Locality-sensitive hashing for protein classification

Determination of sequence similarity is a central issue in computational biology, a problem addressed primarily through BLAST, an alignment based heuristic which has underpinned much of the analysis and annotation of the genomic era. Despite their success, alignment-based approaches scale poorly with increasing data set size, and are not robust under structural sequence rearrangements. Successive waves of innovation in sequencing technologies – so-called Next Generation Sequencing (NGS) approaches – have led to an explosion in data availability, challenging existing methods and motivating novel approaches to sequence representation and similarity scoring, including adaptation of existing methods from other domains such as information retrieval. In this work, we investigate locality-sensitive hashing of sequences through binary document signatures, applying the method to a bacterial protein classification task. Here, the goal is to predict the gene family to which a given query protein belongs. Experiments carried out on a pair of small but biologically realistic datasets (the full protein repertoires of families of Chlamydia and Staphylococcus aureus genomes respectively) show that a measure of similarity obtained by locality sensitive hashing gives highly accurate results while offering a number of avenues which will lead to substantial performance improvements over BLAST..

Potential role of EPB41L3 (Protein 4.1B/Dal-1) as a target for treatment of advanced prostate cancer

Background: Loss of erythrocyte membrane protein band 4.1-like 3 (EPB41L3; aliases: protein 4.1B, differentially expressed in adenocarcinoma of the lung-1 (Dal-1)) expression has been implicated in tumor progression. Objective: To evaluate literature describing the role of EPB41L3 in tumorigenesis and metastasis, and to consider whether targeting this gene would be useful in the treatment of prostate cancer. Methods: A literature review of studies describing EPB41L3 and its aliases was conducted. Online databases (NCBI, SwissProt) were also interrogated to collect further data. Results/conclusion: A growing body of evidence supports a role for loss of EPB41L3 in tumor progression, including in prostate cancer. Therapeutic strategies that could be harnessed to upregulate EPB41L3 gene expression in prostate cancer cells are currently being developed.

Weight-related differences in glucose metabolism and free fatty acid production in two South African population groups

OBJECTIVE The effects of free fatty acids (FFA), leptin, tumour necrosis factor (TNF) alpha and body fat distribution on in vivo oxidation of a glucose load were studied in two South African ethnic groups. DESIGN AND MEASUREMENTS Anthropometric and various metabolic indices were measured at fasting and during a 7h oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis and subcutaneous and visceral fat mass was assessed using a five- and two-level CT-scan respectively. Glucose oxidation was evaluated by measuring the ratio of (13)CO(2) to (12)CO(2) in breath following ingestion of 1-(13)C-labelled glucose. SUBJECTS Ten lean black women (LBW), ten obese black women (OBW), nine lean white women (LWW) and nine obese white women (OWW) were investigated after an overnight fast. RESULTS Visceral fat levels were significantly higher (P < 0.01) in obese white than black women, despite similar body mass indexes (BMIs). There were no ethnic differences in glucose oxidation however; in the lean subjects of both ethnic groups the area under the curve (AUC) was higher than in obese subjects (P < 0.05 for both) and was found to correlate negatively with weight (r = -0.69, P < 0.01) after correcting for age. Basal TNF alpha concentrations were similar in all groups. Percentage suppression of FFAs at 30 min of the OCTT was 24 +/- 12% in OWW and - 38 +/- 23% (P < 0.05) in OBW, ie the 30 min FFA level was higher than the fasting level in the latter group. AUC for FFAs during the late postprandial period (120 - 420 min) was significantly higher in OWW than OBW (P < 0.01) and LWW (P < 0.01) and correlated positively with visceral fat mass independent of age (r = 0.78, P < 0.05) in the OWW only. Leptin levels were higher (P < 0.01) both at fasting and during the course of the OCTT in obese women from both ethnic groups compared to the lean women. CONCLUSIONS Glucose oxidation is reduced in obese subjects of both ethnic groups; inter- and intra-ethnic differences were observed in visceral fat mass and FFA production and it is possible that such differences may play a role in the differing prevalences of obesity-related disorders that have been reported in these two populations.

Ethnic differences in lipid metabolism in two groups of obese South African women

There is a higher prevalence of ischemic heart disease (IHD) in South African white than black women. The objective of this study was to determine biochemical explanations for this prevalence. The study group contained 15 obese black women (OBW) and 14 obese white women (OWW), ah premenopausal, who were examined after an overnight fast. Anthropometric measurements and blood concentrations of glucose, non-esterified fatty acids (NEFAs), catecholamines, plasminogen activator inhibitor-1, C-peptide, proinsulin, lipograms, cortisol, growth hormone, and post-heparin Lipoprotein Lipase activity were measured during an oral glucose tolerance test (OGTT), Body composition was measured using bioelectrical impedance analysis, and subcutaneous and visceral fat mass were assessed with CT-scans. Visceral fat area was higher in OWW (139.7 +/- 10.7 cm(2)) than in OBW (72.3 +/- 3.9 cm(2)) (P < 0.01), as were fasting and 3 h triglyceride concentrations (P < 0.05 for all). OWW also had higher NEFA levels than OBW at 3 and 4 h compared, with OBW (P < 0.05 for both). Fasting cortisol (266 +/- 24 vs. 197 +/- 19 nmol/l; P < 0.05) was higher in OWW than in OBW. These data demonstrate that OWW have higher visceral fat mass than OBW, which may lead to a more atherogenic fasting and postprandial Lipid profile. The higher cortisol levels of the OWW may promote visceral fat deposition. - Punyadeera, C., M-T. van der Merwe, N.J. Crowther, M. Toman, C. P. Schlaphoff, and I. P. Gray. Ethnic differences in lipid metabolism in two groups of obese South African women.

Protein-energy malnutrition exists and is associated with negative outcomes in morbidly obese hospital patients

Prevalence of protein-energy malnutrition (PEM), food intake inadequacy and associated health-related outcomes in morbidly obese (Body Mass Index ≥ 40 kg/m2) acute care patients are unknown. This study reports findings in morbidly obese participants from the Australasian Nutrition Care Day Survey (ANCDS) conducted in 2010. The ANCDS was a cross-sectional survey involving acute care patients from 56 Australian and New Zealand hospitals. Hospital-based dietitians evaluated participants’ nutritional status (defined by Subjective Global Assessment, SGA) and 24-hour food intake (as 0%, 25%, 50%, 75%, and 100% of the offered food). Three months later, outcome data, including length of stay (LOS) and 90-day in-hospital mortality, were collected. Of the 3122 participants, 4% (n = 136) were morbidly obese (67% females, 55 ± 14 years, BMI: 48 ± 8 kg/m2). Eleven percent (n = 15) of the morbidly obese patients were malnourished, and most (n = 11/15, 73%)received standard hospital diets without additional nutritional support. Malnourished morbidly obese patients had significantly longer LOS and greater 90-day in-hospital mortality than well-nourished counterparts (23 days vs. 9 days, p = 0.036; 14% vs. 0% mortality, p = 0.011 respectively). Thirteen morbidly obese patients (10%) consumed only 25% of the offered meals with a significantly greater proportion of malnourished (n = 4, 27%) versus well-nourished (n = 9, 7%) (p = 0.018). These results provide new knowledge on the prevalence of PEM and poor food intake in morbidly obese patients in Australian and New Zealand hospitals. For the first time internationally, the study establishes that PEM is significantly associated with negative outcomes in morbidly obese patients and warrants timely nutritional support during hospitalisation.

Estrogen metabolizing enzymes in endometrium and endometriosis

BACKGROUND Estradiol (E-2) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)l in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS Aromatase and 17 beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17 beta-HSD, EST and STS were readily detectable. Only 17 beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17 beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS In endometriosis lesions, the balance is tilted in favor of enzymes producing E2. This is due to a suppression of types 2 and 4 17 beta-HSD, and an increased expression of aromatase and type 1 17 beta-HSD in ectopic endometrium.

Three-dimensional flow-through protein platform

We have developed a new protein microarray (Immuno-Flow Protein Platform, IFPP) that utilizes a porous nitrocellulose (NC) membrane with printed spots of capture probes. The sample is pumped actively through the NC membrane, to enhance binding efficiency and introduce stringency. Compared to protein microarrays assayed with the conventional incubation-shaking method the rate of binding is enhanced on the IFPP by at least a factor of 10, so that the total assay time can be reduced drastically without compromising sensitivity. Similarly, the sensitivity can be improved. We demonstrate the detection of 1 pM of C-reactive protein (CRP) in 70 mu L of plasma within a total assay time of 7 min. The small sample and reagent volumes, combined with the speed of the assay, make our IFPP also well-suited for a point-of-care/near-patient setting. The potential clinical application of the IFPP is demonstrated by validating CRP detection both in human plasma and serum samples against standard clinical laboratory methods.

One-step homogeneous C-reactive protein assay for saliva

Background: Cardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the body's health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting. Methods: We have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n = 55, ages 20-70 years) as well as from cardiac patients (n = 28, ages 43-86 years). Results: The assay incubation time was optimised from 90 min to 15 mm and generated a positive correlation (n = 29, range 10-2189 pg/mL, r2 = 0.94; Passing Bablok slope 0.885. Intercept 0, p>0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p<0.01). Analysis of CRP concentrations in paired serum and saliva samples from cardiac patients gave a positive correlation (r2 = 0.84, p<0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients. Conclusions: The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events. (C) 2011 Elsevier B.V. All rights reserved.

Complementary surface-enhanced resonance raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b5, whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.

Functionalized Ag nanoparticles with tunable optical properties for selective protein analysis

We present a preparation procedure for small sized biocompatibly coated Ag nanoparticles with tunable surface plasmon resonances. The conditions were optimised with respect to the resonance Raman signal enhancement of heme proteins and to the preservation of the native protein structure....

Novel approaches for SER spectroscopic analysis of protein cofactors

Biomimetic systems employed for biotechnological applications i.e. as biosensors or bio fuel cells, require initial formation of conducting support/protein complexes with controlled properties. The specific interaction of the protein with the support determines important qualities of the device such as electrical communication, long-term stability and catalytic efficiency. In this respect the system parameters have to be chosen in a way that high protein loading on the support is achieved while protein denaturation upon adsorption is prevented. The conditions on the surface have to be adjusted in such a way that the desired surface reaction of the protein i.e. electron transfer to either the electrode or a second redox partner, is still guaranteed. Hence the choice of support, its functionlisation as well as the right adjustment of solution parameters play a crucial role in the rational design of these support/protein constructs.

GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways

GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from 5 donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures were investigated using real time PCR, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred μM baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway.

Development of a transient, high-level expression platform for protein production in plants

Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants.

Imaging intracellular protein dynamics by spinning disk confocal microscopy

The palette of fluorescent proteins (FPs) has grown exponentially over the past decade, and as a result, live imaging of cells expressing fluorescently tagged proteins is becoming more and more mainstream. Spinning disk confocal (SDC) microscopy is a high-speed optical sectioning technique and a method of choice to observe and analyze intracellular FP dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low-noise scientific grade-cooled charge-coupled device cameras, and can achieve frame rates of up to 1000 frames per second. In this chapter, we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy and provide a rationale for specific design choices. We also give guidelines of how other imaging techniques such as total internal reflection microscopy or spatially controlled photoactivation can be coupled with SDC imaging and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction.

Longitudinal performance of polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in large preclinical animal model : 6- versus 12 months

There is strong current interest in the use of biodegradable scaffolds in combination with bone growth factors as a valuable alternative to the current gold standard autograft in spinal fusion surgery Yong et al. (2013). Here we report on 6- vs 12- month data set evaluating the longitudinal performance of a CaP coated polycaprolactone (PCL) scaffold loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within a preclinical ovine thoracic spine. The results of this study demonstrate the efficacy of scaffold-based delivery of rhBMP-2 in promoting higher fusion grades at 6- and 12- months in comparison to the scaffold alone or autograft group within the same time frame. Fusion grades achieved at six months using PCL+rhBMP-2 are not significantly increased at twelve months post surgery.