Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism

<abstract>Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1±3.2 and 243.0±21.6, respectively, while those after thawing were 23.2±2.5 and 105.7±28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.

Hepatitis C virus inhibitor synergism suggests multistepinteractions between heat-shock protein 90 and hepatitis Cvirus replication

AIM: To address the effect of heat-shock protein 90(HSP90) inhibitors on the release of the hepatitis C virus(HCV), a cell culture-derived HCV(JFH1/HCVcc) from Huh-7 cells was examined.METHODS: We quantified both the intracellular and extracellular(culture medium) levels of the components(RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A(Cs A) or interferon. Finally, we statistically examined the combined effect of radicicoland Cs A using the combination index(CI) and graphical representation proposed by Chou and Talalay.RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor(Cs A or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy(CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release.

Hsp90βis critical for the infection of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.

Heat-shock protein 90 in Candida albicans

Researches on Candidal heat-shock protein 90 (HSP90) in recent years are summarized. Candida albicans is a commensal pathogen in human and animals. in immunocompromised individuals it behaves as an opportunist pathogen, giving rise to superficial or systemic infections. Systemic candidosis is a common cause of death among immunocompromised and debilitated patients, in which the mortality is as high as 70%. HSP90 is now recognized as an immunodominant antigen in C. albicans and plays a key role in systemic candidosis as a molecular chaperone. The 47-ku peptide is the breakdown product of HSP90. Patients who has recovered from systemic candidosis produce high titre of antibodies to 47-ku antigen, whereas the fatal cases have little antibody or falling titre? The three commonest epitopes of candidal HSP90 have been mapped, epitopes C, B and H. Epitopes C and H are immunogenic. The antibody probes of both epitopes may be developed into a new serological test agents for systemic candidosis due to rather

Homeopathic Thuja 30C ameliorates benzo(a)pyreneinduced DNA damage,stress and viability of perfused lung cells of mice in vitro

OBJECTIVE： To examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo（a）pyrene （BaP）-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro. METHODS： Perfused normal lung cells from mice were cultured in 5% Roswell Park Memorial Institute medium and exposed to BaP, a potent carcinogen, at the half maximal inhibitory concentration dose （2.2 μmol/L） for 24 h. Thereafter, the intoxicated cells were either treated with Thuja 30C （used against tumor or cancer） or its vehicle media, succussed alcohol 30C. Relevant parameters of study involving reactive oxygen species （ROS） accumulation, total glutathione （GSH） content, and generations of heat shock protein （hsp）-90 were measured; the cell viability and other test parameters were measured after treatment with either Thuja 30C or its vehicle media. Circular dichroism spectroscopy was performed to examine if Thuja 30C directly interacted with calf thymus DNA as target. For ascertaining if DNA damaged by BaP could be partially repaired and restituted by the remedy, 4＇,6-diamidino-2-phenylindole staining was performed. RESULTS： Thuja 30C increased cell viability of BaP-intoxicated cells significantly, as compared to drug-untreated or drug-vehicle control. A minimal dose of Thuja 30C significantly inhibited BaP-induced stress level, by down-regulating ROS and hsp-90, and increasing GSH content. Thuja 30C itself had no DNA-damaging effect, and no direct drug-DNA interaction. However, it showed quite striking ability to repair DNA damage caused by BaP. CONCLUSION： Thuja 30C ameliorates BaP-induced toxicity, stress and DNA damage in perfused lung cells of mice and it apparently has no effect on normal lung cells.

Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency

The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β(Hsp90β)gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection.Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and BglII.Recombinant plasmids were transformed into Escherichia coli,DH5a,and the colonies were picked and grown in the Amp-agarose.The presence of positive clones was checked by the means of endodigestion and sequencing.Three cell strains,HepG2,Human umbilicus vein endothelium cells(HUVEC)and HeK293,were cultured.Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine.The transfection efficiency was measured by detection of enhanced green fluorescence protein(EGFP).The presence of positive recombinant clones were verified by double-digestion and sequencing.The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1,pSuper-Hsp90β2 and pSuper-Hsp90β3.After optimizing the ratio of plasmid to Lipofectamine,we achieved high transfection efficiency in HeK293 cells.Transfection efficiency was still low in the HepG2 cells.In conclusion,the si-RNA-synthesizing plasmids targeting Hsp90βwere constructed and transfected into cells with different transfection efficiency.

Nanoscale covalent organic framework for the low-temperature treatment of tumor growth and lung metastasis

Photothermal therapy(PTT)has shown promising applications in tumor therapies.However,due to laserinduced nonspecific heating and heat diffusion,high levels of hyperthermia(>50℃)in tumor tissues often increase the risk of cancer recurrence and metastasis,which causes the patient pain and destroys the surrounding normal cells and tissues.It is therefore important to develop photothermal strategies that have excellent therapeutic efficiencies under low-temperature conditions(≤45℃).In addition,the heterogeneity and complexity of tumors require the development of combinatorial antitumor treatments as the therapeutic efficiency of monomodal PTT is not currently sufficient.Herein,we have adopted a stepwise synthetic approach to develop a highly efficient multimodal therapeutic agent GA@PCOF@PDA by successive bonding defect functionalization(BDF),guest encapsulation,and surface modification steps.The covalently grafted porphyrinic photosensitizers(Por),encapsulated gambogic acid(GA),and surface-modified PDA film are independently responsible for photodynamic therapy(PDT),heat-shock protein 90(HSP90)down-regulation and chemotherapy(CT),and low-temperature PTT.This proof-ofconcept study illustrates an efficient,generalized approach to design high-performance covalent organic framework(COF)-based nanoagents that can be easily tailored to combine different therapeutic modalities for improved cancer theranostics at low temperatures.

Enhancing the magnetocaloric response of high-entropy metallic-glass by microstructural control

Non-equiatomic high-entropy alloys(HEAs),the second-generation multi-phase HEAs,have been recently reported with outstanding properties that surpass the typical limits of conventional alloys and/or the first-generation equiatomic single-phase HEAs.For magnetocaloric HEAs,non-equiatomic(Gd_(36)Tb_(20)Co_(20)Al_(24))100−xFex microwires,with Curie temperatures up to 108 K,overcome the typical low temperature limit of rare-earth-containing HEAs(which typically concentrate lower than around 60 K).For alloys with x=2 and 3,they possess some nanocrystals,though very minor,which offers a widening in the Curie temperature distribution.In this work,we further optimize the magnetocaloric responses of x=3 microwires by microstructural control using the current annealing technique.With this processing method,the precipitation of nanocrystals within the amorphous matrix leads to a phase compositional difference in the microwires.The multi-phase character leads to challenges in rescaling the magnetocaloric curves,which is overcome by using two reference temperatures during the scaling procedure.The phase composition difference increases with increasing current density,whereby within a certain range,the working temperature span broadens and simultaneously offers relative cooling power values that are at least 2-fold larger than many reported conventional magnetocaloric alloys,both single amorphous phase or multi-phase character(amorphous and nanocrystalline).Among the amorphous rare-earth-containing HEAs,our work increases the working temperature beyond the typical<60 K limit while maintaining a comparable magnetocaloric effect.This demonstrates that microstructural control is a feasible way,in addition to appropriate compositional design selection,to optimize the magnetocaloric effect of HEAs.