The ubiquitously expressed bZIP inhibitor,JDP2, suppresses the transcription of its homologue immediate early gene counterpart,ATF3

JDP2 is a ubiquitously expressed bZIP repressor protein. JDP2 binds TPA response element and cyclic AMP response element located within various promoters. JDP2 displays a high degree of homology to the immediate early gene ATF3. ATF3 plays a crucial role in the cellular adaptive response to multiple stress insults as well as growth stimuli. We have identified ATF3 as a potential target gene for JDP2 repression. JDP2 regulates the ATF3 promoter potentially through binding to both the consensus ATF/CRE site and a non-consensus ATF3 auto-repression DNA-binding element. Expression of ATF3 protein in wild-type mouse embryo fibroblast (MEF) cells is below the detectable levels, whereas, JDP2 disrupted MEF cells display noticeable level of ATF3 protein. Following either serum or ER stress stimulation, ATF3 expression is potentiated in JDP2-KO fibroblast cells as compared with wild-type cells. Mice with either JDP2 over-expression or JDP2 disruption display undetectable level of ATF3 protein. However, ATF3 induction in response to either growth or stress signals is dependent on JDP2 expression level. ATF3 induction is attenuated in JDP2 over-expressing mice whereas is potentiated in JDP2-KO mice as compared with the corresponding wild-type mice. Collectively, the data presented strongly suggest that JDP2 plays a role in the determination of the ATF3 adaptive cellular threshold response to different stress insults and growth stimuli.     

Candida potacharoeniae sp. nov. and Candida spenceri sp. nov., two novel galactose-containing ascomycetous anamorphic yeast species isolated in Thailand

Fifteen strains of anamorphic yeasts isolated from various natural substrates collected in various places in Thailand were found to represent two novel species of anamorphic yeast genus Candida based on the sequence analysis of the D1/D2 domain of the large subunit rRNA genes, chemotaxonomic and conventional properties used for the classification of yeasts. These strains are located in the clade including Candida etchellsii and Candida magnoliae. Fourteen strains represented by ST-490(T) (BCC 15176(T)=NBRC 106439(T)= CBS 11674(T)) are closely related to Candida sorbosivorans in the D1/D2 sequences but 11 nucleotides (2.4%) were substituted. The remaining strain, ST-594(T) (=BCC 15278(T)=NBRC 106446(T)=CBS 11673(T)) showed a close relationship to Candida geochares but 21 nucleotides (4.7%) were substituted. Apparently, these strains represent two novel Candida species of the Starmerella clade. The two species are described as Candida potacharoeniae sp. nov. and Candida spenceri sp. nov. in the present paper. Like the most species of this clade, the two species contain galactose in the cells in addition to glucose and mannose and have high mol% G + C of 54.4-55.9 and 54.9, respectively.     

Real-time monitoring of quality attributes by in-line Fourier transform infrared spectroscopic sensors at ultrafiltration and diafiltration of bioprocess

Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool.     

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning,lateral vessel (STLV) bioreactor

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 × 10⁵ cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.     

CO2 efflux from subterranean nests of ant communities in a seasonal tropical forest,Thailand

Many ant species construct subterranean nests. The presence of their nests may explain soil respiration “hot spots”, an important factor in the high CO₂ efflux from tropical forests. However, no studies have directly measured CO₂ efflux from ant nests. We established 61 experimental plots containing 13 subterranean ant species to evaluate the CO₂ efflux from subterranean ant nests in a tropical seasonal forest, Thailand. We examined differences in nest CO₂ efflux among ant species. We determined the effects of environmental factors on nest CO₂ efflux and calculated an index of nest structure. The mean CO₂ efflux from nests was significantly higher than those from the surrounding soil in the wet and dry seasons. The CO₂ efflux was species‐specific, showing significant differences among the 13 ant species. The soil moisture content significantly affected nest CO₂ efflux, but there was no clear relationship between nest CO₂ efflux and nest soil temperature. The diameter of the nest entrance hole affected CO₂ efflux. However, there was no significant difference in CO₂ efflux rates between single‐hole and multiple‐hole nests. Our results suggest that in a tropical forest ecosystem the increase in CO₂ efflux from subterranean ant nests is caused by species‐specific activity of ants, the nest soil environment, and nest structure.     

Two new ascomycetous anamorphic yeast species related to <Emphasis Type="Italic">Candida friedrichii</Emphasis>—<Emphasis Type="Italic">Candida jaroonii</Emphasis> sp. nov., and <Emphasis Type="Italic">Candida songkhlaensis</Emphasis> sp. nov.—isolated in Thailand

In a study of yeast diversity in Thailand, eight strains of hitherto undescribed anamorphic yeasts were isolated: four from insect frass, two from Marasmius sp. fruiting bodies, one from a flower, and one from jackfruit exudates. Phylogenetic analysis of the D1/D2 domain of 26S ribosomal DNA nucleotide sequences indicated that the eight strains represented two new species related to Candida friedrichii. Genetic separation of the two new species was further supported by DNA-DNA hybridization analysis, which resulted in between-species similarity values of less than 48%, and by electrophoretic karyotyping. The two new species are C. jaroonii sp. nov. (type strain, ST-300(T) = NBRC 103209(T) = BCC 11783(T) = CBS 10790(T)) and C. songkhlaensis sp. nov. (type strain, ST-328(T) = NBRC 103214(T) = BCC 11804(T) = CBS 10791(T)).     

Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors

Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation. For standardized mass EB production, a well defined scale-up platform is necessary. Recently, novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems (spinner flasks), rotating cell culture system and rotary orbital culture have allowed large-scale EB formation. Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods. This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems. Furthermore, an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently, new insights in induced pluripotent stem (iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research. These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity. Hence, culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells, although direct evidence of their use for iPS cells is still limited.     

Candida nonsorbophila sp. nov., a new ascomycetous yeast species isolated in Thailand

Four yeast strains, RS42, SSK10, ST-520 and ST-521, isolated from water in a mangrove forest, bark of a tree and fruit of Ficus sp., respectively, were found to represent a hitherto undescribed anamorphic species. The four strains are related to Candida sinolaborantium in the D1/D2 domain of the large subunit rRNA gene, but differed by eight nucleotide substitutions and two indels, and for this reason are regarded as members of a separate species. Because ascospore formation was not detected, it is described as a new species of Candida, Candida nonsorbophila sp. nov. The type strain is RS42^T (BCC 25963^T=NBRC 103860^T=CBS 10862^T). This species is distinguished from C. sinolaborantium by the inability to assimilate l -sorbose, l -rhamnose and galactitol, and a higher maximum growth temperature.