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.

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.

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.

Mycobacterium tuberculosis Inhibits RAB7 Recruitment to Selectively Modulate Autophagy Flux in Macrophages.

Here we report a novel regulatory mechanism for autophagy-mediated degradation of Mycobacterium tuberculosis (Mtb) and specific strategy exploited by the virulent Mtb to evade it. We show while both avirulent (H37Ra) and virulent (H37Rv) mycobacteria could readily localize to autophagosomes, their maturation into autolysosomes (flux) was significantly inhibited by the latter strain. The inhibition of autophagy flux by the virulent strain was highly selective, as it did not perturb the basal autophagy flux in the macrophages. Selective inhibition of flux of Mtb-containing autophagosomes required virulence regulators PhoP and ESAT-6. We show that the maturation of Mtb-containing autophagosomes into autolysosomes required recruitment of the late endosome marker RAB7, forming the intermediate compartment amphisomes. Virulent Mtb selectively evaded their targeting to the amphisomes. Thus we report a crosstalk between autophagy and phagosome maturation pathway and highlight the adaptability of Mtb, manifested by selective regulation of autophagy flux.

Selective enrichment of mycobacterial proteins from infected host macrophages.

Upon infection, Mycobacterium tuberculosis (Mtb) deploys specialized secretion machinery to deliver virulent proteins with the capacity to modulate a variety of host-cellular pathways. Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host. Here, we introduce an Mtb-selective protein labeling method based on genetic incorporation of azidonorleucine (Anl) through the expression of a mutant methionyl-tRNA synthetase. Exclusive incorporation of Anl, into native Mtb proteins, provided a click handle to pull out low abundant secretory proteins from the lysates of infected cells. Further, temporal secretome profiling, upon infection with strains of varying degree of virulence, revealed the proficiency of virulent Mtb to secrete chaperones. This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence. The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.

Pathogenicity of Mycobacterium tuberculosis is expressed by regulating metabolic thresholds of the host macrophage.

The success of Mycobacterium tuberculosis as a pathogen derives from its facile adaptation to the intracellular milieu of human macrophages. To explore this process, we asked whether adaptation also required interference with the metabolic machinery of the host cell. Temporal profiling of the metabolic flux, in cells infected with differently virulent mycobacterial strains, confirmed that this was indeed the case. Subsequent analysis identified the core subset of host reactions that were targeted. It also elucidated that the goal of regulation was to integrate pathways facilitating macrophage survival, with those promoting mycobacterial sustenance. Intriguingly, this synthesis then provided an axis where both host- and pathogen-derived factors converged to define determinants of pathogenicity. Consequently, whereas the requirement for macrophage survival sensitized TB susceptibility to the glycemic status of the individual, mediation by pathogen ensured that the virulence properties of the infecting strain also contributed towards the resulting pathology.

Express path analysis identifies a tyrosine kinase Src-centric network regulating divergent host responses to Mycobacterium tuberculosis infection.

Global gene expression profiling has emerged as a major tool in understanding complex response patterns of biological systems to perturbations. However, a lack of unbiased analytical approaches has restricted the utility of complex microarray data to gain novel system level insights. Here we report a strategy, express path analysis (EPA), that helps to establish various pathways differentially recruited to achieve specific cellular responses under contrasting environmental conditions in an unbiased manner. The analysis superimposes differentially regulated genes between contrasting environments onto the network of functional protein associations followed by a series of iterative enrichments and network analysis. To test the utility of the approach, we infected THP1 macrophage cells with a virulent Mycobacterium tuberculosis strain (H37Rv) or the attenuated non-virulent strain H37Ra as contrasting perturbations and generated the temporal global expression profiles. EPA of the results provided details of response-specific and time-dependent host molecular network perturbations. Further analysis identified tyrosine kinase Src as the major regulatory hub discriminating the responses between wild-type and attenuated Mtb infection. We were then able to verify this novel role of Src experimentally and show that Src executes its role through regulating two vital antimicrobial processes of the host cells (i.e. autophagy and acidification of phagolysosome). These results bear significant potential for developing novel anti-tuberculosis therapy. We propose that EPA could prove extremely useful in understanding complex cellular responses for a variety of perturbations, including pathogenic infections.

Delineation of key regulatory elements identifies points of vulnerability in the mitogen-activated signaling network.

Drug development efforts against cancer are often hampered by the complex properties of signaling networks. Here we combined the results of an RNAi screen targeting the cellular signaling machinery, with graph theoretical analysis to extract the core modules that process both mitogenic and oncogenic signals to drive cell cycle progression. These modules encapsulated mechanisms for coordinating seamless transition of cells through the individual cell cycle stages and, importantly, were functionally conserved across different cancer cell types. Further analysis also enabled extraction of the core signaling axes that progressively guide commitment of cells to the division cycle. Importantly, pharmacological targeting of the least redundant nodes in these axes yielded a synergistic disruption of the cell cycle in a tissue-type-independent manner. Thus, the core elements that regulate temporally distinct stages of the cell cycle provide attractive targets for the development of multi-module-based chemotherapeutic strategies.

Cellular phosphatases facilitate combinatorial processing of receptor-activated signals.

BACKGROUND: Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response. RESULTS: To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR) under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors. CONCLUSION: Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile.

Mycobacterium tuberculosis secreted antigen (MTSA-10) modulates macrophage function by redox regulation of phosphatases.

Macrophages are the primary host cells for Mycobacterium tuberculosis (Mtb). Although macrophages can mount a strong inflammatory response to dispose of invading microbial pathogens, the immune dysfunction of the Mtb-infected macrophage constitutes the hallmark of mycobacterial pathogenesis. A 10-kDa, Mtb secretory antigen (MTSA-10), encoded by ORF Rv3874, is one of the predominant members of the 'region of difference 1' locus of Mtb genome that has been strongly implicated in mycobacterial virulence. In this study, we investigated the possible role of MTSA-10 in modulating the macrophage dysfunction in a mouse macrophage cell line J774.1. We found that recombinant MTSA-10 caused extensive protein dephosphorylation in J774.1 cells as revealed by two-dimensional gel electrophoresis analysis. We also observed that MTSA-10 treatment downregulated the reactive oxygen species levels in the cells leading to activation of cellular protein phosphatases putatively responsible for the dephosphorylation phenomenon. This implied a direct role of MTSA-10 in the disruption of host cell signaling, resulting in downregulation of transcription of several genes essential for macrophage function.

The strength of receptor signaling is centrally controlled through a cooperative loop between Ca2+ and an oxidant signal.

Activation of cell-surface receptors stimulates generation of intracellular signals that, in turn, direct the cellular response. However, mechanisms that ensure combinatorial control of these signaling events are not well understood. We show here that the Ca2+ and reactive oxygen intermediates generated upon BCR activation rapidly engage in a cooperative interaction that acts in a feedback manner to amplify the early signal generated. This cooperativity acts by regulating the concentration of the oxidant produced. The latter exerts its influence through a pulsed inactivation of receptor-coupled phosphatases, where the amplitude of this pulse is determined by oxidant concentration. The extent of phosphatase inhibition, in turn, dictates what proportion of receptor-proximal kinases are activated and, as a result, the net strength of the initial signal. It is the strength of this initial signal that finally determines the eventual duration of BCR signaling and the rate of its transmission through downstream pathways.

A GPI-linked isoform of the IgD receptor regulates resting B cell activation.

The induction of a humoral response depends upon efficient cross-linking by antigen of surface immunoglobulin on primary B lymphocytes. We demonstrate here the presence of a glycosylphosphatidylinositol-linked isoform of membrane IgD (mIgD) receptors on murine resting B cells. This subset was constitutively localized to cell membrane raft microdomains. Its stimulation resulted in the activation of cAMP-dependent signaling pathways, which integrated with signals derived from the transmembrane mIgD receptors. This, in turn, provided a mechanism by which the activation status of the target cells could be variably regulated. Thus, by partitioning receptor activity, preimmune B cells can moderate the extent to which they are activated, depending upon the strength of the antigenic stimulus.

The primary antibody repertoire represents a linked network of degenerate antigen specificities.

In this study, germline Abs were used to select clones from a random dodecapeptide phage-display library. This revealed a much greater heterogeneity of binders than could be obtained with mutated daughter Abs that presumably had been selected in vivo by nominal Ag during active immune responses. We demonstrate that the pluripotency of germline Abs can subsequently be optimized by binding interactions that correlate with thermodynamic changes indicative of structural adaptations at the interface. This singular feature confers on each Ab a distinct window of Ag specificities, where the entropic space explored constitutes a thermodynamic signature of that particular Ab. Combining site plasticity may facilitate overlaps in such windows, with independent Abs converging onto common determinants with near identical binding affinities. In addition to providing for an amplified recognition potential, this networking of individual spectra of Ag specificities simultaneously facilitates the rapid recognition of Ag. Importantly, it also ensures that the primary response is composed of Abs with a high degree of "evolvability."

Epitope recognition by diverse antibodies suggests conformational convergence in an antibody response.

Crystal structures of distinct mAbs that recognize a common epitope of a peptide Ag have been determined and analyzed in the unbound and bound forms. These Abs display dissimilar binding site structures in the absence of the Ag. The dissimilarity is primarily expressed in the conformations of complementarity-determining region H3, which is responsible for defining the epitope specificity. Interestingly, however, the three Abs exhibit similar complementarity-determining region conformations in the Ag binding site while recognizing the common epitope, indicating that different pathways of binding are used for Ag recognition. The epitope also exhibits conformational similarity when bound to each of these Abs, although the peptide Ag was otherwise flexible. The observed conformational convergence in the epitope and the Ag binding site was facilitated by the plasticity in the nature of interactions.