Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.
Secretory phospholipase A2-IIA (sPLA2-IIA) is one of the key enzymes causing lipoprotein modification and vascular inflammation. Maslinic acid is a pentacyclic triterpene which has potential cardioprotective and anti-inflammatory properties. Recent research showed that maslinic acid interacts with sPLA2-IIA and inhibits sPLA2-IIA-mediated monocyte differentiation and migration. This study elucidates the potential of maslinic acid in modulating sPLA2-IIA-mediated inflammatory effects in THP-1 macrophages. We showed that maslinic acid inhibits sPLA2-IIA-mediated LDL modification and suppressed foam cell formation. Further analysis revealed that sPLA2-IIA only induced modest LDL oxidation and that inhibitory effect of maslinic acid on sPLA2-IIA-mediated foam cells formation occurred independently of its anti-oxidative properties. Interestingly, maslinic acid was also found to significantly reduce lipid accumulation observed in macrophages treated with sPLA2-IIA only. Flow cytometry analysis demonstrated that the effect observed in maslinic acid might be contributed in part by suppressing sPLA2-IIA-induced endocytic activity, thereby inhibiting LDL uptake. The study further showed that maslinic acid suppresses sPLA2-IIA-induced up-regulation of PGE2 levels while having no effects on COX-2 activity. Other pro-inflammatory mediators TNF-a and IL-6 were not induced in sPLA2-IIA-treated THP-1 macrophages. The findings of this study showed that maslinic acid inhibit inflammatory effects induced by sPLA2-IIA, including foam cells formation and PGE2 production.
Chondrosenescence (chondrocyte senescence) and subchondral bone deterioration in osteoarthritic rats were analyzed after treatment with the estrogenic herb Labisia pumila (LP) or diclofenac. Osteoarthritis (OA) was induced in bilaterally ovariectomized (OVX) rats by injecting mono-iodoacetate into the right knee joints. Rats were grouped (n = 8) into nontreated OVX+OA control, OVX+OA + diclofenac (5 mg/kg) (positive control), OVX+OA + LP leaf extract (150 and 300 mg/kg) and healthy sham control. After 8 weeks' treatment, their conditions were evaluated via serum biomarkers, knee joint histology, bone histomorphometry, protein and mRNA expressions. The LP significantly reduced cartilage erosion, femur bone surface alteration, bone loss and porosity and increased trabecular bone thickness better than diclofenac and the non-treated OA. The cartilage catabolic markers' (matrix metalloproteinase (MMP)-13, RUNX2, COL10a, ERa, CASP3 and HIF-2 alpha) mRNA expressions were down-regulated and serum bone formation marker, PINP, was increased by LP in a dose-dependent manner. The LP (containing myricetin and gallic acid) showed protection against chondrosenescence, chondrocyte death, hypoxia-induced cartilage catabolism and subchondral bone deterioration. The bone and cartilage protective effects were by suppressing proteases (collagen break-down), bone resorption and upregulating subchondral bone restoration. The cartilage ER alpha over-expression showed a strong positive correlation with MMP-13, COL10 alpha1, histological, micro-computed tomography evidence for cartilage degradation and chondrosenescence.
This study investigated the association of Uncoupling Protein 2 gene (UCP2) 45-bp I/D polymorphism with obesity and adiposity in 926 Malaysian subjects (416 males;265 obese; 102/672/152 Malays/Chinese/Indians). The overall minor allele frequency (MAF) was 0.14, while MAFs according to Malay/Chinese/Indian were 0.17/0.12/0.21. The polymorphism was associated with ethnicity, obesity and overall adiposity (total body fat percentage, TBF), but not gender and central adiposity (waist-hip ratio, WHR). Gender- and ethnicity-stratified analysis revealed that within males, the polymorphism was not associated with ethnicity and anthropometric classes. However, within females, significantly more Indians, obese and those with high TBF carried I allele. Logistic regression analysis among females further showed the polymorphism was associated with obesity and overall adiposity; however, when adjusted for age and ethnicity, this association was abolished for obesity but remained significant for overall adiposity [Odds Ratio (OR) for ID genotype = 2.02 (CI=1.18, 3.45; p=0.01); I allele =1.81 (CI=1.15, 2.84; p=0.01)]. Indeed, covariate analysis controlling for age and ethnicity also showed that those carrying ID genotype or I allele had significantly higher TBF than the rest. In conclusion, UCP2 45-bp I/D polymorphism is associated with overall adiposity among Malaysian women.
DARPP-32 (dopamine and adenosine 3', 5'-monophosphate-regulated phosphoprotein of 32 kDa), which belongs to PPP1R1 gene family, is known to act as an important integrator in dopamine-mediated neurotransmission via the inhibition of protein phosphatase-1 (PP1). Besides its neuronal roles, this protein also behaves as a key player in pathological and pharmacological aspects. Use of bioinformatics and phylogenetics approaches to further characterize the molecular features of DARPP-32 can guide future works. Predicted phosphorylation sites on DARPP-32 show conservation across vertebrates. Phylogenetics analysis indicates evolutionary strata of phosphorylation site acquisition at the C-terminus, suggesting functional expansion of DARPP-32, where more diverse signalling cues may involve in regulating DARPP-32 in inhibiting PP1 activity. Moreover, both phylogenetics and synteny analyses suggest de novo origination of PPP1R1 gene family via chromosomal rearrangement and exonization.
Traumatic brain injury (TBI) causes significant mortality in most developing countries worldwide. At present, it is imperative to identify a treatment to address the devastating post-TBI consequences. Therefore, the present study has been performed to assess the specific effect of immediate exposure to normabaric hyperoxia (NBO) after fluid percussion injury (FPI) in the striatum of mice. To execute FPI, mice were anesthetised and sorted into (i) a TBI group, (ii) a sham group without injury and (iii) a TBI group treated with immediate exposure to NBO for 3 h. Afterwards, brains were harvested for morphological assessment. The results revealed no changes in morphological and neuronal damage in the sham group as compared to the TBI group. Conversely, the TBI group showed severe morphological changes as well as neuronal damage as compared to the TBI group exposed to NBO for 3 h. Interestingly, our findings also suggested that NBO treatment could diminish the neuronal damage in the striatum of mice after FPI. Neuronal damage was evaluated at different points of injury and the neighbouring areas using morphology, neuronal apoptotic cell death and pan-neuronal markers to determine the complete neuronal structure. In conclusion, immediate exposure to NBO following FPI could be a potential therapeutic approach to reduce neuronal damage in the TBI model.
An extensive guide on practicable and significant quantitative proteomic approaches in neuroscience research is important not only because of the existing overwhelming limitations but also for gaining valuable understanding into brain function and deciphering proteomics from the workbench to the bedside. Early methodologies to understand the functioning of biological systems are now improving with high-throughput technologies, which allow analysis of various samples concurrently, or of thousand of analytes in a particular sample. Quantitative proteomic approaches include both gel-based and non-gel-based methods that can be further divided into different labelling approaches. This review will emphasize the role of existing technologies, their advantages and disadvantages, as well as their applications in neuroscience. This review will also discuss advanced approaches for targeted proteomics using isotope-coded affinity tag (ICAT) coupled with laser capture microdissection (LCM) followed by liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. This technology can further be extended to single cell proteomics in other areas of biological sciences and can be combined with other 'omics' approaches to reveal the mechanism of a cellular alterations. This approach may lead to further investigation in basic biology, disease analysis and surveillance, as well as drug discovery. Although numerous challenges still exist, we are confident that this approach will increase the understanding of pathological mechanisms involved in neuroendocrinology, neuropsychiatric and neurodegenerative disorders by delivering protein biomarker signatures for brain dysfunction.
Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and downregulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.
Proteus vulgaris is a rod-shaped Gram-negative bacterium known to be the member of Enterobacteriaceae that is able to cause disease in human being. Generally, non-protein-coding RNAs (npcRNAs) do not code for proteins, but they play a vital role in gene regulation at the RNA level including pathogenicity. The present study aims at elucidating homologous npcRNAs from other bacteria in Proteus vulgaris. A comparative genomic analysis was carried out to identify npcRNA homolog of other Enterobacteriaceae pathogens in Proteus vulgaris. A total of 231 npcRNAs previously reported in Salmonella typhi, Salmonella typhimurium and Escherichia coli were screened using BLASTn tool against Proteus vulgaris genome. Interestingly, 33 npcRNAs are homologs to Proteus vulgaris. Northern blot analysis of 6 out of 33 npcRNA candidates confirmed their expression and showed that most of them are differentially expressed during lag, exponential and stationary growth phases. This study is the first approach of identification and characterization of npcRNAs in P. vulgaris. Hence, this could be a pioneer study to further validate the regulatory functions of these npcRNAs to fill the gaps in understanding of the pathogenicity of P. vulgaris.
Psoriasis is a chronic skin disease characterized by thickening and disorganization of the skin's protective barrier. Although current models replicate some aspects of the disease, development of therapeutic strategies have been hindered by absence of more relevant models. This study aimed to develop and characterize an in vitro psoriatic human skin equivalent (HSE) using human keratinocytes HaCat cell line grown on fibroblasts-derived matrices (FDM). The constructed HSEs were treated with cytokines (IL-1α, TNF-α, IL-6, and IL22) to allow controlled induction of psoriasis-associated features. Histological stainings showed that FDMHSE composed of a fully differentiated epidermis and fibroblast-populated dermis comparable to native skin and rat tail collagen-HSE. Hyperproliferation (CK16 and Ki67) and inflammatory markers (TNF-α and IL-6) expression were significantly enhanced in the cytokine-induced FDM- and rat tail collagen HSEs compared to non-treated HSE counterparts. The characteristics were in line with those observed in psoriasis punch biopsies. Treatment with all-trans retinoic acid (ATRA) has shown to suppress these effects, where HSE models treated with both ATRA and cytokines exhibit histological characteristics, hyperproliferation and differentiation markers expression like non-treated control HSEs. Cytokine-induced FDM-HSE, constructed entirely from human cell lines, provides an excellent opportunity for psoriasis research and testing new therapeutics.
Identification of plant variety and cultivar is pivotal in the agricultural sector due to the abundance of plant varieties and cultivars developed in many crop species. However, plant variety and cultivar identification via basic morphological features is problematic and challenging when differentiating closely related species not only due to their limited differences but also due to technical limitations of the process being time-consuming, labour-intensive and costly, and statistically imprecise information being available due to phenotypic plasticity. Therefore, it is imperative to have rapid and highly efficient techniques to mitigate these limitations. This review provides an overview and summarization of the development and application of molecular markers such as Random Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeats (SSR), Inter-simple sequence repeats (ISSR), Amplified Fragment Length Polymorphism (AFLP), Single nucleotide polymorphism (SNP) and DNA barcoding, High-resolution melting (HRM) and biosensor technology as potential tools in the identification of plant variety and cultivar.