Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3+CD56+ Subset through the Co-Culturing Dendritic Cells

Cytokine-induced killer (CIK) cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs). We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6) in DCs at the expense of Th2 inducing phenotype (IL-13) and regulatory phenotype (PD-L1, IDO). Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet) and the downregulation of the Th2 signature (GATA-3) and the Th17 marker (RORC) on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.     

Benzene Exposure at Gasoline Stations: Health Risk Assessment

This study aimed to assess the health risk of benzene exposure among gasoline stations workers. There were 98 workers from each zone of gasoline station in Muang Khon Kaen,Thailand. The benzene concentrations were monitored via personal air sampling and analyzed with a gas chromatography flame-ionization detector (GC-FID). Information about adverse symptoms were obtained by an interview questionnaire. Levels of health risk were calculated from the likelihood level of exposure and the adverse symptoms level, which were summarized in a risk matrix. Most workers had experienced adverse symptoms related to benzene toxicity (68.4%). The health risk of benzene exposure was higher than an acceptable level in 15.3% of workers and in a similarly proportion of fueling workers and cashiers. Moreover, the proportion of risk group was found highest in urban zone (18%) and the risk scores had a significant difference across all zones. This study showed that over 15% of workers were under health risk conditions on benzene exposure and over 60% of workers had experienced adverse symptoms related to benzene toxicity. Therefore, findings suggest the need of the health surveillance program and safety training for workers to increase awareness of benzene exposure.     

Locational Diversity of Alpha Satellite DNA and Intergeneric Hybridization Aspects in the Nomascus and Hylobates Genera of Small Apes

Recently, we discovered that alpha satellite DNA has unique and genus-specific localizations on the chromosomes of small apes. This study describes the details of alpha satellite localization in the genera Nomascus and Hylobates and explores their usefulness in distinguishing parental genome sets in hybrids between these genera. Fluorescence in situ hybridization was used to establish diagnostic criteria of alpha satellite DNA markers in discriminating small ape genomes. In particular we established the genus specificity of alpha satellite distribution in three species of light-cheeked gibbons (Nomascus leucogenys, N. siki, and N. gabriellae) in comparison to that of Hylobates lar. Then we determined the localization of alpha satellite DNA in a hybrid individual which resulted from a cross between these two genera. In Nomascus the alpha satellite DNA blocks were located at the centromere, telomere, and four interstitial regions. In Hylobates detectable amounts of alpha satellite DNA were seen only at centromeric regions. The differences in alpha satellite DNA locations between Nomascus and Hylobates allowed us to easily distinguish the parental chromosomal sets in the genome of intergeneric hybrid individuals found in Thai and Japanese zoos. Our study illustrates how molecular cytogenetic markers can serve as diagnostic tools to identify the origin of individuals. These molecular tools can aid zoos, captive breeding programs and conservation efforts in managing small apes species. Discovering more information on alpha satellite distribution is also an opportunity to examine phylogenetic and evolutionary questions that are still controversial in small apes.     

Neuroprotective effect of Danshensu derivatives as anti-ischaemia agents on SH-SY5Y cells and rat brain

Novel Danshensu derivatives (3–8) were designed and synthesized to improve bioactivity based on the strategy of ‘medicinal chemical hybridization’. Our previous studies indicated that these compounds exhibited noticeable cardioprotective activities. Here, we investigate whether these novel Danshensu derivatives exert neuroprotective activities. An in vitro study revealed that these compounds could increase cell viability and reduce LDH (lactate dehydrogenase) leakage. Moreover, Danshensu-cysteine derivative compounds 6 and 8 could significantly inhibit lipid peroxidation of cell membrane and regulate the expression of apoptosis-related protein (Bcl-2, Bax and caspase 3). An in vivo study demonstrated that treatment with compound 6 at 30 mg/kg markedly decreased the infarct volume of MCAO (middle cerebral artery occlusion) insulted rat brain. Furthermore, treatment with compound 6 showed the antioxidant capacity by increasing the activity of SOD (superoxide dismutase) and GPx (glutathione peroxidase) and decreasing the level of MDA (malondialdehyde) and the ROS (reactive oxygen species) production significantly. These results suggested that these novel conjugates exert significant neuroprotective effects as anti-ischaemia agents and those with high potential merit further investigation.     

Gracilaria phuquocensis sp. nov., a new flattened Gracilaria species (Gracilariales,Rhodophyta), previously recognized as G. mammillaris,from the southern coast of Vietnam

Flattened Gracilaria species are widely distributed along the coasts of the South China Sea with more than 20 species recorded. Within the South China Sea, Gracilaria mammillaris has only been reported from Vietnam, but this species is likely restricted to the western Atlantic. This study aimed to reevaluate the taxonomic status of Vietnamese specimens of ‘G. mammillaris’ using combined morphological and molecular data. Our data clearly indicated that Vietnamese specimens were morphologically and genetically distinct from authentic G. mammillaris from the western Atlantic, and also other described flat Gracilaria species. We, therefore, propose that specimens from Vietnam originally identified as G. mammillaris be designated as a new species, Gracilaria phuquocensis sp. nov. Morphologically, G. phuquocensis can be distinguished from other flat Gracilaria species by its small thallus size, narrower blades, many medullary layers, abundant basal nutritive filaments within mature cystocarps, and tetrasporangial nemathecium. Our rbcL sequence analyses showed that the new species was sister to Gracilaria rhodymenioides from Thailand, and these two species formed a clade with cylindrical Gracilaria species. Our study contributes to clarification of the taxonomic status of misidentified specimens attributed to the flattened Gracilaria species in the South China Sea.     

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

Background

Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.

Results

A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val₃₂₇ - Asn₃₅₆) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp₂₉₈-Val₃₁₃ peptide was shortened to Asp₂₉₈-Arg₃₁₀ in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.

Conclusions

The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.     

Tracing the origin of a cryptic invader: phylogeography of the Euwallacea fornicatus (Coleoptera: Curculionidae: Scolytinae) species complex

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The perforated stones of the Doi Pha Kan burials (Northern Thailand): A Mesolithic singularity?

Throughout continental Southeast Asia, the Hoabinhian techno-complex stands out in clear contrast with the universal chrono-cultural model essentially established on the basis of western prehistory. Following this model, early authors considered perforated stones and associated lithic artefacts as markers of what was then believed to pertain to a Southeast Asian Mesolithic. However, Southeast Asian Mesolitithic has progressively been abandoned in favour of a ubiquitous Hoabinhian spanning from 30,000 to 3000 BP. Here, we present and discuss the discovery of perforated stones at the Doi Pha Kan site in northern Thailand. Perforated stones have almost never been found in undisturbed stratigraphic conditions nor dated with any sufficient degree of certainty. At Doi Pha Kan site, such a kind of artefacts was found in burials intersecting sedimentary layers that could be ascertained as Hoabinhian. In contrast with similar perforated stones described in the literature, that found at Doi Pha Kan are well-dated (13,000 BP), thus providing a time-reference for a putative Southeast Asian Mesolithic. We therefore advocate that such non-Hoabinhian artefacts support the early authors’ hypothesis of the existence of a Southeast Asian Mesolithic. Finally, the funerary practices, the unusually high stature of individuals found at Doi Pha Kan in conjunction with the particular lithic assemblages further contributes to raise the question of the co-occurrence of several cultures or populations at the Pleistocene–Holocene interface in continental Southeast Asia.