Sittiruk Roytrakul

Silver nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent pathways

Silver nanoparticles (AgNPs) are incorporated into a large number of consumer and medical products. Several experiments have demonstrated that AgNPs can be toxic to the vital organs of humans and especially to the lung. The present study evaluated the in vitro mechanisms of AgNP (<100 nm) toxicity in relationship to the generation of reactive oxygen species (ROS) in A549 cells. AgNPs caused ROS formation in the cells, a reduction in their cell viability and mitochondrial membrane potential (MMP), an increase in the proportion of cells in the sub-G1 (apoptosis) population, S phase arrest and down-regulation of the cell cycle associated proliferating cell nuclear antigen (PCNA) protein, in a concentration- and time-dependent manner. Pretreatment of the A549 cells with N-acetyl-cysteine (NAC), an antioxidant, decreased the effects of AgNPs on the reduced cell viability, change in the MMP and proportion of cells in the sub-G1population, but had no effect on the AgNP-mediated S phase arrest or down-regulation of PCNA. These observations allow us to propose that the in vitro toxic effects of AgNPs on A549 cells are mediated via both ROS-dependent (cytotoxicity) and ROS-independent (cell cycle arrest) pathways.

Identification of prohibitin as a Chikungunya virus receptor protein

Chikungunya virus (CHIKV) has recently re‐emerged causing millions of infections in countries around the Indian Ocean. While CHIKV has a broad host cell range and productively infects a number of different cell types, macrophages have been identified as a potential viral reservoir serving to increase the duration of symptoms. To date no CHIKV interacting protein has been characterized and this study sought to identify CHIKV binding proteins expressed on target cell membranes. Two‐dimensional virus overlay identified prohibitin (PHB) as a microglial cell expressed CHIKV binding protein. Co‐localization, co‐immunoprecipitation as well as antibody and siRNA mediated infection inhibition studies all confirmed a role for PHB in mediating internalization of CHIKV into microglial cells. PHB is the first identified CHIKV receptor protein, and this study is evidence that PHB may play a role in the internalization of multiple viruses. J. Med. Virol. 84:1757–1770, 2012.

Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon

Isolation and characterization of genes involving gonadal development are an initial step towards understanding reproductive maturation and sex determination of the giant tiger shrimp (Penaeus monodon). In the present study, 896 clones from the testis cDNA library were sequenced. A total of 606 ESTs (67.6%) significantly matched sequences in the GenBank (E-value <1e-04) whereas 290 ESTs (32.4%) were newly unidentified transcripts. The full length cDNA of genes functionally involved in testicular development including cyclophilin A (PMCYA), small ubiquitin-like modifier 1 (PMSUMO-1), ubiquitin conjugating enzyme E2, dynactin subunit 5, cell division cycle 2 (cdc2) and mitotic checkpoint BUB3 were discovered. In addition, Tra-2, a gene involving sex determination cascades, was successfully characterized by RACE-PCR and first reported in crustaceans. Expression analysis indicated that a homologue of low molecular weight neurofilament protein XNF-L (termed P. monodon testis-specific transcript 1, PMTST1; N=8 for each sex) was only expressed in testes but not ovaries. PMCYA, thyroid hormone receptor-associated protein complex 240 kDa component (Trap240), multiple inositol polyphosphate phosphatase 2 (MIPP2) and heat shock-related 70 kDa protein 2 (HSP70-2), but not PMSUMO-1, PMTra-2 and prohibitin2 were differentially expressed between ovaries and testes of P. monodon. Expression of PMTST1 was up-regulated but that of the remaining genes in testes of P. monodon broodstock was down-regulated after shrimp were molted (P<0.05). Significant reduction of PMSUMO-1 and increment of prohibitin2 transcripts in domesticated broodstock (P<0.05) suggested that these reproductively related genes may be used as biomarkers to evaluate reduced degrees of the reproductive maturation in domesticated P. monodon.

Chikungunya in Southeast Asia: understanding the emergence and finding solutions

In the last few years, chikungunya has become a major problem in Southeast Asia, with large numbers of cases being reported in Singapore, Malaysia, and Thailand. Much of the current epidemic of chikungunya in Southeast Asia is being driven by the emergence of a strain of chikungunya virus that originated in Africa and spread to islands in the Indian Ocean, as well as to India and Sri Lanka, and then onwards to Southeast Asia. There is currently no specific treatment for chikungunya and no vaccine is available for this disease. This review seeks to provide a short update on the reemergence of chikungunya in Southeast Asia and the prospects for control of this disease.

Extraction and characterisation of pepsin‐solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus)

BACKGROUND Fish collagen has been paid increasing attention as an alternative to the mammalian counterpart owing to the abundance of fish skin as a processing by-product. Generally, the low yield of collagen extracted using the typical acid solubilisation process has led to the use of mammalian pepsin as an aid for increasing the yield. Alternatively, fish pepsin, especially from tuna stomach, can be used for the extraction of pepsin-solubilised collagen (PSC). Therefore the objective of this study was to extract and characterise PSC from the skin of bigeye snapper, a fish widely used for surimi production in Thailand. RESULTS PSCs from the skin of two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, were extracted with the aid of tongol tuna (Thunnus tonggol) pepsin and porcine pepsin. PSCs from the skin of both species extracted using porcine pepsin had a higher content of beta-chain but a lower content of alpha-chains compared with those extracted using tuna pepsin. All PSCs contained glycine as the major amino acid and had an imino acid (proline and hydroxyproline) content of 189-193 residues per 1000 residues. Transition temperatures of PSCs were in the range 30.6-31.3 degrees C. Fourier transform infrared spectra revealed some differences in molecular order between PSCs extracted using porcine pepsin and tuna pepsin. Nevertheless, the triple-helical structure of PSCs was not affected by pepsin digestion. Zeta potential analysis indicated that PSCs from P. tayens and P. macracanthus possessed zero net charge at pH 7.15-7.46 and 5.97-6.44 respectively. CONCLUSION Tongol tuna pepsin could be used as a replacement for mammalian pepsin in PSC extraction. However, a slight difference in PSC properties was found.

Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms.

Determination of the Plasmodium vivax schizont stage proteome

With the genome of the malaria parasite Plasmodium vivax sequenced, it is important to determine the proteomes of the parasite in order to assist efforts in antigen and drug target discovery. Since a method for continuous culture of P. vivax parasite is not available, we tried to study the proteome of the erythrocytic stages using fresh parasite isolates from patients. In schizont-enriched samples, 316 proteins were confidently identified by tandem mass spectrometry. Almost 50% of the identified proteins were hypothetical, while other major categories include proteins with binding function, protein fate, protein synthesis, metabolism and cellular transport. To identify proteins that are recognized by host humoral immunity, parasite proteins were separated by two-dimensional gel electrophoresis and screened by Western blot using an immune serum from a P. vivax patient. Mass spectrometry analysis of protein spots recognized by the serum identified four potential antigens including PV24. The recombinant protein PV24 was recognized by antibodies from vivax malaria patients even during the convalescent period, indicating that PV24 could elicit long-lasting antibody responses in P. vivax patients.

Comprehensive proteomic analysis of white blood cells from chikungunya fever patients of different severities

BackgroundChikungunya fever (CHIKF) is a recently re-emerged mosquito transmitted viral disease caused by the chikungunya virus (CHIKV), an Alphavirus belonging to the family Togaviridae. Infection of humans with CHIKV can result in CHIKF of variable severity, although the factors mediating disease severity remain poorly defined.MethodsWhite blood cells were isolated from blood samples collected during the 2009-2010 CHIKF outbreak in Thailand. Clinical presentation and viral load data were used to classify samples into three groups, namely non chikungunya fever (non-CHIKF), mild CHIKF, and severe CHIKF. Five samples from each group were analyzed for protein expression by GeLC-MS/MS.ResultsCHIKV proteins (structural and non-structural) were found only in CHIKF samples. A total of 3505 human proteins were identified, with 68 proteins only present in non-CHIKF samples. A total of 240 proteins were found only in CHIKF samples, of which 65 and 46 were found only in mild and severe CHIKF samples respectively. Proteins with altered expression mapped predominantly to cellular signaling pathways (including toll-like receptor and PI3K-Akt signaling) although many other processes showed altered expression as a result of CHIKV infection. Expression of proteins consistent with the activation of the inflammasome was detected, and quantitation of (pro)-caspase 1 at the protein and RNA levels showed an association with disease severity.ConclusionsThis study confirms the infection of at least a component of white blood cells by CHIKV, and shows that CHIKV infection results in activation of the inflammasome in a manner that is associated with disease severity.

Characterization and expression of cell division cycle 2 (Cdc2) mRNA and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

The meiotic maturation of oocytes is regulated by the maturation-promoting factor (MPF), a complex of Cdc2 (Cdk1) and Cyclin B. Here, the complete open reading frame (ORF) of Cdc2 in Penaeus monodon was characterized. PmCdc2 were 900bp in length corresponding to a polypeptide of 299 amino acids with the conserved Thr14, Tyr15 and Thr161 residues. Quantitative real-time PCR indicated that the expression level of PmCdc2 in wild intact broodstock was significantly increased in stages II (vitellogenic) and III (early cortical rod) ovaries relative to stage I (previtellogenic) ovaries and peaked in stage IV (mature) ovaries (P<0.05). The expression level of PmCdc2 in stages I-IV ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian developmental stages in intact broodstock (P<0.05). Expression levels of PmCdc2 in ovaries of 18-month-old P. monodon upon 5-HT injection (50μg/g body weight) were significantly increased at 1hour post injection (hpi, P<0.05). Recombinant PmCdc2 protein and its polyclonal antibody were successfully produced. Western blot analysis revealed the expected 34kDa band (PmCdc2) along with a smaller band of 23kDa (ribosomal protein S3) in ovaries of juveniles and various ovarian stages of broodstock. Using phospho-Cdc2 (Thr161) polyclonal antibody, the positive signal of 34kDa was observed in all ovarian stages but the most intense signal was found in stage IV ovaries. Results in the present study indicated that PmCdc2 gene/protein plays an important role in the development and maturation of oocytes/ovaries in P. monodon.

Garvieacin Q, a Novel Class II Bacteriocin from Lactococcus garvieae BCC 43578

ABSTRACT Lactococcus garvieae BCC 43578 produces a novel class II bacteriocin, garvieacin Q (GarQ), 70 amino acids in length and containing a 20-amino-acid N-terminal leader peptide. It is cleaved at the Gly-Gly site to generate the mature GarQ (5,339 Da), which is especially inhibitory against Listeria monocytogenes ATCC 19115 and other L. garvieae strains.