kDa Heat Shock Protein Research Articles

Effect of heat shock on embryonic development and its impact on commercial traits of silkworm Bombyx mori. L

The early stage of an organism – embryonic stage, architects all the post embryonic developments which are governed by genetic and environmental conditions, but the effect of hot events during that stage remain obscure in the silkworm Bombyx mori L. Thus, APM1; a multivoltine parental breed of a ruling CB and APHO1 silkworm breed developed through induction of thermotolerance, and APHO1 breed to examine the impact of heat shock (HS) on the embryo and resulta nt larvae. Different developmental stages of embryo were exposed to varied HS temperatures for 2 h followed by a 2 h recovery period. After HS the eggs and the resultant larvae were reared under normal environmental conditions. Interestingly, 450C although determined as lethal temperature yielded vibrant larvae. Whereas APM1 and APHO1 eggs heat shocked at 350C exhibited increased hatching (91.66 and 69.33%), larval weight (1.72 and 3.33 g), effective rate of rearing (72.39 and 81.93%), cocoon weight (1.01 and1.6 g), shell weight (0.12 and 0.29 g), shell ratio (13.11 and 20.52%) and pupal weights (0.87 and1.29g) when compared to control APM1 and APHO1. Besides increased total protein content, expression of 205 kDa, 90 kDa and 70 kDa heat shock proteins and the glycogen content was found more on day - 3 compared to day - 2 in the embryos of APM1 and APHO1 which eventually declined as the embryonic development proceeded to hatching. This work shows that APM1 and APHO1 eggs had shown profound response to HS temperatures exhibiting varied acquired thermotolerance to overcome fluctuating environmental condition.

Tryptophan alleviates lipopolysaccharide-induced liver injury and inflammation by modulating necroptosis and pyroptosis signaling pathways in piglets

The liver plays crucial roles in material metabolism and immune response. Bacterial endotoxin can cause various liver diseases, thereby causing significant economic losses to pig industry. Tryptophan is an essential amino acid in piglets. However, whether tryptophan can alleviate liver injury and inflammation by regulating necroptosis and pyroptosis has not been clarified. This study aimed to investigate whether dietary tryptophan can alleviate lipopolysaccharide (LPS)-induced liver injury in weaned piglets. 18 weaned piglets were randomly distributed to three treatments, each with 6 replicates: (1) control; (2) LPS-challenged control; (3) LPS + 0.2% tryptophan. After feeding with control or 0.2% tryptophan-supplemented diets for 35 d, pigs were intraperitoneally injected with saline or LPS (100 mg/kg body weight). At 4 h post-injection, blood samples and liver were collected. Results indicated that tryptophan reduced alanine aminotransferase, aspartate aminotransferase, decreased the mRNA expression and protein expression of 70-kDa heat shock proteins. Moreover, tryptophan increased the mRNA expression and protein expression of claudin-1, occludin and zonula occludens and decreased hydrogen peroxide and malondialdehyde contents, and increased catalase, glutathione peroxidase and total superoxide dismutase activities and proinflammatory cytokine levels in the liver. Meanwhile, tryptophan inhibited pyroptosis-related and necroptosis-related protein expression in liver. Collectively, tryptophan could relieve liver damage, increased the antioxidant capacity and reduced inflammation by inhibiting pyroptosis and necroptosis signaling pathways.

P02.19.A ASSESSMENT OF HEAT SHOCK PROTEIN HSP70 EXPRESSION IN THE GLIOBLASTOMA STEM-LIKE CELLS

Abstract BACKGROUND Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by poor prognosis. The search of biomarkers for early diagnosis and as targets and for tumor theranostics due to the aggressiveness of the disease is urgently needed. One of the promising targets for therapy of brain tumors is 70 kDa heat shock protein (Hsp70), which has been found on the plasma membrane of various tumor cells (including GBM) but not on normal cells. GBM, like other types of tumors, has cellular heterogeneity. In particular, it contains tumor stem-like cells (TSLCs), which are considered analogues of healthy stem cells and are largely responsible for disease progression, recurrence and mediate resistance to ongoing therapy. In the present work, the histological sections of human GBM were analyzed to identify and quantify the colocalization of the heat shock protein Hsp70 with TSLC markers using the Opal Multiplex analysis system. MATERIAL AND METHODS Histological sections were prepared in accordance with the standard protocol using postoperative material from patients with newly diagnosed GBM (n=15). Zones of necrosis and living tissue were identified on tumor sections. Based on a phenotyping map using the Pathology Views™ fluorescence image analysis software, Hsp70 colocalization was quantified with Nestin and Sox2 in the areas of necrosis and viable tissue employing primary antibodies (anti-Hsp70, anti-Nestin and anti-Sox2). The preparations were stained using an Opal 3-Plex Manual Detection Kit according to the manufacturer's protocol. RESULTS The expression of Hsp70 and TSLC markers (Nestin and Sox2) was detected. The obtained quantitative data did not reveal significant differences in the distribution of cells expressing Hsp70 in the areas of necrosis and viable tissue (36 and 41%, respectively), which may indicate their uniform distribution in the tumor. Sufficiently high colocalization of Hsp70 with Nestin and Sox2 factors was detected mainly in the zone of viable tissue (57 and 30%, respectively). CONCLUSION Colocalization of Hsp70 with TSLC markers indicates the prospects for further studies of Hsp70, as well as its use for targeted therapy of malignant brain tumors.

Inhibition of HSP90 in Driver Oncogene-Defined Lung Adenocarcinoma Cell Lines: Key Proteins Underpinning Therapeutic Efficacy.

The use of 90 kDa heat shock protein (HSP90) inhibition as a therapy in lung adenocarcinoma remains limited due to moderate drug efficacy, the emergence of drug resistance, and early tumor recurrence. The main objective of this research is to maximize treatment efficacy in lung adenocarcinoma by identifying key proteins underlying HSP90 inhibition according to molecular background, and to search for potential biomarkers of response to this therapeutic strategy. Inhibition of the HSP90 chaperone was evaluated in different lung adenocarcinoma cell lines representing the most relevant molecular alterations (EGFR mutations, KRAS mutations, or EML4-ALK translocation) and wild-type genes found in each tumor subtype. The proteomic technique iTRAQ was used to identify proteomic profiles and determine which biological pathways are involved in the response to HSP90 inhibition in lung adenocarcinoma. We corroborated the greater efficacy of HSP90 inhibition in EGFR mutated or EML4-ALK translocated cell lines. We identified proteins specifically and significantly deregulated after HSP90 inhibition for each molecular alteration. Two proteins, ADI1 and RRP1, showed independently deregulated molecular patterns. Functional annotation of the altered proteins suggested that apoptosis was the only pathway affected by HSP90 inhibition across all molecular subgroups. The expression of ADI1 and RRP1 could be used to monitor the correct inhibition of HSP90 in lung adenocarcinoma. In addition, proteins such as ASS1, ITCH, or UBE2L3 involved in pathways related to the inhibition of a particular molecular background could be used as potential response biomarkers, thereby improving the efficacy of this therapeutic approach to combat lung adenocarcinoma.

The transportosome system as a model for the retrotransport of soluble proteins

The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.

Genomic loss of the HSP70cA gene in the vertebrate lineage.

Metazoan 70 kDa heat shock protein (HSP70) genes have been classified into four lineages: cytosolic A (HSP70cA), cytosolic B (HSP70cB), endoplasmic reticulum (HSP70er), and mitochondria (HSP70m). Because previous studies have identified no HSP70cA genes in vertebrates, we hypothesized that this gene was lost on the evolutionary path to vertebrates. To test this hypothesis, the present study conducted a comprehensive database search followed by phylogenetic and synteny analyses. HSP70cA genes were found in invertebrates and in two of the three subphyla of Chordata, Cephalochordata (lancelets) and Tunicata (tunicates). However, no HSP70cA gene was found in the genomes of Craniata (another subphylum of Chordata; lamprey, hagfish, elephant shark, and coelacanth), suggesting the loss of the HSP70cA gene in the early period of vertebrate evolution. Synteny analysis using available genomic resources indicated that the synteny around the HSP70 genes was generally conserved between tunicates but was largely different between tunicates and lamprey. These results suggest the presence of dynamic chromosomal rearrangement in early vertebrates that possibly caused the loss of the HSP70cA gene in the vertebrate lineage.

Analysis and functional relevance of the chaperone TRAP-1 interactome in the metabolic regulation and mitochondrial integrity of cancer cells

The 90 kDa heat shock protein, Hsp90, functions as a cancer chaperone contributing to tumor proliferation. We have encountered the mitochondrial homolog of Hsp90, the TRAP-1, regulating mitochondrial dynamics, metabolism, and tumor metastasis. Although Hsp90 is associated with a broad network of proteins regulating various cellular processes, TRAP-1-mediated cellular networks are unclear. Therefore, using TRAP-1 knockdown (KD) and overexpression (OE) systems, we compared their quantitative transcriptome (RNA Sequencing) and proteomic (LC–MS/MS) patterns to obtain molecular signatures that are altered in response to TRAP-1 KD or OE. We report TRAP-1 modulating vital metabolic pathways such as the tricarboxylic acid cycle, oxidative phosphorylation, electron transport chain, glycolysis, and gluconeogenesis. In addition, TRAP-1 facilitated the pentose phosphate pathway to shunt carbons back to glycolysis or gluconeogenesis, a much-solicited tumor response. Subsequently, we examined the TRAP-1 interactome using the tandem affinity purification system and identified 255 unique proteins. These diverse proteins appear to regulate several cellular processes, including energy metabolism, suggesting that TRAP-1, in addition to metabolic rewiring, maintains mitochondrial integrity. Our study exposes the unknown functions of TRAP-1 in cancer cells. Systematic evaluation of TRAP-1 interactors may uncover novel regulatory mechanisms in disease aggression. Since metabolic inhibitors are emerging as potential anticancer agents, our study gains importance.

Cryptosporidium equi n. sp. (Apicomplexa: Cryptosporidiidae): Biological and genetic characterisations

The horse genotype is one of three common Cryptosporidium spp. in equine animals and has been identified in some human cases. The species status of Cryptosporidium horse genotype remains unclear due to the lack of extensive morphological, biological, and genetic data. In the present study, we have conducted biological and whole genome sequence analyses of an isolate of the genotype from hedgehogs and proposed to name it Cryptosporidium equi n. sp. to reflect its common occurrence in equine animals. Oocysts of C. equi measured 5.12 ± 0.36 μm × 4.46 ± 0.21 μm with a shape index of 1.15 ± 0.08 (n = 50). Cryptosporidium equi was infectious to 3-week-old four-toed hedgehogs (Atelerix albiventris) and mice, with a prepatent period of 2–9 days and a patent period of 30–40 days in hedgehogs. It was not infectious to rats and rabbits. Phylogenetic analyses of small subunit rRNA, 70 kDa heat shock protein, actin, 60 kDa glycoprotein and 100 other orthologous genes revealed that C. equi is genetically distinct from other known Cryptosporidium species and genotypes. The sequence identity between C. equi and Cryptosporidium parvum genomes is 97.9%. Compared with C. parvum, C. equi has lost two MEDLE genes and one insulinase-like protease gene and gained one SKSR gene. In addition, 60 genes have highly divergent sequences (sequence differences ≥ 5.0%), including those encoding mucin-like glycoproteins, insulinase-like peptidases, and MEDLE and SKSR proteins. The genetic uniqueness of C. equi supports its increasing host range and the naming of it as a valid Cryptosporidium species. This is the first known use of whole genome sequence data in delineating new Cryptosporidium species.

Proteomics-based milk whey proteome profiling of Indian Jersey crossbreed cows followed by chromosomal mapping.

Milk contains a massive class of minor proteins that are known for their various biological and molecular functions. Many whey proteins transfer the host defense mechanism to the human body. In this assay, electrophoresis followed by a high-resolution mass spectrometry-based proteomic approach has been applied to identify the whey proteome of Indian Jersey crossbreed bovines. Two search engines, MS Amanda and Sequest HT, have shown more than 29 minor proteins. Chromosomal mapping revealed that chromosomes 5 and 9 are expressing maximum proteins in the whey proteome. The principal component analysis, outlier plots, scree plots, score plots, and loading plots were generated to further assess the results. The majorly expressed ones are glycosylation-dependent cell adhesion molecule-1, ubiquitin, desmoglein, annexin, glycoprotein, arginase, histones, peroxiredoxin, vimentin, desmin, catenin, peripherin, and 70 kDa heat shock protein. © 2023 Society of Chemical Industry.

Protein Antioxidant Complex of Water Extract of Larvae of Black Beetles <i>Ulomoides dermestoides</i>

An aqueous extract from the larvae of black beetles Ulomoides dermestoides grown under controlled conditions was obtained and its proteomic analysis based on one-dimensional electrophoretic separation of proteins, their trypsinolysis in a gel and subsequent chromatography-mass spectrometry was performed. Protein identification was performed using MaxQuant v.1.6.3.4 software. The Uniprot database was used to identify proteins by homology. Catalase activity was determined spectrophotometrically by hydrogen peroxide concentration decrease. Antioxidant activity was evaluated by luminol chemiluminescence quenching. The extract contained proteins that can determine the biological activity of the extract: universal regulators of cellular processes calmodulin (62%), cytochrome c-2 (13.5%), nucleoside diphosphate kinase (11.1%), the enzymatic antioxidants superoxide dismutase, catalase, glutathione S-transferase, peroxiredoxin, glutathione synthetase as well as thioredoxin, 70 and 60 kDa heat shock proteins, chitinase complex (13.4% in total). Catalase activity was 6.3 ± 1.1 µmol H2O2/min/mg protein; antioxidant activity of 1 mg protein per ml extract was equivalent to 1.36 ± 0.3 mM trolox. The prospects for practical use of the extract as a natural antioxidant complex were noted.

Anchovy’s protein as a potential precursor of Angiotensin-I Converting Enzyme (ACE) inhibitory peptide and Dipeptidyl Peptidase-IV (DPP-IV) inhibitory peptide by an in silico approach

Protein from fish is known as the precursor of biologically active peptides that exert various health benefits such as antihypertensive, antitumor, and immunomodulatory properties. Hence, this study aimed to perform an extraction of anchovy, LC-MS/MS analysis and in silico evaluation of the major proteins in anchovies as potential precursors of biologically active peptides in addition to determining whether such peptides can be released by selected proteolytic enzymes. Anchovy was subjected to protein extraction followed by total soluble protein concentration determination using Bradford assay. The sample was subjected to in-solution trypsin digestion, which was then analysed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). A bioinformatic approach by PEAKS Studio was used to identify the protein. A total of four anchovy’s proteins which are myosin light chain 1, V(D)J recombination-activating protein, 60 kDa chaperonin and heat shock protein 90AA1 were identified. Then, the identified proteins were subjected to in silico approach using the BIOPEP database. The biological potential of the theoretically released angiotensin-I converting enzyme (ACE) inhibitory peptides and dipeptidyl peptidase (DPP)-IV inhibitory peptides were predicted by determining the frequency of occurrence of fragments with a given activity. 60 kDa chaperonin and heat shock protein 90AA1 predicted the highest number of biological activities for ACE inhibitory peptides (284 and 264 fragments) and DPP-IV inhibitory peptides (395 and 409 fragments). The most promising enzyme for the generation of bioactive peptides from anchovy protein was anticipated to be pepsin (pH > 2), which theoretically released a high number of DPP-IV inhibitory peptides and ACE inhibitory peptides through the action of in silico proteolysis. Overall, this work highlighted that anchovy protein could be a promising precursor of bioactive peptides that have ACE and DPP-IV inhibitory activities for developing functional food or nutraceutical products.

First‐trimester plasma extracellular heat shock proteins levels and risk of preeclampsia

Preeclampsia (PE) occurs annually in 8% of pregnancies. Patients without risk factors represent 10% of these. There are currently no first-trimester biochemical markers that accurately predict PE. An increase in serum 60- and 70-KDa extracellular heat shock proteins (eHsp) has been shown in patients who developed PE at 34 weeks. We sought to determine whether there is a relationship between first-trimester eHsp and the development of PE. This was a prospective cohort study performed at a third level hospital in Mexico City from 2019 to 2020. eHsp levels were measured during the first-trimester ultrasound in singleton pregnancies with no comorbidities. First-trimester eHsp levels and biochemical parameters of organ dysfunction were compared between patients who developed preeclampsia and those who did not. All statistical analyses and model of correlation (r) between eHsp and clinical parameter were performed using bootstrapping R-software. p-values <0.05 were considered significant. The final analysis included 41 patients. PE occurred in 11 cases. eHsp-60 and eHsp-70 were significantly higher at 12 weeks in patients who developed PE (p= 0.001), while eHsp-27 was significantly lower (p= 0.004). Significant differences in first-trimester eHsp concentration suggest that these are possible early biomarkers useful for the prediction of PE.

HSP70 Family in Cancer: Signaling Mechanisms and Therapeutic Advances.

The 70 kDa heat shock proteins (HSP70s) are a group of highly conserved and inducible heat shock proteins. One of the main functions of HSP70s is to act as molecular chaperones that are involved in a large variety of cellular protein folding and remodeling processes. HSP70s are found to be over-expressed and may serve as prognostic markers in many types of cancers. HSP70s are also involved in most of the molecular processes of cancer hallmarks as well as the growth and survival of cancer cells. In fact, many effects of HSP70s on cancer cells are not only related to their chaperone activities but rather to their roles in regulating cancer cell signaling. Therefore, a number of drugs directly or indirectly targeting HSP70s, and their co-chaperones have been developed aiming to treat cancer. In this review, we summarized HSP70-related cancer signaling pathways and corresponding key proteins regulated by the family of HSP70s. In addition, we also summarized various treatment approaches and progress of anti-tumor therapy based on targeting HSP70 family proteins.

Heat-related biomarkers: Focus on the correlation of troponin I and 70 kDa heat shock protein

Heat-related biomarkers: Focus on the correlation of troponin I and 70 kDa heat shock protein Abstract Introduction: There is intensive research related to the forensic importance of biomarkers that would be the standard for postmortem damage to cardiomyocytes and the mechanism of the resulting damage. The aim of the research was to examinate the forensic-medical significance of serum levels of biomarkers as detectors of terminal hyperthermic damage to the myocardium. Material and Method: 40 laboratory animals were divided into groups: the first group was the control (n = 8) exposed to a physiological temperature of 37 °C, the second group was divided into two subgroups: antemortem (n = 8) and postmortem (n = 8), which included a exposure temperature of 41 °C and the third group was divided into two subgroups: antemortem (n = 8) and postmortem (n = 8), which included a exposure temperature of 44 °C. The concentration of cardiac TnI and Hsp70 was resoluted in serum by immunochemical enzyme-labeled immunoabsorption method. Results: A positive correlation was found between the temperature measured at the time of death and the serum values of cTnI (p = 0.02), in G41, and Hsp70 values did not significantly correlate with the core temperature in this group, p > 0.005. A positive correlation was significant between the concentration of Hsp 70 and the body temperature of rats in the group of rats with a fatal outcome was determined, p = 0.03. Conclusion: Changes in the concentration of cTnI and Hsp70 in rat serum may indicate hyperthermic damage to the myocardium in the Wistar rat model of heat stroke.

Heat shock protein A2 is a novel extracellular vesicle-associated protein

70-kDa Heat Shock Proteins (HSPA/HSP70) are chaperones playing a central role in the proteostasis control mechanisms. Their basal expression can be highly elevated as an adaptive response to environmental and pathophysiological stress conditions. HSPA2, one of poorly characterised chaperones of the HSPA/HSP70 family, has recently emerged as epithelial cells differentiation-related factor. It is also commonly expressed in cancer cells, where its functional significance remains unclear. Previously, we have found that proteotoxic stress provokes a decrease in HSPA2 levels in cancer cells. In the present study we found that proteasome inhibition-related loss of HSPA2 from cancer cells neither is related to a block in the gene transcription nor does it relate to increased autophagy-mediated disposals of the protein. Proteotoxic stress stimulated extracellular release of HSPA2 in extracellular vesicles (EVs). Interestingly, EVs containing HSPA2 are also released by non-stressed cancer and normal cells. In human urinary EVs levels of HSPA2 were correlated with the levels of TSG101, one of the main EVs markers. We conclude that HSPA2 may constitute basic components of EVs. Nevertheless, its specific role in EVs and cell-to-cell communication requires further investigation.

Effects of carnosine on the embryonic development and TiO2 nanoparticles-induced oxidative stress on Zebrafish.

Oxidative stress is due to an unbalance between pro-oxidants, such as reactive oxygen (ROS) and nitrogen (RNS) species, and antioxidants/antioxidant system. Under physiological conditions these species are involved in different cellular processes such as cellular homeostasis and immune response, while an excessive production of ROS/RNS has been linked to the development of various diseases such as cancer, diabetes, and Alzheimer's disease. In this context, the naturally occurring dipeptide carnosine has shown the ability to scavenge ROS, counteract lipid peroxidation, and inhibit proteins oxidation. Titanium dioxide nanoparticles (TiO2-NPs) have been widely used to produce cosmetics, in wastewater treatment, in food industry, and in healthcare product. As consequence, these NPs are often released into aquatic environments. The Danio rerio (commonly called zebrafish) embryos exposure to TiO2-NPs did not affect the hatching rate, but induced oxidative stress. According to this scenario, in the present study, we first investigated the effects of carnosine exposure and of a sub-toxic administration of TiO2-NPs on the development and survival of zebrafish embryos/larvae measured through the acute embryo toxicity test (FET-Test). Zebrafish larvae represent a useful model to study oxidative stress-linked disorders and to test antioxidant molecules, while carnosine was selected based on its well-known multimodal mechanism of action that includes a strong antioxidant activity. Once the basal effects of carnosine were assessed, we then evaluated its effects on TiO2-NPs-induced oxidative stress in zebrafish larvae, measured in terms of total ROS production (measured with 2,7-dichlorodihydrofluorescein diacetate probe) and protein expression by immunohistochemistry of two cellular stress markers, 70 kDa-heat shock protein (Hsp70) and metallothioneins (MTs). We demonstrated that carnosine did not alter the phenotypes of both embryos and larvae of zebrafish at different hours post fertilization. Carnosine was instead able to significantly decrease the enhancement of ROS levels in zebrafish larvae exposed to TiO2-NPs and its antioxidant effect was paralleled by the rescue of the protein expression levels of Hsp70 and MTs. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of pathologies characterized by oxidative stress such as neurodegenerative disorders.

BARX1 promotes osteosarcoma cell proliferation and invasion by regulating HSPA6 expression

Osteosarcoma (OS) is a bone tumour affecting adolescents. Dysregulation of Barx homeobox 1 (BARX1) expression is involved in various cancers, but its function and mechanism in the process of OS are undefined. This study revealed that BARX1 expression is higher in OS tissue than in adjacent normal tissue. Downregulation of BARX1 in OS cells significantly suppressed their proliferation and migration, whereas enforced expression of exogenous BARX1 exerted the opposite effects on OS cells. Subsequently, heat shock 70-kDa protein 6 (HSPA6) expression was clearly increased after BARX1 overexpression in OS cells, as confirmed by RNA sequencing. The dual-luciferase reporter assay confirmed that HSPA6 expression is directly regulated by BARX1. The in vitro assay indicated that silencing HSPA6 expression attenuated OS proliferation and migration induced by BARX1. A dual immunofluorescence labelling assay provided further evidence that BARX1 was overexpressed and associated with HSPA6 overexpression in OS tumour tissue. In conclusion, BARX1 promotes OS cell proliferation and migration by inducing the expression of HSPA6, which plays an oncogenic role in OS. BARX1 and HSPA6 can potentially act as novel therapeutic targets for OS.

Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures.

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

Causes and consequences of endogenous hypoxia on growth and metabolism of developing maize kernels.

Maize (Zea mays) kernels are the largest cereal grains, and their endosperm is severely oxygen deficient during grain fill. The causes, dynamics, and mechanisms of acclimation to hypoxia are minimally understood. Here, we demonstrate that hypoxia develops in the small, growing endosperm, but not the nucellus, and becomes the standard state, regardless of diverse structural and genetic perturbations in modern maize (B73, popcorn, sweet corn), mutants (sweet4c, glossy6, waxy), and non-domesticated wild relatives (teosintes and Tripsacum species). We also uncovered an interconnected void space at the chalazal pericarp, providing superior oxygen supply to the placental tissues and basal endosperm transfer layer. Modeling indicated a very high diffusion resistance inside the endosperm, which, together with internal oxygen consumption, could generate steep oxygen gradients at the endosperm surface. Manipulation of oxygen supply induced reciprocal shifts in gene expression implicated in controlling mitochondrial functions (23.6 kDa Heat-Shock Protein, Voltage-Dependent Anion Channel 2) and multiple signaling pathways (core hypoxia genes, cyclic nucleotide metabolism, ethylene synthesis). Metabolite profiling revealed oxygen-dependent shifts in mitochondrial pathways, ascorbate metabolism, starch synthesis, and auxin degradation. Long-term elevated oxygen supply enhanced the rate of kernel development. Altogether, evidence here supports a mechanistic framework for the establishment of and acclimation to hypoxia in the maize endosperm.

Proteostasis in ice: the role of heat shock proteins and ubiquitin in the freeze tolerance of the intertidal mussel, Mytilus trossulus.

The bay mussel, Mytilus trossulus, is an animal that can survive extracellular ice formation. Depending on air and ocean temperatures, freeze tolerant intertidal organisms, like M. trossulus, may freeze and thaw many times during the winter. Freezing can cause protein denaturation, leading to an induction of the heat shock response with expression of chaperone proteins like the 70kDa heat shock protein (HSP70), and an increase in ubiquitin-conjugated proteins. There has been little work on the mechanisms of freeze tolerance in intertidal species, limiting our understanding of this survival strategy. Additionally, this limited research has focused solely on the effects of single freezing events, but the act of repeatedly crossing the freezing threshold may present novel physiological or biochemical stressors that have yet to be discovered. Mytilus are important ecosystem engineers and provide habitat for other intertidal species, thus understanding their physiology under thermal extremes is important for preserving shoreline health. We predicted that repeated freeze exposures would increase mortality, upregulate HSP70 expression, and increase ubiquitin conjugates in mussels, relative to single, prolonged freeze exposures. Mytilus trossulus from Vancouver, Canada were repeatedly frozen for a combination of 1 × 8h, 2 × 4h, or 4 × 2h. We then compared mortality, HSP70 expression, and the quantity of ubiquitin-conjugated proteins across experimental groups. We found a single 8-h freeze caused significantly more mortality than repeated freeze-thaw cycles. We also found that HSP70 and ubiquitinated protein was upregulated exclusively after freeze-thaw cycles, suggesting that freeze-thaw cycles offer a period of damage repair between freezes. This indicates that freeze-thaw cycles, which happen naturally in the intertidal, are crucial for M. trossulus survival in sub-zero temperatures.