Intracellular ATP Concentration Research Articles

Subminute thermal damage to cell types present in the skin

Introduction Psoriasis is characterized by an increase in the proliferation of keratinocytes and nerve fiber activity, contributing to the typical skin lesions. Pulsed Dye Laser (PDL) treatment is effective for the treatment of psoriatic lesions but its mechanism remains unclear. One hypothesis is that PDL causes thermal damage by the diffusion of heat to neighboring structures in lesional skin. There is limited information on the thermal sensitivity of these neighboring skin cells when exposed to hyperthermia for durations lasting less than a minute. Our study aimed to investigate the cell-specific responses to heat using sub-minute exposure times and moderate to ablative hyperthermia. Materials and Methods Cultured human endothelial cells, smooth muscle cells, neuronal cells, and keratinocytes were exposed to various time (2–20 sec) and temperature (45–70 °C) combinations. Cell viability was assessed by measuring intracellular ATP content 24 h after thermal exposure and this data was used to calculate fit parameters for the Arrhenius model and CEM43 calculations. Results Our results show significant differences in cell survival between cell types (p < 0.0001). Especially within the range of 50–60 °C, survival of neuronal cells and keratinocytes was significantly less than that of endothelial and smooth muscle cells. No statistically significant difference was found in the lethal dose (LT50) of thermal energy between neuronal cells and keratinocytes. However, CEM43 calculations showed significant differences between all four cell types. Conclusion The results imply that there is a cell-type-dependent sensitivity to thermal damage which suggests that neuronal cells and keratinocytes are particularly susceptible to diffusing heat from laser treatment. Damage to these cells may aid in modulating the neuro-inflammatory pathways in psoriasis. These data provide insight into the potential mechanisms of PDL therapy for psoriasis and advance our understanding of how thermal effects may play a role in its effectiveness.

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Analysis of Cellular Stress Assay Parameters and Intracellular ATP in Platelets: Comparison of Platelet Preparation Methods.

Platelets are metabolically active, anucleated and small circulating cells mainly responsible for the prevention of bleeding and maintenance of hemostasis. Previous studies showed that platelets mitochondrial content, function, and energy supply change during several diseases such as HIV/AIDS, COVID-19, pulmonary arterial hypertension, and in preeclampsia during pregnancy. These changes in platelets contributed to the severity of diseases and mortality. In our previous studies, we have shown that the seahorse-based cellular stress assay (CSA) parameters are crucial to the understanding of the mitochondrial performance in peripheral blood mononuclear cells (PBMCS). Moreover, the results of CSA parameters were significantly influenced by the PBMC preparation methods. In this study, we assessed the correlation of CSA parameters and intracellular ATP content in platelets and evaluated the effects of platelet preparation methods on the results of CSA parameters and intracellular ATP content. We compared the results of CSA parameters and intracellular ATP content in platelets isolated by density centrifugation with Optiprep and simple centrifugation of blood samples without Optiprep. Platelets isolated by centrifugation with Optiprep showed a higher spare capacity, basal respiration, and maximal respiration than those isolated without Optiprep. There was a clear correlation between basal respiration and maximal respiration, and the whole-ATP content in both isolation methods. Moreover, a positive correlation was observed between the relative spare capacity and whole-cell ATP content. In conclusion, the results of seahorse-based CSA parameters and intracellular ATP content in platelets are markedly influenced by the platelet isolation methods employed. The results of basal respiration and maximal respiration are hallmarks of cellular activity in platelets, and whole-cell ATP content is a potential hint for basic platelet viability. We recommend further studies to evaluate the role of CSA parameters and intracellular ATP content in platelets as biomarkers for the diagnosis and prediction of disease states.

Lysis Physiology of Pseudomonas aeruginosa Infected with ssRNA Phage PRR1.

The phage PRR1 belongs to the Leviviridae family, a group of ssRNA bacteriophages that infect Gram-negative bacteria. The variety of host cells is determined by the specificity of PRR1 to a pilus encoded by a broad host range of IncP-type plasmids that confer multiple types of antibiotic resistance to the host. Using P. aeruginosa strain PAO1 as a host, we analyzed the PRR1 infection cycle, focusing on cell lysis. PRR1 infection renders P. aeruginosa cells sensitive to lysozyme approximately 20 min before the start of a drop in suspension turbidity. At the same time, infected cells start to accumulate lipophilic anions. The on-line monitoring of the entire infection cycle showed that single-gene-mediated lysis strongly depends on the host cells' physiological state. The blockage of respiration or a reduction in the intracellular ATP concentration during the infection resulted in the inhibition of lysis. The same effect was observed when the synthesis of PRR1 lysis protein was induced in an E. coli expression system. In addition, lysis was strongly dependent on the level of aeration. Dissolved oxygen concentrations sufficient to support cell growth did not ensure efficient lysis, and a coupling between cell lysis initiation and aeration level was observed. However, the duration of the drop in suspension turbidity did not depend on the level of aeration.

FV-429 induces apoptosis by regulating nuclear translocation of PKM2 in pancreatic cancer cells

Of all malignancies, pancreatic ductal adenocarcinoma (PDAC), constituting 90% of pancreatic cancers, has the worst prognosis. Glycolysis is overactive in PDAC patients and is associated with poor prognosis. Drugs that inhibit glycolysis as well as induce cell death need to be identified. However, glycolysis inhibitors often fail to induce cell death. We here found that FV-429, a derivative of the natural flavonoid wogonin, can induce mitochondrial apoptosis and inhibit glycolysis in PDAC in vivo and in vitro. In vitro, FV-429 inhibited intracellular ATP content, glucose uptake, and lactate generation, consequently leading to mitochondrial dysfunction and apoptosis in PDAC cells. Furthermore, it decreased the expression of PKM2 (a specific form of pyruvate kinase) through the ERK signaling pathway and enhanced PKM2 nuclear translocation. TEPP-46, the activator of PKM2, reversed FV-429-induced glycolysis inhibition and mitochondrial apoptosis in the PDAC cells. In addition, FV-429 exhibited significant tumor suppressor activity and high safety in BxPC-3 cell xenotransplantation models. These results thus demonstrated that FV-429 decreases PKM2 expression through the ERK signaling pathway and enhances PKM2 nuclear translocation, thereby resulting in glycolysis inhibition and mitochondrial apoptosis in PDAC in vitro and in vivo, which makes FV-429 a promising candidate for pancreatic cancer treatment.

Activity-based protein profiling technology reveals malate dehydrogenase as the target protein of cinnamaldehyde against Aspergillus niger

Cinnamaldehyde displays strong antifungal activity against fungi such as Aspergillus niger, but its precise molecular mechanisms of antifungal action remain inadequately understood. In this investigation, we applied chemoproteomics and bioinformatic analysis to unveil the target proteins of cinnamaldehyde in Aspergillus niger cells. Additionally, our study encompassed the examination of cinnamaldehyde's effects on cell membranes, mitochondrial malate dehydrogenase activity, and intracellular ATP levels in Aspergillus niger cells. Our findings suggest that malate dehydrogenase could potentially serve as an inhibitory target of cinnamaldehyde in Aspergillus niger cells. By disrupting the activity of malate dehydrogenase, cinnamaldehyde interferes with the mitochondrial tricarboxylic acid (TCA) cycle, leading to a significant decrease in intracellular ATP levels. Following treatment with cinnamaldehyde at a concentration of 1 MIC, the inhibition rate of MDH activity was 74.90 %, accompanied by an 84.5 % decrease in intracellular ATP content. Furthermore, cinnamaldehyde disrupts cell membrane integrity, resulting in the release of cellular contents and subsequent cell demise. This study endeavors to unveil the molecular-level antifungal mechanism of cinnamaldehyde via a chemoproteomics approach, thereby offering valuable insights for further development and utilization of cinnamaldehyde in preventing and mitigating food spoilage.

Abstract 7126: Synergistic combinations and tissue type-specificity of compounds targeting components of the DNA damage response pathway in 2D culture and 3D spheroid cell viability assays

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have changed the landscape of treatment for homologous recombination (HR)-deficient cancers. PARP inhibitors are synthetically lethal with deficiencies in genes involved in the HR pathway, such as BRCA2, but resistance against PARP inhibitors is a major problem in the clinic. The use of new synergistic drug combinations targeting different components of the DNA damage response (DDR) pathway may overcome PARP inhibitor resistance. Sixteen small molecule inhibitors acting on different targets in the DDR pathway (including PARP, Polθ and ATR) were profiled in 2D culture and 3D spheroid cell viability assays. Intracellular ATP content was used as an indirect readout of cell number. Compound synergies were determined by profiling combinations of compounds in a 6-by-6 matrix, and quantifying synergistic effects using the Excess over Bliss score [1]. A pair of isogenic BRCA2-deficient and -proficient DLD-1 colon adenocarcinoma cell lines was used as a model of BRCA2-deficient tumors. In 2D cell viability assays, PARP inhibitors and the Polθ inhibitor ART558 were more active in BRCA2-deficient compared to BRCA2-proficient DLD-1 cells, whereas an ATR inhibitor (ceralasertib) was equally potent in both cell lines. Combining olaparib with ART558 in 2D culture and 3D spheroid studies showed synergistic effects in BRCA2-deficient DLD-1 cells. Moreover, olaparib synergized with ceralasertib in both BRCA2-deficient and -proficient DLD-1 cells. Different DDR-targeting inhibitors were profiled in over a hundred cancer cell lines from diverse tumor tissue origin in 2D assays. This showed a relatively high potency for PARP inhibitors in ovarian cancer cell lines compared to cell lines derived from other tissue types, which is consistent with the clinical application of PARP inhibitors for treatment of ovarian cancers. For ATR inhibitors and ART558, kidney and myeloid cancer cell lines were found to be most sensitive, respectively, suggesting potential therapeutic utility of ATR and Polθ inhibitors for treatment of these cancer types. The genomic determinants of the different sensitivities were investigated by comparing compound potencies in the cell viability assays with the mutation status of more than 500 genes involved in cancer or the DDR pathway in the cell lines. Additionally, cancer cell line sensitivity was correlated with the expression levels of more than 19,000 genes in these cell lines to identify novel RNA signatures capable of predicting response to DDR-targeting inhibitors. This study uncovers new synergistic drug combinations that increase the efficiency of PARP inhibitors, and provides new insights into the determinants of cancer cell line sensitivity to compounds targeting different components of the DDR pathway. [1] Liu et al. Stat. Biopharm. Res. 10, 112-22; 2018 Citation Format: Janneke J. Melis, Daphne J. Kluitmans, Jeffrey J. Kooijman, Jelle Dylus, Sabine Wetemans, Suzan Geerdink-Datema, Jeroen A. de Roos, Yvonne Grobben, Guido J. Zaman. Synergistic combinations and tissue type-specificity of compounds targeting components of the DNA damage response pathway in 2D culture and 3D spheroid cell viability assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7126.

Evaluation of the viability of the Bulgarian BCG vaccine by the adenosine triphosphate assay

We applied a modified assay for the measurement of the intracellular ATP content as an alternative biochemical method for estimating the viability of the BCG vaccine instead of the standard cultural microbiological assay. Eight lyophilized BCG ampoule samples (Lot 287-2 and Lot 254-2, Bulgarian BCG strain SL222 Sofia) were tested on different days. Each calibration curve was measured for the following range of ATP concentrations: 100, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.1562 pmol. The obtained results were compared to the traditional cultural colony counting method for evaluating the viability of the BCG vaccine. The NCP of Lot 287-2 (min 2.325–max 3.300) was higher in comparison to Lot 254-2 (min 1.438–max 3.330). That result corresponded to higher ATP-content registered for Lot 287-2 (min 0.409–max 1.338) than Lot 254-2 (min 0.18–max 1.104). Our results confirmed the observation made by other researchers that the modified ATP assay is a reliable, simple, and quick method for the evaluation of the viability of the BCG vaccine. Additional compiled data about all BCG strains are needed to perform studies on the correlation between the luciferin-luciferase bioluminescent ATP assay and the cultural method for viability and enumeration of the units in the lyophilized BCG vaccine.

Synthesis, structure–activity relationship and biological evaluation of indole derivatives as anti-Candida albicans agents

In this work, we synthesized a series of indole derivatives to cope with the current increasing fungal infections caused by drug-resistant Candida albicans. All compounds were evaluated for antifungal activities against Candida albicans in vitro, and the structure–activity relationships (SARs) were analyzed. The results indicated that indole derivatives used either alone or in combination with fluconazole showed good activities against fluconazole-resistant Candida albicans. Further mechanisms studies demonstrated that compound 1 could inhibit yeast-to-hypha transition and biofilm formation of Candida albicans, increase the activity of the efflux pump, the damage of mitochondrial function, and the decrease of intracellular ATP content. In vivo studies, further proved the anti-Candida albicans activity of compound 1 by histological observation. Therefore, compound 1 could be considered as a novel antifungal agent.

Alpha-synuclein interaction with mitochondria is the final mechanism of ferroptotic death induced by erastin in SH-SY5Y cells

Ferroptosis has been characterized as a form of iron-dependent regulated cell death accompanied by an accumulation of reactive oxygen species and lipid oxidation products along with typical morphological alterations in mitochondria. Ferroptosis is activated by diverse triggers and inhibited by ferrostatin-1 and liproxstatin-1, apart from iron chelators and several antioxidants, and the process is implicated in multiple pathological conditions. There are, however, certain ambiguities about ferroptosis, especially regarding the final executioner of cell death subsequent to the accumulation of ROS. This study uses a typical inducer of ferroptosis such as erastin on SH-SY5Y cells, and shows clearly that ferroptotic death of cells is accompanied by the loss of mitochondrial membrane potential and intracellular ATP content along with an accumulation of oxidative stress markers. All these are prevented by ferrostatin-1 and liproxstatin-1. Additionally, cyclosporine A prevents mitochondrial alterations and cell death induced by erastin implying the crucial role of mitochondrial permeability transition pore (mPTP) activation in ferroptotic death. Furthermore, an accumulation of α-synuclein occurs during erastin induced ferroptosis which can be inhibited by ferrostatin-1 and liproxstatin-1. When the knock-down of α-synuclein expression is performed by specific siRNA treatment of SH-SY5Y cells, the mitochondrial impairment and ferroptotic death of the cells induced by erastin are markedly prevented. Thus, α-synuclein through the involvement of mPTP appears to be the key executioner protein of ferroptosis induced by erastin, but it needs to be verified if it is a generalized mechanism of ferroptosis by using other inducers and cell lines.

Importance of Intracellular Energy Status on High-Hydrostatic-Pressure Inactivation of sake Yeast Saccharomyces cerevisiae.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

The antibacterial activity and mechanism of a novel peptide MR-22 against multidrug-resistant Escherichia coli.

Bacterial infections have become serious threats to human health, and the excessive use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria. E. coli is a human bacterial pathogen, which can cause severe infectious. Antimicrobial peptides are considered the most promising alternative to traditional antibiotics. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and hemolytic activity were determined by the microdilution method. The antimicrobial kinetics of MR-22 against E. coli were studied by growth curves and time-killing curves. The cytotoxicity of MR-22 was detected by the CCK-8 assay. The antimicrobial activity of MR-22 in salt, serum, heat and trypsin was determined by the microdilution method. The antimicrobial mechanism of MR-22 against drug-resistant E. coli was studied by Scanning Electron Microscope, laser confocal microscopy, and Flow Cytometry. The in vivo antibacterial activity of MR-22 was evaluated by the mice model of peritonitis. In this study, MR-22 is a new antimicrobial peptide with good activity that has demonstrated against MDR E. coli. The antimicrobial activity of MR-22 exhibited stability under conditions of high temperature, 10% FBS, and Ca2+. However, a decline of the activity was observed in the presence of Na+, serum, and trypsin. MR-22 had no significant cytotoxicity or hemolysis in vitro. SEM and fluorescent images revealed that MR-22 could disrupt the integrity of cell membrane. DCFH-DA indicated that MR-22 increased the content of reactive oxygen species, while it decreased the content of intracellular ATP. In mice model of peritonitis, MR-22 exhibited potent antibacterial activity in vivo. These results indicated that MR-22 is a potential drug candidate against drug-resistant E. coli.

Identification of polyphenol extracts from flaxseed and study on its bacteriostatic mechanism

In this paper, the polyphenol components of flaxseed were identified, and their antibacterial effect and mechanism of action against pathogenic bacteria were studied. Results showed that 14 phenolic acids and 15 flavonoids were identified in flaxseed. Among them, 5 phenolic acids, including ferulic acid, vanillic acid, sinapic acid, gallic acid, 4-hydroxybenzoic acid, as well as 6 flavonoids, including vitexin, quercitrin, quercetin, apigenin, kaempfero1, (+)-dihydroquercetin, were the main phenolic compounds in flaxseed and have been quantitatively analyzed and determined. The content of sinapic acid and quercetin accounted for over 42.67% of flaxseed polyphenol extract. The Oxford Cup method confirmed the antibacterial effect of flaxseed polyphenol extract on Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Salmonella and Pseudomonas fluorescens. The experimental data showed that flaxseed polyphenol extract had a strong inhibitory effect on the growth of both common food-borne pathogens and non-food-borne pathogens. Flaxseed polyphenols increased the permeability of microbial cell membrane, resulting in microbial protein leakage, cell membrane depolarization, and decreased intracellular ATP content, and increased cell membrane potential and cell contents leakage. The results of this study clarified the composition of flaxseed polyphenol and provided a new way for the comprehensive utilization of flaxseed.

Engineering yeast with a light-driven proton pump system in the vacuolar membrane

BackgroundThe supply of ATP is a limiting factor for cellular metabolism. Therefore, cell factories require a sufficient ATP supply to drive metabolism for efficient bioproduction. In the current study, a light-driven proton pump in the vacuolar membrane was constructed in yeast to reduce the ATP consumption required by V-ATPase to maintain the acidification of the vacuoles and increase the intracellular ATP supply for bioproduction.ResultsDelta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, was expressed and localized in the vacuolar membrane of Saccharomyces cerevisiae by conjugation with a vacuolar membrane-localized protein. Vacuoles with dR were isolated from S. cerevisiae, and the light-driven proton pumping activity was evaluated based on the pH change outside the vacuoles. A light-induced increase in the intracellular ATP content was observed in yeast harboring vacuoles with dR.ConclusionsYeast harboring the light-driven proton pump in the vacuolar membrane developed in this study are a potential optoenergetic cell factory suitable for various bioproduction applications.

Oleic Acid Activates Mitochondrial Energy Metabolism and Reduces Oxidative Stress Resistance in the Pancreatic β-Cell Line INS-1.

Elevated concentration of saturated fatty acids in plasma adversely affects pancreatic β-cells, but the effects of unsaturated fatty acids are controversial. In this study, we examined the effects of oleic acid (OA), a monounsaturated fatty acid, on mitochondrial function, which is important for insulin secretion, using INS-1 cells, a pancreatic β-cell line derived from rats. Observations of mitochondrial membrane potential and intracellular ATP concentration showed that the electron transport chain was enhanced and ATP production increased in cells treated with OA, indicating that the response that occurs from sensing an increase in glucose concentration to the production of ATP was accelerated. Measurements of intracellular reactive oxygen species (ROS) indicated that the rate of increase in ROS after glucose stimulation was significantly higher in OA-treated cells. The mRNA expression levels of superoxide dismutase 1 and 2, which are responsive to ROS and other substances, were significantly increased in OA 1-d treated cells, but decreased in OA 7-d treated cells. It can be inferred that continued exposure to high concentrations of OA reduced ROS processing capacity and increased intracellular ROS levels. The mRNA expression of apoptosis-inducing enzyme Caspase-3 was significantly increased in OA-treated cells, although its activity was not high. However, the apoptosis induction rate after H2O2 stimulation was significantly higher in OA-treated cells. The high OA environment was shown to promote mitochondrial energy metabolism, leading to an increase in glucose sensitivity and a decrease in oxidative stress resistance.

High Production of Nattokinase via Fed-Batch Fermentation of the γ-PGA-Deficient Strain of Bacillus licheniformis

During the production of nattokinase (NK) by Bacillus species, certain by-products such as poly-γ-glutamic acid (γ-PGA) are simultaneously synthesized. The impact of γ-PGA synthesis on NK production remains unclear. In this study, we knocked out the pgsC gene, a component of the γ-PGA synthetase cluster (pgsBCA), and constructed a novel recombinant strain, Bacillus licheniformis BL11. Next, we compared the fed-batch fermentation profiles of BL11 and its parental strain BL10, conducted transcriptional analysis, and measured intracellular ATP content. We also optimized glucose-feeding strategies under varying oxygen supply conditions. Our results indicated that the utilization rates of glucose and soybean meal were both improved in the pgsC-deficient strain BL11, and NK activity was enhanced. Furthermore, the transcriptional levels of genes involved in glycolysis and the TCA cycle were relatively upregulated in BL11. The maximal NK activity reached 2522.2 FU/mL at 54 h of fermentation using a constant glucose-feeding rate of 5.0 g/(L·h) under high oxygen supply conditions. The newly developed recombinant strain B. licheniformis BL11, along with the optimized feeding strategy, shows promise for large-scale NK production.

The Primary Mode of Action of Lippia graveolens Essential Oil on Salmonella enterica subsp. Enterica Serovar Typhimurium.

Essential oils are known to exhibit diverse antimicrobial properties, showing their value as a natural resource. Our work aimed to investigate the primary mode of action of Cuban Lippia graveolens (Kunth) essential oil (EO) against Salmonella enterica subsp. enterica serovar Typhimurium (S. enterica ser. Typhimurium). We assessed cell integrity through various assays, including time-kill bacteriolysis, loss of cell material with absorption at 260 and 280 nm, total protein leakage, and transmission electron microscopy (TEM). The impact of L. graveolens EO on membrane depolarization was monitored and levels of intracellular and extracellular ATP were measured by fluorescence intensity. The minimum inhibitory and bactericidal concentrations (MIC and MBC) of L. graveolens EO were 0.4 and 0.8 mg/mL, respectively. This EO exhibited notable bactericidal effects on treated cells within 15 min without lysis or leakage of cellular material. TEM showed distinct alterations in cellular ultrastructure, including membrane shrinkage and cytoplasmic content redistribution. We also observed disruption of the membrane potential along with reduced intracellular and extracellular ATP concentrations. These findings show that L. graveolens EO induces the death of S. enterica ser. Typhimurium, important information that can be used to combat this foodborne disease-causing agent.

PER2 regulates odontoblastic differentiation of dental papilla cells in vitro via intracellular ATP content and reactive oxygen species levels

Background Dental papilla cells (DPCs) are one of the key stem cells for tooth development, eventually forming dentin and pulp. Previous studies have reported that PER2 is expressed in a 24-hour oscillatory pattern in DPCs in vitro. In vivo, PER2 is highly expressed in odontoblasts (which are differentiated from DPCs). However, whether PER2 modulates the odontogenic differentiation of DPCs is uncertain. This research was to identify the function of PER2 in the odontogenic differentiation of DPCs and preliminarily explore its mechanisms. Methods We monitored the expression of PER2 in DPCs differentiated in vivo. We used PER2 overexpression and knockdown studies to assess the role of PER2 in DPC differentiation and performed intracellular ATP content and reactive oxygen species (ROS) assays to further investigate the mechanism. Results PER2 expression was considerably elevated throughout the odontoblastic differentiation of DPCs in vivo. Overexpressing Per2 boosted levels of odontogenic differentiation markers, such as dentin sialophosphoprotein (Dspp), dentin matrix protein 1 (Dmp1), and alkaline phosphatase (Alp), and enhanced mineralized nodule formation in DPCs. Conversely, the downregulation of Per2 inhibited the differentiation of DPCs. Additionally, downregulating Per2 further affected intracellular ATP content and ROS levels during DPC differentiation. Conclusion Overall, we demonstrated that PER2 positively regulates the odontogenic differentiation of DPCs, and the mechanism may be related to mitochondrial function as shown by intracellular ATP content and ROS levels.

Antimicrobial Activity and Mechanisms of Walnut Green Husk Extract.

Walnut green husks (WGHs), by-products of walnut production, are believed to possess antimicrobial properties, making them a potential alternative to antibiotics. In this study, the antibacterial activities of three extracts, derived from WGH, against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were investigated, and the antibacterial mechanisms of an anhydrous ethanol extract of WGH (WGHa) were examined. The results showed that WGHa exhibited inhibitory effects on all tested bacteria. The ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis revealed that the major active compounds present in WGHa were terpenoids, phenols, and flavonoids. Treatment with WGHa resulted in the leakage of intracellular ions and alkaline phosphatase; a reduction in intracellular ATP content, ATPase activity, and nucleic acid content; as well as cellular metabolic viability. The transmission electron microscopy images showed varying degrees of cell deformation and membrane damage following WGHa treatment. The transcriptome sequencing and differentially expressed gene enrichment analyses revealed an up-regulation in pathways associated with RNA degradation, translation, protein export, and oxidative phosphorylation. Conversely, pathways involved in cell movement and localization, as well as cell wall organization and carbohydrate transport, were found to be down-regulated. These findings suggest that WGHa alters cell membrane permeability and causes damage to the cell wall. Additionally, WGHa interferes with cellular energy metabolism, compromises RNA integrity, and induces DNA replication stress, consequently inhibiting the normal growth and proliferation of bacteria. These findings unveiled the antimicrobial mechanisms of WGHa, highlighting its potential application as an antibiotic alternative.

Abstract B061: Sensitivity and resistance mechanisms of human cancer cell lines treated with PARP, POLθ, and ATR inhibitor combinations in 2D and 3D spheroid cell viability assays

Abstract Poly ADP Ribose Polymerase (PARP) inhibitors have changed the landscape of treatment of homologous recombination (HR) -deficient cancers. The combination of PARP targeting agents and genetic alterations in the HR pathway is known to be synthetically lethal. Currently, four PARP inhibitors are approved for treatment of HR-deficient cancers. In combination with DNA damaging agents, PARP inhibitors synergistically kill tumor cells. Occurrence of resistance to PARP inhibitors is a major problem in the clinic. Targeting alternative DNA repair pathways or the use of new drug combinations may overcome PARP inhibitor resistance. We have determined and compared the effect of inhibitors of PARP, POLθ and ATR on cell viability in a large panel of human cancer cell lines (the Oncolines® panel) in 2D cell viability assays, using intracellular ATP content as an indirect read-out of cell number. The relationship between the mutation and expression status of genes involved in the DNA damage response pathway, or known to be involved in PARP inhibitor resistance, was determined. The effect of co-treatment of PARP inhibitors with inhibitors of POLθ, ATR and DNA damaging, cytotoxic agents was determined in BLISS combination matrix experiments in the colorectal adenocarcinoma cell line DLD1 and an isogenic BRCA2-deficient knock-out derivative. Drug combinations were studied in 2D and in 3D spheroid viability assays. To identify novel, potential mechanisms of resistance to PARP inhibitors, BRCA1-deficient ovarian UWB1.289 and BRCA2-deficient DLD1 cells were treated with sequentially increasing concentrations of PARP inhibitors talazoparib or olaparib. Resistant clones were selected and characterized. Our study reveals insight into the genetic determinants of cancer cell line sensitivity to inhibitors of PARP, POLθ and ATR. We also present the first side-by-side comparative study of the effect of drug combinations targeting the DNA damage response pathway in HR-deficient tumors in 2D and 3D spheroid cell viability assays. Citation Format: Janneke J.T.M. Melis, Jeffrey Kooijman, Sabine Wetemans, Suzan Geerdink-Datema, Jelle Dylus, Jeroen A.D.M. de Roos, Wilhelmina E. van Riel, Guido J.R. Zaman. Sensitivity and resistance mechanisms of human cancer cell lines treated with PARP, POLθ, and ATR inhibitor combinations in 2D and 3D spheroid cell viability assays [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B061.