A comparative proteomic analysis of skin secretions of the tammar wallaby (Macropus eugenii) and the wombat (Vombatus ursinus)

The secretome of the pouch skin of the model marsupial the tammar wallaby, Macropus eugenii has been investigated using techniques of two-dimensional gel electrophoresis, in-gel trypsin digestion followed by nanoliquid chromatography coupled tandem mass spectrometry (LC-MS/MS). Differences in the patterns of secreted proteins were observed in the female pouch at three stages of maturity — reproductively immature; reproductively mature and active and mature, postreproductively active. Skin from the underarm area of mature females had a markedly different secreted protein profile. The greatest diversity of proteins was seen in the mature reproductive pouch and from an opportunistic sample collected from the pouch another mature female marsupial, the common wombat, Vombatus ursinus. A total of 20 proteins were confidently identified from the pouch skin secretions of the tammar wallaby and wombats, whilst 20 proteins were tentatively identified. In all skin secretomes, globins were the most abundant proteins whilst the antimicrobial, dermcidin was detected in the wombat sample. Some proteins such as keratin and actin could be sourced to sloughed and degraded skin cells. A number of proteins were present at such low concentrations that confident identification was not possible. This was compounded by the lack of a comprehensive database of marsupial proteins which constrains the reliability of automated identification protocols.     

In-Vitro and in-Silico characterization of Sophora interrupta plant extract as an anticancer activity

Sophora interrupta belongs to the family of Fabaceae and the species in this genus have a diverse medicinal importance as a folk medicine for preventing many ailments including cancer. In order to evaluate the anticancer activity of S.interrupta, we have performed in vitro anti-oxidant, anti-inflammatory, anti-proliferative, and cell based anticancer activity in MCF-7 and PC-3 cell lines. Secondary metabolites of S.interrupta were used to identify anticancer compounds using Open Eye software. The antioxidant activity of the S.interrupta root ethylacetate (SEA) extract at 100 µg/ml is equal to that of ascorbic acid at 50 µg/ml. The antiinflammatory activity of SEA is half of that of diclofenac at 50 µg/ml. Anticancer activity was detected by measuring the mitochondrial dehydrogenase activity (MTT assay). The half maximal inhibitory concentrations (IC₅₀) for MCF-7 and PC-3 cell lines are 250 and 700 µg/ml respectively. This was supported by the morphological changes such as membrane blebbing, cell detachment and rounded cell morphology when compared to the parental cells. In addition, we observed few green cells (live) over red cells (dead) based on the uptake of acridine orange and ethidium bromide dyes. Kaempferol-3-O-b-D-glucopyranoside, a Secondary metabolite of S.interrupta form 6 hydrogen bond interactions with Arg 202, Gln 207, Gly 227, Gly 229, Thr 231 and Ala 232 human DEAD box RNA helicase, DDX3 protein and is equivalent to crystal structure of adenosine mono phosphate to DDX3. Overall, it suggests that the SEA extract has anticancer compounds, and it can be used to enhance death receptor mediated cancer cell death.     

Proteomic analysis of the neutrophil proteins of the tammar wallaby (Macropus eugenii)

A proteomic analysis of neutrophils from the tammar wallaby, Macropus eugenii, has been performed. Neutrophils were isolated from peripheral blood using density gradient centrifugation with Histopaque-1077, followed by treatment with ammonium chloride to lyse residual erythrocytes. Two-dimensional gel electrophoresis (2-DE) of lysed neutrophils was undertaken followed by in-gel trypsin digest and nanoliquid chromatography coupled tandem mass spectrometry (LC-MS) analysis and database searches. Seventy-seven proteins were isolated, 53 of which could be identified with high confidence as primarily of cytosolic origin. Protein identifications were only possible by matching identical peptide sequences within the NCBInr mammalian database with the Mascot search program. Sequence identities were only deemed acceptable if more than three peptides were identified, the precursor/protein ion tolerances were less than ± 0.25 Da and the total Mowse scores were greater than 100. The validity of this approach was tested using a scrambled database where no single identified peptide showed Mowse scores greater than 55. This is the first report of the neutrophil proteins of any marsupial and represents a first step in examining the identity of proteins involved in innate defence in this marsupial.     

Downregulation of Keratin 76 Expression during Oral Carcinogenesis of Human,Hamster and Mouse

Background

Keratins are structural marker proteins with tissue specific expression; however, recent reports indicate their involvement in cancer progression. Previous study from our lab revealed deregulation of many genes related to structural molecular integrity including KRT76. Here we evaluate the role of KRT76 downregulation in oral precancer and cancer development.

Methods

We evaluated KRT76 expression by qRT-PCR in normal and tumor tissues of the oral cavity. We also analyzed K76 expression by immunohistochemistry in normal, oral precancerous lesion (OPL), oral squamous cell carcinoma (OSCC) and in hamster model of oral carcinogenesis. Further, functional implication of KRT76 loss was confirmed using KRT76-knockout (KO) mice.

Results

We observed a strong association of reduced K76 expression with increased risk of OPL and OSCC development. The buccal epithelium of DMBA treated hamsters showed a similar trend. Oral cavity of KRT76-KO mice showed preneoplastic changes in the gingivobuccal epithelium while no pathological changes were observed in KRT76 negative tissues such as tongue.

Conclusion

The present study demonstrates loss of KRT76 in oral carcinogenesis. The KRT76-KO mice data underlines the potential of KRT76 being an early event although this loss is not sufficient to drive the development of oral cancers. Thus, future studies to investigate the contributing role of KRT76 in light of other tumor driving events are warranted.     

Genomic profiling of advanced-stage oral cancers reveals chromosome 11q alterations as markers of poor clinical outcome

Identifying oral cancer lesions associated with high risk of relapse and predicting clinical outcome remain challenging questions in clinical practice. Genomic alterations may add prognostic information and indicate biological aggressiveness thereby emphasizing the need for genome-wide profiling of oral cancers. High-resolution array comparative genomic hybridization was performed to delineate the genomic alterations in clinically annotated primary gingivo-buccal complex and tongue cancers (n = 60). The specific genomic alterations so identified were evaluated for their potential clinical relevance. Copy-number changes were observed on chromosomal arms with most frequent gains on 3q (60%), 5p (50%), 7p (50%), 8q (73%), 11q13 (47%), 14q11.2 (47%), and 19p13.3 (58%) and losses on 3p14.2 (55%) and 8p (83%). Univariate statistical analysis with correction for multiple testing revealed chromosomal gain of region 11q22.1–q22.2 and losses of 17p13.3 and 11q23–q25 to be associated with loco-regional recurrence (P = 0.004, P = 0.003, and P = 0.0003) and shorter survival (P = 0.009, P = 0.003, and P 0.0001) respectively. The gain of 11q22 and loss of 11q23-q25 were validated by interphase fluorescent in situ hybridization (I-FISH). This study identifies a tractable number of genomic alterations with few underlying genes that may potentially be utilized as biological markers for prognosis and treatment decisions in oral cancers.     

Inferences from the ADMET analysis of predicted inhibitors to Follicle Stimulating Hormone in the context of infertility

Follicle stimulating hormone (FSH) is a glycoprotein secreted by gonadotrophs of the anterior pituitary gland that regulates reproduction in mammals. FSH targets its receptor (FSHR) expressed only on grannulosa cells and induce the maturation of ovarian follicles in females. The levels of both FSH and FSHR rise until the middle of estrus cycle and then falls on level at the time of ovulation. It is associated with stimulated sertoli cell proliferation in testes and supports spermatogenesis in males. The interaction between the polypeptide FSH hormone and its corresponding receptor is highly selective. Therefore, it is of interest to inhibit FSH in the context of infertility. The structure of FSH (PDB ID: 1XWD) is screened using molecular docking techniques against the ZINC database (a database of 2.7 million compounds) with reference to known standard compounds. This exercise identifies compounds with better binding and ADMET (Absorption, Digestion, Metabolism, Excretion and Toxicity) properties compared to known standard compounds. These observations find application for the consideration of such compounds for further validation towards inhibiting the FSH.     

Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces

Candida albicans and mutans streptococci are frequently detected in dental plaque biofilms from toddlers afflicted with early childhood caries. Glucosyltransferases (Gtfs) secreted by Streptococcus mutans bind to saliva-coated apatite (sHA) and to bacterial surfaces, synthesizing exopolymers in situ, which promote cell clustering and adherence to tooth enamel. We investigated the potential role Gtfs may play in mediating the interactions between C. albicans SC5314 and S. mutans UA159, both with each other and with the sHA surface. GtfB adhered effectively to the C. albicans yeast cell surface in an enzymatically active form, as determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the yeast cell surface were more susceptible to dextranase than those synthesized in solution or on sHA and bacterial cell surfaces (P < 0.05), indicating an elevated α-1,6-linked glucose content. Fluorescence imaging revealed that larger numbers of S. mutans cells bound to C. albicans cells with glucans present on their surface than to yeast cells without surface glucans (uncoated). The glucans formed in situ also enhanced C. albicans interactions with sHA, as determined by a novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast cells significantly increased the accumulation of S. mutans on the sHA surface (versus S. mutans incubated alone or mixed with uncoated C. albicans; P < 0.05). These data reveal a novel cross-kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast cell surface. Surface-bound GtfB promotes the formation of a glucan-rich matrix in situ and may enhance the accumulation of S. mutans on the tooth enamel surface, thereby modulating the development of virulent biofilms.