Transcriptional profiling of the early stages of germination in Candida albicans by real-time RT-PCR

By using real-time RT-PCR, we profiled the expression of CGR1, CaMSI3, EFG1, NRG1, and TUP1 in Candida albicans strains JCM9061 and CAI4 under several conditions, including induction of morphological transition, heat shock, and treatment with calcium inhibitors. Expression of CaMSI3 changed under these growth conditions except during heat shock. CGR1 expression increased during the early stages of hyphal growth in JCM9061, while expression was strain-dependent during heat shock. Both EFG1 and NRG1 were similarly expressed under hypha-inducing conditions and heat shock. Expression of TUP1 was slightly different from the expression of EFG1 or NRG1.     

Identification and expression analysis of four heat shock protein genes associated with thermal stress in rice weevil, Sitophilus oryzae

In order to explore the function of heat shock proteins during thermal stress in rice weevil, Sitophilus oryzae, four heat shock protein genes were cloned and characterized. These heat shock protein genes (hsps) were named as Sohsp70–1, Sohsp70–2, Sohsc70, and Sohsp90, respectively. These hsps showed high sequence conservation with the maximum identity with hsps of Tribolium castaneum and other insects. All the four genes showed the highest mRNA expression in pupal stage and the lowest levels in larval stage. The induced expression of the two Sohsp70s (Sohsp70–1 and Sohsp70–2) were reached to the highest levels (15.59-fold and 12.66-fold) after 2?h of incubation at 37?°C, respectively. Expression of Sohsp90 not only was significantly elevated by heat stress but also by cold stress. Whereas, expression level of Sohsc70 was not induced either by heat or cold stress. Furthermore, for rapid heat hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were observed as 2.57, 2.53, 3.33 and 2.33-fold higher than control, respectively; for rapid cold hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were reported as 2.27, 3.02, 3.37 and 2.23-fold higher than control, respectively. Hence, our results revealed that the four Sohsps were associated with temperature adaption under rapid heat or cold hardening.     

An analysis of the Impact of <Emphasis Type="Italic">NRG1</Emphasis> Overexpression on the <Emphasis Type="Italic">Candida albicans</Emphasis> Response to Specific Environmental Stimuli

The ability of the opportunistic fungal pathogen Candida albicans to form filaments has been strongly linked to its capacity to cause disease in humans. We previously described the construction of a strain in which filamentation can be modulated both in vitro and in vivo by placing one copy of the NRG1 gene under the control of a tetracycline-regulatable promoter. To further characterize the role of NRG1 in controlling filamentous growth, and in an attempt to determine whether NRG1 downregulation is a requirement for filamentation per se, or is only necessary under certain environmental conditions, we have conducted an analysis of the growth of the tet-NRG1 strain under a variety of in vitro conditions. Through overexpression of NRG1, we were able to block filamentation of C. albicans in both liquid media and on solid media. Filamentation in response to the low-oxygen environment of embedded growth was also inhibited. In all of these conditions, normal filamentation could be restored by down regulating expression from the tet-NRG1 allele. Interestingly, although elevated NRG1 levels were able to inhibit the formation of true hyphae in response to a wide range of environmental stimuli, elevated NRG1 expression did not affect the formation of pseudohyphae on nitrogen-limiting synthetic low ammonia dextrose (SLAD) medium. This work further illustrates the key role played by NRG1 in the control of filamentation and suggests that, although NRG1 repression plays a key role in regulating true hyphal growth, it apparently does not regulate pseudohyphal growth in the same fashion.     

Heat stress regulates the expression of TPK1 gene at transcriptional and post-transcriptional levels in Saccharomyces cerevisiae

In Saccharomyces cerevisiae cAMP regulates different cellular processes through PKA. The specificity of the response of the cAMP-PKA pathway is highly regulated. Here we address the mechanism through which the cAMP-PKA pathway mediates its response to heat shock and thermal adaptation in yeast. PKA holoenzyme is composed of a regulatory subunit dimer (Bcy1) and two catalytic subunits (Tpk1, Tpk2, or Tpk3). PKA subunits are differentially expressed under certain growth conditions. Here we demonstrate the increased abundance and half-life of TPK1 mRNA and the assembly of this mRNA in cytoplasmic foci during heat shock at 37 °C. The resistance of the foci to cycloheximide-induced disassembly along with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 expression was also evaluated during a recurrent heat shock and thermal adaptation. Tpk1 protein level is significantly increased during the recovery periods. The crosstalk of cAMP-PKA pathway and CWI signalling was also studied. Wsc3 sensor and some components of the CWI pathway are necessary for the TPK1 expression upon heat shock. The assembly in foci upon thermal stress depends on Wsc3. Tpk1 expression is lower in a wsc3? mutant than in WT strain during thermal adaptation and thus the PKA levels are also lower. An increase in Tpk1 abundance in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favourable conditions. Therefore, the regulation of TPK1 expression by thermal stress contributes to the specificity of cAMP-PKA signalling.     

Candida albicans PHO81 is required for the inhibition of hyphal development by farnesoic acid

Farnesoic acid is a signaling molecule that inhibits the transition from budding yeast to filament formation in Candida albicans, but the molecular mechanism regulated by this substance is unknown. In this study, we analyzed the function of CaPHO81, which is induced by farnesoic acid. The pho81Δ mutant cells existed exclusively as filaments under favorable yeast growth conditions. Furthermore, the inhibition of hyphal growth and repression of CPH1, EFG1, HWP1, and GAP1 mRNA expression in response to farnesoic acid were defective in pho81Δ mutant cells. These data suggest a role for CaPHO81 in the inhibition of hyphal development by farnesoic acid.     

Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels

Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.     

Normalization genes for mRNA expression in the marine diatom Ditylum brightwellii following exposure to thermal and toxic chemical stresses

Toxicity assessments using the diatom Ditylum brightwellii are well documented; however, analysis of their toxicogenomics has been little attempted. Currently, quantitative real-time PCR is the most accurate and widely applied method to detect differential gene expression, including that of specific genes induced by environmental contaminants. This method requires internal reference genes to normalize expression levels, and their selection is a critical factor for the correct analysis of the results. Here, we assessed the gene expression stability of nine housekeeping genes (HKGs), including 18S rRNA, ACT, TUA, EF2, MDH, UBQ, UCE, PCNA, and GAPDH, in 28 RNA samples of D. brightwellii. All the tested HKGs displayed different expression patterns under different experimental conditions such as heat shock and exposure to metals and non-metals. Analysis of C _T values showed that at least two genes were required for proper normalization according to the tested conditions. Overall, TUA, followed by ACT, was the most stable gene under all conditions. Furthermore, we examined the expression of the HSP70 gene in D. brightwellii when exposed to heat shock and chemicals by using the most stable references and found that the gene was significantly up-regulated during the stress period. This study has evaluated, for the first time, the normalization genes in D. brightwellii, providing potential references for gene expression studies of diatoms.     

Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat,drought and their combination in Nicotiana tabacum plants

Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations.