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Mpox (MPX), a zoonotic infection reported as early as 1970, is a double-stranded DNA virus, Mpox virus (MPXV), belonging to the genus Orthopoxvirus. MPX, a virus closely related to smallpox, was once considered a geographically restricted infection because it was predominantly endemic in West and Central Africa, except for the Democratic Republic of the Congo (DRC). However, in May 2022, a rare case of MPX was diagnosed in the UK. As of February 25, 2023, 86,127 confirmed cases and 97 deaths have been reported globally. It spreads through close contact with an infected person, infected animal, or an object which an infected person has touched. Although most people infected with MPXV may present with mild symptoms and will recover fully within a few weeks, people can have serious diseases and complications and may need to be hospitalized with it. Although smallpox vaccines and antiviral drugs are also effective against MPX, there is still no specific vaccine or drug for MPXV infection. Therefore, we need to take the right attitude and measures to stop the MPXV outbreak. In this review, we summarize the characteristics, mechanisms, immunology, and transmission of MPXV, as well as current vaccines, diagnostics, and treatments used for MPX, and provide clues for controlling MPX outbreaks and preventing such diseases.

Background Ixodes granulatus is of significant research value as the primary vector of Lyme disease in Asia, yet the bacterial community structure and diversity carried by these ticks have not been reported so far. Part of bacteria in ticks can affect tick development and the transmission of tick-borne pathogens. With the emergence of new pathogens, it is necessary to identify the bacterial microbiome carried by I. granulatus. Methods Ixodes granulatus were collected from rodent animals in the Binchuan, China. The bacterial DNA was extracted from non-engorged adult female I. granulatus. Sequencing of the V3-V4 regions of the 16S rRNA genes was performed using the Illumina NovaSeq sequencing platform. Initial data was assembled in FLASH, QIIME, and UCHIME algorithm. Uparse and MUSCLE software were used to annotate and analyze the effective data. Result The predominant phyla in all samples were Firmicutes, Proteobacteria, Actinobacteria, and Spirochaetota. The major genera were Spiroplasma, Staphylococcus, Candidatus-Lariskella, Corynebacterium, Ralstonia, Borreliella (Borrelia), Vibrio, Bacillus, and Listeria. Staphylococcus xylosus, Ralstonia pickettii, Corynebacterium mastitidis, Bryopsis hypnoides, and Vibrio metschnikovii were dominant bacterial species. The results demonstrated that there were discrepancies in bacterial microbiome richness and diversity among different individual ticks of the same species, and found a large number of pathogenic bacteria and opportunistic pathogens, yet Coxiella and Rickettsia were not detected. Conclusion The results presented here speculate that Spiroplasma is the endosymbiont of I. granulatus and competitively inhibits Borrelia. Our study suggested that Binchuan is at risk of Lyme disease transmission and needs to be taken seriously. These findings may serve fundamental knowledge for developing novel strategies to control ticks and their transmission of diseases.

Shoulder disorders, including frozen shoulder, bursitis, and rotator cuff lesions, are common musculoskeletal problems in patients with Parkinson disease (PD). Because musculoskeletal ultrasound (US) can clearly image shoulder joints, we aimed to evaluate shoulder joints using US in patients with PD and healthy participants and correlation between US and PD severity.This is a prospective case-control study. 50 patients with PD and 50 healthy subjects from the outpatient department were administered US for bilateral shoulders. For data analysis, we chose the more severely affected side in the PD group for matching with the corresponding shoulder in the control group according to age, sex, and body mass index. Pain and disability were measured using the Visual Analogue Scale (VAS) for pain, Shoulder Pain and Disability Index (SPADI), and the Shoulder Disability Questionnaire (SDQ).The PD group had higher VAS pain scores during activity (p = 0.003) and rest (p 0.001), as well as the SPADI and SDQ scores (p 0.001). In US findings, biceps long head tendon sheath effusion (p = 0.001), humeral head cortical irregularity (p = 0.012), and abnormality in the supraspinatus tendon (p = 0.003) were significantly greater in the PD group. Intra-group analysis in the PD group demonstrated a significant difference in passive flexion (p = 0.019) and supraspinatus tendinopathy (p = 0.033) on US examination during different disease stages.Patients with PD had more supraspinatus tendinopathy on US findings than control subjects. The lesion was significantly associated with disease severity.NCT02702232.

Objective To investigate whether combination of corticosteroid subdeltoid injections and physiotherapy was more effective than either treatment alone in chronic subacromial bursitis. Design Prospective, three-arm randomised controlled trial. Setting Rehabilitation department of an academic hospital. Subjects Patients with chronic subacromial bursitis. Interventions Patients were divided into corticosteroid injection (N = 36), physiotherapy (N = 40) and combined (N = 35) groups. Two corticosteroid subdeltoid injections in corticosteroid group, 8-week physical therapy emphasising on therapeutic exercise in physiotherapy group, and combined both treatments in combined group. Main outcome measures The primary outcome measures were pain visual analogue scale and Shoulder Pain and Disability Index at 8 weeks after finishing treatment. The secondary outcome measures were active range of motion, Shoulder Disability Questionnaire, Western Ontario Rotator Cuff Index, patient's evaluation of treatment effect, and symptom recurrence. Results Group comparison showed significant statistical difference in shoulder flexion ( P Conclusion Corticosteroid subdeltoid injection, or combined with physiotherapy, was superior to physiotherapy alone, but the recurrence rate was least in the physiotherapy group.

During mammary development, the transdifferentiation of mammary preadipocytes is one of the important sources for lactating mammary epithelial cells (MECs). However, there is limited knowledge about the mechanisms of dynamic regulation of transcriptome and genome-wide DNA methylation in the preadipocyte transdifferentiation process. Here, to gain more insight into these mechanisms, preadipocytes were isolated from adipose tissues from around the goat mammary gland (GM-preadipocytes). The GM-preadipocytes were cultured on Matrigel in conditioned media made from goat MECs to induce GM-preadipocyte-to-MEC transdifferentiation. The transdifferentiated GM-preadipocytes showed high abundance of keratin 18, which is a marker protein of MECs, and formed mammary acinar-like structures after 8 days of induction. Then, we performed transcriptome and DNA methylome profiling of the GM-preadipocytes and transdifferentiated GM-preadipocytes, respectively, and the differentially expressed genes and differentially methylated genes that play underlying roles in the process of transdifferentiation were obtained. Subsequently, we identified the candidate transcription factors in regulating the GM-preadipocyte-to-MEC transdifferentiation by transcription factor-binding motif enrichment analysis of differentially expressed genes and differentially methylated genes. Meanwhile, the secretory proteome of GM-preadipocytes cultured in conditioned media was also detected. By integrating the transcriptome, DNA methylome, and proteome, three candidate genes, four proteins, and several epigenetic regulatory axes were further identified, which are involved in regulation of the cell cycle, cell polarity establishment, cell adhesion, cell reprogramming, and adipocyte plasticity. These findings provide novel insights into the molecular mechanism of preadipocyte transdifferentiation and mammary development.

The bacterium Aeromonas salmonicida is the pathogen responsible for furunculosis, which is a serious disease of salmonids. This disease has a significant economic impact on the economic benefits of the global salmon farming industry. However, the pathogenesis of this disease in fish is still unknown. Members of the aldehyde dehydrogenase gene (ALDH) superfamily play a key role in the enzyme detoxification of endogenous and exogenous aldehydes. In this study, we obtained a recombinant aldehyde dehydrogenase 7A1 (ALDH7A1) protein to find its functions on Atlantic salmon infected by A. salmonicida. The transcriptional response in the liver of Atlantic salmon (Salmo salar) with differing levels of A. salmonicida infection was analysed and compared in order to reveal mechanisms by which ALDH7A1 may confer infection resistance. With the addition of ALDH7A1 protein, it was found that a total of 13,369 genes were annotated with one or more KEGG and localized to 360 KEGG pathways in the high concentration infection group. The differential expression genes were more enriched in immune signalling pathways such as the Toll-like receptor signalling pathway, NF-kappa B signalling pathway and TNF signalling pathway. On the other hand, at low concentrations of infection, KEGG enriched a smaller number of differential expression genes. However, these differential genes were more concentrated in immune signalling pathways such as the PI3K-Akt signalling pathway, JAK-STAT signalling pathway and complement and coagulation cascades. In addition, several known immune-related genes including HSP90α, HSP70, DNA damage-inducible transcript 4, integrin alpha 5 and microtubule-associated protein 2 were among the differentially expressed transcripts. These data provide the first insights into the host-ALDH7A1 vaccine interactome. The results of this study contribute to identifying the potential resistance mechanisms of Atlantic salmon to A. salmonicida infection and determining future treatment strategies.

In this study, we isolated and characterized HSP70 cDNA from pufferfish (Takifugu rubripes). The 3053 bp full-length TrHSP70 sequence consisted of a 167 bp 5′-UTR (untranslated region), a 2535 bp open reading frame, and a 351 bp 3′-UTR. BLAST analysis revealed that the TrHSP70 shared high similarity with HSP70 sequences in other species. In our study, we set 3 experimental groups as H1 group (20 °C), H2 group (24 °C), and H3 group (28 °C) for checking the expression level of TrHSP70 in T. rubripes. Tissue-specific gene expression results showed that TrHSP70 had higher expression in the intestines than other tissues of the T. rubripes by RT-qPCR. In the experimental group, we found that the expression of TrHSP70 was upregulated in different tissues in the H3 group. The results show that TrHSP70 is a constitutively expressed gene, which plays an important role in maintaining normal physiological function and coping with stress.

Takifugu rubripes is a commercially important fish in the Chinese aquaculture industry due to its excellent meat quality. In eukaryotic cells, heat shock proteins (HSPs) play a crucial role in maintaining intracellular protein homeostasis. They play significant roles in response to adverse environmental stresses in aquatic animals. In this study, we used bioinformatics to comprehensively compare two different full-length cDNA sequences at the molecular level in the HSP family of T. rubripes. We performed various physicochemical studies such as race cloning and RT-qPCR, and found that TrHSP60 (575 amino acids) has a larger polypeptide, followed by TrHSP70–13 (441 amino acids). Homology and phylogenetic studies showed that these sequences have the greatest homology to Takifugu bimaculatus. The mRNA of the two genes were constitutively expressed in all tested tissues of untreated T. rubripes, including brain, muscle, kidney, liver, spleen, gill, and intestine, with highest expression levels in the intestine. Three experimental groups were established in this study, the H1 group (20 °C), H2 group (24 °C) and H3 group (28 °C), in order to analyze the expressed levels of TrHSP70 in T. rubripes. To understand the role of HSPs in temperature stress in T. rubripes, we analyzed the expression levels of HSPs in T. rubripes at different temperatures. The results showed that the mRNA expression of the HSP gene were influenced by temperature stress. Gene expression of TrHSP70 and TrHSP60 were mainly up-regulated at 12 h and 48 h in the H1 group, respectively. Meanwhile, gene expression of TrHSP70 was highest at 48 h in the H3 group and TrHSP60 reached was lowest at 24 h in the H3 group. The results showed that the two genes were constitutively expressed genes, which play an important role in maintaining normal physiological function and stress responses. The results show that TrHSP70 and TrHSP60 are constitutively expressed genes, which plays important role in maintaining normal physiological function and coping with stress.

Aeromonas salmonicida (A. salmonicida) is a pathogenic bacterium that causes serious problems in the global Atlantic salmon aquaculture industry. In this study, we comprehensively analyzed the profiles of lncRNAs, miRNAs and mRNAs in gills of Atlantic salmon at high-dose A. salmonicida infection (3.06 × 10

Renal fibrosis is an inevitable outcome of various manifestations of progressive chronic kidney diseases (CKD). The need for efficacious treatment regimen against renal fibrosis can therefore not be overemphasized. Here we show a novel protective role of Bacteroides fragilis (B. fragilis) in renal fibrosis in mice. We demonstrate decreased abundance of B. fragilis in the feces of CKD patients and unilateral ureteral obstruction (UUO) mice. Oral administration of live B. fragilis attenuates renal fibrosis in UUO and adenine mice models. Increased lipopolysaccharide (LPS) levels are decreased after B. fragilis administration. Results of metabolomics and proteomics studies show decreased level of 1,5-anhydroglucitol (1,5-AG), a substrate of SGLT2, which increases after B. fragilis administration via enhancement of renal SGLT2 expression. 1,5-AG is an agonist of TGR5 that attenuates renal fibrosis by inhibiting oxidative stress and inflammation. Madecassoside, a natural product found via in vitro screening promotes B. fragilis growth and remarkably ameliorates renal fibrosis. Our findings reveal the ameliorative role of B. fragilis in renal fibrosis via decreasing LPS and increasing 1,5-AG levels.

Objective: To investigate the effects of adjuvant danhong injection therapy on nerve injury and platelet activation markers in patients with acute cerebral infarction. Methods: A total of 102 patients with acute cerebral infarction who were treated in our hospital between January 2016 and September 2017 were reviewed and divided into the routine group (n=54) who received conventional therapy and the danhong injection group who received adjuvant danhong injection therapy. The differences in the contents of nerve injury indexes in serum and platelet activation markers in peripheral blood were compared between the two groups before treatment, after 3 d of treatment and after 7 d of treatment. Results: There was no statistically significant difference in the contents of nerve injury indexes in serum and platelet activation markers in peripheral blood between the two groups before treatment. After 3 d of treatment and after 7 d of treatment, copeptin, H-FABP and NSE contents in serum of danhong injection group were lower than those of routine group whereas BDNF and bFGF contents were higher than those of routine group; CD62p, CD42b, PAC-1 and PMA contents in peripheral blood were lower than those of routine group. Conclusion: Conventional therapy combined with adjuvant danhong injection therapy can effectively reduce the degree of nerve injury and inhibit the platelet activation in patients with acute cerebral infarction.

Takifugu rubripes and Dicentrarchus labrax are important commercial fish in China that are under serious threat from Cryptocaryon irritans. C. irritans is a ciliated obligate parasite that causes marine white spot disease and leads to heavy economic losses. We analysed the transcriptome in the gills of T. rubripes and D. labrax to compare differentially expressed genes (DEGs) and pathways during infection with C. irritans. In total, we identified 6,901 and 35,736 DEGs from T. rubripes and D. labrax, respectively. All DEGs were annotated into GO terms; 6,901 DEGs from T. rubripes were assigned into 991 sub-categories, and 35,736 DEGs from D. labrax were assigned into 8,517 sub-categories. We mapped DEGs to the KEGG database and obtained 153 and 350 KEGG signalling pathways from T. rubripes and D. labrax, respectively. Immune-related categories included Toll-like receptors, MAPK, lysosome, C-type lectin receptor and NOD-like receptor signalling pathways were significantly enriched pathways. In immune-related signalling pathways, we found that AP-1, P38, IL-1β, HSP90 and PLA were significantly up-regulated DEGs in T. rubripes, but P38 and PLA were significantly down-regulated in D. labrax. In this study, transcriptome was used to analyse the difference between scaly and non-scaly fish infection by C. irritans, which not only provided a theoretical basis for the infection mechanism of C. irritans, but also laid a foundation for effectively inhibiting the occurrence of this disease. Our work provides further insight into the immune response of host resistance to C. irritans.

Objective: To study the changes of microelement content and erythrocyte immune function in patients with cerebral infarction complicated by pneumonia and their relationship with infection indexes. Methods: Patients with cerebral infarction complicated by pneumonia who were treated in Huangshi Central Hospital between April 2014 and February 2017 were selected as the infection group of the research, and patients with cerebral infarction who were treated in Huangshi Central Hospital during the same period were selected as the control group of the research. Serum was collected to determine the contents of trace elements Zn, Fe, Cu, Se and Ca as well as infection index PCT, and peripheral blood was collected to determine the erythrocyte immune function indexes. Results: Serum PCT content of infection group was greatly higher than that of control group. Serum zinc and Fe contents as well as peripheral blood RBC-ICR, RBC-C3bR, FEER, CD58 and CD59 levels of infection group were greatly lower than those of control group, FEIR level in peripheral blood was obviously higher than that of control group, and Cu, Se and Ca contents were not greatly different from those of control group; serum zinc and Fe contents as well as peripheral blood RBC-ICR, RBC- C3bR, FEER, CD58 and CD59 levels of infection group of patients with high PCT content were remarkably lower than those of patients with low PCT content, FEIR level in peripheral blood was significantly higher than that of patients with low PCT content, and Cu, Se and Ca contents were not greatly different from those of patients with low PCT content. Conclusion: The deficiency of microelements Zn and Fe as well as the weakening of erythrocyte immune function is closely related to the degree of infection in the patients with cerebral infarction complicated by pneumonia.

Purpose: To compare the effect of unloading knee brace with physical therapy (PT) in Asian patients with osteoarthritis (OA) of the knee. Method: This is a non-random, two-group comparative study. Patients with medial compartment knee OA (n = 41) were assigned to either the brace group (n = 20) or PT group (n = 21). Patients in the brace group were fitted with an unloading knee brace for three months and the PT group received a 60-min session of physiotherapy over the affected knee, three times a week, for three months. The primary outcome measures were the pain visual analogue scale (VAS) and the Western Ontario McMaster University Osteoarthritis Index (WOMAC); the second outcome measures were the 36-item Short-Form Health Survey (SF-36) and patient's satisfaction. The patients were evaluated at baseline, and at one month and three months. Results: Group comparison showed no significant difference regarding pain VAS, WOMAC, SF-36, and patient's satisfaction, except stiffness in WOMAC (P = .006) and social functioning in SF-36 (P = .007). Time and group interaction revealed significant differences only in general health (P = .007) and mental health (P = .006) of SF-36. Within-group comparison found that pain VAS and WOMAC decreased significantly at one months and three months in both groups. Conclusion: The effect of brace fitting in patients with knee OA was similar to that of physical therapy. A Western-made unloading knee brace is acceptable in some Asian people with knee OA. Clinical trial registration number: NCT02712710. Keywords: Knee osteoarthritis, Knee brace, Therapeutic exercise, Treatment

Objective The aim of the study was to compare the effects of corticosteroid injection with lidocaine injection in treating tennis elbow. Design It is a prospective, double-blinded, randomized controlled trial. Patients with tennis elbow for more than 1 mo were recruited from a hospital-based rehabilitation outpatient clinic. A total of 70 patients were recruited, and 61 patients completed the study. Patients received an injection of either 10 mg (1 ml) of triamcinolone (corticosteroid group, n = 30) or 1 ml of 1% lidocaine (lidocaine group, n = 31). All of the outcome measures were evaluated before the intervention and at 2 wks and 2 mos after treatment. Results No significant group differences were observed between the corticosteroid and lidocaine groups regarding Patient-Rated Tennis Elbow Evaluation, Disability of the Arm, Shoulder, and Hand, visual analog scale for pain, and grip strength at baseline and at 2 wks and 2 mos after treatment (P > 0.05). However, within-group comparison showed significant improvement after injection with regard to Patient-Rated Tennis Elbow Evaluation, Disability of the Arm, Shoulder, and Hand, visual analog scale for pain, and grip strength in both groups (P > 0.05). Conclusions No differences in the short-term outcomes were found between lidocaine and corticosteroid injection in a small sample of people with tennis elbow with mean duration of 3.8 mos.

Background Colorectal cancer (CRC) is one of the leading cancers worldwide. Several studies have performed microarray data analyses for cancer classification and prognostic analyses. Microarray assays also enable the identification of gene signatures for molecular characterization and treatment prediction. Objective Microarray gene expression data from the online Gene Expression Omnibus (GEO) database were used to to distinguish colorectal cancer from normal colon tissue samples. Methods We collected microarray data from the GEO database to establish colorectal cancer microarray gene expression datasets for a combined analysis. Using the Prediction Analysis for Microarrays (PAM) method and the GSEA MSigDB resource, we analyzed the 14,698 genes that were identified through an examination of their expression values between normal and tumor tissues. Results Ten genes (ABCG2, AQP8, SPIB, CA7, CLDN8, SCNN1B, SLC30A10, CD177, PADI2, and TGFBI) were found to be good indicators of the candidate genes that correlate with CRC. From these selected genes, an average of six significant genes were obtained using the PAM method, with an accuracy rate of 95%. The results demonstrate the potential of utilizing a model with the PAM method for data mining. After a detailed review of the published reports, the results confirmed that the screened candidate genes are good indicators for cancer risk analysis using the PAM method. Conclusions Six genes were selected with 95% accuracy to effectively classify normal and colorectal cancer tissues. We hope that these results will provide the basis for new research projects in clinical practice that aim to rapidly assess colorectal cancer risk using microarray gene expression analysis.

Given advancements in cancer immunity, cancer treatment has gained breakthrough developments. Immune checkpoint inhibitors, such as programmed cell death 1 (PD-1) inhibitors, are the most promising drugs in the field and have been approved to treat various types of cancer, such as metastatic melanoma, head and neck squamous cell carcinoma, and urothelial carcinoma. However, whether PD-1 inhibitors should be administered to renal transplant patients with advanced cancer remains unclear because the T-cells produced after administration of these inhibitors act against not only tumor antigens but also donor alloantigens. Thus, the use of PD-1 inhibitors in kidney-transplanted patients with advanced cancer is limited on account of the high risk of graft failure due to acute rejection. Hence, finding optimal treatment regimens to enhance the tumor-specific T-cell response and decrease T-cell-mediated alloreactivity after administration of a PD-1 inhibitor is necessary. Thus far, no recommendations for the use of PD-1 inhibitors to treat cancer in renal transplant patients are yet available, and very few cases reporting kidney-transplanted patients treated with PD-1 inhibitors are available in the literature. Therefore, in this work, we review the published cases and suggest feasible approaches for renal transplant patients with advanced malignancy treated by a PD-1 inhibitor. Of the 22 cases we obtained, four patients maintained intact grafts without tumor progression after treatment with a PD-1 inhibitor. Among these patients, one maintained steroid dose before initiation of anti-PD1, two received immunosuppressive regimens with low-dose steroid and calcineurin inhibitor (CNI)-elimination with sirolimus before initiation of anti-PD-1 therapy, and one received combined anti-PD-1, anti-vascular endothelial growth factor (VEGF), and chemotherapy with unchanged immunosuppressive regimens. mammalian target of rapamycin (mTOR) inhibitors and anti-VEGF may act as regulators of tumor-specific and allogenic T-cells. However, more studies are necessary to explore the optimal therapy and ensure the safety and efficacy of PD-1 inhibitors in kidney-transplanted patients.

Summary Avoparcin, a vancomycin analogue, was banned as a feed additive in Taiwan in 2000. A nationwide surveillance was conducted to study the prevalence of vancomycin-resistant enterococci (VRE) on chicken farms between 2000 and 2003. Among the 1021 E. faecalis and 967 E. faecium isolates studied, resistance to tetracycline, erythromycin, high-level aminoglycosides, ciprofloxacin and chloramphenicol either increased or remained high except vancomycin. The proportion of VRE decreased, between 2000 and 2003, from 13.7% (22/161) to 3.7% (11/299) for E. faecalis, and 3.4% (4/119) to 0% (0/300) for E. faecium. Only 8.8% (7/80) of the chicken farms surveyed harboured VRE in 2003 compared with 25% (15/60) in 2000. All VRE were resistant to tetracycline and erythromycin. All VRE possess the vanA gene but nearly all (79 of 83 isolates) were susceptible to teicoplanin, indicating VanB phenotype. Some clones were detected from different farms in various regions over the years. We conclude that the frequency of VRE in chicken farms decreased in association with a ban on avoparcin; and the continued presence of VRE may be due to the ability of some strains to persist in the farms, transfer of vancomycin resistance determinants or co-selection by the continued use of other antibiotics.

Gulf war illnesses (GWI) are currently affecting thousands of veterans. To date, the molecular mechanisms underlying the pathogenesis of these illnesses remain unknown. During Gulf war I, military personnel were exposed to multiple stressors, one or more vaccines, pyridostigmine (PY), and other chemicals. In our previous studies, we found that stress induces activation of mitogen activated protein-kinase kinase 4 (MKK4) and c-Jun-N-terminal kinase (JNK) in the mouse brain (Liu et al. 2004). Our working hypothesis is that stress, vaccination, and PY may synergistically induce activation of MKK4 and JNK in the brain, leading to over-activation of these kinases and neurological injuries. To test our hypothesis, we examined the effect of keyhole limpet hemocyanin (KLH) immunization alone or in combination with PY on activation of MKK4 and JNK induced by stress. We found that KLH immunization alone had a small effect on MKK4 or JNK activity but it significantly enhanced and prolonged activation of these kinases induced by stress, from a few hours to several days. Additionally, KLH immunization caused activation of p38MAPK. PY treatment further enhanced and prolonged activation of these kinases induced by stress in combination with KLH immunization and triggered activation of caspase-3. Our current studies suggest that stress, vaccination, and PY may synergistically act on multiple stress-activated kinases in the brain to cause neurological impairments in GWI.

Stress is a part of daily life. However, molecular mechanisms underlying the activation of limbic-hypothalamic-pituitary-adrenal (LHPA) axis remains unknown. In this study, we explored whether activation of the mitogen-activated kinase kinase 4 (MKK4)-c-Jun-N-terminal kinase (JNK) signaling pathway may play a role in the activation of the LHPA axis. We found that forced-swim stress induced elevation of activated MKK4 in the hippocampal formation, amygdala, and hypothalamus. Unlike MKK4, a high basal level of JNK activity is present in many brain areas of unstressed mice. Forced-swim stress significantly elevated JNK activity in the hypothalamus and amygdala and, to a lesser extent, in the cortex, CA1 and CA3 regions, and the dentate gyrus. To further investigate the role of MKK4 and JNK in induction of stress responses, we investigated whether a different stress, namely, restraint stress, induced activation of MKK4 or JNK in the brain. We found that restraint stress also induced elevation of activated MKK4 and JNK in the hippocampal formation, amygdala, and hypothalamus. Because MKK4 and JNK were activated within 5 min following stress, we propose that the MKK4-JNK signaling may be an early neural event in the initiation of neuroendocrine, autonomic and behavioral stress responses.

In our previous studies, we found that expression of polyglutamine-expanded huntingtin in HN33 cells induced sensitization of N-methyl-D-aspartate (NMDA) receptors (Sun, Y., Savinainen, A., and Liu, Y. F. (2001) J. Biol. Chem. 276, 24713-24718). Following this study, we investigated whether tyrosine phosphorylation of NMDA receptors might contribute to the altered property of the receptors. Expression of polyglutamine-expanded huntingtin induced elevation of phosphorylated or activated Src and increased targeting of PSD-95 (post-synaptic density 95) and activated Src to cell surface membrane. Expression of the mutated huntingtin also induced tyrosine phosphorylation of NR2B (NMDA receptor 2B) subunits, and co-expression of PSD-95 enhanced the phosphorylation. Treatment of SU6656 (a specific Src inhibitor) or co-expression of a mutated NR2B subunit with mutations of all three major tyrosine phosphorylation sites significantly attenuated neuronal toxicity induced by the mutated huntingtin. Addition of AP-5 did not further inhibit the neuronal toxicity. Taken together, our studies show that polyglutamine-expanded huntingtin increases tyrosine phosphorylation of NMDA receptors via PSD-95 and Src, and increased tyrosine phosphorylation may contribute to the sensitization of the receptors mediated by polyglutamine-expanded huntingtin.

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial beta-amyloid precursor protein APP-V642I (IAPP) or mutant presenilin-1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild-type amyloid precursor protein or presenilin-1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with beta-amyloid (Abeta) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a gamma-secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Abeta peptide induces actin stress fibers. Because activation of the p38 mitogen-activated protein kinase (p38MAPK)-mitogen-associated protein kinase-associated protein kinase (MAPKAPK)-2-heat-shock protein 27 signaling pathway mediates actin polymerization, we explored whether Abeta peptide activates p38MAPK and MAPKAPK-2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK-2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Abeta peptide, IAPP or mPS1. Moreover, treatment with a gamma-secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK-2. In summary, our data suggest that overproduction of Abeta peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.

Kainate receptor glutamate receptor 6 (GluR6) subunit-deficient and c-Jun N-terminal kinase 3 (JNK3)-null mice share similar phenotypes including resistance to kainite-induced epileptic seizures and neuronal toxicity (Yang, D. D., Kuan, C-Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakis, P., and Flavell, R. (1997) Nature 389, 865-869; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). This suggests that JNK activation may be involved in GluR6-mediated excitotoxicity. We provide evidence that post-synaptic density protein (PSD-95) links GluR6 to JNK activation by anchoring mixed lineage kinase (MLK) 2 or MLK3, upstream activators of JNKs, to the receptor complex. Association of MLK2 and MLK3 with PSD-95 in HN33 cells and rat brain preparations is dependent upon the SH3 domain of PSD-95, and expression of GluR6 in HN33 cells activated JNKs and induced neuronal apoptosis. Deletion of the PSD-95-binding site of GluR6 reduced both JNK activation and neuronal toxicity. Co-expression of dominant negative MLK2, MLK3, or mitogen-activated kinase kinase (MKK) 4 and MKK7 also significantly attenuated JNK activation and neuronal toxicity mediated by GluR6, and co-expression of PSD-95 with a deficient Src homology 3 domain also inhibited GluR6-induced JNK activation and neuronal toxicity. Our results suggest that PSD-95 plays a critical role in GluR6-mediated JNK activation and excitotoxicity by anchoring MLK to the receptor complex.

MAP kinase signaling pathways are important mediators of cellular responses to a wide variety of stimuli. Signals pass along these pathways via kinase cascades in which three protein kinases are sequentially phosphorylated and activated, initiating a range of cellular programs including cellular proliferation, immune and inflammatory responses, and apoptosis. One such cascade involves the mixed lineage kinase, MLK2, signaling through MAP kinase kinase 4 and/or MAP kinase kinase 7 to the SAPK/JNK, resulting in phosphorylation of transcription factors including the oncogene, c-jun. Recently we showed that MLK2 causes apoptosis in cultured neuronal cells and that this effect is dependent on activation of the JNK pathway (Liu, Y. F., Dorow, D. S., and Marshall, J. (2000) J. Biol. Chem. 275, 19035-19040). Furthermore, dominant-negative MLK2 blocked apoptosis induced by polyglutamine-expanded huntingtin protein, the product of the mutant Huntington's disease gene. Here we show that as well as activating the stress-signaling pathway, MLK2 is a target for phosphorylation by activated JNK. Phosphopeptide mapping of MLK2 proteins revealed that activated JNK2 phosphorylates multiple sites mainly within the noncatalytic C-terminal region of MLK2 including the C-terminal 100 amino acid peptide. In addition, MLK2 is phosphorylated in vivo within several of the same C-terminal peptides phosphorylated by JNK2 in vitro, and this phosphorylation is increased by cotransfection of JNK2 and treatment with the JNK activator, anisomycin. Cotransfection of dominant-negative JNK kinase inhibits phosphorylation of kinase-negative MLK2 by anisomycin-activated JNK. Furthermore, we show that the N-terminal region of MLK2 is sufficient to activate JNK but that removal of the C-terminal domain abrogates the apoptotic response. Taken together, these data indicate that the apoptotic activity of MLK2 is dependent on the C-terminal domain that is the main target for MLK2 phosphorylation by activated JNK.

Based on the presence of multiple proline-rich motifs in the huntingtin sequence, we tested its possible association with epidermal growth factor (EGF) receptor signaling complexes through SH3 domain-containing modules. We found that huntingtin is associated with Grb2, RasGAP, and tyrosine-phosphorylated EGF receptor. These associations are regulated by activation of the EGF receptor, suggesting that they may be part of EGF receptor-mediated cellular signaling cascade. In vitro binding studies indicate that SH3 domains of Grb2 or RasGAP are required for their binding to huntingtin. Our results suggest that huntingtin may be a unique adapter protein for EGF receptor-mediated signaling and may be involved in the regulation of Ras-dependent signaling pathways.

Claudin-6 is a protein component of tight junctions and its expression could downregulate the malignant phenotype of breast carcinoma. Here we investigated the mechanisms of claudin-6 induced human MCF-7 breast cancer cells apoptosis, invasion, and migration.Terminal deoxyribonucleotide transferase-mediated nick-end labeling assay and Annexin-V/PI double stain assay were carried out to evaluate apoptosis. Inhibitors of each pathway were used to inactivate the signaling pathways. The expression of claudin-6 and phosphate p38, Erk 1/2 and Akt protein levels was confirmed by Western blotting analysis. Invasive and migratory traits of claudin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay.Cells with high-level expression of claudin-6 had a higher rate of apoptosis than control cells. Western blotting assay showed that by contrast to control groups, p38 pathways were more activated in claudin-6 expressing cells. However, after inhibitor SB203580 treatment, the activation status could be significantly counteracted. Furthermore, by applying inhibitors to the apoptotic rate, invasive and migratory traits were also recovered in cells with claudin-6 expression.Claudin-6 may function through p38 mitogen-activated protein kinase pathway, of which inhibition may reverse claudin-6-induced cell apoptosis, invasion, and migration.

In rat pituitary GH4C1 cells, activation of transfected dopamine D2 receptors (long, D2L, or short, D2S, form) and endogenously expressed somatostatin and muscarinic M4 receptors induced inhibition of cAMP synthesis and of Bay K 8644-induced calcium entry via pertussis toxin-sensitive G proteins. To analyze the role of alpha 0 and alpha i2 in relaying of these signals, alpha 0 or alpha i2 antisense constructs were separately and stably transfected into GH4C1 cells. Reverse transcription-polymerase chain reaction and Western blot analyses indicated specific ablation of alpha 0 or alpha i2 in the antisense transfectant clones. Elimination of alpha 0 selectively abolished receptor-mediated inhibition of calcium entry. Notably, the action of dopamine D2L receptor was partially (about 30%) retained. By contrast, depletion of alpha i2 selectively impaired receptor-mediated inhibition of cAMP accumulation. Inhibition of basal cAMP synthesis by any of the four receptors studied was blocked in alpha i2-depleted clones. Additionally, dopamine D2L, somatostatin and muscarinic M4 receptor-mediated inhibition of vasoactive intestinal peptide-stimulated cAMP formation was also abolished. Remarkably, somatostatin even potentiated (by 30%) the action of vasoactive intestinal peptide in alpha i2-antisense clones. In contrast, the action of dopamine D2S receptor on stimulated cAMP synthesis remained largely unaltered. The results demonstrate that alpha 0 specifically triggers receptor-induced closure of calcium channels, whereas alpha i2 specifically mediates inhibition of adenylyl cyclase in GH4C1 cells. Furthermore, the data suggest that G1 protein specificity in receptor coupling to inhibition of adenylyl cyclase depends critically on the activity state of the enzyme. Moreover, the results indicate an essential difference in coupling of dopamine D2L and D2S receptors to G proteins.

Chemotactic factors bound to receptors of the seven-transmembrane domain family signal leukocytes through associated guanine nucleotide-binding (G) proteins. Human leukocytes of the HL-60 line, which express G protein-coupled receptors for leukotriene B4 (LTB4) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) after differentiation with vitamin D3 and transforming growth factor-beta, were transfected with expression plasmids containing antisense-oriented cDNAs encoding the alpha-chains of Go, Gi1, Gi2, and Gi3. Antisense mRNA for Go and Gi2 alpha-chains suppressed by over 80% the level of the respective G protein. Go-deficient HL-60 cells had depressed functional and intracellular calcium responses to LTB4 and fMLP, but no alterations in the responses of cyclic adenosine 3‘,5‘-monophosphate (cAMP). In contrast, HL-60 cells deficient in Gi2 lost only responses of the intracellular concentration of cAMP. Antisense mRNA suppression of distinct G proteins thus may delineate some transductional requirements for cellular responses.

To explore the role of estrogen in the regulation of the expression of claudin-6 and biological behavior in MCF-7 cells.RT-PCR and immunocytochemistry were conducted to analyze the expression and localization of claudin-6 in MCF-7 cells treated with 17β-estradiol. CCK-8 kit assay and Scratch Test were conducted to analyze the capability of proliferation and migration of 17β-estradiol treated MCF-7 cells.RT-PCR analysis and immunocytochemistry showed that 17β-estradiol induced a concentration-and time-dependent effect on claudin-6. At 5 nmol/L and at 24 h, 17β-estradiol treatment led to an increased level of claudin-6, which was located in the membrane of MCF-7 cells. CCK-8 analysis showed a significant decrease in the capability of proliferation of MCF-7 cells compared with the control group (P0.05). Cells Scratch Test showed decreased migration capability of MCF-7 cells compared with the control group (P0.05).17β-E2 might regulate the expression of claudin-6 and inhibit the proliferation and migration of MCF-7 cells. The inhibitory effects of 17β-E2 on growth and migration of MCF-7 cells may be mediated by claudin-6 expression regulation.

To explore the effect of over-expression of tight junction protein claudin-6 upon the biological characters of breast cancer cell line MCF-7.The MCF-7 subline expressing a high level of claudin-6 was established by transfection with a pcDNA3.1-claudin-6 expression vector. The expression of claudin-6 in mRNA and its protein level were confirmed by RT-PCR, Western blot and immunofluorescent assays. Then the effect of claudin-6 upon cell proliferation was examined by MTT assay. Colony-forming assays were used to examine 2-D and 3-D colony-forming capacities. Invasive and migratory traits of claudin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay. The structure and function of tight junctions in both parental and claudin-6 expression MCF-7 cells were evaluated by measuring transepithelial electrical resistance.Immunofluorescent assays showed that transfected cells expressed claudin-6 on their membrane. Cells with a high level expression of claudin-6 grew slowly than control cells. Anchorage-independent growth, invasive and migratory traits also decreased substantially in cells with claudin-6 expression; whereas the transepithelial electrical resistance increased in the claudin-6 transfected cells.The up-regulation of claudin-6 expression in MCF-7 breast cancer cells suppresses their malignant phenotypes with a correlation with the restoration of tight junction integrity. Claudin-6 may function as a cancer suppressor whose down-regulation contributes to the malignant progression of certain types of breast cancers.

Based on product design systematic and modularization, aiming at an unknown working environment, the research of snake robot creates a new field for bionic robot research. Based on the requirements such as small size, multi-degree of freedom, light, simple, reliable, cheap and easy to install and maintain, we developed a small snake robot. The robot is made up of the same modules and able to complete various movement methods in their special connection ways. The robot can move by serpentine locomotion and cross obstacles. With the small snake robot technology matures, it will be smaller and may into the human body for some diagnosis or treatment work.