Electrophoretic Analysis of Human Parotid Salivary Proteins with Application to the Study of Rheumatoid Arthritis and Sjogren's Syndrome

Human parotid saliva contains many proteins with diverse functions. In the course of a number of diseases, especially where the normal function of the salivary gland is affected, changes may occur in the levels of certain of these. Therefore the analysis of some of these proteins may be of diagnostic significance. This study has focused on the development and refinement of electrophoretic and of protein detection techniques in order to allow the fractionation of proteins in small volumes of human saliva with the minimum of sample preparation. In order to give an example of their possible diagnostic significance, the electrophoretic techniques which were developed were applied to the fractionation and partial characterisation of the anionic salivary proteins associated with connective tissue disorders such as rheumatoid arthritis and Sjogren's Syndrome. The saliva of patients with rheumatoid arthritis and Sjogren's Syndrome contains additional anionic proteins, which are either present in very low levels or below detection limits in the saliva of normal healthy individuals. Research into the identity of these proteins has been largely hindered by the relatively high electrolyte and low protein content of human parotid saliva, making it necessary to desalt and concentrate the saliva samples prior to carrier ampholyte-based isoelectric focusing. Desalting requires relatively large volumes (preferably > 2ml) of saliva, which may be difficult or even impossible to obtain from diseased glands. Also, one-dimensional isoelectric focusing cannot separate these anionic proteins from the acidic proline-rich proteins of human saliva, as both groups of proteins have overlapping isoelectric points. In this study, a hybrid carrier ampholyte-immobilised pH gradient isoelectric focusing technique was developed to analyse human salivary proteins. Immobilised pH gradients (IPG's) of 3 pH ranges were prepared: broad-range (pH 4-9) IPG was used for the general study of human salivary proteins; while 2 narrow, acidic range IPG's (pH 2.8-4.5 and pH 3.5-5.0) were used to analyse proteins of low isoelectric points, such as the anionic salivary proteins associated with rheumatoid arthritis and Sjogren's Syndrome. This method allowed the difficulties involved when conventional carrier ampholyte-based isoelectric focusing is used to be circumvented, thus making it possible to fractionate the proteins in small volumes (approximately 50ul) of human saliva without prior treatment except for centrifugation. Parotid salivary proteins were also analysed by onedimensional SDS-PAGE. SDS-PAGE gels were subjected to im-muno- and lectin affinity-blotting in order to characterise or identify some of the protein bands. Proline-rich proteins were recognised by their characteristic pink-staining with the dye Coommassie Brilliant Blue R250, and some of the bands which were revealed have been correlated with proline-rich proteins which have been isolated and partially characterised by other research groups. SDS-PAGE failed to reveal any obvious differences between the band patterns of normal subjects and those of patients with rheumatoid arthritis or Sjogren's Syndrome. Two-dimensional gel electrophoresis was also carried out using hybrid carrier ampholyte-immobilised pH gradient polyacrylamide gels in the first dimension and thin-layer SDS-polyacrylamide gradient gels in the second. By means of a combination of staining and electroblotting of the two-dimensional gels onto nitrocellulose followed by probing with specific antisera, a two-dimensional map of human parotid salivary proteins, in which most of the major components have been identified, has been obtained. These techniques were applied to the investigation of the nature of the anionic salivary proteins associated with rheumatoid arthritis and Sjogren's Syndrome. Two-dimensional electrophoresis with pH 3.5-5.0 IPG's in the first dimension followed by silver staining revealed these proteins to be heterogeneous (pI's approximately 3.65-4.75) and of a single relative molecular weight (approximately 32,000). In normal healthy controls these silver stained components were less heterogeneous (pI's approximately 3.65-4.25). Incubation with neuraminidase showed that their heterogeneity was largely due to differing contents of sialic acid in their carbohydrate side-chains. In order to attempt to identify these proteins, the one-dimensional IPG and two-dimensional gels were electroblotted and the blots were probed with a variety of antisera. The proteins appeared to be immunoreactive with antisera to human tissue kallikrein, a protein the level of which has often been reported to be elevated in the saliva of patients with Sjogren's Syndrome. (Abstract shortened by ProQuest.)

Penentuan Hasil Tindakbalas Antara Asid Kromotropik Dan Formaldehid

Identiti tindakbalas antara asid kromotropik dan formaldehid berlebihan tanpa asid sulfuric pekat dibuktikan sebagai dimer di mana dua molekul asid kromotropik diikat melalui dua titisan olefinik, berasaskan kepada spectrum N.M.R Proton, mikroanalisisuntuk C,H, N dan S, dan pentitratan Karl Fischer

Investigation of twenty selected medicinal plants from Malaysia for anti-Chikungunya virus activity

Chikungunya virus is a reemerging arbovirus transmitted mainly by Aedes mosquitoes. As there are no specific treatments available, Chikungunya virus infection is a significant public health problem. This study investigated 120 extracts from selected medicinal plants for anti-Chikungunya virus activity. The plant materials were subjected to sequential solvent extraction to obtain six different extracts for each plant. The cytotoxicity and antiviral activity of each extract were examined using African monkey kidney epithelial (Vero) cells. The ethanol, methanol and chloroform extracts of Tradescantia spathacea (Commelinaceae) leaves showed the strongest cytopathic effect inhibition on Vero cells, resulting in cell viabilities of 92.6% ± 1.0% (512 μg/ml), 91.5% ± 1.7% (512 μg/ml) and 88.8% ± 2.4% (80 μg/ml) respectively. However, quantitative RT-PCR analysis revealed that the chloroform extract of Rhapis excelsa (Arecaceae) leaves resulted in the highest percentage of reduction of viral load (98.1%), followed by the ethyl acetate extract of Vernonia amygdalina (Compositae) leaves (95.5%). The corresponding 50% effective concentrations (EC50) and selectivity indices for these two extracts were 29.9 ± 0.9 and 32.4 ± 1.3 μg/ml, and 5.4 and 5.1 respectively. Rhapis excelsa and Vernonia amygdalina could be sources of anti-Chikungunya virus agents. [Int Microbiol 19(3):175-182 (2016)]Keywords: Chikungunya virus · antivirals · cytotoxicity · sequential extraction · medicinal plant

Interactions between Plant Extracts and Cell Viability Indicators during Cytotoxicity Testing: Implications for Ethnopharmacological Studies

Purpose: To compare the cytotoxicity of six medicinal plants (Acmella ciliata, Amaranthus tricolor, Coriandrum sativum, Glebionis coronaria, Kyllinga brevifolia and Tradescantia zebrina) using 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assays.Methods: Hexane, chloroform, ethyl acetate, ethanol, methanol and water extracts were obtained for each plant by sequential solvent extraction. Cytotoxicity was evaluated in triplicate, from 640 to 5 μg/mL, two-fold, serially on monkey kidney epithelial (Vero) cells.Results: The hexane, chloroform and ethyl acetate extracts of the six plants were more toxic to the Vero cells compared to the ethanol, methanol and water extracts. Thirty one percent (11/36) and 75 % (27/36) of the extracts showed significant cytotoxicity (p < 0.05) in MTT and NRU assays, respectively. The 78, 52 and 7 % cytotoxicity levels detected in 27 extracts using the MTT assay were significantly (p < 0.05) underestimated at 640, 320 and 160 μg/mL, respectively, using NRU assay. Nine extracts from five plants exhibited significantly lower (p < 0.05) 50 % cytotoxic concentration (CC50) when NRU assay was employed compared to MTT assay. At 640 μg/mL, 10 of the 21 extracts were also found to react chemically with MTT, causing a 2.0 – 29.1-fold increase in the absorbance value (550 nm) compared to control.Conclusion: The plant extracts of A. ciliata, A. tricolor, C. sativum, G. coronaria, K. brevifolia and T. zebrina show concentration- and extraction method-dependent cytotoxicity using MTT and NRU assays. NRU assay appears to be more sensitive and reliable than MTT assay for cell viability evaluation of the plant extracts.Keywords: Acmella ciliata, Amaranthus tricolor, Coriandrum sativum, Glebionis coronaria, Kyllinga brevifolia and Tradescantia zebrina, Extraction, Medicinal plant, Neutral red uptake assay, Vero cel

Antimicrobial, antiviral and cytotoxic activities of selected marine organisms collected from the coastal areas of Malaysia

Many marine organisms have developed the capability of producing unique metabolites and thus are highly likely to contain anti-infective agents. This study was conducted to investigate extracts of three seaweeds (Caulerpa racemosa, Caulerpa sertularioides, Kappaphycus alvarezii), two soft corals (Lobophytum microlobulatum, Sarcophyton auritum) and a marine sponge (Spheciospongia vagabunda) collected from Malaysian coast for antibacterial, antifungal, antiviral and cytotoxic activities. The samples were subjected to sequential solvent extraction in order to obtain hexane, chloroform, ethyl acetate, ethanol, methanol and water extracts. The antibacterial and antifungal activities were studied using a colorimetric broth microdilution method. The hexane extract of L. microlobulatum had the strongest antibacterial activity and exhibited the lowest minimum inhibitory concentration (0.04 mg/mL) and minimum bactericidal concentration (0.08 mg/mL) against Staphylococcus aureus and Bacillus cereus, respectively. For antifungal activity, the lowest MIC and minimum fungicidal concentration values were produced by the hexane extract of S. auritum against the dimorphic yeast Cryptococcus neoformans, both with 0.04 mg/mL. None of the extracts were active against the filamentous fungus Aspergillus fumigatus. Only the hexane and ethanol extracts of L. microlobulatum and the ethyl acetate extract of S. auritum exhibited strong inhibition on the cytopathic effect induced by the Chikungunya virus (a re-emerging mosquito-borne virus) with 50% effective concentrations of 14.3 0.2, 124.3 1.9 and 176.6 9.7 µg/mL, respectively. Extracts from the two soft corals, L. microlobulatum and S. auritum possessed stronger antibacterial, antifungal and antiviral activities compared to the seaweeds and the sponge

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Identification of novel breast cancer susceptibility loci in meta-analyses conducted among Asian and European descendants

Abstract: Known risk variants explain only a small proportion of breast cancer heritability, particularly in Asian women. To search for additional genetic susceptibility loci for breast cancer, here we perform a meta-analysis of data from genome-wide association studies (GWAS) conducted in Asians (24,206 cases and 24,775 controls) and European descendants (122,977 cases and 105,974 controls). We identified 31 potential novel loci with the lead variant showing an association with breast cancer risk at P < 5 × 10−8. The associations for 10 of these loci were replicated in an independent sample of 16,787 cases and 16,680 controls of Asian women (P < 0.05). In addition, we replicated the associations for 78 of the 166 known risk variants at P < 0.05 in Asians. These findings improve our understanding of breast cancer genetics and etiology and extend previous findings from studies of European descendants to Asian women

Identification of novel breast cancer susceptibility loci in meta-analyses conducted among Asian and European descendants

Abstract: Known risk variants explain only a small proportion of breast cancer heritability, particularly in Asian women. To search for additional genetic susceptibility loci for breast cancer, here we perform a meta-analysis of data from genome-wide association studies (GWAS) conducted in Asians (24,206 cases and 24,775 controls) and European descendants (122,977 cases and 105,974 controls). We identified 31 potential novel loci with the lead variant showing an association with breast cancer risk at P < 5 × 10−8. The associations for 10 of these loci were replicated in an independent sample of 16,787 cases and 16,680 controls of Asian women (P < 0.05). In addition, we replicated the associations for 78 of the 166 known risk variants at P < 0.05 in Asians. These findings improve our understanding of breast cancer genetics and etiology and extend previous findings from studies of European descendants to Asian women

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts