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Tuberculosis is a devastating human disease that kills over 1.2 million people a year. This disease is caused by the bacterial pathogen Mycobacterium tuberculosis ( Mtb ). Mtb excels at surviving in the human host by entering a non-replicating, dormant state. The current work investigated the proteomic changes that Mtb undergoes in response to carbon starvation, a culture condition that models dormancy. The authors found broad effects of carbon starvation on the proteome, with the relative abundance of 37% of proteins significantly altered. Protein changes related to cell wall biosynthesis, metabolism, and drug susceptibility are discussed. Proteins associated with a carbon starvation phenotype are identified, and results are compared to other dormancy models, including hypoxia.

MicroRNAs have emerged as ubiquitous post-transcriptional regulators that coordinate many fundamental processes within cells, including those commonly linked to cancer when dysregulated. Profiling microRNAs across stages of cancer progression provides focus as to which microRNAs are key players in cancer development and are therefore important to manipulate with interventions to delay cancer onset and progression. Calorie restriction is one of the most effective preventive interventions across many types of cancer, although its effects on microRNAs have not been well characterized. We used the dimethylbenz[a]-anthracene-induced model of luminal mammary cancer in Sprague Dawley rats to elucidate which microRNAs are linked to progression in this type of cancer and, subsequently, to study how calorie restriction affects such microRNAs. We identified eight microRNAs (miR-10a, miR-10b, miR-21, miR-124, miR-125b, miR-126, miR-145 and miR-200a) to be associated with DMBA-induced mammary tumor progression. Calorie restriction, which greatly increased tumor-free survival and decreased the overall size of tumors that did develop, significantly decreased the expression of one microRNA, miR-200a, which was positively associated with tumor progression. We further showed that inhibition of miR-200a function, mimicking the effect of calorie restriction on this microRNA, inhibited proliferation in both rat (LA7) and human (MCF7) luminal mammary cancer cell lines. These findings present, for the first time, a stage-specific profile of microRNAs in a rodent model of luminal mammary cancer. Furthermore, we have identified the regulation of miR-200a, a microRNA that is positively associated with progression in this model, as a possible mechanism contributing to the anticancer effects of calorie restriction.

Obesity is associated with increased risk of breast cancer in postmenopausal women and is linked with poor prognosis in pre- and postmenopausal breast cancer patients. The mechanisms underlying the obesity-breast cancer connection are becoming increasingly clear and provide multiple opportunities for primary to tertiary prevention. Several obesity-related host factors can influence breast tumor initiation, progression and/or response to therapy, and these have been implicated as key contributors to the complex effects of obesity on cancer incidence and outcomes. These host factors include components of the secretome, including insulin, insulin-like growth factor-1, leptin, adiponectin, steroid hormones, cytokines, vascular regulators, and inflammation-related molecules, as well as the cellular and structural components of the tumor microenvironment. These secreted and structural host factors are extrinsic to, and interact with, the intrinsic molecular characteristics of breast cancer cells (including breast cancer stem cells), and each will be considered in the context of energy balance and as potential targets for cancer prevention.

Calorie restriction (CR) inhibits triple-negative breast cancer (TNBC) progression in several preclinical models in association with decreased insulin-like growth factor 1 (IGF1) signaling. To investigate the impact of CR on microRNAs (miRs) that target the IGF1/IGF1R pathway, we used the spontaneous murine model of TNBC, C3(1)/SV40 T-antigen (C3-TAg). In C3-TAg mice, CR reduced body weight, IGF1 levels, and TNBC progression. We evaluated the tumoral expression of 10 miRs. CR increased the expression of miR-199a-3p, miR-199a-5p, miR-486, and miR-15b. However, only miR-15b expression correlated with tumorigenicity in the M28, M6, and M6C C3-TAg cell lines of TNBC progression. Overexpressing miR-15b reduced the proliferation of mouse (M6) and human (MDA-MB-231) cell lines. Serum restriction alone or in combination with low levels of recombinant IGF1 significantly upregulated miR-15b expression and reduced Igf1r in M6 cells. These effects were reversed by the pharmacological inhibition of IGFR with BMS754807. In silico analysis using miR web tools predicted that miR-15b targets genes associated with IGF1/mTOR pathways and the cell cycle. Our findings suggest that CR in association with reduced IGF1 levels could upregulate miR-15b to downregulate Igf1r and contribute to the anticancer effects of CR. Thus, miR-15b may be a therapeutic target for mimicking the beneficial effects of CR against TNBC.

The pathogen ( ) can cause severe and difficult to treat chronic lung infections. Despite the rising incidence and clinical concern of infections, treatment options are limited and often ineffective. Treatment is complicated by 's ability to persist in a non-replicative, drug-resistant state. Several β-lactam antibiotics are potently bactericidal against but are underutilized because their molecular mechanisms of action against are incompletely understood. In the current study, we used β-lactam-derived activity-based probes and chemoproteomics to report the first comprehensive list of enzymes in targeted by β-lactams. We compared β-lactam targets across two subspecies in actively replicating and non-replicative cultures, using a new carbon starvation model of persistence. We identified 17 targets that were active in every condition tested, seven of which were previously unknown to bind β-lactams. Lastly, we characterized the β-lactamase activity and β-lactam inhibition profiles of nine enzymes, demonstrating that imipenem inhibits these targets more effectively than cefoxitin. These findings provide clarity on the mechanisms of action of clinically relevant β-lactams in , a crucial step toward fully realizing their potential for treating infections caused by this opportunistic pathogen.

( ) is the causative agent of tuberculosis (TB), the leading cause of infectious-disease related deaths worldwide. TB infections present as a spectrum from active to latent disease. In the human host, faces hostile environments, such as nutrient deprivation, hypoxia, and low pH. Under these conditions, can enter a dormant, but viable, state characterized by a lack of cell replication and increased resistance to antibiotics. These dormant pose a major challenge to curing infections and eradicating TB globally. In the current study, we subjected to carbon starvation (CS), a culture condition that induces growth stasis and mimics nutrient-starved conditions associated with dormancy . We provide a detailed analysis of the proteome in CS compared to replicating samples. We observed extensive proteomic reprogramming, with 36% of identified proteins significantly altered in CS. Many enzymes involved in oxidative phosphorylation and lipid metabolism were retained or upregulated in CS. The cell wall biosynthetic machinery was present in CS, although numerous changes in the abundance of peptidoglycan, arabinogalactan, and mycolic acid biosynthetic enzymes likely result in pronounced remodeling of the cell wall. Many clinically approved anti-TB drugs target cell wall biosynthesis, and we found that these enzymes were largely retained in CS. Lastly, we compared our results to those of other dormancy models and propose that CS produces a physiologically-distinct state of stasis compared to hypoxia in .

The historical model, which posits that β-lactams inhibit bacterial growth while β-lactamase inhibitors (BLIs) merely protect β-lactams from enzymatic degradation, fails to fully explain their activity against Mycobacterium abscessus (Mab). This study demonstrates that synergistic effects extend beyond the traditional one β-lactam+one BLI paradigm, refuting the oversimplified mechanistic framework. First, β-lactam-based BLIs such as clavulanic acid, sulbactam, and tazobactam exhibit intrinsic antibacterial activity against Mab. These agents synergized not only with β-lactams but also with one another, undermining their historical classification as mere β-lactamase inhibitors. The data indicate that their activity is not limited to inhibiting β-lactamases but extends to directly targeting critical bacterial processes. Second, dual β-lactam combinations exhibit synergism against Mab even in the absence of BLIs. For example, despite being rapidly hydrolyzed by the native β-lactamase BlaMab, amoxicillin demonstrates strong synergism with β-lactams such as imipenem or ceftaroline. This suggests that the second β-lactam either acts as a functional BLI surrogate or targets complementary pathways. Supporting this, experiments using penicillin- and carbapenem-based probes revealed that β-lactams bind to multiple Mab proteins simultaneously, reinforcing the idea that their synergy arises from targeting complementary essential proteins. Finally, triple combinations comprising dual β-lactam and one BLI, such as amoxicillin + ceftaroline + avibactam, achieved very high synergy, underscoring the complementary roles of dual β-lactams and BLIs. The evidence in this study necessitates a revised model that can more accurately explain the activities of β-lactams and BLIs and underscores the potential for optimizing β-lactam/BLI regimens against Mab.

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis. Comprehensive profiling of ligand-induced perturbation responses of the MCF10A mammary epithelial cell line provides an exhaustive resource for identification of the molecular basis of cellular phenotypes.

Infections with Mycobacterium tuberculosis (Mtb) cause tuberculosis (TB), which requires at least six months of treatment with multiple antibiotics. There is emergent interest in using β-lactam antibiotics to improve treatment outcomes for patients. These drugs target cell wall biosynthesis, but a comprehensive list of enzymes inhibited by β-lactams in Mtb is lacking. In the current study, we sought to identify and characterize Mtb enzymes inhibited by β-lactam antibiotics using physiological conditions representative of both acute and chronic TB disease. We used new activity-based probes based on the β-lactam antibiotic meropenem due to its approval by the World Health Organization for TB treatment. Activity-based probes label enzymes based on both substrate specificity and catalytic mechanism, enabling precise identification of drug targets. We identified previously undiscovered targets of meropenem in addition to known cell wall biosynthetic enzymes. We validated β-lactam binding and hydrolysis for six newly identified targets: Rv1723, Rv2257c, Rv0309, DapE (Rv1202), MurI (Rv1338), and LipD (Rv1923). Our results demonstrate that there are at least 30 enzymes in Mtb vulnerable to inhibition by meropenem. This is many more β-lactam targets than historically described, suggesting that efficacy in Mtb is a direct result of polypharmacology.Competing Interest StatementThe authors have declared no competing interest.

Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a protein's behavior in situ. Some proteins can be analyzed by fusion to protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in living cells. Labeling is mediated by the formation of a high affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific, with no cross-reactivity between the three VIP tags under cellular conditions. Lastly, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin), highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.Competing Interest StatementThe authors have declared no competing interest.