Dimethyl Sulfoxide Concentration Research Articles

Small molecule co-solvents enhance activity of SARS-CoV-2 main protease through a dielectric effect.

Since the beginning of the pandemic, there has been a surge of interest to learn more about SARS-CoV-2 and its many drug-targetable proteins. The main protease (Mpro) is an excellent candidate for a drug target because of its vital role in coronavirus replication and because there exists no human homologue, minimizing off-target effects. We find that Mpro is active at very high concentrations of dimethyl sulfoxide (DMSO), a common co-solvent for inhibitor candidates. The enzyme has enhanced activity with DMSO, reaching a maximum at 33%: the effect is also observed with other solvent mixtures at a similar dielectric i.e., polyethene glycol. Molecular dynamics simulations suggest DMSO does not bind to a specific site, but rather does a global conformational change of the enzyme, particularly near the active site and at the C-terminus. This work attempts to better understand Mpro dimerization, substrate binding, and interaction with its chemical environment and ultimately provide important details for robust design of inhibitors against Mpro and future variants of its kind.

Quantitative assessment of intracellular/extracellular dimethyl sulfoxide concentrations during freezing with low-temperature confocal Raman micro-spectroscopy.

Although cryopreservation plays an indispensable role in the clinical application of cell therapy, the research on the osmotic behavior of cells during freezing is still at the level of theoretical models, and quantitative experimental data are still lacking. Therefore, the Raman spectra of dimethyl sulfoxide (DMSO) solutions with different standard concentrations (5%-80% v/v) were recorded experimentally to establish a quantitative evaluation method with the intensity ratio of different labeled peaks to the hydrogen bonding peak (as the internal standard) of water molecules in relation to different DMSO concentrations. By using this method, the characteristics of quantitative changes in intra- and extracellular concentrations under three different freezing methods were explored, including direct freezing, ice seeding freezing and vitrification. It was found that the intracellular concentration (@ -50 °C) after the ice seeding (@ -7 °C) freezing (1 °C min-1) method could reach 41.6%-49.2%, significantly higher than that of the direct freezing method (1 °C min-1 to -50 °C) of 32.4%-39.1%. Moreover, it is worth noting that the quantitative values of concentrations (@ -50 °C) of the ice seeding freezing are more consistent with the primary saturation curve of the DMSO solution. Thus, for the first time, it was revealed from the experimental data that the fundamental reason for the improvement of cell survival after ice seeding operation was pre-dehydration, higher concentration and smaller osmotic pressure difference between the inside and outside of the cell. These results also confirmed the validity of the famous two-factor hypothesis and more work will be carried out in depth.

Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration.

The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P<0.05) to the other FS. Regarding CFU, no difference (P>0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P<0.05) comparing to the control, without differ (P>0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS.

Aspects of the microscopic structure of curcumin solutions with water-dimethylsulfoxide solvent. Molecular dynamics computer simulation study

We explore some aspects of the microscopic structure of curcumin solutions with water-dimethylsulfoxide solvent of variable composition. Molecular dynamics computer simulations at isobaric-isothermal conditions are used for this purpose. The model consists of the OPLS-UA type model for the enol conformer of curcumin (J. Mol. Liq., 223, 707, 2016), the OPLS model for the dimethylsulfoxide (DMSO) and the SPC/E water model. Radial distributions for the centers of mass of curcumin molecules are evaluated and the corresponding running coordination numbers are analyzed. The disaggregation of curcumin clusters upon increasing the DMSO content in water-DMSO solvent is elucidated. Changes of the distribution of water and DMSO species around curcumin molecules are investigated. A qualitative comparison of our findings with the results of other authors is performed. A possibility to relate predictions of the model with the experimental observations in terms of the so-called critical water aggregation percentage is discussed.

Effect of Extenders Including High Concentrations Dimethyl Sulfoxide (DMSO) on Post-Thaw Rabbit Sperm Parameters

Cryoprotectants have critical roles to prevent cell damages during cryopreservation. However, the adjustment of cryoprotectant concentration is also very crucial to protect cells from cryoprotectant toxicity. The present study was designed to investigate the effect of extenders including high concentration dimethyl sulfoxide (DMSO) on post-thaw rabbit sperm quality. Pooled rabbit sperm samples (n=7) were diluted and cryopreserved in extenders including 250 mmol/L Tris, 88 mmol/L citric acid, 47 mmol/L glucose, 1% sucrose and different concentrations of DMSO. The presence of high concentration DMSO (12 and 14%) in extender decreased sperm total and progressive motility (P &lt; 0.01). The 8 and 10 percent DMSO supplementations in extender increased live sperm rates (P &lt; 0.01). Live and intact acrosome or intact membrane sperm rates were detected higher in 8 and 10 DMSO groups (P &lt; 0.05). When total intact membrane sperm rates were similar in all groups, total intact acrosome sperm rate was higher in 8 DMSO group compared to 12 DMSO group (P &lt; 0.05). In conclusion, when 12 and 14 percent DMSO additions in extender adversely affected post-thaw sperm parameters, the presence of 8 percent DMSO in extender provided the highest post-thaw sperm quality.

Influence of Two Widely Used Solvents, Ethanol and Dimethyl Sulfoxide, on Human Sperm Parameters.

To study mechanisms involved in fertility, many experimental assays are conducted by incubating spermatozoa in the presence of molecules dissolved in solvents such as ethanol (EtOH) or dimethyl sulfoxide (DMSO). Although a vehicle control group is usually included in such studies, it does not allow to evaluate the intrinsic effect of the solvent on sperm parameters and its potential influence on the outcome of the experiment. In the present study, we incubated human spermatozoa for 4 h in a capacitation medium in the absence or the presence of different concentrations of EtOH and DMSO (0.1, 0.5, 1.0, and 2.0%) to assess the impact of these solvents on sperm motility, vitality, capacitation, and acrosome integrity. The presence of statistically significant relationships between increasing solvent concentrations and the investigated parameters was assessed using linear mixed models. A significant effect was observed with both solvents for total and progressive sperm motilities. We also evaluated the effect of time for these parameters and showed that the influence of the solvents was stable between 0 and 4 h, indicating an almost direct impact of the solvents. While EtOH did not influence sperm vitality and acrosome integrity, a significant effect of increasing DMSO concentrations was observed for these parameters. Finally, regarding capacitation, measured via phosphotyrosine content, although a dose-dependent effect was observed with both solvents, the statistical analysis did not allow to precisely evaluate the intensity of the effect. Based on the results obtained in the present study, and the corresponding linear mixed models, we calculated the concentration of both solvents which would result in a 5% decline in sperm parameters. For EtOH, these concentrations are 0.9, 0.7, and 0.3% for total motility, progressive motility, and capacitation, respectively, while for DMSO they are 1.5, 1.1, >2, 0.3 and >2% for total motility, progressive motility, vitality, capacitation, and acrosome integrity, respectively. We recommend using solvent concentrations below these values to dissolve molecules used to study sperm function in vitro, to limit side effects.

Cosolvent Dimethyl Sulfoxide Influences Protein–Ligand Binding Kinetics via Solvent Viscosity Effects: Revealing the Success Rate of Complex Formation Following Diffusive Protein–Ligand Encounter

Protein-ligand-exchange kinetics determines the duration of biochemical signals and consequently plays an important role in drug design. Binding studies commonly require solubilization of designed ligands in solvents such as dimethyl sulfoxide (DMSO), resulting in residual amounts of DMSO following titration of solubilized ligands into aqueous protein samples. Therefore, it is critical to establish whether DMSO influences protein-ligand binding. Here, we address the general and indirect effect of DMSO on protein-ligand binding caused by solvent viscosity, which is strongly dependent on the relative concentrations of DMSO and water. As a model system, we studied the binding of a drug-like ligand to the carbohydrate recognition domain of galectin-3 in the presence of variable amounts of DMSO. We used isothermal titration calorimetry to characterize binding thermodynamics and 15N NMR relaxation to monitor kinetics. The binding enthalpy is not affected, but we observe a subtle trend of increasingly unfavorable entropy of binding, and consequently decreased affinity, with increasing DMSO concentration. The increasing concentration of DMSO results in a reduced association rate of binding, while the dissociation rate is less affected. The observed association rate is inversely proportional to the viscosity of the DMSO-water mixture, as expected from theory, but significantly reduced from the diffusion-controlled limit. By comparing the viscosity dependence of the observed association rate with that of the theoretical diffusion-controlled association rate, we estimate the success rate of productive complex formation following an initial encounter of proteins and ligands, showing that only one out of several hundred binding "attempts" are successful.

EVALUATION OF TWO CRYOPROTECTANTS USED IN A NEW HUMAN SPERM CRYOPRESERVATION TECHNIQUE.

The aim: To examine the efficiency of different concentrations of Dimethyl sulfoxide (DMSO) and glycerol as a cytoprotectants in protection of human sperms during cryopres¬ervation in this technique. Materials and methods: Thirty oligozoospermic semen samples were used in this study. Samples diagnosed according to WHO 2010 criteria. Sheep's ovarian follicles obtained from local slaughterhouse and prepared by slicing the ovaries and evacuating the follicular fluid and oocyte. Each semen sample divided into six equal parts, and diluted 1:1 with cryosolution contains 5%, 10%, 15% DMSO or glycerol and injected within the emptied follicles. After freezing and thawing, the semen mixture aspired outside the follicles and sperm concentration, progressive motility, total motility, and normal morphology were examined. Results: The best recovery rate of progressive and total motility post-thawing were with the use of 5% glycerol, and the lowest recovery rate of progressive and total motility and normal morphology were with the use of 15% DMSO. Conclusions: In this technique, glycerol was more efficient than DMSO regarding sperm motility. The best concentration of glycerol for cryopreserve human spermatozoa is 5%.

Effect of different solvents on nisin ZP potential as anticancer agent against MG-63 osteosarcoma cells

Nisin, a known food preservative, has antimicrobial and anticancer properties. Nisin is a polycyclic polypeptide with five internal rings; a feature that strongly influence the choice of solvent to attain effectivity of the compound for all cell culture experiments. Previous studies used a number of solvents but comparison between these to identify the best solvent have not been reported. Aim: To investigate the effect of nisin ZP (NZP) when dissolved in four different solvents on the viability of MG63 osteosarcoma cells. Materials and methods: MG-63 cells were incubated overnight at 37°C, 5% CO2 and treated with increasing concentration of dimethyl sulfoxide (DMSO), methanol and acetic acid to observe the cytotoxic effects of these chemicals onto the cells via MTS assay. NZP was then dissolved in distilled water and the three solvents in increasing concentrations and applied to the cultured cells. Cell viability and morphology were documented. Results: Acetic acid, DMSO and methanol at 0.05% maintained more than 90% viability of MG-63. NZP precipitated at higher concentrations (≥ 800µg/ml) when dissolved in DMSO, methanol and distilled water which interfered with the measurement of formazan density in the cell viability assay. Increasing doses of NZP in 0.05% acetic acid showed the least interference with the desirable reduction of the cancer cell viability. Microscopically, as concentration of NZP increases, cell shrinkage and apoptotic bodies were more visible. In addition, brown precipitates were abundantly observed. Conclusion: Acetic acid provides the optimal solubility of NZP and the best solvent to use in experiments involving MG-63 cells.

Cryoprotective Mechanism of DMSO Induced by the Inhibitory Effect on Eutectic NaCl Crystallization.

Cryopreservation is a critical procedure in autologous hematopoietic stem cell transplantation. Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice. Optimization of the cryopreservation protocol in the past revealed a dramatic loss of cell viability associated with a reduction of the DMSO concentration below 2 vol % in the freezing medium. The cryoprotective mechanism of DMSO is usually ascribed to the ability to suppress ice formation and reduce the adverse effects of the freeze-concentrated solution. This work proposes an alternative hypothesis considering the detrimental impact of NaCl eutectic crystallization on cell viability. Thermoanalytical and microstructural analysis of the DMSO effect on eutectic phase transformation of cryoprotective mixtures revealed a correlation between the loss of cell viability and eutectic NaCl crystallization. DMSO inhibits the eutectic crystallization of NaCl and preserves cell viability. Thermodynamic description of the inhibitory action and possible mechanism of cryoinjury are provided.

Short Communication: The use of lactose-astaxanthin to maintain the quality of green junglefowl frozen semen

Abstract. Bebas W, Agustina KK. 2022. Short Communication: The use of lactose-astaxanthin to maintain the quality of green junglefowl frozen semen.Biodiversitas 23: 5759-5764. The Green Junglefowl is Indonesian germplasm that is at risk of population decline. Therefore, efforts are needed to protect its existence and survival. Cryopreservation of spermatozoa in the form of frozen semen is required to preserve this germplasm. This research aims to determine the effect of lactose-astaxanthin in preventing DNA fragmentation and intact plasma membrane of Green Junglefowl spermatozoa during the freezing process. A completely randomized design with a 2x4 factorial pattern, including two treatment groups and four dimethylsulfoxide (DMSO) concentration factors, was used. The treatment groups are T1 and T2, consisting of egg yolk phosphate diluent and egg yolk phosphate diluent added with a combination of 0.6% lactose-0.004% astaxanthin, respectively. The four DMSO concentration factors were 4%, 6%, 8%, and 10% respectively. Subsequently, each treatment was repeated four times. The DNA fragmentation and intact plasma membrane of spermatozoa were observed, and the data obtained afterward were analyzed for variance, followed by the Ducan test. The results showed that the addition of 0.6% lactose-0.004% astaxanthin with 6% DMSO had a significantly better effect (P&lt;0.05) on the quality of post-thawing spermatozoa with 19.75±0.96% DNA fragmentation and 53.75±3.86% intact plasma membranes. Therefore, it is recommended that a combination of 0.6% lactose-0.004% astaxanthin at 6% DMSO concentration should be added to Green Junglefowl semen during the freezing process to prevent DNA fragmentation and intact plasma membranes.

Effects of Alternative Solvents in Experimental Enamel Infiltrants on Bond Strength and Selected Properties.

To evaluate different concentrations of solvents (tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) and monomers on the degree of conversion, microtensile bond strength, and mechanical properties of experimental resin infiltrants. Resin infiltrants were formulated and divided into eleven groups: (1) Icon, (2) 75% TEGDMA (T) +25% UDMA (U), (3) T +25% BIS-EMA (B), (4) T + U +0.5%DMSO, (5) T + U +5% DMSO, (6) T + U +0.5% THF, (7) T + U +5% THF, (8) T + B +0.5% DMSO, (9) T + B +5% DMSO, (10) T + B +0.5% THF, and (11) T + B +5% THF. One hundred and ten bovine mandibular incisors were sectioned, treated, and destined to the degree of conversion, tensile cohesive strength, microtensile bond strength, flexural strength, and elastic modulus. Data were submitted to one-way ANOVA and Tukey's test (α = 0.05). The degree of conversion was lowest for T + B +5%THF (41.9%) and highest for T + U +5%THF (62.1%). In flexural strength and E-modulus, the T + B (96.5 MPa and 0.49 GPa) obtained the highest values and the lowest for T + U +5% DMSO (18.5 MPa and 9.7 GPa). Icon showed the highest bond strength (19.3 MPa) and cohesive strength (62.2 MPa), while T + U +5%DMSO (9.7 MPa) and T + B +5% DMSO (9.8 MPa) the lowest values and T + B +0.5% DMSO (12.3 MPa) the lowest cohesive strength. The addition of lower concentrations of DMSO or THF (0.5%) did not impair bond strength or significantly affect monomer conversion, but reduced the mechanical properties of resin infiltration.

Vitrification of the ovarian tissue in sturgeons

The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me2SO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M Me2SO in the ES, and 1.5 M MeOH and 5.5 M Me2SO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M Me2SO in the ES, and 3 M PG and 3 M Me2SO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons.

Determining the Population Doubling Time of HepG2 and Huh-7 Cells and the Toxic Effect of Dimethyl Sulfoxide (DMSO)

Developing cell lines that carry promising qualities closest to human hepatocytes in drug studies is a dream of many research laboratories. About 90% of drugs are metabolised by the liver. Therefore, most drug discovery studies utilise hepatocytes to understand the basic mechanism of the drug’s breakdown. To assure hepatocytes survival, various solvents were tested for their cytotoxicity on the cell line. Since most drugs are weakly soluble in water, they can be dissolved in an aprotic solvent. To obtain accurate results, the requirements of these solvents are biocompatible and non-toxic to the cells. One of the most commonly used solvents is dimethyl sulfoxide (DMSO). This study aims to understand the cell characteristic of cells doubling time and investigate the toxic effect of DMSO using the cell proliferation measurement technique. The toxicity was measured on liver-derived cell lines HepG2 and Huh-7. Cell growth and morphology were observed with an inverted phase microscope while cell viability was counted using Vi-Call XR 2.03. Results showed that a doubling time of 32 hours would be best for treatments on both cells. A concentration of more than 0.40% DMSO has significant toxicity and caused inhibition of proliferation on both cell lines, resulting in low cell count and higher cell death. Ethanol and methanol were observed as good solvents since they have low toxicity. However, drugs that were only dissolved in a low concentration of DMSO (0.05-0.2%) gave the best result without any harmful effect on cell proliferation.&#x0D;

Application of Photo-Fenton, Electro-Fenton, and Photo-Electro-Fenton processes for the treatment of DMSO and DMAC wastewaters

Biological treatment, due to the formation of hazardous chemicals to remove organic compounds such as dimethyl sulfoxide (DMSO) and N, N-dimethylacetamide (DMAC), has limited potential. Advanced oxidation processes (AOPs) are regarded as a viable alternative for treating molecules containing carbon-hydrogen bonds that cannot be broken down by traditional physico-chemical methods. In this investigation, various AOPs such as Photo-Fenton, Electro-Fenton, and Photo-Electro-Fenton processes were studied to treat wastewaters containing DMSO and DMAC. The effects of the operating parameters, including various initial concentrations of DMSO and DMAC, initial pH, reaction time, different concentrations of Fenton’s reagent, power of UV lamp, different concentrations of electrolytes, the distance between electrodes and current intensity, were investigated. The findings of the experiments revealed that a pH of 3 and a reaction time of 120 min were optimal. At 2000 mg L−1 of DMSO, maximum degradation and the final concentration of TOC were 98.64 % and 256.8 mg L−1, respectively, by the Electro-Fenton process under the optimal conditions. The Electro-Fenton process was successful in determining the maximum degradation of DMAC (96.31 %) and the final TOC concentration (10.03 mg L−1) at 250 mg L−1 of DMAC under optimal conditions. Finally, it can be concluded that the Electro-Fenton process was the best process for the efficient removal of DMSO and DMAC. The second step of the kinetic model follows a pseudo-first-order reaction for 250 and 500 mg L−1 of pollutants and obeyed a pseudo-second-order kinetic model for concentrations of 1000, 2000 mg L−1.

N-acetylcysteine attenuates oxidative stress-mediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production.

Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require long-term cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and off-the-shelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSO-induced cytotoxicity. Furthermore, we examined the potential of antioxidant N-acetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO. NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed. DMSO concentrations ≤1% for 24 h did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24 h) and cell death in a dose- and time-dependent manner. DMSO increased intracellular and mitochondrial ROS (1.9-fold and 3.6-fold respectively after 12 h exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a dose-dependent manner. Tempering ROS through NAC treatment significantly attenuated DMSO-induced oxidative stress and supported maintenance of cell viability. This study demonstrated dose- and time-dependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSO-induced oxidative stress in the freeze-thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products.

Cryopreservation of rabbit semen: impacts of permeable and non-permeable mixture of cryoprotectant, male group individuality, freezing rate, semen package size and antioxidant bovine serum albumin on rabbit semen freezability

In the present study, three experiments were designed to identify the most appropriate technique for freezing rabbit semen. Experiment 1 aimed to determine the optimal levels of dimethyl sulfoxide (DMSO) contents in freezing medium and their effects on individual bucks. Semen ejaculates for each buck (n=15 bucks) were mixed and split into three portions for extension with a freezing medium containing varying concentrations of DMSO (0.75, 1.0, and 1.4 M). Diluted semen samples were packaged in 0.25 mL straws and suspended above liquid nitrogen (LN) for 10 min, then dipped in LN. A few days after freezing, post-thaw semen evaluation was assessed, and according to the results, six bucks and an extender containing 0.75 M of DMSO were used for experiments 2 and 3. In experiment 2, the pooled semen from 6 bucks was divided into two portions for packaging in two straw sizes (0.25 and 0.50 mL). Each straw size was divided into five groups and suspended at different heights above LN (2, 4, 6, 8, and 10 cm) for 10 minutes before being preserved in LN. In experiment 3, the pooled semen was divided into four portions for dilution with freezing medium containing different concentrations of bovine serum albumin (BSA; 0, 2.5, 5.0, and 7.5 mg/mL). Semen samples were packaged in a 0.50 mL straw and suspended 10 min, 4 cm above LN for freezing. Pre-freezing and post-thawing, semen samples were evaluated for semen quality. Results showed that the extender containing 0.75 M DMSO had higher significant values for post-thaw sperm motility, longevity, acrosome integrity and sperm plasma membrane permeability. Bucks’ individuality had significant effects on post-thaw motility, acrosome and sperm plasma membrane integrity. A significant interaction was recorded between DMSO concentrations and bucks’ individuality on sperm longevity. Semen package sizes had no significant effects on the evaluated parameters. Semen was frozen at 2 and 4 cm above LN had significantly better post-thaw quality. BSA at concentrations 5 and 7.5 mg/mL improved recovery rates of acrosome integrity and sperm membrane permeability. DMSO 0.75 M and freezing 4 cm above LN seem to be more adequate for rabbit semen cryopreservation. The appropriate level of DMSO differs between bucks, as the post-thaw sperm longevity is affected. BSA enhanced acrosome and sperm membrane integrity. Results obtained will need further investigation to be confirmed in the field.

Processes taking place during the preparation and use of electrospun PLA fibers and their effect on controlled drug release

PLA fibers containing metronidazole as the active component were produced by electrospinning from a solvent mixture of dichloromethane (DCM) and dimethyl sulfoxide (DMSO). The DMSO content of the spinning solution changed between 0 and 25 vol% in 5 vol% steps. The fibers were dried at different temperatures, and processes taking place during fiber production and drying were followed by thermogravimetric analysis and differential scanning calorimetry. The morphology and structure of the fibers were studied by microscopy and X-ray diffraction. The mechanical properties of fiber mats and the release of the drug were also determined as a function of processing and drying parameters. The results showed that several processes take place during the production and subsequent handling of the fibers including the evaporation of the solvent (DMSO), the crystallization of the polymer, the changing of composition, phase separation and the consequent partitioning of the drug. The crystalline structure of the fibers changes considerably during drying which determines their mechanical properties. The rate of evaporation and crystallization is in the same order of magnitude. The rate of both processes increases considerably with temperature, but does not depend strongly on the amount of DMSO in the spinning solution. Both the amount of the drug released and the rate of release cover a wide range depending on the parameters of the preparation technology. The large range of mechanical and functional properties obtained allows the control of the kinetics of drug release to some extent.

Standardization and validation of novel ex-vivo method for mitochondrial bioenergetics using mitochondrial modulators

Mitochondria is an essential organelle; it produces 95% of the adenine triphosphate (ATP) of cells, their dysfunction is related to the pathogenesis of multiple diseases, such as diabetes mellitus, cardiovascular and neurological disorders. Various pharmacologic agents are known to target mitochondrial function. Moreover, the toxic side effects of multiple drugs used to treat diseases are related to the impairment of mitochondrial function. Thus, there is a need to develop a method to evaluate the effect of pharmacologic agents for their potential and side effects to identify effective mitochondrial-modulating agents. Therefore, the objective of this study was to develop and validate an ex-vivo method for studying the effect of pharmacologic agents on mitochondrial function and rescue of dysfunction. Dimethyl sulfoxide (DMSO) concentrations that drugs were soluble in and maintained mitochondrial function were determined. Metformin (MET) is a known mitochondrial complex-1 inhibitor tested for its ability to compromise mitochondrion function. Coenzyme Q10 (Q10) and Resveratrol (RSV), which are known to enhance mitochondrial function, were added alone and dose-dependent, tested for the ability to rescue metformin-induced mitochondrial dysfunction. Ex-vivo liver and brain mitochondrial function was assessed using an oxytherm Clark-type oxygen electrode. DMSO was found to be toxic above 10% and drugs insoluble below 5%. The addition of 0.5 mg/ml MET decreased liver and brain mitochondrial respiratory control rate (RCR). At the same time, Q10 improved RCR in normal mitochondria and a concentration-dependent manner in MET-induced dysfunctional mitochondria. RSV was added in the last step of the experiment to confirm that compromised function is due to MET. Hence this method can be used to screen pharmacological agents for their potential therapeutics or toxic effect on mitochondria.

Out of control: The need for standardised solvent approaches and data reporting in antibiofilm assays incorporating dimethyl-sulfoxide (DMSO)

Bacteria in biofilm formations are up to 1000 times less susceptible to antibiotics than their planktonic counterparts. Recognition of the role of biofilms in ∼80% of chronic infections, their contribution to bacterial tolerance and development of antimicrobial resistance, and thus the search for compounds with antibiofilm properties, has increased greatly in recent years. The need for robust experimental methods is therefore critical but currently undermined by inappropriate controls when dimethyl-sulfoxide (DMSO) is used to enhance test compound solubility. DMSO is known to have a limited effect on planktonic growth, but emerging data indicates that the solvent can affect biofilm formation even at low concentrations. Here, we present both a literature review on the application of DMSO in in vitro antibiofilm studies, as well as a series of experiments and Bayesian hormetic dose-response modelling to define the effects of DMSO alone and in combination with standard antibiotics using two clinically important biofilm-forming bacteria. DMSO has been used in 76 published studies to solubilise a wide variety of synthesised and natural products, including plant extracts, isolated secondary metabolites, modified lead molecules and proteins, in in vitro antibiofilm assays. DMSO solvent concentrations to which biofilms were exposed ranged between <1 and 100% but unfortunately, 35% of articles did not specify the DMSO concentrations used, 50% of articles did not include solvent controls and, of those that did, 26% did not specify control concentrations, 47% did not report or discuss control data, and 53% omitted media controls. In a further 12 studies, DMSO is used as a biofilm treatment, demonstrating the antibiofilm properties of this solvent at higher concentrations. We provide evidence that DMSO (between 0.03 and 25%) significantly inhibits biofilm formation in Pseudomonas aeruginosa, but not Streptococcus pneumoniae, and acts synergistically with standard antibiotics at very low concentrations (<1%). Interestingly, intermediate concentrations of DMSO (∼6%) strongly promote the growth of P. aeruginosa biofilms. As the research community strives to identify bioactive antimicrobial compounds, there is a need for increased scientific rigour when using DMSO as a solvent in antibiofilm assays.