Effects of different surface treatments on surface topography and bond strength in the repair of fiber-reinforced dentin composite.

OBJECTIVE: This study aimed to evaluate the effects of different surface treatments on the repair bond strength between a fiber-reinforced dentin composite and a posterior composite. METHODS: Forty fiber-reinforced dentin composite resin blocks (4 mm × 4 mm × 4 mm) were separated into eight groups (n = 5) according to the surface preparation methods: (G1) negative control group, (G2) adhesive application, (G3) 50% dimethylsulfoxide (DMSO) application, (G4) 50% DMSO + adhesive application, (G5) 37% phosphoric acid etch + adhesive application, (G6) air abrasion + adhesive application, (G7) 37% phosphoric acid etch + 50% DMSO application + adhesive application, and (G8) air abrasion +50% DMSO application + adhesive application group. The composite surfaces were repaired in two layers with a posterior composite. Composite sticks were subjected to a micro tensile bond strength (µTBS) test. Fractured surfaces were evaluated using a stereomicroscope (×25). Short fiber-reinforced composite samples' surfaces were investigated by scanning electron microscope (SEM). Shapiro Wilk, one-way ANOVA, and Tukey HSD tests were used for statistical evaluation. RESULTS: The highest average (µTBS) values were observed in G8, whereas the lowest mean µTBS values were evident in the G1 group. Statistically significant µTBS values were found in all adhesive-applied groups when compared with the negative control group. Notably, the application of 50% DMSO without adhesive did not lead to a statistically significant increase in µTBS values. SEM images demonstrated that acid etching partially eliminated residues on the composite surface, while air abrasion had a detrimental effect on the integrity of fiber structures. CONCLUSION: In the repair of fiber-reinforced dentin composite with a posterior composite, adhesive application is an effective approach. The treatment of 50% DMSO without adhesive did not confer a statistically significant advantage, and the supplemental use of acid etch or air abrasion did not show an additional benefit compared to adhesive-only repairs. CLINICAL SIGNIFICANCE: Adhesive application emerges as a potent and effective strategy for the repair of bur-roughened fiber-reinforced dentin composites. With its limitations, the study highlights the efficacy of adhesive-only repairs without the necessity for additional surface treatments.

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor.

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.

Effect of PAR irradiance intensity on Phaeocystis antarctica (Prymnesiophyceae) growth and DMSP, DMSO, and acrylate concentrations.

Phaeocystis antarctica forms extensive spring blooms in the Southern Ocean that coincide with high concentrations of dimethylsulfoniopropionate (DMSP), dimethylsulfoxide (DMSO), dimethylsulfide (DMS), and acrylate. We determined how concentrations of these compounds changed during the growth of axenic P. antarctica cultures exposed to light-limiting, sub-saturating, and saturating PAR irradiances. Cellular DMSP concentrations per liter cell volume (CV) ranged between 199 and 403 mmol · LCV -1 , with the highest concentrations observed under light-limiting PAR. Cellular acrylate concentrations did not change appreciably with a change in irradiance level or growth, ranging between 18 and 45 mmol · LCV -1 , constituting an estimated 0.2%-2.8% of cellular carbon. Both dissolved acrylate and DMSO increased substantially with irradiance during exponential growth on a per-cell basis, ranging from 0.91 to 3.15 and 0.24 to 1.39 fmol · cell-1 , respectively, indicating substantial export of these compounds into the dissolved phase. Average cellular DMSO:DMSP ratios increased 7.6-fold between exponential and stationary phases of batch growth, with a 3- to 13-fold increase in cellular DMSO likely formed from abiotic reactions of DMSP and DMS with reactive oxygen species (ROS). At mM levels, cellular DMSP and acrylate are proposed to serve as de facto antioxidants in P. antarctica not regulated by oxidative stress or changes in ROS. Instead, cellular DMSP concentrations are likely controlled by other physiological processes including an overflow mechanism to remove excess carbon via acrylate, DMS, and DMSO during times of unbalanced growth brought on by physical stress or nutrient limitation. Together, these compounds should aid P. antarctica in adapting to a range of PAR irradiances by maintaining cellular functions and reducing oxidative stress.

Mechanism of acitretin-induced relaxations in isolated rat thoracic aorta preparations.

Acitretin is a member of vitamin A-derived retinoids, and its effect on vascular smooth muscle had not yet been studied. The aim of this study is to investigate the effect of acitretin, a retinoid, on vascular smooth muscle contractility. Thoracic aorta preparations obtained from 34 male Sprague-Dawley rats (355 ± 15 g) were studied in isolated organ baths containing Krebs-Henseleit solution. The relaxation responses were obtained with acitretin (10-12-10-4 M) in endothelium-preserved and endothelium-denuded aorta preparations precontracted with submaximal concentration of phenylephrine (10-6 M). The role of retinoic acid receptors (RARs), nitric oxide, adenylyl, and guanylyl cyclase enzymes, and potassium channels in these relaxation responses were investigated. Acitretin produced concentration-dependent relaxations, which were independent of its solvent dimethylsulfoxide (DMSO), in endothelium-denuded phenylephrine-precontracted thoracic aorta preparations. While incubation with the RAR antagonist (AGN193109, 10-5 M) had no effect on these relaxations; nitric oxide synthase inhibitor (L-NG-Nitro arginine methyl ester (L-NAME), 10-4 M), adenylyl cyclase inhibitor (SQ2253, 10-5 M), guanylyl cyclase inhibitor (oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), 10-6 M), and potassium channel blocker (tetraethylammonium (TEA), 10-2 M) significantly eliminated the relaxation responses induced by acitretin. Acitretin induces relaxation in rat isolated thoracic aorta preparations without endothelium, which may be mediated by nitric oxide, cyclic adenosine monophosphate, and cyclic guanosine monophosphate-dependent kinases and potassium channels.

Cryopreservation of human amniotic membrane for ocular surface reconstruction: a comparison between protocols.

PURPOSE: To compare the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -180 °C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80 °C. METHODS: Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -180 °C, dimethyl sulfoxide at -80 °C, dextran-based medium at -180 °C, dextran-based medium at -80 °C and dry freezing at -80 °C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. RESULTS: None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. CONCLUSIONS: Switching from liquid nitrogen cryopreservation to -80 °C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.

Assessing the Functional and Structural Stability of the Met80Ala Mutant of Cytochrome c in Dimethylsulfoxide.

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c.

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science.

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride.

Disaccahrides-Based Cryo-Formulant Effect on Modulating Phospho/Mitochondrial Lipids and Biological Profiles of Human Leukaemia Cells.

BACKGROUND/AIMS: The use of novel cryo-additive agents to increase cell viability post-cryopreservation is paramount to improve future cell based-therapy treatments. We aimed to establish the Human Leukemia (HL-60) cells lipidomic and biological patterns when cryo-preserved in DMSO alone and with 300 µM Nigerose (Nig), 200 µM Salidroside (Sal) or a combination of Nig (150 µM) and Sal (100 µM). METHODS: HL-60 cells were pre-incubated with Nig/Sal prior, during and post cryopreservation, and subjected to global lipidomic analysis. Malondialdeyhde (MDA), released lactate dehydrogenase (LDH) and reactive oxygen scavenger (ROS) measurements were also carried out to evaluate levels of lipid peroxidation and cytotoxicity. RESULTS: Cryopreserving HL-60 cells in DMSO with Nig and Sal provided optimal protection against unsaturated fatty acid oxidation. Post-thaw, cellular phospholipids and mitochondrial cardiolipins were increased by Nig/Sal as the ratio of unsaturated to saturated fatty acids 2.08 +/- 0.03 and 0.95 +/- 0.09 folds respectively in comparison to cells cryopreserved in DMSO alone (0.49 +/- 0.05 and 0.86 +/- 0.10 folds). HL-60 lipid peroxidation levels in the presence of DMSO + Nig and Sal combined were significantly reduced relative to pre-cryopreservation levels (10.91 +/- 2.13 nmole) compared to DMSO (17.1 +/- 3.96 nmole). DMSO + Nig/Sal combined also significantly reduced cell cytotoxicity post-thaw (0.0128 +/- 0.00182 mU/mL) in comparison to DMSO (0.0164 +/- 0.00126 mU/mL). The combination of Nig/Sal also reduced significantly ROS levels to the levels of prior cryopreservation of HL-60. CONCLUSION: Overall, the establishment of the cryopreserved HL-60 cells lipidomic and the corresponding biological profiles showed an improved cryo-formulation in the presence of DMSO with the Nig/Sal combination by protecting the, mitochondrial inner membrane, unsaturated fatty acid components (i. e. Cardiolipins) and total phospholipids.

Cryopreservation of adipose tissue with and without cryoprotective agent addition for breast lipofilling: A cytological and histological study.

In the second reconstructive phase of the breast after mastectomy, lipofilling is often necessary. Currently, lipofilling occurs immediately after autologous adipose tissue harvesting procedure, but most of the patients, usually, require multiple sessions to obtain a satisfactory result. Therefore, the need of repeated surgical harvesting outputs implies high risk of patients' morbidity and discomfort as well as increasing medical time and costs. The aim of our pilot study was to find out a feasible method to cryopreserve adipose tissue, in order to avoid reiterated liposuctions. Lipoaspirates samples have been harvested from 10 women and preserved by three methods: (1) the first one, using 10% Me2SO and 20% human albumin from human plasma as cryoprotective agents; (2) the second one, adding 5% Me2SO as cryoprotective agent; 3) the last one, without any cryoprotective agent. Fresh and cryopreserved fat samples, obtained through the aforementioned processes, have been analyzed ex vivo. The efficiency of the cryopreservation methods used was determined by adipocyte viability and the expression of adipocytes surface markers. Lipoaspirates stored at -196 °C for 3 months, after thawing, retained comparable adipocyte viability and histology to fresh tissue and no significant differences were found between the three methods used. Although the current results, differences between the methodologies in terms of viability may not become evident until breast lipofilling using frozen-thawed cryopreserved tissue.

Inhibition of urease activity in the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis by dimethylsulfoxide (DMSO).

AIMS: Urease is a virulence factor for the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis. Dimethylsulfoxide (DMSO) is structurally similar to urea, used as a solvent for urease inhibitors, and an effective treatment for interstitial cystitis/bladder pain syndrome (IC/BPS). The aims of this study were to test DMSO as a urease inhibitor and determine its physiological effects on S. saprophyticus and P. mirabilis. METHODS AND RESULTS: Urease activity in extracts and whole cells was measured by the formation of ammonium ions. Urease was highly sensitive to noncompetitive inhibition by DMSO (Ki about 6 mmol l-1 ). DMSO inhibited urease activity in whole cells, limited bacterial growth in media containing urea, and slowed the increase in pH which occurred in artificial urine medium. CONCLUSIONS: DMSO should be used with caution as a solvent when testing plant extracts or other potential urease inhibitors. Because it can inhibit bacterial growth and delay an increase in pH, it may be an effective treatment for urinary tract infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study of the inhibition of urease by DMSO. Dimethylsulfoxide may be used to treat urinary tract infections that are resistant to antibiotics or herbal remedies.

Non-toxic freezing media to retain the stem cell reserves in adipose tissues.

Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) and adipose-derived stromal/stem cells (ASCs) that are inherently multipotent and exhibit regenerative properties. In current practice, lipoaspirate specimens harvested from liposuction surgeries are routinely discarded as a biohazard waste due to a lack of simple, cost effective, and validated cryopreservation protocols. The aim of this study is to develop a xenoprotein-free cryoprotective agent cocktail that will allow for short-term (up to 6 months) preservation of lipoaspirate tissues suitable for fat grafting and/or stromal/stem cell isolation when stored at achievable temperatures (-20 °C or -80 °C). Lipoaspirates donated by three consenting healthy donors undergoing elective cosmetic liposuction surgeries were suspended in five freezing media (FM1: 10% DMSO and 35% BSA; FM2: 2% DMSO and 43% BSA; FM3: 10% DMSO and 35% lipoaspirate saline; FM4: 2% DMSO and 6% HSA; and FM5: 40% lipoaspirate saline and 10% PVP) all suspended in 1X DMEM/F12 and frozen using commercially available freezers (-20 °C or -80 °C) and stored at least for a 1 month. After 1 month of freezing storage, SVF cells and ASCs were isolated from the frozen-thawed lipoaspirates by digestion with collagenase type I. Cell viability was evaluated by fluorescence microscopy after staining with acridine orange and ethidium bromide. The SVF isolated from lipoaspirates frozen at -80 °C retained comparable cell viability with the tested freezing media (FM2, FM3, FM4) comparable with the conventional DMSO and animal serum media (FM1), whereas the FM5 media resulted in lower viability. In contrast, tissues frozen and stored at -20 °C did not yield live SVF cells after thawing and collagenase digestion. The surface marker expression (CD90, CD29, CD34, CD146, CD31, and CD45) of ASCs from frozen lipoaspirates at -80 °C in different cryoprotectant media were also evaluated and no significant differences were found between the groups. The adipogenic and osteogenic differentiation potential were studied by histochemical staining and gene expression by qRT-PCR. Oil Red O staining for adipogenesis revealed that the CPA media FM1, FM4 and FM5 displayed robust differentiation. Alizarin Red S staining for osteogenesis revealed that FM1 and FM4 media displayed superior differentiation in comparison to other tested media. Measurement of adipogenic and osteogenic gene expression by qRT-PCR provided similar outcomes and indicated that FM4 CPA media comparable with FM1 for adipogenesis and osteogenesis.

Biotransformation of cladribine by a magnetic immobilizated biocatalyst of Lactobacillus animalis.

A stable biocatalyst with magnetic properties based on immobilized Lactobacillus animalis ATCC 35,046 to obtain 2-chloroadenine-2'-deoxyriboside, known as cladribine, is reported for the first time. This nucleoside analogue is an antitumor agent used in the treatment of a wide variety of types of leukemia. In this study, an eco-compatible and alternative bioprocess to obtain cladribine was developed. Product conversion was close to 90% at 2 h in optimized nonconventional reaction media. The microscale biosynthesis of the compound of interest afforded a total productivity close to 370 mg/L/h in the presence of DMSO, and it was stable at least for 30 days in storage conditions.

Polyphenolic extract from extra virgin olive oil inhibits the inflammatory response in IL-1ß-activated synovial fibroblasts.

The polyphenolic extract (PE) from extra virgin olive oil (EVOO) has been shown to possess important anti-inflammatory and joint protective properties in murine models of rheumatoid arthritis (RA). This study was designed to evaluate the effects of PE on IL-1ß-activated human synovial fibroblasts SW982 cell line. PE from EVOO treatment inhibited IL-1ß-induced matrix metalloproteases (P<0·001), TNF-α and IL-6 production (P<0·001). Similarly, IL-1ß-induced cyclo-oxygenase-2 and microsomal PGE synthase-1 up-regulations were down-regulated by PE (P<0·001). Moreover, IL-1ß-induced mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation were ameliorated by PE (P<0·001). These results suggest that PE from EVOO reduces the production of proinflammatory mediators in human synovial fibroblasts; particularly, these protective effects could be related to the inhibition of MAPK and NF-κB signalling pathways. Taken together, PE from EVOO probably could provide an attractive complement in management of diseases associated with over-activation of synovial fibroblasts, such as RA.

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage, access and transport.

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me2SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me2SO and EG, respectively. Concentration changes in EG/Me2SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.

7-Hydroxymatairesinol improves body weight, fat and sugar metabolism in C57BJ/6 mice on a high-fat diet.

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro.

Cell density, dimethylsulfoxide concentration and needle gauge affect hydrogel-induced bone marrow mesenchymal stromal cell viability.

Mesenchymal stromal cells (MSCs) have shown potential therapeutic benefits for a range of medical disorders and continue to be a focus of intense scientific investigation. Transplantation of MSCs into injured tissue can improve wound healing, tissue regeneration and functional recovery. However, implanted cells rapidly lose their viability or fail to integrate into host tissue. Hydrogel-seeded bone marrow (BM)-MSCs offer improved viability in response to mechanical forces caused by syringe needles, cell density and dimethylsulfoxide (DMSO) concentration, which in turn, will help to clarify which factors are important for enhancing biomaterial-induced cell transplantation efficiency and provide much needed guidance for clinical trials. In this study, under the control of cell density (<2 × 107 cells/mL) and final DMSO concentration (<0.5%), hydrogel-induced BM-MSC viability remained >82% following syringe needle passage by 25- or 27-gauge needles, providing improved cell therapeutic approaches for regenerative medicine.

Reliable and practical methods for cryopreservation of embryogenic cultures and cold storage of somatic embryos of Norway spruce.

Somatic embryogenesis (SE) is considered as the most-effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a cryopreservation method able to handle large numbers of embryogenic tissues (ETs) reliably and at low costs is needed. The aim of the present study was to compare pretreatments, cryoprotectants and slow-cooling devices for cryopreservation of Norway spruce ETs, with 12 variations of methods and a total of 136 spruce genotypes. Secondly, possible applications for cold storage of mature somatic embryos were studied with the aim of developing a flexible time window for embling production. At best, 100% of the embryogenic lines were recovered following cryopreservation, but the results varied among the sets of lines. Also physiological condition of the tissues, pre-treatment and cryoprotectant applied, as well as the slow-cooling device used were found to affect the recovery. The best option for cryopreservation of Norway spruce is to select fresh growth from young ETs as samples, pretreat them on semi-solid medium with increasing sucrose concentration (0.1 M for 24 h; 0.2 M for another 24 h), apply a mixture of polyethylene glycol 6000, glucose, and dimethylsulfoxide, 10% w/v each, as cryoprotectant and use a programmable freezer with a slow cooling rate (0.17 °C/min). On average, 87% of the genotypes can be recovered, without any effect on their genetic fidelity, as shown by microsatellite markers and embryo production capacity. Mature somatic embryos of Norway spruce can also be safely cold-stored at +4 °C, without adverse effects on their germination ability.

Determination of residual dimethylsulfoxide in cryopreserved cardiovascular allografts.

Dimethylsulfoxide (DMSO) is a solvent which protects the structure of allografts during the cryopreservation and thawing process. However, several toxic effects of DMSO in patients after transplantation of cryopreserved allografts have been described. The aim of this study is to determine the residual DMSO in the cardiovascular allografts after thawing and preparation of cryopreserved allografts for clinical application following guidelines of the European Pharmacopoeia for DMSO detection. Four types of EHB allografts (aortic valve-AV, pulmonary valve-PV, descending thoracic aorta-DA, and femoral artery-FA) are cryopreserved using as cryoprotecting solution a 10% of DMSO in medium 199. Sampling is carried out after thawing, after DMSO dilution and after delay of 30 min from final dilution (estimated delay until allograft implantation). After progressive thawing in sterile water bath at 37-42 °C (duration of about 20 min), DMSO dilution is carried out by adding consecutively 33, 66 and 200 mL of saline. Finally, tissues are transferred into 200 mL of a new physiologic solution. Allograft samples are analysed for determination of the residual DSMO concentration using a validated Gas Chromatography analysis. Femoral arteries showed the most important DMSO reduction after the estimated delay: 92.97% of decrease in the cryoprotectant final amount while a final reduction of 72.30, 72.04 and 76.29% in DMSO content for AV, PV and DA, was found, respectively. The residual DMSO in the allografts at the moment of implantation represents a final dose of 1.95, 1.06, 1.74 and 0.26 mg kg-1 in AV, PV, DA and FA, respectively, for men, and 2.43, 1.33, 2.17 and 0.33 mg kg-1 for same tissues for women (average weight of 75 kg in men, and 60 kg in women). These results are seriously below the maximum recommended dose of 1 g DMSO kg-1 (Regan et al. in Transfusion 50:2670-2675, 2010) of weight of the patient guaranteeing the safety and quality of allografts.

Functional amplification and preservation of human gut microbiota.

Background: The availability of fresh stool samples is a prerequisite in most gut microbiota functional studies. Objective: Strategies for amplification and long-term gut microbiota preservation from fecal samples would favor sample sharing, help comparisons and reproducibility over time and between laboratories, and improve the safety and ethical issues surrounding fecal microbiota transplantations. Design: Taking advantage of in vitro gut-simulating systems, we amplified the microbial repertoire of a fresh fecal sample and assessed the viability and resuscitation of microbes after preservation with some common intracellular and extracellular acting cryoprotective agents (CPAs), alone and in different combinations. Preservation efficiencies were determined after 3 and 6 months and compared with the fresh initial microbiota diversity and metabolic activity, using the chemostat-based Environmental Control System for Intestinal Microbiota (ECSIM) in vitro model of the gut environment. Microbial populations were tested for fermentation gas, short-chain fatty acids, and composition of amplified and resuscitated microbiota, encompassing methanogenic archaea. Results: Amplification of the microbial repertoire from a fresh fecal sample was achieved with high fidelity. Dimethylsulfoxide, alone or mixed with other CPAs, showed the best efficiency for functional preservation, and the duration of preservation had little effect. Conclusions: The amplification and resuscitation of fecal microbiota can be performed using specialized in vitro gut models. Correct amplification of the initial microbes should ease the sharing of clinical samples and improve the safety of fecal microbiota transplantation. Abbreviations: CDI, Clostridium difficile infection; CPA, cryoprotective agent; D, DMSO, dimethylsulfoxide; FMT, fecal microbiota transplantation; G, glycerol; IBD, inflammatory bowel disease; P, PEG-4000, polyethylene glycol 4000 g.mol-1; SCFA, short-chain fatty acid; SNR, signal-to-noise ratio.

Genotype-dependent responsivity to inflammatory pain: A role for TRPV1 in the periaqueductal grey.

Negative affective state has a significant impact on pain, and genetic background is an important moderating influence on this interaction. The Wistar-Kyoto (WKY) inbred rat strain exhibits a stress-hyperresponsive, anxiety/depressive-like phenotype and also displays a hyperalgesic response to noxious stimuli. Transient receptor potential subfamily V member 1 (TRPV1) within the midbrain periaqueductal grey (PAG) plays a key role in regulating both aversive and nociceptive behaviour. In the present study, we investigated the role of TRPV1 in the sub-columns of the PAG in formalin-evoked nociceptive behaviour in WKY versus Sprague-Dawley (SD) rats. TRPV1 mRNA expression was significantly lower in the dorsolateral (DL) PAG and higher in the lateral (L) PAG of WKY rats, compared with SD counterparts. There were no significant differences in TRPV1 mRNA expression in the ventrolateral (VL) PAG between the two strains. TRPV1 mRNA expression significantly decreased in the DLPAG and increased in the VLPAG of SD, but not WKY rats upon intra-plantar formalin administration. Intra-DLPAG administration of either the TRPV1 agonist capsaicin, or the TRPV1 antagonist 5'-Iodoresiniferatoxin (5'-IRTX), significantly increased formalin-evoked nociceptive behaviour in SD rats, but not in WKY rats. The effects of capsaicin were likely due to TRPV1 desensitisation, given their similarity to the effects of 5'-IRTX. Intra-VLPAG administration of capsaicin or 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, and similar effects were seen with 5'-IRTX in WKY rats. Intra-LPAG administration of 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, but not in WKY rats. These results indicate that modulation of inflammatory pain by TRPV1 in the PAG occurs in a sub-column-specific manner. The data also provide evidence for differences in the expression of TRPV1, and differences in the effects of pharmacological modulation of TRPV1 in specific PAG sub-columns, between WKY and SD rats, suggesting that TRPV1 expression and/or functionality in the PAG plays a role in hyper-responsivity to noxious stimuli in a genetic background prone to negative affect.