Morphological and molecular characteristics of a Trypanosoma sp. from triatomines (Triatoma rubrofasciata) in China.

BACKGROUND: Triatomines (kissing bugs) are natural vectors of trypanosomes, which are single-celled parasitic protozoans, such as Trypanosoma cruzi, T. conorhini and T. rangeli. The understanding of the transmission cycle of T. conorhini and Triatoma rubrofasciata in China is not fully known. METHODS: The parasites in the faeces and intestinal contents of the Tr. rubrofasciata were collected, and morphology indices were measured under a microscope to determine the species. DNA was extracted from the samples, and fragments of 18S rRNA, heat shock protein 70 (HSP70) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) were amplified and sequenced. The obtained sequences were then identified using the BLAST search engine, followed by several phylogenetic analyses. Finally, laboratory infections were conducted to test whether Tr. rubrofasciata transmit the parasite to rats (or mice) through bites. Moreover, 135 Tr. rubrofasciata samples were collected from the Guangxi region and were used in assays to investigate the prevalence of trypanosome infection. RESULTS: Trypanosoma sp. were found in the faeces and intestinal contents of Tr. rubrofasciata, which were collected in the Guangxi region of southern China and mostly exhibited characteristics typical of epimastigotes, such as the presence of a nucleus, a free flagellum and a kinetoplast. The body length ranged from 6.3 to 33.9 µm, the flagellum length ranged from 8.7 to 29.8 µm, the nucleus index was 0.6 and the kinetoplast length was -4.6. BLAST analysis revealed that the 18S rRNA, HSP70 and gGAPDH sequences of Trypanosoma sp. exhibited the highest degree of similarity with those of T. conorhini (99.7%, 99.0% and 99.0%, respectively) and formed a well-supported clade close to T. conorhini and T. vespertilionis but were distinct from those of T. rangeli and T. cruzi. Laboratory experiments revealed that both rats and mice developed low parasitaemia after inoculation with Trypanosoma sp. and laboratory-fed Tr. rubrofasciata became infected after feeding on trypanosome-positive rats and mice. However, the infected Tr. rubrofasciata did not transmit Trypanosoma sp. to their offspring. Moreover, our investigation revealed a high prevalence of Trypanosoma sp. infection in Tr. rubrofasciata, with up to 36.3% of specimens tested in the field being infected. CONCLUSIONS: Our study is the first to provide a solid record of T. conorhini from Tr. rubrofasciata in China with morphological and molecular evidence. This Chinese T. conorhini is unlikely to have spread through transovarial transmission in Tr. rubrofasciata, but instead, it is more likely that the parasite is transmitted between Tr. rubrofasciata and mice (or rats). However, there was a high prevalence of T. conorhini in the Tr. rubrofasciata from our collection sites and numerous human cases of Tr. rubrofasciata bites were recorded. Moreover, whether these T. conorhini strains are pathogenic to humans has not been investigated.

Synergistic Effect of Dietary Supplementation with Sodium Butyrate, ß-Glucan and Vitamins on Growth Performance, Cortisol Level, Intestinal Microbiome and Expression of Immune-Related Genes in Juvenile African Catfish (Clarias gariepinus).

The effect of dietary supplementation with sodium butyrate, ß-glucan and vitamins (A, D3, E, K, C) on breeding indicators and immune parameters of juvenile African catfish was examined. The fish were fed with unenriched (group C) and enriched feed with a variable proportion of sodium butyrate/ß-glucan, and constant content of vitamins (W1-W3). After the experiment, blood and the middle gut were collected. The microbiome of the gut was determined using Next Generation Sequencing (NGS). Liver tissue was collected for determination of expression of immune-related genes (HSP70, IL-1ß, TNFα). W2 and W3 were characterized by the most favorable values of breeding indicators (p < 0.05). The highest blood cortisol concentration was in group C (71.25 ± 10.45 ng/mL), and significantly the lowest in W1 (46.03 ± 7.01 ng/ mL) (p < 0.05). The dominance of Cetobacterium was observed in all study groups, with the largest share in W3 (65.25%) and W1 (61.44%). Gene expression showed an increased number of HSP70 genes in W1. IL-1ß and TNFα genes peaked at W3. The W3 variant turns out to be the most beneficial supplementation, due to the improvement of breeding and immunological parameters. The data obtained can be used to create a preparation for commercial use in the breeding of this species.

Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

BACKGROUND: Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. METHODS AND RESULTS: PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies.

The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.

Thermotolerance in S. cerevisiae as a model to study extracellular vesicle biology.

The budding yeast Saccharomyces cerevisiae is a proven model organism for elucidating conserved eukaryotic biology, but to date its extracellular vesicle (EV) biology is understudied. Here, we show yeast transmit information through the extracellular medium that increases survival when confronted with heat stress and demonstrate the EV-enriched samples mediate this thermotolerance transfer. These samples contain vesicle-like particles that are exosome-sized and disrupting exosome biogenesis by targeting endosomal sorting complexes required for transport (ESCRT) machinery inhibits thermotolerance transfer. We find that Bro1, the yeast ortholog of the human exosome biomarker ALIX, is present in EV samples, and use Bro1 tagged with green fluorescent protein (GFP) to track EV release and uptake by endocytosis. Proteomics analysis reveals that heat shock protein 70 (HSP70) family proteins are enriched in EV samples that provide thermotolerance. We confirm the presence of the HSP70 ortholog stress-seventy subunit A2 (Ssa2) in EV samples and find that mutant yeast cells lacking SSA2 produce EVs but they fail to transfer thermotolerance. We conclude that Ssa2 within exosomes shared between yeast cells contributes to thermotolerance. Through this work, we advance Saccharomyces cerevisiae as an emerging model organism for elucidating molecular details of eukaryotic EV biology and establish a role for exosomes in heat stress and proteostasis that seems to be evolutionarily conserved.

Circulating mortalin in blood and activation of the alternative complement pathway as risk indicators in COVID-19 infection.

Background: Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70. It protects cells from various types of damages and from senescence. Our goal was to determine whether COVID-19 patients have circulating mortalin in their blood and to assess its prognostic value in anticipating disease severity. Methods: Mortalin was determined by ELISA in the sera of 83 COVID-19 patients enrolled in the study. Patients were categorized into 4 groups: critical patients who died (FATAL) or required intensive care and survived (ICU), patients of mild severity (hospitalized but not critical) who required nasal oxygen support (HOSP+O2), and patients who did not need oxygen therapy (HOSP). Results: The mortalin concentration in the serum of all COVID-19 patients in the cohort was 194-2324 pg/mL. A comparison of the mortalin levels by peak severity among the various patient groups showed a highly significant difference between the HOSP and FATAL groups and a significant difference between the HOSP and the ICU groups. COVID-19 patients who eventually failed to survive had at hospitalization a markedly higher level of mortalin in their sera. Cox regression analysis revealed a high mortality hazard (HR=3.96, p<0.01) in patients with high mortalin circulating levels (above the median, ≥651 pg/mL). This was confirmed in survival curve analysis (Kaplan-Meier; p=0.0032, log-rank test). Mortalin remained an independent predictor of mortality even after adjusting for age and sex or various complement activation products. Complement activation data collected in an earlier study in the same cohort was compared regarding the mortalin levels. Patients with higher circulating mortalin levels also had higher levels of complement C3a but reduced levels of properdin. Discussion: This is the first report on circulating mortalin in COVID-19 patients. Higher mortalin levels were associated with more severe illnesses and a higher risk of death. We claim that quantifying the blood levels of mortalin and activated complement proteins will provide important information on the prognosis of COVID-19 patients and will serve as a useful tool for guiding their clinical management and treatment.

Phytomediated stress modulates antioxidant status, induces overexpression of CYP6M2, Hsp70, α-esterase, and suppresses the ABC transporter in Anopheles gambiae (sensu stricto) exposed to Ocimum tenuiflorum extracts.

The incorporation of phytoactive compounds in the management of malarial vectors holds promise for the development of innovative and efficient alternatives. Nevertheless, the molecular and physiological responses that these bioactive substances induce remain underexplored. This present study investigated the toxicity of different concentrations of aqueous and methanol extracts of Ocimum tenuiflorum against larvae of Anopheles gambiae (sensu stricto) and unraveled the possible underlying molecular pathways responsible for the observed physiological effects. FTIR and GCMS analyses of phytoactive compounds in aqueous and methanol crude extracts of O. tenuiflorum showed the presence of OH stretching vibration, C = C stretching modes of aromatics and methylene rocking vibration; ring deformation mode with high levels of trans-ß-ocimene, 3,7-dimethyl-1,3,6-octatriene in aqueous extract and 4-methoxy-benzaldehyde, 1,3,5-trimethyl-cyclohexane and o-cymene in methanol extract. The percentage mortality upon exposure to methanol and aqueous extracts of O. tenuiflorum were 21.1% and 26.1% at 24 h, 27.8% and 36.1% at 48 h and 36.1% and 45% at 72 h respectively. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), down-regulation of ABC transporter, overexpression of CYP6M2, Hsp70, and α-esterase, coupled with significantly increased levels of SOD, CAT, and GSH, were observed in An. gambiae (s.s.) exposed to aqueous and methanol extracts of O. tenuiflorum as compared to the control. Findings from this study have significant implications for our understanding of how An. gambiae (s.s.) larvae detoxify phytoactive compounds.

Challenges for heat stress: Intestinal culturable bacteria of Lohmann Brown chickens.

This study aimed to assess how heat stress, specifically within the range of 35-38 °C, affects the populations of culturable intestinal lactobacilli, enterococci, and Escherichia coli, as well as the expression of Heat Shock Proteins (HSP70), in Lohmann Brown chickens. It also explored the influence of the chickens' blood transferrin and ceruloplasmin genotypes on these responses. Thirty chickens underwent eight hours of heat stress, maintained at an average temperature of 37 °C and a relative humidity of 75-80%, with continuous access to food and water. Behavioral monitoring was conducted throughout to prevent excessive heat-related mortality. The Lohmann Brown chickens from the Yerevan "Arax" poultry farm were initially classified based on their blood transferrin and ceruloplasmin genotypes to investigate potential correlations between intestinal bacterial composition and variations in these polymorphisms. A significant correlation was found between heat stress and the abundance of culturable enterococci within the intestinal microbiota, regardless of chicken TfAB, TfBC, CpAB, CpCC and TfAB, TfBC, CpAB, CpCD genotypes. Heat stress led to nearly double the HSP70 levels in chicken blood, along with a reduction in the culturable enterococci population by at least 10,000-fold in the intestinal microbiota. These findings are significant for targeted management strategies to mitigate heat stress in chicken populations.

Tertiary structure and conformational dynamics of the anti-amyloidogenic chaperone DNAJB6b at atomistic resolution.

DNAJB6b is a molecular chaperone of the heat shock protein network, shown to play a crucial role in preventing aggregation of several disease-related intrinsically disordered proteins. Using homology modeling and microsecond-long all-atom molecular dynamics (MD) simulations, we show that monomeric DNAJB6b is a transiently interconverting protein cycling between three states: a closed state, an open state (both abundant), and a less abundant extended state. Interestingly, the reported regulatory autoinhibitory anchor between helix V in the G/F1 region and helices II/III of the J-domain, which obstructs the access of Hsp70 to the J-domain remains present in all three states. This possibly suggests a mechanistically intriguing regulation in which DNAJB6b only becomes exposed when loaded with substrates that require Hsp70 processing. Our MD results of DNAJB6b carrying mutations in the G/F1 region that are linked to limb-girdle muscular dystrophy type D1 (LGMDD1) show that this G/F1 region becomes highly dynamic, pointing towards a spontaneous release of the autoinhibitory helix V from helices II/III. This would increase the probability of non-functional Hsp70 interactions to DNAJB6b without substrates. Our cellular data indeed confirm that non-substrate loaded LGMDD1 mutants have aberrant interactions with Hsp70.

[Identification and expression analysis of the HSP70 gene family under abiotic stresses in Litchi chinensis].

HSP70 protein, as an important member of the heat shock protein (HSP) family, plays an important role in plant growth, development, and response to biotic and abiotic stresses. In order to explore the role of HSP70 gene family members in Litchi chinensis under low temperature, high temperature, drought, and salt stress, bioinformatics methods were used to identify the HSP70 gene family members within the entire L. chinensis genome. The expression of these genes under various abiotic stresses was then detected using quantitative real-time PCR (qRT-PCR). The results showed that the LcHSP70 gene family consisted of 18 members, which were unevenly distributed across ten L. chinensis chromosomes. The LcHSP70 protein contained 479-851 amino acids, with isoelectric points ranging from 5.07 to 6.95, and molecular weights from 52.44 kDa to 94.07 kDa. The predicted subcellular localization showed that LcHSP70 protein was present in the nucleus, cytoplasm, endoplasmic reticulum, mitochondria, and chloroplast. Phylogenetic analysis divided the LcHSP70 proteins into five subgroups, namely Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅵ. The promoter regions of the LcHSP70 genes contained various cis-acting elements related to plant growth, development, hormone response, and stress response. Moreover, the expression of LcHSP70 genes displayed distint tissue-specific expression level, categorized into universal expression and specific expression. From the selected 6 LcHSP70 genes (i.e., LcHSP70-1, LcHSP70-5, LcHSP70-10, LcHSP70-14, LcHSP70-16, and LcHSP70-18), their relative expression levels were assessed under different abiotic stresses using qRT-PCR. The results indicated that the gene family members exhibited diverse responses to low temperature, high temperature, drought, and salt stress, with significant variations in their expression levels across different time periods. These results provide a foundation for further exploration of the function of the LcHSP70 gene family.

The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase.

Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during, e.g., food processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.

The Ability of Clinically Relevant Chemotherapeutics to Induce Immunogenic Cell Death in Squamous Cell Carcinoma.

BACKGROUND: Immunogenic cell death (ICD) is a crucial mechanism for triggering the adaptive immune response in cancer patients. Damage-associated molecular patterns (DAMPs) are critical factors in the detection of ICD. Chemotherapeutic drugs can cause ICD and the release of DAMPs. The aim of this study was to assess the potential for paclitaxel and platinum-based chemotherapy regimens to induce ICD in squamous cell carcinoma (SCC) cell lines. In addition, we examined the immunostimulatory effects of clinically relevant chemotherapeutic regimens utilized in the treatment of SCC. METHODS: We screened for differentially expressed ICD markers in the supernatants of three SCC cell lines following treatment with various chemotherapeutic agents. The ICD markers included Adenosine Triphosphate (ATP), Calreticulin (CRT), Annexin A1 (ANXA 1), High Mobility Group Protein B1 (HMGB1), and Heat Shock Protein 70 (HSP70). A vaccination assay was also employed in C57BL/6J mice to validate our in vitro findings. Lastly, the levels of CRT and HMGB1 were evaluated in Serum samples from SCC patients. RESULTS: Addition of the chemotherapy drugs cisplatin (DDP), carboplatin (CBP), nedaplatin (NDP), oxaliplatin (OXA) and docetaxel (DOC) increased the release of ICD markers in two of the SCC cell lines. Furthermore, mice that received vaccinations with cervical cancer cells treated with DDP, CBP, NDP, OXA, or DOC remained tumor-free. Although CBP induced the release of ICD-associated molecules in vitro, it did not prevent tumor growth at the vaccination site in 40% of mice. In addition, both in vitro and in vivo results showed that paclitaxel (TAX) and LBP did not induce ICD in SCC cells. CONCLUSION: The present findings suggest that chemotherapeutic agents can induce an adjuvant effect leading to the extracellular release of DAMPs. Of the agents tested here, DDP, CBP, NDP, OXA and DOC had the ability to act as inducers of ICD.

Sublethal effects of chlorantraniliprole on immunity in Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae): Promote encapsulation by upregulating a heat shock protein 70 family gene SfHSP68.1.

As an agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, poses a severe threat to agriculture in China. Chlorantraniliprole has been widely used to control this pest. In our previous studies, we discovered that LD10, LD20, and LD30 chlorantraniliprole promoted encapsulation in the 4th instar larvae of the FAW, with LD30 chlorantraniliprole having the most significant effect. To further investigate the molecular mechanism underlying the sublethal effects of chlorantraniliprole on encapsulation in the FAW, this study conducted the effects of encapsulation in 4th instar larvae of the FAW exposed to LD30 chlorantraniliprole. Then, we analyzed the transcriptome of the FAW hemolymph treated with LD30 chlorantraniliprole and identified genes related to encapsulation using RNAi. Our results showed that the encapsulation in the FAW was enhanced at 6, 12, 18, 24, and 48 h after exposure to LD30 chlorantraniliprole. Additionally, LD30 chlorantraniliprole significantly affected the expression of certain immune-related genes, with the heat shock protein 70 family gene SfHSP68.1 showing the most significant upregulation. Subsequent interference with SfHSP68.1 resulted in a significant inhibition of encapsulation in FAW. These findings suggested that LD30 chlorantraniliprole can promote encapsulation in the FAW by upregulating SfHSP68.1 expression. This study provides valuable insights into the sublethal effects of chlorantraniliprole on encapsulation in the FAW and the interaction between encapsulation and heat shock proteins (HSPs).

OMA1 competitively binds to HSPA9 to promote mitophagy and activate the cGAS-STING pathway to mediate GBM immune escape.

BACKGROUND: Immunotherapy with checkpoint inhibitors, especially those targeting programmed death receptor 1 (PD-1)/PD-1 ligand (PD-L1), is increasingly recognized as a highly promising therapeutic modality for malignancies. Nevertheless, the efficiency of immune checkpoint blockade therapy in treating glioblastoma (GBM) is constrained. Hence, it is imperative to expand our comprehension of the molecular mechanisms behind GBM immune escape (IE). METHODS: Protein chip analysis was performed to screen aberrantly expressed OMA1 protein in PD-1 inhibitor sensitive or resistant GBM. Herein, public databases and bioinformatics analysis were employed to investigate the OMA1 and PD-L1 relation. Then, this predicted relation was verified in primary GBM cell lines through distinct experimental methods. To investigate the molecular mechanism behind OMA1 in immunosuppression, a series of experimental methods were employed, including Western blotting, co-immunoprecipitation (Co-IP), mass spectrometry (MS), immunofluorescence, immunohistochemistry, and qRT-PCR. RESULTS: Our findings revealed that OMA1 competitively binds to HSPA9 to induce mitophagy and mediates the IE of GBM. Data from TCGA indicated a significant correlation between OMA1 and immunosuppression. OMA1 promoted PD-L1 levels in primary cells from patients with GBM. Next, the results of Co-IP and MS conducted on GBM primary cells revealed that OMA1 interacts with HSPA9 and induces mitophagy. OMA1 promoted not only cGAS-STING activity by increasing mitochondrial DNA release but also PD-L1 transcription by activating cGAS-STING. Eventually, OMA1 has been found to induce immune evasion in GBM through its regulation of PD-1 binding and PD-L1 mediated T cell cytotoxicity. CONCLUSIONS: The OMA1/HSPA9/cGAS/PD-L1 axis is elucidated in our study as a newly identified immune therapeutic target in GBM.

Heat shock factor 1 inhibition enhances the effects of modulated electro hyperthermia in a triple negative breast cancer mouse model.

Female breast cancer is the most diagnosed cancer worldwide. Triple negative breast cancer (TNBC) is the most aggressive type and there is no existing endocrine or targeted therapy. Modulated electro-hyperthermia (mEHT) is a non-invasive complementary cancer therapy using an electromagnetic field generated by amplitude modulated 13.56 MHz frequency that induces tumor cell destruction. However, we have demonstrated a strong induction of the heat shock response (HSR) by mEHT, which can result in thermotolerance. We hypothesized that inhibition of the heat shock factor 1 (HSF1) can synergize with mEHT and enhance tumor cell-killing. Thus, we either knocked down the HSF1 gene with a CRISPR/Cas9 lentiviral construct or inhibited HSF1 with a specific small molecule inhibitor: KRIBB11 in vivo. Wild type or HSF1-knockdown 4T1 TNBC cells were inoculated into the mammary gland's fat pad of BALB/c mice. Four mEHT treatments were performed every second day and the tumor growth was followed by ultrasound and caliper. KRIBB11 was administrated intraperitoneally at 50 mg/kg daily for 8 days. HSF1 and Hsp70 expression were assessed. HSF1 knockdown sensitized transduced cancer cells to mEHT and reduced tumor growth. HSF1 mRNA expression was significantly reduced in the KO group when compared to the empty vector group, and consequently mEHT-induced Hsp70 mRNA upregulation diminished in the KO group. Immunohistochemistry (IHC) confirmed the inhibition of Hsp70 upregulation in mEHT HSF1-KO group. Demonstrating the translational potential of HSF1 inhibition, combined therapy of mEHT with KRIBB11 significantly reduced tumor mass compared to either monotherapy. Inhibition of Hsp70 upregulation by mEHT was also supported by qPCR and IHC. In conclusion, we suggest that mEHT-therapy combined with HSF1 inhibition can be a possible new strategy of TNBC treatment with great translational potential.

Chaperone Hsp70 helps Salmonella survive infection-relevant stress by reducing protein synthesis.

In all domains of life, Hsp70 chaperones preserve protein homeostasis by promoting protein folding and degradation and preventing protein aggregation. We now report that the Hsp70 from the bacterial pathogen Salmonella enterica serovar Typhimurium-termed DnaK-independently reduces protein synthesis in vitro and in S. Typhimurium facing cytoplasmic Mg2+ starvation, a condition encountered during infection. This reduction reflects a 3-fold increase in ribosome association with DnaK and a 30-fold decrease in ribosome association with trigger factor, the chaperone normally associated with translating ribosomes. Surprisingly, this reduction does not involve J-domain cochaperones, unlike previously known functions of DnaK. Removing the 74 C-terminal amino acids of the 638-residue long DnaK impeded DnaK association with ribosomes and reduction of protein synthesis, rendering S. Typhimurium defective in protein homeostasis during cytoplasmic Mg2+ starvation. DnaK-dependent reduction in protein synthesis is critical for survival against Mg2+ starvation because inhibiting protein synthesis in a dnaK-independent manner overcame the 10,000-fold loss in viability resulting from DnaK truncation. Our results indicate that DnaK protects bacteria from infection-relevant stresses by coordinating protein synthesis with protein folding capacity.

Mrj is a chaperone of the Hsp40 family that regulates Orb2 oligomerization and long-term memory in Drosophila.

Orb2 the Drosophila homolog of cytoplasmic polyadenylation element binding (CPEB) protein forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation is dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2A's prion-like oligomerization forms long-term memory but fails to maintain it over time. Since this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here, we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Among these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, Mrj knockout exhibits a deficit in long-term memory and our observations suggest Mrj is needed in mushroom body neurons for the regulation of long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating ribosomes.

Sensitive detection of HSP70 using a current-amplified biosensor based on antibody-loaded PS-AuNPs@Cys/Au modified ITO chip.

A biosensing electrochemical platform for heat shock protein 70 (HSP70) has been developed by integrating a three-electrode indium tin oxide (ITO) on a chip. The platform includes modifications to the reference electrode and working electrode for the detection of HSP70. The new platform is constructed by assembly of HSP70 antibody on PS-AuNPs@Cys/Au indium tin oxide (ITO) electrode to create a high HSP70 sensitive surface. The PS-AuNPs@Cys/Au indium tin oxide (ITO) electrode is obtained by immersing the ITO electrode into the PS-AuNPs@Cys solution and performing constant potential deposition at -1.4 V (Ag/AgCl). The PS-AuNPs@Cys/Au film deposited on ITO glass provides a desirable substrate for the immobilization of the HSP70 antibody and improves the loading of antibody between PS-AuNPs@Cys/Au and the electrode resulting in a significant amplification. Under optimal conditions, the fabricated sensor demonstrates a linear range extending from 0.1 ng mL- 1 to 1000 ng mL- 1, with an impressive detection limit of 25.7 pg mL- 1 (S/N = 3). The developed immunoassay method successfully detected the HSP70 content in normal human blood samples and outperformed the ELISA method commonly used for clinical sample analysis.

Biochemical and molecular-level effects of co-exposure to chlorpyrifos and lambda-cyhalothrin on the earthworm (Eisenia fetida).

Farmland soil organisms frequently encounter pesticide mixtures presented in their living environment. However, the underlying toxic mechanisms employed by soil animals to cope with such combined pollution have yet to be explored. This investigation aimed to reveal the changes in cellular and mRNA levels under chlorpyrifos (CPF) and lambda-cyhalothrin (LCT) co-exposures in earthworms (Eisenia fetida). Results exhibited that the combination of CPF and LCT triggered an acute synergistic influence on the animals. Most exposures resulted in significant alterations in the activities of total superoxide dismutase (T-SOD), copper/zinc superoxide dismutase (Cu/Zn-SOD), caspase 3, and carboxylesterase (CarE) compared to the basal level. Moreover, when exposed to chemical mixtures, the transcription levels of four genes [heat shock protein 70 (hsp70), gst, sod, and calreticulin (crt)] also displayed more pronounced changes compared with their individual exposures. These changes in determined parameters indicated the occurrence of oxidative stress, cell death, detoxification dysfunction, and endoplasmic reticulum damage after co-exposure to CPF and LCT in E. fetida. The comprehensive examination of mixture toxicities of CPF and LCT at different endpoints would help to understand the overall toxicity they cause to soil invertebrates. The augmented deleterious effect of these pesticides in a mixture suggested that mixture toxicity assessment was necessary for the safety evaluation and application of pesticide mixtures.