The High Sensitivity of the Multi-Cancer Detection Test ONCOVERYX-F Offers a Promising Platform for Ovarian Cancer Screening.

We evaluated the potential relevance of our multi-cancer detection test, OncoVeryx-F, for ovarian cancer screening. For this, we compared its accuracy with that of CA125-based screening. We demonstrate here that, in contrast to CA125-based detection, OncoVeryx-F detected ovarian cancer with very high sensitivity and specificity. Importantly here, Stage I cancers too could be detected with an accuracy of >98%. Furthermore, again unlike CA 125, the detection accuracy of OncoVeryx-F remained comparable in both Caucasian and South Asian/Indian women. Thus, the robustness and accuracy of OncoVeryx-F, particularly for early-stage detection, underscores its potential utility for ovarian cancer screening.

Temporal Profiling of Host Proteome against Different M. tuberculosis Strains Reveals Delayed Epigenetic Orchestration.

Apart from being preventable and treatable, tuberculosis is the deadliest bacterial disease afflicting humankind owing to its ability to evade host defence responses, many of which are controlled by epigenetic mechanisms. Here, we report the temporal dynamics of the proteome of macrophage-like host cells after infecting them for 6, 18, 30, and 42 h with two laboratory strains (H37Ra and H37Rv) and two clinical strains (BND433 and JAL2287) of Mycobacterium tuberculosis (MTB). Using SWATH-MS, the proteins characterized at the onset of infection broadly represented oxidative stress and cell cytoskeleton processes. Intermediary and later stages of infection are accompanied by a reshaping of the combination of proteins implicated in histone stability, gene expression, and protein trafficking. This study provides strain-specific and time-specific variations in the proteome of the host, which might further the development of host-directed therapeutics and diagnostic tools against the pathogen. Also, our findings accentuate the importance of proteomic tools in delineating the complex recalibration of the host defence enabled as an effect of MTB infection. To the best of our knowledge, this is the first comprehensive proteomic account of the host response to avirulent and virulent strains of MTB at different time periods of the life span of macrophage-like cells. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD022352.

A non-invasive method for concurrent detection of multiple early-stage cancers in women.

Untargeted serum metabolomics was combined with machine learning-powered data analytics to develop a test for the concurrent detection of multiple cancers in women. A total of fifteen cancers were tested where the resulting metabolome data was sequentially analysed using two separate algorithms. The first algorithm successfully identified all the cancer-positive samples with an overall accuracy of > 99%. This result was particularly significant given that the samples tested were predominantly from early-stage cancers. Samples identified as cancer-positive were next analysed using a multi-class algorithm, which then enabled accurate discernment of the tissue of origin for the individual samples. Integration of serum metabolomics with appropriate data analytical tools, therefore, provides a powerful screening platform for early-stage cancers.

A non-invasive method for concurrent detection of early-stage women-specific cancers.

We integrated untargeted serum metabolomics using high-resolution mass spectrometry with data analysis using machine learning algorithms to accurately detect early stages of the women specific cancers of breast, endometrium, cervix, and ovary across diverse age-groups and ethnicities. A two-step approach was employed wherein cancer-positive samples were first identified as a group. A second multi-class algorithm then helped to distinguish between the individual cancers of the group. The approach yielded high detection sensitivity and specificity, highlighting its utility for the development of multi-cancer detection tests especially for early-stage cancers.

Co-Administration of Anticancer Candidate MK-2206 Enhances the Efficacy of BCG Vaccine Against Mycobacterium tuberculosis in Mice and Guinea Pigs.

The failure of M. bovis BCG to induce long-term protection has been endowed to its inability to escape the phagolysosome, leading to mild activation of CD8+ mediated T cell response. Induction of apoptosis in host cells plays an important role in potentiating dendritic cells-mediated priming of CD8+ T cells, a process defined as "cross-priming." Moreover, IL-10 secretion by infected cells has been reported to hamper BCG-induced immunity against Tuberculosis (TB). Previously, we have reported that apoptosis of BCG-infected macrophages and inhibition of IL-10 secretion is FOXO3 dependent, a transcription factor negatively regulated by the pro-survival activated threonine kinase, Akt. We speculate that FOXO3-mediated induction of apoptosis and abrogation of IL-10 secretion along with M. bovis BCG immunization might enhance the protection imparted by BCG. Here, we have assessed whether co-administration of a known anti-cancer Akt inhibitor, MK-2206, enhances the protective efficacy of M. bovis BCG in mice model of infection. We observed that in vitro MK-2206 treatment resulted in FOXO3 activation, enhanced BCG-induced apoptosis of macrophages and inhibition of IL-10 secretion. Co-administration of M. bovis BCG along with MK-2206 also increased apoptosis of antigen-presenting cells in draining lymph nodes of immunized mice. Further, MK-2206 administration improved BCG-induced CD4+ and CD8+ effector T cells responses and its ability to induce both effector and central memory T cells. Finally, we show that co-administration of MK-2206 enhanced the protection imparted by M. bovis BCG against Mtb in aerosol infected mice and guinea pigs. Taken together, we provide evidence that MK-2206-mediated activation of FOXO3 potentiates BCG-induced immunity and imparts protection against Mtb through enhanced innate immune response.

Outlining the Grb2 interactome data and its interacting partners in HEK293 cells in absence and presence of epidermal growth factor.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein involved in the signal transduction pathways. This dataset enlists proteins which interact with Grb2 in the presence and absence of a mitogenic stimulus. Grb2 expressing HEK293 cells were cultured in light and heavy labeled SILAC media. Normal lysine and arginine were incorporated as light labels while 8 and 10 Da heavier labels of respective isotopes were used for heavy labeling. While light labeled cells were used to enrich basal Grb2 interactome, the heavy labeled cells were stimulated in presence of epidermal growth factor (EGF) to investigate the altered Grb2 interactome dynamics. Equal number of EGF stimulated and non-stimulated cells was pooled, lysed and subjected to affinity purification coupled to mass spectrometry (AP-MS). The variety of Grb2 protein partners changed as a consequence of EGF stimulation. Additionally, SILAC labeling helped in quantitative estimation of altered association of a few interactors with the bait protein. Data are available via PRIDE repository with the dataset identifier PXD012957 (https://www.ebi.ac.uk/pride/archive/projects/PXD012957).

Concurrent interactome and metabolome analysis reveals role of AKT1 in central carbon metabolism.

OBJECTIVE: Signal transduction not only initiates entry into the cell cycle, but also reprograms the cell's metabolism. To control abnormalities in cell proliferation, both the aspects should be taken care of, thus pleiotropic signaling molecules are considered as crucial modulators. Considering this, we investigated the role of AKT1 in central carbon metabolism. The role of AKT1 has already been established in the process of cell cycle, but its contribution to the central carbon metabolism is sparsely studied. RESULTS: To address this, we combined the metabolomics and proteomics approaches. In accordance to our hypothesis, we found that the AKT1 kinase activity is regulating the levels of acetyl CoA through pyruvate dehydrogenase complex. Further, the decreased levels of acetyl CoA and dependency of acetyl CoA acetyl transferase protein on AKT1 kinase activity was also found to perturb the synthesis rate of palmitic acid which is a representative of fatty acid. This was analyzed in the present study using lipid labeling method through mass spectrometry.

Rb interactome data and its modulations during cell cycle progression in HEK 293 cells.

The Rb protein is a tumor suppressor protein that regulates the key G1S checkpoint consequently blocking the progression of cell cycle into S-phase. Despite its pertinent role in cell cycle regulation, comprehensive information on its interacting partners across cell cycle progression is lacking. Here, we aim to submit a comprehensive set of Rb interactors as the cell progresses from G0 through G1 and S into G2 phase in HEK 293 cell line. Affinity purification of HA-tagged Rb protein along with its interactors was analyzed by mass spectrometry (AP-MS). SILAC labeling enabled differentiation of Rb interactors in different cell cycle stages as well as their quantification - G0 cells were labeled with light labels of lysine and arginine (K0R0), cells in G1S transition were labeled with heavy labels (K8R10) while the G2 cells were labeled with medium labels (K6R6). LC-MS/MS analysis resulted in 6 wiff files which were submitted to protein pilot software for peptide identification and quantification. Here we submit the dataset which clearly captures the changing interacting partners of the Rb protein as the cell cycle progressed from G0 through G1S checkpoint into G2 phase. Data is publicly available via ProteomeXchange with identifier PXD007708.

Defining the Akt1 interactome and its role in regulating the cell cycle.

Cell growth and proliferation are two diverse processes yet always linked. Akt1, a serine/threonine kinase, is a multi-functional protein implicated in regulation of cell growth, survival and proliferation. Though it has a role in G1/S progression, the manner by which Akt1 controls cell cycle and blends cell growth with proliferation is not well explored. In this study, we characterize the Akt1 interactome as the cell cycle progresses from G0 to G1/S and G2 phase. For this, Akt1-overexpressing HEK293 cells were subjected to AP-MS. To distinguish between individual cell cycle stages, cells were cultured in the light, medium and heavy labelled SILAC media. We obtained 213 interacting partners of Akt1 from these studies. GO classification revealed that a significant number of proteins fall into functional classes related to cell growth or cell cycle processes. Of these, 32 proteins showed varying association with Akt1 in different cell cycle stages. Further analyses uncovered a subset of proteins showing counteracting effects so as to tune stage-specific progression through the cycle. Thus, our study provides some novel perspectives on Akt1-mediated regulation of the cell cycle and offers the framework for a detailed resolution of the downstream cellular mechanisms that are mediated by this kinase.

Dataset to delineate changes in association between Akt1 and its interacting partners as a function of active state of Akt1 protein.

Akt1 is a multi-functional protein, implicated in multiple human solid tumors. Pertaining to its key role in cell survival, Akt1 is under focus for development of targeted therapies. Functional diversity of Akt1 is a result of its interactions with other proteins; which changes with changing context. This investigation was designed to capture the dynamics of Akt1 Interactome as a function of its active state. Delineating dynamic changes in association of Akt1 with its interactors could help us comprehend how it changes as a function of inhibition of its active form. Similar information on changes in Akt1 interactome as of now is not well explored. Akt1 expressing HEK293 cells were cultured in light and heavy labeled SILAC media. Normal lysine and arginine were incorporated as light labels while for heavy labeling the isotopes were 8 and 10 Da heavier. Light labeled cells represented the indigenous state of Akt1 interactome while heavy labeled cells represented Akt1 interactome in presence of its allosteric inhibitor, MK-2206. Equal number of cells from both conditions were pooled, lysed and subjected to Affinity Purification coupled to Mass Spectroscopy (AP-MS). Additionally, SILAC labeling aided in quantitative estimation of changing association of a number of proteins which were common to the two experimental conditions, with Akt1. Data are available via ProteomeXchange with identifier PXD005976.

Defining the Akt1 interactome data and delineating alterations in its composition as a function of cell cycle progression.

Akt1 is a multi-functional protein implicated in key cellular processes including regulation of proliferation, survival, metabolism and protein synthesis. Its functional diversity results through interactions with other proteins which change with changing context. This study was designed to capture proteins, which interact with Akt1 as the cell cycle progresses from G0 to G1S and then G2 phase. Such an insight might help us understand the role of Akt1 in cell cycle, which as of now is not well explored. Akt1 expressing HEK 293 cells were cultured in light, medium and heavy labeled SILAC media. Normal lysine and arginine were incorporated as light labels; 6 Da (Dalton) heavier isotopes of the same amino acids were used as medium labels; while for heavy labeling the isotopes were 8 and 10 Da heavier. Light labeled cells were arrested in G0 phase while medium and heavy labeled cells were arrested in G2 and G1S phases, respectively. Equal number of cells from each phase was pooled, lysed and subjected to Affinity Purification coupled to Mass Spectroscopy (AP-MS). The obtained Akt1 protein partners were observed to change as the cell cycle progressed from G0 to G1S and then to G2 phase. Additionally, SILAC labeling aided in quantitative estimation of changing association of a number of proteins which were common to two or more phases, with Akt1. Data are available via ProteomeXchange with identifier PXD005557.

A mathematical model predicting host mitochondrial pyruvate transporter activity to be a critical regulator of Mycobacterium tuberculosis pathogenicity.

Modulation of host metabolic machinery by Mycobacterium tuberculosis is a well established phenomenon. In our earlier study (Mehrotra et al., 2014), we observed a marked increase in acetyl-CoA levels in cells bearing virulent M. tuberculosis infections compared to host cells harbouring avirulent infections. The difference was observed inspite of similar levels of total host cellular pyruvate in both infection types. The present study aimed in capturing the cause for such a phenomenon that defines the pathogenicity of M. tuberculosis. Through mathematical model, we dissected the relative importance of virulence mediated effect on Pyruvate dehydrogenase (PDH) activity, rate of acetyl-CoA consumption and mitochondrial pyruvate transporter (MPC) activity in causing the observed outcomes. Simulation results exhibit MPC to be the key regulatory junction perturbed by virulent strains of M. tuberculosis leading to alteration of mitochondrial metabolic flux and regulation of acetyl-CoA formation. As an experimental validation, drug mediated inhibition of MPC activity was sufficient to reduce virulent bacillary loads, pointing towards a possible mechanistic target for drug discovery.

Dataset generated using hyperplexing and click chemistry to monitor temporal dynamics of newly synthesized macrophage secretome post infection by mycobacterial strains.

Here we provide data for SILAC and iTRAQ based hyperplexing combined with BONCAT based click chemistry for selective enrichment of newly synthesized proteins secreted by THP1 macrophages at various time points after infection with four different strains of Mycobacterium tuberculosis. The macrophages were infected with H37Ra, H37Rv, BND433 and JAL2287 strains of M. tuberculosis. Newly-synthesized secreted host proteins were observed, starting from six hours post-infection till 26 h, at 4 h intervals. We have combined BONCAT with hyperplexing (18-plex), which blends SILAC and iTRAQ, for the first time. Two sets of triplex SILAC were used to encode the strains of M. tuberculosis - H37Ra & H37Rv in one and BND433 & JAL2287 in another with a control in each. BONCAT was used to enrich the secretome for newly synthesized proteins while 6-plex iTRAQ labeling was employed to quantify the temporal changes in the captured proteome. Each set of 18-plex was run in 4 MS replicates with two linear and two non-linear separation modes. This new variant of hyperplexing method, combining triplex SILAC with 6-plex iTRAQ, achieves 18-plex quantitation in a single MS run. Hyperplexing enables large scale spatio-temporal systems biology studies where large number of samples can be processed simultaneously and in quantitative manner. Data are available via ProteomeXchange with identifier ProteomeXchange: PXD004281.

Dissecting host factors that regulate the early stages of tuberculosis infection.

Incomplete understanding of mechanisms involved in the host-pathogen interactions constrains our efforts to eliminate tuberculosis. In many individuals, resulting from immune response to mycobacterial infection organised structures called granulomas are formed. To identify host responses that may control at least the early stages of infection, we employed an in vitro granuloma model. Here, human PBMCs were infected with live Mycobacterium tuberculosis in culture, and the appearance of granuloma-like structures was monitored over the next several days. Production of cytokines and chemokines in culture supernatants was monitored at various times, and the resulting temporal profiles were examined for possible correlations with either granuloma formation, or bacterial growth. While a positive association of TNF-α and IFN-γ secretion levels with extent of granuloma formation could clearly be identified, we were, however, unable to detect any statistically significant relationship between any cytokine/chemokine and bacterial growth. Examination of specific host cellular biochemical pathways revealed that either modulation of neutral lipid homeostasis through inhibition of the Gi-protein coupled receptor GPR109A, or regulation of host metabolic pathways through addition of vitamin D, provided a more effective means of controlling infection. A subsequent genotypic analysis for a select subset of genes belonging to pathways known to be significant for TB pathology revealed associations of polymorphisms with cytokine secretions and bacterial growth independently. Collectively therefore, the present study supports that key metabolic pathways of the host cell, rather than levels of relevant cytokines/chemokines might be more critical for regulating the intracellular mycobacterial load, in the context of granuloma formation.

Comparative Proteomic Analyses of Avirulent, Virulent, and Clinical Strains of Mycobacterium tuberculosis Identify Strain-specific Patterns.

Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv, and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 protein groups covering ∼54% of the predicted M. tuberculosis proteome. Label-free quantification analysis of the data revealed 257 differentially expressed protein groups. The differentially expressed protein groups could be classified into seven K-means cluster bins, which broadly delineated strain-specific variations. Analysis of the data for possible mechanisms responsible for drug resistance phenotype of JAL suggested that it could be due to a combination of overexpression of proteins implicated in drug resistance and the other factors. Expression pattern analyses of transcription factors and their downstream targets demonstrated substantial differential modulation in JAL, suggesting a complex regulatory mechanism. Results showed distinct variations in the protein expression patterns of Esx and mce1 operon proteins in JAL and BND strains, respectively. Abrogating higher levels of ESAT6, an important Esx protein known to be critical for virulence, in the JAL strain diminished its virulence, although it had marginal impact on the other strains. Taken together, this study reveals that strain-specific variations in protein expression patterns have a meaningful impact on the biology of the pathogen.

Mycobacterial escape from macrophage phagosomes to the cytoplasm represents an alternate adaptation mechanism.

Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage.

Towards understanding the biological function of the unusual chaperonin Cpn60.1 (GroEL1) of Mycobacterium tuberculosis.

The 60 kDa heat shock proteins, also known as Cpn60s (GroELs) are components of the essential protein folding machinery of the cell, but are also dominant antigens in many infectious diseases. Although generally essential for cellular survival, in some organisms such as Mycobacterium tuberculosis, one or more paralogous Cpn60s are known to be dispensable. In M. tuberculosis, Cpn60.2 (GroEL2) is essential for cell survival, but the biological role of the non-essential Cpn60.1 (GroEL1) is still elusive. To understand the relevance of Cpn60.1 (GroEL1) in M. tuberculosis physiology, detailed transcriptomic analyses for the wild type H37Rv and cpn60.1 knockout (groEL1-KO) were performed under in vitro stress conditions: stationary phase, cold shock, low aeration, mild cold shock and low pH. Additionally, the survival of the groEL1-KO was assessed in macrophages at multiplicity of infection (MOI) of 1:1 and 1:5. We observed that survival under low aeration was significantly compromised in the groEL1-KO. Further, the gene expression analyses under low aeration showed change in expression of several key virulence factors like two component system PhoP/R and MprA/B, sigma factors SigM and C and adversely affected known hypoxia response regulators Rv0081, Rv0023 and DosR. Our work is therefore suggestive of an important role of Cpn60.1 (GroEL1) for survival under low aeration by affecting the expression of genes known for hypoxia response.

Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions.

Differential proteomics approach to identify putative protective antigens of Mycobacterium tuberculosis presented during early stages of macrophage infection and their evaluation as DNA vaccines.

Unsatisfactory performance of the existing BCG vaccines, especially against the adult pulmonary disease, has urged the need for an effective vaccine against tuberculosis (TB). In this study, we employed differential proteomics to obtain a list of antigens as potential vaccine candidates. Bacterial epitopes being presented at early stages on MHC class I and class II molecules of macrophages infected with Mycobacterium tuberculosis (M. tb) were identified using iTRAQ labelling and reverse phase LC-MS/MS. The putative vaccine candidates, thus identified, were tested as plasmid DNA vaccines in mice to ascertain their protective efficacy against the aerosolized M. tb challenge, based on their ability to reduce the bacterial load in the lungs of infected mice. Here, we observed that 4 out of the 17 selected antigens imparted significant protection against the challenge of M. tb. The four shortlisted antigens were further assessed in a more stringent guinea pig model, where too, they demonstrated.significant protection. It concludes that combining a proteomics approach with the in vivo assessment of vaccine candidates in animal models can be valuable in identifying new potential candidates to expand the antigenic repertoire for novel vaccines against TB.

M. tuberculosis Secretory Protein ESAT-6 Induces Metabolic Flux Perturbations to Drive Foamy Macrophage Differentiation.

The Foamy Macrophage (FM) differentiation forms a major component of the host dependent survival axis of M. tuberculosis. The FM which are characterized by the intracellular accumulation of lipid bodies (LBs), ensure a privileged existence for the bacilli through ready provision of nutrients and by conferring protection against bactericidal pathways. The mycobacterial secretory protein ESAT-6 has been identified as the molecular mediator of the FM differentiation process although little is known about the mechanism through which it induces this process. In the present study, we show that ESAT-6 induces GLUT-1 mediated enhanced glucose uptake by macrophages which is coupled to metabolic flux perturbations in the glycolytic pathway caused by differential rates of reaction at several steps in the pathway. Two major changes identified were the simultaneous buildup of DHAP (for Triglyceride synthesis) and AcCoA (for synthesis of 3-HB, ligand for the anti-lipolytic GPR109A). We also show that part of the observed effects involve protein- protein interactions between ESAT-6 and the macrophage glycolytic enzymes, Enolase1 and Phosphoglycerate kinase1.