Heat Shock Protein Hsp Research Articles

Effective Antitumor Synergistic Treatment with Fiber-Photothermal Therapy and Heat Shock Protein Inhibitors.

Effective treatment of malignant tumors remains a thorny issue in current medicine. As a new type of anticancer strategy, photothermal therapy (PTT) has attracted tremendous attention due to its favorable therapeutic effectiveness, high spatial-temporal controllability, and low occurrence of side effects. However, the efficacy of PTT is significantly reduced due to the limited penetration of light and heat-induced overexpression of heat shock protein (Hsp). Herein, we propose an antitumor synergistic therapy that combines fiber-optic PTT and Hsp inhibitors. A rare-earth-doped optical fiber was used as the PTT actuator, and the Hsp inhibitor AT533 was loaded on the fiber surface by use of a hydrogel layer. PTT fibers can be guided to reach tumor lesions directly without being subject to the light penetration limit. The Hsp inhibitor can be released upon the softening of the hydrogel layer under photoheating to deactivate Hsp in the tumor and thus reduce the resistance of the tumor to PTT. This synergistic treatment enhanced the effect of PTT and successfully eradicated tumors in colorectal cancer (CRC) xenograft mouse models, providing a feasible way to realize antitumor and antirecurrence treatment. More importantly, the success of the synergistic treatment of PTT and Hsp inhibition opens new avenues for the development of multimodal and multitype synergistic fiber-optic treatments, which offer pronounced enhancement of therapeutic effectiveness for treating cancer.

Cloning and functional analysis of heat shock protein Hsp70 from Sclerotinia sclerotiorum

To clarify the roles of the heat shock protein gene Hsp70 in the sclerotial formation and pathogenicity of Sclerotinia sclerotiorum, we employed reverse transcription PCR (RT-PCR) to clone Hsp70 from S. sclerotiorum and performed sequence analysis. Quantitative real-time PCR (qRT-PCR) was employed to determine the relative expression levels of Hsp70 at different growth stages and under the stress induced by cyclic adenosine monophosphate (cAMP) and low and high temperatures. The thermal stability of Hsp70 was measured. The Agrobacterium-mediated method was employed to construct the Hsp70-silenced strain. The pathogenicity and fungicide resistance of strains were tested by inoculation in detached rapeseed leaves and cultivation in the media containing procymidone and thiophanate-methyl, respectively. The results showed that the cloned Hsp70 had a total length of 1 890 bp and close relationship with the Hsp70 gene of Ciborinia. Hsp70 showcased the highest expression level in sclerotia, which was more than 30 times higher than that in hyphae. The cAMP stress significantly induced the expression of Hsp70. The expression level of Hsp70 showed an increasing-decreasing-increasing trend at 40 ℃ and no significant change at 4 ℃. Recombinant strain with high expression of Hsp70 showed good thermal stability. The Hsp70-silenced transformant did not form sclerotia, with decreased pathogenicity and fungicide resistance. This study reveals that Hsp70 plays an important role in the sclerotial formation and stress resistance of S. sclerotium, providing reference for further in-depth research on the biological roles of Hsp70 in S. sclerotium.

BiP Proteins from Symbiodiniaceae: A “Shocking” Story

More than four decades ago, the discovery of a companion protein of immunoglobulins in myeloma cells and soon after, of their ability to associate with heavy chains, made the term immunoglobulin binding protein (BiP) emerge, prompting a tremendous amount of effort to understand their versatile cellular functions. BiPs belong to the heat shock protein (Hsp) 70 family and are crucial for protein folding and cellular stress responses. While extensively studied in model organisms such as Chlamydomonas, their roles in dinoflagellates, especially in photosynthetic Symbiodiniaceae, remain largely underexplored. Given the importance of Symbiodiniaceae-cnidarian symbiosis, critical for the sustaining of coral reef ecosystems, understanding the contribution of Hsps to stress resilience is essential; however, most studies have focused on Hsps in general but none on BiPs. Moreover, despite the critical role of light in the physiology of these organisms, research on light effects on BiPs from Symbiodiniaceae has also been limited. This review synthesizes the current knowledge from the literature and sequence data, which reveals a high degree of BiP conservation at the gene, protein, and structural levels in Symbiodiniaceae and other dinoflagellates. Additionally, we show the existence of a potential link between circadian clocks and BiP regulation, which would add another level of regulatory complexity. The evolutionary relationship among dinoflagellates overall suggests conserved functions and regulatory mechanisms, albeit expecting confirmation by experimental validation. Finally, our analysis also highlights the significant knowledge gap and underscores the need for further studies focusing on gene and protein regulation, promoter architecture, and structural conservation of Symbiodiniaceae and dinoglagellate BiPs in general. These will deepen our understanding of the role of BiPs in the Symbiodiniaceae-cnidarian interactions and dinoflagellate physiology.

The arylbipyridine platinum (II) complex increases the level of ROS and induces lipid peroxidation in glioblastoma cells.

Here we present the biological properties of the arylbipyridine platinum (II) complex (arylbipy-Pt) and describe the potential mechanism of its antitumor action which differs from that of the well-known cisplatin. Leading to the oxidative stress and lipid peroxidation, the arylbipyridine platinum (II) complex showcases the significant cytotoxicity against the glioblastoma cells as shown by the MTT test. Using the 5-ethyl-2-deoxyuridine we study the proliferative activity of glioblastoma cells to affirm that arylbipyridine platinum (II) complex does not impede cell division or DNA replication. Staining by the MitoCLox dye and 2',7'-dichlorodihydrofluorescein diacetate demonstrates that the glioblastoma cells treated with arylbipy-Pt exhibit a strong increase of the lipid peroxidation and the stimulation of the reactive oxygen species formation. The hypothesis that arylbipy-Pt promotes oxidative death of tumor cells is confirmed by control experiments using N-acetyl-L-cysteine as an antioxidant. Further evidence for the oxidative mechanism of action is provided by real-time PCR, which shows high expression levels for genes associated with the heat shock proteins HSP27 and HSP70, which can be used as markers of tumor cell ferroptosis. To elucidate the chemical nature of the arylbipy-Pt complex activity, we perform 195Pt NMR spectroscopy and cyclic voltammetry measurements under biologically relevant conditions. The results obtained clearly indicate the structural transformation of the arylbipy-Pt complex in the DMSO-saline mixture, which is crucial for its further antitumor activity via the oxidative pathway. The found correlation between the molecular structure of arylbipy-Pt and its effect on the tumor cell cycle paves the way for the rational design of Pt complexes possessing the alternative mechanism of antitumor activity as compared to DNA intercalation, providing possible solutions to the major problems such as toxicity and drug resistance.

Treatment with Tau fibrils impact Huntington's disease-related phenotypes in cell and mouse models

There is now compelling evidence for the presence of pathological forms of Tau in tissues of both patients and animal models of Huntington's disease (HD). While the root cause of this illness is a mutation within the huntingtin gene, a number of studies now suggest that HD could also be considered a secondary tauopathy. However, the contributory role of Tau in the pathogenesis and pathophysiology of this condition, as well as its implications in cellular toxicity and consequent behavioral impairments are largely unknown. We therefore performed intracerebral stereotaxic injections of recombinant human Tau monomers and fibrils into the knock-in zQ175 mouse model of HD. Tau fibrils induced cognitive and anxiety-like phenotypes predominantly in zQ175 mice and increased the number and size of insoluble mutant huntingtin (mHTT) aggregates in the brains of treated animals. To better understand the putative mechanisms through which Tau could initiate and/or contribute to pathology, we incubated StHdh striatal cells, an in vitro model of HD, with the different Tau forms and evaluated the effects on cell functionality and heat shock proteins Hsp70 and Hsp90. Calcium imaging experiments showed functional impairments of HD StHdh cells following treatment with Tau fibrils, as well as significant changes to the levels of both heat shock proteins which were found trapped within mHTT aggregates. The accumulation of Hsp70 and 90 within aggregates was also present in mouse tissue which suggests that alteration of molecular chaperone-dependent protein quality control may influence aggregation, implicating proteostasis in the mHTT-Tau interplay.

Isolation and Characterization of Heat Shock Protein 70-Ipek 1 from Toxoplasma gondii

Toxoplasma gondii is a common intracellular parasite that causes the toxoplasmosis. Heat shock proteins (Hsps) have a critical role in pathogenesis of toxoplasmosis. Hsps are highly conserved proteins in evolution among living organisms. This protein family responsible for a wide range of biological processes such as protein folding, protein translocation, protein aggregation. In the present study, Hsp70, a member of the Hsp family, was isolated from T. gondii and its sequence and motifs were determined by PCR, cloning, sequencing and homology modelling analysis. ATP hydrolysis, luciferase folding, and luciferase aggregation experiments were performed for determination of its chaperone activity while the stability and secondary structure of the Hsp70 were discovered by using biophysical experiments (FTIR, florescence and quenching experiment). In addition, in silico analysis were used to determine the physicochemical characteristics of Hsp70. The results revealed that Hsp70 protein obtained from T. gondii (Hsp70-IPEK1) is similar to Hsp70s from other organisms. Also, the chaperone activity, stability and secondary structure of Hsp70-IPEK1 were determined. Hsp70-IPEK1 together with other chaperones in the presence of nucleotide were dramatically increased protein folding and aggregation. According to these results, it is thought that Hsp70 has a potential to contribute many research areas such as pharmaceutical analysis

A potential trade-off between reproduction and enhancement of thermotolerance in Liriomyza trifolii populations driven by thermal acclimation

The invasive pest, Liriomyza trifolii, poses a significant threat to ornamental and vegetable plants. It spreads rapidly and causes large-scale outbreaks with pronounced thermotolerance. In this study, we developed L. trifolii strains adapted to high temperatures (strains designated 35 and 40); these were generated from a susceptible strain (designated S) by long-term thermal acclimation to 35 °C and 40 °C, respectively. Age-stage, two-sex life tables, thermal preferences, critical thermal limits, knockdown behaviors, eclosion and survival rates as well as expression of genes encoding heat shock proteins (Hsps) were compared for the three strains. Our findings indicated that the thermotolerance of L. trifolii was enhanced after long-term thermal acclimation, which suggested an adaptive plastic response to thermal stress. A trade-off between reproduction and thermotolerance was observed under thermal stress, potentially improving survival of the population and fostering adaptionary changes. Acclimation at 35 °C improved reproductive performance and population density of L. trifolii, particularly by enhancing the fecundity of female adults and accelerating the speed of development. Although the 40 strain exhibited the highest developmental speed and greater thermotolerance, it incurred a larger reproductive cost. This study provides a theoretical framework for monitoring and controlling leafminers and understanding their evolutionary adaptation to environmental changes.

Genome-Wide Association Study Reveals Marker–Trait Associations for Heat-Stress Tolerance in Sweet Corn

Sweet corn (Zea mays var. rugosa Bonaf.) is a crop with a high economic benefit in tropical and subtropical regions. Heat tolerance analysis and heat-tolerant gene mining are of great significance for breeding heat-resistant varieties. By combining improved genotyping using targeted sequencing (GBTS) with liquid chip (LC) technology, a high-density marker array containing 40 K multiple single polynucleotide polymorphisms (mSNPs) was used to genotype 376 sweet corn inbred lines and their heat-stress tolerance was evaluated in the spring and summer of 2019. In general, plant height, ear height and the number of lateral branches at the first level of the male flowers were reduced by 24.0%, 36.3%, and 19.8%, respectively. High temperatures in the summer accelerated the growth process of the sweet corn, shortening the days to shedding pollen by an average of 21.6% compared to the spring. A genome-wide association study (GWAS) identified 85 significant SNPs distributed on 10 chromosomes. Phenotypes in the spring and summer were associated with the 21 and 15 loci, respectively, and significant phenotypic differences between the two seasons caused by the temperature change were associated with the 49 SNP loci. The seed setting rate (SSR) was more susceptible to heat stress. An annotation analysis identified six candidate genes, which are either heat shock transcription factors (Hsfs) or heat shock proteins (Hsps) in Arabidopsis and rice (Oryza sativa), and these candidate genes were directly and indirectly involved in the heat-resistant response in the sweet corn. The current findings provide genetic resources for improving the heat-stress tolerance of sweet corn by molecular breeding.

Peroxyredoxin 6 Protects RIN-M5F Pancreatic Beta Cells Against Streptozotocin-Induced Senescence.

There are evidences that a decrease in the functional activity of pancreatic β-cells under type 2 diabetes conditions may be associated with their senescence, therefore, senotherapy may be a prospective strategy for the diabetes treatment. The senotherapeutic potential of peroxiredoxin 6 (PRDX6) was studied in RIN-m5F pancreatic β-cells with streptozotocin-induced senescence by measuring markers, associated with senescence. Exposure to streptozotocin (STZ) resulted in the senescence of the β-cells. The addition of PRDX6 to the culture medium of RIN-m5F β-cells before treatment with STZ decreased the levels of the following senescence markers: the percentage of SA-β-Gal-positive cells, the phosphorylation of histone H2AX and p21 proteins, and the secretion of the proinflammatory cytokine IL-6 but not the anti-inflammatory cytokine IL-10. These effects were accompanied by a decrease in the production of reactive oxygen species (ROS) and the restoration of impaired NF-κB activation. In addition, PRDX6 altered the production of the heat shock protein HSP90: the production of the constitutive form of HSP90-beta decreased, while the level of inducible HSP90-alpha increased. PRDX6 prevented the senescence of RIN-m5F cells in response to the DNA damage-inducing agent streptozotocin, indicating a potential protective role of PRDX6 in type 2 diabetes mellitus.

Model-free inference of memory in conformational dynamics of a multi-domain protein

Abstract Single-molecule experiments provide insight into the motion (conformational dynamics) of individual protein molecules. Usually, a well-defined but coarse-grained intramolecular coordinate is measured and subsequently analysed with the help of hidden Markov models to deduce the kinetics of protein conformational changes. Such approaches rely on the assumption that the microscopic dynamics of the protein evolve according to a Markov-jump process on some network. However, the manifestation and extent of memory in the dynamics of the observable strongly depends on the chosen underlying Markov model, which is generally not known and therefore can lead to misinterpretations. Here, we combine extensive single-molecule plasmon ruler experiments on the heat shock protein Hsp90, computer simulations, and theory to infer and quantify memory in a model-free fashion. Our analysis is based on the bare definition of non-Markovian behaviour and does not require any underlying model. In the case of Hsp90 probed by a plasmon ruler, the Markov assumption is found to be clearly and conclusively violated on timescales up to roughly 50 s, which corresponds roughly to ∼50% of the inferred correlation time of the signal. The extent of memory is striking and reaches biologically relevant timescales. This implies that memory effects penetrate even the slowest observed motions. We provide clear and reproducible guidelines on how to test for the presence and duration of memory in experimental single-molecule data.

Heat shock protein (Hsp27)-ceramide synthase (Cers1) protein-protein interactions provide a new avenue for unexplored anti-cancer mechanism and therapy

Hsp27 is a member of the small heat-shock proteins (sHSPs) – the known cellular line of defence against abnormal protein folding behaviors. Nevertheless, its upregulation is linked to a variety of pathological disorders, including several types of cancers. The ceramide synthases (CerS) mediate the synthesis of ceramide, a critical structural and signaling lipid. Functionally, downstream ceramide metabolites are implicated in the apoptosis process and their abnormal functionality has been linked to anticancer resistance. Studies showed that CerS1 are possibly inhibited by Hsp27 leading to biochemical anticancer effects in vitro. Nevertheless, the nature of such protein-protein interaction (PPI) has not been considerably investigated in molecular terms, hence, we present the first description of the dynamics CerS1-Hsp27 interaction landscapes using molecular dynamics simulations. Time-scale molecular dynamics simulation analysis indicated a system-wide conformational events of decreased stability, increased flexibility, reduced compactness, and decreased folding of CerS1. Analysis of binding energy showed a favorable interaction entailing 56 residues at the interface and a total stabilizing energy of −158 KJ/mol. The CerS1 catalytic domain experienced an opposite trend compared to the protein backbone. Yet, these residues adopted a highly compact conformation as per DCCM and DSSP analysis. Furthermore, conserved residues (SER 212, ASP 213, ALA 240, GLY 243, ASP 319) comprising the substrate shuttling machinery showed notable rigidity implying a restrained ceramide precursor access and assembly; hence, a possible inhibitory mechanism. Findings from this report would streamline a better molecular understanding of CerS1-Hsp27 interactions and decipher its potential avenue toward unexplored anti-cancer mechanisms and therapy.

Long-term thermal acclimation enhances heat resistance of Hong Kong catfish (Clarias fuscus) by modulating gill tissue structure, antioxidant capacity and immune metabolic pathways

The rapid temperature changes caused by global warming significantly challenge fish survival by affecting various biological processes. Fish generally mitigate stress through physiological plasticity, but when temperature changes exceed their tolerance limits, even adaptable species like Siluriformes can experience internal disruptions. This study investigates the effects of extreme thermal climate on Hong Kong catfish (Clarias fuscus), native to tropical and subtropical regions. C. fuscus were exposed to normal temperature (NT, 26 ℃) or high temperature (HT, 34 ℃) condition for 90 days. Subsequently, histological, biochemical, and transcriptomic changes in gill tissue were observed after exposure to acute high temperatures (34 ℃) and subsequent temperature recovery (26 ℃). Histological analysis revealed that C. fuscus in the HT group exhibited less impact from sudden temperature shifts compared to the NT group, as they adapted by reducing the interlamellar cell mass (ILCM) and lamellae thickness (LT) of gill tissue, thereby mitigating the aftermath of acute heat shock. Biochemical analysis showed that catalase (CAT) activity in the high temperature group continued to increase, while malondialdehyde (MDA) levels decreased, suggesting establishment of a new oxidative balance and enhanced environmental adaptability. Transcriptome analysis identified 520 and 463 differentially expressed genes in the NT and HT groups, respectively, in response to acute temperature changes. Enrichment analysis highlighted that in response to acute temperature changes, the NT group inhibited apoptosis and ferroptosis by regulating the activity of alox12, gclc, and hmox1a, thereby attenuating the adverse effects of heat stress. Conversely, the HT group increased the activity of pfkma and pkma to provide sufficient energy for tissue repair. The higher degree of heat shock protein (Hsp) response in NT group also indicated more severe heat stress injury. These findings demonstrate alterations in gill tissue structure, regulation of oxidative balance, and the response of immune metabolic pathways to acute temperature fluctuations in C. fuscus following thermal exposure, suggesting potential avenues for further exploration into the thermal tolerance plasticity of fish adapting to global warming.

COPPER AND MICROPLASTIC EXPOSURE AFFECTS THE GILL GENE EXPRESSION OF COMMON CARP DURING SALTWATER CHALLENGE

The aim of the present study was to assess if pre-exposure to water copper and/or polyvinyl chloride microparticles affects the transcriptional responses of common carp, Cyprinus carpio, to saltwater exposure. Fish were exposed to 0.25 mg/L copper alone (Cu) or in the presence of 0.5 mg/L polyvinyl chloride microparticles (Cu-MPVC) for 14 days, followed by 72 hours of salt water exposure (0 to 13 ppt NaCl). The copper content in the gills and the expression of heat shock protein (hsp70) and cytochrome P450 family 1 (cyp1a) transcripts were examined. The results showed that gill copper levels increased significantly (P = 0.008) in the Cu and Cu-MPVC treatments after 14 days of exposure, compared to the control fish; the Cu and Cu-MPVC treatments had similar gill copper levels. After 14 days of exposure, branchial expression of the hsp70 and cyp1a genes was significantly up-regulated in the Cu and Cu-MPVC treatments. Exposure to salt water led to a significant down-regulation of the gene transcripts in all treatments after 24 h of exposure. At this point, the Cu and Cu-MPVC treatments showed transcripts similar to those of the control fish prior to saltwater exposure. The fish treated with Cu-MPVC showed significantly higher hsp70 expression 72 h after saltwater exposure than the other treatments. At this time point, the control and Cu fish had significantly lower cyp1a expression than before saltwater exposure. In conclusion, the present data suggest that copper exposure induces stress in the fish gills, and the presence of MPCV in the water hampers normal transcriptomic responses of the fish gills to saltwater exposure.

Understanding Interactions between a Potential Antimalarial 'MAL2-11B' and its Targets using In Silico Methods.

The 70 kDa heat shock proteins (Hsp70) are ubiquitous molecules that play central roles in protein homeostasis. Their nucleotide-binding domains (NBD) are associated with the J domains of 40 kDa co-chaperone 'HSP40' in performing their functions. Interruption of this interaction significantly impacts the critical ATPase activity of Hsp70s, making them dysfunctional. MAL2-11B is a dihydropyrimidine derivative that blocks Hsp70-Hsp40 interaction and hence holds the potential to be used as a drug. This Hsp70 inhibitor is a structural analogue of MAL3-101 that has proven anti-cancer and antiparasitic activity. MAL2-11B is predicted to have better drug-likeness, solubility, and absorption properties than MAL3-101. In the present study, we have therefore explored the potential of MAL2-11B as an antimalarial by using in silico tools. Molecular docking of MAL2-11B with all Plasmodium falciparum Hsp70 (PfHsp70) proteins revealed its preferential affinity for two out of four homologs at the nucleotide-binding site. Detailed analysis of the docked complexes helped us to predict the kind of protein-inhibitor interactions and specific amino acid residues involved in binding. After in vitro validation, these data may be used as the groundwork for the design and development of new inhibitors and drugs against malaria.

Expressional respones of hsp70 genes against abiotic and entomopathgenic stresses in four different noctuid larval species (Lepidoptera: Noctidae)

Heat shock proteins (Hsps) are stress response proteins. In a previous study, host larval Hsp70s were identified as the structural proteins of virions of Heliothis virescens ascovirus 3h (HvAV-3h), an insect virus that mainly infects noctuid larvae. To investigate the response of hsp70s of healthy Mythimna separata, Spodoptera exigua, Spodoptera frugiperda, and Spodoptera litura larvae to various abiotic or entomopathogenic stresses, quantitative PCR was used to detect larval hsp70s expression patterns. Results showed distinct expression patterns of hsp70s in response to different abiotic stresses. Notably, Mshsp70 expression pattern resembled Slhsp70 under most treatments. In healthy larvae, no tissue tropism was observed concerning the relative expression of Mshsp70, Sfhsp70, and Slhsp70. After infection with HvAV-3h, the expression of hsp70s in all dissected tissues of all tested larval species increased. Significant differences were found in the fat bodies of M. separata, S. exigua, and S. litura as well as in the hemolymph of S. exigua and S. litura. Subsequent silencing of Slhsp70, resulted in a significant decrease in DNA replication levels of HvAV-3h in S. litura larvae at 24 and 72 h post RNA interference, indicating that Slhsp70 is necessary for DNA replication in HvAV-3h. These data can provide references for the studying on the stress response of noctuid larvae to different environmental factors.

SlWRKY55 coordinately acts with SlVQ11 to enhance tomato thermotolerance by activating SlHsfA2.

High temperature (HT) severely restricts plant growth, development, and productivity. Plants have evolved a set of mechanisms to cope with HT, including the regulation of heat stress transcription factors (Hsfs) and heat shock proteins (Hsps). However, it is not clear how the transcriptional and translational levels of Hsfs and Hsps are controlled in tomato. Here, we reported that the HT-induced transcription factor SlWRKY55 recruited SlVQ11 to coordinately regulate defense against HT. SlWRKY55 directly bound to the promoter of SlHsfA2 and promoted its expression, which was increased by SlVQ11. Moreover, both SlWRKY55 and SlVQ11 physically interacted with SlHsfA2 to enhance the transcriptional activity of SlHsfA2. Thus, our results revealed a molecular mechanism that the SlWRKY55/SlVQ11-SlHsfA2 cascade enhanced thermotolerance and provided potential target genes for improving the adaptability of crops to HT.

Colistin Resistance Mechanisms and Molecular Epidemiology of Enterobacter cloacae Complex Isolated from a Tertiary Hospital in Shandong, China.

Enterobacter cloacae complex (ECC), which includes major nosocomial pathogens, causes urinary, respiratory, and bloodstream infections in humans, for which colistin is one of the last-line drugs. This study aimed to analyse the epidemiology and resistance mechanisms of colistin-resistant Enterobacter cloacae complex (ECC) strains isolated from Shandong, China. Two hundred non-repetitive ECC strains were collected from a tertiary hospital in Shandong Province, China, from June 2020 to June 2022. Whole-genome sequencing and bioinformatics analyses were performed to understand the molecular epidemiology of the colistin-resistant ECC strains. The nucleotide sequences of heat shock protein (hsp60) were analyzed by using BLAST search to classify ECC. The gene expression levels of ramA, soxS, acrA, acrB, phoP, and phoQ were assessed using RT-qPCR. MALDI-TOF MS was used to analyse the modification of lipid A. Twenty-three colistin-resistant strains were detected among the 200 ECC clinical strains (11.5%). The hsp60 cluster analysis revealed that 20 of the 23 ECC strains belonged to heterogeneous resistance clusters. Variants of mgrB, phoPQ, and pmrAB, particularly phoQ and pmrB, were detected in the 23 ECC strains. The soxS and acrA genes were significantly overexpressed in all 23 colistin-resistant ECC strains (P < 0.05). Additionally, all 23 ECC strains contained modified lipid A related to colistin resistance, which showed five ion peaks at m/z 1876, 1920, 1955, 2114, and 2158. Among the 23 ECC strains, 6 strains possessed a phosphoethanolamine (pETN) moiety, 16 strains possessed a 4-amino-4-deoxy-L-arabinose (-L-Ara4N) moiety, and one strain had both pETN and -L-Ara4N moieties. This study suggests that diverse colistin resistance existed in ECC, including unknown resistance mechanisms, exist in ECC. Mechanistic investigations of colistin resistance are warranted to optimise colistin use in clinical settings and minimise the emergence of resistance.

Mild heat shock at 40°C increases levels of autophagy: Role of Nrf2.

The exposure to low doses of stress induces an adaptive survival response that involves the upregulation of cellular defense systems such as heat shock proteins (Hsps), anti-apoptosis proteins, and antioxidants. Exposure of cells to elevated, non-lethal temperatures (39-41°C) is an adaptive survival response known as thermotolerance, which protects cells against subsequent lethal stress such as heat shock (>41.5°C). However, the initiating factors in this adaptive survival response are not understood. This study aims to determine whether autophagy can be activated by heat shock at 40°Cand if this response is mediated by the transcription factor Nrf2. Thermotolerant cells, which were developed during 3h at 40°C, were resistant to caspase activation at 42°C. Autophagy was activated when cells were heated from 5 to 60min at 40°C. Levels of acidic vesicular organelles (AVOs) and autophagy proteins Beclin-1, LC3-II/LC3-I, Atg7, Atg5, Atg12-Atg5, and p62 were increased. When Nrf2 was overexpressed or depleted in cells, levels of AVOs and autophagy proteins were higher in unstressed cells, compared to the wild type. Stress induced by mild heat shock at 40°C further increased levels of most autophagy proteins in cells with overexpression or depletion of Nrf2. Colocalization of p62 and Keap1 occurred. When Nrf2 levels are low, activation of autophagy would likely compensate as a defense mechanism to protect cells against stress. An improved understanding of autophagy in the context of cellular responses to physiological heat shock could be useful for cancer treatment by hyperthermia and the protective role of adaptive responses against environmental stresses.

Genome-wide identification and coexpression network analysis of heat shock protein superfamily in Apolygus lucorum.

Heat shock proteins (Hsp) function as crucial molecular chaperones, playing pivotal roles in insects' response to stress stimuli. Apolygus lucorum, known for its broad spectrum of host plants and significant crop damage potential, presents a compelling subject for understanding stress response mechanisms. Hsp is important for A. lucorum to tolerate temperature and insecticide stress and may be involved in the formation of resistance to the interactive effects of temperature and insecticide. Here, we employed comprehensive genomic approaches to identify Hsp superfamily members in its genome. In total, we identified 42 Hsp genes, including 3 Hsp90, 16 Hsp70, 13 Hsp60, and 10 Hsp20. Notably, we conducted motif analysis and gene structures for Hsp members, which suggested the same families are relatively conserved. Furthermore, leveraging the weighted gene coexpression network analysis, we observed diverse expression patterns of different Hsp types across various tissues, with certain Hsp70 showing tissue-specific bias. Noteworthy among the highly expressed Hsp genes was testis-specific, which may serve as a pivotal hub gene regulating the gene network. Our findings shed light on the molecular evolutionary dynamics and temperature stress response mechanisms of Hsp genes in A. lucorum, offering insights into its adaptive strategies and potential targets for pest management.

Heat stress induced piRNA alterations in pachytene spermatocytes and round spermatids

BackgroundSpermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells.MethodsWe generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs.ResultsAs a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts.ConclusionsIn heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.