Proteomic analysis of human osteoarthritis synovial fluid

Background

Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography.

Results

We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples.

Conclusions

We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

160-fold acceleration of the Smith-Waterman algorithm using a field programmable gate array (FPGA)

Background  

To infer homology and subsequently gene function, the Smith-Waterman (SW) algorithm is used to find the optimal local alignment between two sequences. When searching sequence databases that may contain hundreds of millions of sequences, this algorithm becomes computationally expensive.     

Frequency and clinical patterns of stroke in Iran - Systematic and critical review

Background  

Cerebrovascular disease is the second commonest cause of death, and over a third of stroke deaths occur in developing countries. To fulfil the current gap on data, this systematic review is focused on the frequency of stroke, risk factors, stroke types and mortality in Iran.     

Traction force on a kinetochore at metaphase acts as a linear function of kinetochore fiber length

We are investigating the relation between the force pulling a kinetochore poleward and the length of the corresponding kinetochore fiber. It was recognized by Ostergren in 1950 (Hereditas 36:1-19) that the metaphase position of a chromosome could be achieved by a balance of traction forces were proportional to the distance from kinetochore to pole. For the typical chromosome (i.e., a meiotic bivalent or mitotic chromosome) with a single kinetochore fiber extending to each pole, the resultant force (RF) would equal zero when the chromosome lay at the midpoint between the two poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles, Ostergren’s proposal suggests that RF = 0 when the chromosome is shifted closer to the pole toward which the greater number of kinetochore fibers are pulling. We have measured the force-length relationship in living spindles by analyzing the metaphase positions of experimentally generated multivalent chromosomes having three or four kinetochore fibers. Multivalent chromosomes of varied configurations were generated by γ-irradiation of nymphs of the grasshopper melanoplus differentialis, and their behavior was analyzed in living first meiotic spermocytes. The lengths of kinetochore fibers were determined from time-lapse photographs by measuring the kinetochore-to-pole distances for fully congressed chromosomes just before the onset of anaphase. In our analysis, force (F) along a single kinetochore fiber is expressed by: F = kL(exp), where k is a length-independent proportionality constant, L represents the kinetochore fiber length, and exp is an unknown exponent. The RF on a chromosome is then given by: RF = σk(i)L(i)(exp), where kinetochore fiber lengths in opposite half- spindles are given opposite sign. If forces on a metaphase chromosome are at equilibrium (RF = 0), then for asymmetrical orientations of multivalents we can measure the individual kinetochore fiber lengths (L(i)) and solve for the exponent that yields a resultant force of zero. The value of the exponent relates how the magnitude of force along a kinetochore fiber varies with its length. For six trivalents and one naturally occurring quadrivalent we calculated an average value of exp = 1.06 +/- 0.18. This result is consistent with Ostergren’s hypothesis and indicates that the magnitude of poleward traction force along a kinetochore fiber is directly proportional to the length of the fiber. Our finding suggests that the balance of forces along a kinetochore fiber may be a major factor regulating the extent of kinetochore microtubule assembly.     

Molecular docking analysis of glycogen phosphorylase with inhibitors from Cissampelos pareira Linn.

Cissampelos pareira Linn. is a climbing herb known in Indian traditional medicine as laghupatha. It belongs to the Menispermaceae family. The enzyme glycogen phosphorylase (GP) is a promising target for the treatment of type-2 diabetes (T2DM). A variety of natural product inhibitors with both pharmaceutical and nutraceutical potential have been reported in the search for powerful, selective and drug-like GP inhibitors that could lead to hypoglycemic medicines. Therefore, it is of interest to document the molecular docking analysis data of glycogen phosphorylase with compounds from Cissampelos pareira Linn. We report the optimal binding features of 4 compounds namely Trans-N-feruloyltyramine, Coclaurine, Magnoflorine, and Curine with the target protein for further consideration in the context of T2DM.     

A third variant of glucose phosphate isomerase in pigs

A new variant of glucose phosphate isomerase (GPI), also known as phosphohexose isomerase (PHI), was detected in a primitive pig population.