High Concentrations Of Dimethyl Sulfoxide Research Articles

Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at -80 °C: An Effective Option in Resource-Limited Settings

Long-term cryopreservation of peripheral blood stem cells (PBSCs) is highly useful in the setting of tandem/multiple transplantations or treatment of relapse in the autologous hematopoietic stem cell transplantation (HSCT) setting. Even in allogeneic HSCT, donor lymphocyte infusions may be stored for months to years if excess stem cells are collected from donors. Cryopreservation is a delicate, complex, and costly procedure, and higher concentrations of dimethyl sulfoxide (DMSO), a commonly used cryoprotectant, can be toxic to cells and cause adverse effects in the recipient during infusions. In this study, we examined the effect of long-term cryopreservation using 4.35% DMSO (as final concentration) with methyl cellulose and uncontrolled rate freezing in a mechanical freezer (-80 °C) on the viability and colony-forming ability of CD34+ human PBSCs. For patients undergoing autologous HSCT, PBSCs were cryopreserved using DMSO (final concentration of 4.35%) with methyl cellulose. The post-thaw viability of PBSCs was determined using Trypan blue exclusion and flow cytometry-based 7-amino-actinomycin-D (FC-7AAD) methods. Concentrations of CD34+ stem cells and immune cell subsets in post-thaw PBSC harvest samples were assessed using multicolor flow cytometry, and the clonogenic potential of post-thaw stem cells was studied using a colony-forming unit (CFU) assay. CD34+ stem cell levels were correlated with the prestorage CD34 levels using the Pearson correlation test. The viability results in the Trypan blue dye exclusion method and the flow cytometry-based method were compared using Bland-Altman plots. We studied 26 PBSC harvest samples with a median cryopreservation duration of 6.6 years (range, 3.8 to 11.5 years). The median viability of post-thaw PBSCs was >80% using both methods, with a weak agreement between them (r=.03; P=.5). The median CD34+ stem cell count in the post-thaw samples was 9.13×106/kg (range, .44 to 26.27×106/kg). The CFU assay yielded a good proliferation and differentiation potential in post-thaw PBSCs, with a weak correlation between granulocyte macrophage CFU and CD34+ stem cell levels (r=.4; P=.05). Two samples that had been cryopreserved for >8 years showed low viability. Cryopreservation of PBSCs using 4.35% DMSO with methyl cellulose and uncontrolled freezing in a mechanical freezer at -80 °C allows the maintenance of long-term viability of PBSC for up to 8 years.

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.

Redefining the Molecular Interplay between Dimethyl Sulfoxide, Lipid Bilayers, and Dehydration.

The potentially damaging action of dimethyl sulfoxide (DMSO) on phospholipid bilayers remains a matter of controversy. We have conducted a series of long-scale molecular dynamics simulations of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers at various levels of hydration in the presence of variable quantities of DMSO. These simulations provide evidence for a non-destructive dehydrating mechanism of action for DMSO on DOPC bilayers across a wide concentration range and levels of hydration. Specifically, under full- and low-hydration conditions, the bilayer underwent a minor lateral contraction, coinciding with surface dehydration in the presence of dilute DMSO solutions (XDMSO < 0.3). At higher DMSO concentrations, this bilayer structure was retained despite a progressive deterioration of the hydration structure at the interface. A similar convergence of bilayer structural properties was observed under dehydration conditions for 0.3 < XDMSO < 0.7. Destabilization occurred for dehydrated bilayers in the presence of XDMSO ≥ 0.7, suggesting the existence of a DMSO concentration and/or dehydration threshold. However, such DMSO concentrations far exceed those established as toxic to other cellular components. Our findings represent a computational model for DMSO-DOPC interactions that is consistent with a range of experimental characterizations, offering new molecular insights into the cryoprotective mechanisms of action of DMSO.

How cryoprotectants work: hydrogen-bonding in low-temperature vitrified solutions.

Dimethyl sulfoxide (DMSO) increases cell and tissue viability at low temperatures and is commonly used as a cryoprotectant for cryogenic storage of biological materials. DMSO disorders the water hydrogen-bond networks and inhibits ice-crystal growth, though the specific DMSO interactions with water are difficult to characterize. In this study, we use a combination of Fourier Transform infrared spectroscopy (FTIR), molecular dynamics simulations, and vibrational frequency maps to characterize the temperature-dependent hydrogen bonding interactions of DMSO with water from 30 °C to −80 °C. Specifically, broad peaks in O–D stretch vibrational spectra of DMSO and deuterated water (HDO) cosolvent systems show that the hydrogen bond networks become increasingly disrupted compared to pure water. Simulations demonstrated that these disrupted hydrogen bond networks remain largely localized to the first hydration shell of DMSO, which explains the high DMSO concentrations needed to prevent ice crystal formation in cryopreservation applications.

DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo Developmental Toxicity Assay.

Dimethyl sulfoxide (DMSO) is a popular solvent for developmental toxicity testing of chemicals and pharmaceuticals in zebrafish embryos. In general, it is recommended to keep the final DMSO concentration as low as possible for zebrafish embryos, preferably not exceeding 100 μL/L (0.01%). However, higher concentrations of DMSO are often required to dissolve compounds in an aqueous medium. The aim of this study was to determine the highest concentration of DMSO that can be safely used in our standardized Zebrafish Embryo Developmental Toxicity Assay (ZEDTA). In the first part of this study, zebrafish embryos were exposed to different concentrations (0–2%) of DMSO. No increase in lethality or malformations was observed when using DMSO concentrations up to 1%. In a follow-up experiment, we assessed whether compounds that cause no developmental toxicity in the ZEDTA remain negative when dissolved in 1% DMSO, as false positive results due to physiological disturbances by DMSO should be avoided. To this end, zebrafish embryos were exposed to ascorbic acid and hydrochlorothiazide dissolved in 1% DMSO. Negative control groups were also included. No significant increase in malformations or lethality was observed in any of the groups. In conclusion, DMSO concentrations up to 1% can be safely used to dissolve compounds in the ZEDTA.

Hydrophobic Cargo Encapsulation into Virus Protein Cages by Self-Assembly in an Aprotic Organic Solvent.

While extensive studies of virus capsid assembly in environments mimicking in vivo conditions have led to an understanding of the thermodynamic driving forces at work, applying this knowledge to virus assembly in other solvents than aqueous buffers has not been attempted yet. In this study, Brome mosaic virus (BMV) capsid proteins were shown to preserve their self-assembly abilities in an aprotic polar solvent, dimethyl sulfoxide (DMSO). This facilitated protein cage encapsulation of nanoparticles and dye molecules that favor organic solvents, such as β-NaYF4-based upconversion nanoparticles and BODIPY dye. Assembly was found to be robust relative to a surprisingly broad range of DMSO concentrations. Cargos with poor initial stability in aqueous solutions were readily encapsulated at high DMSO concentrations and then transferred to aqueous solvents, where they remained stable and preserved their function for months.

Seasonality of dimethylated sulfur compounds cycling in north China marginal seas

Dimethylated sulfur compounds play an important role in global sulfur cycle. We investigated the seasonality of dimethylsulfoniopropionate (DMSP), dimethylsulfoxide (DMSO), dimethylsulfide (DMS) and associated processes in two north China marginal seas during 2014 and 2016. High concentrations of DMS, DMSP and DMSO occurred in summer/spring, while the lowest were observed in winter. This clear seasonality was primarily driven by biomass abundance and phytoplankton communities, reflected in chlorophyll a concentrations and the composition/ratios of diatoms and dinoflagellates. The spring maximum was attributed to the annual occurrence of algal bloom. The sea-to-air fluxes of DMS also varied largely between seasons, with an average of 8.84, 11.87, 10.50 and 2.14 μmol m−2 day−1 in spring, summer, autumn and winter, respectively. Given the seasonal uncertainty of sea-to-air flux, the seasonality or situations where specific blooms occur regularly should be considered for accurate estimation of annual global DMS emission.

Antifungal activity of dimethyl sulfoxide against Botrytis cinerea and phytotoxicity on tomato and lettuce plants

For the first time the antifungal activity of dimethyl sulfoxide (DMSO) was evaluated against Botrytis cinerea, that it is one of the phytopathogenic fungi which causes the greatest damage in agriculture. In in-vitro tests, the greatest inhibitory effect of DMSO on fungal grow was recorded at pH 6. A significant growth inhibition was caused by 0.5% DMSO at 96 h post-inoculation. With higher DMSO concentrations, significant effects were recorded starting from 48 h post-inoculation. As the medium pH decreased, the inhibitory effect of DMSO also decreased. At pH 4 and 5 significant growth inhibition was caused by 1% DMSO starting from 72 h post-inoculation. At all tested pH values, a total growth inhibition was caused by ≥ 2% DMSO. On tomato leaves infected with B. cinerea, 2% DMSO significantly decreased the extent of damaged leaf area. The administration of DMSO at concentrations ranging from 0.5 to 2% through nebulization on leaves of young Solanum lycopersicum and Lactuca sativa plants did not change the chlorophyll fluorescence (Fv/Fm and ΦPSII) at any of the experimental times. Overall, the data obtained suggest that, at the concentrations tested, DMSO is toxic to B. cinerea, while it is well tolerated by lettuce and tomato plants.

Dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO) cell quotas variations arising from sea ice shifts of salinity and temperature in the Prymnesiophyceae Phaeocystis antarctica

Environmental contextDimethylsulfoniopropionate and dimethylsulfoxide could have a climatic influence especially in the polar areas. We investigate the effect of sea ice salinity and temperature on the production of these two sulfur metabolites by a polar microalga, and suggest their potential roles of osmoregulator and cryoprotectant. These results bring new information about the sulfur cycle in sea ice that is useful for climate models. AbstractThe Southern Ocean, which includes the seasonal ice zone (SIZ), is a source of large sea-air fluxes of dimethylsulfide (DMS), a climate active gas involved in Earth cooling processes. In this area, the prymnesiophyte Phaeocystis antarctica (P. antarctica) is one of the main producers of dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO), two metabolites that are precursors of DMS. These algae are also present in sea ice and contribute substantially to the high DMSP and DMSO concentrations observed in this habitat. DMSP and DMSO production in sea ice by P. antarctica is proposed to be promoted by its ability to live in extreme environmental conditions. We designed cell culture experiments to test that hypothesis, focusing on the impact of shifts of temperature and salinity on the DMSP and DMSO cell quotas. Our experiments show an increase in DMSP and DMSO cell quotas following shifts in salinity (34 to 75, at 4°C), which suggests a potential osmoregulator function for both DMSP and DMSO. Stronger salinity shifts (up to 100) directly impact cell growth and induce a crash of the cultures. Combining the salinity (34 to 75) and temperature (4°C to –2.3°C) shifts induces higher increases of DMSP and DMSO cell quotas that also suggests an implication of both metabolites in a cryoprotectant system. Experimental cell quotas (including diatom Fragilariopsis cylindrus quotas from a previous study) are then used to reconstruct DMSP and DMSO profiles in sea ice based on the biomass and taxonomy. Finally, the complexity of the transposition of rates obtained in the experimental domain to the real world is discussed.

DMSO induced dehydration of heterogeneous lipid bilayers and its impact on their structures.

Recently, we have reported that higher concentrations of dimethyl sulfoxide (DMSO) exhibit an enhancement in the structural ordering of the homogeneous N-palmitoyl-sphingomyelin (PSM) bilayer, whereas the presence of DMSO at lower concentrations leads to minor destabilization of the PSM bilayer structure. In this study, we aim to understand how these two modes of action of DMSO diversify for heterogeneous bilayers by employing atomistic molecular dynamic simulations. A binary bilayer system comprising PSM and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a ternary bilayer system consisting of cholesterol along with PSM and POPC are the two heterogeneous biomimetic bilayers studied herein. We have simulated both the mixed lipid bilayer systems at 323 K, which is above the main phase transition temperature of the PSM lipid. This study reveals that DMSO exerts contrasting effects on the structure and stability of mixed bilayer systems, depending on its concentration. At 5 mol% of DMSO, the binary bilayer system shows slight disordering of lipid tails in conjunction with an appreciable increase in the area per lipid (APL), whereas for the ternary bilayer system, the orientational ordering of the lipid tails does not alter much; however, a slight expansion in the APL is observed. On the other hand, at 20 mol% of DMSO, an appreciable increase in the ordering of lipid tails for both the mixed bilayer systems occurs, depicting an enhancement in the structural stability of the bilayers. Furthermore, the H-bond analysis reveals that water-lipid H-bonding interaction decreases with increasing concentration of DMSO. We also observe contraction of the water-lipid interfacial region, pointing out DMSO induced dehydration at the lipid head-group region, and the dehydration effect is prominent for 20 mol% of DMSO. Furthermore, the computed free energies suggest that the free energy required for the transfer of a DMSO molecule from the lipid head-group region to the lipid head-tail interface is higher for the cholesterol containing ternary bilayer.

Stepped vitrification technique for human ovarian tissue cryopreservation

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants.

The Effect of Dimethyl Sulfoxide on the Lysozyme Unfolding Kinetics, Thermodynamics, and Mechanism.

The thermal stability of proteins in the presence of organic solvents and the search for ways to increase this stability are important topics in industrial biocatalysis and protein engineering. The denaturation of hen egg-white lysozyme in mixtures of water with dimethyl sulfoxide (DMSO) with a broad range of compositions was studied using a combination of differential scanning calorimetry (DSC), circular dichroism (CD), and spectrofluorimetry techniques. In this study, for the first time, the kinetics of unfolding of lysozyme in DMSO–water mixtures was characterized. In the presence of DMSO, a sharp decrease in near-UV CD and an increase in the fluorescence signal were observed at lower temperatures than the DSC denaturation peak. It was found that differences in the temperatures of the CD and DSC signal changes increase as the content of DMSO increases. Changes in CD and fluorescence are triggered by a break of the tertiary contacts, leading to an intermediate state, while the DSC peak corresponds to a subsequent complete loss of the native structure. In this way, the commonly used two-state model was proven to be unsuitable to describe the unfolding of lysozyme in the presence of DMSO. In kinetic studies, it was found that even high concentrations of DMSO do not drastically change the activation energy of the initial stage of unfolding associated with a disruption of the tertiary structure, while the enthalpy of denaturation shows a significant dependence on DMSO content. This observation suggests that the structure of the transition state upon unfolding remains similar to the structure of the native state.

ВИКОРИСТАННЯ ТРОМБОЦИТАРНОГО ЛІЗАТУ ЯК КОМПОНЕНТА КРІОЗАХИСНИХ СРЕДОВИЩ ДЛЯ КРІОКОНСЕРВУВАННЯ МЕЗЕНХІМАЛЬНИХ СТРОМАЛЬНИХ КЛІТИН

Mesenchymal stromal cells (MSCs) due to their unique properties are widely used in regenerative medicine. Standard cryopreservation methods that are based on the use of penetrating cryoprotectant dimethyl sulfoxide (DMSO) and fetal bovine serum (FS) can ensure high cell survival, but limit the possibility of therapeutic application because of the risk of adverse reactions. The toxicity of high DMSO concentrations and FS immunogenicity require significant optimization of cryopreservation approaches. In the current study, freezing of human MSCs in cryoprotective media (CPM) with different compositions was performed. Twenty-four hours prior to freezing, cells were pretreated by addition of 100 mM sucrose into the culture medium. CPM with 200 mM sucrose were supplemented with 10% FS or 10% platelet lysate (PL) in the presence or absence of 1% DMSO. The cells frozen without any cryoprotectants were used as a negative control. The MSCs cryopreserved in media containing 10% DMSO and 10% FS were chosen as a positive control group. The MSCs were frozen in cryogenic vials with a cooling rate of 1 deg/min to -80°C with the following immersion into liquid nitrogen. The cell survival was determined by trypan blue staining; metabolic activity was assessed using the Alamar Blue test. It was revealed that viability of MSCs after freezing in CPM containing 200 mM sucrose, 10% FS or 10% PL without DMSO addition were 59±3.3% and 58±2.5%, respectively. The metabolic activity of cells in the PL group exceeded the results of the FS group by 12%. When 1% DMSO was added into the CPM containing 200 mM sucrose and 10% PL, the cell survival rate and metabolic activity were by 7% and 13% higher than in the presence of 10% FS. The obtained results indicate that replacement of FS with PL in the CPM without penetrating cryoprotectant DMSO allows to maintain MSCs viability and increase their metabolic activity after freeze-thawing.

CD4+ T cell activation, function, and metabolism are inhibited by low concentrations of DMSO

Dimethyl sulfoxide (DMSO) is a polar organic solvent used in a wide range of biological applications. DMSO is routinely used as a cryoprotectant for long-term cell freezing as well as to dissolve peptides and drugs for immune cell functional assays. Here, human CD4+ T cell activation, cytokine production, proliferation, and metabolism were investigated after stimulation in the presence of 0.01% to 1%, DMSO, representing concentrations commonly used in vitro. Surface expression of the activation markers CD69, CD25 and CD154 after polyclonal activation of CD4+ T cells was inhibited by 0.25% or higher concentrations of DMSO. The frequencies of IL-21+, IL-4+, and IL-22+ CD4+ T cells, following polyclonal activation were variably inhibited by DMSO at concentrations ranging from 0.25% to 1%, whereas IFNγ+ cells were unaffected. CD4+ T cell proliferation after anti-CD3 or antigen stimulation was inhibited by 0.5% DMSO and abolished by 1% DMSO. After polyclonal stimulation, glucose uptake was inhibited in the presence of 1% DMSO, but only minor effects on CD4+ T cell respiration were observed. Consistent with the immune effects, the gene expression of early signaling and activation pathways were inhibited in CD4+ T cells in the presence of 1% DMSO. Our study revealed that DMSO at concentrations generally used for in vitro studies of T cells impacts multiple features of T cell function. Therefore, we urge care when adding DMSO-containing preparations to T cell cultures.

Annual cycle of dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO) related to phytoplankton succession in the Southern North Sea

The influence of abiotic and biotic variables on the concentration of dimethyl sulfide (DMS), dimethylsulfoniopropionate (DMSP), and dimethylsulfoxide (DMSO), were investigated during an annual cycle in 2016 in the Belgian Coastal Zone (BCZ, North Sea). We reported strong seasonal variations in the concentration of these compounds linked to the phytoplankton succession with high DMS(P,O) producers (mainly Phaeocystis globosa) occurring in spring and low DMS(P,O) producers (various diatoms species) occurring in early spring and autumn. Spatial gradients of DMS and DMSP were related to those of phytoplankton biomass itself related to the inputs of nutrients from the Scheldt estuary. However, the use of a relationship with Chlorophyll-a (Chl-a) concentration is not sufficient to predict DMSP. Accounting for the phytoplankton composition, two different DMSP versus Chl-a correlations could be established, one for diatoms and another one for Phaeocystis colonies. We also reported high nearshore DMSO concentrations uncoupled to Chl-a and DMSP concentrations but linked to high suspended particulate matter (SPM) presumably coming from the Scheldt estuary as indicated by the positive relationship between annual average SPM and salinity.

Effect of dimethyl sulfoxide on dentin collagen

ObjectivesInfiltration of adhesive on dentin matrix depends on interaction of surface and adhesive. Interaction depends on dentin wettability, which can be enhanced either by increasing dentin surface energy or lowering the surface energy of adhesive. The objective was to examine the effect of dimethyl sulfoxide (DMSO) on demineralized dentin wettability and dentin organic matrix expansion. MethodsAcid-etched human dentin was used for sessile drop contact angle measurement to test surface wetting on 1–5% DMSO-treated demineralized dentin surface, and linear variable differential transformer (LVDT) to measure expansion/shrinkage of dentinal matrix. DMSO-water binary liquids were examined for surface tension changes through concentrations from 0 to 100% DMSO. Kruskal–Wallis and Mann–Whitney tests were used to test the differences in dentin wettability, expansion and shrinkage, and Spearman test to test the correlation between DMSO concentration and water surface tension. The level of significance was p<0.05. ResultsPretreatment with 1–5% DMSO caused statistically significant concentration-dependent increase in wetting: the immediate contact angles decreased by 11.8% and 46.6% and 60s contact angles by 9.5% and 47.4% with 1% and 5% DMSO, respectively. DMSO-water mixtures concentration-dependently expanded demineralized dentin samples less than pure water, except with high (≥80%) DMSO concentrations which expanded demineralized dentin more than water. Drying times of LVDT samples increased significantly with the use of DMSO. SignificanceIncreased dentin wettability may explain the previously demonstrated increase in adhesive penetration with DMSO-treated dentin, and together with the expansion of collagen matrix after drying may also explain previously observed increase in dentin adhesive bonding.

Solvent stimulated actuation of polyurethane-based shape memory polymer foams using dimethyl sulfoxide and ethanol

Solvent exposure has been investigated to trigger actuation of shape memory polymers (SMPs) as an alternative to direct heating. This study aimed to investigate the feasibility of using dimethyl sulfoxide (DMSO) and ethanol (EtOH) to stimulate polyurethane-based SMP foam actuation and the required solvent concentrations in water for rapid actuation of hydrophobic SMP foams. SMP foams exhibited decreased Tg when submerged in DMSO and EtOH when compared to water submersion. Kinetic DMA experiments showed minimal or no relaxation for all SMP foams in water within 30 min, while SMP foams submerged in EtOH exhibited rapid relaxation within 1 min of submersion. SMP foams expanded rapidly in high concentrations of DMSO and EtOH solutions, where complete recovery over 30 min was observed in DMSO concentrations greater than 90% and in EtOH concentrations greater than 20%. This study demonstrates that both DMSO and EtOH are effective at triggering volume recovery of polyurethane-based SMP foams, including in aqueous environments, and provides promise for use of this actuation technique in various applications.

The SecB-like chaperone Rv1957 from Mycobacterium tuberculosis: crystallization and X-ray crystallographic analysis.

Protein export is important in all bacteria, and bacteria have evolved specialized export machineries to fulfil this task. In Mycobacterium tuberculosis, the causative agent of tuberculosis, the general secretion pathway (Sec pathway) is conserved and is essential in performing the export of proteins. The bacterial Sec pathway post-translationally exports unfolded proteins out of the cytoplasm, and the core of the Sec pathway is composed of a heterotrimeric membrane-embedded channel, SecYEG, and two cytosolic components, SecA and SecB. SecB functions by stabilizing unfolded proteins, maintaining them in an export-competent state. Although SecB is mainly found in Proteobacteria, a SecB-like protein, Rv1957, that controls a stress-response toxin-antitoxin system, is found in M. tuberculosis. Rv1957 can also functionally replace the Escherichia coli SecB chaperone both in vivo and in vitro. In this work, the production, crystallization and X-ray crystallographic analysis of Rv1957 are reported. Notably, diffraction-quality crystals were obtained only at high concentrations of dimethyl sulfoxide, i.e. about 12%(v/v). The crystals of Rv1957 belonged to space group P212121, with unit-cell parameters a = 64.5, b = 92.0, c = 115.4 Å.

Protein Structural Memory Influences Ligand Binding Mode(s) and Unbinding Rates.

The binding of small molecules (e.g., natural ligands, metabolites, and drugs) to proteins governs most biochemical pathways and physiological processes. Here, we use molecular dynamics to investigate the unbinding of dimethyl sulfoxide (DMSO) from two distinct states of a small rotamase enzyme, the FK506-binding protein (FKBP). These states correspond to the FKBP protein relaxed with and without DMSO in the active site. Since the time scale of ligand unbinding (2-20 ns) is faster than protein relaxation (100 ns), a novel methodology is introduced to relax the protein without having to introduce an artificial constraint. The simulation results show that the unbinding time is an order of magnitude longer for dissociation from the DMSO-bound state (holo-relaxed). That is, the actual rate of unbinding depends on the state of the protein, with the protein having a long-lived memory. The rate thus depends on the concentration of the ligand as the apo and holo states reflect low and high concentrations of DMSO, respectively. Moreover, there are multiple binding modes in the apo-relaxed state, while a single binding mode dominates the holo-relaxed state in which DMSO acts as hydrogen bond acceptor from the backbone NH of Ile56, as in the crystal structure of the DMSO/FKBP complex. The solvent relaxes very fast (∼1 ns) close to the NH of Ile56 and with the same time scale of the protein far away from the active site. These results have implications for high-throughput docking, which makes use of a rigid structure of the protein target.

Label-free concentration of viable neurons, hESCs and cancer cells by means of acoustophoresis.

Concentration of viable cell populations in suspension is of interest for several clinical and pre-clinical applications. Here, we report that microfluidic acoustophoresis is an effective method to efficiently concentrate live and viable cells with high target purity without any need for protein fluorescent labeling using antibodies or over-expression. We explored the effect of the acoustic field acoustic energy density and systematically used different protocols to induce apoptosis or cell death and then determined the efficiency of live and dead cell separation. We used the breast cancer cell line MCF-7, the mouse neuroblastoma N2a as well as human embryonic stem cells (hESCs) to demonstrate that this method is gentle and can be applied to different cell populations. First, we induced cell death by means of high osmotic shock using a high concentration of PBS (10×), the protein kinase inhibitor staurosporine, high concentrations of dimethyl sulfoxide (DMSO, 10%), and finally, cell starvation. In all the methods employed, we successfully induced cell death and were able to purify and concentrate the remaining live cells using acoustophoresis. Importantly, the concentration of viable cells was not dependent on a specific cell type. Further, we demonstrate that different death inducing stimuli have different effects on the intrinsic cell properties and therefore affect the efficiency of the acoustophoretic separation.