slowest growth and feed utilization efficiency. FCR of shrimp fed grade 2 FM and imported FM was significantly the poorest (p

A novel cDNA of a SF21-like protein or NDR-like protein (EgNDL) from Elaeis guineensis Jacq. is 1,044 bp in length and encodes a putative protein with a 347-amino-acid open reading frame. The EgNDL showed 93% identity to the pollenspecific SF21-like protein of Phoenix dactylifera and also showed 79% identity to the NDL protein of Theobroma cacao. Expression analysis of the EgNDL gene in various tissues showed that EgNDL was expressed in the anthers, pistils, mesocarp and leaves. The high expression of EgNDL among three oil palm varieties was significantly expressed in Pisifera (P

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1–72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 μM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

The phagocytosis activating protein (PAP) gene was isolated from Penaeus monodon infected with the white spot syndrome virus (WSSV). The endocytosis pathway of GST–PAP (glutathione- S -transferase–PAP) that caused activation of phagocytosis of shrimp haemocytes was investigated. In this study, a yeast two-hybrid screening assay of a WSSV-infected P. monodon cDNA library, was performed using PAP as bait to test for the interaction between PAP and other proteins in WSSV-infected shrimp. An alpha-2-macroglobulin (α2M) was one of the proteins that interacted with PAP. A GST pull-down assay showed that GST–PAP was capable of co-precipitating α2M whereas GST itself was not. An intramuscular injection with inactivated WSSV caused a significant increase in the expression of both the PAP and α2M gene in the haemolymph. The highest expression of both genes was detected at 24 h post-injection and remained constant for 1 week. Moreover, the uptake of GST–PAP by shrimp haemocytes was higher in the presence of α2M than in its absence. The results indicated that α2M may facilitate the entry of GST–PAP into phagocytic cells and increase the survival rate of the shrimp after being infected with WSSV.

A black tiger shrimp (Penaeus monodon) caspase cDNA homologue (PmCasp) has been identified from a hemocyte library using a previously identified caspase homologue from the banana shrimp (Penaeus merguiensis) as a probe. The full-length PmCasp was 1202 bp with a 954 bp open reading frame, encoding 317 amino acids. The deduced protein contained a potential active site (QACRG pentapeptide) conserved in most caspases. It had 83% identity with caspase of P. merguiensis and 30% identity with drICE protein of Drosophila melanogaster, and it exhibited caspase-3 activity in vitro. PmCasp was cloned and expressed in Escherichia coli and a rabbit polyclonal antiserum was produced. In Western blots, the antiserum reacted with purified recombinant PmCasp and with lysates of E. coli containing the expressed plasmid. In crude protein extracts from normal shrimp, the antiserum reacted with 36 and 26 kDa bands likely to correspond to inactive pro-caspase and its proteolytic intermediate form, respectively. PmCasp expression was measured in normal shrimp and in white spot syndrome virus (WSSV)-infected shrimp at 24 and 48 h post-injection (p.i.) by semi-quantitative RT-PCR, Western blot analysis, and immunohistochemistry. Semi-quantitative RT-PCR analysis revealed up-regulation of PmCasp at 48 h p.i. and expression remained high up to the moribund state. These results were supported by Western blot analysis showing increased PmCasp protein levels at 24 and 48 h p.i. when compared to normal control shrimp. Immunohistochemical analysis of gills from the WSSV-infected shrimp revealed immunoreactivity localized in the cytoplasm of both normal and apparently apoptotic cells. In summary, a caspase-3 like gene is conserved in P. monodon and is up-regulated after WSSV infection.

Shrimp respond to viral infection with up- and down-regulation of many critical genes. In our previous work, we showed that white spot syndrome virus ( WSSV ) infection of Penaeus monodon ( Pm ) caused cellular syntenin levels to increase. In order to further explore the signal transduction pathway of syntenin, we constructed the cDNA library of WSSV -infected shrimp and performed a yeast two-hybrid screening of the library using syntenin as bait. Here we report that syntenin specifically binds eukaryotic translation initiation factor 5A (eIF5A), a 157 amino acid polypeptide that is implicated in cell proliferation and survival. GST-pull-down assays showed the presence of specific interaction between Pm -syntenin and eIF5A. Message analyses revealed that syntenin expression remained normal when shrimp were infected with WSSV but did not show any gross signs of disease. Syntenin expression, however, increased 1.4-fold when shrimp became dead. In contrast, eIF5A expression increased markedly during the stage of WSSV infection when there were no gross signs of disease, i.e., prior to the moribund stage. At the moribund stage, eIF5A expression was down-regulated. This data suggest that syntenin and eIF5A physically interact with each other in the setting of WSSV infection and that these two molecules exhibit dynamic changes in their expression patterns as infection progresses from grossly normal, infected shrimp to full-blown phase.

Apoptosis plays a critical role in many diseases including cancer and infectious diseases. The assessment of apoptosis occurring in the human body would allow clinicians to diagnose and treat these...

A large-scale expressed sequence tag (EST) sequencing project was undertaken for the purpose of gene discovery in the black tiger shrimp Penaeus monodon. Initially, 15 cDNA libraries were constructed from different tissues (eyestalk, hepatopancrease, haematopoietic tissue, haemocyte, lymphoid organ, and ovary) of shrimp, reared under normal or stress conditions, to identify tissue-specific genes and genes responding to infection and heat stress. A total of 10,100 clones were analyzed by single-pass sequencing from the 5' end. Clustering and assembling of these ESTs resulted in a total of 4845 unique sequences with 917 overlapping contigs and 3928 singletons. The redundancy of each cDNA library ranged from 13.4% to 61.3% with an overall redundancy of 61.1%. About half of these ESTs (2365 clones, 48.8%) showed significant homology (BLASTX, e-values

Ovarian development in prawns is not well understood. The up-regulated prawn ovarian translationally controlled tumor protein (Pmer-TCTP) of the banana prawn (Penaeus merguiensis) was quantitated by real-time PCR. The results showed that the gene has a significantly increased expression (P

White spot syndrome virus (WSSV) is a highly virulent and infectious virus to farmed shrimps and represents a serious threat to aquatic industries. Fortilin, also known as translationally controlled tumor protein (TCTP), protects mammalian cells under stress from cell death. A subtraction hybridization screening in our laboratory between healthy and moribund WSSV-infected Penaeus mondon shrimps has identified a fortilin gene whose messages critically decrease during the terminal stage of the WSSV-induced illness. Fortilin/TCTPs are highly conserved throughout the animal and plant kingdoms, and shrimp fortilin has a 64% identity in amino acid composition with human fortilin. In our previous work, the data clearly suggested that fortilin in shrimp protected WSSV-infected shrimps from death. Although human fortilin has a role in apoptosis regulation, it is not known if shrimp fortilin has any role in apoptosis regulation. We report that fortilin is greatly upregulated in shrimp haemolymph during the early phase of WSSV infection and that its expression abruptly decreases as the shrimp becomes moribund. Strikingly, shrimp fortilin, when overexpressed in mammalian cells, protected them from cell death induced by etoposide, staurosporine, cisplatin, hydroxyurea, and 5-fluorouracil (5-FU). These data suggest that shrimp fortilin, like mammalian fortilin, can protect cells under toxic conditions from death. In addition, since shrimp fortilin was capable of protecting cells in a mammalian environment, this indicates that shrimp and human fortilin use a common cellular pathway to achieve this. Shrimp fortilin may play a critical role in their response to WSSV-infection, through regulation of a cell death pathway that is common to shrimp and humans.

The ribosomal protein L26 (RPL26) gene, a macrophage activator, isolated from Penaeus monodon infected with the white spot syndrome virus (WSSV) had a deduced amino acid sequence that had 98.6% and a 63.1% similarity to the sequences of RPL26 isolated from Penaeus japonicus and Mus musculus, respectively. As no macrophage has been reported in shrimp, the shrimp macrophage activating protein was named phagocytosis activating protein (PAP). In this study, an intramuscular injection with inactivated Vibrio harveyi (IVH), formalin-inactivated WSSV and fucoidan produced a significantly increased expression (p b 0.05) of the RPL26 gene in P. monodon. Activation was detected by RT-PCR with maximum expression occurring after 72 h (3-fold), 1 week (3-fold) and 2 weeks (4.8-fold) post-injection of the immunostimulants, respectively. The survival rates of shrimps challenged with a 7 × 10 − 6 and 9 × 10 − 6 dilution of WSSV, 72 h after an intramuscular injection of IVH were 60.00% and 53.33%, respectively. An oral administration of IVH at 225, 300 and 375 mg/kg of body weight/day also protected shrimps from WSSV infection (survival rates were 33.33%, 80% and 40%, respectively). In addition, the recombinant phagocytosis activator protein (rGST-PAP) showed a 2 fold increased percentage of haemocyte phagocytosis compared with a non activated or rGST activated haemocyte. Therefore, the results indicate that immuno-stimulation with IVH will induce the PAP gene and facilitate the protective defenses against WSSV infection. Moreover, the expression level of the PAP gene might act as an indicator of the immune response of cultured shrimp.

The ribosomal DNA internal transcribed spacer 1 (ITS1) was investigated in the search for additional genetic marker that is suitable for population studies of the penaeid shrimps. The sequence variations of the ITS1 were determined and found to be informative in estimating phylogenies in that they differentiate four species of penaeid shrimps, namely Penaeus merguiensis , Penaeus silasi , Penaeus monodon and Penaeus semisalcatus and the populations of P. merguiensis collected in the Gulf of Thailand and the Andaman Sea . The length of the ITS1 ranged from 499 to 772 bp, with a GC content of 63.30–67.37%. Four microsatellite loci are found in the ITS1 at 5′ end and the middle of region and seem to be associated with sequence divergence and size variation in Penaeus species. Some microsatellites were found in only one specie, (GCGA) 4 in P. semisalcatus and (CGGA) 4–9 in P. monodon . These microsatellite regions are considerably long enough and the level of intragenomic variation in P. merguiensis is less than that between different species, hence, provide a great potential use in the population studies.

Apoptosis is a fundamental cellular process that plays a critical role in normal development and tissue homeostasis, eliminating from an organism unwanted cells, including damaged and virus-infected cells. In order to investigate the significance of apoptosis in response to viral infections in crustacean, we have cloned a caspase gene (cap-3) from the white shrimp, Penaeus merguiensis infected with white spot syndrome virus (WSSV). The deduced amino acid sequence of the protein named CAP-3 was 320 amino acids in length and contained the highly conserved pentapeptide QACRG recognized as the active site of caspase. CAP-3 showed similarities to the insect caspase, Spodoptera frugiperda Sf caspase-1 and the caspases of Spodoptera littoralis, Bombyx mori, Drosophila melanogaster and Anopheles gambiae, which in turn belong to the group of human caspase-3, caspase-7 and CED-3. The recombinant CAP-3 protein prepared from Escherichia coli had enzymatic activity when assayed using a luminescent substrate specific for the cleavage target site for human caspase-3, implying that CAP-3 functioned as caspase-3. There was a high level of expression of this gene product in moribund shrimp infected with WSSV.

Shrimp exhibit a diverse response to viral infection that is manifested in drastic up- and down-regulations of a variety of genes. In our previous work, we identified syntenin of the shrimp Penaeus monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection, its message being greatly upregulated in the acute phase of the infection. In order to further explore the link between Pm-syntenin and viral infection, we performed a yeast two-hybrid screening of a P. monodon cDNA library, using Pm-syntenin as bait. One of the molecules that specifically interacted with Pm-syntenin was the receptor-binding domain of alpha-2-macroglobulin (alpha2M). A GST pull-down assay showed that GST-alpha2M, but not GST alone, was capable of co-precipitating syntenin. Another GST pull-down assay showed that GST-syntenin, but not GST alone, was capable of co-precipitating alpha2M. In addition, mutant analyses showed that the N-terminal 131 amino acids of syntenin were both necessary and sufficient to bind the C-terminus receptor-binding domain of alpha2M. Furthermore, WSSV-infected Pm showed a significant upregulation of the alpha2M message, suggesting that both syntenin and its protein partner alpha2M are upregulated in the acute phase of a WSSV infection. Taken together with a previous report showing the co-localization of alpha2M and syntenin in the exosome of a dendritic cell line, it is likely that syntenin, through its interaction with alpha2M, plays an important role in the immune defense mechanisms of viral infections of shrimps.

Polymorphisms of nuclear DNA markers were used to study the genetic variation and differentiation among populations of Penaeus merguiensis in Thailand. The data consisted of three nuclear loci analysed for 163 individuals from five populations collected from the Gulf of Thailand (Trad, Surat Thani and Songkhla) and the Andaman Sea (Satun and Trang). The multilocus FST estimated at three nuclear loci revealed great and highly significant differentiation between the Gulf of Thailand and Andaman Sea (FST=0.203, P

Fortilin, a potent 172-amino acid antiapoptotic polypeptide (Li, F., Zhang, D., and Fujise, K. (2001) J. Biol. Chem. 276, 47542-47549), binds MCL1, a protein of the antiapoptotic Bcl-2 family. The fortilin-MCL1 interaction stabilizes and increases the half-life of fortilin but not necessarily of MCL1 (Zhang, D., Li, F., Weidner, D., Mnjoyan, Z. H., and Fujise, K. (2002) J. Biol. Chem. 277, 37430-37438). It is not known to what extent each protein depends on the other for its apoptotic activity. Here, we present evidence that fortilin and MCL1 are capable of functioning as antiapoptotic proteins independently of each other. Using a robust small interfering RNA (siRNA)-mediated gene silencing system developed in our laboratory, we analyzed the cytoprotective effects of fortilin and MCL1 together and apart in U2OS cell lines exposed to 5-fluorouracil (5-FU) in both monoclonal and polyclonal cell populations. When MCL1 was silenced by MCL1-targeted siRNA, fortilin was still able to protect cells from 5-FU-induced cytotoxicity in a dose-dependent manner. Conversely, when fortilin was silenced by fortilin-targeted siRNA, MCL1 was also able to protect cells from 5-FU-induced cytotoxicity in a dose-dependent manner. Together, these data clearly suggest that fortilin and MCL1 can exert their cytoprotective activities independently of each other. The silencing of fortilin and MCL1 did not qualitatively change the subcellular localization of MCL1 and fortilin, respectively. The biological significance of fortilin-MCL1 interaction may be that it increases cellular resistance to apoptosis by allowing MCL1, an independently antiapoptotic protein, to stabilize another independently antiapoptotic protein, fortilin.

Oral administration of crude fucoidan (CF) extracted from Sargassum polycystum can reduce the impact of White Spot Syndrome virus (WSSV) infection in Penaeus monodon. Crude fucoidan mixed with diet for shrimps weighing of 5–8 and 12–15 g was fed before and after WSSV infection. After 10 days of infection, the maximum survival rates of shrimps of 5–8 and 12–15 g were 46% and 93%, respectively. The crude fucoidan also inhibited the growth of Vibrio harveyi, Staphylococcus aureus and Escherichia coli at minimal inhibition concentrations (MIC) of 12.0, 12.0 and 6.0 mg/ml, respectively. D 2004 Elsevier B.V. All rights reserved.

Closely related species, Penaeus merguiensis and Penaeus silasi from Thai waters, were genetically examined using variation observed in 558 base pairs (bp) of sequence from cytochrome oxidase subunit I (COI) gene of mtDNA. The sequence divergences of COI between P. merguiensis and other Penaeus species were 5.76–6.15% (P. silasi), 13.17–13.97% (Penaeus indicus), 16.43% (Penaeus vannamei), 16.63% (Penaeus monodon), and 18.37% (Penaeus japonicus). From the alignment reported that there were four clades on phylogenetic tree, the distinction of the two monophyletic clades was referred as P. merguiensis, one monophyletic clade within P. silasi and P. indicus. These results point toward the possibility of P. merguiensis being a complex of two cryptic species or a single species with strong phylogeographic subdivision.

Background Billions of cells undergo apoptosis each day in the average normal adult. The ability to readily assess the degree of apoptosis in human diseases is hampered by the lack of sensitive and specific serum biomarkers of apoptosis. Fortilin is a novel prosurvival molecule that protects cells against various noxious stimuli. While fortilin is secreted into the extracellular space under certain conditions, the relationship between the serum concentration of fortilin and the presence and extent of apoptosis in vivo remains unknown. Methods & results Using a newly developed fortilin ELISA system, we show here that fortilin exists in the normal human and mouse circulation. We further demonstrate that fortilin serum levels are significantly elevated in patients with solid cancer, in response to anti-cancer chemo- or radiation therapy. The elevation of fortilin serum levels is more robust and sensitive than that of such previously-reported serum biomarkers of apoptosis as fragmented cytokeratin-18, cytochrome c, and nucleosomal DNA. In addition, targeted apoptotic liver damage induced by Jo2 anti-Fas (CD95) antibody consistently and significantly increased serum fortilin levels in C57BL/6J mice. Finally, when challenged by anti-human-Fas IgM antibody, Jurkat leukemic T cells apoptosed and released fortilin into the medium before plasma membrane integrity was compromised. Conclusions Taken together, these data suggest that serum fortilin levels reflect the degree and extent of apoptosis occurring in vivo. General significance Fortilin is a viable serum biomarker of in vivo apoptosis and can be utilized to noninvasively assess the status of in vivo apoptosis in humans., Graphical abstract, Highlights • Ultra-sensitive fortilin ELISA has been developed. Fortilin circulates in blood. • Fortilin serum levels become highly elevated after apoptosis-inducing therapy. • Fortilin is more robust and sensitive than other serum apoptosis markers. • Fortilin is actively secreted before plasma membrane becomes disrupted. • Fortilin is an excellent serum biomarker of in vivo apoptosis.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) has been demonstrated to be involved in the immune response to bacterial challenge in various organisms. However, little is known about GILT function in innate immunity. Drosophila has been commonly used as a model for the study of the innate immune response of invertebrates. Here, we identify the CG9796, CG10157, and CG13822 genes of fruit fly Drosophila melanogaster as GILT homologues. All deduced Drosophila GILT coding sequences contained the major characteristic features of the GILT protein family: the GILT signature CQHGX2ECX2NX4C sequence and the active site CXXC or CXXS motif. The mRNA transcript levels of the Drosophila GILT genes were up-regulated after Gram-negative bacteria Escherichia coli DH5α infection. Moreover, a bacterial load assay showed that over-expression of Drosophila GILT in fat body or hemocytes led to a low bacterial colony number whereas knock-down of Drosophila GILT in fat body or hemocytes led to a high bacterial colony number when compared to a wild-type control. These results indicate that the Drosophila GILTs are very likely to play a role in the innate immune response upon bacterial challenge of Drosophila host defense. This study may provide the basis for further study on GILT function in innate immunity.

Hirschsprung disease (HSCR) is a congenital developmental defect of the enteric nervous system known to be associated with the RET-protooncogene and other candidates. Recently, a genome-wide association study has added NRG1, a regulator of the development of the enteric ganglia precursors, as a new candidate gene. The aim of this study is to validate the association of the RET-protooncogene and the NRG1 in HSCR in Thai patients. The study used TaqMan single-nucleotide polymorphism (SNP) genotyping and PCR–restriction fragment length polymorphism for genotyping of 10 SNPs within the RET-protooncogene and four SNPs within the NRG1, in 68 Thai sporadic HSCR cases and 120 ethnic-matched controls. On univariate disease association analysis, 9 of 10 RET-protooncogene SNPs and all four NRG1 SNPs showed an association with HSCR. The rs2435357 (RET-protooncogene) and rs2439305 (NRG1) showed the strongest associations with the disease at P-values of 8.17E-09 (odds ratio (OR)=6.43, 95% confidence intervals (CI)=3.33–12.40) and 6.94E-03 (OR=3.28, 95% CI=1.28–8.38), respectively. The RET-protooncogene rs2435357 (TT genotype) in combination with the NRG1 rs2439305 (GG genotype) was strongly associated with an increased risk of HSCR with a P-value of 1.99E-04 (OR=20.34, 95% CI; 2.54–162.78) when compared with a single SNP of the RET-protooncogene or NRG1. Genetic variation of the RET-protooncogene and NRG1 is involved in the risk of HSCR development in the Thai population. Moreover, the study also detected a combined effect of SNPs by SNP–SNP interaction, which may help in predicting HSCR risk.