BISCUIT: an efficient, standards-compliant tool suite for simultaneous genetic and epigenetic inference in bulk and single-cell studies.

Data from both bulk and single-cell whole-genome DNA methylation experiments are under-utilized in many ways. This is attributable to inefficient mapping of methylation sequencing reads, routinely discarded genetic information, and neglected read-level epigenetic and genetic linkage information. We introduce the BISulfite-seq Command line User Interface Toolkit (BISCUIT) and its companion R/Bioconductor package, biscuiteer, for simultaneous extraction of genetic and epigenetic information from bulk and single-cell DNA methylation sequencing. BISCUIT's performance, flexibility and standards-compliant output allow large, complex experimental designs to be characterized on clinical timescales. BISCUIT is particularly suited for processing data from single-cell DNA methylation assays, with its excellent scalability, efficiency, and ability to greatly enhance mappability, a key challenge for single-cell studies. We also introduce the epiBED format for single-molecule analysis of coupled epigenetic and genetic information, facilitating the study of cellular and tissue heterogeneity from DNA methylation sequencing.

Dupsifter: a lightweight duplicate marking tool for whole genome bisulfite sequencing.

SUMMARY: In whole genome sequencing data, polymerase chain reaction amplification results in duplicate DNA fragments coming from the same location in the genome. The process of preparing a whole genome bisulfite sequencing (WGBS) library, on the other hand, can create two DNA fragments from the same location that should not be considered duplicates. Currently, only one WGBS-aware duplicate marking tool exists. However, it only works with the output from a single tool, does not accept streaming input or output, and requires a substantial amount of memory relative to the input size. Dupsifter provides an aligner-agnostic duplicate marking tool that is lightweight, has streaming capabilities, and is memory efficient. AVAILABILITY AND IMPLEMENTATION: Source code and binaries are freely available at https://github.com/huishenlab/dupsifter under the MIT license. Dupsifter is implemented in C and is supported on macOS and Linux.

Activation of DAF-16/FOXO by reactive oxygen species contributes to longevity in long-lived mitochondrial mutants in Caenorhabditis elegans.

Mild deficits in mitochondrial function have been shown to increase lifespan in multiple species including worms, flies and mice. Here, we study three C. elegans mitochondrial mutants (clk-1, isp-1 and nuo-6) to identify overlapping genetic pathways that contribute to their longevity. We find that genes regulated by the FOXO transcription factor DAF-16 are upregulated in all three strains, and that the transcriptional changes present in these worms overlap significantly with the long-lived insulin-IGF1 signaling pathway mutant daf-2. We show that DAF-16 and multiple DAF-16 interacting proteins (MATH-33, IMB-2, CST-1/2, BAR-1) are required for the full longevity of all three mitochondrial mutants. Our results suggest that the activation of DAF-16 in these mutants results from elevated levels of reactive oxygen species. Overall, this work reveals an overlapping genetic pathway required for longevity in three mitochondrial mutants, and, combined with previous work, demonstrates that DAF-16 is a downstream mediator of lifespan extension in multiple pathways of longevity.

Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence.

The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.

Mitochondrial unfolded protein response transcription factor ATFS-1 promotes longevity in a long-lived mitochondrial mutant through activation of stress response pathways.

BACKGROUND: The mitochondrial unfolded protein response (mitoUPR) is a stress response pathway activated by disruption of proteostasis in the mitochondria. This pathway has been proposed to influence lifespan, with studies suggesting that mitoUPR activation has complex effects on longevity. RESULTS: Here, we examined the contribution of the mitoUPR to the survival and lifespan of three long-lived mitochondrial mutants in Caenorhabditis elegans by modulating the levels of ATFS-1, the central transcription factor that mediates the mitoUPR. We found that clk-1, isp-1, and nuo-6 worms all exhibit an ATFS-1-dependent activation of the mitoUPR. While loss of atfs-1 during adulthood does not affect lifespan in any of these strains, absence of atfs-1 during development prevents clk-1 and isp-1 worms from reaching adulthood and reduces the lifespan of nuo-6 mutants. Examining the mechanism by which deletion of atfs-1 reverts nuo-6 lifespan to wild-type, we find that many of the transcriptional changes present in nuo-6 worms are mediated by ATFS-1. Genes exhibiting an ATFS-1-dependent upregulation in nuo-6 worms are enriched for transcripts that function in stress response and metabolism. Consistent, with this finding, loss of atfs-1 abolishes the enhanced stress resistance observed in nuo-6 mutants and prevents upregulation of multiple stress response pathways including the HIF-1-mediated hypoxia response, SKN-1-mediated oxidative stress response and DAF-16-mediated stress response. CONCLUSIONS: Our results suggest that in the long-lived mitochondrial mutant nuo-6 activation of the mitoUPR causes atfs-1-dependent changes in the expression of genes involved in stress response and metabolism, which contributes to the extended longevity observed in this mutant. This work demonstrates that the mitoUPR can modulate multiple stress response pathways and suggests that it is crucial for the development and lifespan of long-lived mitochondrial mutants.

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism.

Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.

Diagnostic Performance of High Sensitivity Compared with Contemporary Cardiac Troponin I for the Diagnosis of Acute Myocardial Infarction.

BACKGROUND: We examined the diagnostic performance of high-sensitivity cardiac troponin I (hs-cTnI) vs contemporary cTnI with use of the 99th percentile alone and with a normal electrocardiogram (ECG) to rule out acute myocardial infarction (MI) and serial changes (deltas) to rule in MI. METHODS: We included consecutive patients presenting to a US emergency department with serial cTnI onclinical indication. Diagnostic performance for acute MI, including MI subtypes, and 30-day outcomes were examined. RESULTS: Among 1631 patients, MI was diagnosed in 12.9% using the contemporary cTnI assay and in 10.4% using the hs-cTnI assay. For ruling out MI, contemporary cTnI ≤99th percentile at 0, 3, and 6 h and a normal ECG had a negative predictive value (NPV) of 99.5% (95% CI, 98.6-100) and a sensitivity of 99.1% (95% CI, 97.4-100) for diagnostic and safety outcomes. Serial hs-cTnI measurements ≤99th percentile at 0 and 3 h and a normal ECG had an NPV and sensitivity of 100% (95% CI, 100-100) for diagnostic and safety outcomes. For ruling in MI, contemporary cTnI measurements had specificities of 84.4% (95% CI, 82.5-86.3) at presentation and 78.7% (95% CI, 75.4-82.0) with serial testing at 0, 3, and 6 h, improving to 89.2% (95% CI, 87.1-91.3) by using serial cTnI changes (delta, 0 and 6 h) >150%. hs-cTnI had specificities of 86.9% (95% CI, 85.1-88.6) at presentation and 85.7% (95% CI, 83.5-87.9) with serial testing at 0 and 3 h, improving to 89.3% (95% CI, 87.3-91.2) using a delta hs-cTnI (0 and 3 h) >5 ng/L. CONCLUSIONS: hs-cTnI and contemporary cTnI assays are excellent in ruling out MI following recommendations predicated on serial testing and the 99th percentile with a normal ECG. For ruling in MI, deltas improve the specificity. ClinicalTrials.gov Identifier: NCT02060760.

Discordance between ICD-Coded Myocardial Infarction and Diagnosis according to the Universal Definition of Myocardial Infarction.

BACKGROUND: International Classification of Diseases (ICD) coding is the standard diagnostic tool for healthcare management. At present, type 2 myocardial infarction (T2MI) classification by the Universal Definition of Myocardial Infarction (MI) remains ignored in the ICD system. We determined the concordance for the diagnosis of MI using ICD-9 coding vs the Universal Definition. METHODS: Cardiac troponin I (cTnI) was measured by both contemporary (cTnI) and high-sensitivity (hs-cTnI) assays in 1927 consecutive emergency department (ED) patients [Use of TROPonin In Acute coronary syndromes (UTROPIA) cohort] who had cTnI ordered on clinical indication. All patients were adjudicated using both contemporary and hs-cTnI assays. The Kappa index and McNemar test were used to assess concordance between ICD-9 code 410 and type 1 MI (T1MI) and type 2 MI (T2MI). RESULTS: Among the 249 adjudicated MIs using the contemporary cTnI, only 69 (28%) were ICD-coded MIs. Of 180 patients not ICD coded as MI, 34 (19%) were T1MI and 146 (81%) were T2MI. For the ICD-coded MIs, 79% were T1MI and 21% were T2MI. A fair Kappa index, 0.386, and a McNemar difference of 0.0892 (P < 0.001) were found. Among the 207 adjudicated MIs using the hs-cTnI assay, 67 (32%) were ICD coded as MI. Of the 140 patients not ICD coded as MI, 27 (19%) were T1MI and 113 (81%) were T2MI. For the ICD-coded MIs, 85% were T1MI and 15% T2MI. A moderate Kappa index, 0.439, and a McNemar difference of 0.0674 (P < 0.001) were found. CONCLUSIONS: ICD-9-coded MIs captured only a small proportion of adjudicated MIs, primarily from not coding T2MI. Our findings emphasize the need for an ICD code for T2MI.

Change Your Ways: Fostering Health Attitudes Toward Change Through Selective Exposure to Online Health Messages.

Health information is often sought online, despite varying credibility of online sources, and may shape health behaviors. This investigation builds on the Selective Exposure Self- and Affect-Management model to examine selective exposure to online health information from low- and high-credibility sources and subsequent effects on attitudes toward health behaviors. In a lab study, 419 participants accessed online search results about health topics. The display varied messages in a 4 × 2 × 2 all within-subjects design, with topic as a four-step factor (organic food, coffee, fruit and vegetable consumption, physical exercise) and source credibility (low vs. high) and issue stance (promoting vs. opposing health behavior) as two-step factors. Displayed messages either promoted or opposed the related behavior. Results showed that perceiving greater standard-behavior discrepancy (between recommended behavior standards and own behavior) fostered behavior-related attitudes through selective exposure to messages promoting that behavior. The effects from selective exposure to health messages on attitudes occurred regardless of associated source credibility.

SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

BACKGROUND: Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. RESULTS: To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. CONCLUSIONS: SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with limited bioinformatics experience to analyze their data and integrate next generation sequencing (NGS) technologies into the classroom. The SPARTA software and tutorial are available at sparta.readthedocs.org.

Internal medicine point-of-care ultrasound assessment of left ventricular function correlates with formal echocardiography.

PURPOSE: Although focused cardiac ultrasonographic (FoCUS) examination has been evaluated in emergency departments and intensive care units with good correlation to formal echocardiography, accuracy for the assessment of left ventricular systolic function (LVSF) when performed by internal medicine physicians still needs independent evaluation. METHODS: This prospective observational study in a 640-bed, academic, quaternary care center, included 178 inpatients examined by 10 internal medicine physicians who had completed our internal medicine bedside ultrasound training program. The ability to estimate LVSF with FoCUS as "normal," "mild to moderately decreased," or "severely decreased" was compared with left ventricular ejection fraction (>50%, 31-49%, and <31%, respectively) from formal echocardiography interpreted by a cardiologist. RESULTS: Sensitivity and specificity of FoCUS for any degree of LVSF impairment were 0.91 (95% confidence interval [CI] 0.80, 0.97) and 0.88 (95% CI 0.81, 0.93), respectively. The interrater agreement between internal medicine physician-performed FoCUS and formal echocardiography for any LVSF impairment was "good/substantial" with κ = 0.77 (p < 0.001), 95% CI (0.67, 0.87). Formal echocardiography was classified as "technically limited due to patient factors" in 20% of patients; however, echogenicity was sufficient in 100% of FoCUS exams to classify LVSF. CONCLUSIONS: Internal medicine physicians using FoCUS identify normal versus decreased LVSF with high sensitivity, specificity, and "good/substantial" interrater agreement when compared with formal echocardiography. These results support the role of cardiac FoCUS by properly trained internal medicine physicians for discriminating normal from reduced LVSF.

Slow growth of Mycobacterium tuberculosis at acidic pH is regulated by phoPR and host-associated carbon sources.

During pathogenesis, Mycobacterium tuberculosis (Mtb) colonizes environments, such as the macrophage or necrotic granuloma, that are acidic and rich in cholesterol and fatty acids. The goal of this study was to examine how acidic pH and available carbon sources interact to regulate Mtb physiology. Here we report that Mtb growth at acidic pH requires host-associated carbon sources that function at the intersection of glycolysis and the TCA cycle, such as pyruvate, acetate, oxaloacetate and cholesterol. In contrast, in other tested carbon sources, Mtb fully arrests its growth at acidic pH and establishes a state of non-replicating persistence. Growth-arrested Mtb is resuscitated by the addition of pyruvate suggesting that growth arrest is due to a pH-dependent checkpoint on metabolism. Additionally, we demonstrate that the phoPR two-component regulatory system is required to slow Mtb growth at acidic pH and functions to maintain redox homeostasis. Transcriptional profiling and functional metabolic studies demonstrate that signals from acidic pH and carbon source are integrated to remodel pathways associated with anaplerotic central metabolism, lipid anabolism and the regeneration of oxidized cofactors. Because phoPR is required for Mtb virulence in animals, we suggest that pH-driven adaptation may be critical to Mtb pathogenesis.

The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence.

Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

Visual presentation of statistical concepts in diagnostic testing: the 2 × 2 diagram.

OBJECTIVE: The purpose of this article is to present a visual conceptual framework for important statistical concepts in radiology, and to provide an online application to facilitate this visualization. CONCLUSION: Statistical measures such as sensitivity, specificity, and predictive values are ubiquitous in medical literature, yet thinking fluidly about these concepts is not always easy. The 2 × 2 diagram is a helpful guide.

Cell State of Origin Impacts Development of Distinct Endometriosis-Related Ovarian Carcinoma Histotypes.

Clear cell ovarian carcinoma (CCOC) and endometrioid ovarian carcinoma (ENOC) are ovarian carcinoma histotypes, which are both thought to arise from ectopic endometrial (or endometrial-like) cells through an endometriosis intermediate. How the same cell type of origin gives rise to two morphologically and biologically different histotypes has been perplexing, particularly given that recurrent genetic mutations are common to both and present in nonmalignant precursors. We used RNA transcription analysis to show that the expression profiles of CCOC and ENOC resemble those of normal endometrium at secretory and proliferative phases of the menstrual cycle, respectively. DNA methylation at the promoter of the estrogen receptor (ER) gene (ESR1) was enriched in CCOC, which could potentially lock the cells in the secretory state. Compared with normal secretory-type endometrium, CCOC was further defined by increased expression of cysteine and glutathione synthesis pathway genes and downregulation of the iron antiporter, suggesting iron addiction and highlighting ferroptosis as a potential therapeutic target. Overall, these findings suggest that while CCOC and ENOC arise from the same cell type, these histotypes likely originate from different cell states. This "cell state of origin" model may help to explain the presence of histologic and molecular cancer subtypes arising in other organs. SIGNIFICANCE: Two cancer histotypes diverge from a common cell of origin epigenetically locked in different cell states, highlighting the importance of considering cell state to better understand the cell of origin of cancer.

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Social comparison and envy on social media: A critical review.

There is both public and scholarly concern that (passive) social media use decreases well-being by providing a fertile ground for harmful (upward) social comparison and envy. The present review critically summarizes evidence on this assumption. We first comprehensively synthesize existing evidence, including both prior reviews and the most recent publications (2019-2021). Results show that earlier research finds social comparison and envy to be common on social media and linked to lower well-being. Yet, increasingly, newer studies contradict this conclusion, finding positive links to well-being as well as heterogeneous, person-specific, conditional, and reverse or reciprocal effects. The review identifies four critical conceptual and methodological limitations of existing evidence, which offer new impulses for future research.

Therapeutic efficacy of antimalarial drugs targeting DosRS signaling in Mycobacterium abscessus.

A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis, is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS. OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.

The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins.

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo-like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

Macrophage Infection Models for Mycobacterium tuberculosis.

Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages, HoxB8 conditionally immortalized myeloid cells, Max Planck Institute alveolar macrophage-like cells, and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.