A pyrene-based chromo-fluorogenic probe for specific detection of sarin gas mimic, diethylchlorophosphate.

Nerve agents are becoming serious issues for the healthy and sustainable environment of modern civilization. Therefore, its detection and degradation are of paramount importance to the scientific community. In the present contribution, we have introduced a chromo-fluorogenic pyrene-based  probe, (E)-2-methoxy-3-(pyren-1-ylimino)-3,8a-dihydro-2H-chromen-4-ol (PMCO) to detect sarin stimulant diethylchlorophosphate (DCP) in solution and gaseous phases. On inserting DCP in PMCO solution, a visual colorimetric change from yellow to clear colourless in daylight and highly intensified blue fluorescence was observed instantly under a 365 nm portable UV lamp light. PMCO has outstanding selectivity and high sensitivity with a limit of detection of 1.32 µM in dimethyl sulfoxide (DMSO) medium and 77.5 nM in 20% H2O-DMSO. A handy strained paper strip-based experiment was demonstrated to recognize DCP in a mixture of similar toxic analytes. A dip-stick experiment was performed to identify DCP vapour, and may be used as an effective photonic tool. We also demonstrated real sample analysis utilizing different DCP-spiked water samples and validating DCP detection even in various types of soils such as sand, field, and mud. Therefore, this present study provides an effective chemosensor for instant and on-site detection of toxic nerve agents in dangerous circumstances.

DMSO synergized with sugars: optical clearing agent modulation of optical coherence tomography of skin tissues.

The stratum corneum of the outermost skin is an important barrier impeding transdermal permeation, and permeation enhancers can reduce the barrier resistance of the stratum corneum and enhance the permeation of drugs in tissues. The optical imaging depth, signal intensity, and scattering coefficient variation rules of skin tissues in time dimension are obtained by using optical coherence tomography (OCT). The effect of optical clearing agents (OCAs) on OCT imaging is obtained by quantitatively analyzing the changes in the optical properties of tissues. D-fructose, one of the monosaccharides, and sucrose, one of the disaccharides, were selected for the ex vivo optical clearing experiments on pig skin tissues utilizing the dimethyl sulfoxide (DMSO) carrier effect. We find that DMSO synergized with sugars applied to skin tissue has a more significant increase in the optical imaging depth and signal intensity, and a reduction in the scattering coefficient with an increasing concentration of DMSO. DMSO with a high concentration and D-fructose with saturated concentration (10:1; v/v) effectively reduce light attenuation in OCT imaging and improve the image quality. This operation will also shorten the application time to minimize skin damage from hyperosmotic agents.

Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation.

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

[Deciphering cellular processes responding to lethality of 17ß-estradiol by quantitative phosphoproteomics].

17ß-Estradiol (E2), an important endocrine hormone in the mammalian body, participates in the regulation of the physiological functions of the reproductive system, mammary glands, bone, and cardiovascular system, among others. Paradoxically, despite the physiological actions of endogenous E2 (0.2-1.0 nmol/L), numerous clinical and experimental studies have demonstrated that high-dose E2 treatment can cause tumor regression and exert pro-apoptotic actions in multiple cell types; however, the underlying mechanism remains undescribed. In particular, little information of the cellular processes responding to the lethality of E2 is available. In the present study, we attempted to characterize the cellular processes responding to high-dose (µmol/L) E2 treatment using quantitative phosphoproteomics to obtain a better understanding of the regulatory mechanism of E2-induced cell death. First, the cell phenotype induced by high-dose E2 was determined by performing Cell Counting Kit-8 assay (CCK8), cell cytotoxicity analysis by trypan blue staining, and microscopic imaging on HeLa cells treated with 1-10 µmol/L E2 or dimethyl sulfoxide (DMSO) for 1-3 d. E2 inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Compared with the DMSO-treated HeLa cells, the cells treated with 5 µmol/L E2 for 2 d demonstrated >74% growth inhibition and approximately 50% cell death. Thus, these cells were used for quantitative phosphoproteomic analysis. Next, a solid-phase extraction (SPE)-based immobilized titanium ion affinity chromatography (Ti4+-IMAC) phosphopeptide-enrichment method coupled with data-independent acquisition (DIA)-based quantitative proteomics was employed for the in-depth screening of high-dose E2-regulated phosphorylation sites to investigate the intracellular processes responding to high-dose E2 treatment. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified over 10000 phosphorylation sites regulated by E2 and DMSO in HeLa cells. In comparison with the DMSO-treated cells, the cells treated with 5 µmol/L E2 showed 537 upregulated phosphorylation sites and 387 downregulated phosphorylation sites, with a threshold of p<0.01 and |log2(fold change)|≥1. A total of 924 phosphorylation sites on 599 proteins were significantly regulated by high-dose E2, and these sites were subjected to enrichment analysis. In addition, 453 differently regulated phosphorylation sites on 325 proteins were identified only in the E2- or DMSO-treated cell samples. These phosphorylation sites may be phosphorylated or dephosphorylated in response to high-dose E2 stimulation and were subjected to parallel enrichment analyses. Taken together, 1218 phosphorylation sites on 741 proteins were significantly regulated by high-dose E2 treatment. The functional phosphoproteins in these two groups were then analyzed using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) to determine the biological processes in which they participate and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Consistent with the cell-phenotype data, cell cycle-related proteins were highly enriched in the two groups of E2-regulated phosphoproteins (p<0.05), indicating that high-dose E2 treatment can regulate cell proliferation. In addition, E2-regulated phosphoproteins were highly enriched in the cellular processes of ribosome biogenesis, nucleocytoplasmic transport, and messenger ribonucleic acid (mRNA) processing/splicing (p<0.05), indicating that the activation of these processes may contribute to high-dose E2-induced cell death. These results further confirm that high-dose E2 treatment inhibits protein translation and induces cell death. Furthermore, the significant upregulation of multiple phosphorylation sites associated with epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPKs) MAPK1, MAPK4, and MAPK14 by high-dose E2 indicates that the EGFR and MAPK signaling pathways are likely involved in the regulation of E2-induced cell death. These phosphorylation sites likely play vital roles in E2-induced cell death in HeLa cells. Overall, our phosphoproteomic data could be a valuable resource for uncovering the regulatory mechanisms of E2 in the micromolar range.

Resveratrol prevents cognitive deficits induced by sleep deprivation via modulating sirtuin 1 associated pathways in the hippocampus.

Accumulating evidence confirms that sleep insufficiency is a high risk factor for cognitive impairment, which involves inflammation and synaptic dysfunction. Resveratrol, an agonist of the Sirt1, has demonstrated anti-inflammation and neuroprotective effects in models of Alzheimer's disease, Parkinson's disease, and schizophrenia. However, the beneficial effects of resveratrol on sleep deprivation-induced cognitive deficits and its underlying molecular mechanisms are unclear. In the present study, thirty-two male C57BL/6 J mice were randomly divided into a Control+DMSO group, Control+Resveratrol group, SD+DMSO group, and SD+Resveratrol group. The mice in the SD+Resveratrol group underwent 5 days of sleep deprivation after pretreatment with resveratrol (50 mg/kg) for 2 weeks, while the mice in the SD+DMSO group only underwent sleep deprivation. After sleep deprivation, we evaluated spatial learning and memory function using the Morris water maze test. We used general molecular biology techniques to detect changes in levels of pro-inflammatory cytokines and Sirt1/miR-134 pathway-related synaptic plasticity proteins. We found that resveratrol significantly reversed sleep deprivation-induced learning and memory impairment, elevated interleukin-1ß, interleukin-6, and tumor necrosis factor-α levels, and decreased brain-derived neurotrophic factor, tyrosine kinase receptor B, postsynaptic density protein-95, and synaptophysin levels by activating the Sirt1/miR-134 pathway. In conclusion, resveratrol is a promising agent for preventing sleep deprivation-induced cognitive dysfunction by reducing pro-inflammatory cytokines and improving synaptic function via the Sirt1/miR-134 pathway.

Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis.

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. the advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

[Quantitative 31P-NMR for Purity Determination of Organophosphorus Compounds (Pharmaceuticals)].

Quantitative NMR (qNMR), particularly 1H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for quantification is difficult, because of the overlap and complexity of organic molecules. Therefore, we focused on the 31P nucleus, owing to the simplicity of its signals, and investigated the 31P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible 31P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for 31P-qNMR], hygroscopicity and solution stability of the analyte and RS, and qNMR measurements-such as acquisition time, relaxation delay time, and spectral width-were examined. The CP purities determined using 31P-qNMR agreed well with those for the established 1H-qNMR method in D2O. In contrast, the SOF purity determined using 31P-qNMR was 1.6% higher than that for 1H-qNMR in the protic solvent CD3OD. Therefore, using a protic solvent, such as CD3OD, was not suitable for 31P-qNMR; the deuterium exchange with the RS for 31P-qNMR (i.e., phosphonoacetic acid) resulted in a small integrated intensity. Consequently, the aprotic solvent DMSO-d6 was employed to determine the SOF purity. The data revealed that the SOF purities determined using 31P-qNMR agreed well with the established 1H-qNMR values, indicating that the absolute quantification of SOF using both 31P-qNMR and 1H-qNMR is possible in DMSO-d6. Thus, we established an optimized and reproducible 31P-qNMR method in validation study across multiple laboratories.

Effects of vehicles on the physical properties and biocompatibility of premixed calcium silicate cements.

Premixed calcium silicate cements (pCSCs) contain vehicles which endow fluidity and viscosity to CSCs. This study aimed to investigate the effects of three vehicles, namely, polyethylene glycol (PEG), propylene glycol (PG), and dimethyl sulfoxide (DMSO), on the physicochemical properties and biocompatibility of pCSCs. The setting time, solubility, expansion rate, and mechanical strength of the pCSCs were evaluated, and the formation of calcium phosphate precipitates was assessed in phosphate-buffered saline (PBS). The effects of pCSC extracts on the osteogenic differentiation of mesenchymal stem cells (MSCs) were investigated. Finally, the tissue compatibility of pCSCs in rat femurs was observed. CSC containing PEG (CSC-PEG) exhibited higher solubility and setting time, and CSC-DMSO showed the highest expansion rate and mechanical strength. All pCSCs generated calcium phosphate precipitates. The extract of CSC-PG induced the highest expressions of osteogenic markers along with the greatest calcium deposites. When implanted in rat femurs, CSC-PEG was absorbed considerably, whereas CSC-PG remained relatively unaltered inside the femur.

Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

Quercetin improves epithelial regeneration from airway basal cells of COPD patients.

BACKGROUND: Airway basal cells (BC) from patients with chronic obstructive pulmonary disease (COPD) regenerate abnormal airway epithelium and this was associated with reduced expression of several genes involved in epithelial repair. Quercetin reduces airway epithelial remodeling and inflammation in COPD models, therefore we examined whether quercetin promotes normal epithelial regeneration from COPD BC by altering gene expression. METHODS: COPD BC treated with DMSO or 1 µM quercetin for three days were cultured at air/liquid interface (ALI) for up to 4 weeks. BC from healthy donors cultured at ALI were used as controls. Polarization of cells was determined at 8 days of ALI. The cell types and IL-8 expression in differentiated cell cultures were quantified by flow cytometry and ELISA respectively. Microarray analysis was conducted on DMSO or 1 µM quercetin-treated COPD BC for 3 days to identify differentially regulated genes (DEG). Bronchial brushings obtained from COPD patients with similar age and disease status treated with either placebo (4 subjects) or 2000 mg/day quercetin (7 subjects) for 6 months were used to confirm the effects of quercetin on gene expression. RESULTS: Compared to placebo-, quercetin-treated COPD BC showed significantly increased transepithelial resistance, more ciliated cells, fewer goblet cells, and lower IL-8. Quercetin upregulated genes associated with tissue and epithelial development and differentiation in COPD BC. COPD patients treated with quercetin, but not placebo showed increased expression of two developmental genes HOXB2 and ELF3, which were also increased in quercetin-treated COPD BC with FDR < 0.001. Active smokers showed increased mRNA expression of TGF-ß (0.067) and IL-8 (22.0), which was reduced by 3.6 and 4.14 fold respectively after quercetin treatment. CONCLUSIONS: These results indicate that quercetin may improve airway epithelial regeneration by increasing the expression of genes involved in epithelial development/differentiation in COPD. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov on 6-18-2019. The study number is NCT03989271.

Toxicity of organic solvents and surfactants to the sea urchin embryos.

A comparative toxicity of widely applied organic solvents (methanol, ethanol, n-propanol, i-propanol, n-butanol, 2-butanol, i-butanol, t-butanol, 3-methoxy-3-methylbutanol-1 (MMB), ethylene glycol, diethylene glycol, 2-methoxyethanol, 2-ethoxyethanol, glycerol, ethyl acetate, acetonitrile, benzene, dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, 2-pyrrolidone, and N-methyl-2-pyrrolidone) and surfactants (PEG 300, PEG 6000, Tween 20, Tween 80, miramistin, and Cremophor EL) was studied using a sea urchin embryo model. Sea urchin embryo morphological alterations caused by the tested chemicals were described. The tested molecules affected P. lividus embryo development in a concentration-dependent manner. The observed phenotypic anomalies ranged from developmental delay and retardation of plutei growth to formation of aberrant blastules and gastrules, cleavage alteration/arrest, and embryo mortality. Discernible morphological defects were found after embryo exposure with common pharmaceutical ingredients, such as glycerol, Tween 80, and Cremophor EL. In general, solvents were less toxic than surfactants. PEG 6000 PEG 300, DMSO, ethanol, and methanol were identified as the most tolerable compounds with minimum effective concentration (MEC) values of 3.0-7.92 mg/mL. Previously reported MEC value of Pluronic F127 (4.0 mg/mL) fell within the same concentration range. Toxic effects of methanol, ethanol, DMSO, 2-methoxyethanol, 2-ethoxyethanol, Tween 20, and Tween 80 on P. lividus embryos correlated well with their toxicity obtained using other cell and animal models. The sea urchin embryos could be considered as an appropriate test system for toxicity assessment of solvents and surfactants for their further application as solubilizers of hydrophobic molecules in conventional in vitro cell-based assays and in vivo mammalian models. Nevertheless, to avoid adverse effect of a solubilizing agent in ecotoxicological and biological experiments, the preliminary assessment of its toxicity on a chosen test model would be beneficial.

Systematic cryopreservation study of cardiac myoblasts in suspension.

H9c2 myoblasts are a cell line derived from embryonic rat heart tissue and demonstrate the ability to differentiate to cardiac myotubes upon reduction of the serum concentration (from 10% to 1%) and addition of all-trans retinoic acid in the growth medium. H9c2 cells are increasingly being used as an easy-to-culture proxy for some functions of cardiomyocytes. The cryobiology of cardiac cells including H9c2 myoblasts has not been studied as extensively as that of some cell types. Consequently, it is important to characterize the cryobiological response and systematically develop well-optimized cryopreservation protocols for H9c2 cells to have optimal and consistent viability and functionality after thaw for high quality studies with this cell type. In this work, an interrupted slow cooling protocol (graded freezing) was applied to characterize H9c2 response throughout the cooling profile. Important factors that affect the cell response were examined, and final protocols that provided the highest post-thaw viability are reported. One protocol uses the common cryoprotectant dimethyl sulfoxide combined with hydroxyethyl starch, which will be suitable for applications in which the presence of dimethyl sulfoxide is not an issue; and the other protocol uses glycerol as a substitute when there is a desire to avoid dimethyl sulfoxide. Both protocols achieved comparable post-thaw viabilities (higher than 80%) based on SYTO 13/GelRed flow cytometry results. H9c2 cells cryopreserved by either protocol showed ability to differentiate to cardiac myotubes comparable to fresh (unfrozen) H9c2 cells, and their differentiation to cardiac myotubes was confirmed with i) change in cell morphology, ii) expression of cardiac marker troponin I, and iii) increase in mitochondrial mass.

Repeatability quantification of brain diffusion-weighted imaging for future clinical implementation at a low-field MR-linac.

BACKGROUND: Longitudinal assessments of apparent diffusion coefficients (ADCs) derived from diffusion-weighted imaging (DWI) during intracranial radiotherapy at magnetic resonance imaging-guided linear accelerators (MR-linacs) could enable early response assessment by tracking tumor diffusivity changes. However, DWI pulse sequences are currently unavailable in clinical practice at low-field MR-linacs. Quantifying the in vivo repeatability of ADC measurements is a crucial step towards clinical implementation of DWI sequences but has not yet been reported on for low-field MR-linacs. This study assessed ADC measurement repeatability in a phantom and in vivo at a 0.35 T MR-linac. METHODS: Eleven volunteers and a diffusion phantom were imaged on a 0.35 T MR-linac. Two echo-planar imaging DWI sequence variants, emphasizing high spatial resolution ("highRes") and signal-to-noise ratio ("highSNR"), were investigated. A test-retest study with an intermediate outside-scanner-break was performed to assess repeatability in the phantom and volunteers' brains. Mean ADCs within phantom vials, cerebrospinal fluid (CSF), and four brain tissue regions were compared to literature values. Absolute relative differences of mean ADCs in pre- and post-break scans were calculated for the diffusion phantom, and repeatability coefficients (RC) and relative RC (relRC) with 95% confidence intervals were determined for each region-of-interest (ROI) in volunteers. RESULTS: Both DWI sequence variants demonstrated high repeatability, with absolute relative deviations below 1% for water, dimethyl sulfoxide, and polyethylene glycol in the diffusion phantom. RelRCs were 7% [5%, 12%] (CSF; highRes), 12% [9%, 22%] (CSF; highSNR), 9% [8%, 12%] (brain tissue ROIs; highRes), and 6% [5%, 7%] (brain tissue ROIs; highSNR), respectively. ADCs measured with the highSNR variant were consistent with literature values for volunteers, while smaller mean values were measured for the diffusion phantom. Conversely, the highRes variant underestimated ADCs compared to literature values, indicating systematic deviations. CONCLUSIONS: High repeatability of ADC measurements in a diffusion phantom and volunteers' brains were measured at a low-field MR-linac. The highSNR variant outperformed the highRes variant in accuracy and repeatability, at the expense of an approximately doubled voxel volume. The observed high in vivo repeatability confirms the potential utility of DWI at low-field MR-linacs for early treatment response assessment.

Impact of cryopreservation on elastomuscular artery mechanics.

Low temperatures slow or halt undesired biological and chemical processes, protecting cells, tissues, and organs during storage. Cryopreservation techniques, including controlled media exchange and regulated freezing conditions, aim to mitigate the physical consequences of freezing. Dimethyl sulfoxide (DMSO), for example, is a penetrating cryoprotecting agent (CPA) that minimizes ice crystal growth by replacing intracellular water, while polyvinyl alcohol (PVA) is a nonpenetrating CPA that prevents recrystallization during thawing. Since proteins and ground substance dominate the passive properties of soft biological tissues, we studied how different freezing rates, storage temperatures, storage durations, and the presence of cryoprotecting agents (5% [v/v] DMSO + 1 mg/mL PVA) impact the histomechanical properties of the internal thoracic artery (ITA), a clinically relevant blood vessel with both elastic and muscular characteristics. Remarkably, biaxial mechanical analyses failed to reveal significant differences among the ten groups tested, suggesting that mechanical properties are virtually independent of the cryopreservation technique. Scanning electron microscopy revealed minor CPA-independent delamination in rapidly frozen samples, while cryoprotected ITAs had better post-thaw viability than their unprotected counterparts using methyl thiazole-tetrazolium (MTT) metabolic assays, especially when frozen at a controlled rate. These results can be used to inform ongoing and future studies in vascular engineering, physiology, and mechanics.

Bovine serum albumin uptake and polypeptide disaggregation studies of hypoglycemic ruthenium(II) uracil Schiff-base complexes.

Our prior studies have illustrated that the uracil ruthenium(II) diimino complex, [Ru(H3ucp)Cl(PPh3)] (1) (H4ucp = 2,6-bis-((6-amino-1,3-dimethyluracilimino)methylene)pyridine) displayed high hypoglycemic effects in diet-induced diabetic rats. To rationalize the anti-diabetic effects of 1, three new derivatives have been prepared, cis-[Ru(bpy)2(urdp)]Cl2 (2) (urdp = 2,6-bis-((uracilimino)methylene)pyridine), trans-[RuCl2(PPh3)(urdp)] (3), and cis-[Ru(bpy)2(H4ucp)](PF6)2 (4). Various physicochemical techniques were utilized to characterize the structures of the novel ruthenium compounds. Prior to biomolecular interactions or in vitro studies, the stabilities of 1-4 were monitored in anhydrous DMSO, aqueous phosphate buffer containing 2% DMSO, and dichloromethane (DCM) via UV-Vis spectrophotometry. Time-dependent stability studies showed ligand exchange between DMSO nucleophiles and chloride co-ligands of 1 and 3, which was suppressed in the presence of an excess amount of chloride ions. In addition, the metal complexes 1 and 3 are stable in both DCM and an aqueous phosphate buffer containing 2% DMSO. In the case of compounds 2 and 4 with no chloride co-ligands within their coordination spheres, high stability in aqueous phosphate buffer containing 2% DMSO was observed. Fluorescence emission titrations of the individual ruthenium compounds with bovine serum albumin (BSA) showed that the metal compounds interact non-discriminately within the protein's hydrophobic cavities as moderate to strong binders. The metal complexes were capable of disintegrating mature amylin amyloid fibrils. In vivo glucose metabolism studies in liver (Chang) cell lines confirmed enhanced glucose metabolism as evidenced by the increased glucose utilization and glycogen synthesis in liver cell lines in the presence of complexes 2-4.

Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Busulfan administration replicated the characteristics of the epididymal initial segment observed in mice lacking testis-epididymis lumicrine signaling.

The physiological functions of the mammalian epididymis are typically regulated by the testes. In addition to sex steroids secreted by testicular Leydig cells, which act on the epididymis in an endocrine manner, there is a non-sex-steroidal signaling pathway known as the lumicrine pathway. This lumicrine signaling pathway involves ligand proteins secreted from germ cells within the testicular seminiferous tubules traversing the male reproductive tract, which induce epithelial differentiation in the epididymis. These findings prompted an inquiry into whether treatments influencing testis physiology can disrupt epididymal function by interfering with testis-epididymis communication. Busulfan, an alkylating agent commonly used to deplete testicular germ cells in reproductive biology, has not been sufficiently explored because of its effects on the epididymis. This study investigated the effects of busulfan administration on the proximal epididymis using histological and transcriptomic analyses. Notably, busulfan, as opposed to the vehicle dimethyl sulfoxide (DMSO), altered the morphology of the initial segment of the epididymis, leading to a reduction in the cell height of the luminal epithelium. RNA sequencing identified 185 significantly downregulated genes in the proximal epididymis of busulfan-administered mice compared to DMSO-administered mice. Comparative transcriptome analyses revealed similarities between the epididymal transcriptome of busulfan-administered mice and lumicrine-deficient mice, such as efferent-duct-ligated W/Wv and Nell2-/- mice. However, this differed from that of bilaterally orchidectomized mice, in which both the endocrine and lumicrine signaling pathways were simultaneously ablated. Collectively, these results suggested that the harmful effects of busulfan on the proximal epididymis are secondary consequences of the ablation of testis-epididymis lumicrine signaling.

Optimization and degradation studies of cellulose transesterification to palmitate esters in superbase ionic liquid.

Cellulose palmitates (CPs) were synthesized with varying degrees of substitution (DS) via a catalyst-free, homogeneous transesterification of cellulose in a novel superbase ionic liquid (SB-IL) system, specifically 5-methyl-1,5,7-triaza-bicyclo[4.3.0]non-6-enium acetate [mTBNH][OAc], combined with dimethyl sulfoxide (DMSO) as a co-solvent, using vinyl palmitate as the acylating agent. We examined the influence of reaction temperature, reaction time, and the molar ratio of vinyl palmitate to anhydroglucose unit (AGU) on the DS, which ranged from 0.5 to 2.3 under the given conditions. Notably, the reaction order of the three hydroxy groups was C6-OH > C2-OH > C3-OH. To elucidate the chemical structure of CPs and confirm the transesterification process, various spectroscopic techniques including 1H nuclear magnetic resonance (NMR), 13C NMR, heteronuclear single quantum correlation (HSQC), and solid-state NMR were employed. Higher reaction temperatures and extended reaction times led to a decrease in the DS of CPs, potentially due to the degradation of some of the involved chemicals during the transesterification process. We also investigated the stability of the pure ionic liquid (IL) and the IL + DMSO solvent system at elevated temperatures by heating them at 100 °C for 5 h, confirming their chemical integrity through 1H NMR analysis. Additionally, we assessed the compatibility between the solvent system and cellulose by subjecting a mixture of cellulose and the solvent system to 100 °C for 5 h. To compare the structures of untreated cellulose and regenerated cellulose, Fourier transform infrared (FT-IR) spectroscopy was employed. Furthermore, we determined the molar mass of both untreated cellulose and regenerated cellulose, as well as CPs synthesized at higher reaction temperatures and longer durations, using intrinsic viscosity measurements. Lastly, we examined the solubility properties of CPs.

Fatty acid production and associated gene pathways are altered by increased salinity and dimethyl sulfoxide treatments during cryopreservation of Symbiodinium pilosum (Symbiodiniaceae).

The Symbiodinium genus is ancestral among other Symbiodiniaceae lineages with species that are both symbiotic and free living. Changes in marine ecosystems threaten their existence and crucial ecological roles. Cryopreservation offers an avenue for their long-term storage for future habitat restoration after coral bleaching. In our previous study we demonstrated that high salinity treatments of Symbiodiniaceae isolates led to changes in their fatty acid (FA) profiles and higher cell viabilities after cryopreservation. In this study, we investigated the role of increased salinity on FA production and the genes involved in FA biosynthesis and degradation pathways during the cryopreservation of Symbiodinium pilosum. Overall, there was a twofold increase in mass of FAs produced by S. pilosum after being cultured in medium with increased salinity (54 parts per thousand; ppt). Dimethyl sulfoxide (Me2SO) led to a ninefold increase of FAs in standard salinity (SS) treatment, compared to a fivefold increase in increased salinity (IS) treatments. The mass of the FA classes returned to baseline during recovery. Transcriptomic analyses showed an acyl carrier protein gene was significantly upregulated after Me2SO treatment in the SS cultures. Cytochrome P450 reductase genes were significantly down regulated after Me2SO addition in SS treatment preventing FA degradation. These changes in the expression of FA biosynthesis and degradation genes contributed to more FAs in SS treated isolates. Understanding how increased salinity changes FA production and the roles of specific genes in regulating FA pathways will help improve current freezing protocols for Symbiodiniaceae and other marine microalgae.