Heat Shock Stress Research Articles

Roles of Three HSF Domain-Containing Proteins in Mediating Heat-Shock Protein Genes and Sustaining Asexual Cycle, Stress Tolerance, and Virulence in Beauveria bassiana.

Heat-shock transcription factors (HSFs) with a HSF domain are regulators of fungal heat-shock protein (HSP) genes and many others vectoring heat-shock elements, to which the domain binds in response to heat shock and other stress cues. The fungal insect pathogen Beauveria bassiana harbors three HSF domain-containing orthologous to Hsf1, Sfl1, and Skn7 in many fungi. Here, we show that the three proteins are interrelated at transcription level, play overlapping or opposite roles in activating different families of 28 HSP genes and mediate differential expression of some genes required for asexual developmental and intracellular Na+ homeostasis. Expression levels of skn7 and sfl1 largely increased in Δhsf1, which is evidently lethal in some other fungi. Hsf1 was distinct from Sfl1 and Skn7 in activating most HSP genes under normal and heat-shocked conditions. Sfl1 and Skn7 played overlapping roles in activating more than half of the HSP genes under heat shock. Each protein also activated a few HSP genes not targeted by two others under certain conditions. Deletion of sfl1 resulted in most severe growth defects on rich medium and several minimal media at optimal 25°C while such growth defects were less severe in Δhsf1 and minor in Δskn7. Conidiation level was lowered by 76% in Δskn7, 62% in Δsfl1, and 39% in Δhsf1. These deletion mutants also showed differential changes in cell wall integrity, antioxidant activity, virulence and cellular tolerance to osmotic salt, heat shock, and UV-B irradiation. These results provide a global insight into vital roles of Hsf1, Sfl1, and Skn7 in B. bassiana adaptation to environment and host.

Identification of heat shock-inducible transcripts and Hop (Hsp-organizing protein)-interacting proteins in orchardgrass (Dactylis glomerata L.)

Plants are continuously exposed to temperature changes and biotic/abiotic stresses. Current global warming dynamics cause extreme temperature fluctuations that severely affect crop productivity. Orchardgrass (Dactylis glomerata L.) is one of the most important perennial grasses adapted for cool temperatures. Orchardgrass displays rapid establishment, vigorous growth, and rapid recovery after cutting or grazing, but it is sensitive to heat stress. In this study, we isolated 23 heat shock-inducible partial gene fragments from orchardgrass using a differential-display RT-PCR and a reverse northern blotting. Those full-length cDNAs encode major molecular chaperones (Hsp90s, Hsp70, and small Hsp) and Hsp90 co-chaperones (Hop, p23, and Aha) were identified from a heat-treated orchardgrass cDNA library and analyzed those expressions by northern blot analysis. In addition, we further characterized Hop, which plays as an important adapter that organizes the Hsp90 and Hsp70 complex in the Hsp90-associated heteromultimeric chaperone complex. Orchardgrass Hop (DgHop) transcript levels were enhanced by heat shock stress and dramatically reduced by cold, salt, or dehydration stress. DgHop contains three conserved tetratricopeptide repeat domains, which are involved in interactions with major Hsps. Yeast two-hybrid and pull-down analysis indicated that the major DgHop client proteins were Hsp90 and Hsp70, and DgHop directly interacted with DgHsp90 and DgHsp70. Our data provide new genetic information on heat shock responses in orchardgrass. We suggest that this information contribute to generating heat shock-tolerant forage crops.

Abstract 461: An essential role of the iron-sulfur cluster assembly enzyme isu-1 in the aging and stress responses of C. elegans

Abstract Iron is essential for animal cellular homeostasis by acting in the biogenesis of two important redox-reactive prosthetic groups of enzymes: iron sulfur clusters (ISC) and heme. Previous studies have indicated that the misregulated iron metabolism is associated with multiple aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. However, how iron metabolism modulates the aging process remains largely elusive.In the current study, we aim to reveal the roles and mechanisms of iron metabolism in the aging process of C. elegans, a well-established genetic model organism for aging research. Through our preliminary studies, we have found that the iron-sulfur cluster assembly gene isu-1 plays an important role in the lifespan modulation and stress resistance of C. elegans. Specifically, RNAi knocking-down of isu-1 causes a significantly upregulated mitochondrial unfolded protein response (mitoUPR). Furthermore, the isu-1 RNAi-treated worms are significantly more resistant to heat shock and oxidative stress. Lastly, isu-1 RNAi significantly extends lifespan (~25.7%).Currently, we are examining the transcription factors downstream of isu-1 in regulating aging and stress responses. Surprisingly, the isu-1 RNAi-triggered lifespan extension is independent of insulin and IGF pathway, an evolutionarily conserved master regulator of animal growth and aging. Next, we will characterize the mechanisms underlying the effects of iron-sulfur cluster in animal aging and stress response using various genetic and biochemistry approaches. Citation Format: Rui Xiao, Yi Sheng. An essential role of the iron-sulfur cluster assembly enzyme isu-1 in the aging and stress responses of C. elegans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 461.

Predicted Glycerol 3-Phosphate Dehydrogenase Homologs and the Glycerol Kinase GlcA Coordinately Adapt to Various Carbon Sources and Osmotic Stress in Aspergillus fumigatus.

Glycerol plays an important role in the adaptation of fungi to various microenvironments and stressors, including heat shock, anoxic conditions and osmotic stress. Glycerol 3-phosphate dehydrogenase (G3PDH) is able to catalyze dihydroxyacetone phosphate to glycerol 3-phosphate (G3P), which is subsequently dephosphorylated into glycerol. However, current knowledge about the functions of G3PDH homologs in glycerol biosynthesis in Aspergillus fumigatus is limited. Here, we show that the A. fumigatus G3PDH gene, gfdA, is crucial for normal colony growth in glucose media under both normoxic and hypoxic conditions. In addition, failure of the overexpression of the gfdA homolog, gfdB, to rescue the phenotype of a gfdA null mutant suggests that gfdA plays a predominant role in the synthesis of G3P and glycerol. However, in a wild-type background, overexpressing either gfdA or gfdB is able to significantly enhance biomass production of mycelia, suggesting that gfdA and gfdB have similar functions in promoting the use of glucose. Interestingly, overexpression of the gene encoding the predicted glycerol kinase, GlcA, which is capable of phosphorylating glycerol to form G3P, significantly rescues the growth defects of gfdA null mutants in glucose media, indicating that the growth defects of gfdA null mutants might be due to the absence of G3P rather than glycerol. Moreover, Western blotting analysis revealed that gfdA is inducibly expressed by osmotic mediators. However, in the absence of gfdA, osmotic stress can rescue colony growth defects and allow colonies to partially bypass the gfdA requirement in a high osmolarity glycerol pathway-dependent manner. Therefore, the findings of this study elucidate how saprophytic filamentous fungi have developed pathways distinct from those of budding yeasts to adapt to varied carbon sources and survive environmental stresses.

Tracking Gene Expression via Light Sheet Microscopy and Computer Vision in Living Organisms.

Automated tracking of spatiotemporal gene expression using in vivo microscopy images have given great insight into understanding developmental processes in multicellular organisms. Many existing analysis tools rely on the fluorescent tagging of cell wall or cell nuclei localized proteins to assess position, orientation, and overall shape of an organism; information necessary for determining locations of gene expression activity. Particularly in plants, organism lines that have fluorescent tags can take months to develop, which can be time consuming and costly. We propose an automated solution for analyzing spatial characteristics of gene expression without the necessity of fluorescent tagged cell walls or cell nuclei. Our solution indicates, segments, and tracks gene expression using a fluorescent imaging channel of a light sheet microscope while determining gene expression location within an organism from a Brightfield (non-fluorescent) imaging channel. We use the images obtained from the Arabidopsis thaliana root as a proof of concept for our solution by studying the effects of heat shock stress on CYCLIN B1 protein production.

Modification of N6-methyladenosine RNA methylation on heat shock protein expression.

This study was conducted to investigate effect of N6-methyladenosine (m6A) RNA methylation on Heat shock proteins (HSPs) and dissect the profile of HSP RNA methylation. The results showed that m6A methyltransferases METTL3 mRNA was decreased in responses to heat shock stress in HepG2 cells, but m6A-specific binding protein YTHDF2 mRNA was upregulated in a manner similar to HSP70 induction. Immunofluorescence staining showed that the majority of YTHDF2 was present in the cytosol, however, nearly all YTHDF2 translocated from the cytosol into the nucleus after heat shock. METTL3 knockdown significantly changed HSP70, HSP60, and HSP27 mRNA expression in HepG2 cells using siRNA, however, mRNA lifetime was not impacted. Silence of YTHDF2 using siRNA did not change expression of HSP70, but significantly increased HSP90, HSP60, and HSPB1 mRNA expression. In addition, m6A-seq revealed that HSP m6A methylation peaks are mainly enriched on exons and around stop codons, and shows a unique distribution profile in the 5’UTR and 3’UTR. Knockdown of METTL3 changed the methylation patterns of HSPs transcript. In conclusion, m6A RNA methylation regulates HSP gene expression. Differential expression of HSPs modulated by m6A may depend on the m6A site and abundance of the target gene. This finding provides insights into new regulatory mechanisms of HSPs in normal and stress situations.

DdrI, a cAMP Receptor Protein Family Member, Acts as a Major Regulator for Adaptation of Deinococcus radiodurans to Various Stresses

The DNA damage response ddrI gene encodes a transcription regulator belonging to the cAMP receptor protein (CRP) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects and sensitivity to DNA-damaging agents and to oxidative stress. Here, we show that the absence of the DdrI protein also confers sensitivity to heat shock treatment, and several genes involved in heat shock response were shown to be upregulated in a DdrI-dependent manner. Interestingly, expression of the Escherichia coli CRP partially compensates for the absence of the DdrI protein. Microscopic observations of ΔddrI mutant cells revealed an increased proportion of two-tetrad and anucleated cells in the population compared to the wild-type strain, indicating that DdrI is crucial for the completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. The in silico prediction of putative DdrI binding sites in the D. radiodurans genome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, the DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and it acts as a dimer. All these data underline the major role of DdrI in D. radiodurans physiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes, including central metabolism and specific responses to diverse harmful environments.IMPORTANCEDeinococcus radiodurans has been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, and diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights into the crucial contribution of the transcription factor DdrI for the D. radiodurans ability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock, and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.

Holothuria scabra extracts possess anti-oxidant activity and promote stress resistance and lifespan extension in Caenorhabditis elegans.

Holothuria scabra is a sea cucumber that is mostly found in the Indo-Pacific region including Thailand. Extracts from many sea cucumbers possess pharmacological activities proposed to benefit human health. In this study, we investigated the anti-oxidant and anti-ageing activities of extracts from H. scabra by using Caenorhabditis elegans as a model organism. Parts of H. scabra were solvent-extracted and divided into nine fractions including whole body-hexane (WBHE), whole body-ethyl acetate (WBEA), whole body-butanol (WBBU), body wall-hexane (BWHE), body wall-ethyl acetate (BWEA), body wall-butanol (BWBU), viscera-hexane (VIHE), viscera-ethyl acetate (VIEA), and viscera-butanol (VIBU). All fractions of the extracts were tested for anti-oxidant activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays and for anti-ageing effects by lifespan assays using C. elegans as a model. The results showed anti-oxidant properties in all fractions with the highest activity shown by the DPPH assay in WBBU (EC50 = 3.12 ± 0.09 mg/ml), and by the ABTS assay in WBHE (EC50 = 0.31 ± 0.10 mg/ml). In lifespan assays the highest anti-ageing effect was detected in WBBU- and BWEA-treated C. elegans with increased mean lifespans of 8.12% and 4.77%, respectively. Furthermore, WBBU and BWEA-treated C. elegans exhibited significantly higher resistance against heat shock and paraquat-induced oxidative stresses than controls. By using LC-MS/MS, both extracts were characterized to contain triterpene glycosides as the main bioactive components. To explore mechanisms of H. scabra extracts on longevity and stress resistance, worms with genetic mutations in anti-ageing pathways were analyzed and showed that WBBU and BWEA did not prolong the lifespan of daf-16, age-1, sir-2.1, jnk-1, sek-1, and osr-1 mutants, suggesting that these genetic pathways are involved in mediating the anti-ageing effects of the H. scabra extracts. Moreover, WBBU and BWEA enhanced the nuclear translocation of the FoxO/DAF-16 transcription factor, and increased mRNA expression of this gene and its downstream targets sod-3, hsp12.3, and hsp16.2. In conclusion, this study strongly demonstrates anti-oxidant and anti-ageing properties of H. scabra extracts containing triterpene glycosides, which, in the C. elegans model, may be mediated via the insulin/IGF-1 signaling (IIS)-DAF-16 pathway.

Different Expression Levels of Human Mutant Ubiquitin B+1 (UBB+1) Can Modify Chronological Lifespan or Stress Resistance of Saccharomyces cerevisiae.

The ubiquitin-proteasome system (UPS) is the main pathway responsible for the degradation of misfolded proteins, and its dysregulation has been implicated in several neurodegenerative diseases, including Alzheimer’s disease (AD). UBB+1, a mutant variant of ubiquitin B, was found to accumulate in neurons of AD patients and it has been linked to UPS dysfunction and neuronal death. Using the yeast Saccharomyces cerevisiae as a model system, we constitutively expressed UBB+1 to evaluate its effects on proteasome function and cell death, particularly under conditions of chronological aging. We showed that the expression of UBB+1 caused inhibition of the three proteasomal proteolytic activities (caspase-like (β1), trypsin-like (β2) and chymotrypsin-like (β5) activities) in yeast. Interestingly, this inhibition did not alter cell viability of growing cells. Moreover, we showed that cells expressing UBB+1 at lower level displayed an increased capacity to degrade induced misfolded proteins. When we evaluated cells during chronological aging, UBB+1 expression at lower level, prevented cells to accumulate reactive oxygen species (ROS) and avert apoptosis, dramatically increasing yeast life span. Since proteasome inhibition by UBB+1 has previously been shown to induce chaperone expression and thus protect against stress, we evaluated our UBB+1 model under heat shock and oxidative stress. Higher expression of UBB+1 caused thermotolerance in yeast due to induction of chaperones, which occurred to a lesser extent at lower expression level of UBB+1 (where we observed the phenotype of extended life span). Altering UPS capacity by differential expression of UBB+1 protects cells against several stresses during chronological aging. This system can be valuable to study the effects of UBB+1 on misfolded proteins involved in neurodegeneration and aging.

Systematic biology analysis on photosynthetic carbon metabolism of maize leaf following sudden heat shock under elevated CO2

Plants would experience more complex environments, such as sudden heat shock (SHS) stress combined with elevated CO2 in the future, and might adapt to this stressful condition by optimizing photosynthetic carbon metabolism (PCM). It is interesting to understand whether this acclimation process would be altered in different genotypes of maize under elevated CO2, and which metabolites represent key indicators reflecting the photosynthetic rates (PN) following SHS. Although B76 had greater reduction in PN during SHS treatment, our results indicated that PN in genotype B76, displayed faster recovery after SHS treatment under elevated CO2 than in genotype B106. Furthermore, we employed a stepwise feature extraction approach by partial linear regression model. Our findings demonstrated that 9 key metabolites over the total (35 metabolites) can largely explain the variance of PN during recovery from SHS across two maize genotypes and two CO2 grown conditions. Of these key metabolites, malate, valine, isoleucine, glucose and starch are positively correlated with recovery pattern of PN. Malate metabolites responses to SHS were further discussed by incorporating with the activities and gene expression of three C4 photosynthesis-related key enzymes. We highlighted the importance of malate metabolism during photosynthesis recovery from short-term SHS, and data integration analysis to better comprehend the regulatory framework of PCM in response to abiotic stress.

Influence of ex vivo perfusion on the biomolecular profile of rat lungs.

Despite increasing clinical adoption, biologic influences of ex vivo lung perfusion (EVLP) remain insufficiently elucidated. The aim of the current study was to investigate biomolecular changes induced by EVLP in rat lungs. EVLP was maintained for 180 min. Hyaluronan, mediators, and cells were assessed in the perfusate. Gene expression, signaling pathways, and ATP content were investigated in lung tissue. EVLP induced the release of medium-high molecular weight hyaluronan and transcription of hyaluronan synthases ( P < 0.001). Increasing concentrations of inflammatory mediators were detected in the perfusate ( P < 0.001). Perfused lungs exhibited a distinctive transcriptional signature compared with organs examined before or after surgery/procurement ( P = 0.003). Up-regulated genes were involved in inflammation and its regulation, apoptosis/survival, heat shock, and oxidative stress response ( q = 0). Down-regulated genes were related to lymphocyte function ( q = 0). The NF-κB, signal transducer and activator of transcription 3, ERK1/2, p38, Akt, and stress-activated protein kinase/JNK signaling pathways were modulated by EVLP ( P < 0.05). Most of these biomolecular changes were examined and confirmed in additional experiments that were performed in lungs procured from donation after cardiocirculatory death after 180 min of warm ischemia. The current study demonstrates that EVLP broadly affects the lung biomolecular phenotype. These findings improve our comprehension of the effects exerted by the procedure and encourage additional research in preclinical models to implement therapeutic interventions.-Lonati, C., Bassani, G. A., Brambilla, D., Leonardi, P., Carlin, A., Faversani, A., Gatti, S., Valenza, F. Influence of ex vivo perfusion on the biomolecular profile of rat lungs.

Temperature stress response of heat shock protein 90 (Hsp90) in the clam Paphia undulata

The Heat shock proteins (HSPs) are a group of molecular chaperones that play a crucial role in cell response to various stresses. A full-length cDNA of the heat shock protein 90 (PuHsp90) was cloned and sequenced from the clam Paphia undulata. Phylogenetic analysis revealed that PuHsp90 grouped with the Hsp90 from other metazoan species. Expression of PuHsp90 was highly detected in the gonad, followed by digest gland, gills and heart but was found poorly expressed in mantle, adductor muscle and hemocytes. After a heat shock stress at 32 °C up-regulation of PuHsp90 was detected in the mantle, adductor muscle, gills and hemocytes. Maximal expression levels occurred at 4 h after the heat shock and up-regulation is indicative of protein denaturation and of an increase in energy consumption. In contrast after the heat shock, PuHsp90 was continuously down-regulated in the digestive gland and in the gonad suggesting modifications of the biochemical pathways and energy budgets involved in the synthesis of other protein, such as catalase and of other Hsp proteins. These results reveal that PuHsp90 may play an important role in the clam response to a temperature stress.

Polyamines protect nucleic acids against depurination.

Depurination is accelerated by heat and reactive oxygen species under physiological conditions. We previously reported that polyamines are involved in mitigation of heat shock and oxidative stresses through stimulation of the synthesis of heat shock and antioxidant proteins. This time, we investigated whether polyamines are directly involved in protecting nucleic acids from thermal depurination induced by high temperature. The suppressing efficiencies of depurination of DNA by spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 50%, 60% and 80%, respectively. Mg2+ also protected nucleic acids against depurination but to a lesser degree than polyamines. Longer unusual polyamines were more effective at protecting DNA against depurination compared to standard polyamines. The tRNA depurination suppressing efficiencies of spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 60%, 70% and 80%, respectively. Standard polyamines protected tRNA and ribosomes more effectively than DNA against thermal depurination. Branched polyamines such as mitsubishine and tetrakis(3-aminopropyl)ammonium also protected RNA more effectively than DNA against depurination. These results suggest that the suppressing effect of depurination of nucleic acids (DNA and RNA) depends on the types of polyamines: i.e. to maintain functional conformation of nucleic acids at high temperature, longer and branched polyamines play important roles in protecting nucleic acids from depurination compared to standard polyamines and Mg2+.

Anatomically distinct representatives of Cactaceae Juss. family have different response to acute heat shock stress

In order to escape extreme environmental conditions plants have developed different survival strategies. The study of plant response to high temperatures is especially interesting in representatives of varied cacti families that have to resist big temperature oscillations. In the present study plants of Aylostera, Echinocactus and Mammillaria genera were exposed to temperatures of 40 or 50 °C for 3 h and peroxidase and superoxide dismutase activity, photosynthetic pigments and flavonoids concentration, and lipid peroxide oxidation level in the stem were determined. Plants with different anatomical structure showed distinct patterns of response to high temperature. Plants of Echinocactus grusonii proved the highest drought and heat resistant with strong antioxidant response and the most expressed xerophytic features of anatomical structures (epidermis and cuticle thickening, calcium oxalate accumulation, water-storage tissue development). Heat stress induced a quick increase of peroxidase activity and flavonoid concentration in this species. Relative heat resistance (40 °C) in Mammillaria bocasana is also facilitated by increased flavonoid concentration, chlorophyll a and carotenoid concentration and also high superoxide dismutase activity under normal conditions.We conclude that the development of antioxidant reactions depends on anatomical protection of plants. More effective anatomical conformation to the stress conditions may be accompanied by rapid response to stress at the antioxidant biochemical level. Less effective protection at the anatomical level to hyperthermia is accompanied by the presence of a larger activity of antioxidant enzymes in conditions without short-term high-temperature stress. Plants which do not possess these qualities, Aylostera flavistyla in particular, suffer more from short-term high-temperature stress.

Mycobacterium tuberculosis GrpE, A Heat-Shock Stress Responsive Chaperone, Promotes Th1-Biased T Cell Immune Response via TLR4-Mediated Activation of Dendritic Cells

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is an extremely successful pathogen with multifactorial ability to control the host immune response. Insights into the Mtb factors modulating host response are required for the discovery of novel vaccine antigen targets as well as a better understanding of dynamic interactions between the bacterial factors and host cells. Here, we exploited the functional role of Mtb GrpE, a cofactor of heat-shock protein 70 (HSP70), in promoting naïve CD4+/CD8+T cell differentiation toward Th1-type T-cell immunity through interaction with dendritic cells (DCs). GrpE functionally induced DC maturation by up-regulating the expression of cell surface molecules (CD80, CD86, and MHC class I and II) and production of several pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12p70) in DCs. These effects of GrpE in DC activation were initiated upon binding to Toll-like receptor 4 (TLR4) followed by activation of downstream MyD88-, TRIF-, MAPK-, and NF-κB-dependent signaling pathways. GrpE-activated DCs displayed an excellent capacity to effectively polarize naïve CD4+ and CD8+ T cells toward Th1-type T-cell immunity with the dose-dependent secretion of IFN-γ and IL-2 together with increased levels of CXCR3 expression. Notably, GrpE-stimulated DCs induced the proliferation of GrpE-specific Th1-type effector/memory CD4+/CD8+CD44highCD62Llow T cells from the spleen of Mtb-infected mice in a TLR4-dependent manner. Collectively, these results demonstrate that GrpE is a novel immune activator that interacts with DCs, in particular, via TLR4, to generate Th1-biased memory T cells in an antigen-specific manner. GrpE may contribute to the enhanced understanding of host-pathogen interactions as well as providing a rational basis for the discovery of new potential targets to develop an effective tuberculosis vaccine.

Rhodobacter sp. Rb3, an aerobic anoxygenic phototroph which thrives in the polyextreme ecosystem of the Salar de Huasco, in the Chilean Altiplano.

The Salar de Huasco is an evaporitic basin located in the Chilean Altiplano, which presents extreme environmental conditions for life, i.e. high altitude (3800m.a.s.l.), negative water balance, a wide salinity range, high daily temperature changes and the occurrence of the highest registered solar radiation on the planet (> 1200Wm-2). This ecosystem is considered as a natural laboratory to understand different adaptations of microorganisms to extreme conditions. Rhodobacter, an anoxygenic aerobic phototrophic bacterial genus, represents one of the most abundant groups reported based on taxonomic diversity surveys in this ecosystem. The bacterial mat isolate Rhodobacter sp. strain Rb3 was used to study adaptation mechanisms to stress-inducing factors potentially explaining its success in a polyextreme ecosystem. We found that the Rhodobacter sp. Rb3 genome was characterized by a high abundance of genes involved in stress tolerance and adaptation strategies, among which DNA repair and oxidative stress were the most conspicuous. Moreover, many other molecular mechanisms associated with oxidative stress, photooxidation and antioxidants; DNA repair and protection; motility, chemotaxis and biofilm synthesis; osmotic stress, metal, metalloid and toxic anions resistance; antimicrobial resistance and multidrug pumps; sporulation; cold shock and heat shock stress; mobile genetic elements and toxin-antitoxin system were detected and identified as potential survival mechanism features in Rhodobacter sp. Rb3. In total, these results reveal a wide set of strategies used by the isolate to adapt and thrive under environmental stress conditions as a model of polyextreme environmental resistome.

The Entamoeba histolytica TBP and TRF1 transcription factors are GAAC-box binding proteins, which display differential gene expression under different stress stimuli and during the interaction with mammalian cells

BackgroundEntamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown.MethodsEMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed.ResultsBoth transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The KD values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10-12 M to 10-11 M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites.ConclusionsTo our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.

Bacillus subtilis Type I antitoxin SR6 Promotes Degradation of Toxin yonT mRNA and Is Required to Prevent Toxic yoyJ Overexpression.

yonT/SR6 is the second type I toxin-antitoxin (TA) system encoded on prophage SPβ in the B. subtilis chromosome. The yonT ORF specifying a 58 aa toxin is transcribed on a polycistronic mRNA under control of the yonT promoter. The antitoxin SR6 is a 100 nt antisense RNA that overlaps yonT at its 3′ end and the downstream gene yoyJ encoding a second, much weaker, toxin at its 5′ end. SR6 displays a half-life of >60 min, whereas yonT mRNA is less stable with a half-life of ≈8 min. SR6 is in significant excess over yonT mRNA except in minimal medium with glucose. It interacts with the 3′ UTR of yonT mRNA, thereby promoting its degradation by RNase III. By contrast, SR6 does not affect the amount or half-life of yoyJ mRNA. However, in its absence, a yoyJ overexpression plasmid could not be established in Bacillus subtilis suggesting that SR6 inhibits yoyJ translation by directly binding to its ribosome-binding site. While the amounts of both yonT RNA and SR6 were affected by vancomycin, manganese, heat-shock and ethanol stress as well as iron limitation, oxygen stress decreased only the amount of SR6.

Reverse phase protein arrays for the identification/validation of biomarkers of beef texture and their use for early classification of carcasses

The validation of biomarkers and tools for the prediction of beef texture remains a challenging task. In this study, reverse phase protein arrays (RPPA) quantified 29 protein biomarkers in the m. Longissimus thoracis of Charolais cattle sampled early post-mortem. Myosin heavy chain 1 (MHC1, slow-oxidative fibers) and Retinal dehydrogenase 1 (ALDH1A1, oxidative enzyme) discriminated between tender and juicy vs. tough meat with residues classes and are validated as prime biomarkers of beef texture. Several proteins belonging to energy metabolism, heat shock and oxidative stress, cytoskeletal, cell signaling and apoptosis were related with tenderness. Among the unusual proteins, Four and a half LIM domains 1 (FHL1) and Tripartite motif protein 72 (TRIM72) correlated respectively negatively and positively with beef tenderness. Principal component regression was used for the first time to explain beef texture traits using biomarkers. The results are very promising as they revealed sophisticated mechanisms behind the tenderizing process.

Inhibition of Abeta Proteotoxicity by Paeoniflorin in Caenorhabditis elegans Through Regulation of Oxidative and Heat Shock Stress Responses.

Alzheimer's disease (AD) is a common form of dementia and amyloid-β peptide (Aβ) aggregation is considered to be one of its main causes. Paeoniflorin has been previously shown to attenuate cognitive damage inflicted by exogenous Aβ protein. Using transgenic Caenorhabditis elegans models expressing human Aβ1-42, we demonstrate here that paeoniflorin can delay progressive paralysis caused by endogenous Aβ expression and reduce the amount of toxic Aβ oligomers in vivo, although it has no effect on Aβ aggregation in vitro. Paeoniflorin does not, however, affect the lifespan of either wild-type or AD-like nematodes, implying a mechanism independent of a general antiaging effect. We then demonstrate that paeoniflorin can reduce reactive oxygen species levels in C. elegans AD models, which may contribute to its in vivo suppression of Aβ toxicity. Moreover, paeoniflorin is shown to upregulate the expression of the small heat shock protein HSP-16.2 as it is capable of increasing the hsp-16.2 transcript level in wild-type as well as AD-like nematodes and enhancing the fluorescence intensity in hsp-16.2::GFP nematodes. Taken together, our findings demonstrate the underlying mechanisms of the protective effect of paeoniflorin against age-onset Aβ proteotoxicity, which are, in part, connected with oxidative and heat shock stress responses.