2D-DIGE based urinary proteomics and functional enrichment studies to reveal novel Mycobacterium tuberculosis and human protein biomarker candidates for pulmonary tuberculosis.

In the absence of a sensitive and specific diagnostic modality capable of detecting all forms of tuberculosis (TB), proteomics may identify specific Mycobacterium tuberculosis (M.tb) proteins in urine, with a potential as biomarkers. To identify candidate biomarkers for TB, proteome profile of urine from pulmonary TB patients was compared with non-disease controls (NDC) and disease controls (DC, Streptococcus pneumonia infected patients) using a combination of two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LCMS/MS). Eleven differentially expressed host proteins and Eighteen high abundant M.tb proteins were identified. Protein-protein interactome (PPI) and functional enrichment analyses like Gene Ontologies, Reactome pathway etc. demonstrated that the human proteins mainly belong to extracellular space and show physiological pathways for immune response and hematological disorders. Whereas, M.tb proteins belong to the cell periphery, plasma membrane and cell wall, and demonstrated catalytic, nucleotide binding and ATPase activities along with other functional processes. The study findings provide valuable inputs about the biomarkers of TB and shed light on the probable disease consequences as an outcome of the bacterial pathogenicity.

Biomineralization process in hard tissues: The interaction complexity within protein and inorganic counterparts.

Biomineralization can be considered as nature's strategy to produce and sustain biominerals, primarily via creation of hard tissues for protection and support. This review examines the biomineralization process within the hard tissues of the human body with special emphasis on the mechanisms and principles of bone and teeth mineralization. We describe the detailed role of proteins and inorganic ions in mediating the mineralization process. Furthermore, we highlight the various available models for studying bone physiology and mineralization starting from the historical static cell line-based methods to the most advanced 3D culture systems, elucidating the pros and cons of each one of these methods. With respect to the mineralization process in teeth, enamel and dentin mineralization is discussed in detail. The key role of intrinsically disordered proteins in modulating the process of mineralization in enamel and dentine is given attention. Finally, nanotechnological interventions in the area of bone and teeth mineralization, diseases and tissue regeneration is also discussed. STATEMENT OF SIGNIFICANCE: This article provides an overview of the biomineralization process within hard tissues of the human body, which encompasses the detailed mechanism innvolved in the formation of structures like teeth and bone. Moreover, we have discussed various available models used for studying biomineralization and also explored the nanotechnological applications in the field of bone regeneration and dentistry.

Mapping the Inorganic and Proteomic Differences among Different Types of Human Teeth: A Preliminary Compositional Insight.

In recent years, studies on mineralized tissues are becoming increasingly popular not only due to the diverse mechanophysical properties of such materials but also because of the growing need to understand the intricate mechanism involved in their assembly and formation. The biochemical mechanism that results in the formation of such hierarchical structures through a well-coordinated accumulation of inorganic and organic components is termed biomineralization. Some prime examples of such tissues in the human body are teeth and bones. Our current study is an attempt to dissect the compositional details of the inorganic and organic components in four major types of human teeth using mass spectrometry-based approaches. We quantified inorganic materials using inductively coupled plasma resonance mass spectrometry (ICP-MS). Differential level of ten different elements, Iron (Fe), Cadmium (Cd), Potassium (K), Sulphur (S), Cobalt (Co), Magnesium (Mg), Manganese (Mn), Zinc (Zn), Aluminum (Al), and Copper (Cu) were quantified across different teeth types. The qualitative and quantitative details of their respective proteomic milieu revealed compositional differences. We found 152 proteins in total tooth protein extract. Differential abundance of proteins in different teeth types were also noted. Further, we were able to find out some significant protein-protein interaction (PPI) backbone through the STRING database. Since this is the first study analyzing the differential details of inorganic and organic counterparts within teeth, this report will pave new directions to the compositional understanding and development of novel in-vitro repair strategies for such biological materials.

Accelerated identification of serine racemase inhibitor from Centella asiatica.

Serine racemase (SR) converts the free form of L-serine into D-serine (DS) in the mammalian brain. The DS functions as a co-agonist of N-methyl D-aspartate (NMDA) receptor. The over- activation of NMDA receptor leads to many neurological disorders like stroke, amyotrophic lateral sclerosis, Alzheimer's disease and an effective inhibitor of SR could be a corrective method for the receptor over-activation. We report for the first time here a rapid way of purifying and identifying an inhibitor from medicinal plants known to have the neuro-protective effect. We have purified SR inhibitor from the methanolic extract of Centella asiatica by affinity method. High resolution mass spectrometry and infrared spectroscopy were used to identify the ligand to be madecassoside. We have shown the madecassoside binding in silico and its inhibition of recombinant human serine racemase in vitro and ex vivo.

Disorder-to-order transition in PE-PPE proteins of Mycobacterium tuberculosis augments the pro-pathogen immune response.

A growing body of evidence supports the hypothesis that intrinsically disordered proteins often mediate host-pathogen interactions and modulate host functions for pathogen survival and virulence. Mycobacterium tuberculosis (M.tb) has evolved largely through reductive evolution, with a few exceptions such as the glycine-alanine-rich PE-PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, our analyses of the M.tb proteome and secretome revealed that the PE-PGRS subfamily is enriched for disordered regions and disordered binding sites, pointing to their importance in host-pathogen interactions. As a case study, the secondary structure of PE35-PPE68 and PE32-PPE65 of the pathogenesis-related RD1 and RD8 regions was analyzed through Fourier-transform infrared spectroscopy. These disordered proteins displayed a considerable structural shift from disordered to ordered while engaged in the formation of complexes. While these proteins are immunogenic individually and enhance the pro-pathogen response, their corresponding complexes enhanced the responses manifold as displayed here by PE35 and PPE68. It is likely that M.tb exploits such disorder-order structural dynamics as a strategy to mount a pro-pathogen response and subvert host defense for productive infection. This functional gain also serves as a means to compensate genomic content loss due to reductive evolution.

Publisher Correction: Characterization of protein extracts from different types of human teeth and insight in biomineralization.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of protein extracts from different types of human teeth and insight in biomineralization.

The present study describes an efficient method for isolation and purification of protein extracts from four types of human teeth i.e. molar, premolar, canine, and incisor. Detailed structural characterization of these protein extracts was done by Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) which showed that a major fraction of the proteins present are unstructured in nature including primarily random coils in addition to the other structures like extended beta (ß) structure, poly-l-proline-type II (PPII) helix, turns, with only a small fraction constituting of ordered structures like alpha (α) helix and ß sheets. These resultant labile structures give the proteins the necessary flexibility that they require to interact with a variety of substrates including different ions like calcium and phosphates and for other protein-protein interactions. We also did initial studies on the mineralization of calcium phosphate with the protein extracts. Nanoparticle tracking analysis (NTA) show an increase in the size of calcium phosphate accumulation in the presence of protein extracts. We propose that protein extracts elevate the crystallization process of calcium phosphate. Our current biophysical study provides novel insights into the structural characterization of proteins from human teeth and their implications in understanding the tooth biomineralization. As per our knowledge, this is the first report which focuses on the whole protein extraction from different types of human teeth as these extracts imitate the in vivo tooth mineralization.

Structural insights into the substrate binding mechanism of novel ArgA from Mycobacterium tuberculosis.

The Mycobacterium tuberculosis (Mtb) Rv2747 gene encodes for a functional protein known as ArgA, which plays an important role in the first step of the l-arginine biosynthesis pathway. ArgA transfers the acetyl group from the acetyl-CoA to either l-glutamate or l-glutamine, which are the known substrates. Here, we present two crystal structures of ArgA: one complexed with CoA and product bound N-acetylglutamine and the other complexed with acetyl-CoA and the inhibitor l-arginine at 2.3 and 3.0 Šresolution respectively. The Mtb ArgA protomer was found to have a "V" cleft and a "ß" bulge, archetypal of a classical GCN5-related N-acetyltransferase superfamily of proteins. The product bound form implies that ArgA can also acetylate l-glutamine like l-glutamate. The active site is strongly inhibited by l-arginine resulting in a closed conformation of ArgA and both l-arginine and N-acetylglutamine were found to occupy at the same active site. Together with structural analysis, molecular docking studies, microscale thermophoresis and enzyme inhibition assays, we conclude that l-glutamine, l-glutamate and l-arginine, all occupy at the same active site of ArgA. Furthermore in case of Mtb ArgA, l-arginine does not act as an allosteric inhibitor unlike other N-acetylglutamate synthase family of proteins.

Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein.

Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion-an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis.IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells.

Role of Infrared Spectroscopy and Imaging in Cancer Diagnosis.

BACKGROUND: Cancer is a major global health issue. It causes extensive individual suffering and gives a huge burden on the health care in society. Despite extensive research and different tools have been developed it still remains a challenge for early detection of this disease. FTIR imaging has been used to diagnose and differentiate the molecular differences between normal and diseased tissues. METHODS: Fourier Transform Infrared Spectroscopy (FTIR) is able to measure biochemical changes in tissue, cell and biofluids based on the vibrational signature of their components. This technique enables to the distribution and structure of lipids, proteins, nucleic acids as well as other metabolites. These differences depended on the type and the grade of cancer. RESULTS: We emphasize here, that the FTIR spectroscopy and imaging can be considered as a promising technique and will find its place on the detection of this dreadful disease because of high sensitivity, accuracy and inexpensive technique. Now the medical community started using and accepting this technique for early stage cancer detection. We discussed this technique and the several challenges in its application for the diagnosis of cancer in regards of sample preparations, data interpretation, and data analysis. The sensitivity of chemotherapy drugs on individual specific has also discussed. CONCLUSION: So far progressed has done with the FTIR imaging in understanding of cancer disease pathology. However, more research is needed in this field and it is necessary to understand the morphology and biology of the sample before using the spectroscopy and imaging because invaluable information to be figured out.

Studies on Liquefaction Time and Proteins Involved in the Improvement of Seminal Characteristics in Dromedary Camels (Camelus dromedarius).

Semen was collected from six dromedary camels using artificial vagina during rutting season. Liquefaction of the viscous semen occurred in 23.89 ± 1.49 h. During liquefaction, proteins with molecular masses of 24.55 kDa and 22.07 kDa appeared in conjunction with the disappearance of intact 26.00 kDa protein after 18-24 h. These proteins were identified as ß-nerve growth factors (ß-NGFs) in liquefied camel semen. Guanidine-HCL improves the rheological characteristics of dromedary camel semen along with significant (P < 0.01) increase in sperm motility. No significant differences were found in viability of spermatozoa indicating no visible detrimental effects on spermatozoa. The cause of semen viscosity, as well as proteins that are present in liquefied dromedary camel seminal plasma, is described for the first time.

Proteome analysis of the macroscopically affected colonic mucosa of Crohn's disease and intestinal tuberculosis.

Differentiation between intestinal tuberculosis (ITB) and Crohn's disease (CD) is challenging in geographical regions where both these diseases are prevalent. There is a need of biomarkers for differentiation between these two disorders. Colonic biopsies from inflamed mucosa of treatment-naive patients with ITB, CD and controls were used for analysis. Protein extracted from biopsies was digested with trypsin and resulting peptides were labeled with iTRAQ reagents. The peptides were subsequently analyzed using LC-MS/MS for identification and quantification. Gene ontology annotation for proteins was analyzed in PANTHER. Validation experiments were done for six differentially expressed proteins using immunohistochemistry. 533 proteins were identified and 241 proteins were quantified from 5 sets of iTRAQ experiments. While 63 were differentially expressed in colonic mucosa of patients with CD and ITB in at least one set of iTRAQ experiment, 11 proteins were differentially expressed in more than one set of experiments. Six proteins used for validation using immunohistochemistry in a larger cohort of patients; none of them however was differentially expressed in patients with ITB and CD. There are differentially expressed proteins in tissue proteome of CD and ITB. Further experiments are required using a larger cohort of homogeneous tissue samples.

Identification of invasion proteins of Cryptosporidium parvum.

Host cell interactions and invasion by Cryptosporidium is a complex process mediated by zoites ligand-host cell receptors. Knowledge of proteins involved in this process will enable entry level inhibitors to be tried as therapeutic agents. In the present study, invasion proteins of Cryptosporidium parvum were studied in vitro. Cryptosporidium sporozoites membrane proteins were isolated and Cy5 dye labelled. They were then allowed to interact with the intact host cells. The interacting proteins were identified using 2-dimensional gel electrophoresis followed by mass spectrometry analysis. Sixty-one proteins were identified including twenty-seven previously reported invasion proteins. The newly identified proteins such as serine/threonine protein kinase, PI4 kinase, Hsp105 and coiled coil may have their roles in the parasitic invasion process. Thus, a new approach was used in the study to identify the probable proteins involved in invasion and/or host-parasite interactions. The advantage of this method is that it takes only a months' time instead of decades to identify these proteins involved in invasion process.

Antioxidant and anticoagulant activity of sulfated polysaccharide from Gracilaria debilis (Forsskal).

Sulfated polysaccharide was isolated from Gracilaria debilis and purified through gel chromatography and their molecular weight was determined through AGE and PAGE. The total sugars in the crude, fractionated and purified polysaccharide were estimated as 52.65%, 59.70% and 67.60%, respectively. The ash and moisture content of crude and purified polysaccharide was found to be 14.2% and 23.5% and the polysaccharide was free from protein contamination. The sulfate and uronic acid contents in the crude, fractionated and purified were estimated as 14.08%, 15.33% and 16.01% and 10.12%, 13.56%, 16.70%. The elemental composition including carbon (crude - 23.12%, purified - 21.05%), hydrogen (crude - 3.4%, purified - 4.13%) and nitrogen (crude - 1.22%, purified - 0.56%) were also analyzed. The anticoagulant activity of the sulfated polysaccharide through APTT and PT was estimated at 14.11 and 8.23IU/mg. The purified polysaccharide with the molecular mass of 20kDa showed highest antioxidant activity (38.57%, 43.48% and 38.88%) in all the assays tested such as DPPH hydroxyl radical, superoxide radical, hydroxyl radical scavenging activities and the structural property was analyzed through FT-IR and (1)H NMR spectrum. The results together suggest that the isolated low molecular weight sulfated polysaccharide will demonstrate as a enormously available alternative natural source of antioxidant for industrial uses.

Comparative proteomic analysis of Taurine, Indicine, and crossbred (Bos taurus × Bos indicus) bull spermatozoa for identification of proteins related to sperm malfunctions and subfertility in crossbred bulls.

Subfertility is one of the most common problems observed among Taurine × Indicine crossbred bulls in tropical countries; however, the etiology remain unknown in most of the cases. In present study, we compared the proteomic profile of spermatozoa from crossbred bulls (Bos taurus × Bos indicus) against their purebred parent lines (Holstein Friesian [Taurine] and Tharparkar [Indicine]) to find out alteration in expressions of proteins, if any. The proteomic profiles of freshly ejaculated spermatozoa from these breeds were compared by two-dimensional difference gel electrophoresis, and differentially expressed proteins were identified through mass spectrometry. It was observed that compared to Holstein Friesian, nine proteins were underexpressed and eight proteins were overexpressed (P < 0.05) in the spermatozoa of crossbred bulls. Similarly, four proteins were overexpressed and four proteins were underexpressed (P < 0.05) in the spermatozoa of crossbred bulls compared to Tharparkar bulls. In concurrent three breed comparison, 14 proteins were found to be differentially expressed (P < 0.05) between these breeds. From the findings of the study, it is apparent that the expression levels of several functionally significant proteins are either upregulated or downregulated in spermatozoa of crossbred bulls, which might be related to high incidence of subfertility in these bulls.

Structural characterization and bioactivities of sulfated polysaccharide from Monostroma oxyspermum.

Sulfated polysaccharide was isolated from Monostroma oxyspermum through hot water extraction, anion-exchange and gel permeation column chromatography. The sulfated polysaccharide contained 92% of carbohydrate, 0% of protein, 7.8% of uronic acid, 22% of ash and 33% of moisture respectively. The elemental composition was analyzed using CHNS/O analyzer. The molecular weight of sulfated polysaccharide determined through PAGE was found to be as 55 kDa. Monosaccharides analysis revealed that sulfated polysaccharide was composed of rhamnose, fructose, galactose, xylose, and glucose. The structural features of sulfated polysaccharide were analyzed by NMR spectroscopy. Further the sulfated polysaccharide showed total antioxidant and DPPH free radical scavenging activity were as 66.29% at 250 µg/ml and 66.83% at 160 µg/ml respectively. The sulfated polysaccharide also showed ABTS scavenging ability and reducing power were as 83.88% at 125 µg/ml and 15.81% at 400 µg/ml respectively. The anticoagulant activity was determined for human plasma with respect to Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) was 20.09 IU and 1.79 IU at 25 µg/ml respectively. These results indicated that the sulfated polysaccharide from M. oxyspermum had potent antioxidant and anticoagulant activities.

MALDI-TOF mass spectrometry for rapid identification of clinical fungal isolates based on ribosomal protein biomarkers.

This study aimed to evaluate the identification of clinical fungal isolates (yeast and molds) by protein profiling using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS). A total of 125 clinical fungal culture isolates (yeast and filamentous fungi) were collected. The test set included 88 yeast isolates (Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida parapsilosis, Candida rugosa, Candida tropicalis and Cryptococcus neoformans) and 37 isolates of molds (Alternaria spp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cunninghamella spp., Histoplasma capsulatum, Microsporum gypseum, Microsporum nanum, Rhizomucor spp. and Trichophyton spp.). The correlation between MALDI TOF MS and conventional identification for all these 125 fungal isolates included in the study was 87.2% at the species level and 90.4% at the genus level. MALDI TOF MS results revealed that the correlation in yeast (n=88) identification was 100% both at the genus and species levels whereas, the correlation in mold (n=37) identification was more heterogeneous i.e. 10.81% isolates had correct identification up to the genus level, 56.7% isolates had correct identification both at the genus and species levels, whereas 32.42% isolates were deemed Not Reliable Identification (NRI). But, with the modification in sample preparation protocol for molds, there was a significant improvement in identification. 86.4% isolates had correct identification till the genus and species levels whereas, only 2.7% isolates had Not Reliable Identification. In conclusion, this study demonstrates that MALDI-TOF MS could be a possible alternative to conventional techniques both for the identification and differentiation of clinical fungal isolates. However, the main limitation of this technique is that MS identification could be more precise only if the reference spectrum of the fungal species is available in the database.

Crystal structure of the VapBC-15 complex from Mycobacterium tuberculosis reveals a two-metal ion dependent PIN-domain ribonuclease and a variable mode of toxin-antitoxin assembly.

Although PIN (PilT N-terminal)-domain proteins are known to have ribonuclease activity, their specific mechanism of action remains unknown. VapCs form a family of ribonucleases that possess a PIN-domain assembly and are known as toxins. The activities of VapCs are impaired by VapB antitoxins. Here we present the crystal structure of the VapBC-15 toxin-antitoxin complex from Mycobacterium tuberculosis determined to 2.1Å resolution. The VapB-15 and VapC-15 components assemble into one heterotetramer (VapB2C2) and two heterotrimers (VapBC2) in each asymmetric unit of the crystal. The active site of VapC-15 toxin consists of a cluster of acidic amino acid residues and two divalent metal ions, forming a well organised ribonuclease active site. The distribution of the catalytic-site residues of the VapC-15 toxin is similar to that of T4 RNase H and of Methanococcus jannaschii FEN-1, providing strong evidence that these three proteins share a similar mechanism of activity. The presence of both VapB2C2 and VapBC2 emphasizes the fact that the same antitoxin can bind the toxin in 1:1 and 1:2 ratios. The crystal structure determination of the VapBC-15 complex reveals for the first time a PIN-domain ribonuclease protein that shows two metal ions at the active site and a variable mode of toxin-antitoxin assembly. The structure further shows that VapB-15 antitoxin binds to the same groove meant for the binding of putative substrate (RNA), resulting in the inhibition of VapC-15's toxicity.

Delineation of the Structural Elements of Oriental Liver Fluke PLA2 Isoforms for Potent Drug Designing.

Clonorchis sinensis or the Chinese liver fluke is one of the most prevalent parasites affecting a major population in the oriental countries. The parasite lacks lipid generating mechanisms but is exposed to fatty acid rich bile in the liver. A secretory phospholipase A2, an enzyme that breaks down complex lipids, is important for the growth of the parasite. The enzyme is also implicated in the pathogenesis leading up to the hepatic fibrosis and its complications including cancer. The five isoforms of this particular enzyme from the parasite therefore qualify as potential drug targets. In this study, a detailed structural and ligand binding analysis of the isoforms has been done by modeling. The overall three dimensional structures of the isoforms are well conserved with three helices and a ß-wing stabilized by four disulfide bonds. There are characteristic differences at the calcium binding loop, hydrophobic channel and the C-terminal domain that can potentially be exploited for drug binding. But the most significant feature pertains to the catalytic site where the isoforms exhibit three variations of either a histidine-aspartate-tyrosine or histidine-glutamate-tyrosine or histidine-aspartate-phenylalanine. Molecular docking studies show that isoform specific residues and their conformations in the substrate binding hydrophobic channel make unique interactions with certain inhibitor molecules resulting in a perfect tight fit. The proposed ligand molecules have a predicted affinity in micro-molar to nano-molar range. Interestingly, few of the ligand binding interaction patterns is in accordance to the phylogenetic studies to thereby establish the usefulness of evolutionary mechanisms in aiding ligand design. The molecular diversity of the parasitic PLA2 described in this study provides a platform for personalized medicine in the therapeutics of clonorchiasis.