Prognosis value of heat-shock proteins in esophageal and esophagogastric cancer:A systematic review and meta-analysis

BACKGROUND Heat shock proteins(HSPs)are molecular chaperones that play an important role in cellular protection against stress events and have been reported to be overex-pressed in many cancers.The prognostic significance of HSPs and their regulatory factors,such as heat shock factor 1(HSF1)and CHIP,are poorly understood.AIM To investigate the relationship between HSP expression and prognosis in esophageal and esophagogastric cancer.METHODS A systematic review was conducted in accordance with PRISMA recommend-ations(PROSPERO:CRD42022370653),on Embase,PubMed,Cochrane,and LILACS.Cohort,case-control,and cross-sectional studies of patients with eso-phagus or esophagogastric cancer were included.HSP-positive patients were compared with HSP-negative,and the endpoints analyzed were lymph node metastasis,tumor depth,distant metastasis,and overall survival(OS).HSPs were stratified according to the HSP family,and the summary risk difference(RD)was calculated using a random-effect model.RESULTS The final selection comprised 27 studies,including esophageal squamous cell carcinoma(21),esophagogastric adenocarcinoma(5),and mixed neoplasms(1).The pooled sample size was 3465 patients.HSP40 and 60 were associated with a higher 3-year OS[HSP40:RD=0.22;95%confidence interval(CI):0.09-0.35;HSP60:RD=0.33;95%CI:0.17-0.50],while HSF1 was associated with a poor 3-year OS(RD=-0.22;95%CI:-0.32 to-0.12).The other HSP families were not associated with long-term survival.HSF1 was associated with a higher probability of lymph node metastasis(RD=-0.16;95%CI:-0.29 to-0.04).HSP40 was associated with a lower probability of lymph node dissemination(RD=0.18;95%CI:0.03-0.33).The expression of other HSP families was not significantly related to tumor depth and lymph node or distant metastasis.CONCLUSION The expression levels of certain families of HSP,such as HSP40 and 60 and HSF1,are associated with long-term survival and lymph node dissemination in patients with esophageal and esophagogastric cancer.

Expression profiles of two small heat shock proteins and antioxidant enzyme activity in Mytilus galloprovincialis exposed to cadmium at environmentally relevant concentrations

Small heat shock proteins encompass a widespread but diverse class of proteins,which play key roles in protecting organisms from various stressors.In the present study,the full-length cDNAs of two small heat shock proteins(MgsHSP22 and MgsHSP24.1)were cloned from Mytilus galloprovincialis,which encoded peptides of 181 and 247 amino acids,respectively.Both MgsHSP22 and MgsHSP24.1 were detected in all tissues examined by real-time PCR,with the highest expression being observed in muscle and gonad tissues.The real-time PCR results revealed that Cd signifi cantly inhibited MgsHSP22 expression at 24 h and MgsHSP24.1 at 24 and 48 h under 5 μg/L Cd 2 + exposure.MgsHSP24.1 expression was also signifi cantly inhibited after 50 μg/L Cd 2+ exposure for 48 h.With regard to antioxidant enzymes,increased GPx and CAT activity were detected under Cd 2+ stress(5 and 50 μg/L),while no signifi cant difference in SOD activity was observed throughout the experiment.Overall,both MgsHsps and antioxidant enzymes revealed their potential as Cd stress biomarkers in M.galloprovincialis.

Structural modeling of proteins by integrating small-angle x-ray scattering data

Elucidating the structure of large biomolecules such as multi-domain proteins or protein complexes is challenging due to their high flexibility in solution. Recently, an ＂integrative structural biology＂ approach has been proposed, which aims to determine the protein structure and characterize protein flexibility by combining complementary high- and lowresolution experimental data using computer simulations. Small-angle x-ray scattering（SAXS） is an efficient technique that can yield low-resolution structural information, including protein size and shape. Here, we review computational methods that integrate SAXS with other experimental datasets for structural modeling. Finally, we provide a case study of determination of the structure of a protein complex formed between the tandem SH3 domains in c-Cb1-associated protein and the proline-rich loop in human vinculin.

Protein kinase small molecule inhibitors for rheumatoid arthritis: Medicinal chemistry/clinical perspectives

Medicinal chemistry strategies have contributed to the development, experimental study of and clinical trials assessment of the first type of protein kinase small molecule inhibitor to target the Janus kinase/Signal Transducers and Activators of Transcription(JAK/STAT) signaling pathway. The orally administered small molecule inhibitor, tofacitinib, is the first drug to target the JAK/STAT pathway for entry into the armamentarium of the medical therapy of rheumatoid arthritis. The introduction of tofacitinib into general rheumatologic practice coupled with increasing understanding that additional cellular signal transduction pathways including the mitogen-activated protein kinase and phosphatidylinositide-3-kinase/Akt/mammalian target of rapa-mycin pathways as well as spleen tyrosine kinase also contribute to immune-mediated inflammatory in rheumatoid arthritis makes it likely that further development of orally administered protein kinase small molecule inhibitors for rheumatoid arthritis will occur in the near future.

Expression analysis of two reverse duplicated small heat shock protein genes in rice(Oryza sativa L.)

supported by grants from the National Natural Science Foundation of China （30671178）;the Shanxi Province Science Foundation for Youths, China （2014021029-2）

Analysis and Review of Downregulated Actin Cytoskeletal Proteins in Non-Small Cell Lung Cancer

Actin, a highly conserved protein, plays a dominant role in Non-small cell lung cancer (NSCLC). Late diagnosis and the aggressive nature of NSCLC pose a significant threat. Studying the clinic pathological properties of NSCLC proteins is a potential alternative for developing treatment strategies. Towards this, 35 downregulated actin cytoskeletal proteins on NSCLC prognosis and treatment were studied by examining their protein-protein interactions, gene ontology enrichment terms, and signaling pathways. Using PubMed, various proteins in NSCLC were identified. The protein-protein interactions and functional associations of these proteins were examined using the STRING database. The focal adhesion signaling pathway was selected from all available KEGG and Wiki pathways because of its role in regulating gene expression, facilitating cell movement and reproduction, and significantly impacting NSCLC. The protein-protein interaction network of the 35 downregulated actin cytoskeleton proteins revealed that ACTG1, ACTR2, ACTR3, ANXA2, ARPC4, FLNA, TLN1, CALD1, MYL6, MYH9, MYH10, TPM1, TPM3, TPM4, PFN1, IQGAP1, MSN, and ZXY exhibited the highest number of interactions. Whereas HSPB1, CTNNA1, KRT17, KRT7, FLNB, SEPT2, and TUBA1B displayed medium interactions, while UTRN, TUBA1B, and DUSP23 had relatively fewer interactions. It was discovered that focal adhesions are critical in connecting membrane receptors with the actin cytoskeleton. In addition, protein kinases, phosphatases, and adapter proteins were identified as key signaling molecules in this process, greatly influencing cell shape, motility, and gene expression. Our analysis shows that the focal adhesion pathway plays a crucial role in NSCLC and is essential for developing effective treatment strategies and improving patient outcomes.

PnSCR82,a small cysteine-rich secretory protein of Phytophthora nicotianae,can enhance defense responses in plants

A number of plant pathogenic species of Phytophthora are known to produce different classes of secretory proteins during interactions with their hosts.Although several small cysteine-rich(SCR)secretory proteins,conserved in oomycete pathogens,have been identified in Phytophthora,their specific involvement in these interactions remains unknown.In this study,an SCR effector encoded by Pn SCR82 in P.nicotianae was identified and shown to have similarities to P.cactorum phytotoxic protein,Pc F(Phytophthora cactorum Fragaria).Agroinfection with potato virus X vector,Pn SCR82,was capable of inducing plant hypersensitive cell death in Nicotiana benthamiana and Solanum lycopersicum.Real-time PCR results indicated that transiently expressed Pn SCR82 in N.benthamiana leaves activated the jasmonate,salicylic acid and ethylene signaling pathways.Transient expression of Pn SCR82 enhanced plant resisitance to P.capsici.In summary,our results demonstrated that P.nicotianae Pn SCR82 elicits defensive responses in N.benthamiana and may potentially play a significant role in future crop protection programs.

Subcellular Localization of Small GTP-binding Protein DsRab in Dunaliella salina

With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.

Discovery of a small-molecule bromodomain-containing protein 4 inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer

OBJECTIVE To discover a small-molecule bromodomain-containing protein 4(BRD4)inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer and exploreits potential mechanisms.METHODS BRD4 interactors were analyzed by PPI network prediction and The Cancer Genome Atlas(TCGA)analysis.The interaction between BRD4 and AMPK was confirmed by co-immunoprecipitation assay.Novel BRD4 inhibitors were designed and synthesized based upon pharmacophore analysis of BRD4(1),then screened by antiproliferative activity and Alpha Screen of BRD4(1).The selectivity of the best candidate compound 8f was validated by co-crystallization,FRET assay and co-immuno precipitation assay.The mechanisms of 8f were investigated by fluorescence microscopy,electron microscopy,Western blotting,immunocytochemistry,si RNA and GFP-m RFP-LC3 plasmid transfections,as well as immunohistochemistry and immunofluorescence.Potential mechanisms were discovered by i TRAQ-based proteomics analysis and the therapeutic effect of 8f was assessed by xenograft breast cancer mouse and zebrafish models.RESULTS We identified that BRD4 interacted with AMPK,which was remarkably downregulated in breast cancer.We next designed and synthesized 49 candidate compounds,and eventually discovered a selective small-molecule inhibitor of BRD4(8f).Subsequently,8f was discovered to induce autophagyassociated cell death(ACD)by BRD4-AMPK interaction,and thus activating AMPK-m TOR-ULK1-modulated autophagic pathway in breast cancer cells.Interestingly,the i TRAQ-based proteomics analyses revealed that 8f induced ACD pathways,involved in HMGB1,VDAC1/2 and e EF2.Moreover,8f displayed a therapeutic potential on both xenograft breast cancer mouse and zebrafish models.CONCLUSION We discovered a novel small-molecule inhibitor of BRD4 that induces BRD4-AMPK-modulated ACD in breast cancer,which may provide a candidate drug for future cancer therapy.

Two Small Molecules, ZCL278 and AZA197 Show Promise in Influencing Protein Interactions Involving the Ras-Related Protein Cell division cycle 42 [Cdc42] to Modulate Its Oncogenic Potential

Cdc42 is a member of the Rho subfamily of Ras-related proteins, which were among the first oncogenic proteins to be identified as playing a sig-nificant role in a variety of cellular events [Barbacaid, 1987, Ann. Rev. Biochem]. Equally important, Protein-Protein Interactions [PPIs] involving Cdc42 continue to highlight the role of Ras-related proteins’ relevance to cancer. As these proteins have been considered incapable of being “druggable”, due to a perceived lack of binding surface[s] that are amenable to small molecule targeting, there remains limited development of therapies to tackle diseased states caused by Cdc42-stimulated hyperactivity. Thusly, it has become important to characterize molecular details, including dynamics, of PPIs involving Cdc42 that may lend themselves as potential targets for therapeutic approaches. Recently, two small molecules, ZCL278 and AZA197, have shown promise in directly targeting Cdc42 to influence PPIs that are capable of causing Cdc42-stimulated abnormal signaling. In this editorial, we highlight recent studies that show case how these two small molecules may influence Cdc42-protein interactions.

Plant and animal positive-sense single-stranded RNA viruses encode small proteins important for viral infection in their negative-sense strand

Positive-sense single-stranded RNA(+ssRNA)viruses,the most abundant viruses of eukaryotes in nature,require the synthesis of negative-sense RNA(-RNA)using their genomic(positive-sense)RNA(+RNA)as a template for replication.Based on current evidence,viral proteins are translated via viral+RNAs,whereas-RNA is considered to be a viral replication intermediate without coding capacity.Here,we report that plant and animal+ssRNA viruses contain small open reading frames(ORFs)in their-RNA(reverse ORFs[rORFs]).Using turnip mosaic virus(TuMV)as a model for plant+ssRNA viruses,we demonstrate that small proteins encoded by rORFs display specific subcellularlocalizations,and confirm the presence of rORF2 in infected cells through mass spectrometry analysis.The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes.The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase,and mutation of rORF2 completely abolishes virus infection,whereas ectopic expression of rORF2 rescues the mutant virus.Furthermore,we show that several rORFs in the-RNA of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have the ability to suppress type l interferon production and facilitate the infection of ve-sicular stomatitis virus.In addition,we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs.Taken together,these findings indicate that the-RNA of+ssRNA vi-ruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection,revealing a viral proteome larger than previously thought.

Challenges in diagnosing adhesive small bowel obstruction

Adhesive small bowel obstruction(ASBO)is the most frequently encountered surgical disorder of the small intestine.Up to 80%of ASBO cases resolve spontaneously and do not require invasive treatment.It is important to identify such patients that will benefit from conservative treatment in order to prevent unnecessarily exposing them to the risks associated with surgical intervention,such as morbidity and further adhesion formation.For the remaining ASBO patients,timely surgical intervention is necessary to prevent small bowel strangulation,which may cause intestinal ischemia and bowel necrosis.While early identification of these patients is key to decreasing ASBO-related morbidity and mortality,the non-specific signs and laboratory findings upon clinic presentation limit timely diagnosis and implementation of appropriate clinical management.Combining the clinical presentation findings with those from other diagnostic imaging modalities,such as abdominal X-ray,computed tomography-scan and water-soluble contrast studies,will improve diagnosis of ASBO and help clinicians to better evaluate the potential of conservative management as a safe strategy for a particular patient.Nonetheless,patients who present with moderate findings by all these approaches continue to represent a challenge.A new diagnostic strategy is urgently needed to further improve our ability to identify early signs of strangulated bowel,and this diagnostic modality should be able to indicate when surgical management is required.A number of potential serum markers have been proposed for this purpose,including intestinal fatty acid binding protein andα-glutathione S transferase.On-going research is attempting to clearly define their diagnostic utility and to optimize their potential role in determining which patients should be managed surgically.

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80％ at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0～60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.

Phenotypic changes of Schwann cells on the proximal stump of injured peripheral nerve during repair using small gap conduit tube

Dedifferentiation of Schwann cells is an important feature of the response to peripheral nerve injury and specific negative myelination reg- ulators are considered to have a major role in this process. However, most experiments have focused on the distal nerve stump, where the Notch signaling pathway is strongly associated with Schwann cell dedifferentiation and repair of the nerve. We observed the phenotypic changes of Schwann cells and changes of active Notch signaling on the proximal stump during peripheral nerve repair using small gap conduit tubulization. Eighty rats, with right sciatic nerve section of 4 mm, were randomly assigned to conduit bridging group and control group （epineurium suture）. Glial fibrillary acidic protein expression, in myelinating Schwann cells on the proximal stump, began to up-reg- ulate at 1 day after injury and was still evident at 5 days. Compared with the control group, Notchl mRNA was expressed at a higher level in the conduit bridging group during the first week on the proximal stump. Hesl mRNA levels in the conduit bridging group significantly increased compared with the control group at 3, 5, 7 and 14 days post-surgery. The change of the Notch intracellular domain shared a simi- lar trend as Hesl mRNA expression. Our results confirmed that phenotypic changes of Schwann cells occurred in the proximal stump. The differences in these changes between the conduit tubulization and epineurium suture groups correlate with changes in Notch signaling. This suggests that active Notch signaling might be a key mechanism during the early stage of neural regeneration in the proximal nerve stump.

Effect of growth hormone on small intestinal homeostasis relation to cellular mediators IGF-I and IGFBP-3

AIM:To evaluate the effects of growth hormone(GH) on the histology of small intestines which might be related to the role of insulin like growth factor(IGF)-I, IGF-binding protein 3(IGFBP-3)and its receptors. METHODS:Twelve week-old adult male Wistar albino rats were divided into two groups.The study group(n =10),received recombinant human growth hormone (rGH)at a dose of 2 mg/kg per day subcutaneously for 14 d and the control group(n=10)received physiologic serum.Paraffin sections of jejunum were stained with periodic acid shift(PAS)and hematoxylin and eosin(HE) for light microscopy.They were also examined for IGF-I, IGFBP-3 and IGF-receptor immunoreactivities.Staining intensity was graded semi-quantitatively using the HS- CORE. RESULTS:Goblet cells and the cells in crypt epitheliawere significantly increased in the study group compared to that of the control group.We have demonstrated an increase of IGF-I and IGFBP-3 immunoreactivities in surface epithelium of the small intestine by GH application.IGF-I receptor immunoreactivities of crypt,villous columnar cells,enteroendocrine cells and muscularis mucosae were also more strongly positive in the study group compared to those of in the control group. CONCLUSION:These findings confirm the important trophic and protective role of GH in the homeostasis of the small intestine.The trophic effect is mediated by an increase in IGF-I synthesis in the small intestine, but the protective effect is not related to IGF-I.

Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation

INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but failed.We introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinalepithelial cell by electron microscopy,immunihistochemistry and enzymatic histoch-emistry. It has been applied to some relatedresearch work[17-21]. It was found that manyfactors were involved in the culture system. Ourpresent study focuses on the culture method and theinfluencing factors on IEC-6.

Cross-talk between Smad4 and P38 Proteins in Non-small Cell Lung Cancer

Objective： Impaired signal transduction is associated with tumorigenesis and progression of various kinds of human cancers. Transforming growth factor （TGF）-beta/Smad and ras-mitogen activated protein kinase （MAPK） are two major signal transduction pathways for adjusting cell proliferation and differentiation. Little is known about TGF-beta/Smad4 in non-small cell lung cancer （NSCLC）. Hereby, we investigated the expression of Smad4 in NSCLC, its correlation with MAPK proteins （including p38, ERK1 and JNK1 proteins） and their clinical significance in NSCLC. Methods： The expressions of Smad4, p38, ERK1 and JNK1 were detected at protein level with Western blotting and immunohistochemistry, at transcription level with RT-PCR. Statistical analysis was performed for the comparisons of expressions of Smad4, p38, ERK1 and JNK1, and their correlation with various clinicopathological parameters and the prognosis of NSCLC. Results： The levels of protein and mRNA expression of Smad4 in lung cancer tissues were significantly lower than in normal tissues （P〈0.05）. All these four proteins were associated with TNM staging. There was a strongly negative correlation between p38 and Smad4. Expressions of Smad4, p38 and JNK1, as well as tumor differentiation and staging were significantly correlated with the prognosis of NSCLC by univariate analysis. By multivariate analysis, only Smad4, p38, tumor differentiation and staging were correlated with the prognosis. Taken together, the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC. Conclusion： Smad4 could be of vital importance for the initiation and development of NSCLC. The expression of Smad4 might be inhibited by p38, supporting a cross-talk between main proteins of TGF-beta/Smad and ras-MAPK signal transduction pathways. Smad4 and p38 could be possible prognostic factors for NSCLC.

Heat Treatment of Small Heat Shock Proteins α-Crystallin and Hsp16.3: Structural Changes vs. Chaperone-like Activity

Both α crystallin from bovine eye lens and Hsp16.3 from Mycobacterium tuberculosis are members of the small heat shock protein family. They were preincubated at 100 ℃ for 15 min and then cooled on ice immediately. The chaperone like activities of preheated proteins were measured at 37 ℃ using DTT treated insulin B chains as substrates. Both preheated proteins exhibited greatly enhanced chaperone like activities, accompanied with almost unchanged secondary structures and surface hydrophobicity but with a minor change in tertiary structures. The dramatically enhanced chaperone like activities of preheated α crystallin and Hsp16.3 may have resulted from the irreversible change in the tertiary structure as detected by near UV CD spectra.

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.

Overexpression of a tobacco small G protein gene NtRop1 causes salt sensitivity and hydrogen peroxide production in transgenic plants

The small GTPases of Rop/Rho family is central regulators of important cellular processes in plants. Tobacco small G protein gene NtRop1 has been isolated; however, its roles in stress responses were unknown. In the present study, the genomic sequence of NtRop1 was cloned, which has seven exons and six introns, similar to the Rop gene structure from Arabidopsis. The NtRop1 gene was constitutively expressed in the different organs whereas the other six Rop genes from tobacco had differential expression patterns. The expression of the NtRop1 gene was moderately induced by methyl viologen, NaCl, and ACC treatments, but slightly inhibited by ABA treatment, with no significant induction by NAA treatment. The transgenic Arabidopsis plants overexpressing the NtRop1 showed increased salt sensitivity as can be seen from the reduced root growth and elevated relative electrolyte leakage. The hydrogen peroxide production was also promoted in the NtRop1-trangenic plants in comparison with wild type plants. These results imply that the NtRop1 may confer salt sensitivity through activation of H2O2 production during plant response to salt stress.