Biphasic Curve Research Articles

Enzyme kinetics model for the coronavirus main protease including dimerization and ligand binding.

The coronavirus main protease (MPro) plays a pivotal role in viral replication and is the target of several antivirals against SARS-CoV-2. In some species, CRCs of MPro enzymatic activity can exhibit biphasic behavior in which low ligand concentrations activate the enzyme whereas higher ones inhibit it. While this behavior has been attributed to ligand-induced dimerization, quantitative enzyme kinetics models have not been fit to it. Here, we develop a kinetic model integrating dimerization and ligand binding. We perform a Bayesian regression to globally fit the model to multiple types of biochemical and biophysical data. The reversible covalent inhibitor GC376 strongly induces dimerization and binds to the dimer with no cooperativity. In contrast, the fluorescent peptide substrate has a minor effect on dimerization but binds to the dimer with positive cooperativity. The biphasic concentration response curve occurs because compared to substrate, the inhibitor accelerates turnover in the opposite catalytic site.

Use of imidazo[1,5-a]quinoline scaffold as the pharmacophore in the design of bivalent ligands of central benzodiazepine receptors

The imidazo[1,5-a]quinoline scaffold of central benzodiazepine receptor (CBR) ligands was used as the pharmacophore in the design of bivalent ligands bearing spacers showing variable length and different physicochemical features. The newly designed compounds were synthesized along with the corresponding reference monovalent compounds bearing the corresponding spacers terminated with a tert-butoxycarbonyl group. The novel compounds were tested in binding assays with different CBR preparations such as the cerebral cortex from male CD-1 albino mice or the human recombinant α1β3γ2 and α2β3γ2 γ-aminobutyric acid type A receptors (GABAARs) stably expressed in mouse L(tk-) cells. The tested compounds showed IC50 values from the sub-micromolar up to the nanomolar range with very similar inhibition constants values for the two isoforms of GABAARs. The similarity in the affinity between the bivalent ligands and the corresponding monovalent ones appeared to rule out any bivalent interactions of these ligands with the two isoforms of GABAARs. Similarly, both series were able to inhibit the binding of radiolabeled flumazenil to GABAARs in cortical membranes of albino CD-1 mice, but most of the tested compounds showed biphasic inhibition curves, suggesting the existence of two well-distinct populations of binding sites. Finally, some CBR ligands selected from the bivalent ligands (i.e. 6a,c) and from the reference monovalent ligands (i.e. 7a) were then tested in vivo for their potential pharmacological effects, evaluating four classical benzodiazepine actions such as anticonvulsant, anxiolytic, locomotor, and anti-amnesic activities. All the tested compounds showed anticonvulsant and anxiolytic properties with neither muscle relaxant effect nor learning and memory impairments.

New potent muscarinic receptor ligands bearing the 1,4-dioxane nucleus: Investigation on the nature of the substituent in position 2.

A new series of muscarinic acetylcholine receptor (mAChR) ligands obtained by inserting different substituents in position 2 of the potent 6,6-diphenyl-1,4-dioxane antagonists 4 and 5 was designed and synthesized to investigate the influence of steric bulk on the mAChR affinity. Specifically, the insertion of a 2-methyl group, affording compounds 6 and 9, resulted as the most favorable modification in terms of affinity for all muscarinic subtypes. As supported by computational studies performed on the hM1 receptor, this substituent may contribute to stabilize the ligand within the binding site by favoring the formation of stable interactions between the cationic head of the ligand and the residue D105. The increase of steric bulk, obtained by replacing the methyl group with an ethyl (7 and 10) and especially a phenyl substituent (8 and 11), caused a marked decrease of mAChR affinity, demonstrating the crucial role played by the steric bulk of the 2-substituent in the mAChR interaction. The most intriguing result was obtained with the tertiary amine 9, which, surprisingly, showed two different pKi values for all mAChRs, with preferential subpicomolar affinities for the M1, M3, and M4 subtypes. Interestingly, biphasic curves were also observed with both the eutomer (S)-(-)-9 and the distomer (R)-( + )-9.

Differences in the physiology of Pichia pastoris grown on glycerol/glucose using polyurethane foam slabs as solid support

AbstractBACKGROUNDGrowth physiology and metabolic flux of Pichia pastoris were analyzed using glass columns containing slabs of polyurethane foam as a solid support and culture medium with glycerol or glucose as a carbon source.RESULTSMedia with glycerol produced an average of 30.91% more biomass than media where glucose was used. However, the specific growth rate registered higher values for media with glucose than those with glycerol at low substrate concentrations: 0.46 versus 0.22 h−1. Media with glycerol produced a large amount of d‐arabitol, whereas ethanol and citrate were the predominant metabolites in media with glucose. Both types of culture systems generated biphasic curves in terms of the respiratory quotient value, whose inflection point coincided with the transition from aerobic metabolism to a mixed one.CONCLUSIONAdequate oxygen transfer occurred in both culture systems; therefore, the differences found in growth physiology can be explained by the transport mechanism that internalizes glycerol, the access point to primary metabolism, and the degree of glycerol oxidation. The arrangement of cells in the polyurethane matrix allowed an attenuation of the repression phenomenon due to excess substrate for both carbon sources. This is the first work to study metabolic flux comparisons using this system. © 2024 Society of Chemical Industry (SCI).

Diagnostic Utility of Pre-Genomic Hepatitis B RNA in the Evaluation of HBV/HIV Coinfection.

Newer biomarkers of Hepatitis B virus (HBV) infection and treatment response have not been well-characterized in individuals with HBV/HIV coinfection. Pre-genomic RNA (pgRNA) and quantitative HBsAg (qHBsAg) were used to evaluate the associations with baseline characteristics. Participants included two separate groups - 236 with HBV/HIV coinfection enrolled in a cross-sectional cohort in Ghana and 47 from an HBV nucleoside/nucleotide treatment trial comparing tenofovir to adefovir in the United States. In both cohorts, HBe antigenemia was highly associated with pgRNA and HBV DNA levels. In the treatment cohort, pre-treatment pgRNA serum concentration was 7.0 log10 U/mL, and mean qHBsAg was 201,297 IU/mL. The observed treatment-associated decrease in pgRNA was consistent with a biphasic decline curve that reached second-phase kinetics following treatment week 12. Changes from baseline were significantly correlated with changes in serum ALT (r = - 0.518; P = 0.023) but not with changes in HBV DNA (r = 0.132, P = NS). qHBsAg also correlated with ALT change (r = - 0.488, P = 0.034). pgRNA and qHBsAg represent newer biomarkers of HBV replication that may help monitor response and treatment outcomes. HBV pgRNA is highly associated with both HBeAg and ALT and may predict both active replication from the closed circular DNA (cccDNA) template as well as hepatic injury.

A high-throughput assay identifies molecules with antimicrobial activity against persister cells.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Encapsulation of Cinnamaldehyde and Vanillin as a Strategy to Increase Their Antimicrobial Activity.

Many studies have suggested that the encapsulation of natural antimicrobials increases their antimicrobial activity. In this sense, the objective was to study the inactivation of microorganisms with encapsulated cinnamaldehyde and vanillin (E-CIN and E-VN), in comparison with the unencapsulated antimicrobials (CIN and VN) in protein beverages. Additionally, the microbial response was quantified through mathematical modeling. Cinnamaldehyde and vanillin were encapsulated using whey protein concentrate (WPC) as the encapsulating agent. The effectiveness at inactivating Escherichia coli, Listeria innocua, and Saccharomyces cerevisiae was evaluated in a protein-apple juice beverage during storage (4 °C). Encapsulation increased the effectiveness of cinnamaldehyde, reaching reductions of 1.8, 3.3, and 5.3 log CFU/mL in E. coli, L. innocua, and S. cerevisiae, respectively, while vanillin encapsulation had little effect on antimicrobial activity, reducing by 0.5, 1.4, and 1.1 log cycles, respectively. The combined treatments (E-CIN + E-VN) had an additive effect in reducing E. coli and a synergistic effect against S. cerevisiae. The Gompertz model was more versatile and better described the biphasic curves, whereas the Weibull model complemented the information regarding the spectrum of resistances within the microbial population. In conclusion, the encapsulation of cinnamaldehyde with WPC enhanced its activity. However, further studies are necessary to improve the antimicrobial activity of vanillin.

Modeling HepaRG metabolome responses to pyrrolizidine alkaloid exposure for insight into points of departure and modes of action

The new challenges in toxicology demand novel and innovative in vitro approaches for deriving points of departure (PODs) and determining the mode of action (MOA) of chemicals. Therefore, the aim of this original study was to couple in vitro studies with untargeted metabolomics to model the concentration-response of extra- and intracellular metabolome data on human HepaRG cells treated for 48 h with three pyrrolizidine alkaloids (PAs): heliotrine, retrorsine and lasiocarpine. Modeling revealed that the three PAs induced various monotonic and, importantly, biphasic curves of metabolite content. Based on unannotated metabolites, the endometabolome was more sensitive than the exometabolome in terms of metabolomic effects, and benchmark concentrations (BMCs) confirmed that lasiocarpine was the most hepatotoxic PA. Regarding its MOA, impairment of lipid metabolism was highlighted at a very low BMC (first quartile, 0.003 µM). Moreover, results confirmed that lasiocarpine targets bile acids, as well as amino acid and steroid metabolisms. Analysis of the endometabolome, based on coupling concentration-response and PODs, gave encouraging results for ranking toxins according to their hepatotoxic effects. Therefore, this novel approach is a promising tool for next-generation risk assessment, readily applicable to a broad range of compounds and toxic endpoints.

Investigation of Seasonal Dynamism of Peroxidase from Strains of Aspergillus sp.

Peroxidases A and B production were carried out from Aspergillus tamari and fumigates respectively isolated from petroleum hydrocarbon spilled soil. Physicochemical properties of the respective soil showed pH of 4.45 and 6.5 for soils from point 1 and II respectively and higher conductivity of 613 and 1013 (Ω-1 Cm-1), respectively when compared with the control sample. Dissolved mineral of Cl-, SO4, K, Ca, Mg in the respective soil samples from the petroleum spilled sites was significantly high when compared with the control experiment except for soil sample I which showed a relative low phosphate concentration of 1,23 in the presence of the control experiment, respectively. TOC and TOM contents were 87.91, 119.04; 108.13 and 146.42 mg/g for soil sample I, and III, respectively. In all the tested parameters, the experimented soils were significantly high than the control soil sample. Molecular tests (18S rDNA.) were used to identify the pure isolates of Aspergillia. Studies on the effect of the incubation period on the production of peroxidase from strains of ofAspergillus tamarrii sp. and Aspergillusshowed that the highest peroxidase (A and B) activity representing peroxidase from dry and wet conditions were obtained on the day 6th and 5th of the fermentation time figure peroxidase A activity peaked at pH 5 while that of peroxidase B peaked at pH 6.0; proteins with highest peroxidase activity was peak precipitated at 60% and 80% saturation of the salt for peroxidase A and B, respectively. The gel chromatogram showed single almost superimposed peaks of enzyme activity for peroxidase A and B respectively. Peroxidase A and B activity peaked at pH 4.5 and 5.0. Optimum temperature for the enzyme activity was at 50 and 60°C respectively. Km and V max of 3.45mM and 280 μmole/min; 2.44mM and 305μmole/min were extrapolated from the reciprocal curve of Lineweaver-burke at various concentrations of 2,6 DMP for peroxidase A. Fe, Ca, Co and Mn selected as their notable impact in the active site of peroxidase guided the selected were assayed in the presence of the enzymes, respectively. The stability curve obtained for the peroxidases was single biphasic which represents the first order; Peroxidase B maintained greater stability than A at its optimum pH and pH 7. The enzymes maintained greater than 50% of their activity after 30 min of incubation as activity progressively decreased up to 40% after 60 min of incubation. Thermal stability of peroxidase A and B at their respective optimum temperatures (50 and 60°C) and at 70°C showed a biphasic stability curve of peroxidase A but single phase in peroxidase B. Peroxidase B maintained greater stability than A at its optimum temperature and at 70°C. The enzymes maintained greater than 50% of their activity after 60 min of incubation. Stability curve of peroxidase A and B at 70°C showed a maximum activity of the enzymes after 30 min of incubation. However, Peroxidase B maintained greater stability than A after 60 min of incubation. Peroxidase B maintained 57.89% of its activity after 60 min while peroxidase A from fig 19 showed a residual activity of 41.2%.

Organoids as a biomarker for personalized treatment in metastatic colorectal cancer: drug screen optimization and correlation with patient response

BackgroundThe inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies.MethodsWe optimized drug screen methods on 5–11 PDOs per condition of the complete set of 23 PDOs from patients treated for metastatic colorectal cancer (mCRC). PDOs were exposed to 5-fluorouracil (5-FU), irinotecan- and oxaliplatin-based chemotherapy. We compared medium with and without N-acetylcysteine (NAC), different readouts and different combination treatment set-ups to capture the strongest association with patient response. We expanded the screens using the optimized methods for all PDOs. Organoid sensitivity was correlated to the patient’s response, determined by % change in the size of target lesions. We assessed organoid sensitivity in relation to prior exposure to chemotherapy, mutational status and sidedness.ResultsDrug screen optimization involved excluding N-acetylcysteine from the medium and biphasic curve fitting for 5-FU & oxaliplatin combination screens. CellTiter-Glo measurements were comparable with CyQUANT and did not affect the correlation with patient response. Furthermore, the correlation improved with application of growth rate metrics, when 5-FU & oxaliplatin was screened in a ratio, and 5-FU & SN-38 using a fixed dose of SN-38. Area under the curve was the most robust drug response curve metric. After optimization, organoid and patient response showed a correlation coefficient of 0.58 for 5-FU (n = 6, 95% CI -0.44,0.95), 0.61 for irinotecan- (n = 10, 95% CI -0.03,0.90) and 0.60 for oxaliplatin-based chemotherapy (n = 11, 95% CI -0.01,0.88). Median progression-free survival of patients with resistant PDOs to oxaliplatin-based chemotherapy was significantly shorter than sensitive PDOs (3.3 vs 10.9 months, p = 0.007). Increased resistance to 5-FU in patients with prior exposure to 5-FU/capecitabine was adequately reflected in PDOs (p = 0.003).ConclusionsOur study emphasizes the critical impact of the screening methods for determining correlation between PDO drug screens and mCRC patient outcomes. Our 5-step optimization strategy provides a basis for future research on the clinical utility of PDO screens.

Study on drug separation in Two-Phase aqueous systems using deep eutectic solvent consisting of choline chloride and 1,2 propanediol

Biphasic-aqueous systems have been the subject of extensive research due to their strength to simultaneously separate and purify active pharmaceutical molecules with high efficiency and purity. Aqueous Two-Phase Systems (ATPS) have been used to extract and purify cells, nucleic acids, proteins, antibodies, and tiny molecules due to their intermolecular interaction and chemical makeup. We used a deep eutectic solvent containing (choline chloride and 1,2 propanediol), K2HPO4, and water in the initial investigation of the biphasic curve of this system at the temperature of 298.15 K and atmospheric pressure. Furthermore, we used the ATPSs to study the separation of the selected pharmaceuticals. As new findings, we examined the partition coefficient and the impact of variables such as salt and the deep eutectic solvent concentration on the partitioning of the drugs. Based on the present finding, the pharmaceuticals have the propensity to be extracted in the top phase, which is rich in deep eutectic solvents. Finally, we conducted thermodynamic modeling of the present aqueous two-phase system (ATPS) using experimental liquid–liquid equilibrium data employing the nonrandom two-liquid nonrandom factor (NRTL-NRF) and NRTL models. The NRTL-NRF model can accurately correlate the aqueous two-phase system's thermodynamic behavior with the average relative deviation percent of 4.22.

Diagnosis of the venous duct in fetuses of the early period of human ontogeny

To carry out an analysis of data from the world literature regarding the role of Doppler assessment of blood flow in the venous duct of the fetus in chromosomal abnormalities in the first trimester of pregnancy, as a marker – the spectral form of wave blood flow and its pulsation, which is generated by changes in the pressure and volume of blood to the heart of the fetus. Prenatal diagnosis of venous blood flow makes it possible to assess the state of the fetal cardiovascular system. Blood flow in the ductus venosus is an integrative indicator that reflects the formation of blood circulation of both the uteroplacental and placental-fetal components of gestational hemodynamics during physiological and complicated pregnancy. The performance of this test is 63%, and in combination with the assessment of the thickness of the clavicle – 89.1%. When reverse values of blood flow in the ductus venosus are detected in the phase of contraction of the atrium and dilated cervical space, it is necessary to carry out karyotyping to rule out chromosomal abnormalities. Dopplerography allows us to objectively assess the functioning state of the fetus during physiological and complicated pregnancy based on the study of the state of blood flow in the vessels of the placenta, in the middle cerebral artery, thoracic aorta, renal arteries, inferior vena cava and venous duct. The spectrum of venous blood flow reflects changes in volume and pressure in the atria during the cardiac cycle. With normal fetal development, blood flow in the venous duct has a pulsating character in the form of a unidirectional three-phase curve that corresponds to different periods of the cardiac cycle. The spectrum of normal blood flow in the venous duct in a healthy newborn has a biphasic curve, with the direction from the portal sinus to the inferior vena cava. During three weeks of regular examinations in most babies, obliteration of the venous duct occurs, and the ductus turns into a venous connection. Two-dimensional echocardiogram, color and pulsed dopplerography allow recording the waveforms of blood flow in several structures of the heart. Doppler analysis of blood flow in the venous duct is important to perform in fetuses of the first trimester, as it increases the sensitivity of the combined test in assessing the risk of chromosomal disorders. Assessment of blood flow in the ductus venosus provides an understanding of physiological and pathological processes in the fetal body and identifies fetuses that are prone to or at risk of disease.

A Biphasic Glucose Response during an Oral Glucose Tolerance Test Is Associated with Greater Plasma Insulin and GLP-1 Responses and a Reduction in 1-Hour Glucose but Does Not Relate to the Rate of Gastric Emptying in Healthy, Older Adults.

The pattern of the plasma glucose response curve during an oral glucose tolerance test (OGTT) is of prognostic significance with "biphasic" when compared with "monophasic" patterns being associated with greater insulin sensitivity/secretion and a reduced risk of progression to diabetes. The relationships of the glucose response curves with gastric emptying and incretin hormone secretion are not known. Thirty-six adults (age > 65 years) without known diabetes consumed a 300 mL drink containing 75 g glucose and 150 mg C13-acetate at baseline and follow-up after 5.8 ± 0.1 years. Plasma glucose, glucagon-like peptide-1 (GLP-1), glucose independent insulinotropic polypeptide (GIP) and insulin were measured, and participants classified according to the pattern of their glucose response. Gastric emptying was measured on breath samples (stable isotope breath test). At baseline, 22 participants had a "monophasic" and 14 a "biphasic" glucose response. The 1 h plasma glucose response curve was greater and the GLP-1 AUC0-120 min and insulin secretion lower in the monophasic group. There were no differences in gastric emptying, GIP or insulin sensitivity. At the follow-up, the 1 h glucose response curve was greater again, while GLP-1 AUC0-120 min was lower in the monophasic group. A biphasic curve is associated with a higher 60 min glucose response curve and increases in GLP-1, but no difference in either GIP or gastric emptying.

Antioxidant and drought-acclimation responses in UV-B-exposed transgenic Nicotiana tabacum displaying constitutive overproduction of H2O2.

Hydrogen peroxide (H2O2) is an important molecule that regulates antioxidant responses that are crucial for plant stress resistance. Exposure to low levels of ultraviolet-B radiation (UV-B, 280-315nm) can also activate antioxidant defenses and acclimation responses. However, how H2O2 and UV-B interact to promote stress acclimation remains poorly understood. In this work, a transgenic model of Nicotiana tabacum cv Xanthi nc, with elevated Mn-superoxide dismutase (Mn-SOD) activity, was used to study the interaction between the constitutive overproduction of H2O2 and a 14-day UV-B treatment (1.75kJm-2 d-1 biologically effective UV-B). Subsequently, these plants were subjected to a 7-day moderate drought treatment to evaluate the impact on drought resistance of H2O2- and UV-dependent stimulation of the plants' antioxidant system. The UV-B treatment enhanced H2O2 levels and altered the antioxidant status by increasing the epidermal flavonol index, Trolox Equivalent Antioxidant Capacity, and catalase, peroxidase and phenylalanine ammonia lyase activities in the leaves. UV-B also retarded growth and suppressed acclimation responses in highly H2O2-overproducing transgenic plants. Plants not exposed to UV-B had a higher drought resistance in the form of higher relative water content of leaves. Our data associate the interaction between Mn-SOD transgene overexpression and the UV-B treatment with a stress response. Finally, we propose a hormetic biphasic drought resistance response curve as a function of leaf H2O2 content in N. tabacum cv Xanthi.

Treatment-Resistant Depression (TRD): Is the Opioid System Involved?

About 30% of major depression disorder patients fail to achieve remission, hence being diagnosed with treatment-resistant major depression (TRD). Opium had been largely used effectively to treat depression for centuries, but when other medications were introduced, its use was discounted due to addiction and other hazards. In a series of previous studies, we evaluated the antinociceptive effects of eight antidepressant medications and their interaction with the opioid system. Mice were tested with a hotplate or tail-flick after being injected with different doses of mianserin, mirtazapine, trazodone, venlafaxine, reboxetine, moclobemide, fluoxetine, or fluvoxamine to determine the effect of each drug in eliciting antinociception. When naloxone inhibited the antinociceptive effect, we further examined the effect of the specific opioid antagonists of each antidepressant drug. Mianserin and mirtazapine (separately) induced dose-dependent antinociception, each one yielding a biphasic dose-response curve, and they were antagonized by naloxone. Trazodone and venlafaxine (separately) induced a dose-dependent antinociceptive effect, antagonized by naloxone. Reboxetine induced a weak antinociceptive effect with no significant opioid involvement, while moclobemide, fluoxetine, and fluvoxamine had no opioid-involved antinociceptive effects. Controlled clinical studies are needed to establish the efficacy of the augmentation of opiate antidepressants in persons with treatment-resistant depression and the optimal dosage of drugs prescribed.

Long-Term Photobiomodulation of Cellular and Mitochondrial Activities in IMR-32 Cells Using an 810 nm Light-Emitting Diode

In photobiomodulation (PBM), biological tissues are stimulated using low-energy light. Mitochondria are thought to be integral to the process, and PBM effects vary with light parameters. Biphasic curves are commonly used to describe the dose response of PBM but do not fit in some cases. Dose-dependent PBM effects, especially mitochondrial responses, must be understood to optimize clinical therapies. However, most previous studies have reported short-term mitochondrial activity changes, neglecting long-term effects or dose-dependent changes in whole transcriptomes. We studied the effects of 3-day PBM with an 810 nm circular LED beam on human neuroblastoma IMR-32 cells. Two light doses with opposite effects on mitochondrial DNA copy number were chosen to analyze total RNA, protein content and function of mitochondrial respiratory complexes, and mitochondrial morphology. Daily 15-minute 810 nm light treatment enhanced drug resistance to the mitochondrial inhibitors and uncoupling agent via two distinct mechanisms: 1.40 mW/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> light increased cell proliferation and the proportion of cells in the cell cycle S phase; 1.95 mW/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> light enhanced oxidative phosphorylation at the RNA, protein, and functional levels and the proportion of network-like mitochondria. Our findings reveal the complexity of PBM dose responses and provide insights into light-cell interactions.

Prophage Gifsy-1 Induction in Salmonella enterica Serovar Typhimurium Reduces Persister Cell Formation after Ciprofloxacin Exposure.

Persister cells are drug-tolerant bacteria capable of surviving antibiotic treatment despite the absence of heritable resistance mechanisms. It is generally thought that persister cells survive antibiotic exposure through the implementation of stress responses and/or energy-sparing strategies. Exposure to DNA gyrase-targeting antibiotics could be particularly detrimental for bacteria that carry prophages integrated in their genomes. Gyrase inhibitors are known to induce prophages to switch from their dormant lysogenic state into the lytic cycle, causing the lysis of their bacterial host. However, the influence of resident prophages on the formation of persister cells has only been recently appreciated. Here, we evaluated the effect of endogenous prophage carriage on the generation of bacterial persistence during Salmonella enterica serovar Typhimurium exposure to both gyrase-targeting antibiotics and other classes of bactericidal antibiotics. Results from the analysis of strain variants harboring different prophage combinations revealed that prophages play a major role in limiting the formation of persister cells during exposure to DNA-damaging antibiotics. In particular, we present evidence that prophage Gifsy-1 (and its encoded lysis proteins) are major factors limiting persister cell formation upon ciprofloxacin exposure. Resident prophages also appear to have a significant impact on the initial drug susceptibility, resulting in an alteration of the characteristic biphasic killing curve of persister cells into a triphasic curve. In contrast, a prophage-free derivative of S. Typhimurium showed no difference in the killing kinetics for β-lactam or aminoglycoside antibiotics. Our study demonstrates that induction of prophages increased the susceptibility toward DNA gyrase inhibitors in S. Typhimurium, suggesting that prophages have the potential for enhancing antibiotic efficacy. IMPORTANCE Bacterial infections resulting from antibiotic treatment failure can often be traced to nonresistant persister cells. Moreover, intermittent or single treatment of persister cells with β-lactam antibiotics or fluoroquinolones can lead to the formation of drug-resistant bacteria and to the emergence of multiresistant strains. It is therefore important to have a better understanding of the mechanisms that impact persister formation. Our results indicate that prophage-associated bacterial killing significantly reduces persister cell formation in lysogenic cells exposed to DNA-gyrase-targeting drugs. This suggests that therapies based on gyrase inhibitors should be favored over alternative strategies when dealing with lysogenic pathogens.

Hormesis Responses of Photosystem II in Arabidopsis thaliana under Water Deficit Stress.

Since drought stress is one of the key risks for the future of agriculture, exploring the molecular mechanisms of photosynthetic responses to water deficit stress is, therefore, fundamental. By using chlorophyll fluorescence imaging analysis, we evaluated the responses of photosystem II (PSII) photochemistry in young and mature leaves of Arabidopsis thaliana Col-0 (cv Columbia-0) at the onset of water deficit stress (OnWDS) and under mild water deficit stress (MiWDS) and moderate water deficit stress (MoWDS). Moreover, we tried to illuminate the underlying mechanisms in the differential response of PSII in young and mature leaves to water deficit stress in the model plant A. thaliana. Water deficit stress induced a hormetic dose response of PSII function in both leaf types. A U-shaped biphasic response curve of the effective quantum yield of PSII photochemistry (ΦPSII) in A. thaliana young and mature leaves was observed, with an inhibition at MiWDS that was followed by an increase in ΦPSII at MoWDS. Young leaves exhibited lower oxidative stress, evaluated by malondialdehyde (MDA), and higher levels of anthocyanin content compared to mature leaves under both MiWDS (+16%) and MoWDS (+20%). The higher ΦPSII of young leaves resulted in a decreased quantum yield of non-regulated energy loss in PSII (ΦNO), under both MiWDS (-13%) and MoWDS (-19%), compared to mature leaves. Since ΦNO represents singlet-excited oxygen (1O2) generation, this decrease resulted in lower excess excitation energy at PSII, in young leaves under both MiWDS (-10%) and MoWDS (-23%), compared to mature leaves. The hormetic response of PSII function in both young and mature leaves is suggested to be triggered, under MiWDS, by the intensified reactive oxygen species (ROS) generation, which is considered to be beneficial for activating stress defense responses. This stress defense response that was induced at MiWDS triggered an acclimation response in A. thaliana young leaves and provided tolerance to PSII when water deficit stress became more severe (MoWDS). We concluded that the hormesis responses of PSII in A. thaliana under water deficit stress are regulated by the leaf developmental stage that modulates anthocyanin accumulation in a stress-dependent dose.

Amplified Electrochemiluminescence of the Luminol‐Amisulpride System and its Application for the Ultrasensitive Detection of Amisulpride

AbstractElectrochemiluminescence (ECL) has emerged as a powerful tool in bioassays due to its unique versatility and controllability, but it is still a challenge to amplify ECL signals without utilizing complex synthetic processes, sophisticated nanomaterials, and catalysts. Herein, a new ECL system with high sensitivity and selectivity is reported for the detection of amisulpride (AMS). The AMS was beneficial for enhancing the weak ECL emission of luminol, wherein co‐reaction effect of AMS promoted the generation of superoxide anion radicals (O2⋅−), which significantly improved the ECL efficiency of luminol (emitter). Under the optimized experimental conditions, this anodic ECL system achieved AMS detection (a biphasic curve) with linear concentration ranges of 0.2–5.0 μm and 5.0–100.0 μm. Impressively, the developed system exhibited a low limit of detection (LOD) and quantification (LOQ) of 10.2 nm and 50.5 nm for AMS, respectively. This luminol‐based ECL system is further applied for sensitive AMS detection in real (bulk) drug and pharmaceutical tablet form. Moreover, statistical comparison between the developed and comparison method showed good precision and accuracy of the developed method.

The Glycemic Curve during the Oral Glucose Tolerance Test: Is It Only Indicative of Glycoregulation?

In 1262 subjects (1035 women, 227 men) with a wide range of glucose tolerance, glycemic curves were categorized into four groups: monophasic, biphasic, triphasic, and multiphasic. The groups were then monitored in terms of anthropometry, biochemistry, and timing of the glycemic peak. Most curves were monophasic (50%), then triphasic (28%), biphasic (17.5%), and multiphasic (4.5%). Men had more biphasic curves than women (33 vs. 14%, respectively), while women had more triphasic curves than men (30 vs. 19%, respectively) (p < 0.01). Monophasic curves were more frequent in people with impaired glucose regulation and MS compared to bi-, tri-, and multiphasic ones. Peak delay was the most common in monophasic curves, in which it was also most strongly associated with the deterioration of glucose tolerance and other components of MS. The shape of the glycemic curve is gender dependent. A monophasic curve is associated with an unfavorable metabolic profile, especially when combined with a delayed peak.