Brightfield multiplex immunohistochemistry with multispectral imaging.

Brightfield microscopy is the preferred method of pathologists for diagnosing solid tumors, utilizing common staining techniques such as hematoxylin and eosin staining and immunohistochemistry (IHC). However, as our understanding of the complex tumor microenvironment grows, there is increasing demand for multiplexed biomarker detection. Currently, multiplexed IHC assays are almost exclusively based on immunofluorescence because brightfield techniques are limited by the broad spectral absorption of chromogens and a reliance on conventional 3-channel color cameras.

Covalently deposited dyes: a new chromogen paradigm that facilitates analysis of multiple biomarkers in situ.

Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated.

Lysosomal positioning coordinates cellular nutrient responses.

mTOR (mammalian target of rapamycin) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes, including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates.

Delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties.

Autophagy allows cells to self-digest portions of their own cytoplasm for a multitude of physiological purposes, including innate and adaptive immunity functions. In one of its innate immunity manifestations, autophagy, is known to contribute to the killing of intracellular microbes, including Mycobacterium tuberculosis, although the molecular mechanisms have been unclear. Here, we delineated sequential steps of the autophagic pathway necessary to control intracellular M.

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking.

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery.

The Mycobacterium tuberculosis phagosome.

Tuberculosis is currently the most devastating human bacterial disease, causing millions of deaths annually and infecting an overwhelming percentage of the global population. Its success as a scourge lies in the ability of Mycobacterium tuberculosis to prevent normal phagolysosome biogenesis, essential to the destruction of invading microorganisms, inside macrophages. Recent work has identified host GTPases involved in the block of normal phagolysosome biogenesis during mycobacterial infection and has provided a set of methods, in particular efficient macrophage transfection, which will prove essential in examining the role of host effectors in this process.

Autophagic proteolysis of long-lived proteins in nonliver cells.

Autophagy is a cellular homeostasis pathway used to sustain cellular anabolic needs during times of nutrient or energy deprivation. Autophagosomes sequester cytoplasmic constituents, including macromolecules such as long-lived proteins. Upon fusion of autophagosomes with lysosomes, the engulfed cargo is degraded.

T helper 2 cytokines inhibit autophagic control of intracellular Mycobacterium tuberculosis.

Autophagy is a recently recognized immune effector mechanism against intracellular pathogens. The role of autophagy in innate immunity has been well established, but the extent of its regulation by the adaptive immune response is less well understood. The T helper 1 (Th1) cell cytokine IFN-gamma induces autophagy in macrophages to eliminate Mycobacterium tuberculosis.

Expression, production and release of the Eis protein by Mycobacterium tuberculosis during infection of macrophages and its effect on cytokine secretion.

The eis gene of Mycobacterium tuberculosis has been shown to play a role in the survival of the avirulent Mycobacterium smegmatis within the macrophage. In vitro and in vivo analysis of Deltaeis deletion mutants and complemented strains showed no effect on survival of M. tuberculosis in U-937 macrophages or in a mouse aerosol infection model, respectively.

Higher order Rab programming in phagolysosome biogenesis.

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5-Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion.

Bacterial luciferase is naturally destabilized in Mycobacterium tuberculosis and can be used to monitor changes in gene expression.

Reporter systems efficient at monitoring temporal gene expression in slow-growing mycobacteria would significantly aid the characterization of gene expression in specific environments. Bacterial luciferase is a reporter that has not been widely used to study gene expression in mycobacteria. This report describes the determination of the degradation of bacterial luciferase in Mycobacterium tuberculosis H37Ra and its utility as a reporter of temporal gene expression in this slow-growing mycobacterium.

Molecular characterization of the eis promoter of Mycobacterium tuberculosis.

To further understand Mycobacterium tuberculosis pathogenesis, the regulation of potential virulence genes needs to be investigated. The eis gene of M. tuberculosis H37Rv enhances the intracellular survival of Mycobacterium smegmatis, which does not contain eis, within macrophages (J.