Background: Mitragyna speciosa Korth or Kratom is widely used traditionally for its medicinal values. The major alkaloid content of kratom leaves is mitragynine, which binds to opioid receptors to give opioid-like effects. This study aimed to analyse the brain proteome of animals that displayed addictive behaviors. Design and Methods: Six groups (n=6-8) of rats made up of negative control, positive control using morphine (10 mg/kg), and treatment groups at low (1mg/kg) and high doses of mitragynine (30 mg/kg) for 1 and 4 days. The rats' behaviors were evaluated and subsequently the rats' brains were harvested for proteomic analysis that was performed by using 2D gel electrophoresis and LC/MS/MS. Results: The rats developed physical dependence only on day 4 following morphine and mitragynine (1 and 30mg/kg) treatments. Among the proteins that were up-regulated in treatment groups were four calcium-binding proteins, namely calretinin, F-actin, annexin A3 and beta-centractin. Conclusions: Upregulation of calretinin acted as low Ca2+ buffering upon the blockage of Ca2+ ion channel by mitragynine in the brain, which subsequently caused a reduction of GABA released and inversely increased the dopamine secretions that contributed to dependence indicators.
The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.
Cell migration and invasion are modulated by epithelial-to-mesenchymal transition (EMT) and the reverse MET process. Despite the detection of microRNA-362 (miR-362, both the miR-362-5p and -3p species) in cancers, none of the identified miR-362 targets is a mesenchymal or epithelial factor to link miR-362 with EMT/MET and metastasis. Focusing on the TGF-β/SMAD signaling pathway in this work, luciferase assays and western blot data showed that miR-362 targeted and negatively regulated expression of SMAD4 and E-cadherin, but not SNAI1, which is regulated by SMAD4. However, miR-362 knockdown also down-regulated SMAD4 and SNAI1, but up-regulated E-cadherin expression. Wound-healing and transwell assays further showed that miR-362 knockdown suppressed cell migration and invasion, effects which were reversed by over-expressing SMAD4 or SNAI1, or by knocking down E-cadherin in the miR-362 knockdown cells. In orthotopic mice, miR-362 knockdown inhibited metastasis, and displayed the same SMAD4 and E-cadherin expression profiles in the tumors as in the in vitro studies. A scheme is proposed to integrate miR-362 negative regulation via SMAD4, and to explain miR-362 positive regulation of SMAD4 via miR-362 targeting of known SMAD4 suppressors, BRK and DACH1, which would have resulted in SMAD4 depletion and annulment of subsequent involvement in TGF-β signaling actions. Hence, miR-362 both negatively and positively regulates SMAD4 expression in TGF-β/SMAD signaling pathway to suppress cell motility and invasiveness and metastasis, and may explain the reported clinical association of anti-miR-362 with suppressed metastasis in various cancers. MiR-362 knockdown in miR-362-positive cancer cells may be used as a therapeutic strategy to suppress metastasis.
Chronic psychological stress affects many body systems, including the skeleton, through various mechanisms. This review aims to provide an overview of the factors mediating the relationship between psychological stress and bone health. These factors can be divided into physiological and behavioural changes induced by psychological stress. The physiological factors involve endocrinological changes, such as increased glucocorticoids, prolactin, leptin and parathyroid hormone levels and reduced gonadal hormones. Low-grade inflammation and hyperactivation of the sympathetic nervous system during psychological stress are also physiological changes detrimental to bone health. The behavioural changes during mental stress, such as altered dietary pattern, cigarette smoking, alcoholism and physical inactivity, also threaten the skeletal system. Psychological stress may be partly responsible for epigenetic regulation of skeletal development. It may also mediate the relationship between socioeconomic status and bone health. However, more direct evidence is required to prove these hypotheses. In conclusion, chronic psychological stress should be recognised as a risk factor of osteoporosis and stress-coping methods should be incorporated as part of the comprehensive osteoporosis-preventing strategy.
Bone loss in women commences before the onset of menopause and oestrogen deficiency. The increase of follicle-stimulating hormone (FSH) precedes oestrogen decline and may be a cause for bone loss before menopause. This review summarizes the current evidence on the relationship between FSH and bone derived from cellular, animal and human studies. Cellular studies found that FSH receptor (FSHR) was present on osteoclasts, osteoclast precursors and mesenchymal stem cells but not osteoblasts. FSH promoted osteoclast differentiation, activity and survival but exerted negligible effects on osteoblasts. Transgenic FSHR or FSH knockout rodents showed heterogenous skeletal phenotypes. Supplementation of FSH enhanced bone deterioration and blocking of FSH action protected bone of rodents. Human epidemiological studies revealed a negative relationship between FSH and bone health in perimenopausal women and elderly men but the association was attenuated in postmenopausal women. In conclusion, FSH may have a direct action on bone health independent of oestrogen by enhancing bone resorption. Its effects may be attenuated in the presence of overt sex hormone deficiency. More longitudinal studies pertaining to the effects of FSH on bone health, especially on fracture risk, should be conducted to validate this speculation.
Menopause is the leading cause of osteoporosis for elderly women due to imbalanced bone remodelling in the absence of oestrogen. The ability of tocotrienol in reversing established bone loss due to oestrogen deficiency remains unclear despite the plenitude of evidence showcasing its preventive effects. This study aimed to investigate the effects of self-emulsified annatto tocotrienol (SEAT) on bone histomorphometry and remodelling in ovariectomised rats. Female Sprague Dawley rats (n=36) were randomly assigned into baseline, sham, ovariectomised (OVX) control, OVX-treated with annatto tocotrienol (AT) (60 mg/kg), SEAT (60 mg/kg) and raloxifene (1 mg/kg). Daily treatment given through oral gavage was started two months after castration. The rats were euthanised after eight weeks of treatment. Blood was collected for bone biomarkers. Femur and lumbar bones were collected for histomorphometry and remodelling markers. The results showed that AT and SEAT improved osteoblast numbers and trabecular mineralisation rate (p<0.05 vs untreated OVX). AT also decreased skeletal sclerostin expression in OVX rats (p<0.05 vs untreated OVX). Similar effects were observed in the raloxifene-treated group. Only SEAT significantly increased bone formation rate and reduced RANKL/OPG ratio (p<0.05 vs untreated OVX). However, no changes in osteoclast-related parameters were observed among the groups (p>0.05). In conclusion, SEAT exerts potential skeletal anabolic properties by increasing bone formation, suppressing sclerostin expression and reducing RANKL/OPG ratio in rats with oestrogen deficiency.
Introduction: Orchidectomy is currently the preferred method to induce bone loss in preclinical male osteoporosis model. Gonadotropin-releasing hormone (GnRH) agonists used in prostate cancer treatment can induce testosterone deficiency but its effects on bone in preclinical male osteoporosis model are less studied. Objective: This study aimed to evaluate the skeletal effect of buserelin (a GnRH agonist) in male rats and compare it with orchidectomy. Methods: Forty-six three-month-old male Sprague-Dawley rats were divided into three experimental arms. The baseline arm (n=6) was sacrificed at the onset of the study. In the buserelin arm, the rats received a daily subcutaneous injection of either normal saline (n=8), buserelin acetate at 25 µg/kg (n=8) or 75 µg/kg (n=8). In the orchidectomy arm, the rats were either sham-operated (n=8) or orchidectomized (n=8). All groups underwent in-vivo X-ray micro-computed tomography scanning at the left proximal tibia every month. Blood was collected at the beginning and the end of the study for testosterone level evaluation. The rats were euthanized after the three-month treatment. The femurs were harvested for biomechanical strength and bone calcium determination. Results: The results showed that buserelin at both doses caused a significant decline in testosterone level and deterioration in bone microstructure (p<0.05), but did not affect bone calcium content (p>0.05). Buserelin at 25 µg/kg decreased displacement and strain of the femur significantly (p<0.05). Similar changes were observed in the orchidectomized group compared to the sham-operated group but without any significant changes in biomechanical strength (p>0.05). Conclusion: Buserelin can induce testosterone deficiency and the associated deterioration of bone microarchitecture similar to orchidectomy in three months. However, it may require a longer time to show significant effects on bone strength and mineral content.
The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5(+)/6(+)) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients.
Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer.
Nitric oxide is postulated to be involved in the pathophysiology of neurological disorders due to hypoxia/ anoxia in brain due to increased release of glutamate and activation of N-methyl-D-aspartate receptors. Reactive oxygen species have been implicated in pathophysiology of many neurological disorders and in brain function. To understand their role in anoxia (hypobaric hypoxia) and reperfusion (reoxygenation), the nitric oxide synthase, argininosuccinate synthetase, argininosuccinate lyase, glutamine synthetase and arginase activities along with the concentration of nitrate /nitrite, thiobarbituric acid reactive substances and total antioxidant status were estimated in cerebral cortex, cerebellum and brain stem of rats subjected to anoxia and reperfusion. The results of this study clearly demonstrated the increased production of nitric oxide by increased activity of nitric oxide synthase. The increased activities of argininosuccinate synthetase and argininosuccinate lyase suggest the increased and effective recycling of citrulline to arginine in anoxia, making nitric oxide production more effective and contributing to its toxic effects. The decreased activity of glutamine synthetase may favor the prolonged availability of glutamic acid causing excitotoxicity leading to neuronal damage in anoxia. The increased formation of thiobarbituric acid reactive substances and decreased total antioxidant status indicate the presence of oxidative stress in anoxia and reperfusion. The increased arginase and sustained decrease of GS activity in reperfusion group likely to be protective.
Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity.
Dengue virus (DENV) infection usually presents with mild self-limiting dengue fever (DF). Few however, would present with the more severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the present study, the association between IL-12B, IL-10 and TNF-α gene polymorphisms and dengue severity was investigated.
METHODS: A case-control study was performed on a total of 120 unrelated controls, 86 DF patients and 196 DHF/DSS patients. The polymorphisms in IL-12B, IL-10 and TNF-α genes were genotyped using PCR-RFLP and PCR-sequencing methods.
RESULTS: A protective association of TNF-α -308A allele and -308GA genotype against DHF/DSS was observed, while TNF-α -238A allele and -238GA genotype were associated with DHF/DSS. A combination of TNF-α -308GA+AA genotype and IL-10 non-GCC haplotypes, IL-12B pro homozygotes (pro1/pro1, pro2/pro2) and IL-12B 3'UTR AC were significantly correlated with protective effects against DHF/DSS. An association between the cytokine gene polymorphisms and protection against the clinical features of severe dengue including thrombocytopenia and increased liver enzymes was observed in this study.
CONCLUSION: The overall findings of the study support the correlation of high-producer TNF-α genotypes combined with low-producer IL-10 haplotypes and IL-12B genotypes in reduced risk of DHF/DSS.
KEYWORDS: Infectious disease; cytokine; dengue; genetics; polymorphism.; tropical
A family of PI3Ks is the lipid kinases, which enhance intracellular pools of phosphatidyl inositol 3,4,5-tri-phosphate (PIP3) through phosphorylating its precursor. Amplifications and deletions of genes, as well as somatic missense of the PIK3CA gene have been described in many human cancer varieties, including of the brain, colon, liver, lung and stomach. Immunohistochemistry and Real-time quantitative PCR tests were used to determine the PIK3CA gene amplification (gene copy number) and to detect protein expression, respectively. The results obtained were analysed and the ratio of PIK3CA to β-actin gene copy number was calculated. Positive gene amplification of PIK3CA was appointed as a copy number of ≥4. Also, PI3K p110α protein expression was scored from 0 to 3+ and the scores of 2+ and 3+ were considered as positive for PI3K p110α protein expression. We studied 50 breast carcinoma samples for PI3K p110α protein expression and PIK3CA gene copy numbers. In general, 36 out of 50 (72%) breast carcinoma samples showed a significant increase in PIK3CA gene amplification. 12 out of 50 (24%) showed positive staining, and 38 out of 50 (76%) showed negative staining for PI3K p110α expression. We have identified no significant relationship between PIK3CA amplification, race (p= 0.630) and histological type (p=0. 731) in breast carcinoma, but correlation of PIK3CA amplification and age showed a significant relationship (p=0. 003) between them. No significant relationship has been identified in correlation of PI3K p110α protein expression compared to age (p=0. 284), race (p=0. 546) and histological type (p=0. 285). Amplification of PIK3CA was frequent in breast carcinoma and occurs in stages of breast carcinoma. Our result shows that there is a relationship between gene amplification and age in breast carcinoma. We suggest that PIK3CA is significant in breast tumorigenesis serve as a prevalent mechanism contributes to the oncogenic activation pathway of PIK3CA in breast cancer.