Kinetics Of Cell Death Research Articles

Functional genomics reveals an off-target dependency of drug synergy in gastric cancer therapy

BackgroundIntegrating molecular-targeted agents into combination chemotherapy is transformative for enhancing treatment outcomes in cancer. However, realizing the full potential of this approach requires a clear comprehension of the genetic dependencies underlying drug synergy. While the interactions between conventional chemotherapeutics are well-explored, the interplay of molecular-targeted agents with conventional chemotherapeutics remains a frontier in cancer treatment. Hence, we leveraged a powerful functional genomics approach to decode genomic dependencies that drive synergy in molecular-targeted agent/chemotherapeutic combinations in gastric adenocarcinoma, addressing a critical need in gastric cancer therapy.MethodsWe screened pharmacological interactions between fifteen molecular-targeted agent/conventional chemotherapeutic pairs in gastric adenocarcinoma cells, and examined the genome-scale genetic dependencies of synergy integrating genome-wide CRISPR screening with the shRNA-based signature assay. We validated the synergy in cell death using fluorescence-based and lysis-dependent inference of cell death kinetics assay, and validated the genetic dependencies by single-gene knockout experiments.ResultsOur combination screen identified SN-38/erlotinib as the drug pair with the strongest synergism. Functional genomics assays unveiled a genetic dependency signature of SN-38/erlotinib identical to SN-38. Remarkably, the enhanced cell death with improved kinetics induced by SN-38/erlotinib was attributed to erlotinib’s off-target effect, inhibiting ABCG2, rather than its on-target effect on EGFR.ConclusionIn the era of precision medicine, where emphasis on primary drug targets prevails, our research challenges this paradigm by showcasing a robust synergy underpinned by an off-target dependency. Further dissection of the intricate genetic dependencies that underlie synergy can pave the way to developing more effective combination strategies in gastric cancer therapy.

A meta-analysis of microbial thermal inactivation in low moisture foods

Microbial thermal inactivation in low moisture foods is challenging due to enhanced thermal resistance of microbes and low thermal conductivity of food matrices. In this study, we leveraged the body of previous work on this topic to model key experimental features that determine microbial thermal inactivation in low moisture foods. We identified 27 studies which contained 782 mean D-values and developed linear mixed-effect models to assess the effect of microorganism type, matrix structure and composition, water activity, temperature, and inoculation and recovery methods on cell death kinetics. Intraclass correlation statistics (I2) and conditional R2 values of the linear mixed effects models were: E. coli (R2-0.91, I2-83%), fungi (R2-0.88, I2-85%), L. monocytogenes (R2-0.84, I2-75%), Salmonella (R2-0.69, I2-46%). Finally, global response surface models (RSM) were developed to further study the non-linear effect of aw and temperature on inactivation. The fit of these models varied by organisms from R2 0.88 (E. coli) to 0.35 (fungi). Further dividing the Salmonella data into individual RSM models based on matrix structure improved model fit to R2 0.90 (paste-like products) and 0.48 (powder-like products). This indicates a negative relationship between data diversity and model performance.

Mathematical modelling of Alzheimer's disease biomarkers: Targeting Amyloid beta, Tau protein, Apolipoprotein E and Apoptotic pathways.

Introduction: The kinetics of brain cell death in Alzheimer's disease (AD) is being studied using mathematical models. These mathematical models utilize techniques like differential equations, stochastic processes, and network theory to explore crucial signalling pathways and interactions between different cell types. One crucial area of research is the intentional cell death known as apoptosis, which is crucial for the nervous system. The main purpose behind the mathematical modelling of this is for identification of which biomarkers and pathways are most influential in the progression of AD. In addition, we can also predict the natural history of the disease, by which we can make early diagnosis. Mathematical modelling of AD: Current mathematical models include the Apolipoprotein E (APOE) Gene Model, the Tau Protein Kinetics Model, and the Amyloid Beta Peptide Kinetic Model. The Bcl-2 and Bax apoptosis theories postulate that the balance of pro- and anti-apoptotic proteins in cells determines whether a cell experiences apoptosis, where the Bcl-2 model, depicts the interaction of pro- and anti-apoptotic proteins, it is also being used in research on cell death in a range of cell types, including neurons and glial cells. How peptides are produced and eliminated in the brain is explained by the Amyloid beta Peptide (Aβ) Kinetics Model. The tau protein kinetics model focuses on production, aggregation, and clearance of tau protein processes, which are hypothesized to be involved in AD. The APOE gene model investigates the connection between the risk of Alzheimer's disease and the APOE gene. These models have been used to predict how Alzheimer's disease would develop and to evaluate how different inhibitors will affect the illness's course. Conclusion: These mathematical models reflect physiological meaningful characteristics and demonstrates robust fits to training data. Incorporating biomarkers like Aβ, Tau, APOE and markers of neuronal loss and cognitive impairment can generate sound predictions of biomarker trajectories over time in Alzheimer's disease.

Construction of a novel kinetic model for the production process of a CVA6 VLP vaccine in CHO cells.

The online version contains supplementary material available at 10.1007/s10616-023-00598-8.

377-OR: Stabilizing 14-3-3 and ChREBP Cytoplasmic Interaction to Rescue ß-Cells from Glucolipotoxicity

Carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor with two splice isoforms (α and β). ChREBPα remains cytoplasmic by binding to 14-3-3. Increased glucose metabolism allows ChREBPα to enter the nucleus and induce ChREBPβ, a hyperactive isoform that is a key mediator of glucolipotoxicity. Using structure-guided stabilizer optimization, we have developed novel drugs that stabilize the protein-protein interaction between ChREBPα and 14-3-3, with a goal to prevent ChREBPα nuclear translocation, induction of ChREBPβ, and glucolipotoxicity. One hit compound, K391, has an EC50 of 0.3±0.1μM by fluorescent polarization assay and a Kd of 0.12μM by isotitration calorimetry. Using dual label analysis of the kinetics of cell growth and cell death, we found that K391 significantly reduced β-cell death by 31.8±3% and enabled proliferation in glucolipotoxic conditions in INS-1 cells (20mM glucose and 1mM palmitate). In response to glucose, 76.4±3.5 % of the cells stained for nuclear ChREBP. In the presence of K391, 39.75±5.3 % fewer cells displayed nuclear ChREBP in high glucose concentrations and 65.7±1.6% less in glucolipotoxic conditions, indicating that K391 retains ChREBPα in the cytoplasm and prevents transcription of ChREBPβ. Time course with an antibody against ChREBPα showed that ChREBPα transiently translocated to the nucleus at 30 min and then cleared out of the nucleus in high glucose, an effect blocked by K391 (N=3). In glucolipotoxic conditions, ChREBPα remained nuclear over time, an effect significantly attenuated by K391 (N=3). Txnip is an important mediator of glucose toxicity and a target gene of ChREBPβ. Importantly, K391 blocked the glucose response of the human Txnip promoter in a luciferase assay by 2±0.2 folds. We conclude that the K391 retains ChREBPα in the cytoplasm in high glucose and in glucolipotoxic conditions by stabilizing the ChREBPα and 14-3-3 interaction and thereby rescues β-cells from glucolipotoxicity. Disclosure L.S.Katz: None. I.L.Tse: None. E.Visser: None. S.Baumel-alterzon: None. M.Kaiser: None. L.M.Brunsveld: Consultant; Novartis, Ambagon Therapeutics, Research Support; Roche Diagnostics. M.Pennings: None. C.Ottmann: None. D.Scott: None. Funding National Institutes of Health (R01DK130300)

1723-P: Dual-Label Kinetic Assay to Monitor ß-Cell Death and Proliferation

A deficit of β-cell mass is common to all types of diabetes. Thus, many research efforts are dedicated to developing therapies to preserve and expand β-cell mass. Current methods assessing β-cell proliferation and cell death are limited due to reliance on a single time point, and usually cannot measure proliferation and death simultaneously. Here we have developed and optimized a novel method, based on the single-cell and population-level analyses using real-time kinetic labeling (SPARKL), to study kinetics of cell proliferation and death in β cells. SPARKL uses two fluorescent dyes - SYTO21 labels all cells in green, and YOYO3, a red cell-impermeable viability dye that stains dead cells. We continuously imaged cells every few hours over numerous days and have optimized growth conditions and dye concentrations to monitor INS1 or primary rodent or human islet cells on single cell and population levels. As a first step, we compared growth under 6, 11 and 20 mM glucose and glucolipotoxicity (20 mM glucose and 1 mM palmitate). We found a doubling time of 52±2, 48±4, 44±3 and >120 h, respectively, in INS1 cells. At 48 h, fewer than 16% of the cells are dead in varying glucose concentrations compared to 69±5% dead cells in the glucolipotoxic condition (N=8). Harmine, a known mitogen of β-cells, enhanced proliferation in all glucose concentrations by 5-15% and protected from glucolipotoxic cell death (N=3). In primary islets, as expected, the proliferation rates were extremely low and often masked by cell death in varying glucose concentrations. Glucolipotoxicity led to >80% cell death in mouse and human islets (N=3). Finally, we screened a library of 73 epigenetic modifiers to screen for compounds that rescued INS1 cells from glucolipotoxic-like stress (overexpression of ChREBPβ). Our screen yielded four epigenetic modifiers that reduced cell death to less than 2% at 48 h. In conclusion, SPARKL is a novel and useful method that is accurate, unbiased, and requires minimal handling to simultaneously measure β-cell proliferation and death. Disclosure L.S.Katz: None. P.Wang: None. H.U.Kaniskan: None. J.D.Gelles: None. J.Chipuk: None. A.F.Stewart: None. D.Scott: None. Funding National Institutes of Health (R01DK130300)

Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine Macrophages.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 106 CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.

Performance Comparison of Recombinant Baculovirus and Rabies Virus-like Particles production Using Two Culture Platforms.

This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM's multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Innovative Insights into In Vitro Activity of Colloidal Platinum Nanoparticles against ESBL-Producing Strains of Escherichia coli and Klebsiella pneumoniae.

Growing morbidity and mortality rates due to increase in the number of infections caused by MDR (multi-drug resistant) microorganisms are becoming some of the foremost global health issues. Thus, the need to search for and find novel approaches to fight AMR (antimicrobial resistance) has become obligatory. This study aimed to determine the antimicrobial properties of commercially purchased colloidal platinum nanoparticles by examining the existence and potency of their antibacterial effects and investigating the mechanisms by means of which they express these activities. Antimicrobial properties were investigated with respect to standard laboratory ATCC (American Type Cell Culture) and clinical extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia (E.) coli and Klebsiella (K.) pneumoniae. Standard microbiological methods of serial microdilution, modulation of microbial cell death kinetics (“time–kill” assays), and biofilm inhibition were used. Bacterial cell wall damage and ROS (reactive oxygen species) levels were assessed in order to explore the mechanisms of platinum nanoparticles’ antibacterial activities. Platinum nanoparticles showed strong antibacterial effects against all tested bacterial strains, though their antibacterial effects were found to succumb to time kinetics. Antibiofilm activity was modest overall and significantly effective only against E. coli strains. By measuring extracellular DNA/RNA and protein concentrations, induced bacterial cell wall damage could be assumed. The determination of ROS levels induced by platinum nanoparticles revealed their possible implication in antibacterial activity. We conclude that platinum nanoparticles exhibit potent antibacterial effects against standard laboratory and resistant strains of E. coli and K. pneumoniae. Both, cell wall damage and ROS induction could have important role as mechanisms of antibacterial activity, and, require further investigation.

In vitro antifungal activity of Morinda citrifolia (noni) extract against Candida albicans

Candida albicans is the main agent of the most common fungal infection, Candidiasis. It is an opportunistic and dangerous pathogen, especially in immunosuppressed patients. The biological properties of Morinda citrifolia (noni) make it a potent antifungal. In this study, antifungal effect of M. citrifolia was evaluated to verify its effect on human cells. Extract of M. citrifolia was used against strains of C. albicans (cEC 1291). Glucose consumption in C. albicans biofilm was determined at different concentrations of M. citrifolia, and germ tube formation was evaluated in the presence and absence of M. citrifolia. Fungicidal activity was determined by the kinetics of fungal cell death. THP-1 and HeLa cells were used for cell viability and apoptosis, and cell proliferation assays, respectively. Cells treated with M. citrifolia maintained higher concentration of glucose than the control group (p < 0.05). Germ tube formation was inhibited in cells treated with M. citrifolia (p < 0.05). M. citrifolia exerted a cytotoxic effect on C. albicans cells with 99.99% lethality after 6.82 h (1:1 and 1:2), and reduced the viability of THP-1 cells by 25% and 67% after 12 and 36 h, respectively. Annexin V expression in THP-1 increased in groups that received higher concentrations of M. citrifolia (p < 0.05), reducing the proliferation of THP-1 and HeLa cells (2.8-fold). A greater cytotoxic effect was observed in fungal cells. These results indicate that M. citrifolia exerts biological activity against C. albicans and reduces the viability and proliferation of human cells.

In vitro Susceptibility to β-Lactam Antibiotics and Viability of Neisseria gonorrhoeae Strains Producing Plasmid-Mediated Broad- and Extended-Spectrum β-Lactamases.

Neisseria gonorrhoeae plasmids can mediate high-level antimicrobial resistance. The emergence of clinical isolates producing plasmid β-lactamases that can hydrolyze cephalosporins, the mainstay treatment for gonorrhea, may be a serious threat. In this work, N. gonorrhoeae strains producing plasmid-mediated broad- and extended-spectrum β-lactamases (ESBLs) were obtained in vitro, and their viability and β-lactam antibiotic susceptibility were studied. Artificial pblaTEM-1 and pblaTEM-20 plasmids were constructed by site-directed mutagenesis from a pblaTEM-135 plasmid isolated from a clinical isolate. Minimum inhibitory concentration (MIC) values for a series of β-lactam antibiotics, including benzylpenicillin, ampicillin, cefuroxime, ceftriaxone, cefixime, cefotaxime, cefepime, meropenem, imipenem, and doripenem, were determined. The N. gonorrhoeae strain carrying the pblaTEM-20 plasmid exhibited a high level of resistance to penicillins and second–fourth-generation cephalosporins (MIC ≥2 mg/L) but not to carbapenems (MIC ≤0.008 mg/L). However, this strain stopped growing after 6 h of culture. The reduction in viability was not associated with loss of the plasmid but can be explained by the presence of the plasmid itself, which requires additional reproduction costs, and to the expression of ESBLs, which can affect the structure of the peptidoglycan layer in the cell membrane. Cell growth was mathematically modeled using the generalized Verhulst equation, and the reduced viability of the plasmid-carrying strains compared to the non-plasmid-carrying strains was confirmed. The cell death kinetics of N. gonorrhoeae strains without the pblaTEM-20 plasmid in the presence of ceftriaxone can be described by a modified Chick–Watson law. The corresponding kinetics of the N. gonorrhoeae strain carrying the pblaTEM-20 plasmid reflected several processes: the hydrolysis of ceftriaxone by the TEM-20 β-lactamase and the growth and gradual death of cells. The demonstrated reduction in the viability of N. gonorrhoeae strains carrying the pblaTEM-20 plasmid probably explains the absence of clinical isolates of ESBL-producing N. gonorrhoeae.

Blood, toil, and taxoteres: Biological determinants of treatment-induced ctDNA dynamics for interpreting tumor response.

Collection and analysis of circulating tumor DNA (ctDNA) is one of the few methods of liquid biopsy that measures generalizable and tumor specific molecules, and is one of the most promising approaches in assessing the effectiveness of cancer care. Clinical assays that utilize ctDNA are commercially available for the identification of actionable mutations prior to treatment and to assess minimal residual disease after treatment. There is currently no clinical ctDNA assay specifically intended to monitor disease response during treatment, partially due to the complex challenge of understanding the biological sources of ctDNA and the underlying principles that govern its release. Although studies have shown pre- and post-treatment ctDNA levels can be prognostic, there is evidence that early, on-treatment changes in ctDNA levels are more accurate in predicting response. Yet, these results also vary widely among cohorts, cancer type, and treatment, likely due to the driving biology of tumor cell proliferation, cell death, and ctDNA clearance kinetics. To realize the full potential of ctDNA monitoring in cancer care, we may need to reorient our thinking toward the fundamental biological underpinnings of ctDNA release and dissemination from merely seeking convenient clinical correlates.

Knockout of Sf-Caspase-1 generates apoptosis-resistant Sf9 cell lines: Implications for baculovirus expression.

The Sf9 cell line, originally isolated from the insect Spodoptera frugiperda, is commonly used alongside the baculovirus expression vector system (BEVS) to produce recombinant proteins and other biologics. As more BEVS-derived vaccines and therapeutics are approved by regulators and manufactured at scale, there is increasing interest in improving the Sf9 cell line to improve bioprocess robustness and increase product yields. CRISPR-Cas9 is a powerful genome-editing tool with great potential to improve cell line characteristics. Nevertheless, reports of genome-editing in Sf9 cells are scarce, and targets for engineering are elusive. To evaluate the effectiveness of CRISPR-Cas9 to improve BEVS yields, we generated Sf9 cell lines with functional knockouts in the Sf-Caspase-1 gene, which encodes an effector caspase involved in the execution of apoptosis. Deletion of Sf-Caspase-1 abolished the hallmarks of apoptotic cell death including plasma membrane blebbing and effector caspase activity. Following infection of Sf-Caspase-1 knockout Sf9 cultures with a recombinant baculovirus expressing β-galactosidase, we did not observe any differences in cell death kinetics or increases in productivity. Similar results were obtained when Sf-Caspase-1 expression was suppressed via RNA interference. We anticipate that the CRISPR-Cas9 workflow reported here will spur future efforts to rationally engineer Sf9 cells for improved baculovirus expression.

Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae β-Hemolysin/Cytolysin-Induced Cell Death.

ABSTRACTPore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT β-hemolysin/cytolysin (βhc). Little is understood about the cellular factors involved in βhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to βhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for βhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of βhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on βhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that βhc does not require a single nonessential host factor to mediate target cell death.IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to βhc, a toxin required for GBS virulence. These data suggest that βhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.

Abstract P5-01-08: Mechanistic differences between abrupt and gradual involution of mouse mammary gland

Abstract Objective: Epidemiological studies indicate a direct relation between length of breast feeding and protection against risk of triple negative breast cancer (TNBC), an aggressive subtype. While prolonged breastfeeding allows gradual involution (GI) of the breast, short-term or no breast feeding leads to abrupt involution (AI). We modelled AI and GI of breast in mice, and showed that mice subjected to GI have better protection from tissue remodeling associated injuries in the mammary gland. Our data revealed late development of ductal hyperplasia aided by pro-tumorigenic microenvironment in the AI glands1. Detailed mechanism of mammary gland involution immediately following cessation of lactation has been studied in the past, but only in the AI setting, that is after abrupt removal of pups at the peak of lactation2,3. The goal of this study is to conduct stepwise comparison of the mechanism of GI vs. AI early on to understand the protective effect of GI against breast cancer risk.Methods: Wild-type FVB mice were used in all our studies. Females were mated at 8 weeks of age and uniparous mice were allowed to nurse (6 pups/dam) for 7 days. Females were then assigned randomly to AI or GI cohort. All pups were removed from the AI dams on postnatal day7 (PND7). Three pups each were removed from GI dams on day28 and 31. Mammary glands were harvested intermittently between PND7 and PND35. H&amp;E stained sections were used for histological studies. Unstained FFPE sections were used for immunohistochemistry and TUNEL assay. Total RNA and protein from whole mammary gland was used for qPCR and western blot respectively. Results: GI glands transitioned from fully active lactating to near involuted glands over a period of 8 days (PND17- PND25), while for AI glands it took &amp;lt; 4 days (PND8.5-PND12). The shrinkage and flattening of tall epithelia and loss of acini was gradual in GI glands as opposed to rapid breakdown of acini and adipocyte repopulation in AI gland. Apoptotic cell count peaked on PND11 (5%) in AI vs. PND25 (3%) in GI glands. The pStat3Y705+ cells were highest on PND8.5 (25%) in AI vs. on PND25 (11%) in GI glands. Macrophage infiltration (F4/80+) peaked on PND11 (35%) and remained elevated at ~24% till PND25, while in GI glands increase was gradual from PND17 through PND25 (27%). Expression of key genes identified in AI mice2,3 have markedly different expression pattern in GI mice (Table). While some peaked at a later time point in GI vs. AI coinciding with maximum cell death, expression of some are significantly low or undetectable in GI glands at both RNA and protein level. Conclusions: We show for the first time that kinetics of cell death, adipocyte repopulation, immune cell infiltration and inflammatory state of glands undergoing abrupt vs. gradual involution are markedly different. Several genes known to play a key role during AI are either not expressed or barely detectable in the GI glands at any time point during involution. These data suggests that not only the kinetics, but mechanism of GI and AI are not identical. We conclude that orchestrated cell death in GI protects from drastic lysosomal, and immune cell activities that predisposes mammary glands to higher risk of neoplastic changes.Significance: Epidemiological data highlights the benefits of prolonged breastfeeding in protecting against breast cancer, particularly, TNBC, an aggressive subtype prevalent in the African American women. Our study highlights the mechanism underlying the benefits of gradual involution of breast. GeneFold Change compared to PND7 (GI vs. AI) Peaked on (GI vs. AI)Stat34.0 vs. 5.9PND25 vs. PND8.5Ctsb2.8 vs. 3.4PND25 vs. PND8.5CD143.4 vs. 20PND25 vs. PND8.5Orm11.7 vs. 10PND25 vs. PND12Lrg130 vs. 216PND25 vs. PND12MMP215.8 vs. 27PND25 vs. PND12Chi3L11350 vs. 572, 859PND28 vs. PND11, 28Cebpδnone vs. 4.4None vs. PND8.5CtsLnone vs. 5.7None vs. PND8.5Orm2none vs. 370None vs. PND12Slpinone vs. 92None vs. PND8.5 Citation Format: Bhuvaneswari Ramaswamy, Neelam Shinde, Morgan Bauer, Maria Cuitino, Saba Mehra, Resham Mawalkar, Mustafa Basree, Allen Zhang, Kirti Kaul, Xiaoli Zhang, Ramesh Ganju, Sarmila Majumder. Mechanistic differences between abrupt and gradual involution of mouse mammary gland [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-08.

Fluorescence Time-lapse Imaging of Entosis Using Tetramethylrhodamine Methyl Ester Staining.

Entosis is a process where a living cell launches an invasion into another living cell's cytoplasm. These inner cells can survive inside outer cells for a long period of time, can undergo cell division, or can be released. However, the fate of most inner cells is lysosomal degradation by entotic cell death. Entosis can be detected by imaging a combination of membrane, cytoplasmic, nuclear, and lysosomal staining in the cells. Here, we provide a protocol for detecting entosis events and measuring the kinetics of entotic cell death by time-lapse imaging using tetramethylrhodamine methyl ester (TMRM) staining. This protocol was validated in: J Cell Biol (2021), DOI: 10.1083/jcb.202010030.

Real-time transport kinetics of drug encapsulated nanoparticles into apoptotic cancer cells inside microchannels

Development of nanocomposites as drug delivery vectors is a burgeoning field of research. However, the usage of such newly invented nanomatrices are often limited by the shortcomings associated with the testing of their real-life efficacy. Many drugs fail because a monolayer framework of in vitro cell line screening method does not adequately mimic the in vivo three-dimensional microenvironments. In this direction, the study unveils the development of a continuous flow microreactor wherein the cellulose acetate nanoparticles (CANPs) with varying sizes are prepared before encapsulating them with an anticancer drug—doxorubicin (DOX). Subsequently, an in vitro microfluidic drug delivery model has been introduced in which the HeLa cells specific to cervical cancer is treated with the DOX encapsulated CANPs—DOX@CANPs. Thereafter, the transport of the drugs from the fluidic to cellular environment, their transport inside the cell, and the real-time kinetics of the cancer cell apoptosis have been analyzed systematically to uncover the real-time efficacy and cytotoxic effects of the nanocomposite. Interestingly, experiments reveal, (i) ∼89.4% DOX loading on the nanocomposite owing to a facile electrostatic interaction, (ii) a pH-dependent controlled release of drug during the transport with the cancer cells, and (iii) cell apoptosis after the diffused inoculation of the drug. A mathematical model has been developed to emulate the drug transport from the surrounding fluid to the cancer cells. Experiments together with the mathematical model uncover that the kinetics of cancer cell death is limited by the reaction at the cell-nucleus. The microfluidic model has shown significant potential to be translated as a useful tool for the real-time and on-demand in vitro screening of the cancer drugs.

STACKing the odds for discoveries.

Characterization of cell death kinetics identifies new on- and off-target small-molecule modulators of ferroptosis and reveals a role of amino acid metabolism in regulating ferroptosis sensitivity.

Ferroptotic pores induce Ca2+ fluxes and ESCRT-III activation to modulate cell death kinetics

Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca2+ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of a few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca2+ fluxes during ferroptosis induce the activation of the ESCRT-III-dependent membrane repair machinery, which counterbalances the kinetics of cell death and modulates the immunological signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained increase of cytosolic Ca2+ prior to plasma membrane rupture is a common feature of regulated types of necrosis and position ESCRT-III activation as a general protective mechanism in these lytic cell death pathways.

Cytotoxic Activity of Cu/TiO₂ Nanoparticles on Uterine-Cervical Cancer Cells.

Nanoparticles based on metal oxides serve as carrier matrices for molecules of biological interest. In this work, we used different copper complexes that were coupled to TiO₂ nanoparticles. Nanoparticles were prepared with the sol-gel method. The Cu/TiO₂ nanoparticles were characterized through ultraviolet-visible and Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, nitrogen physisorption analysis, and scanning electron microscopy. Their biological activity was determined through DNA degradation and their cytotoxic effect on HeLa cells. The Cu/TiO₂ nanoparticles presented a pore size between 2 and 6 nm, the size of nanoparticles agglomerates was between 100 and 500 nm. The nanoparticles of Cu/TiO₂ degraded DNA starting at 15 min. The half maximal inhibitory concentration in HeLa cells depends on the used cooper complexes, the kinetics of cell death is of first order. Results revealed that these nanoparticles could be applied in uterine-cervical cancer treatment.