Hsp70 Expression Research Articles

Heat shock protein 70 enhances viral replication by stabilizing Senecavirus A nonstructural proteins L and 3D

Senecavirus A (SVA) is an emerging pathogen that causes idiopathic vesicular infections in pig herds, posing a potential threat to their production performance. Heat shock protein 70 (Hsp70) is a molecular chaperone that plays an important role in host homeostasis under both physiological and stress conditions. However, the effects of Hsp70 on SVA infection and its underlying regulatory mechanisms remain unclear. Here, we confirmed that Hsp70 expression promotes SVA infection, as evidenced by the expression of viral proteins, viral titers, and the number of rSVA-eGFP-infected cells. This positive regulatory role of Hsp70 is mainly involved in post-entry stages of SVA. Viral proteins that interacted with Hsp70 were screened, and co-immunoprecipitation (co-IP) shows an interaction between Hsp70 and SVA L and 3D proteins. Subsequently, we determined that the expression of Hsp70 is beneficial for the stability of the SVA L and 3D proteins. Additionally, the substrate-binding domain (SBD) of Hsp70 plays an important role in the interaction between Hsp70 and SVA L or 3D proteins; and the deletion of this domain results in the loss of the stabilizing effect of Hsp70 on SVA L and 3D proteins and the positive regulatory effect of Hsp70 on SVA replication. These results reveal that Hsp70 promotes SVA infection by stabilizing viral L and 3D proteins and provides a strategy for preventing and controlling SVA infection.

Feedback control of the heat shock response by spatiotemporal regulation of Hsp70.

Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted the induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing the expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity.

The effects of crocin on fear condition memory impairment caused by morphine in relation to gene expression of amygdala HSP70, HSP90 and NF-kB

BackgroundSome studies have shown that morphine causes memory impairment through different mechanisms. The roles of inflammation/NF-κB and BDNF/TrkB/CREB pathways are significant in the memory deficits caused by morphine. Aim: In this study, the effect of crocin on the impairment of fear memory caused by morphine was investigated, and the expression of HSP70, HSP90, and NF-kB genes that play an important role in memory in male Wistar rats was measured. Material and methodThe fear conditioning test was used to check the fear memory of rats, and total Freezing time was considered as an index of memory. Rats (96 Wistar rats) were randomly divided into 12 groups. The rats were injected with saline (1 ml/kg) or different doses of morphine (0.5, 2.5, and 5 mg/kg) 5 min after injection of saline (1 ml/kg) or subthreshold doses of crocin (60 mg/kg). Real-time PCR method was used for HSP70, HSP90, and NF-kB gene expression in the Amygdala. ResultAdministration of morphine 5 mg/kg decreased the total freezing and increased the latency freezing (P < 0.001). Also, the results showed that crocin 60 mg/kg reduced memory impairment induced by morphine. Real-time PCR analysis showed that morphine 5 mg/kg significantly increased the expression of the HSP70, HSP90, and NF-kB genes. However, the subthreshold dose of crocin (60 mg/kg) reduced the increase in gene expression. ConclusionThe study showed that morphine impacts memory and boosts gene expression of HSP70, HSP90, and NF-kB, while crocin could potentially reverse this effect by regulating these amygdala genes, suggesting avenues for future research.

Radiosensitizing Capacity of Fenofibrate in Glioblastoma Cells Depends on Lipid Metabolism

Despite advances in multimodal therapy approaches such as resection, chemotherapy and radiotherapy, the overall survival of patients with grade 4 glioblastoma (GBM) remains extremely poor (average survival time <2 years). Altered lipid metabolism, which increases fatty acid synthesis and thereby contributes to radioresistance in GBM, is a hallmark of cancer. Therefore, we explored the radiosensitizing effect of the clinically approved, lipid-lowering drug fenofibrate (FF) in different GBM cell lines (U87, LN18). Interestingly, FF (50 μM) significantly radiosensitizes U87 cells by inducing DNA double-strand breaks through oxidative stress and impairing mitochondrial membrane integrity, but radioprotects LN18 cells by reducing the production of reactive oxygen species (ROS) and stabilizing the mitochondrial membrane potential. A comparative protein and lipid analysis revealed striking differences in the two GBM cell lines: LN18 cells exhibited a significantly higher membrane expression density of the fatty acid (FA) cluster protein transporter CD36 than U87 cells, a higher expression of glycerol-3-phosphate acyltransferase 4 (GPAT4) which supports the production of large lipid droplets (LDs), and a lower expression of diacylglycerol O-acyltransferase 1 (DGAT1) which regulates the formation of small LDs. Consequently, large LDs are predominantly found in LN18 cells, whereas small LDs are found in U87 cells. After a combined treatment of FF and irradiation, the number of large LDs significantly increased in radioresistant LN18 cells, whereas the number of small LDs decreased in radiosensitive U87 cells. The radioprotective effect of FF in LN18 cells could be associated with the presence of large LDs, which act as a sink for the lipophilic drug FF. To prevent uptake of FF by large LDs and to ameliorate its function as a radiosensitizer, FF was encapsulated in biomimetic cell membrane extracellular lipid vesicles (CmEVs) which alter the intracellular trafficking of the drug. In contrast to the free drug, CmEV-encapsulated FF was predominantly enriched in the lysosomal compartment, causing necrosis by impairing lysosomal membrane integrity. Since the stability of plasma and lysosomal membranes is maintained by the presence of the stress-inducible heat shock protein 70 (Hsp70) which has a strong affinity to tumor-specific glycosphingolipids, necrosis occurs predominantly in LN18 cells having a lower membrane Hsp70 expression density than U87 cells.In summary, our findings indicate that the lipid metabolism of tumor cells can affect the radiosensitizing capacity of FF when encountered either as a free drug or as a drug loaded in biomimetic lipid vesicles.

Potential Probiotic Bacillus Strains with Antioxidant and Antimutagenic Activity Increased Weight Gain and Altered hsp70, cxc, tnfα, il1β, and lysC Gene Expression in Clarias gariepinus

The potential probiotic properties of three Bacillus strains were studied. A probiotic supplement for the African catfish Clarias gariepinus was produced via the solid-state fermentation protocol and incorporated into the fish feed for a period of seven weeks. Since the 36th day of the experiment, all experimental groups had a statistically significant increase in their weight gain than the control group. The maximum weight gain observed in fish fed the probiotic-supplemented feed was 29.16% higher than that of the control group, and the maximum feed conversion rate improvement was 24%. Cell-free extracts from these strains showed antioxidant (11.55–27.40%) and DNA-protective (45.33–61.83%) activity in a series of in vitro biosensor tests. Further investigation into the antimutagenic activity of the strains revealed that two of them reduced the level of induced mutagenesis in an Escherichia coli model (by 33.58% and 54.35%, respectively). We also assessed the impact of probiotic strains on the expression of several key genes in the host (C. gariepinus), including hsp70, cxc, tnfα, il1β, and lysC. More than a 10-fold increase in expression rates was observed for hsp70 in gonads and liver; for cxc in muscles and gonads; for tnfα in brain, gills, and liver; for il1β in the brain, gills, gonads, and liver; and for lysC in gills, gonads, liver, and muscles. This study provides evidence that probiotics exhibiting antioxidant and antimutagenic properties can provide significant benefits in vivo within aquaculture systems. The molecular effects of these probiotics appear to be complex and tissue-specific, with both upregulation and downregulation of immune system genes observed. Nevertheless, at the organismal level, the impact was unequivocally positive in terms of aquaculture objectives, manifested as enhanced body weight gain in the fish. Consequently, these Bacillus strains warrant serious consideration as potential probiotics for this species.

Establishment of a cell culture from Daphnia magna as an in vitro model for (eco)toxicology assays: Case study using Bisphenol A as a representative cytotoxic and endocrine disrupting chemical

Bisphenol A (BPA) is a widely used industrial compound found in polycarbonate plastics, epoxy resin, and various polymer materials, leading to its ubiquitous presence in the environment. The toxicity of BPA to aquatic organisms has been well documented following in vivo exposure scenarios, with known cytotoxic and endocrine-disrupting effects. As such, BPA was used in this study as a well-characterized chemical to implement more ethical and resource-efficient scientific practices in toxicity testing through new approach methods (NAMs). Due to the frequent use of Daphnia spp. as a model organism in toxicology, we developed an in vitro cell culture system from Daphnia magna embryos, with optimized medium to support cell longevity. The cultures were maintained for up to two months, demonstrating their stability and suitability for cytotoxicity studies. Using this novel system, lethal concentration 50 (LC50) values were determined at the 24 and 48 h time points following BPA exposure. Subsequently, oxidative stress, endocrine disruption, and DNA damage were assessed through gene expression, activity assays, and a comet assay in BPA-exposed cells. LC50 values of 52 µM and 20 µM BPA were calculated after 24 and 48 h exposures, respectively. BPA cells exposed to 20 and 52 µM had significantly increased GSH, GPx, and GST activity levels. mRNA expression analysis revealed significant upregulations in the mRNA expression of hsp70, hsp90, gst, gpx, vtg1, and cyp4, with downregulations of sod, cat, and ecr following BPA exposure. Furthermore, comet assays showed a significantly higher level of DNA damage induced by BPA compared to controls, with greater comet and tail lengths. This study established a novel in vitro Daphnia model, using BPA as a case study for determining toxic effects, further highlighting the importance and applicability of utilizing alternative methods in ecotoxicological research through reducing animal use.

Effects of alpha-lipoic acid and sildenafil citrate on sperm quality in asthenozoospermic men during freezing-thawing processes

We aimed to investigate the combined effects of alpha-lipoic acid (ALA) and sildenafil citrate (SC) on sperm quality during freeze-thawing in asthenozoospermic men. We categorized thirty semen samples from asthenozoospermic into five different groups for analysis: Control (fresh), Freeze, Freeze + SC, Freeze + ALA, and Freeze + SC + ALA. We evaluated sperm parameters in each group, including motility, viability, morphology, plasma membrane integrity, DNA fragmentation, mitochondrial membrane potential, protamine deficiency, acrosome integrity, malondialdehyde, tumor necrosis factor-alpha (TNF-α), antioxidants (Superoxide dismutase, Glutathione, Catalase), and gene expression of HSP70 and Bcl-2. The Freeze group showed significant declines in viability, motility, morphology, membrane integrity, antioxidants, and mitochondrial membrane potential, with increases in protamine deficiency, abnormalities, DNA fragmentation, TNF-α, and malondialdehyde compared to control (p = 0.000). These effects were significantly reversed in all treatment groups (p = 0.000). Bcl-2 and HSP70 expression increased significantly in all groups (p = 0.000). Acrosomal integrity decreased significantly (p = 0.000) in the Freeze group compared to controls and in the Freeze + SC group compared to the Freeze group. However, acrosomal integrity significantly increased (p = 0.000) in the Freeze + SC + ALA group compared to the Freeze + SC group and in the Freeze + ALA group compared to other treatments. Our findings indicate that the simultaneous addition of SC and ALA in the freezing media yielded increased sperm quality. This enhancement was evidenced by reductions in oxidative stress, inflammation, and apoptosis, along with partial prevention of premature acrosome reactions induced by SC.

Abstract PR018: Inflammation-induced mechanotransduction is necessary and sufficient to create pre-cancerous squamous lung metaplasias and necessary to drive progression to dysplasia

Abstract Chronic exposure to tobacco smoke triggers chronic inflammation, fostering a fibrotic microenvironment and promoting the focal replacement of columnar human bronchial epithelial cells (hBECs) with cells exhibiting squamous morphology, termed metaplasia. How squamous metaplasia progresses to squamous dysplasia and, ultimately, squamous lung carcinoma is under intense investigation. Using human bronchial tissues, we demonstrate that, both in vitro and in vivo, cellular stress associated with chronic inflammation (e.g., oxidative stress, DNA damage) enhances TGF-β signaling and creates a fibroblast state exhibiting upregulated HSP47 expression, driving increased collagen fiber alignment, elevated tissue stiffness, and subsequent activation of YAP-dependent mechanotransduction in adjacent hBECs. These Stress/Tension-producing Instructive Fibroblasts (STIFs) alone were sufficient to induce hBECs to squamous metaplasia, precursors to tumor formation. Furthermore, when tumor suppressor function was compromised in hBECs, STIFs induced dysplastic phenotypes. Mechanistically, STIFs act in a dominant manner modulating epithelial cell identity and malignant phenotypes via extracellular matrix (ECM)-dependent mechanotransduction. Inhibition of HSP47-dependent collagen processing effectively prevented fibroblast-induced squamous metaplasia and dysplasia and was capable of reversing fibroblast-induced metaplasia, restoring bronchial epithelial cell identity. Citation Format: Thea D. Tisty, Deng Pan, Philippe Gascard, Joseph Caruso, Lorenzo Ferri, Chira Chen-Tanyolac. Inflammation-induced mechanotransduction is necessary and sufficient to create pre-cancerous squamous lung metaplasias and necessary to drive progression to dysplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR018.

KCl enhances the cryoablation-induced antitumor immune response: A hepatocellular carcinoma murine model research

Cryoablation is a valuable treatment for liver cancer. To investigate the effect of KCl solution on the immunological response post cryoablation, we created a tumor-bearing mice model by subcutaneously implanting Hepal-6 cells in adult Balb/c mice. Subsequently, the mice were randomly assigned to three groups: group A (sham cryoablation), group B (cryoablation), and group C (cryoablation plus KCl solution). Mice were sacrificed on days 0, 7, and 14 post-treatment. Immune cell populations were assessed using flow cytometry. Blood samples were analyzed for serum IL-4, HSP70, and TGF-β1 levels with ELISA assays. Ablated tissues stained with immunohistochemistry were utilized to evaluate Ki67 expression at the margins of the ablation site. Our findings revealed higher HSP70 expression levels in groups B and C compared to group A. Cryoablation triggered an immune response, which was enhanced by KCl. On days 0, 7, and 14, the percentages of CD4+ T cells, CD8+ T cells, and NK cells in the spleen of group C were significantly increased compared with groups A and B. Additionally, the Th1/Th2 ratio was significantly increased in group C. Serum TGF-β1 expression was elevated after cryoablation, but KCl solution reduced the high TGF-β1 expression after cryoablation and decreased the invasiveness of cancer cells. Finally, the proliferative activity of untreated tumor tissue was significantly reduced in group C compared to groups A and B. In summary, Cryoablation triggered a systemic immune response in tumor-bearing mice, which was further boosted by combining cryoablation with a KCl solution.

Selenium mitigated cadmium-induced ovarian retardation in female Procambarus clarkii by regulating vitellogenin synthesis and transfer in the hepatopancreas

Cadmium (Cd) is prevalent in aquatic ecosystems and accumulates in various tissues of aquatic organisms, leading to severe biological toxicity. Selenium (Se) is recognized for mitigating heavy metal toxicity, though its protective effects against Cd in aquatic crustaceans remain underexplored. This study, therefore, assessed the effects of dietary Cd (15 mg/kg) exposure and Se (6 mg/kg) supplementation on the hepatopancreas and ovaries of female crayfish to uncover the mechanisms of Cd toxicity and the protective role of Se. The results showed that Cd accumulation in the hepatopancreas caused a reduced hepatopancreas index (HPI), decreased protein content, histopathological damage, and oxidative stress, while Se supplementation reduced Cd levels, mitigated damage, and restored tissue integrity and antioxidant defenses. Transcriptomic analysis further revealed significant alterations in gene expression related to detoxification, lipid metabolism, and energy production in response to Cd exposure, which were partially or fully restored by Se supplementation. Additionally, Se alleviated Cd-induced inhibition of ovarian development, as evidenced by improved ovary index, enhanced oocyte development, and normalization of essential trace element levels. Mechanistically, Se restored the Cd-disrupted vitellogenin (Vtg) synthesis in the hepatopancreas via regulating the mRNA expression of hsp70 and genes related to the molt-inhibiting hormone (MIH) (mih, rxr, and ecr). Overall, these findings indicate that Se supplementation mitigated Cd-induced hepatopancreatic dysfunction, restored Vtg synthesis, and consequently counteracted the inhibition of ovarian development in adult female crayfish.

Design, synthesis and biological studies of new isoxazole compounds as potent Hsp90 inhibitors

Heat shock protein 90 (Hsp90), a molecular chaperone, contributes to the preservation of folding, structure, stability, and function proteins. In this study, novel compounds comprising isoxazole structure were designed, synthesized and their potential ability as Hsp90 inhibitors was validated through docking studies. The active site-based compounds were prepared through a multi-step synthesis process and their chemical structures were characterized employing FT-IR, NMR, and mass spectrometry analysis. Cytotoxic and Hsp90 inhibition activities of synthesized compounds were assessed by MTT assay and ELISA kit, respectively. Based on the obtained results, compound 5 exhibited the highest cytotoxicity (IC50; 14 µM) against cancer cells and reduced Hsp90 expression from 5.54 ng/mL in untreated (normal cells) to 1.56 ng/mL in cancer cells. Moreover, molecular dynamics (MD) simulation results indicated its high affinity to target protein and approved its excellent stability which is essential for exerting an inhibitory effect on cancer cell proliferation.

Heat Shock Proteins expression in malaria and dengue vector.

The survival of mosquitoes under changing climatic conditions particularly temperature, is known to be supported by Heat Shock Proteins (HSPs). In view of climate change, it is imperative to know whether the mosquito vectors will be able to withstand the increased temperatures or perish. Therefore, the present study was undertaken on the expression of HSPs' gene in An. stephensi and Ae. aegypti by exposing them to temperatures ranging from 5 to 45°C for 15-180-minutes for once and continuously or with rest in between. We compared the temperature-tolerance of both the vectors in terms of expression of HSP83, HSP70, and HSP26 genes at varying degrees of temperature and duration. HSP70 and HSP26 were found distinctively expressed in both the vectors as compared to HSP83. With continuous exposure up to 180-minutes at 35°C and 40°C, HSP70 was found upregulated up to 35 and 47 folds in Ae. aegypti while in An. stephensi, the expression was only 1 fold. Between the genes, HSP70 was highly expressed at different temperatures followed by HSP26 and HSP83. The manifold up-regulation of HSP genes in Ae. aegypti than An. stephensi may be attributed to the robustness of Aedes vector in terms of temperature tolerance. This study has shown that Ae. aegypti and An. stephensi can withstand considerable temperature stress by expressing HSPs when exposed to variable temperature and duration. In view of changing climate, the study provides a clue that the vector of dengue and zika virus will be difficult to control.

Dietary Malondialdehyde Impair Intestinal Health and Fillet Quality of Hybrid Grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂).

Aquafeed kept at elevated temperatures and humidity can result in malondialdehyde (MDA) formation, adversely affecting aquafeed quality and triggering negative reactions in fish. To investigate the detrimental effects of dietary MDA on fish, six experimental diets with varying MDA levels (ranging from 0.03 to 17.72 mg/kg, on dietary crude lipid basis) were administered to three replicates of hybrid grouper for 8 weeks. Dietary inclusion of 4.43 mg/kg MDA significantly decreased serum complement 4 content and lysozyme activity, along with intestinal complement 3, complement 4, and immunoglobulin M contents. Furthermore, dietary inclusion of 8.86 mg/kg MDA significantly increased the activities of interleukin-1 receptor-associated kinase, ubiquitin-protein ligase, p38 mitogen-activated protein kinase, and tumor necrosis factor-α, downregulated the relative expression of Occludin but upregulated the relative expression of HSP70 in the hindgut. Additionally, the highest inclusion of MDA (17.72 mg/kg) significantly upregulated the relative levels of pro-inflammatory cytokines (IL-1β and TNF-α), caused intestinal inflammation, and damaged the intestinal microbial structure and fish fillet texture. This study demonstrated a dose-dependent response of MDA on hybrid grouper. A low dietary dose of MDA (<2.21 mg/kg) exhibited minimal impact on immune response and fillet quality. However, higher inclusion levels (≥4.43 mg/kg) impaired the intestinal health and fillet quality. Consequently, the safety limit for MDA content in the diet for hybrid grouper has been established at 4.43 mg/kg based on dietary crude lipid basis.

A Multifunctional Low-Temperature Photothermal Nanomedicine for Melanoma Treatment via the Oxidative Stress Pathway Therapy.

Melanoma is a highly aggressive and dangerous malignant skin tumor and there is an urgent need to develop effective therapeutic approaches against melanoma. The main objective of this study was to construct a multifunctional nanomedicine (GNR@PEG-Qu) to investigate its therapeutic effect on melanoma from the oxidative stress pathway. First, the nanomedicine GNR@PEG-Qu was synthesized and characterized, and its photothermal and antioxidant properties were confirmed. In addition, in vivo imaging capabilities were observed. Finally, the tumor inhibitory effects of GNR@PEG-Qu in vivo and in vitro as well as its biosafety were observed. GNR@PEG-Qu shows good photothermal and anti-oxidation properties. Following exposure to 1064 nm laser irradiation in the second near-infrared II (NIR-II) window, GNR@PEG-Qu shows anti-tumor ability through low-temperature photothermal therapy (PTT) adjuvant drug chemotherapy. GNR@PEG-Qu makes full use of the antioxidant capacity of quercetin, reduces ROS levels in melanoma, alleviates oxidative stress state, and achieves "oxidative stress avoidance" at the tumor site. Quercetin can also downregulate the expression of the heat shock protein Hsp70, which will improve the thermal sensitivity of the tumor site and enhance the efficacy of low-temperature PTT. GNR@PEG-Qu nanoagent exhibits synergistic treatment and high tumor inhibition effects, which is a promising strategy developed to achieve oxidative stress avoidance and synergistic therapy of melanoma using quercetin (Qu)-coated gold nanorod (GNR@PEG).

Neuroprotective properties of a thiazolidine-2,4-dione derivative as an inhibitory agent against memory impairment and phosphorylated tau: In vitro and in vivo investigations

Alzheimer’s disease (AD) is the most common form of neurodegeneration that results in memory disorders and cognitive impairment. The present study investigated the neuroprotective effects of the synthesized thiazolidine-2,4-dione derivative, (E)-5-(4-chlorobenzylidene)-3-(2-oxo-2-phenylethyl)thiazolidine-2,4-dione (TZ4C), an inhibitor of p-Tau and memory impairment, using a SH-SY5Y cell model of methamphetamine-induced tauopathy and a scopolamine-induced memory impairment model in Wistar rats.In the present study, the neuroprotective effect of TZ4C was studied in a SH-SY5Y cellular model of methamphetamine-induced (2 mM) tauopathy and a scopolamine-induced (1.5 mg/kg/day) memory impairment model in male Wistar rats (n = 48). The memory functions and learning abilities of the rats were evaluated using the Morris water maze (MWM) and passive avoidance tests. Additionally, AChE activity in the rat hippocampus was quantified, and the expression of p-Tau, HSP70, and caspase-3 in both in vitro and in vivo samples was evaluated through Western blot analysis.TZ4C (0.1–1000 µM) did not exhibit significantly toxic effects on SH-SY5Y cell viability. Western blot results indicated that TZ4C led to reduced expression of p-Tau, HSP70, and cleaved caspase-3 in SH-SY5Y cells (3 and 10 µM) and the rat hippocampus (2 and 4 mg/kg). Additionally, the findings suggested that TZ4C enhanced memory function in rats with scopolamine-induced impairment and decreased acetylcholinesterase (AChE) specific activity.The comprehensive analysis of in vitro and in vivo experiments underscores the neuroprotective potential (improved neuropathology and reduced memory impairment) of TZ4C. These findings highlight the promise of TZ4C as a candidate for drug discovery programs to identify effective therapies for AD.

Glucose and glutamine drive hepatitis E virus replication.

Viruses have undergone evolutionary adaptations to tune their utilization of carbon sources, enabling them to extract specific cellular substrates necessary for their replication. The lack of a reliable cell culture system and a small-animal model has hampered our understanding of the molecular mechanism of replication of hepatitis E virus (HEV) genotype 1. Our recent identification of a replicative ensemble of mutant HEV RNA libraries has allowed us to study the metabolic prerequisites for HEV replication. Initial assessments revealed increased glucose and glutamine utilization during HEV replication. Inhibition of glycolysis and glycolysis + glutaminolysis reduced the levels of HEV replication to similar levels. An integrated analysis of protein-metabolite pathways suggests that HEV replication markedly alters glycolysis, the TCA cycle, and glutamine-associated metabolic pathways. Cells supporting HEV replication showed a requirement for fructose-6-phosphate and glutamine utilization through the hexosamine biosynthetic pathway (HBP), stimulating HSP70 expression to facilitate virus replication. Observations of mannose utilization and glutamine dependence suggest a crucial role of the HBP in supporting HEV replication. Inhibition of glycolysis and HSP70 activity or knockdown of glutamine fructose-6-phosphate amidotransferase expression led to a substantial reduction in HEV RNA and ORF2 expression accompanied by a significant decrease in HSP70 levels. This study demonstrates that glucose and glutamine play critical roles in facilitating HEV replication.

The Effect of Variegated Grasshopper Meal (Zonocerous variegatus) on Growth, Nutrient Utilization and Gene Expression Study in Juvenile Clarias gariepinus (Burchell, 1822)

Over the years there has been research on how to improve fish yield. This study investigated the effects of variegated grasshopper meal (Zonocerous variegatus) on growth performance, nutrient utilization, and gene expressions in juvenile Clarias gariepinus (African catfish). The variegated grasshopper meal diets (VGDMs) were incorporated with the other feed ingredients at 25% (VGDM1), 50% (VGDM2), 75% (VGDM2), and 100% (VGDM3) diets while the remaining group served as control. Growth performance, nutrient utilization, and gene expression study were evaluated. The mRNA expression of Growth hormone (GH), Heat Shock Protein (HSP70), Melatonin (MEL 1C), tumour necrosis factor-alpha (TNF-α), and interleukin 1 beta (IL-1β) were investigated in the liver and muscle of C. gariepinus fed with varying doses of grasshopper meals (VGDMs) using reverse transcriptase–polymerase chain reaction. There were significant (p&gt;0.05) variations in all parameters in all the treatments. There was a significant upregulation (p&lt;0.05) in the gene expression of GH, HSP70, MEL 1C, and TNF-α in VGDM1 compared to control in both tissue groups. In addition, there was significant downregulation (p&lt;0.05) in interleukin 1 (IL-1β) gene expression in liver VGDM2. This study suggests that dietary variations involving VGMD have the potential to boost growth and improve the immune responses of this fish species.

Yam (Rhizoma Dioscoreae) polysaccharide affects growth performance, immunity, and intestinal microbial community in Bulatmai barbel (LucioBarbus capito)

Polysaccharides from yam (Rhizoma Dioscoreae) can be used as immunopotentiators in aquaculture; however, the protective mechanism of adding them to feed for Bulatmai barbel (LucioBarbus capito) remains unclear. This study aimed to investigate the effects of adding yam polysaccharide (YP) to feed for 8 weeks on the growth performance, antioxidant capacity, gene expression, and intestinal flora of the Bulatmai barbel. The experimental groups were fed a diet including 0.1 %, 0.2 %, 0.4 %, and 0.8 % YP, whereas the diet of the control group contained no added YP, with three replicates per group. The results showed that the addition of YP significantly increased the weight gain rate and specific growth rate of the Bulatmai barbel, which were significantly higher than those of the control group (P < 0.05). The growth effect of the 0.4 % YP (279.77 ± 17.43 g) experimental group was significantly better than that of the other experimental groups, and the feed coefficient (49.35 ± 4.67 g) was significantly lower than that of the control group (P < 0.05). The addition of YP significantly improved immune activity and antioxidant capacity of the liver; HSP70 expression decreased and AKP and SOD expression levels significantly increased in the 0.40 % YP group (P < 0.05). In addition, the relative abundance of intestinal bacteria increased for Fusobacteriota and Firmicutes but decreased in Proteobacteria in the experimental groups compared with the control group. At the genus level, Cetobacterium was the dominant bacterium. With an increase in YP concentration, the relative abundance of Cetobacterium increased, whereas that of Pseudomonas and Phreatobacter decreased. In conclusion, dietary supplementation with 0.4 % YP significantly improved growth performance, antioxidant capacity, and gene expression; and regulated intestinal microbial diversity and floral structure in the Bulatmai barbel.

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.

Anti-inflammatory properties of Bacillus pumilus TS1 in lipopolysaccharide-induced inflammatory damage in broilers

This study investigates whether Bacillus pumilus TS1 improves growth performance and alleviates inflammatory damage in broilers and explored its feasibility as an antibiotic alternative. We divided 240 one-day-old AA308 white-finned broilers into five groups (con, LPS, TS1L + LPS, TS1M + LPS and TS1H + LPS). The TS1L + LPS, TS1M + LPS and TS1H + LPS groups were fed TS1 for 15 days by gavage. The LPS, TS1L + LPS, TS1M + LPS and TS1H + LPS groups were injected intraperitoneally with 1 mg/kg LPS for three days. We investigated the probiotic and anti-inflammatory activities by measuring body weight, sequencing the intestinal flora and examining the structure of tissues by using pathological stain, real-time PCR, Western blotting and immunohistochemical detection. TS1 could improve growth performance and intestinal flora composition, also reduced different organ damage and inflammatory cytokine expression in serum and organs. The mechanism may involve upregulating HSP60 and HSP70 expression, targeting and regulating Nrf2 and P38 MAPK and modulating NF-κB and HO-1 expression at the transcriptional level in different organs. B. pumilus TS1 alleviated Inflammatory injury caused by LPS and attenuated the inflammatory response in broilers, and these effects were achieved through MAPK and Nrf2 regulation of HSPs/HO-1 in different organs. The above results suggested broilers fed with TS1 could release the LPS caused organ damage, and the most suggested dosage was 1.4 × 108 CFU/mL.