Identification of heat shock protein hsp70 family genes from Rana amurensis and its expression profiles upon infection / 生物工程学报

Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.

STIP1 correlates with tumor immune infiltration and prognosis as a potential immunotherapy target: a pan-cancer bioinformatics analysis / 南方医科大学学报

OBJECTIVE@#To investigate the correlation of stress-inducible phosphoprotein 1 (STIP1) expression level with prognosis of different cancers and its potential role in immunotherapy.@*METHODS@#TCGA, TARGET and GTEx databases were used for bioinformatic analysis of STIP1 expression level and its prognostic value in different cancers. We also detected STIP1 expression immunohistochemically in 10 pairs of colorectal cancer and adjacent tissues. We further analyzed the correlation of STIP1 expression level with tumor mutational burden, microsatellite instability, immune cell infiltration, immune regulators and outcomes of different cancers. STIP1- related proteins were identified using protein- protein interaction (PPI) network analysis, and functional enrichment analysis was performed to analyze the regulatory pathways involving STIP1.@*RESULTS@#Bioinformatics analysis showed that STIP1 was highly expressed in most tumors compared with the normal tissues (P < 0.05), which was confirmed by immunohistochemistry of the 10 pairs of colorectal cancer tissues. STIP1 expression level was correlated with clinical stages of multiple cancers (P < 0.05), and in some cancer types, an upregulated STIP1 expression was correlated with a poor prognosis of the patients in terms of overall survival, disease-specific survival, disease-free survival and progression-free survival (P < 0.05). STIP1 expression was significantly correlated with tumor mutational burden, microsatellite instability, immune cell infiltration and immunomodulatory factors in most tumors (P < 0.05). PPI network analysis indicated that STIP1-related proteins included HSPA4, HSPA8, and HSP90AA1. KEGG enrichment analysis suggested that the high expression of STIP1 in liver cancer was related mainly with valerate metabolism, tryptophan metabolism, and butyrate metabolism pathways; HALLMARK enrichment analysis suggested high STIP1 expression in liver cancer was involved in bile acid and fatty acid metabolism.@*CONCLUSION@#STIP1 is up-regulated in multiple cancer types and its expression level is correlated with clinical tumor stage, tumor mutational burden, microsatellite instability, immune cell infiltration and immunomodulatory factors.

Genetic analysis of a child with Charlevoix-Saguenay spastic ataxia due to variant of SACS gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical feature and genetic variant of a child with autosomal recessive Charlevoix-Saguenay type spastic ataxia (ARSACS).@*METHODS@#Clinical data of a child who was admitted to the West China Second Hospital of Sichuan University on April 30, 2021 was collected. Whole exome sequencing (WES) was carried out for the child and his parents. Candidate variants were verified by Sanger sequencing and bioinformatic analysis based on the guidelines from the American College of Medical Genetics and Genomics (ACMG).@*RESULTS@#The child, a 3-year-and-3-month-old female, had a complain of "walking instability for over a year". Physical and laboratory examination revealed progressive and aggravated gait instability, increased muscle tone of the right limbs, peripheral neuropathy of the lower limbs, and thickening of retinal nerve fiber layer. The results of WES revealed that she has harbored a maternally derived heterozygous deletion of exons 1 to 10 of the SACS gene, in addition with a de novo heterozygous c.3328dupA variant in exon 10 of the SACS gene. Based on the ACMG guidelines, the exons 1-10 deletion was rated as likely pathogenic (PVS1+PM2_Supporting), and the c.3328dupA was rated as a pathogenic variant (PVS1_Strong+PS2+PM2_Supporting). Neither variant was recorded in the human population databases.@*CONCLUSION@#The c.3328dupA variant and the deletion of exons 1-10 of the SACS gene probably underlay the ARSACS in this patient.

What is the role of heat shock protein in abdominal organ transplantation?

ABSTRACT Ischemia-reperfusion injury is a pathophysiological event occuring after abdominal organ transplantation, and has a significant influence on prognosis and survival of the graft. It is involved in delaying the primary function or non-functioning of the graft. The objective of this study was to provide information on heat shock protein mechanisms in ischemia-reperfusion injuries in abdominal organ transplantations, and to indicate the possible factors involved that may influence the graft outcome. Several classes of heat shock proteins are part of the ischemia and reperfusion process, both as inflammatory agonists and in protecting the process. Studies involving heat shock proteins enhance knowledge on ischemia-reperfusion injury mitigation processes and the mechanisms involved in the survival of abdominal grafts, and open space to support therapeutic future clinical studies, minimizing ischemia and reperfusion injuries in abdominal organ transplantations. Expression of heat shock proteins is associated with inflammatory manifestations and ischemia-reperfusion injuries in abdominal organ transplantations and may influence graft outcomes.

Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates / Papel de las proteínas de choque térmico en la infección de la línea celular de adenocarcinoma humano MCF-7 por aislados de rotavirus adaptados a tumores

Abstract Background: Viruses are being used as alternative and complementary tools for treating cancers. Oncolytic viruses exhibit tumor tropism, ability to enhance anti-tumor immunity and ability to be used in combination with conventional chemotherapy and radiotherapy. We have recently selected some rotavirus isolates which are adapted to efficiently infect and kill tumor cell lines. Aim: We tested five tumor cell-adapted rotavirus isolates for their ability to infect the human adenocarcinoma cell line MCF-7. Methods: Cell surface membrane-associated proteins mediating virus particle attachment were characterized using ELISA, immunoprecipitation, FACS analysis, and antibody blocking. Results: It was found that heat shock proteins (HSPs) such as Hsp90, Hsp70, Hsp60, and Hsp40 are expressed on the cell surface forming complexes with protein disulfide isomerase (PDI), integrin β3, and heat shock cognate protein 70 (Hsc70) in lipid raft microdomains. Interaction of rotavirus isolates with these cellular proteins was further confirmed by a competition assay and an inhibition assay involving the HSPs tested. Conclusion: Our findings suggest that the tumor cell-adapted rotavirus isolates studied here offer a promising tool for killing tumor cells, thus encouraging further research into this topic, including animal models.

Resumen Antecedentes: Los virus se utilizan como herramientas alternativas y complementarias para el tratamiento del cáncer. Los virus oncolíticos exhiben tropismo por tumores, capacidad para intensificar la inmunidad antitumoral y la capacidad para utilizarse en combinación con quimioterapia y radioterapia convencionales. Recientemente, hemos seleccionado algunos aislamientos de rotavirus que están adaptados para infectar y eliminar de manera eficiente líneas de células tumorales. Objetivo: Se ensayaron cinco aislamientos de rotavirus adaptados a células tumorales para determinar su capacidad para infectar la línea celular de adenocarcinoma humano MCF-7. Métodos: Las proteínas asociadas a la membrana de la superficie celular que median la unión de partículas de virus se caracterizaron mediante ELISA, inmunoprecipitación, análisis FACS y bloqueo de anticuerpos. Resultados: Se encontró que las proteínas de choque térmico (HSPs) como Hsp90, Hsp70, Hsp60 y Hsp40 se expresan en la superficie celular formando complejos con la proteína disulfuro isomerasa (PDI), la integrina β3 y la proteína análoga de choque térmico 70 (Hsc70) en microdominios lipídicos (rafts). La interacción de los aislamientos de rotavirus con estas proteínas celulares se confirmó adicionalmente mediante un ensayo de competición y un ensayo de inhibición que incluía las HSP ensayadas. Conclusión: Nuestros hallazgos sugieren que los aislamientos de rotavirus adaptados a las células tumorales estudiados aquí ofrecen una herramienta prometedora para eliminar las células tumorales, lo que estimula más investigaciones sobre este tema, incluidos los modelos animales.

Rare variants of HSPB1 are probably associated with amyotrophic lateral sclerosis / 南方医科大学学报

OBJECTIVE@#To explore the association between rare HSPB1 variants and amyotrophic lateral sclerosis (ALS).@*METHODS@#We performed next-generation sequencing for 166 Chinese ALS patients to screen for possible pathogenic rare variants of HSPB1. The control individuals were obtained from 1000 Genome Project and an in-house whole-exome sequencing database. The Sequence Kernel Association Test (SKAT) and the SKAT-optimal test (SKAT-O) were used to identify the association between rare HSPB1 variants and ALS.@*RESULTS@#We identified 3 possible pathogenic rare variants of HSPB1 (all were missenses), including c.379C>T (p.R127W), c.446A>C (p.D149A) and c.451A>C (p.T151P). Compared with 1000 Genome Project, SKAT p=3.61×10@*CONCLUSIONS@#Rare variants of HSPB1 are probably associated with the pathogenesis of ALS.

N-glycosylation modification of heat shock protein gp96 affects its immunological function / 生物工程学报

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status / Resposta de choque térmico em perda auditiva induzida por ruído: efeitos da suplementação com dipeptídeos de alanil-glutamina sobre o estado das proteínas de choque térmico

Abstract Introduction: The 72 kDa heat shock protein, HSP72, located intracellularly provides cochlear cytoprotective and anti-inflammatory roles in the inner ear during stressful noise challenges. The expression of intracellular HSP72 (iHSP72) can be potentiated by alanyl-glutamine dipeptide supplementation. Conversely, these proteins act as pro-inflammatory signals in the extracellular milieu (eHSP72). Objective: We explore whether noise-induced hearing loss promotes both intracellular and extracellular HSP72 heat shock response alterations, and if alanyl-glutamine dipeptide supplementation could modify heat shock response and prevent hearing loss. Methods: Female 90 day-old Wistar rats (n = 32) were randomly divided into four groups: control, noise-induced hearing loss, treated with alanyl-glutamine dipeptide and noise-induced hearing loss plus alanyl-glutamine dipeptide. Auditory brainstem responses were evaluated before noise exposure (124 dB SPL for 2 h) and 14 days after. Cochlea, nuclear cochlear complex and plasma samples were collected for the measurement of intracellular HSP72 and extracellular HSP72 by a high-sensitivity ELISA kit. Results: We found an increase in both iHSP72 and eHSP72 levels in the noise-induced hearing loss group, which was alleviated by alanyl-glutamine dipeptide treatment. Furthermore, H-index of HSP72 (plasma/cochlea eHSP72/iHSP72 ratio) was increased in the noise-induced hearing loss group, but prevented by alanyl-glutamine dipeptide treatment, although alanyl-glutamine dipeptide had no effect on auditory threshold. Conclusions: Our data indicates that cochlear damage induced by noise exposure is accompanied by local and systemic heat shock response markers. Also, alanyl-glutamine reduced stress markers even though it had no effect on noise-induced hearing loss. Finally, plasma levels of 72 kDa heat shock proteins can be used as a biomarker of auditory stress after noise exposure.

Resumo Introdução: A proteína de choque térmico de 72 kDa, HSP72 localizada intracelularmente, tem papéis citoprotetores e anti-inflamatórios cocleares na orelha interna durante situações de ruído estressantes. A expressão dessa proteína pode ser potencializada pela suplementação com dipeptídeo de alanil-glutamina. Por outro lado, essas proteínas atuam como sinais pró-inflamatórios no meio extracelular. Objetivo: Investigar se a perda auditiva induzida por ruído promove alterações tanto das proteínas HSP72 intracelulares quanto extracelulares na resposta de choque térmico e se a suplementação com alanil-glutamina pode modificar a resposta de choque térmico e evitar a perda auditiva. Método: Ratos Wistar fêmeas, com 90 dias de idade (n = 32), foram divididos aleatoriamente em quatro grupos: controle, perda auditiva induzida por ruído, tratados com alanil-glutamina e perda auditiva induzida por ruído mais alanil-glutamina. Os potenciais evocados auditivos do tronco encefálico foram avaliados antes da exposição ao ruído (124 dB NPS por 2 h) e 14 dias após. A cóclea, o complexo nuclear coclear e amostras de plasma foram coletadas para mensuração de HSP72 intra e extracelular com um kit Elisa de alta sensibilidade. Resultados: Houve um aumento nos níveis de HSP72 intra e extracelular no grupo perda auditiva induzida por ruído, que foi minimizado pelo tratamento com alanil-glutamina. Além disso, o índice H das HSP72 (razão HSP72 extracelular/HSP72intracelular plasma/cóclea) aumentou no grupo perda auditiva induzida por ruído, mas foi limitado pelo tratamento com alanil-glutamina, embora o alanil-glutamina não tenha efeito no limiar auditivo. Conclusões: Nossos dados indicam que o dano coclear induzido pela exposição ao ruído é acompanhado por marcadores da resposta de choque térmico locais e sistêmicos. Além disso, alanil-glutamina reduziu os marcadores de estresse, mesmo não tendo efeito sobre a perda auditiva induzida por ruído. Finalmente, os níveis plasmáticos de proteínas de choque térmico de 72 kDa podem ser usados como biomarcador do estresse auditivo, após a exposição ao ruído.

Effects of melatonin against thioacetamide-induced testicular toxicity in rats / Efectos de la melatonina contra la toxicidad testicular inducida por tioacetamida en ratas

SUMMARY: This study aimed to investigate the changes in testis tissue of thioacetamide-induced rats and the effect of melatonin on these changes. Thirty-five male Wistar Albino rats were divided into five groups. Group I; Control (n=7), Group II; Melatonin (Mel) (10 mg/kg) a single dose (i.p)(n=7), Group III; Thioacetamide (TAA) (300 mg/kg) (i.p) 2 times with 24 hour intervals (n=7), Group IV; TAA (300 mg/kg) was administered at 24-hour intervals, afterwards of 10 mg/kg single dose of Mel (n=7), Group V; Mel was administered 10 mg/kg a single dose 24 hours before the administration of TAA (n=7). Testis was evaluated histologically, immunohistochemically (Heat Shock Proteins (HSP) 70 and 90), blood serum testosterone, total antioxidant status(TAS) and total oxidant status(TOS) in tissue. The tissue sections of Group III decreased seminiferous tubule diameters, and germinal epithelium spills were observed. HSP70 and HSP90 expressions were increased. There wasn't a statistically significant change in testosterone levels among the groups. While TAS levels decreased in Group III compared to control, TOS levels didn't change. HSP70 and HSP90 decreased in groups with Mel-treated. Mel was found to have both protective and therapeutic effects. According to our results, the therapeutic effect of Mel in thioacetamide-induced acute testicular injury is greater than its protective effect.

RESUMEN: Este estudio tuvo como objetivo investigar los cambios en el tejido testicular de ratas inducidas por tioacetamida y el efecto de la melatonina en estos cambios. Treinta y cinco ratas macho Wistar Albino se dividieron en cinco grupos. Grupo I; Control (n = 7), Grupo II; Melatonina (Mel) (10 mg / kg) una dosis única (i.p) (n = 7), Grupo III; Tioacetamida (TAA) (300 mg / kg) (i.p) 2 veces con intervalos de 24 horas (n = 7), Grupo IV; TAA (300 mg / kg) se administró a intervalos de 24 horas, luego de una dosis única de 10 mg / kg de Mel (n = 7), Grupo V; Mel recibió 10 mg / kg de una dosis única 24 horas antes de la administración de TAA (n = 7). Los testículos se evaluaron histológicamente, inmunohistoquímicamente (proteínas de choque térmico (PCT) 70 y 90), testosterona en suero sanguíneo, estado antioxidante total (EAT) y estado oxidante total (EOT) en el tejido. En secciones de tejido del Grupo III se observó disminución de los diámetros de los túbulos seminíferos y derrames en el epitelio germinal. Se aumentaron las expresiones HSP70 y HSP90. No hubo un cambio estadísticamente significativo en los niveles de testosterona entre los grupos. Mientras que los niveles de EAT disminuyeron en el Grupo III en comparación con el control, los niveles de EOT no cambiaron. HSP70 y HSP90 disminuyeron en los grupos tratados con Mel. Se descubrió que Mel tenía efectos protectores y terapéuticos. Según nuestros resultados, el efecto terapéutico de Mel en la lesión testicular aguda inducida por tioacetamida es mayor que su efecto protector.

In silico cloning and bioinformatics analysis of HSP21 in Manihot esculenta / 生物工程学报

HSP21 gene is a key gene to respond high temperature stress in plant and plays an important role in preventing protein denaturation, protecting cell structure and maintaining normal growth and development. Therefore, cloning HSP21 gene is the basis for revealing the molecular mechanism of resistance to high temperature stress in cassava. To obtain cassava HSP21 homologous gene and analyze the properties of predicted protein, electronic cloning technology was used to assemble and derivate new gene in this study, and bioinformatics analysis method was used to analyze the primary to highest structure, hydrophilicity/hydrophobicity, signal peptide, protein homology and phylogenetic evolution of expressed protein. HSP21 gene was 969 bp, its open reading frame was 705 bp, and the predicted protein contains 234 amino acids. The predicted protein is a non-transmembrane protein that is alkaline and hydrophilic, and is mainly localized in the chloroplast. Through multiple sequence alignment and phylogenetic analysis, it was found that the cassava HSP21 protein has high homology with other plants such as Hevea brasiliensis, Ricinus communis, and Jatropha curcas. The results could provide reference for the study of cloning and transformation of this gene.

Glutamine protects intestine against ischemia-reperfusion injury by alleviating endoplasmic reticulum stress induced apoptosis in rats

Abstract Purpose Glutamine, as an essential part of enteral nutrition and parenteral nutrition agent, has been widely recognized to be a kind of important intestinal mucosa protectant in clinical practice and experimental research. However, the mechanisms of its protective effects are still not fully understand. Consequently, this study aimed to explore the potential mechanism of glutamine on ischemia-reperfusion (I/R) injury induced endoplasmic reticulum (ER) stress in intestine. Methods An experimental model of intestinal I/R in rats was established by 1 hour occlusion of the superior mesenteric artery followed by 3 hours of reperfusion. Morphologic changes of intestinal mucosa, apoptosis of epithelial cells, and expression of intestinal Grp78, Gadd153, Caspase-12, ATF4, PERK phosphorylation (P-PERK) and elF2αphosphorylation(P-elF2α) were determined. Results After I/R, the apoptotic index of intestinal mucosa epithelial cells observably increased with notable necrosis of intestinal mucosa, and the expressions of Grp78, Gadd153, Caspase-12, ATF4, P-PERK and P-elF2αall were increased. However, treatment with glutamine could significantly relieve intestinal I/R injury and apoptosis index. Moreover, glutamine could clearly up-regulate the expression of Grp78, restrain P-PERK and P-elF2α, and reduce ATF4, Gadd153 and Caspase-12 expressions. Conclusion Glutamine may be involved in alleviating ER stress induced intestinal mucosa cells apoptosis.

Análise da expressão gênica das proteínas de choque térmico 60 e 70 e dos receptores Toll-like 2 e 4 em diferentes graus de displasia em portadores de queilite actínica e carcinoma epidermóide de lábio / Analysis of the gene expression of heat shock proteins 60 and 70 and Toll-like receptors 2 and 4 in different degrees of dysplasia in patients with actinic cheilitis and lip squamous cell carcinoma

A queilite actínica (QA) é a desordem potencialmente maligna oral mais frequente e precede a maioria dos casos de carcinoma epidermóide (CEC) de lábio em pacientes não fumantes. Acomete principalmente indivíduos do gênero masculino, leucodermas, acima dos 40 anos, com histórico de exposição crônica à radiação ultravioleta (UV). O objetivo deste estudo foi avaliar a expressão gênica das proteínas de choque térmico (HSP) 60 e 70 e dos receptores Toll-like 2 e 4 com lesões de QA e CEC de lábio por meio de RT-PCR em tempo real, a partir de amostras coletadas durante o procedimento cirúrgico de biópsia incisional. Conduziu-se um estudo caso-controle com 40 pacientes portadores de QA e 6 com CEC de lábio, submetendo o material de biópsia aos procedimentos laboratoriais de análise histopatológica e de extração de RNA para estudo da expressão dos TLR e das HSP por meio da técnica de RT-PCR em tempo real. Foram excluídas 7 amostras após o processo de extração de RNA e outras 15 amostras após a amplificação por PCR em tempo real, modificando a casuística para 18 amostras de QA e 6 de CEC de lábio. Os resultados foram analisados pelo método CT comparativo, pelo teste Shappiro-Wilk e pelo teste T-Student. Houve maior expressão da HSP60 e TLR4 na QA em relação ao CEC de lábio, porém não houve diferença estatisticamente significativa (p>0.05). Já a HSP70 foi mais expressa na QA do que no CEC de lábio com diferença estatisticamente significativa (p=0.034) e o TLR2 mostrou-se mais expresso no CEC de lábio do que na QA, também com diferença estatisticamente significativa (p=0.003). Houve correlação significativa entre o aumento da expressão da HSP70 e os hábitos de tabagismo e exposição solar crônica em pacientes com QA. Estudos mais aprofundados são necessários, visto que não se encontram outros publicados com esses genes em QA e CEC de lábio até a presente data.

High Mobility Group Box 1 and Heat Shock Protein-70 Expression Post (-)-Epigallocatechin-3-Gallate in East Java Green Tea Methanolic Extract Administration During Orthodontic Tooth Movement in Wistar Rats

Abstract Objective: To investigate the expression of High Mobility Group Box 1 (HMGB1) and Heat Shock Protein-70 (HSP-70) during orthodontic tooth movement (OTM) after (-)- Epigallocatechin-3-Gallate (EGCG) in East Java Green Tea (Camelia Sinensis) Methanolic Extract (GTME) administration in vivo. Material and Methods: 28 Wistar rats (Rattus Novergicus) was used and divided into 4 groups accordingly: K- without EGCG and OTM; K+ with OTM, without EGCG for 14 days; T1with OTM for 14 days and EGCG for 7 days; treatment group 2 (T2) with OTM and EGCG for 14 days. OTM animal model was achieved through the installation of the OTM device by means of NiTi close coil spring with 10g force placed between the first incisor and first maxillary molars. The samples were terminated on Day 14. The pre-maxillary was isolated for the immunohistochemical examination. Analysis of Variance (ANOVA) then continued with Tukey Honest Significant Difference (HSD) (p<0.05) was performed to analyze the data. Results: The highest HMGB1 and HSP-70 expression were found in the K+ group pressure side, meanwhile the lowest HMGB1 and HSP-70 expression were found in K- group tension side in the alveolar bone. There was a significant decrease of HMGB1 and HSP-70 expression in T2 compared to T1 and K+ with significant between groups (p<0.05; p=0.0001). Conclusion: The decreased expression of HMGB1 and HSP-70 in alveolar bone of OTM wistar rats due to post administration of GTME that consisted EGCG.

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

Background: In industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze­thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain. Results: Elite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze­thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/ v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield. Conclusions: A multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze­thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.

Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse / Estudio inmunohistoquímico de proteínas de unión de la amelogenina en un ratón con mutación puntual de amelogenina

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.

La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.

S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells / 生理学报

The aim of this study was to investigate the role of S100 calcium binding protein A16 (S100A16) in lipid metabolism in hepatocytes and its possible biological mechanism. HepG2 cells (human hepatoma cell line) were cultured with fatty acid to establish fatty acid culture model. The control model was cultured without fatty acid. Each model was divided into three groups and transfected with S100a16 over-expression, shRNA and vector plasmids, respectively. The concentration of triglyceride (TG) in the cells was measured by kit, and the lipid droplets was observed by oil red O staining. Immunoprecipitation and mass spectrometry were used to find the interesting proteins interacting with S100A16, and the interaction was verified by immunoprecipitation. The further mechanism was studied by Western blot and qRT-PCR. The results showed that the intracellular lipid droplet and TG concentrations in the fatty acid culture model were significantly higher than those in the control model. The accumulation of intracellular fat in the S100a16 over-expression group was significantly higher than that in the vector plasmid transfection group. There was an interaction between heat shock protein A5 (HSPA5) and S100A16. Over-expression of S100A16 up-regulated protein expression levels of HSPA5, inositol-requiring enzyme 1α (IRE1α) and pIREα1, which belong to endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway. Meanwhile, over-expression of S100A16 up-regulated the mRNA expression levels of adipose synthesis-related gene Srebp1c, Acc and Fas. In the S100a16 shRNA plasmid transfection group, the above-mentioned protein and mRNA levels were lower than those of vector plasmid transfection group. These results suggest that S100A16 may promote lipid synthesis in HepG2 cells through endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway.

G protein-coupled estrogen receptor alleviates cerebral ischemia-reperfusion injury through inhibiting endoplasmic reticulum stress / 生理学报

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.

Effect of HSPB9 on Apoptosis of DF-1 Cells / 生物医学与环境科学（英文）

OBJECTIVE@#Our aim was to explore whether heat stress protein (HSP) 9 preferentially expresses under heat stress and affects the expression of other heat stress proteins as well as to explore the effect of HSPB9 overexpression and knockdown on apoptosis in DF-1.@*METHODS@#We used gene cloning to construct an overexpression vector of the target gene, and synthesized the target gene interference fragment to transfect the chicken fibroblast cell line. Gene and protein expression, as well as apoptosis, were detected by RT-qPCR, Western blot, and flow cytometry.@*RESULTS@#Chicken DF-1 cells showed an early state of apoptosis in the early stages of HSPB9 overexpression. In the later stages, as HSPB9 expression increased, the cells showed inhibition of apoptosis. When the cells were under heat stress, HSPB9 expression was much higher and earlier than the expression of HSPB1 and HSPA2. In addition, high expression of HSPB9 had a negative effect on HSPB1 and HSPA2 expression. This negative feedback decreased the percentage of early stages of apoptotic cells and promoted cell survival.@*CONCLUSION@#HSPB9 expression, although rapid, is detrimental to cell survival early during its overexpression. In heat stress, HSPB9 overexpression, while inhibiting the expression of HSPA2 and HSPB1, is beneficial to cell survival.

Analysis of SACS mutation in a family affected with autosomal recessive spastic ataxia of Charlevoix-Saguenay / 中华医学遗传学杂志

OBJECTIVE@#To carry out mutation analysis for a Chinese family affected with autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).@*METHODS@#Whole exome sequencing (WES) was used to screen potential mutations within genomic DNA extracted from the proband. Suspected mutation was validated by combining clinical data and results of Sanger sequencing.@*RESULTS@#A homozygous deletional mutation c.3665_3675delGTGCTGTCTTA (p.S1222fs) was found in the proband, for which her parents were both heterozygous carriers.@*CONCLUSION@#WES is capable of detecting mutation underlying this disorder and facilitating genetic counseling and prenatal diagnosis for the affected family. A novel pathogenic mutation of the SACS gene was discovered.

Heat shock protein 70 increases cell proliferation, neuroblast differentiation, and the phosphorylation of CREB in the hippocampus / 한국실험동물학회지

In the present study, we investigated the effects of heat shock protein 70 (HSP70) on novel object recognition, cell proliferation, and neuroblast differentiation in the hippocampus. To facilitate penetration into the blood–brain barrier and neuronal plasma membrane, we created a Tat-HSP70 fusion protein. Eight-week-old mice received intraperitoneal injections of vehicle (10% glycerol), control-HSP70, or Tat-HSP70 protein once a day for 21 days. To elucidate the delivery efficiency of HSP70 into the hippocampus, western blot analysis for polyhistidine was conducted. Polyhistidine protein levels were significantly increased in control-HSP70- and Tat-HSP70-treated groups compared to the control or vehicle-treated group. However, polyhistidine protein levels were significantly higher in the Tat-HSP70-treated group compared to that in the control-HSP70-treated group. In addition, immunohistochemical study for HSP70 showed direct evidences for induction of HSP70 immunoreactivity in the control-HSP70- and Tat-HSP70-treated groups. Administration of Tat-HSP70 increased the novel object recognition memory compared to untreated mice or mice treated with the vehicle. In addition, the administration of Tat-HSP70 significantly increased the populations of proliferating cells and differentiated neuroblasts in the dentate gyrus compared to those in the control or vehicle-treated group based on the Ki67 and doublecortin (DCX) immunostaining. Furthermore, the phosphorylation of cAMP response element-binding protein (pCREB) was significantly enhanced in the dentate gyrus of the Tat-HSP70-treated group compared to that in the control or vehicle-treated group. Western blot study also demonstrated the increases of DCX and pCREB protein levels in the Tat-HSP70-treated group compared to that in the control or vehicle-treated group. In contrast, administration of control-HSP70 moderately increased the novel object recognition memory, cell proliferation, and neuroblast differentiation in the dentate gyrus compared to that in the control or vehicle-treated group. These results suggest that Tat-HSP70 promoted hippocampal functions by increasing the pCREB in the hippocampus.