Persistence Of Cells Research Articles

Thermosensitive hydrogel loaded with biosynthesized silver nanoparticles using Eucalyptus camaldulensis leaf extract as an alternative treatment for microbial biofilms and persistent cells in tissue infections

Biofilms play a key role in the development of chronic tissue infections and antimicrobial resistance. In situ-forming thermosensitive hydrogel containing biosynthesized silver nanoparticles using aqueous Eucalyptus camaldulensis leaf extract (bio-AgNPs) was developed as an efficient anti-biofilm agent with a sustained release for tissue infection treatment. The formulation with 24% poloxamer 407, 5% poloxamer 188, and 1% hydroxypropyl methylcellulose demonstrated gelation temperature at 33 °C and homogeneous dispersion of bio-AgNPs throughout three-dimensional networks. Pharmaceutical properties of bio-AgNPs-loaded hydrogel presented shear-thinning behavior and zero-order release profile. The formulation showed broad-spectrum antimicrobial activity and comparable effects with existing antibiotics against important pathogens, including Gram-positive, Gram-negative bacteria, and fungi. The formulation at 1/8–1/2 minimum inhibitory concentrations (MIC) significantly inhibited biofilm production in all tested pathogens (p < 0.05). Confocal laser scanning microscopy images clearly illustrated dead cells within mature biofilm upon the treatment with the formulation at 2MIC for 4 h. The formulation effectively scavenged free radicals and decreased nitric oxide production up to 98%. Decrease in mRNA levels of inflammation-related enzymes and pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 cells confirmed the formulation as a potential anti-inflammatory agent. The findings suggested that bio-AgNPs-loaded hydrogel represents a promising therapeutic approach for tissue infections.

Sustained Oncogenic Signaling in the Cytostatic State Enables Targeting of Nonproliferating Persistent Cancer Cells.

This study finds that sustained oncogenic signaling in therapy-induced cytostatic cancer cells confers targetable vulnerabilities to deplete persistent cancer cell populations and reduce cancer recurrence.

Persistent red blood cells retain their ability to move in microcapillaries under high levels of oxidative stress

Oxidative stress is one of the key factors that leads to red blood cells (RBCs) aging, and impairs their biomechanics and oxygen delivery. It occurs during numerous pathological processes and causes anaemia, one of the most frequent side effects of cancer chemotherapy. Here, we used microfluidics to simulate the microcirculation of RBCs under oxidative stress induced by tert-Butyl hydroperoxide. Oxidative stress was expected to make RBCs more rigid, which would lead to decrease their transit velocity in microfluidic channels. However, single-cell tracking combined with cytological and AFM studies reveals cell heterogeneity, which increases with the level of oxidative stress. The data indicates that the built-in antioxidant defence system has a limit exceeding which haemoglobin oxidation, membrane, and cytoskeleton transformation occurs. It leads to cell swelling, increased stiffness and adhesion, resulting in a decrease in the transit velocity in microcapillaries. However, even at high levels of oxidative stress, there are persistent cells in the population with an undisturbed biophysical phenotype that retain the ability to move in microcapillaries. Developed microfluidic analysis can be used to determine RBCs’ antioxidant capacity for the minimization of anaemia during cancer chemotherapy.

Molecular Basis of Beckwith-Wiedemann Syndrome Spectrum with Associated Tumors and Consequences for Clinical Practice.

Simple SummaryBeckwith–Wiedemann syndrome (BWS, OMIM 130650) is an inborn overgrowth disorder caused by molecular alterations in chromosome 11p15.5. These molecular changes affect so-called imprinted genes, i.e., genes which underlie a complex regulation which is linked to the parental origin of the gene copy. Thus, either the maternal gene copy is expressed or the paternal, but this balanced regulation is prone to disturbances. In fact, different types of molecular variants have been identified in BWS, resulting in a variable phenotype; thus, it was consented that the syndromic entity was extended to the Beckwith–Wiedemann spectrum (BWSp). Some molecular subgroups of BWSp are associated with an increased embryonic tumor risk and have different likelihoods for specific tumors. Therefore, the precise determination of the molecular subgroup is needed for precise monitoring and treatment, but the molecular diagnostic procedure has several limitations and challenges which have to be considered.Beckwith–Wiedemann syndrome (BWS, OMIM 130650) is a congenital imprinting condition with a heterogenous clinical presentation of overgrowth and an increased childhood cancer risk (mainly nephroblastoma, hepatoblastoma or neuroblastoma). Due to the varying clinical presentation encompassing classical, clinical BWS without a molecular diagnosis and BWS-related phenotypes with an 11p15.5 molecular anomaly, the syndromic entity was extended to the Beckwith–Wiedemann spectrum (BWSp). The tumor risk of up to 30% depends on the molecular subtype of BWSp with causative genetic or epigenetic alterations in the chromosomal region 11p15.5. The molecular diagnosis of BWSp can be challenging for several reasons, including the range of causative molecular mechanisms which are frequently mosaic. The molecular basis of tumor formation appears to relate to stalled cellular differentiation in certain organs that predisposes persisting embryonic cells to accumulate additional molecular defects, which then results in a range of embryonal tumors. The molecular subtype of BWSp not only influences the overall risk of neoplasia, but also the likelihood of specific embryonal tumors.

Abstract 5349: Single-cell analysis sheds light on the resistance mechanisms of a novel pan-HER inhibitor, pyrotinib, in non-small cell lung cancer

Abstract Recently, a novel oral, irreversible pan-HER tyrosine kinase inhibitor, pyrotinib, has shown anticancer effects on HER2-mutant non-small cell lung cancer (NSCLC) patients, but the explicit responses of lung cancer cells towards pyrotinib treatment are barely investigated. In our current study, we employed single-cell RNA sequencing to profile the longitudinal transcriptomic dynamics of lung cancer cells during the pyrotinib treatment, unraveling the potential resistance mechanisms. We treated a lung adenocarcinoma cell line, H358, with pyrotinib at doses of 100nM, 200nM, 500nM, and 1000nM every four days, respectively. Then, the untreated cells (P0) as well as longitudinal samples after each administration (P4, P8, P12, P16) were harvested for further analysis. After quality control, a total of 41,804 cells were sequenced and included for further analysis. The differentially expressed genes were determined using MAST and the differentially enriched cancer hallmark signatures based on single-sample Gene Set Variation Analysis (ssGSVA) were identified. The single-cell data revealed that the untreated cells (Sample P0) and persistent cells (Sample P16) had stable and distinct gene expression patterns, while cells during the course of treatment shared similar expression features at a transitional state. Several cancer driver genes were found to be aberrantly regulated during the administration, like EPHA2, CDKN2A, CDK4, PTEN, and MYC. Also, some pathways related to immune response were suppressed after pyrotinib treatment, including interferon alpha response, interferon gamma response, TNFA signaling via NF-κB, and inflammatory response. Intriguingly, CD274 (the gene encoding PD-L1) and LGALS9 were found to significantly decrease after pyrotinib treatment, suggesting that pyrotinib may be associated with outcomes of immunotherapy. With the large cell numbers analyzed, we distinguished an isolated, small subpopulation of cells characterized by the enriched expression of MGP, COL1A1, COL1A2, SPARC, and COL3A1 along with the elevated EMT and angiogenesis scores, suggesting its critical role in pyrotinib resistance. Collectively, our study for the first time reveals the transcriptomic evolution of NSCLC cells in response to pyrotinib administration at the single-cell resolution, providing new insights into potential mechanisms of resistance to this novel regimen in NSCLC. Citation Format: Xinfeng Wang, Yuan Li, Runsen Jin, Nan Sun, Jie He. Single-cell analysis sheds light on the resistance mechanisms of a novel pan-HER inhibitor, pyrotinib, in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5349.

Abstract 657: Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung cancer

Abstract Acquired drug resistance to even the most effective anti cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We have seen that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug tolerant cancer cells persisting during therapy. Preventing therapy induced A3A mutagenesis by gene deletion delayed the emergence of drug resistance. Here, we show that therapy induced A3A mutagenesis contributes to tumor evolution in NSCLC. Whole genome sequencing revealed that resistant clones that evolved from persistent drug tolerant cells (late clones) harbored more A3A mutations compared to the parental cell population than pre existing resistant clones (early clones). In a subset of NSCLC patients who received targeted therapies, we observed A3A mutations accompanied clonal evolution during treatment. Comparison of APOBEC mutation fractions in short vs long term responders suggests that short responders with acquired resistance mechanisms that evolved from pre existing resistant clones have less accumulation of APOBEC mutations. Collectively, these findings insist that an increase in mutagenic processes drives tumor evolution during targeted therapy treatment and leads to acquired resistance. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to halt the evolution of resistant clones and prevent acquired resistance to lung cancer targeted therapy. Citation Format: Hideko Isozaki, Ammal Abbasi, Naveed Nikpour, Marcello Stanzione, Ramin Sakhtemani, Susanna L. Monroe, Alice T. Shaw, Jessica J. Lin, Lecia V. Sequist, Zofia Piotrowska, Rémi Buisson, Michael S. Lawrence, Aaron N. Hata. Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 657.

Abstract 3710: Synergistic cell death induction in ovarian cancer by cisplatin and ABT-199 is mediated by expression of NOXA

Abstract Ovarian cancer is among the top ten leading causes of cancer death in women worldwide with a mortality rate of roughly 5% among all cancer deaths. So far, there are no recommended screening tests for the early detection of ovarian cancer. Therefore, patients are frequently diagnosed at late stages of ovarian cancer, which is associated with unfavorable 5-year survival. Patients suffering from ovarian cancer often show a good response to initial therapy with platinum-based compounds and/or taxanes subsequent to resection of the affected tissue. Unfortunately, ovarian cancer shows a high frequency of relapse and after chemotherapy persistent tumor cells re-expand and successively evolve into therapy-resistant tumors. We recently reported that cisplatin (cisPt) treatment of ovarian cancer cells enhances mitochondrial content and mitochondrial reactive oxygen species (Kleih et al. 2019). We therefore speculated that the enhanced mitochondrial content primes cisPt-treated cells for apoptosis induced by BH3 mimetic drugs, such as Venetoclax (ABT-199), that act on anti-apoptotic Bcl-2 proteins at the outer mitochondrial membrane. To explore this hypothesis, we investigated whether the combined administration of cisPt and ABT-199 leads to a synergistic cell death induction in ovarian cancer cells with different cisPt resistance and mitochondrial content. Detection of cell death by flow cytometric measurements of the mitochondrial membrane potential and exposure of phosphatidylserine demonstrated a synergistic cell death induction by cisPt and ABT-199 in OVCAR4 and OVCAR8 cells, irrespective of the cisPt resistance. Interestingly, Western blot analysis revealed that ABT-199 induced the accumulation of the BH3-only protein NOXA in both cancer cell lines. Accumulation of NOXA was dependent on ABT-199-mediated activation of the stress-responsive transcription factor ATF4. Knock-down experiments showed that apoptosis of cisPt-resistant OVCAR8 cells by the combined treatment with ABT-199 and cisPt was entirely dependent on NOXA. Moreover, also cisPt-sensitive OVCAR4 cells revealed an initially reduced apoptosis induction by the NOXA knock-down. Our data therefore elucidate the molecular mechanism of the synergistic efficacy of cisPt/ABT-199 combination therapies, which should be promising especially for the treatment of recurrent tumors developing reduced sensitivity to cisPt-based treatments. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias B. Beigl, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in ovarian cancer by cisplatin and ABT-199 is mediated by expression of NOXA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3710.

Abstract 5364: The TEAD autopalmitoylation inhibitor VT3989 improves efficacy and increases durability of efficacy of osimertinib in preclinical EGFR mutant tumor models

Abstract The Hippo-YAP/TAZ pathway is involved in the regulation of cell proliferation, survival, and cell migration. The major effectors of the Hippo pathway are the TEAD transcription factors, which become transcriptionally activated upon YAP/TAZ binding. Genetic alterations of pathway components have been reported in a variety of human malignancies, resulting in YAP/TAZ constitutive nuclear localization and TEAD activation. The TEAD transcription factors have been shown to autopalmitoylate, and palmitoylation is required for its interaction with YAP/TAZ and hence activation of transcriptional activity. We have identified potent, selective, and orally available small molecule compounds that directly interact with TEAD and block its autopalmitoylation (Tang et al, 2021, Mol Cancer Ther. 20(6):986-998). These TEAD inhibitors disrupt YAP/TAZ-TEAD protein interaction, suppress TEAD transcriptional activity, and selectively block proliferation of NF2-deficient mesothelioma in vitro and inhibit NF2 mutant xenograft tumor growth in vivo. Nuclear YAP accumulation and functional requirement have also been linked to resistance to targeted therapies and cancer relapse in BRAF-mutant, KRAS-mutant, EGFR-mutant, and ALK-rearranged non-small cell lung cancers (NSCLC). Hence, as TEAD transcription factors are the main drivers for YAP/TAZ recruitment to chromatin, we evaluated the effect of our TEAD inhibitor VT3989, currently in clinical testing, on emerging drug tolerant persistent cancer cells and drug resistance during EGFR-tyrosine kinase inhibitor osimertinib treatment in combination studies. In in vitro cell proliferation assays, VT3989 showed strong to very strong synergy in combination with osimertinib in several EGFR mutant NSCLC cell lines tested. In vivo, we observed strong VT3989 and osimertinib combination effect in NCI-H1975 and HCC827 cell line-derived xenograft models. We also tested the combination in several EGFR mutant NSCLC patient-derived xenograft models and demonstrated that the addition of VT3989 enhanced the efficacy of osimertinib and delayed tumor regrowth compared to osimertinib alone. Citation Format: Tracy T. Tang, Leonard Post. The TEAD autopalmitoylation inhibitor VT3989 improves efficacy and increases durability of efficacy of osimertinib in preclinical EGFR mutant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5364.

Abstract SY31-02: Revisiting NK cell immunity to prevent metastasis

Abstract Metastases arising in distant tissues continue to cause the majority of all cancer-related deaths. The uncertainty of whether and when metastases will occur poses a major challenge to treatment effectiveness. In many cancer patients, persistent disseminated tumor cells (DTCs) enter a state of dormancy upon arrival at the distant tissue, only to awaken years or even decades afterwards and initiate deadly metastases. How are DTCs kept dormant, what prompts them to awaken, and how can dormant DTCs be targeted are pressing questions in cancer research. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Recently, we developed a tool to follow dormant DTCs, and surveyed the anatomical distribution of dormant reservoirs in models of breast cancer metastasis. We discovered that DTCs laid preferentially dormant in the liver, and spatially distinct liver sub-microenvironments allow coexistence of dormant DTCs and rare growing metastases even within the same tissue. To explore whether the distinct fates of DTCs stemmed from intra-organ microenvironmental differences, we performed global transcriptomic profiling on hepatic milieus corresponding to dormancy and metastasis. We found that stromal cells clustered on the basis of disease stage, and genes that were upregulated in dormancy samples were associated with host defense processes and natural killer (NK) cell-mediated response. A thorough examination of viable populations of immune cells in each hepatic milieu revealed that NK cells were the only type of immune cell that increased in dormancy milieus. Gain- and loss-of-function experiments in vivo further indicated that the pool of NK cells encountered by DTCs is decisive on their awakening. Remarkably, adjuvant interleukin-15-based immunotherapy ensured an abundant pool of NK cells that sustained dormancy, thereby preventing hepatic metastases and prolonging survival. In addition, we demonstrated in vivo and in liver-like co-cultures that NK cells control dormant DTCs through IFN-γ-induced quiescence. Thus, we uncovered an alternative cytostatic IFNγ-mediated mechanism of NK cell immunity that is essential to the control of breast cancer dormancy. This finding reroutes the importance of IFNγ to much earlier stages of disease, and encourages its therapeutic use for establishing the non-permissive cytokine milieu that is necessary to limit the emergence of DTCs from dormancy. Another key finding of our work is that liver injury limits NK cell expansion, thus triggering the switch from dormancy to liver metastasis. Using a model of chemically-induced liver injury, we found that activated hepatic stellate cells (aHSCs, which activate from quiescent HSCs to proliferative myofibroblasts) orchestrate a stromal response that hampers NK cell-mediated immunity and precipitates metastasis. Mechanistically, we show that aHSCs secrete the chemokine CXCL12, inducing NK cell quiescence via its cognate receptor CXCR4. This NK cell-inhibitory function of CXCL12 adds to its canonical effect as an inducer of DTC proliferation, and contributes to metastatic outgrowth. Our findings revive clinical interest on the use of CXCR4 inhibitors in the treatment of patients with cancer, but suggest that they should be repurposed to earlier stages to prevent progression of dormant disease. Because the size of the NK cell pool can itself determine metastatic outgrowth, a decrease of NK cells in patients with cancer who are apparently disease-free might identify individuals who are at risk of recurrence and who would benefit from CXCR4 inhibition therapy - a question that we will address in a clinical trial that we are currently setting up at the University Hospital Basel. All this work, which I am the first and corresponding author of, was recently published in Nature (Correia et al. Nature, 2021). In summary, our study shows that DTC dormancy is achieved by preserving normal tissue physiology—particularly tissue innate immunity—and that disruptions of the latter present DTCs with an opportunity for reactivation. In patients, this is reflected in the invariable decline in host defense capabilities that occurs during ageing, but also in the increased risk that recurrent infections and lifestyle-related injury triggers (such as alcohol, obesity and smoking) bring to the sprouting of site-specific metastases. We envision adjuvant NK cell immunotherapy as a means to preserve tissue physiology and prevent metastatic disease. Citation Format: Ana Luisa Correia. Revisiting NK cell immunity to prevent metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY31-02.

Induction of autophagy-dependent ferroptosis to eliminate drug-tolerant human retinoblastoma cells

Carboplatin is the most used first-line drug for the treatment of human retinoblastoma (RB), a rare form of cancer in infancy and childhood. However, the clinical application of carboplatin is restricted due to the emergence of acquired multi-drug resistance (MDR) after long-term treatment. Here, we report a new strategy to eliminate MDR RB cells by inducing autophagy-dependent ferroptosis. Compared with parent cells, carboplatin-resistant human RB cells have higher autophagy activity, which drives the formation of MDR to other chemotherapeutic drugs (e.g., etoposide and vincristine). In addition to confirming the traditional strategy of inhibiting autophagy to overcome MDR, we also establish an approach of inducing selective ferritinophagy to eliminate drug-resistant cells. We evaluate the effectiveness and safety of 4-octyl itaconate, a cell-permeable derivative of the metabolite itaconate, in inducing ferritinophagy-dependent ferroptosis in the treatment of MDR RB cells in vitro and in xenograft mouse models. These findings may provide essential clues for initiating clinical trials that target autophagy-dependent ferroptosis to kill drug-tolerant persistent cells during RB therapy.

Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.

Single-cell transcriptomics identifies Mcl-1 as a target for senolytic therapy in cancer

Cells subjected to treatment with anti-cancer therapies can evade apoptosis through cellular senescence. Persistent senescent tumor cells remain metabolically active, possess a secretory phenotype, and can promote tumor proliferation and metastatic dissemination. Removal of senescent tumor cells (senolytic therapy) has therefore emerged as a promising therapeutic strategy. Here, using single-cell RNA-sequencing, we find that senescent tumor cells rely on the anti-apoptotic gene Mcl-1 for their survival. Mcl-1 is upregulated in senescent tumor cells, including cells expressing low levels of Bcl-2, an established target for senolytic therapy. While treatment with the Bcl-2 inhibitor Navitoclax results in the reduction of metastases in tumor bearing mice, treatment with the Mcl-1 inhibitor S63845 leads to complete elimination of senescent tumor cells and metastases. These findings provide insights on the mechanism by which senescent tumor cells survive and reveal a vulnerability that can be exploited for cancer therapy.

Boosting ferroptosis via abplatin(iv) for treatment of platinum-resistant recurrent ovarian cancer

Platinum resistance represents a huge difficulty in ovarian cancer treatment, resulting in disease recurrence and deaths in patients. However, platinum-resistant, recurrent diseases still lack fundamentally effective treatment. Herein, four platinum(IV) (Pt(IV)) prodrugs were developed from the most widely used cisplatin (CisPt) and oxaliplatin (OxaPt), which were subsequently loaded with human serum albumin (HSA) to form nanoparticles (NP1-NP4). We found that NP1, also named Abplatin(iv), showed the best anticancer activity in six ovarian cancer cell lines of various pathological types. Hence, Abplatin(iv) was further evaluated in terms of translational potential. We established patient-derived cells (PDCs) and patient-derived organoids (PDOs) and observed enhanced antitumor effects of Abplatin(iv) in these ex vivo models. Furthermore, in mice bearing orthotopic ovarian cancer, intraperitoneal administration of Abplatin(iv) resulted in targeted drug accumulation in tumors and decreased tumor burden more effectively than CisPt, without causing any detectable side effects. In addition, by analyzing single-cell transcriptome sequencing results, we found a phenotypic shift toward stem-like cells in ovarian cancers undergoing chemotherapy. Therefore, cancer stem cells (SKOV3–3rd) mimicking such persistent tumor cells in postchemotherapeutic residual lesions were established. Abplatin(iv) exhibited a significantly improved antitumor effect on SKOV3–3rd cells both in vitro and in vivo. Finally, RNA sequencing revealed that Abplatin(iv) worked by boosting cell ferroptosis compared with CisPt. Taken together, Abplatin(iv) exhibits low systemic toxicity and high anticancer activity. In particular, it shows robust efficacy against platinum-resistant ovarian cancer derived from patients, indicating its great clinical translation potential.

Antipersister strategies against stress induced bacterial persistence

The increase in antibiotic non-responsive bacteria is the leading concern in current research oriented to eliminate pathogens. Nowadays, the excess use of antibiotics without specifically understanding the potentiality of killing pathogens and bacterial survival patterns has helped bacteria emerge indefatigably. Bacteria use various mechanisms such as resistance, persistence, and tolerance to ensure survival. Among these, persistence is a mechanism by which bacteria reside in their dormant state, bypassing the effects of treatments, making it crucial for bacterial survival. Persistent bacterial cells arise from the normal bacterial population as a slow-growing subset of bacteria with no metabolic flux. This behavior renders it to survive for a longer duration and at higher concentrations of antibiotics. They are one of the underlying causes of recurrence of bacterial infections. The present article explains the detailed molecular mechanisms and strategies of bacterial persistence, including the toxin-antitoxin modules, DNA damage, the formation of inactive ribosomal complexes, (p)ppGpp network, antibiotic-induced persistence, which are triggered by drug-induced stress. The article also comprehensively covers the epigenetic memory of persistence in bacteria, and anti-persistent therapeutics like antimicrobial molecules, synthetic peptides, acyldepsipeptide antibiotics, and endolysin therapy to reduce persister cell formation and control their frequency. These strategies could be utilized in combating the pathogenic bacteria undergoing persistence.

A metabolic perspective into antimicrobial tolerance and resistance

A metabolic perspective into antimicrobial tolerance and resistance

Autophagy Targeting and Hematological Mobilization in FLT3-ITD Acute Myeloid Leukemia Decrease Repopulating Capacity and Relapse by Inducing Apoptosis of Committed Leukemic Cells.

Simple SummaryOne of the most frequent molecular anomalies in acute myeloid leukemia (AML) is the mutation of the fms-like receptor tyrosine kinase 3 through internal tandem duplications, giving rise to a constitutive proliferative signaling. Even though clinical trials have shown that targeting this mutated kinase is of interest and well tolerated, there is still a high frequency of relapse. The emergence of AML cells upon treatment is linked to their maintenance through resistance and persistence mechanisms. Because FLT3-ITD AML cells require autophagy, we explored the consequence of autophagy inhibition by blocking the PI3-kinase class III, Vps34, when AML cells were committed. Results in vitro, ex vivo and in vivo suggest that remission with low minimal residual disease in FLT3-ITD AML offers a promising therapeutic window to target persistent leukemic cells.Targeting FLT3-ITD in AML using TKI against FLT3 cannot prevent relapse even in the presence of complete remission, suggesting the resistance and/or the persistence of leukemic-initiating cells in the hematopoietic niche. By mimicking the hematopoietic niche condition with cultures at low oxygen concentrations, we demonstrate in vitro that FLT3-ITD AML cells decrease their repopulating capacity when Vps34 is inhibited. Ex vivo, AML FLT3-ITD blasts treated with Vps34 inhibitors recovered proliferation more slowly due to an increase an apoptosis. In vivo, mice engrafted with FLT3-ITD AML MV4-11 cells have the invasion of the bone marrow and blood in 2 weeks. After 4 weeks of FLT3 TKI treatment with gilteritinib, the leukemic burden had strongly decreased and deep remission was observed. When treatment was discontinued, mice relapsed rapidly. In contrast, Vps34 inhibition strongly decreased the relapse rate, and even more so in association with mobilization by G-CSF and AMD3100. These results demonstrate that remission offers the therapeutic window for a regimen using Vps34 inhibition combined with mobilization to target persistent leukemic stem cells and thus decrease the relapse rate.

Towards efficient GPGPU Cellular Automata model implementation using persistent active cells

Natural complex phenomena simulation relies on the application of advanced numerical models. Nevertheless, due to their inherent temporal and spatial computational complexity, efficient parallel computing algorithms are required in order to speed up simulation execution times. In this paper, we apply the Nvidia CUDA architecture to the simulation of a groundwater hydrological model based on the Cellular Automata formalism. Different implementations, using different memory access patterns and optimizations, regarding the application of persistent active cells (i.e., once a cell is activated, it remains such throughout a simulation), are presented and evaluated. The obtained results have demonstrated the full suitability of the approach in speeding up simulation times, thus resulting in a valid support for complex system modeling.

Disinfectant resistance of Salmonella in in vitro contaminated poultry house models and investigation of efficient disinfection methods using these models.

Salmonellaenterica subsp. enterica (Salmonella) shows disinfectant resistance by forming biofilms on solid surfaces. However, efficient disinfection methods to eliminate Salmonella biofilms from farms have not yet been examined in detail. In this study, more than 80% of Salmonella strains from farms in Yamagata prefecture, Japan, were biofilm producers. Regardless of the extent of their biofilm formation ability, their biofilms were highly resistant to hypochlorous acid on plastic surfaces. To establish efficient disinfection methods in farms, we developed in vitro Salmonella-contaminated poultry house models by depositing dust on ceramic and stainless-steel carriers in poultry houses for one month and culturing a representative Salmonella strain on the carriers. Biofilm-like structures, including Salmonella-like cells, were observed on the models by scanning electron microscopy. Salmonella was not efficiently removed from the models even by cleaning with a surfactant at 25/65°C and disinfection with quaternary ammonium compound or hypochlorous acid at 25°C; on the contrary, viable Salmonella cells increased in some tests under these conditions, suggesting that these models successfully simulate the highly persistent characteristics of Salmonella in farms. However, the persistent bacterial cells were markedly decreased by soaking in 65°C surfactant followed by rinsing with 80°C water, additional cleaning using chlorine dioxide or disinfection with dolomitic lime, suggesting the effectiveness of these methods against Salmonella in farms. Since many different disinfection conditions may be easily tested in laboratories, our models will be useful tools for establishing effective and practical disinfection methods in farms.

TRNA biogenesis and specific aminoacyl-tRNA synthetases regulate senescence stability under the control of mTOR.

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks. Using experimental models of senescence escape, we now described that the stability of this proliferative arrest relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this tumor suppressive pathway.

The Rise and the Death of Senescent Cells: From Mechanisms to Interventions

Abstract Aging is at the root of age-related diseases and therapies targeting basic age-associated mechanisms have the potential to extend healthy lifespan. A common feature of older organisms is the accumulation of senescent cells – cells that have irreversibly lost the capacity to undergo replication. Senescent cells are characterized by an irreversible cell cycle arrest and by the Senescence-Associated Secretory Phenotype (SASP), which include many tissue remodeling and pro-inflammatory factors. Senescent cells are intermittently present during embryogenesis and in young organisms. On the contrary senescent cells accumulate and persist in aging tissues. Significantly, these persistent senescent cells can drive low-grade chronic inflammation, and their genetic or pharmacological elimination is sufficient to delay a number of diseases and to improve health span. Here, I will discuss the mechanisms by which senescent cells can promote tissue aging and dysfunction and the potential of targeting senescent cells to delay human aging.