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Apocynum venetum L. belongs to the Apocynaceae family and is a plant that is highly resistant to stress. It is important in the fields of ecology, feeding, industry and medicine. The molecular mechanism underlying salt tolerance has not been elucidated. In this study, RNA-seq based transcriptome sequencing of A. venetum leaves after 0, 2, 6, 12, 24 and 48 h of treatment with 300 mM NaCl was performed. We conducted a comprehensive analysis of the transcriptome expression profiles of A. venetum under salt stress using the WGCNA method and identified red, black, and brown as the core modules regulating the salt tolerance of A. venetum. A co-expression regulatory network was constructed to identify the core genes in the module according to the correlations between genes. The genes TRINITY_DN102_c0_g1 (serine carboxypeptidase), TRINITY_DN3073_c0_g1 (SOS signaling pathway) and TRINITY_DN6732_c0_g1 (heat shock transcription factor) in the red module were determined to be the core genes. Two core genes in the black module, TRINITY_DN9926_c0_g1 and TRINITY_DN7962_c0_g1, are pioneer candidate salt tolerance-associated genes in A. venetum. The genes in the brown module were mainly enriched in two pathways, namely photosynthesis and osmotic balance. Among them, the TRINITY_DN6321_c0_g2 and TRINITY_DN244_c0_g1 genes encode aquaporin, which is helpful for maintaining the cell water balance and plays a protective role in defending A. venetum under abiotic stress. Our findings contribute to the identification of core genes involved in the response of A. venetum to salt stress. [ABSTRACT FROM AUTHOR]

The HEAT domains are a family of helical hairpin repeat domains, composed of four or more hairpins. HEAT is derived from the names of four family members: huntingtin, eukaryotic translation elongation factor 3 (eEF3), protein phosphatase 2 regulatory A subunit (PP2A), and mechanistic target of rapamycin (mTOR). HEAT domain-containing proteins play roles in a wide range of cellular processes, such as protein synthesis, nuclear transport and metabolism, and cell signaling. The PCI domains are a related group of helical hairpin domains, with a "winged-helix" (WH) subdomain at their C-terminus, which is responsible for multi-subunit complex formation with other PCI domains. The name is derived from the complexes, where these domains are found: the 26S Proteasome "lid" regulatory subcomplex, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). We noted that in structure similarity searches using HEAT domains, sometimes PCI domains appeared in the search results ahead of other HEAT domains, which indicated that the PCI domains could be members of the HEAT domain family, and not a related but separate group, as currently thought. Here, we report extensive structure similarity analysis of HEAT and PCI domains, both within and between the two groups of proteins. We present evidence that the PCI domains as a group have greater structural similarity with individual groups of HEAT domains than some of the HEAT domain groups have among each other. Therefore, our results indicate that the PCI domains have evolved from a HEAT domain that acquired a WH subdomain. The WH subdomain in turn mediated self-association into a multi-subunit complex, which eventually evolved into the common ancestor of the Proteasome lid/CSN/eIF3. [ABSTRACT FROM AUTHOR]

Among the complications observed after allogeneic hematopoietic stem cell transplantation, graft-versus-host disease (GVHD) is the primary cause of post-transplant mortality. The oral cavity is the second most affected organ target in chronic GVHD. Tissue damage results from the upregulation of inflammatory mediators, which play a critical role in the immunopathogenesis of the disease. This case series observational study aims to evaluate the participation of cytokines, chemokines, transcription factors, and heat shock proteins in the pathogenesis of oral GVHD (oGVHD), describing the mRNA expression of 28 genes selected. Peripheral blood mononuclear cells were isolated from six participants with oGVHD and two without GVHD, and relative expression of transcripts with established roles as inflammatory mediators was determined in triplicate using the human RT2 Profiler™ PCR Array. The gene expression levels in the group with oGVHD were mainly up-regulated compared to those without GVHD. PBMC from oGVDH expressed consistently higher IFN-γ, TNF, IL-1β, CCL2, HSP60 (HSPD1) and HSP90 (HSP90B1). These results can provide a basis for developing new molecular diagnostics and targets therapies for the clinical management of oGVHD. [ABSTRACT FROM AUTHOR]

Around the world, pansies are one of the most popular garden flowers, but they are generally sensitive to high temperatures, and this limits the practicality of planting them during the warmest days of the year. However, a few pansy germplasms with improved heat tolerance have been discovered or bred, but the mechanisms of their heat resistance are not understood. In this study, we investigated the transcript profiles of a heat-tolerant pansy inbred line, DFM16, in response to high temperatures using RNAseq. Approximately 55.48 Gb of nucleotide data were obtained and assembled into 167,576 unigenes with an average length of 959 bp, of which, 5,708 genes were found to be differentially expressed after heat treatments. Real-time qPCR was performed to validate the expression profiles of the selected genes. Nine metabolic pathways were found to be significantly enriched, in the analysis of the differentially expressed genes. Several potentially interesting genes that encoded putative transcription regulators or key components involving heat shock protein (HSP), heat shock transcription factors (HSF), and antioxidants biosynthesis, were identified. These genes were highlighted to indicate their significance in response to heat stress and will be used as candidate genes to improve pansy heat-tolerance in the future. [ABSTRACT FROM AUTHOR]

The nematode Caenorhabditis elegans has been used to examine the influence of epicatechin (EC), an abundant flavonoid in the human diet, in some stress biomarkers (ROS production, lipid peroxidation and protein carbonylation). Furthermore, the ability of EC to modulate the expression of some key genes in the insulin/IGF-1 signaling pathway (IIS), involved in longevity and oxidative or heat shock stress response, has also been explored. The final aim was to contribute to the elucidation of the mechanisms involved in the biological effects of flavonoids. The results showed that EC-treated wild-type C. elegans exhibited increased survival and reduced oxidative damage of biomolecules when submitted to thermal stress. EC treatment led to a moderate elevation in ROS levels, which might activate endogenous mechanisms of defense protecting against oxidative insult. The enhanced stress resistance induced by EC was found to be mediated through the IIS pathway, since assays in daf-2, age-1, akt-1, akt-2, sgk-1, daf-16, skn-1 and hsf-1 loss of function mutant strains failed to show any heat-resistant phenotype against thermal stress when treated with EC. Consistently, EC treatment upregulated the expression of some stress resistance associated genes, such as gst-4, hsp-16.2 and hsp-70, which are downstream regulated by the IIS pathway. [ABSTRACT FROM AUTHOR]

The heat shock response (HSR) pathway is a highly conserved cellular stress response and mediated by its master regulator HSF1. Activation of the pathway results in the expression of chaperone proteins (heat shock proteins; HSP) to maintain protein homeostasis. One of the genes strongest upregulated upon stress is HSPA1A (HSP72). Heavy metals are highly toxic to living organisms and known as environmental contaminants, due to industrialisation. Furthermore, many of them are well-described inducers of the HSR pathway. Here we compare the effect of different heavy metals, concerning their potential to activate HSF1 with a sensitive artificial heat shock reporter cell line, consisting of heat shock elements (HSE). In general the responses of the artificial promoter to heavy metal stress were in good agreement with those of well-established HSF1 target genes, like HSPA1A. Nevertheless, differences were observable when effects of heat and heavy metal stress were compared. Whereas heat stress preferentially activated the HSE promoter, heavy metals more strongly induced the HSPA1A promoter. We therefore analysed the HSPA1A promoter in more detail, by isolating and mutating the HSEs. The results indicate that the importance of the individual binding sites for HSF1 is determined by their sequence similarity to the consensus sequence and their position relative to the transcription start site, but they were not differentially affected by heat or heavy metal stress. In contrast, we found that other parts of the HSPA1A promoter have different impact on the response under different stress conditions. In this work we provide deeper insights into the regulation of HSP72 expression as a well as a method to quantitatively and sensitively evaluate different stressor on their potential to activate HSF1. [ABSTRACT FROM AUTHOR]

Embryos of the crustacean, Artemia franciscana, may undergo oviparous development, forming encysted embryos (cysts) that are released from females and enter diapause, a state of suppressed metabolism and greatly enhanced stress tolerance. Diapause-destined embryos of A. franciscana synthesize three small heat shock proteins (sHsps), p26, ArHsp21 and ArHsp22, as well as artemin, a ferritin homologue, all lacking in embryos that develop directly into nauplii. Of these diapause-specific molecular chaperones, p26 and artemin are important contributors to the extraordinary stress tolerance of A. franciscana cysts, but how their synthesis is regulated is unknown. To address this issue, a cDNA for heat shock factor 1 (Hsf1), shown to encode a protein similar to Hsf1 from other organisms, was cloned from A. franciscana. Hsf1 was knocked down by RNA interference (RNAi) in nauplii and cysts of A. franciscana. Nauplii lacking Hsf1 died prematurely upon release from females, showing that this transcription factor is essential to the survival of nauplii. Diapause cysts with diminished amounts of Hsf1 were significantly less stress tolerant than cysts containing normal levels of Hsf1. Moreover, cysts deficient in Hsf1 possessed reduced amounts of p26, ArHsp21, ArHsp22 and artemin, revealing dependence on Hsf1 for expression of their genes and maximum stress tolerance. The results demonstrate an important role for Hsf1, likely in concert with other transcription factors, in the survival and growth of A. franciscana and in the developmentally regulated synthesis of proteins responsible for the stress tolerance of diapausing A. franciscana cysts. [ABSTRACT FROM AUTHOR]

Phosphine is the only general use fumigant for the protection of stored grain, though its long-term utility is threatened by the emergence of highly phosphine-resistant pests. Given this precarious situation, it is essential to identify factors, such as stress preconditioning, that interfere with the efficacy of phosphine fumigation. We used Caenorhabditis elegans as a model organism to test the effect of pre-exposure to heat and cold shock, UV and gamma irradiation on phosphine potency. Heat shock significantly increased tolerance to phosphine by 3-fold in wild-type nematodes, a process that was dependent on the master regulator of the heat shock response, HSF-1. Heat shock did not, however, increase the resistance of a strain carrying the phosphine resistance mutation, dld-1(wr4), and cold shock did not alter the response to phosphine of either strain. Pretreatment with the LD50 of UV (18 J cm-2) did not alter phosphine tolerance in wild-type nematodes, but the LD50 (33 J cm-2) of the phosphine resistant strain (dld-1(wr4)) doubled the level of resistance. In addition, exposure to a mild dose of gamma radiation (200 Gy) elevated the phosphine tolerance by ~2-fold in both strains. [ABSTRACT FROM AUTHOR]

The transcription factor, heat shock factor 1 (HSF1), influences the expression of heat shock proteins as well as other activities like the induction of tumor suppressor genes, signal transduction pathway, and glucose metabolism. We hypothesized that single nucleotide polymorphisms (SNPs) in HSF1 gene might affect its expression or function which might have an influence on the development of breast cancer. The study group included 242 individuals (146 breast cancer patients and 96 healthy controls). From the cancer patients, genomic DNA was extracted from 96 blood samples and 50 Formalin-Fixed Paraffin Embedded (FFPE) tissues, while from the controls DNA were extracted from blood only. Genotype was carried out for four SNPs in the HSF1 gene (rs78202224, rs35253356, rs4977219 and rs34404564) using Taqman genotyping assay method. The HSF1 expression was investigated using immunohistochemistry on FFPE tissues (cancer tissue and adjacent normal tissue). The SNP rs78202224 (G>T) was significantly associated with increased risk of breast cancer. The combined TT + GT genotype (OR: 6.91; p: 0.035) and the T allele showed high risk (OR: 5.81; p:0.0085) for breast cancer development. The SNP rs34404564 (A>G) had a protective effect against the development of breast cancer. The genotype AG (OR: 0.41; p = 0.0059) and GG+AG (OR: 0.52; p: 0.026) occurred at a significantly lower frequency in the breast cancer patients compared to the frequency in healthy controls. No significant relationship was identified between either rs35253356 (A>G) or rs4977219 (A>C) and breast cancer in Saudi. The HSF1 protein expression was higher in all invasive and in situ breast carcinoma compared to the normal tissue. A stronger positive staining for HSF1 was found in the nucleus compared to the cytoplasm. Our results show that HSF1 gene expression is elevated in breast cancer tissue and two of the studied SNPs correlate significantly with cancer development. [ABSTRACT FROM AUTHOR]

Hsp90 is a molecular chaperone involved in the regulation and maturation of kinases and transcription factors. In Caenorhabditis elegans, it contributes to the development of fertility, maintenance of muscle structure, the regulation of heat-shock response and dauer state. To understand the consequences of Hsp90-depletion, we studied Hsp90 RNAi-treated nematodes by DNA microarrays and mass spectrometry. We find that upon development of phenotypes the levels of chaperones and Hsp90 cofactors are increased, while specific proteins related to the innate immune response are depleted. In microarrays, we further find many differentially expressed genes related to gonad and larval development. These genes form an expression cluster that is regulated independently from the immune response implying separate pathways of Hsp90-involvement. Using fluorescent reporter strains for the differentially expressed immune response genes skr-5, dod-24 and clec-60 we observe that their activity in intestinal tissues is influenced by Hsp90-depletion. Instead, effects on the development are evident in both gonad arms. After Hsp90-depletion, changes can be observed in early embryos and adults containing fluorescence-tagged versions of SEPA-1, CAV-1 or PUD-1, all of which are downregulated after Hsp90-depletion. Our observations identify molecular events for Hsp90-RNAi induced phenotypes during development and immune responses, which may help to separately investigate independent Hsp90-influenced processes that are relevant during the nematode’s life and development. [ABSTRACT FROM AUTHOR]

Circadian clocks drive biological rhythms with a period of approximately 24 hours and keep in time with the outside world through daily resetting by environmental cues. While this external entrainment has been extensively investigated in the suprachiasmatic nuclei (SCN), the role of internal systemic rhythms, including daily fluctuations in core temperature or circulating hormones remains debated. Here, we show that lactating mice, which exhibit dampened systemic rhythms, possess normal molecular clockwork but impaired rhythms in both heat shock response gene expression and electrophysiological output in their SCN. This suggests that body rhythms regulate SCN activity downstream of the clock. Mathematical modeling predicts that systemic feedback upon the SCN functions as an internal oscillator that accounts for in vivo and ex vivo observations. Thus we are able to propose a new bottom-up hierarchical organization of circadian timekeeping in mammals, based on the interaction in the SCN between clock-dependent and system-driven oscillators. [ABSTRACT FROM AUTHOR]

Background: Chickens are animals that are sensitive to thermal stress, which may decrease their production level in terms that it affects feed intake and thus, decreasing body weight gain. The Heat Shock Factors (HSF) and Heat Shock Proteins (HSP) genes are involved in the key cellular defense mechanisms during exposure in hot environments. Aimed with this study to analyze the expression of HSF1, HSF3, HSP70 and HSP90 genes in two local breeds (Peloco and Caneluda) and a commercial broiler line (Cobb 500®) to verify differences in resistance of these chicken to Heat stress treatment. Chicken were submitted to heat stress under an average temperature of 39°C ± 1. Results: Under stress environment, the HSP70 and HSP90 genes were more expressed in backyard chickens than in broiler. There was a difference in HSP70 and HSP90 expression between Caneluda and Cobb and between Peloco and Cobb under stress and comfort environment respectively. HSP70 expression is higher in local breeds during heat stress than in a commercial broiler line. No significant differences were observed in the expression of HSF1 and HSF3 genes between breeds or environments. Conclusions: HSP70 and HSP90 genes are highly expressed, HSF1 and HSF3 genes did not have high expression in all genetic groups. HSP70 and HSP90 are highly expressed in Peloco and Caneluda within heat stress, these breeds proved to be very resistant to high temperature. [ABSTRACT FROM AUTHOR]

The ability of an organism to sense and adapt to environmental stressors is essential for proteome maintenance and survival. The highly conserved heat shock response is a survival mechanism employed by all organisms, including the nematode Caenorhabditis elegans, upon exposure to environmental extremes. Transcriptional control of the metazoan heat shock response is mediated by the heat shock transcription factor HSF-1. In addition to regulating global stress-responsive genes to promote stress-resistance and survival, HSF-1 has recently been shown to regulate stress-independent functions in controlling development, metabolism, and longevity. However, the indirect role of HSF-1 in coordinating stress-dependent and -independent processes through post-transcriptional regulation is largely unknown. MicroRNAs (miRNAs) have emerged as a class of post-transcriptional regulators that control gene expression through translational repression or mRNA degradation. To determine the role of HSF-1 in regulating miRNA expression, we have performed high-throughput small RNA-sequencing in C. elegans grown in the presence and absence of hsf-1 RNAi followed by treatment with or without heat shock. This has allowed us to uncover the miRNAs regulated by HSF-1 via heat-dependent and -independent mechanisms. Integrated miRNA/mRNA target-prediction analyses suggest HSF-1 as a post-transcriptional regulator of development, metabolism, and longevity through regulating miRNA expression. This provides new insight into the possible mechanism by which HSF-1 controls these processes. We have also uncovered oxidative stress response factors and insulin-like signaling factors as a common link between processes affected by HSF-1-regulated miRNAs in stress-dependent and -independent mechanisms, respectively. This may provide a role for miRNAs in regulating cross-talk between various stress responses. Our work therefore uncovers an interesting potential role for HSF-1 in post-transcriptionally controlling gene expression in C. elegans, and suggests a mechanism for cross-talk between stress responses. [ABSTRACT FROM AUTHOR]

Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of HSP expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog HSF genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs. [ABSTRACT FROM AUTHOR]

The heat shock protein 70s (Hsp70s) and heat shock factors (Hsfs) play key roles in protecting plant cells or tissues from various abiotic stresses. Brachypodium distachyon, recently developed an excellent model organism for functional genomics research, is related to the major cereal grain species. Although B. distachyon genome has been fully sequenced, the information of Hsf and Hsp70 genes and especially the regulatory network between Hsfs and Hsp70s remains incomplete. Here, a total of 24 BdHsfs and 29 BdHsp70s were identified in the genome by bioinformatics analysis and the regulatory network between Hsfs and Hsp70s were performed in this study. Based on highly conserved domain and motif analysis, BdHsfs were grouped into three classes, and BdHsp70s divided into six groups, respectively. Most of Hsf proteins contain five conserved domains: DBD, HR-A/B region, NLS and NES motifs and AHA domain, while Hsp70 proteins have three conserved domains: N-terminal nucleotide binding domain, peptide binding domain and a variable C-terminal lid region. Expression data revealed a large number of BdHsfs and BdHsp70s were induced by HS challenge, and a previous heat acclimation could induce the acquired thermotolerance to help seedling suffer the severe HS challenge, suggesting that the BdHsfs and BdHsp70s played a role in alleviating the damage by HS. The comparison revealed that, most BdHsfs and BdHsp70s genes responded to multiple abiotic stresses in an overlapping relationship, while some of them were stress specific response genes. Moreover, co-expression relationships and predicted protein-protein interaction network implied that class A and B Hsfs played as activator and repressors, respectively, suggesting that BdHsp70s might be regulated by both the activation and the repression mechanisms under stress condition. Our genomics analysis of BdHsfs and BdHsp70s provides important evolutionary and functional characterization for further investigation of the accurate regulatory mechanisms among Hsfs and Hsp70s in herbaceous plants. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of roduction of nthocyanin igment 1 (PAP1) and rabidopsis thaliana acuolar H+-yrophosphatase (AVP1). The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels. [ABSTRACT FROM AUTHOR]

Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is possible to formulate mechanism-based hypotheses on the cytotoxic properties of the tested chemicals. [ABSTRACT FROM AUTHOR]

Compensatory growth (CG) is utilised worldwide in beef production systems as a management approach to reduce feed costs. However the underlying biology regulating the expression of CG remains to be fully elucidated. The objective of this study was to examine the effect of dietary restriction and subsequent re-alimentation induced CG on the global gene expression profile of ruminal epithelial papillae. Holstein Friesian bulls (n = 60) were assigned to one of two groups: restricted feed allowance (RES; n = 30) for 125 days (Period 1) followed by ad libitum access to feed for 55 days (Period 2) or (ii) ad libitum access to feed throughout (ADLIB; n = 30). At the end of each period, 15 animals from each treatment were slaughtered and rumen papillae harvested. mRNA was isolated from all papillae samples collected. cDNA libraries were then prepared and sequenced. Resultant reads were subsequently analysed bioinformatically and differentially expressed genes (DEGs) are defined as having a Benjamini-Hochberg P value of <0.05. During re-alimentation in Period 2, RES animals displayed CG, growing at 1.8 times the rate of their ADLIB contemporary animals in Period 2 (P < 0.001). At the end of Period 1, 64 DEGs were identified between RES and ADLIB, with only one DEG identified at the end of Period 2. When analysed within RES treatment (RES, Period 2 v Period 1), 411 DEGs were evident. Genes identified as differentially expressed in response to both dietary restriction and subsequent CG included those involved in processes such as cellular interactions and transport, protein folding and gene expression, as well as immune response. This study provides an insight into the molecular mechanisms underlying the expression of CG in rumen papillae of cattle; however the results suggest that the role of the ruminal epithelium in supporting overall animal CG may have declined by day 55 of re-alimentation. [ABSTRACT FROM AUTHOR]

The heat shock response (HSR) is a well-conserved, cytoprotective stress response that activates the HSF1 transcription factor. During severe stress, cells inhibit mRNA splicing which also serves a cytoprotective function via inhibition of gene expression. Despite their functional interconnectedness, there have not been any previous reports of crosstalk between these two pathways. In a genetic screen, we identified SF3B1, a core component of the U2 snRNP subunit of the spliceosome, as a regulator of the heat shock response in Caenorhabditis elegans. Here, we show that this regulatory connection is conserved in cultured human cells and that there are at least two distinct pathways by which SF3B1 can regulate the HSR. First, inhibition of SF3B1 with moderate levels of Pladienolide B, a previously established small molecule inhibitor of SF3B1, affects the transcriptional activation of HSF1, the transcription factor that mediates the HSR. However, both higher levels of Pladienolide B and SF3B1 siRNA knockdown also change the concentration of HSF1, a form of HSR regulation that has not been previously documented during normal physiology but is observed in some forms of cancer. Intriguingly, mutations in SF3B1 have also been associated with several distinct types of cancer. Finally, we show that regulation of alternative splicing by SF3B1 is sensitive to temperature, providing a new mechanism by which temperature stress can remodel the transcriptome. [ABSTRACT FROM AUTHOR]

Vibrio cholerae experiences a highly hostile environment at human intestine which triggers the induction of various heat shock genes. The hchA gene product of V. cholerae O395, referred to a hypothetical intracellular protease/amidase VcHsp31, is one such stress-inducible homodimeric protein. Our current study demonstrates that VcHsp31 is endowed with molecular chaperone, amidopeptidase and robust methylglyoxalase activities. Through site directed mutagenesis coupled with biochemical assays on VcHsp31, we have confirmed the role of residues in the vicinity of the active site towards amidopeptidase and methylglyoxalase activities. VcHsp31 suppresses the aggregation of insulin in vitro in a dose dependent manner. Through crystal structures of VcHsp31 and its mutants, grown at various temperatures, we demonstrate that VcHsp31 acquires two (Type-I and Type-II) dimeric forms. Type-I dimer is similar to EcHsp31 where two VcHsp31 monomers associate in eclipsed manner through several intersubunit hydrogen bonds involving their P-domains. Type-II dimer is a novel dimeric organization, where some of the intersubunit hydrogen bonds are abrogated and each monomer swings out in the opposite directions centering at their P-domains, like twisting of wet cloth. Normal mode analysis (NMA) of Type-I dimer shows similar movement of the individual monomers. Upon swinging, a dimeric surface of ~400Å2, mostly hydrophobic in nature, is uncovered which might bind partially unfolded protein substrates. We propose that, in solution, VcHsp31 remains as an equilibrium mixture of both the dimers. With increase in temperature, transformation to Type-II form having more exposed hydrophobic surface, occurs progressively accounting for the temperature dependent increase of chaperone activity of VcHsp31. [ABSTRACT FROM AUTHOR]

Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L.) x creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease), antioxidant defense (catalase and glutathione-S-transferase), energy metabolism (glyceraldehyde-3-phosphate dehydrogenase), cell expansion (expansin), and stress protection (heat shock proteins HSP26, HSP70, and HSP101). Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection. [ABSTRACT FROM AUTHOR]

A moderate heat shock induces Neurospora crassa to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. However, wild type cells do not survive when carbohydrate deprivation is added to heat shock. In contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. In order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins in the mutant and wild type strain during dual stress, and we have identified gene transcripts in both strains whose quantities change in response to heat shock or dual stress. These data and supportive experimental evidence point to reasons for survival of the mutant strain. By using alternative respiratory mechanisms, these cells experience less of the oxidative stress that proves damaging to wild type cells. Of central importance, mutant cells recycle limited resources during dual stress by undergoing autophagy, a process that we find utilized by both wild type and mutant cells during heat shock. Evidence points to inappropriate activation of TORC1, the central metabolic regulator, in wild type cells during dual stress, based upon behavior of an additional signaling mutant and inhibitor studies. [ABSTRACT FROM AUTHOR]

Zerumbone is a sesquiterpene present in Zinger zerumbet. Many studies have demonstrated its marked anti-inflammatory and anti-carcinogenesis activities. Recently, we showed that zerumbone binds to numerous proteins with scant selectivity and induces the expression of heat shock proteins (HSPs) in hepatocytes. To dampen proteo-toxic stress, organisms have a stress-responsive molecular machinery, known as heat shock response. Heat shock factor 1 (HSF1) plays a key role in this protein quality control system by promoting activation of HSPs. In this study, we investigated whether zerumbone-induced HSF1 activation contributes to its anti-inflammatory functions in stimulated macrophages. Our findings showed that zerumbone increased cellular protein aggregates and promoted nuclear translocation of HSF1 for HSP expression. Interestingly, HSF1 down-regulation attenuated the suppressive effects of zerumbone on mRNA and protein expressions of pro-inflammatory genes, including inducible nitric oxide synthase and interlukin-1β. These results suggest that proteo-stress induced by zerumbone activates HSF1 for exhibiting its anti-inflammatory functions. [ABSTRACT FROM AUTHOR]

Sieve elements of legumes contain forisomes—fusiform protein bodies that are responsible for sieve-tube occlusion in response to damage or wound signals. Earlier work described the existence of tailless and tailed forisomes. This study intended to quantify and compare location and position of tailless (in Vicia faba) and tailed (in Phaseolus vulgaris) forisomes inside sieve elements and to assess their reactivity and potential mobility in response to a remote stimulus. Location (distribution within sieve elements) and position (forisome tip contacts) of more than altogether 2000 forisomes were screened in 500 intact plants by laser scanning confocal microscopy in the transmission mode. Furthermore, we studied the dispersion of forisomes at different locations in different positions and their positional behaviour in response to distant heat shocks. Forisome distribution turned out to be species-specific, whereas forisome positions at various locations were largely similar in bushbean (Phaseolus) and broadbean (Vicia). In general, the tailless forisomes had higher dispersion rates in response to heat shocks than the tailed forisomes and forisomes at the downstream (basal) end dispersed more frequently than those at the upstream end (apical). In contrast to the tailless forisomes that only oscillate in response to heat shocks, downstream-located tailed forisomes can cover considerable distances within sieve elements. This displacement was prevented by gentle rubbing of the leaf (priming) before the heat shock. Movement of these forisomes was also prohibited by Latrunculin A, an inhibitor of actin polymerization. The apparently active mobility of tailed forisomes gives credence to the idea that at least the latter forisomes are not free-floating, but connected to other sieve-element structures. [ABSTRACT FROM AUTHOR]

The alternative sigma factor σE functions to maintain bacterial homeostasis and membrane integrity in response to extracytoplasmic stress by regulating thousands of genes both directly and indirectly. The transcriptional regulatory network governed by σE in Salmonella and E. coli has been examined using microarray, however a genome-wide analysis of σE–binding sites in Salmonella has not yet been reported. We infected macrophages with Salmonella Typhimurium over a select time course. Using chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq), 31 σE–binding sites were identified. Seventeen sites were new, which included outer membrane proteins, a quorum-sensing protein, a cell division factor, and a signal transduction modulator. The consensus sequence identified for σE in vivo binding was similar to the one previously reported, except for a conserved G and A between the -35 and -10 regions. One third of the σE–binding sites did not contain the consensus sequence, suggesting there may be alternative mechanisms by which σE modulates transcription. By dissecting direct and indirect modes of σE-mediated regulation, we found that σE activates gene expression through recognition of both canonical and reversed consensus sequence. New σE regulated genes (greA, luxS, ompA and ompX) are shown to be involved in heat shock and oxidative stress responses. [ABSTRACT FROM AUTHOR]

Causes of lower induction of Hsp70 in neurons during heat shock are still a matter of debate. To further inquire into the mechanisms regulating Hsp70 expression in neurons, we studied the activity of Heat Shock Factor 1 (HSF1) and histone posttranslational modifications (PTMs) at the hsp70 promoter in rat cortical neurons. Heat shock induced a transient and efficient translocation of HSF1 to neuronal nuclei. However, no binding of HSF1 at the hsp70 promoter was detected while it bound to the hsp25 promoter in cortical neurons during heat shock. Histone PTMs analysis showed that the hsp70 promoter harbors lower levels of histone H3 and H4 acetylation in cortical neurons compared to PC12 cells under basal conditions. Transcriptomic profiling data analysis showed a predominant usage of cryptic transcriptional start sites at hsp70 gene in the rat cerebral cortex, compared with the whole brain. These data support a weaker activation of hsp70 canonical promoter. Heat shock increased H3Ac at the hsp70 promoter in PC12 cells, which correlated with increased Hsp70 expression while no modifications occurred at the hsp70 promoter in cortical neurons. Increased histone H3 acetylation by Trichostatin A led to hsp70 mRNA and protein induction in cortical neurons. In conclusion, we found that two independent mechanisms maintain a lower induction of Hsp70 in cortical neurons. First, HSF1 fails to bind specifically to the hsp70 promoter in cortical neurons during heat shock and, second, the hsp70 promoter is less accessible in neurons compared to non-neuronal cells due to histone deacetylases repression. [ABSTRACT FROM AUTHOR]