Unraveling CRP/cAMP-Mediated Metabolic Regulation In Escherichia coli Persister Cells.

A substantial gap persists in our comprehension of how bacterial metabolism undergoes rewiring during the transition to a persistent state. Also, it remains unclear which metabolic mechanisms become indispensable for persister cell survival. To address these questions, we directed our efforts towards persister cells in Escherichia coli that emerge during the late stationary phase. These cells have been recognized for their exceptional resilience and are commonly believed to be in a dormant state. Our results demonstrate that the global metabolic regulator Crp/cAMP redirects the metabolism of these antibiotic-tolerant cells from anabolism to oxidative phosphorylation. Although our data indicates that persisters exhibit a reduced metabolic rate compared to rapidly growing exponential-phase cells, their survival still relies on energy metabolism. Extensive genomic-level analyses of metabolomics, proteomics, and single-gene deletions consistently emphasize the critical role of energy metabolism, specifically the tricarboxylic acid (TCA) cycle, electron transport chain (ETC), and ATP synthase, in sustaining the viability of persisters. Altogether, this study provides much-needed clarification regarding the role of energy metabolism in antibiotic tolerance and highlights the importance of using a multipronged approach at the genomic level to obtain a broader picture of the metabolic state of persister cells.

Exploring the links between SOS response, mutagenesis, and resistance during the recovery period.

Although the mechanistic connections between SOS-induced mutagenesis and antibiotic resistance are well established, our current understanding of the impact of SOS response levels, recovery durations, and transcription/translation activities on mutagenesis remains relatively limited. In this study, when bacterial cells were exposed to mutagens like ultraviolet light for defined time intervals, a compelling connection between the rate of mutagenesis and the RecA-mediated SOS response levels became evident. Our observations also indicate that mutagenesis primarily occurs during the subsequent recovery phase following the removal of the mutagenic agent. When transcription/translation was inhibited or energy molecules were depleted at the onset of treatment or during the early recovery phase, there was a noticeable decrease in SOS response activation and mutagenesis. However, targeting these processes later in the recovery phase does not have the same effect in reducing mutagenesis, suggesting that the timing of inhibiting transcription/translation or depleting energy molecules is crucial for their efficacy in reducing mutagenesis. Active transcription, translation, and energy availability within the framework of SOS response and DNA repair mechanisms appear to be conserved attributes, supported by their consistent manifestation across diverse conditions, including the use of distinct mutagens such as fluoroquinolones and various bacterial strains.

Dosing-time, feeding, and sex-dependent variations of everolimus pharmacokinetics in mice.

BACKGROUND: Everolimus is an oral mammalian target of rapamycin (mTOR) inhibitor used as an immunosuppressant and anticancer. Its pharmacokinetics is highly variable, it has a narrow therapeutic window and shows chronotoxicity with the best time at ZT13 and worst time at ZT1 (ZT; Zeitgeber time, time after light onset) in the preclinical setting. OBJECTIVES: In the present study, we aimed to investigate whether the pharmacokinetics of everolimus vary according to dosing time and whether sex and feeding status interfere with the chronopharmacokinetics. METHOD: A single dosage of 5 mg/kg everolimus was administered orally to C57BL/6J male and female mice, in fed or fasted states at ZT1-rest and ZT13-activity times and blood and tissue samples were collected at 0.5, 1, 2, 4, 12, and 24 h following drug administration. Ileum, liver, plasma, and thymus concentrations of everolimus were determined. RESULTS: Females had a greater ileum AUC0-24h than males when fed (P = 0.043). Everolimus AUC0-24h in the liver was substantially greater at ZT1 than at ZT13 in a fasted state (P = 0.001). Plasma Cmax , AUC0-24h , and AUCtotal were not statistically significant between the groups (P = 0.098). In one of the target organs of everolimus, the thymus, males had considerably higher amounts at ZT1 than females (P = 0.029). CONCLUSION: Our findings imply that the pharmacokinetics of everolimus in mice may differ according to dosing time, sex, and feeding. Greater tissue distribution of everolimus at ZT1 may be associated with the worst tolerated time of everolimus. Our research suggests that oral chronomodulated everolimus therapy may be more effective and safer for cancer patients.

Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance.

Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Gram-positive bacteria by targeting their ribosomes. Herein, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase of Escherichia coli cells. Given that aminoglycoside uptake relies on the energy-driven electrochemical potential across the cytoplasmic membrane, our initial expectation was that these genetic perturbations would decrease the proton motive force (PMF), subsequently affecting the uptake of aminoglycosides. However, our results did not corroborate this assumption. We found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences in the mutant strains compared to the wild type. Additionally, intracellular concentrations of fluorophore-labeled gentamicin remained similar across all strains. To uncover the mechanism responsible for the observed tolerance in mutant strains, we employed untargeted mass spectrometry to quantify the proteins within these mutants and subsequently compared them to their wild-type counterparts. Our comprehensive analysis, which encompassed protein-protein association networks and functional enrichment, unveiled a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research has the potential to uncover mechanisms behind aminoglycoside tolerance, paving the way for novel strategies to combat such cells.

Expression of Toll-Like Receptors in the Lung Tissue of Mouse Fetuses Generated by in vitro Embryo Culture and Embryo Transfer.

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO2-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro (n = 18) and EGin vivo (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Systematic design of pulse dosing to eradicate persister bacteria.

A small fraction of infectious bacteria use persistence as a strategy to survive exposure to antibiotics. Periodic pulse dosing of antibiotics has long been considered a potentially effective strategy towards eradication of persisters. Recent studies have demonstrated through in vitro experiments that it is indeed feasible to achieve such effectiveness. However, systematic design of periodic pulse dosing regimens to treat persisters is currently lacking. Here we rigorously develop a methodology for the systematic design of optimal periodic pulse dosing strategies for rapid eradication of persisters. A key outcome of the theoretical analysis, on which the proposed methodology is based, is that bactericidal effectiveness of periodic pulse dosing depends mainly on the ratio of durations of the corresponding on and off parts of the pulse. Simple formulas for critical and optimal values of this ratio are derived. The proposed methodology is supported by computer simulations and in vitro experiments.

Suppression of amyloid-ß fibril growth by drug-engineered polymorph transformation.

Fibrillization of the protein amyloid ß is assumed to trigger Alzheimer's pathology. Approaches that target amyloid plaques, however, have garnered limited clinical success, and their failures may relate to the scarce understanding of the impact of potential drugs on the intertwined stages of fibrillization. Here, we demonstrate that bexarotene, a T-cell lymphoma medication with known antiamyloid activity both in vitro and in vivo, suppresses amyloid fibrillization by promoting an alternative fibril structure. We employ time-resolved in situ atomic force microscopy to quantify the kinetics of growth of individual fibrils and supplement it with structure characterization by cryo-EM. We show that fibrils with structure engineered by the drug nucleate and grow substantially slower than "normal" fibrils; remarkably, growth remains stunted even in drug-free solutions. We find that the suppression of fibril growth by bexarotene is not because of the drug binding to the fibril tips or to the peptides in the solution. Kinetic analyses attribute the slow growth of drug-enforced fibril polymorph to the distinctive dynamics of peptide chain association to their tips. As an additional benefit, the bexarotene fibrils kill primary rat hippocampal neurons less efficiently than normal fibrils. In conclusion, the suggested drug-driven polymorph transformation presents a mode of action to irreversibly suppress toxic aggregates not only in Alzheimer's but also potentially in myriad diverse pathologies that originate with protein condensation.

Characteristics and long-term survival of patients with left ventricular non-compaction cardiomyopathy.

AIMS: Left ventricular non-compaction cardiomyopathy (LVNC) is a poorly understood entity resulting in heart failure. Whether it is a distinct form of cardiomyopathy or an anatomical phenotype is a subject of discussion. The current diagnosis is based on morphologic findings by comparing the compacted to non-compacted myocardium. The study aimed to compare demographic and prognostic variables of patients with dilated cardiomyopathy (DCM) and LVNC. Emphasis was given to cardiac magnetic resonance (CMR) imaging analysis. Data on survival were also assessed. METHODS AND RESULTS: We retrospectively evaluated the characteristics and outcomes of 262 non-ischaemic cardiomyopathy patients with LVNC and DCM phenotypes. Petersen's CMR criteria of non-compacted to the compacted myocardial ratio 2.3 were used to diagnose LVNC. The primary endpoint was a composite endpoint of major adverse cardiovascular events comprising cardiovascular-related death, left ventricular assisted device implantation, or heart transplantation. A total of 262 patients with CMR data were included in the study. One hundred fifty-five patients who fulfilled CMR criteria were diagnosed as LVNC. CMR findings revealed that LVNC patients had higher left ventricular end-diastolic (137.2 ± 51.6, 116.8 ± 44.6, P = 0.002) and systolic volume index (98.4 ± 49.5, 85.9 ± 42.7, P = 0.049). Cardiac haemodynamics, cardiac output (5.61 ± 2.03, 4.96 ± 1.83; P = 0.010), stroke volume (73.9 ± 28.8, 65.1 ± 25.1; P = 0.013), and cardiac index (2.85 ± 1.0, 2.37 ± 0.72; P < 0.0001), were higher in LVNC patients. Of all the 249 patients, 102 (40.9%) patients demonstrated late gadolinium enhancement (LGE). According to Petersen's criteria, the Kaplan-Meier survival outcome did not reveal significant differences (hazard ratio [HR]: 1.53, 95% confidence interval [CI]: [0.89-2.63], P = 0.11). The presence or pattern of LGE did not show significant importance for endpoint-free survival. Most of the sub-epicardial LGE pattern was found in LVNC patients (94.4%). When receiver operator characteristics analysis was applied to NC/C ratio to discriminate the primary endpoint, a higher NC/C ratio of 2.57 was associated with adverse events (HR: 1.90, 95% CI: [1.12-3.24], P = 0.016). CONCLUSIONS: Our study questions the criteria being used for the diagnosis of LVNC. Further evaluation of CMR variables and association of these findings with demographic variables and survival is mandatory.

Electrocardiographic variables associated with underlying Brugada syndrome or drug-induced Type 1 Brugada pattern in patients with slow/fast atrioventricular nodal reentrant tachycardia.

Background: The coexistence of clinical atrioventricular nodal reentrant tachycardia (AVNRT) and drug-induced type 1 Brugada pattern (DI-Type 1 BrP) has been previously reported. The present study was designed to determine the 12-lead ECG characteristics at baseline and during AVNRT and to identify a subset of 12-lead ECG variables of benefit associated with underlying Brugada syndrome (BrS)/DI-Type 1 BrP among patients with slow/fast AVNRT. Methods: A total of 40 (11 numerical/29 categorical) 12-lead ECG parameters were analyzed and compared between patients with (n = 69) and without (n = 104) BrS/DI-Type1-BrP matched for age, female gender, body mass index, left ventricular ejection fraction and comorbid conditions. Five distinct types of ECG pattern (Type A/B/C/D/E) in V1-V2 leads during AVNRT were defined. Results: A total of nine electrocardiographic variables, four at baseline, and five during AVNRT were identified. At baseline, patients with BrS/DI-Type 1 BrP had higher prevalence of interatrial block, leftward shift of frontal plane QRS axis, the absence of normal QRS pattern (the presence of rSr' pattern or type 2/3 Brugada pattern) in V1-V2 and QRS fragmentation in inferior leads compared to patients without BrS/DI-Type 1 BrP. During AVNRT, patients with BrS/DI-Type 1 BrP had higher prevalence of Type A ECG pattern ("coved-type" ST-segment elevation) in V1-V2, Type C ECG pattern (pseudo-r' deflection in V1 and "RBBB-like" pattern in V2), pseudo-r' deflection in V1, QRS fragmentation in inferior leads and "isolated" QRS fragmentation/notching/slurring in aVL compared to patients without BrS/DI-Type 1 BrP. Conclusions: We identify several electrocardiographic variables that point to an underlying type 1 BrP among patients with slow/fast AVNRT.

Proton Motive Force Inhibitors Are Detrimental to Methicillin-Resistant Staphylococcus aureus Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are tolerant of conventional antibiotics, making them extremely dangerous. Previous studies have shown the effectiveness of proton motive force (PMF) inhibitors at killing bacterial cells; however, whether these agents can launch a new treatment strategy to eliminate antibiotic-tolerant cells mandates further investigation. Here, using known PMF inhibitors and two different MRSA isolates, we showed that the bactericidal potency of PMF inhibitors seemed to correlate with their ability to disrupt PMF and permeabilize cell membranes. By screening a small chemical library to verify this correlation, we identified a subset of chemicals (including nordihydroguaiaretic acid, gossypol, trifluoperazine, and amitriptyline) that strongly disrupted PMF in MRSA cells by dissipating either the transmembrane electric potential (ΔΨ) or the proton gradient (ΔpH). These drugs robustly permeabilized cell membranes and reduced MRSA cell levels below the limit of detection. Overall, our study further highlights the importance of cellular PMF as a target for designing new bactericidal therapeutics for pathogens. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) emerged as a major hypervirulent pathogen that causes severe health care-acquired infections. These pathogens can be multidrug-tolerant cells, which can facilitate the recurrence of chronic infections and the emergence of diverse antibiotic-resistant mutants. In this study, we aimed to investigate whether proton motive force (PMF) inhibitors can launch a new treatment strategy to eliminate MRSA cells. Our in-depth analysis showed that PMF inhibitors that strongly dissipate either the transmembrane electric potential or the proton gradient can robustly permeabilize cell membranes and reduce MRSA cell levels below the limit of detection.

Efficacy of citalopram on stroke recurrence: A randomized clinical trial.

Post-stroke depression is one of the main causes of cerebrovascular and cardiovascular diseases. The aim of the present study was to investigate the efficacy of citalopram on stroke recurrence. A 52-week, randomized, double-blind, studyinvolved 440 ischemic stroke patients with depression. Patients with depression who met depression criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV and V) and Hamilton Depression Rating Scale ≥ 8 (HAM-DRS) were dichotomized into patients receiving citalopram (225 patients), titrated according to clinical response, and patients with placebo (215 patients) for 52 weeks. The primary outcome measure was stroke recurrence and the secondary outcome measures were cardiovascular events and mortality. Stroke recurrence (66% vs 34%; P = 0.001) and cardiovascular events (76% vs. 24%; P = o.oo1) were significantly higher in the placebo group compared to those treated with citalopram. Multivariable analysis showed that hypertension, atrial fibrillation, and large-artery disease were significantly associated with stroke recurrence. Executive processing disorder was more associated with stroke recurrence than other neuropsychological disorders (OR, 1.74; CI95%, 1.04-2.89; P = 0.035). Survival analysis showed that treatment for depression interacted with time to reduce stroke recurrence by nearly half (39% vs. 61%; P = 0.05). The current study supports the importance of depression treatment in protecting the patients from recurrent strokes. This result warrants further studies to demonstrate the efficacy of depression treatment on stroke recurrence.

Pleiotropic actions of phenothiazine drugs are detrimental to Gram-negative bacterial persister cells.

Bacterial persister cells are temporarily tolerant to bactericidal antibiotics but are not necessarily dormant and may exhibit physiological activities leading to cell damage. Based on the link between fluoroquinolone-mediated SOS responses and persister cell recovery, we screened chemicals that target fluoroquinolone persisters. Metabolic inhibitors (e.g., phenothiazines) combined with ofloxacin (OFX) perturbed persister levels in metabolically active cell populations. When metabolically stimulated, intrinsically tolerant stationary phase cells also became OFX-sensitive in the presence of phenothiazines. The effects of phenothiazines on cell metabolism and physiology are highly pleiotropic: at sublethal concentrations, phenothiazines reduce cellular metabolic, transcriptional, and translational activities; impair cell repair and recovery mechanisms; transiently perturb membrane integrity; and disrupt proton motive force by dissipating the proton concentration gradient across the cell membrane. Screening a subset of mutant strains lacking membrane-bound proteins revealed the pleiotropic effects of phenothiazines potentially rely on their ability to inhibit a wide range of critical metabolic proteins. Altogether, our study further highlights the complex roles of metabolism in persister cell formation, survival and recovery, and suggests metabolic inhibitors such as phenothiazines can be selectively detrimental to persister cells.

Effect of Coronary Thrombus Aspiration in Non ST Elevation Acute Coronary Syndrome Patients on Three-Year Survival- Does it add any Benefit?

We assessed the effect of thrombus aspiration (TA) during percutaneous coronary intervention (PCI) on in-hospital and 3-year mortality in consecutive non-ST segment elevation myocardial infarction (n = 189) and unstable angina pectoris (n = 148) patients (n = 337) between 2011 and 2016. In total, 153 patients (45.4%) underwent TA. The number of patients with postoperative thrombolysis in terms of myocardial infarction grade 3 blood flow (P < .001) and myocardial blush grade 3 (P < .001) were significantly higher in all TA groups. At 6-, 12- and 24-month post-PCI, the mean left ventricular ejection fraction was significantly higher in the all TA groups versus the stand alone PCI group (P < .001). Thrombus aspiration was associated with a significant improvement both in epicardial flow, myocardial perfusion and left ventricular ejection fraction. Thrombus aspiration during PCI in all acute coronary syndrome (except ST segment elevation) patients was associated with better survival compared with stand alone PCI group at 3-year follow-up (P = .019).

High-Throughput Screening of a Promoter Library Reveals New Persister Mechanisms in Escherichia Coli.

Persister cells are a small subpopulation of phenotypic variants that survive high concentrations of bactericidal antibiotics. Their survival mechanisms are not heritable and can be formed stochastically or triggered by environmental stresses such as antibiotic treatment. In this study, high-throughput screening of an Escherichia coli promoter library and subsequent validation experiments identified several genes whose expression was upregulated by antibiotic treatment. Among the identified genes, waaG, guaA, and guaB were found to be important in persister cell formation in E. coli as their deletion significantly enhanced the sensitivity of cells to various antibiotics. The GuaA and GuaB enzymes form the upstream reactions of ppGpp (a global persister molecule) biosynthesis, and the deletion of guaA and guaB drastically perturbs the ppGpp regulon in E. coli. WaaG, a lipopolysaccharide glucosyltransferase, plays an important role in shaping the outer membrane structure, and the deletion of waaG dissipates the proton gradient (ΔpH) component of cellular proton motive force (PMF), perturbs cellular ATP production, and reduces type I persister formation in stationary phase. Active respiration in the stationary phase, which drives the PMF, was previously shown to play a critical role in type I persister formation, and our results associated with the waaG deficient strain further corroborate these findings. IMPORTANCE Persistence is a nonheritable trait by which normal growing cells switch phenotypically to antibiotic tolerant persister cells. This transient state enables persister cells to recover and grow into an antibiotic-sensitive population. Persister cells have been observed in many pathogenic and nonpathogenic bacteria. Previous studies highlight the complexity and diversity of bacterial persister-cell mechanisms, many of which still remain to be elucidated. Here, using promoter and knockout cell libraries in Escherichia coli, we have identified genes that reveal novel persister mechanisms. As persistence is a critical survival strategy that evolved in many bacteria, our study will enhance the current molecular-level understanding of this conserved mechanism.

lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli.

Bacterial persisters are nongrowing cells highly tolerant to bactericidal antibiotics. However, this tolerance is reversible and not mediated by heritable genetic changes. Lon, an ATP-dependent protease, has repeatedly been shown to play a critical role in fluoroquinolone persistence in Escherichia coli. Although lon deletion (Δlon) is thought to eliminate persister cells via accumulation of the cell division inhibitor protein SulA, the exact mechanism underlying this phenomenon is not yet elucidated. Here, we show that Lon is an important regulatory protein for the resuscitation of the fluoroquinolone persisters in E. coli, and lon deletion impairs the ability of persister cells to form colonies during recovery through a sulA- and ftsZ-dependent mechanism. Notably, this observed "viable but nonculturable" state of antibiotic-tolerant Δlon cells is transient, as environmental conditions, such as starvation, can restore their culturability. Our data further indicate that starvation-induced SulA degradation or expression of Lon during recovery facilitates Z-ring formation in Δlon persisters, and Z-ring architecture is important for persister resuscitation in both wild-type and Δlon strains. Our in-depth image analysis clearly shows that the ratio of cell length to number of FtsZ rings for each intact ofloxacin-treated cell predicts the probability of resuscitation and, hence, can be used as a potential biomarker for persisters. IMPORTANCE The ATP-dependent Lon protease is one of the most studied bacterial proteases. Although deletion of lon has been frequently shown to reduce fluoroquinolone persistence, the proposed mechanisms underlying this phenomenon are highly controversial. Here, we have shown that lon deletion in Escherichia coli impairs the ability of persister cells to form colonies during recovery and that this reduction of persister levels in lon-deficient cells can be transient. We also found that altered Z-ring architecture is a key biomarker in both wild-type and lon-deficient persister cells transitioning to a normal cell state. Collectively, our findings highlight the importance of differentiating persister formation mechanisms from resuscitation mechanisms and underscore the critical role of the nonculturable cell state in antibiotic tolerance.

Cellular Self-Digestion and Persistence in Bacteria.

Cellular self-digestion is an evolutionarily conserved process occurring in prokaryotic cells that enables survival under stressful conditions by recycling essential energy molecules. Self-digestion, which is triggered by extracellular stress conditions, such as nutrient depletion and overpopulation, induces degradation of intracellular components. This self-inflicted damage renders the bacterium less fit to produce building blocks and resume growth upon exposure to fresh nutrients. However, self-digestion may also provide temporary protection from antibiotics until the self-digestion-mediated damage is repaired. In fact, many persistence mechanisms identified to date may be directly or indirectly related to self-digestion, as these processes are also mediated by many degradative enzymes, including proteases and ribonucleases (RNases). In this review article, we will discuss the potential roles of self-digestion in bacterial persistence.

BRAF-Inhibitor-Induced Metabolic Alterations in A375 Melanoma Cells.

Acquired drug tolerance has been a major challenge in cancer therapy. Recent evidence has revealed the existence of slow-cycling persister cells that survive drug treatments and give rise to multi-drug-tolerant mutants in cancer. Cells in this dynamic persister state can escape drug treatment by undergoing various epigenetic changes, which may result in a transient metabolic rewiring. In this study, with the use of untargeted metabolomics and phenotype microarrays, we characterize the metabolic profiles of melanoma persister cells mediated by treatment with vemurafenib, a BRAF inhibitor. Our findings demonstrate that metabolites associated with phospholipid synthesis, pyrimidine, and one-carbon metabolism and branched-chain amino acid metabolism are significantly altered in vemurafenib persister cells when compared to the bulk cancer population. Our data also show that vemurafenib persisters have higher lactic acid consumption rates than control cells, further validating the existence of a unique metabolic reprogramming in these drug-tolerant cells. Determining the metabolic mechanisms underlying persister cell survival and maintenance will facilitate the development of novel treatment strategies that target persisters and enhance cancer therapy.

Monitoring Persister Resuscitation with Flow Cytometry.

Persister cells are defined as a small fraction of phenotypic variants in a cell population that are temporarily tolerant to bactericidal antibiotics. Persisters are not mutant cells; they generally survive lethal concentrations of antibiotics due to their transient nongrowing state. Persister cells have the ability to resuscitate after the end of antibiotic treatment. Despite significant advancements in the understanding of the molecular mechanisms underlying persister formation, we still have little information about their resuscitation mechanisms. In this chapter, we describe a method to detect and monitor persister resuscitation at the single-cell level using flow cytometry analysis. This method enables us to not only assess the resuscitation characteristics of persisters but also determine and quantify various subpopulations in antibiotic-treated cultures, including viable but nonculturable (VBNC) and dead cells.