Identification and Evaluation of Serum Protein Biomarkers That Differentiate Psoriatic Arthritis From Rheumatoid Arthritis.

OBJECTIVE: To identify serum protein biomarkers that might distinguish patients with early inflammatory arthritis (IA) with psoriatic arthritis (PsA) from those with rheumatoid arthritis (RA) and may be used to support appropriate early intervention. METHODS: The serum proteome of patients with PsA and patients with RA was interrogated using nano-liquid chromatography mass spectrometry (nano-LC-MS/MS) (n = 64 patients), an aptamer-based assay (SomaScan) targeting 1,129 proteins (n = 36 patients), and a multiplexed antibody assay (Luminex) for 48 proteins (n = 64 patients). Multiple reaction monitoring (MRM) assays were developed to evaluate the performance of putative markers using the discovery cohort (n = 60 patients) and subsequently an independent cohort of PsA and RA patients (n = 167). RESULTS: Multivariate machine learning analysis of the protein discovery data from the 3 platforms revealed that it was possible to differentiate PsA patients from RA patients with an area under the curve (AUC) of 0.94 for nano-LC-MS/MS, 0.69 for bead-based immunoassay measurements, and 0.73 for aptamer-based analysis. Subsequently, in the separate verification and evaluation studies, random forest models revealed that a subset of proteins measured by MRM could differentiate PsA and RA patients with AUCs of 0.79 and 0.85, respectively. CONCLUSION: We present a serum protein biomarker panel that can separate patients with early-onset IA with PsA from those with RA. With continued evaluation and refinement using additional and larger patient cohorts, including those with other arthropathies, we suggest that the panel identified here could contribute to improved clinical decision making.

Identification of Differentially Expressed Proteins from Smokeless Tobacco Addicted Patients Suffering from Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the eight most common malignancy worldwide with an incidence rate of 40% in south-east Asia. Lack of effective diagnostic tools at early stage and disease recurrence despite extensive treatments are main reasons for high mortality and low survival rates. The aim of current study was to identify differentially expressed proteins to explore potential candidate biomarkers having diagnostic significance. We performed comparative proteomic analysis of paired protein samples (cancerous buccal mucosa and adjacent normal tissue) from OSCC patients using a combination of two dimensional gel electrophoresis and Mass spectrometric analysis. On the basis of spot intensity, seventeen proteins were found to be consistently differentially expressed among most of the samples which were identified through mass spectrometry. For validation of identified proteins, expression level of stratifin was determined using immuno-histochemistry and Western blot analysis. All identified proteins were analyzed by STRING to explore their interaction. Among uniquely identified proteins in this study, at least two candidate markers (Ig Kappa chain C region and Isoform 2 of fructose bisphosphate aldolase A) were found to be novel with respect to OSCC which can be explored further. Results presented in current study are likely to contribute in understanding the involvement of these molecules in carcinogenesis apart from their plausible role as diagnostic/prognostic markers.

Potential mechanisms of calcium dependent regulation of the mammalian cell cycle revealed by comprehensive unbiased label-free nLC-MS/MS quantitative proteomics.

Calcium (Ca2+) controls progression through the mammalian cell cycle by engaging a diverse range of molecular pathways. While the essential role of spatio-temporal Ca2+ signalling in the cell cycle is well established, the precise mechanisms by which it regulates cell cycle entry and progression through G1 are not particularly well understood. Here, high-resolution label-free semi-quantitative nLC-MS/MS analysis has been used to support a highly reproducible unbiased analysis of Ca2+ influx dependent growth factor induced protein expression early in G1 in human fibroblasts. Using this strategy a panel of 182 proteins whose expression was Ca2+ dependent were identified. Pathway analysis has indicated that Ca2+ likely regulates cell proliferation via PI3K/AKT pathway and its downstream target mTOR. In addition to cell proliferation found proteins were involved in the regulation of cell morphology and cellular assembly and organization, the environmental clues, which are known to regulate G1 progression. Reported here data represents one of the most comprehensive proteomic datasets of growth factor and Ca2+ dependent protein expression in the mammalian cell cycle and provides a rich source of publically available data to support continued investigation of the role of Ca2+ in G1 progression at both the molecular and systems level. BIOLOGICAL SIGNIFICANCE: The results of this study provide new insight into Ca2+ dependent regulation of cell cycle. This manuscript reports first to date global analysis of Ca2+ regulated protein expression changes early in G1 in non-transformed human fibroblast cell line. It also highlights canonical signalling pathways and biological processes that are regulated by the inhibition of Ca2+ influx. Importantly, it appears that Ca2+ may be the factor that links cell division with environmental cues, cell morphology and cellular assembly and organization, on which cell proliferation depends. Hence, the findings presented here provide numerous opportunities for more detailed investigations of the mechanism of Ca2+ dependent regulation of cell cycle at the molecular and systemic level.

Proteomes, Their Compositions and Their Sources.

Biological samples of human and animal origin are utilized in research for many purposes and in a variety of scientific fields, including mass spectrometry-based proteomics. Various types of samples, including organs, tissues, cells, body fluids such as blood, plasma, cerebrospinal fluid, saliva and semen, can be collected from humans or animals and processed for proteomics analysis. Depending on the physiological state and sample origin, collected samples are used in research and diagnostics for different purposes. In mass spectrometry-based proteomics, body fluids and tissues are commonly used in discovery experiments to search for specific protein markers that can distinguish physiological from pathophysiological states, which in turn offer new diagnosis strategies and help developing new drugs to prevent disease more efficiently. Cell lines in combination with technologies such as Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) have broader application and are used frequently to investigate the mechanism of a disease or to investigate for the mechanism of a drug function. All of these are important components for defining the mechanisms of disease, discovering new pharmaceutical treatments and finally testing side effects of newly discovered drugs.

The relationship of omental and subcutaneous adipocyte size to metabolic disease in severe obesity.

OBJECTIVE: Several studies have reported the existence of a subgroup of obese individuals with normal metabolic profiles. It remains unclear what factors are responsible for this phenomenon. We proposed that adipocyte size might be a key factor in the protection of metabolically healthy obese (MHO) individuals from the adverse effects of obesity. SUBJECTS: Thirty-five patients undergoing bariatric surgery were classified as MHO (n = 15) or metabolically unhealthy obese (MUO, n = 20) according to cut-off points adapted from the International Diabetes Federation definition of the metabolic syndrome. Median body mass index (BMI) was 48 (range 40-71). RESULTS: There was a moderate correlation between omental adipocyte size and subcutaneous adipocyte size (r = 0.59, p<0.05). The MHO group had significantly lower mean omental adipocyte size (80.9+/-10.9 microm) when compared with metabolically unhealthy patients (100.0+/-7.6 microm, p<0.0001). Mean subcutaneous adipocyte size was similar between the two groups (104.1+/-8.5 microm versus 107.9+/-7.1 microm). Omental, but not subcutaneous adipocyte size, correlated with the degree of insulin resistance as measured by HOMA-IR (r = 0.73, p<0.0005), as well as other metabolic parameters including triglyceride/HDL-cholesterol ratio and HbA1c. Twenty-eight patients consented to liver biopsy. Of these, 46% had steatohepatitis and fibrosis. Fifty percent (including all the MHO patients) had steatosis only. Both omental and subcutaneous adipocyte size were significantly associated with the degree of steatosis (r = 0.66, p<0.0001 and r = 0.63, p<0.005 respectively). However, only omental adipocyte size was an independent predictor of the presence or absence of fibrosis. CONCLUSION: Metabolically healthy individuals are a distinct subgroup of the severely obese. Both subcutaneous and omental adipocyte size correlated positively with the degree of fatty liver, but only omental adipocyte size was related to metabolic health, and possibly progression from hepatic steatosis to fibrosis.

Are natural killer cells protecting the metabolically healthy obese patient?

With the emerging obesity pandemic, identifying those who appear to be protected from adverse consequences such as type 2 diabetes and certain malignancies will become important. We propose that the circulating immune system plays a role in the development of these comorbidities. Clinical data and blood samples were collected from 52 patients with severe obesity attending a hospital weight-management clinic and 11 lean healthy controls. Patients were classified into metabolically "healthy obese" (n = 26; mean age 42.6 years, mean BMI 46.8 kg/m(2)) or "unhealthy obese" (n = 26; mean age 45 years, mean BMI 47.5 kg/m(2)) groups, based upon standard cutoff points for blood pressure, lipid profile, and fasting glucose. Circulating lymphoid populations and phenotypes were assessed by flow cytometry. Obese patients had significantly less circulating natural killer (NK) and cytotoxic T lymphocytes (CTL) compared to lean controls. There were significantly higher levels of NK cells and CTLs in the healthy obese group compared to the unhealthy obese group (NK: 11.7% vs. 6.5%, P < 0.0001, CD8 13.4% vs. 9.3%, P = 0.04), independent of age and BMI and these NK cells were also less activated in the healthy compared to the unhealthy group (CD69, 4.1% vs. 11.8%, P = 0.03). This is the first time that quantitative differences in the circulating immune system of obese patients with similar BMI but different metabolic profiles have been described. The significantly higher levels of CTLs and NK cells, which express fewer inhibitory molecules, could protect against malignancy, infection, and metabolic disease seen in obesity.

Distribution of androgen receptor and steroid hormone concentrations in ovaries of immature bank voles: effect of photoperiod.

Immunolocalisation of androgen receptor (AR) and steroid contents were analyzed in the ovaries of 7- and 14-day-old bank voles, reared in a long (LD) and short (SD) photoperiod. The strongest AR immunoreaction was found in the stromal cells, especially in the ovaries of 7-day-old animals, and in the granulosa cells of all types of ovarian follicles. Oocytes and the cells of surface epithelium were AR positive. The amount of ovarian androgens was relatively high, whereas the level of estradiol was negligible. This finding, and the presence of numerous ARs in various ovarian compartments, suggest that aromatization was very low during development and the primary function of androgens was hormonal action via a receptor-mediated pathway. Age- and photoperiod-related differences in ovarian progesterone (P4) levels were higher in animals kept in LD than in SD, rising significantly on day 14. Androgen content tended to be lower in LD voles and slightly decreased on day 14. Photoperiod-related differences concerning AR immunolabeling were apparent only in 14-day-old animals. In LD, ovaries already possessed early antral follicles, showing strong AR immunolabeling in the cumulus cells. Immunoreaction of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) showed that the primary interstitial and theca cells were the first to be active in ovarian steroidogenesis. In conclusion, AR is present in juvenile vole ovaries as early as day 7. The influence of the photoperiod on their number is observed beginning on day 14. Differences in steroid contents due to LD conditions occur in 7-day-old, and progresses in 14-day-old animals.

Androgen receptor in the ovary of postnatal bank vole females.

In this experiment, we investigated the influence of the photoperiod upon androgen receptor (AR) distribution and steroid concentrations in ovaries of 21-day-old bank vole females. Sections (6 mum) were assayed immunohistochemically. ARs were localized in the nuclei of granulosa cells. The strongest immunoreaction was observed in primary and early antral follicles, and declined during follicular development. P(4), A, and E(2) contents were measured by RIAs in ovarian homogenates from animals kept in two photoperiods. Ovaries of animals kept in a long photoperiod (18:6 light:dark), which stimulates gonadal activity, contained more P(4) than those kept in a short one (6:18 light:dark). There was no difference in levels of A and E(2).