Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.

Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.

High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Toxicokinetics and bioavailability of indoxacarb enantiomers and their new metabolites in rats.

Indoxacarb is a chiral insecticide that consists of two enantiomers, S-(+)-indoxacarb and R-(-)-indoxacarb, of which only S-(+)-indoxacarb has insecticidal activity. Previous enantioselective toxicology studies of indoxacarb focused mostly on simple environmental model organisms. The lack of a toxicology evaluation of indoxacarb conducted in a mammalian system could mean that the extent of the potential health risk posed by the insecticide to humans is not adequately known. In this study, we reported on a new pair of enantiomers, S-IN-RM294 and R-IN-RM294, derived from the metabolic breakdown of S-(+)-indoxacarb and R-(-)-indoxacarb, respectively, in rats. The toxicokinetics of S-(+)-indoxacarb, R-(-)-indoxacarb, S-IN-RM294, and R-IN-RM294 in rats were evaluated to provide a more comprehensive risk assessment of these molecules. The bioavailability and excretion rates of both S-(+)-indoxacarb and R-(-)-indoxacarb were relatively low, which may be due to their faster metabolism and accumulation in the tissues. In addition, there were significant differences in the metabolism and distribution between the two indoxacarb enantiomers and their metabolites in vivo. S-(+)-Indoxacarb was found to be more easily metabolized in the blood compared with R-(-)-indoxacarb, as shown by the differences in pharmacokinetic parameters between oral and intravenous administration. Analysis of their tissue distribution showed that S-(+)-indoxacarb was less likely to accumulate in most tissues. The results obtained for the two metabolites were consistent with those of the two parent compounds. S-IN-RM294 was more readily cleared from the blood and less likely to accumulate in the tissues compared with R-IN-RM294. Therefore, whether from the perspective of insecticidal activity or from the perspective of mammalian and environmental friendliness, the application of optically pure S-(+)-indoxacarb in agriculture may be a more efficient and safer strategy.

Resazurin to determine the minimum inhibitory concentration on antifungal susceptibility assays for Fonsecaea sp. using a modified EUCAST protocol.

Chromoblastomycosis is a fungal chronic disease, which affects humans, especially in cutaneous and subcutaneous tissues. There is no standard treatment for Chromoblastomycosis, and it is a therapeutic challenge, due natural resistance of their causative agents, inadequate response of patients and common cases of relapse. Protocols for determination of antifungal drugs susceptibility are not standardized for chromoblastomycosis agents and endpoint definition is usually based on visual inspection, which depends on the analyst, making it sometimes inaccurate. We presented a colorimetric and quantitative methodology based on resazurin reduction to resofurin to determine the metabolic status of viable cells of Fonsecaea sp. Performing antifungal susceptibility assay by a modified EUCAST protocol allied to resazurin, we validated the method to identify the minimum inhibitory concentrations of itraconazole, fluconazole, amphotericin B, and terbinafine for eight Fonsecaea clinical isolates. According to our data, resazurin is a good indicator of metabolic status of viable cells, including those exposed to antifungal drugs. This work aimed to test resazurin as an indicator of the metabolic activity of Fonsecaea species in susceptibility assays to antifungal drugs. Species of this genus are the main causative agents of Chromoblastomycosis, which affects humans.

Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.

The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.

Microbial degradation mechanism and pathway of the insecticide thiamethoxam by isolated Bacillus Cereus from activated sludge.

The high water solubility and ecotoxicity of thiamethoxam (TMX) is a potential hazard to ecosystems and human health. Here, a strain of Bacillus cereus with high TMX degradation activity was isolated from the sediment of the A2O process in the wastewater treatment plant and was able to utilize TMX as its sole carbon source. Under different environmental conditions, the degradation efficiency of TMX by Bacillus cereus-S1 (strain S1) ranged from 41.0% to 68.9% after 216 h. The optimum degradation conditions were DO = 3.5 mg/L and pH 9.0. The addition of an appropriate carbon-to-nitrogen ratio could accelerate the degradation of TMX. A plausible biodegradation pathway has been proposed based on the identified metabolites and their corresponding degradation pathways. TMX can be directly converted into Clothianidin (CLO), TMX-dm-hydroxyl and TMX-Urea by a series of reactions such as demethylation, oxadiazine ring cleavage and C=N substitution by hydroxy group. The main products were TMX-dm-hydroxyl and TMX-Urea, the amount of CLO production is relatively small. This study aims to provide a new approach for efficient degradation of TMX; furthermore, strain S1 is a promising biological source for in situ remediation of TMX contamination.

Structure-Based Optimization of Coumestan Derivatives as Polyketide Synthase 13-Thioesterase(Pks13-TE) Inhibitors with Improved hERG Profiles for Mycobacterium tuberculosis Treatment.

Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antitubercular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC50 = 0.52 µM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039-0.0078 µg/mL) and exhibited limited hERG inhibition (IC50 ≥ 25 µM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.

Higher quality rather than superior quantity of oocytes determine the amount of fertilizable oocytes in two outbred Dummerstorf high-fertility mouse lines.

Dummerstorf fertility lines FL1 and FL2 represent two models of enhanced fertility characterized by the doubling of the litter size compared with an unselected control population (ctrl line, Dummerstorf FztDU). Both biodiverse FLs managed to reach this goal by increasing the ovulation rate per cycle, even showing decreased pregnancy rate and irregular oestrous cycle and metabolic hormone levels, compared with ctrl. The aim of the present study was to analyse oocytes in terms of quality and quantity by comparing the entire pool of oocytes per ovary, with those from the antral follicles within the same animal. We performed Brilliant Cresyl Blue staining as a non-invasive marker of oocyte quality in combination with an analysis of additional morphological indicators, e.g. cytoplasm clarity, cumulus cell layers, nuclear anatomy, size and shape. We compared our fertility lines with the unselected control population and with another independent line selected from the same founder population, showing lower litter size (DU6P). Our results suggest that fertility lines show decreased number of oocytes per ovary compared with DU6P but increased number of high-quality oocytes before ovulation. Hence, the raise in the ovulation rate and litter size of those super fertile mouse lines are not associated with an increased number of oocytes per ovary but rather with an increased number of higher quality fertilizable oocytes per cycle. In addition, the most conspicuous method to acquire oocytes with the highest quality in our lines is to assess their morphology, rather than their status after staining. All these discoveries together may be of fundamental importance for further studies in livestock farm animals showing some similar characteristics, e.g. irregular cycle or hormonal misbalances, to improve production while lowering costs, and in humans to increase the possibilities of successful pregnancies for couples undergoing in vitro fertilization (IVF).

Indoxacarb resistance-associated mutation of Liriomyza trifolii in Hainan, China.

Liriomyza trifolii, which has been recently prevalent in China, harms more than 300 plant species, especially cowpea in Hainan. This pest also affects the quality and production of vegetables in winter. Indoxacarb is the first commercial oxadiazine pesticide, which is a new efficient insecticide used to control pests of Diptera, including L. trifolii. The unique mechanism of indoxacarb is that indenyl is transformed into N-demethoxycarbonyl metabolite (DCJW) in insects and acts on inactivated sodium channel; DCJW could then destroy the conduction of nerve impulses, which leads to movement disorders, feeding stoppage, paralysis, and eventually the death of pests. The field population of L. trifolii developed resistance by 769 times higher than the sensitive population in Sanya, Hainan. Results revealed the existence of a mutation (i.e., V1848I) in the sixth transmembrane segment of Domain IV of the sodium channel in the field population. The homozygous resistant genotype frequency for the V1848I mutation was 10-15% among the three field-collected populations. This paper reports for the first time the presence of the kdr mutation V1848I in resistant populations of L. trifolii to indoxacarb. The present study will contribute to the understanding of the evolution of indoxacarb resistance and contribute to the development of resistance management practices for winter vegetables in Hainan.

Structural Optimization and Structure-Activity Relationship Studies of 6,6-Dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one Derivatives as A New Class of Potent Inhibitors of Pan-Trk and Their Drug-Resistant Mutants.

Tropomyosin receptor kinases (TrkA, TrkB, and TrkC) are attractive therapeutic targets for multiple cancers. Two first-generation small-molecule Trks inhibitors, larotrectinib and entrectinib, have just been approved to use clinically. However, the drug-resistance mutations of Trks have already emerged, which calls for new-generation Trks inhibitors. Herein, we report the structural optimization and structure-activity relationship studies of 6,6-dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one derivatives as a new class of pan-Trk inhibitors. The prioritized compound 11g exhibited low nanomolar IC50 values against TrkA, TrkB, and TrkC and various drug-resistant mutants. It also showed good kinase selectivity. 11g displayed excellent in vitro antitumor activity and strongly suppressed Trk-mediated signaling pathways in intact cells. In in vivo studies, compound 11g exhibited good antitumor activity in BaF3-TEL-TrkA and BaF3-TEL-TrkCG623R allograft mouse models without exhibiting apparent toxicity. Collectively, 11g could be a promising lead compound for drug discovery targeting Trks and deserves further investigation.

Decoding Cinnabarinic Acid-Specific Stanniocalcin 2 Induction by Aryl Hydrocarbon Receptor.

Aryl hydrocarbon receptor (AhR) is a ligand-mediated transcription factor known for regulating response to xenobiotics, including prototypical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the activation of CYP1A1 expression. Upon ligand-binding, AhR translocates to the nucleus, interacts with the AhR nuclear translocator, and binds to xenobiotic response elements (XREs; GCGTG) present in the promoter region of AhR-regulated genes. Recently, we identified a novel tryptophan catabolite, cinnabarinic acid (CA), as an endogenous AhR agonist capable of activating expression of AhR target gene stanniocalcin 2 (stc2). The CA-driven stc2 induction bestowed cytoprotection against hepatotoxicity in an AhR-dependent manner. Interestingly, only CA but not TCDD was able to induce stc2 expression in liver, and CA was unable to upregulate the TCDD responsive cyp1a1 gene. In this report, we identified CA-specific histone H4 lysine 5 acetylation and H3 lysine 79 methylation at the AhR-bound stc2 promoter. Moreover, histone H4 lysine 5 acetylation writer, activating transcription factor 2 (Atf2), and H3 lysine 79 methylation writer, disruptor of telomeric silencing 1-like histone lysine methyltransferase (Dot1l), were interacting with the AhR complex at the stc2 promoter exclusively in response to CA treatment concurrent with the histone epigenetic marks. Suppressing Atf2 and Dot1l expression using RNA interference confirmed their role in stc2 expression. CRISPR/Cas9-assisted replacement of cyp1a1 promoter-encompassing XREs with stc2 promoter XREs resulted in CA-dependent induction of cyp1a1, underlining a fundamental role of quaternary structure of XRE sequence in agonist-specific gene regulation. In conclusion, CA-driven recruitment of specific chromatin regulators to the AhR complex and resulting histone epigenetic modifications may serve as a molecular basis for agonist-specific stc2 regulation by AhR. SIGNIFICANCE STATEMENT: Results reported here provide a mechanistic explanation for the agonist-specific differential gene regulation by identifying interaction of aryl hydrogen receptor with specific chromatin regulators concomitant with unique histone epigenetic marks. This study also demonstrated that the agonist-specific target-gene expression can be transferred with the gene-specific promoter xenobiotic response element-sequence in the context of chromatin architecture.

Cytotoxicity of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide in multidrug-resistant cancer cells through activation of PERK/eIF2α/ATF4 pathway.

After decades of research, multidrug resistance (MDR) remains a huge challenge in cancer treatment. In this study, the cytotoxic of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide (MCC1734) has been investigated towards multidrug-resistant cancer cell lines. MCC1734 exerted cytotoxicity on cell lines expressing different mechanisms of drug resistance (P-glycoprotein, BCRP, ABCB5, EGFR, p53 knockout) to a different extent. Interestingly, sensitive CCRF-CEM cells and multidrug-resistant P-gp-overexpressing CEM/ADR5000 cells represented similar sensitivity towards MCC1734, indicating MCC1734 can bypass P-gp-mediated resistance. Microarray-based mRNA expression revealed that MCC1734 affected cells by multiple pathways, including cell cycle regulation, mitochondrial dysfunction, apoptosis signaling, and EIF2 signaling. MCC1734 stimulated the generation of excessive reactive oxygen species and the collapse of mitochondria membrane potential in CCRF-CEM cells, companied by the arrest of the cell cycle in the G2M phase and apoptosis induction as determined by flow cytometry. In addition, our immunoblotting analysis highlighted that MCC1734 triggered endoplasmic reticulum (ER) stress, evidenced by the activation of p-PERK, p-eIF2α, ATF4 and CHOP. The anti-cancer effects of MCC1734 were further observed in vivo using human xenograft tumors transplanted to zebrafish, providing further support for MCC1734 as a promising new candidate for cancer drug development.

Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.

Ribosomal S6 protein kinase 4 (RSK4) was identified to be a promising target for the treatment of esophageal squamous cell carcinoma (ESCC) in our previous research, whose current treatments are primarily chemotherapy and radiotherapy due to the lack of targeted therapy. However, few potent and specific RSK4 inhibitors are reported. In this study, a series of 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones derivatives were designed and synthesized as novel and potent RSK4 inhibitors. Compound 14f was identified with potent RSK4 inhibitory activity both in vitro and in vivo. 14f significantly inhibited the proliferation and invasion of ESCC cells in vitro with IC50 values of 0.57 and 0.98 µM, respectively. It dose dependently inhibited the phosphorylation of RSK4 downstream substrates while exerting little effect on the substrates of RSK1-3 in ESCC cells. The markedly suppressed tumor growth and no observed toxicity to main organs in the ESCC xenograft mouse model suggested 14f to be a promising RSK4-targeting agent for ESCC treatment.

Eco-Friendly UPLC-MS/MS Method for Determination of a Fostamatinib Metabolite, Tamatinib, in Plasma: Pharmacokinetic Application in Rats.

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid-liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an AcquityTM CSH C18 (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1-1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method.

Emerging solvatochromic push-pull dyes for monitoring the lipid order of biomembranes in live cells.

Solvatochromic dyes have emerged as a new class of fluorescent probes in the field of lipid membranes due to their ability to identify the lipid organization of biomembranes in live cells by changing the colour of their fluorescence. This type of solvatochromic function is useful for studying the heterogeneous features of biomembranes caused by the uneven distribution of lipids and cholesterols in live cells and related cellular processes. Therefore, a variety of advanced solvatochromic dyes have been rapidly developed over the last decade. To provide an overview of the works recently developed solvatochromic dyes have enabled, we herein present some solvatochromic dyes, with a particular focus on those based on pyrene and Nile red. As these dyes exhibit preferable photophysical properties in terms of fluorescence microscopy applications and unique distribution/localization in cellular compartments, some have already found applications in cell biological and biophysical studies. The goal of this review is to provide information to researchers who have never used solvatochromic dyes or who have not discovered applications of such dyes in biological studies.

Physiologically Relevant Concentrations of Dolutegravir, Emtricitabine, and Efavirenz Induce Distinct Metabolic Alterations in HeLa Epithelial and BV2 Microglial Cells.

Microglia, the resident brain phagocytes, likely play a key role in human immunodeficiency virus (HIV) infection of the central nervous system (CNS) and subsequent neuropathogenesis; however, the nature of the infection-induced changes that yield damaging CNS effects and the stimuli that provoke microglial activation remains elusive, especially in the current era of using antiretroviral (ARV) drugs for ARV therapy (ART). Altered microglial metabolism can modulate cellular functionality and pathogenicity in neurological disease. While HIV infection itself alters brain energy metabolism, the effect of ARV drugs, particularly those currently used in treatment, on metabolism is understudied. Dolutegravir (DTG) and emtricitabine (FTC) combination, together with tenofovir (TAF or TDF), is one of the recommended first line treatments for HIV. Despite the relatively good tolerability and safety profile of FTC, a nucleoside reverse transcriptase inhibitor, and DTG, an integrase inhibitor, adverse side effects have been reported and highlight a need to understand off-target effects of these medications. We hypothesized that similar to previous ART regimen drugs, DTG and FTC side effects involve mitochondrial dysfunction. To increase detection of ARV-induced mitochondrial effects, highly glycolytic HeLa epithelial cells were forced to rely on oxidative phosphorylation by substituting galactose for glucose in the growth media. We assessed ATP levels, resazurin oxidation-reduction (REDOX), and mitochondrial membrane potential following 24-hour exposure (to approximate effects of one dose equivalent) to DTG, FTC, and efavirenz (EFV, a known mitotoxic ARV drug). Further, since microglia support productive HIV infection, act as latent HIV cellular reservoirs, and when dysfunctional likely contribute to HIV-associated neurocognitive disorders, the experiments were repeated using BV2 microglial cells. In HeLa cells, FTC decreased mitochondrial REDOX activity, while DTG, similar to EFV, impaired both mitochondrial ATP generation and REDOX activity. In contrast to HeLa cells, DTG increased cellular ATP generation and mitochondrial REDOX activity in BV2 cells. Bioenergetic analysis revealed that DTG, FTC, and EFV elevated BV2 cell mitochondrial respiration. DTG and FTC exposure induced distinct mitochondrial functional changes in HeLa and BV2 cells. These findings suggest cell type-specific metabolic changes may contribute to the toxic side effects of these ARV drugs.

Enzyme Mimic Facilitated Artificial Cell to Mammalian Cell Signal Transfer.

Catalyzing biochemical reactions with enzymes and communicating with neighboring cells via chemical signaling are two fundamental cellular features that play a critical role in maintaining the homeostasis of organisms. Herein, we present an artificial enzyme (AE) facilitated signal transfer between artificial cells (ACs) and mammalian HepG2 cells. We synthesize metalloporphyrins (MPs) based AEs that mimic cytochrome P450 enzymes (CYPs) to catalyze a dealkylation and a hydroxylation reaction, exemplified by the conversion of resorufin ethyl ether (REE) to resorufin and coumarin (COU) to 7-hydroxycoumarin (7-HC), respectively. The AEs are immobilized in hydrogels to produce ACs that generate the two diffusive fluorophores, which can diffuse into HepG2 cells and result in dual intracellular emissions. This work highlights the use of AEs to promote AC to mammalian signal transfer, which opens up new opportunities for integrating the synthetic and living world with a bottom-up strategy.

Characterization of neutral sphingomyelinase activity and isoform expression in rodent skeletal muscle mitochondria.

Skeletal muscle is composed of fiber types that differ in mitochondrial content, antioxidant capacity, and susceptibility to apoptosis. Ceramides have been linked to oxidative stress-mediated apoptotic intracellular signalling and the enzyme neutral sphingomyelinase (nSMase) is, in part, responsible for generating these ceramides through the hydrolysis of sphingomyelin. Despite the role of ceramides in mediating apoptosis, there is a gap in the literature regarding nSMase in skeletal muscle mitochondria. This study aimed to characterize total nSMase activity and individual isoform expression in isolated subsarcolemmal (SS) mitochondria from soleus, diaphragm, plantaris, and extensor digitorum longus (EDL). Total nSMase activity did not differ between muscle types. nSMase2 content was detectable in all muscles and higher in EDL, soleus, and plantaris compared to diaphragm whereas nSMase3 was undetectable in all muscles. Finally, total nSMase activity positively correlated to nSMase2 protein content in soleus but not the other muscles. These findings suggest that nSMase associated with SS mitochondria may play a role in intracellular signalling processes involving ceramides in skeletal muscle and nSMase2 may be the key isoform, specifically in slow twitch muscle like soleus. Further studies are needed to fully elucidate the specific contribution of nSMase, along with the role of the various isoforms and mitochondrial subpopulation in generating mitochondrial ceramides in skeletal muscle, and its potential effects on mediating apoptosis.

Impact of switching to TAF/FTC/RPV, TAF/FTC/EVG/cobi and ABC/3TC/DTG on cardiovascular risk and lipid profile in people living with HIV: a retrospective cohort study.

BACKGROUND: We aimed to assess the overall cardiovascular and metabolic effect of the switch to three different single tablet regimens (STRs) [tenofovir alafenamide/emtricitabine/rilpivirine (TAF/FTC/RPV), TAF/FTC/elvitegravir/cobi (TAF/FTC/EVG/cobi) and ABC/lamivudine/dolutegravir (ABC/3TC/DTG)] in a cohort of people living with HIV/AIDS (PLWH) under effective ART. METHODS: All PLWH aged above 18 years on antiretroviral treatment with an HIV-RNA < 50 cp/mL at the time of the switch to TAF/FTC/RPV, TAF/FTC/EVG/cobi and ABC/3TC/DTG were retrospectively included in the analysis. Framingham risk score modification after 12 months from the switch such as lipid profile and body weight modification were assessed. The change from baseline to 12 months in mean cardiovascular risk and body weight in each of the STR's group were assessed by means of Wilcoxon signed-rank test whereas a mixed regression model was used to assess variation in lipid levels. RESULTS: Five-hundred and sixty PLWH were switched to an STR regimen of whom 170 (30.4%) to TAF/FTC/EVG/cobi, 191 (34.1%) to TAF/FTC/RPV and 199 (35.5%) to ABC/3TC/DTG. No difference in the Framingham cardiovascular risk score was observed after 12 months from the switch in each of the STR's groups. No significant overtime variation in mean total cholesterol levels from baseline to 12 months was observed for PLWH switched to ABC/3TC/DTG [200 (SD 38) mg/dl vs 201 (SD 35) mg/dl; p = 0.610] whereas a significant increment was observed in PLWH switched to TAF/FTC/EVG/cobi [192 (SD 34) mg/dl vs 208 (SD 40) mg/dl; p < 0.0001] and TAF/FTC/RPV [187 (SD 34) mg/dl vs 195 (SD 35) mg/dl; p = 0.027]. In addition, a significant variation in the mean body weight from baseline to 12 months was observed in PLWH switched to TAF/FTC/EVG/cobi [72.2 (SD 13.5) kilograms vs 74.6 (SD 14.3) kilograms; p < 0.0001] and TAF/FTC/RPV [73.4 (SD 11.6) kilograms vs 75.6 (SD 11.8) kilograms; p < 0.0001] whereas no difference was observed in those switched to ABC/3TC/DTG [71.5 (SD 12.8) kilograms vs 72.1 (SD 12.6) kilograms; p = 0.478]. CONCLUSION: No difference in the cardiovascular risk after 1 year from the switch to these STRs were observed. PLWH switched to TAF/FTC/EVG/cobi and TAF/FTC/RPV showed an increase in total cholesterol levels and body weight 12 months after the switch.

Antioxidant and Cytoprotective Properties of Plant Extract from Dry Flowers as Functional Dyes for Cosmetic Products.

Nowadays, natural dyes are expected by the cosmetic and food industries. In contrast to synthetic dyes, colorants derived from natural sources are more environmentally friendly and safer for human health. In this work, plant extracts from Gomphrena globasa L., Clitoria ternatea L., Carthamus tinctorius L., Punica granatum L. and Papaver rhoeas L. as the natural and functional dyes for the cosmetics industry were assessed. Cytotoxicity on keratinocyte and fibroblast cell lines was determined as well as antioxidant and anti-aging properties by determining their ability to inhibit the activity of collagenase and elastase enzymes. In addition, the composition of the extracts was determined. The obtained extracts were also applied in face cream formulation and color analyses were performed. It has been shown that the obtained extracts were characterized by no cytotoxicity and a high antioxidant potential. The extracts also show strong ability to inhibit the activity of collagenase and moderate ability to inhibit elastase and provide effective and long-lasting hydration after their application on the skin. Application analyses showed that the extracts of P. rhoeas L., C. ternatea L. and C. tinctorius L. can be used as effective cosmetic dyes that allow for attainment of an intense and stable color during the storage of the product. The extracts of P. granatum L. and G. globasa L., despite their beneficial effects as active ingredients, did not work effectively as cosmetic dyes, because cosmetic emulsions with these extracts did not differ significantly in color from emulsions without the extract.