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Receptor interacting serine/threonine kinase 1 (RIPK1) mediates apoptosis by regulating the classic proapoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Although Bcl-2-related ovarian killer (Bok) is structurally similar to Bak and Bax, it is unclear whether it mediates apoptosis in skeletal muscle ischemia reperfusion (IR) injury. We hypothesized that by regulating Bok-mediated apoptosis, inhibiting RIPK1 with necrostatin-1 would reduce skeletal muscle IR injury.Rats were randomized into four groups: sham (SM), IR, IR treated with necrostatin-1 (NI), or vehicle dimethyl sulfoxide (DI). For the IR group, the right femoral artery was clamped for 4 hours and then reperfused for 4 hours, and for the NI and DI groups, necrostatin-1 (1.65 mg/kg) and the equal volume of dimethyl sulfoxide were intraperitoneally administered prior to IR induction. The structural damage of muscle tissue and protein expression of Bok, Bcl-2, and cleaved caspase-3 were investigated, and apoptotic cells were identified with terminal dUTP nick-end labeling (TUNEL) staining. In vitro, human skeletal muscle cells (HSMCs) were exposed to 6 hours of oxygen-glucose deprivation followed by normoxia for 6 hours to establish an oxygen-glucose deprivation/reoxygenation (OGD/R) model. To determine the role of Bok, cell viability, lactate dehydrogenase (LDH) release, and flow cytometry were examined to demonstrate the effects of necrostatin-1 and Bok knockdown on the OGD/R insult of HSMCs.Necrostatin-1 pretreatment markedly reduced IR-induced muscle damage and RIPK1, Bok, and cleaved caspase-3 expression, whereas upregualted Bcl-2 expression (p0.05). Furthermore, necrostatin-1 prevented mitochondrial damage and decreased TUNEL-positive muscle cells (p0.05). In vitro, HSMCs treated with necrostatin-1 showed reduced Bok expression, increased cell viability, and reduced LDH release in response to OGD/R (p0.05), and Bok knockdown significantly blunted the OGD/R insult in HSMCs.Necrostatin-1 prevents skeletal muscle from IR injury by regulating Bok-mediated apoptosis.

In the present study, we explored the effects of a novel xylanase from camel rumen metagenome (CrXyn) on wheat bran hydrolysis. CrXyn was heterologously expressed in Escherichia coli and showed maximum activity at 40 °C and pH 7.0. Furthermore, CrXyn exhibited preferential hydrolysis of xylan, but no obvious activity toward other substrates, including carboxymethylcellulose and Avicel. Using wheat straw xylan as a substrate, the Km and Vmax values for CrXyn were 5.98 g/L and 179.9 μmol xylose/min/mg protein, respectively. Mn

The increasing prevalence and complications related to liver diseases (caused by infection, toxic agents, or metabolic syndrome), together with insufficient existence of treatments, make evident the need for better therapeutic alternatives. Therefore, the aim of this study was to determine the effect of 4-hydroxychalcone (4-HC) as preventive and curative treatment in acute and chronic liver injury, respectively. Liver damage was induced with carbon tetrachloride (CCl4) in Wistar rats. Rats were divided into two groups: (1) acute liver injury and (2) chronic liver injury. In turn, each group was divided into four subgroups: (i) control (water); (ii) dimethyl sulfoxide 10%; (iii) CCl4; and (iv) 4-HC. The pre-treatment with 4-HC decreased transaminases, IL-6 serum levels, and hepatic malondialdehyde, increased IL-10 serum levels and hepatic glutathione, and decreased liver damage (necrosis, steatosis, and inflammatory infiltrate). In contrast, treatment with 4-HC after the induction of chronic liver injury decreased IL-6 serum levels and liver damage (steatosis, inflammatory infiltrate, ballooning cells, steatofibrosis, and fibrosis degree). Thus, the 4-HC treatment is proposed as a preventive treatment against acute liver injury; moreover, these results suggested the potential of 4-HC as a curative treatment against chronic liver injury, but other scheme treatments must be evaluated in future.

Cartilage transplantation is an effective way to repair, reconstruct, and replace damaged articular cartilage (AC) but its use is limited by the inability to preserve AC for long periods of time. Vitrification is an ideal choice for long-term storage of AC, and multiple cryoprotective agents (CPAs) with high concentration are usually used. To obtain high cell viability, chondrocytes at all locations inside AC should be protected properly by the CPAs during cooling and rewarming. Hence, it is important to know the mass transport properties of multiple CPAs as they synergistically infiltrate AC. In this study, a mathematical model to describe the mass transport behavior of multiple CPAs in AC was developed based on the mixture-averaged diffusion model. In addition, a methodology for the simultaneous determination of dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol by carbon-13 nuclear magnetic resonance was established. The model is applicable for predicting single- and multiple-CPA permeation into AC, and its accuracy was verified by a massive experimental dataset. Simulation results showed reverse diffusion in the multiple-CPA permeation process, which was not found in the single-CPA permeation process. This curious phenomenon shows the sharp contrast between the diffusion behavior of a binary mixture and a multicomponent mixture.

Vitrification is a promising cryopreservation technique for complex specimens such as tissues and organs. However, it is challenging to identify mixtures of cryoprotectants (CPAs) that prevent ice formation without exerting excessive toxicity. In this work, we developed a multi-CPA toxicity model that predicts the toxicity kinetics of mixtures containing five of the most common CPAs used in the field (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide). The model accounts for specific toxicity, non-specific toxicity, and interactions between CPAs. The proposed model shows reasonable agreement with training data for single and binary CPA solutions, as well as ternary CPA solution validation data. Sloppy model analysis was used to examine the model parameters that were most important for predictions, providing clues about mechanisms of toxicity. This analysis revealed that the model terms for non-specific toxicity were particularly important, especially the non-specific toxicity of propylene glycol, as well as model terms for specific toxicity of formamide and interactions between formamide and glycerol. To demonstrate the potential for model-based design of vitrification methods, we paired the multi-CPA toxicity model with a published vitrification/devitrification model to identify vitrifiable CPA mixtures that are predicted to have minimal toxicity. The resulting optimized vitrification solution composition was a mixture of 7.4 molal glycerol, 1.4 molal DMSO, and 2.4 molal formamide. This demonstrates the potential for mathematical optimization of vitrification solution composition and sets the stage for future studies to optimize the complete vitrification process, including CPA mixture composition and CPA addition and removal methods.

In the recent years, the use of constant volume (isochoric) cryopreservation, in medicine and biotechnology has captured more attention from the research community and now there is an increasing interest in the use of this new technology. It has been established that the thermodynamics of isochoric freezing is different from that of isobaric (constant pressure) freezing. This study provides researchers in the field experimental results for various compositions of cryoprotectants commonly used in isobaric cryopreservation, in terms of temperature-pressure-molar concentration correlation. It also reveals experimental isochoric thermodynamic data for the following cryoprotectants, commonly used in isobaric cryopreservation: dimethyl sulfoxide, trehalose, ethylene glycol and diethylene glycol. Currently, the data on the pressure-temperature correlation in an isochoric system of cryoprotectants used in isobaric cryopreservation is not available. Our new experimental results indicate that the studied concentrations for each of the CPAs, lower and expands the range of temperatures in which cryopreservation by isochoric freezing can be safely practiced. We consider that these experiments will aid researchers developing new isochoric cryopreservation protocols.

Yueming Guo,1 Pengpeng Li,2,3 Zongliang Wang,4 Peibiao Zhang,4 Xiaodong Wu2 1Department of Orthopaedics, Foshan Hospital of Traditional Chinese Medicine, Foshan, 528000, People’s Republic of China; 2Xuzhou Central Hospital, Xuzhou, 221009, People’s Republic of China; 3Graduate School of Bengbu Medical College, Bengbu, 233030, People’s Republic of China; 4Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, People’s Republic of ChinaCorrespondence: Xiaodong Wu; Peibiao Zhang, Email xiaodongwu2009@163.com; zhangpb@ciac.ac.cnIntroduction: As a popular dietary supplement containing sulfur compound, methylsulfonylmethane (MSM) has been widely used as an alternative oral medicine to relieve joint pain, reduce inflammation and promote collagen protein synthesis. However, it is rarely used in developing bioactive scaffolds in bone tissue engineering.Methods: Three-dimensional (3D) hydroxyapatite/poly (lactide-co-glycolide) (HA/PLGA) porous scaffolds with different doping levels of MSM were prepared using the phase separation method. MSM loading efficiency, in vitro drug release as well as the biological activity of MSM-loaded scaffolds were investigated by incubating mouse pre-osteoblasts (MC3T3-E1) in the uniform and interconnected porous scaffolds.Results: Sustained release of MSM from the scaffolds was observed, and the total MSM release from 1% and 10% MSM/HA/PLGA scaffolds within 16 days was up to 64.9% and 68.2%, respectively. Cell viability, proliferation, and alkaline phosphatase (ALP) activity were significantly promoted by incorporating 0.1% of MSM in the scaffolds. In vivo bone formation ability was significantly enhanced for 1% MSM/HA/PLGA scaffolds indicated by the repair of rabbit radius defects which might be affected by a stimulated release of MSM by enzyme systems in vivo.Discussion: Finding from this study revealed that the incorporation of MSM would be effective in improving the osteogenesis activity of the HA/PLGA porous scaffolds.Graphical abstract: Keywords: biopolymer, scaffold, drug delivery, sustained release, osteogenesis

We report transarterial Onyx embolization with flow control using rapid ventricular pacing (RVP) in a middle-aged male patient with tentorial dural arteriovenous fistulas (TDAVFs). The patient completed angiographic obliteration in one session without any complications, and the 6-month postangiographic obliteration follow-up showed no evidence of residual or recurrent dural arteriovenous fistulas. RVP may be a novel treatment option of flow control to facilitate the embolic agent penetrating into the venous side and to achieve complete cure in transarterial embolization of TDAVFs.

Aim. To investigate the mechanism of progesterone inhibiting the scorch death of SH-SY5Y cells induced by exogenous adenosine triphosphate (ATP). Methods. SH-SY5Y cells with good logarithmic growth were used in the experiment. The cells were randomly divided into 5 groups: normal control group, DMSO group, BBG group, ATP group, and ATP+progesterone group. The cell survival rate of each group was measured by CCK-8 method. The expressions of P2X7 receptor, caspase-1, caspase-11, and IL-1β were detected by western blotting. Results. (1) After SH-SY5Y cells were treated with ATP at different concentrations (1, 3, 6, and 9 mmol/L) for 2 hours, the cell survival rate decreased in a concentration-dependent manner compared with the normal blank group. The results showed that the optimal lethal concentration of ATP was 6 mmol/L. SH-SY5Y cells were preincubated with progesterone at different concentrations (3, 10, 30, and 100 nmol/L) for 30 minutes and then incubated with 6 mmol/L ATP. The cell survival rate of this group was significantly improved ( P < 0.01 ). The optimal concentration of progesterone to improve cell survival and inhibit cell death was 30 nmol/L. (2) Compared to the control group, there was no significant difference ( P > 0.05 ) in P2X7 receptor, caspase-1, caspase-11, and IL-1β with the DMSO group (0.001% DMSO, 24 h) and BBG group (bbg1 mmol/L, 24 h). (3) In the ATP group, the expression of P2X7 receptor and caspase-1 (the key protein of classical cell death pathway) increased significantly ( P < 0.01 ), which was related to inflammatory factor IL-1β with consistent performance ( P < 0.01 ). There was no significant change in caspase-11 (the key protein of nonclassical focal death pathway) ( P > 0.05 ). (4) The expression of P2X7 receptor, caspase-1, and inflammatory factor IL-1β in the progesterone+ATP group was significantly downregulated ( P < 0.01 ). There was no significant change in caspase-11 ( P > 0.05 ). Conclusion. Certain dose of progesterone can inhibit the focal death of SH-SY5Y cells induced by extracellular high concentration ATP. It can reduce the expression of P2X7 receptor, inhibit the conduction pathway of cell death, reduce the release of inflammatory factor IL-1β, and improve cell survival.

Cryoprotectants are crucial factors in cell cryopreservation. Trehalose (Tre), a nontoxic, nonreducing, and natural disaccharide, has the potential to protect cells as a cryoprotectant. As an inducer of autophagy, Tre can influence the development of many diseases and may also have an effect on cell cryopreservation through this mechanism. In this study, human aortic endothelial cells were preserved in different cryopreservation fluids with or without dimethyl sulfoxide and Tre was added. Subsequently, the expression of the main autophagy-related genes LC3, BECN, and P62, cell death and apoptosis, and the proliferation rate were measured in different groups after cryopreservation. Our data showed that Tre can improve the expression of the autophagy-related genes LC3 and BECN and reduce the expression of P62. Dead/alive staining and flow cytometry showed that cell death and cell apoptosis were reduced during cryopreservation with Tre. In addition, the cell proliferation rate after thawing was increased in the Tre group when compared with others. These results all indicated that there might be a connection between Tre-triggered autophagy and the protective role of Tre in cell cryopreservation. Furthermore, strategies to regulate autophagy to reduce apoptosis in this process should be investigated in future research.

Porcine skin-derived stem cells (pSDSCs) are a type of adult stem cells (ASCs) that retain the ability to self-renew and differentiate. Currently, pSDSCs research has entered an intense period of development; however there has been no research regarding methods of cryopreservation. In this paper, we explored an efficient cryopreservation method for pSDSCs. Our results demonstrated that cryopreserving 50 μm diameter pSDSCs aggregates resulted in a lower apoptosis rate and a greater ability to proliferate to form larger spherical cell aggregates than during single-cell cryopreservation. To further optimize the cryopreservation method, we added different concentrations of melatonin (N-acetyl-5-methoxytryptamine, MLT) and trehalose (d-trehalose anhydrous, TRE) to act as cryoprotectants (CPAs) for the pSDSCs. After comparative experiments, we found that the cryopreservation efficiency of 50 mM TRE was superior. Further experiments demonstrated that the reason why 50 mM TRE improved cryopreservation efficiency was that it reduced the intracellular oxidative stress and mitochondrial damage caused by cryopreservation. Taken together, our results suggest that cryopreserving 50 μm diameter pSDSCs aggregates in F12 medium with 10% dimethyl sulfoxide (DMSO) and 50 mM TRE promotes the long-term storage of pSDSCs.

Benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon compound, is a carcinogen that causes head and neck cancers. Despite intensive research, the molecular mechanism of BaP in the development of oral squamous cell carcinoma (OSCC) remains largely unknown. In the present study, the SCC-9 human OSCC cell line was cultured in vitro, separated into treatment groups, and treated with dimethyl sulfoxide or BaP at various concentrations. The malignant behavior ascribed to the BaP treatment was investigated by cell proliferation, clony formation assay, and Transwell assays. Furthermore, transcriptome sequencing was performed to detect the differentially expressed genes, followed by quantitative real-time PCR to measure the expression levels of nine of these genes. Moreover, the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed the biological processes and signaling pathways in which the target genes were involved. Significant effects on SCC-9 cell proliferation, tumorigenicity, cell migration, and invasion were observed after exposure to 8 μM BaP. Additional results revealed that BaP inhibited apoptosis in a dose-dependent manner. The transcriptome sequencing results showed 137 upregulated genes and 135 downregulated genes induced by BaP, associated with tumor-related biological processes and signaling pathways, mainly including transcriptional dysregulation in cancer, the tumor necrosis factor signaling pathway, metabolism of xenobiotics by cytochrome P450, mitogen-activated protein kinase signaling pathway, and so forth. Our study demonstrates that BaP may regulate the expression of certain genes involved in tumor-associated signaling pathways, thereby promoting the proliferative, tumorigenic, and metastatic behaviors of OSCC cells while suppressing their apoptosis.

An in-situ compatibilized starch (St) and polyacrylonitrile (PAN) composite spinning solution was designed by preparing starch-graft-polyacrylonitrile (St-g-PAN) through graft copolymerizing acrylonitrile from soluble starch and using ammonium cerium nitrate (CAN) as initiator. As dimethyl sulfoxide (DMSO) was used as the solvent, St/St-g-PAN/PAN/DMSO spinning solution was prepared and St/St-g-PAN/PAN composite fibers were obtained by dry-wet spinning technique. The effects of air gap, coagulation bath, hot drawing and stretching, and thermal-setting process were studied in detail. Fourier transform infrared spectroscopy (FT-IR), solid state nuclear magnetic resonance (

What is the central question of this study? Do transmembrane member 16A (TMEM16A) blockers modulate the activation of heat loss responses of sweating and cutaneous vasodilatation? What are the main finding and its importance? Relative to the vehicle control site, TMEM16A blockers T16Ainh-A01 and benzbromarone had no effect on sweat rate or cutaneous vascular conductance during whole-body heating inducing a 1.1 ± 0.1°C increase in core temperature above baseline resting levels. These results suggest that TMEM16A blockers T16Ainh-A01 and benzbromarone do not modulate the regulation of sweating and cutaneous vasodilatation during whole-body heat stress.Animal and in vitro studies suggest that transmembrane member 16A (TMEM16A), a Ca

The testis is susceptible to ionizing radiation, and male infertility and sexual dysfunction are prevalent problems after whole-body or local radiation exposure. Currently, there is no approved agent for the prevention or treatment of radiation-induced testicular injury. Herein, we investigated the radioprotective effect of dimethyl sulfoxide (DMSO), an organosulfur compound that acts as a free radical scavenger, on testicular injury. Treatment of mice with a single dose of DMSO prior to 5 Gy irradiation restored sex hormones and attenuated the reduction in testis weight. Histological analyses revealed that DMSO alleviated the distorted architecture of seminiferous tubules and promoted seminiferous epithelium regeneration following irradiation. Moreover, DMSO provided quantitative and qualitative protection for sperm and preserved spermatogenesis and fertility in male mice. Mechanistically, DMSO treatment enhanced GFRα-1+ spermatogonial stem cell and c-Kit+ spermatogonial survival and regeneration after radiation. DMSO also alleviated radiation-induced oxidative stress and suppressed radiation-induced germ cell apoptosis in vivo and in vitro. Additionally, DMSO efficiently reduced DNA damage accumulation and induced the expression of phosph-BRCA1, BRCA1, and RAD51 proteins, indicating that DMSO facilitates DNA damage repair with a bias toward homologous recombination. In summary, our findings demonstrate the radioprotective efficacy of DMSO on the male reproductive system, which warrants further studies for future application in the preservation of male fertility during conventional radiotherapy and nuclear accidents.

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane - EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

Changes in mitochondrial dynamics have been recognized as being one of the mechanisms related to cardiotoxicity following a high cumulative dose of doxorubicin (DOX). A mitochondrial division inhibitor-1 (Mdivi-1) and fusion promoter (M1) have been shown to be cardioprotective in a variety of cardiovascular settings, however, their anticardiotoxic efficacy against DOX therapy remains unclear. We therefore investigated whether treatment with Mdivi-1 and M1 protects the heart against DOX-induced cardiotoxicity via mitochondria-targeted pathways. Male Wistar rats (n=40) received DOX (3 mg/kg, six doses, n=32) or 3% dimethylsulfoxide (DMSO) in the normal saline solution (NSS) (n=8) as a control. DOX-injected rats were given one of four treatments beginning with the first DOX injection via intraperitoneal injection: 1) 3% DMSO in NSS (n=8), 2) Mdivi-1 (1.2 mg/kg per day, n=8), 3) M1 (2 mg/kg per day, n=8), and 4) Mdivi-1+M1 (n=8) for 30 days. Cardiac function, mitochondrial function, oxidative stress, myocardial injury, and protein expression associated with inflammation, autophagy, mitophagy, apoptosis, and mitochondrial dynamics were determined. DOX caused a significant deterioration in mitochondrial function and dynamic regulation, and an increase in markers of oxidative stress, inflammation, myocardial injury, apoptosis, autophagy, and mitophagy, resulting in impaired cardiac function. Cotreatment of DOX with Mdivi-1, M1, or a combination of the two mitigated these detrimental effects of DOX. These findings imply that either inhibiting fission or promoting fusion of mitochondria protects the heart from DOX-induced myocardial damage. Modulation of mitochondrial dynamics could be a novel therapeutic target in alleviating DOX-induced cytotoxic effects without compromising its anticancer efficacy.

The effects of astaxanthin (AST) were evaluated on oxidative mediators, neuronal apoptosis, and autophagy in functional motor recovery after spinal cord injury (SCI). Rats were divided into three groups of sham, SCI + DMSO (dimethyl sulfoxide), and SCI + AST. Rats in the sham group only underwent a laminectomy at thoracic 8-9. While, the SCI + DMSO and SCI + AST groups had a compression SCI with an aneurysm clip. Then, this groups received an intrathecal (i.t.) injection of 5% DMSO and AST (10 μl of 0.005 mg/kg), respectively. The rat motor functions were assessed weekly until the 28th day using a combined behavioral score (CBS). Total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were measured in spinal tissue to evaluate oxidative stress-related parameters. Besides, autophagy-related proteins (P62, LC3B, and Beclin1) and apoptosis-associated proteins (Bax and Bcl2) were determined using western blotting on the 1st and 7th days after surgery. Hematoxylin-eosin and Fluoro-Jade B staining were performed to detect the histological alterations and neuronal degeneration. As the result, treatment with AST potentially attenuated rat CBS scores (p 0.001) towards a better motor performance. AST significantly reduced the spinal level of oxidative stress by increasing TAC, SOD, and GPx, while decreasing MDA (p 0.001). Furthermore, AST treatment remarkably upregulated expression of LC3B (p 0.001), and Beclin1 (p 0.05) in the spinal cord, but downregulated P62 (p 0.05) and the Bax/Bcl2 ratio (p 0.001). Consequently, AST reduced SCI-induced histological alterations and neuronal degeneration (p 0.001). In conclusion, AST can improve motor function after SCI by reducing oxidative stress/apoptosis and increasing neuronal autophagy.

Domestic pigs have become increasingly popular as a model for human diseases such as neurological diseases. Drug discovery platforms have increasingly been used to identify novel compounds that combat neurodegeneration. Currently, bioactive molecules such as melatonin have been demonstrated to offer a neuroprotective effect in several studies. However, a neurodegenerative platform to study novel compounds in a porcine model has not been fully established. In this study, characterized porcine induced neural stem cells (iNSCs) were used for evaluation of the protective effect of melatonin against chemical and pathogenic stimulation. First, the effects of different concentrations of melatonin on the proliferation of porcine iNSCs were studied. Second, porcine iNSCs were treated with the appropriate concentration of melatonin prior to induced degeneration with dimethyl sulfoxide or Zika virus (ZIKV). The results demonstrated that the percentages of Ki67 expression in porcine iNSCs cultured in 0.1, 1, and 10 nM melatonin were not significantly different from that in the control groups. Melatonin at 1 nM protected porcine iNSCs from DMSO-induced degeneration, as confirmed by a dead cell exclusion assay and mitochondrial membrane potential (ΔΨm) analysis. In addition, pretreatment with melatonin reduced the percentage of dead porcine iNSCs after ZIKV infection. Melatonin increased the ΔΨm, resulting in a decrease in cell degeneration. However, pretreatment with melatonin was unable to suppress ZIKV replication in porcine iNSCs. In conclusion, the present study demonstrated the anti-degenerative effect of melatonin against DMSO- and ZIKV-induced degeneration in porcine iNSCs.

To prospectively evaluate the safety and efficacy of embolization using ethylene vinyl alcohol copolymer (OnyxThis prospective study was approved by the institutional ethics review board. Ten clinical parameters were retained for evaluation of PeVD (pelvic pain, dyspareunia, post-coital pain, menstruation pain, lower limbs pain, difficulty walking, aesthetic discomfort, impact on daily working life, psychological impact and impact on daily life), measured on a visual analogue scale (VAS) between 0 and 10, and a global score out of 100 was noted before embolization, after 3 months during the imaging follow-up, and at the end of follow-up by phone call. The main criterion was clinical efficacy of embolization defined by an impairment score40/100 and a 50% decrease in overall score. Complications were recorded. Visualization of OnyxBetween July 2017 and May 2019, 73 consecutive women (mean age ± SD [range]: 41 ± 11 years [25-77]) treated by embolization with OnyxEmbolization using Onyx• Embolization using Onyx

Algae, which may be unicellular or multicellular, can carry out photosynthesis just like plants as they effectively utilize light energy. They contain various physiologically active substances and are, therefore, widely used commercially to produce healthy food and feed additives, cosmetics, and energy supplements. For useful applications, the cryopreservation technique has been used in various fields. Recently, to develop suitable cryopreservation methods for algal applications, various studies have been performed. However, adequate investigations have not been conducted to understand the mechanism underlying algal cryopreservation at the molecular level. Therefore, this study examined the profile alteration of the proteome using cryopreservation with various cryoprotectants (CPAs). Trichormus variabilis was cultured and then cryopreserved with 10% dimethyl sulfoxide, methanol, and glycerol, after which, proteome profiling was done. Finally, signaling pathway search was performed, and a new signaling pathway was established based on differentially expressed proteins. As a result, the expression levels of 17 proteins were observed. Additionally, it was confirmed that the differentially expressed proteins were related to 16 signaling pathways and that they were capable of interacting with each other. The findings suggest that the differentially expressed proteins may be applied as biomarkers for algal cryopreservation and to understand the mechanism underlying T. variabilis cryopreservation. Moreover, it is anticipated that the results from this study would be useful in selecting suitable CPAs and in upgrading the cryopreservation techniques.

Sperm fertility decreases significantly after freezing. Providing a suitable and useful diluent compound for freezing ram sperm can increase the efficiency of artificial insemination and consequently, the reproductive performance of sheep. Various biological properties such as antibacterial, anti-cancer, immunosuppressive, antioxidant and reproductive properties of royal jelly (RJ) are well known. The aim of this study was to investigate the possible synergistic effect of royal jelly in combination with glycerol and dimethyl sulfoxide (DMSO) in sperm cryopreservation extender of Romanov ram. The pooled semen samples from 5 Romanov rams were allocated into 3 experiments. The effect of 6% DMSO, 6% glycerol and a combination of 3% DMSO +3% glycerol co-supplemented with 1, 2 and 3% RJ was evaluated in 3 experiments. Samples were frozen by conventional slow freezing method and post-thaw parameters of total motility, progressive motility, plasma membrane integrity, DNA damage, apoptosis, enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), total antioxidant capacity (TAC) and malondialdehyde (MDA) were evaluated. The results showed that the percentage of motility, progressive motility, TAC, GPx, SOD and all sperm kinematic parameters except LIN in the group containing 2% RJ + 6% DMSO was higher than the control group (p 0.05). Some parameters such as progressive motility, sperm membrane integrity, TAC, GPx, VAP, VCL, STR and SRT in the group containing 2% RJ + 6% DMSO were more than (p 0.05) in the sperm group containing 1% RJ + 6% DMSO. MDA values in sperm groups containing 2% RJ + 6% DMSO were significantly (p 0.05) lower than the sperm containing 1% royal jelly and the control group. In the sperm group containing 2% RJ + 6% glycerol, sperm membrane integrity, TAC, GPx, SOD, progressive motility and all sperm kinematic parameters except VAP were higher and MDA values and sperm abnormalities were lower than the control group (p 0.05). The sperm group containing 1% RJ and 3% DMSO +3% glycerol had higher motility, progressive motility, membrane integrity, and all sperm kinematic parameters except VSL; and lower sperm abnormalities, DNA damage, apoptosis and MDA than the control group (p 0.05). As a general conclusion of this study, the addition of 2% RJ + 3% DMSO and 3% glycerol to the freezing extender improved microscopic and biochemical ram sperm parameters after the freeze-thaw process. Hence, moderate concentrations of royal jelly (2%) are sufficient to protect sperm from freezing damage, and high (3%) and low (1%) concentrations do not have a good cryoprotective effect.

Reproductive diseases in mares are a significant cause of subfertility and profound economic loss in the equine industry. Utilizing a 3D in vitro cell culture system that recapitulates the in vivo physiology will reduce time, cost, and welfare concerns associated with in vivo reproductive research in mares. If this 3D model is combined with effective cryopreservation, reproductive research on mares can occur year-round, which is not currently possible in this seasonal species. Endometrial organoids, 3D in vitro cell clusters that exhibit in vivo uterine physiology, have been established in mice, women, and mares. Here we report the first comprehensive assessment of cryopreservation of endometrial organoids in the domestic mare. Organoid growth rate was not affected by the type of freezing media. However, growth rate varied among non-cryopreserved controls, organoids cryopreserved at passage 0 (P0), and organoids cryopreserved at passage 3 (P3). Additionally, there was no difference in organoid viability among freezing media or freezing timepoint (passages). Furthermore, fresh and frozen-thawed organoids displayed positive immunohistochemical staining for ZO-1, which is a marker for intercellular tight junctions, and for periodic acid-Schiff staining as marker for organoid function through mucin production. Results demonstrate that equine endometrial organoids can be cryopreserved with 10% dimethyl sulfoxide with minimal detrimental effects while maintaining intercellular tight junctions (ZO-1) and secretory function. Availability of cryopreserved endometrial organoids may permit expanded research on uterine pathologies that negatively affect mare fertility and improve efficiency, reduce cost, and minimize animal welfare concerns associated with in vivo research in the domestic mare.

To find the gold standard out of three pre-established routine slow freezing (SF) methods, ovarian cortex tissues of nine transsexual individuals were cryopreserved and compared to each other, as well as the control (fresh) samples. Histological, genomic, and endocrinological effects of the SFs were assessed post-thawing and after a seven-day culture period. SF1 included 10% dimethyl-sulfoxide (Me2SO) in the base medium (BM), SF2 had 1.5 M/L ethylene-glycol (EG) and 0.1 M/L sucrose in the BM, and SF3 consisted of 6% Me2SO, 6% EG and 0.15 M/L sucrose in the BM. The cortical tissue strips went under a programmed cooling process and were stored in liquid nitrogen. Histological criteria (tissue damage and follicular quality), as well as gene expression levels, were assessed in the thawed and control tissues. Half of the thawed and control tissues were cultured for seven days and their histology, genetic profile, and hormonal status were examined as the reflection of the avascular tension effect. Post-thawing tissue damage was similar between all groups but significantly increased post-culture (P 0.05). The percentages of high-quality follicles diminished in all SFs after thawing and culture (P 0.05) except for the similarity of post-thawing SF3, compared to control. The genetic profile of the tissue after thawing and culture suggested quiescence/activation balance in SF1 and 2 and significant down-regulation in SF3, compared to the control specimens (P 0.05). Post-thawing BAX:BCL2 was higher than control in SF1 and SF3 (P 0.05), while this ratio in SF2 was similar to the control. However, after culture this ratio was similar to that of control in SF3 and diminished in SF1 and 2 (P 0.05). The expression levels of gap-junction genes showed dramatic pre- and post-thawing fluctuations in all groups. After culture, estradiol in SF3 was significantly higher than SF1 and 2 (P 0.05). In addition, progesterone in SF3 was similar to control but significantly lower in SF1 and 2 (P 0.05). In conclusion, all SFs showed advantages and disadvantages, and the follicular quality and its function depend on the type of cryoprotectant and the speed of thawing. The effects of freezing/thawing continue to appear during the seven days of culture. According to the results of this study, SF3 seems to be more promising in keeping the follicles functional and safe from cell damage during culture.

OBJECTIVE The natural course of dural arteriovenous fistulas (DAVFs) is unfavorable. Transarterial embolization with Onyx is currently the therapeutic method of choice, although the long-term stability of Onyx has been questioned. The literature reports a significant difference in the recurrence rate after complete DAVF occlusion and lacks larger series with long-term follow-up. The authors present the largest series to date with a long-term follow-up to determine the stability of Onyx, prospectively comparing magnetic resonance angiography (MRA) and digital subtraction angiography (DSA) as follow-up diagnostic methods. METHODS Demographics, clinical symptomatology, length of follow-up, diagnostic methods, and angiographic findings of DAVFs were recorded and retrospectively evaluated in 112 patients. A prospective group of 15 patients with more than 5 years of follow-up after complete DAVF occlusion was established. All 15 patients in the prospective group underwent a clinical examination and MRA; 10 of these patients also underwent DSA. The recurrences and the correlation between the two diagnostic methods were evaluated. RESULTS Among the 112 patients, 71 were men and 41 were women, with an average age of 60 years. Intracranial hemorrhage (40%) was the most common clinical presentation of DAVF. At the last follow-up, 73% of the patients experienced clinical improvement, 21% remained unchanged, and 6% worsened. Overall, 87.5% of the DAVFs were occluded entirely with endovascular treatment, and 93% of the DAVFs were classified as cured at the last follow-up (i.e., completely embolized DAVFs and DAVFs that thrombosed spontaneously or after Gamma Knife surgery). Two recurrences of DAVFs were recorded in the entire series. Both were first diagnosed by MRA and confirmed with DSA. The mean follow-up was 27.7 months. In the prospective group, a small asymptomatic recurrence was diagnosed. The mean follow-up of the prospective group was 96 months. CONCLUSIONS Onyx is a stable embolic material, although recurrence of seemingly completely occluded DAVFs may develop because of postembolization hemodynamic changes that accentuate primarily graphically absent residual fistula. These residuals can be diagnosed with MRA at follow-up. The authors’ data suggest that MRA could be sufficient as the follow-up diagnostic method after complete DAVF occlusion with Onyx. However, larger prospective studies on this topic are needed.

In this paper we report a hemicyanine dye that is used to distinguish cancer cells from normal cells with its ability to target different organelles. Probe 1, a red emission hemicyanine functional dye, was connected to oxazolo[4,5

The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency. We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro. After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo.

This study was designed to investigate the roles of retinoic acid (RA) in transdifferentiation of primary fetal alveolar epithelial type II cells (AECIIs) into alveolar epithelial type I cells (AECIs). Primary fetal AECIIs isolated from rats at a gestational of 19 days were divided into: (i) DMSO group treated using 0.1% DMSO; (ii) RA group, treated with 1 μM RA; and (iii) RA+BMS493 group treated with 1 μM RA and 10

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.

Cryopreservation has facilitated considerable advances in both medical technology and scientific research. However, further developments have been limited by the relatively low number of effective cryoprotective agents. Even after fifty years of research, most protocols rely on the same two toxic agents, i.e. dimethylsulfoxide or glycerol. Ionic liquids are a class of promising solvents which are known glass formers and may offer a less-toxic alternative. The research presented here investigates ten protic ionic liquids as potential cryoprotective agents. The liquids are screened for key properties including cellular toxicity, permeability and thermal behaviour. The most promising, ethylammonium acetate, was then tested as a cryoprotective agent on a model cell line and was found to be as effective as the common cryoprotectant, dimethylsulfoxide. This work reports the first use of a protic ionic liquid as an effective cryoprotective agent for a mammalian cell line. This will inform the development of a suite of potential new ionic liquid-based cryoprotectants that could potentially allow the cryopreservation of new cell types.

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.

Ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, has emerged as a therapeutic target for cancer and Alzheimer's disease (AD). To inhibit ODC, α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, has been widely used. However, due to its poor pharmacokinetics, the need for discovery of better ODC inhibitors is inevitable. For high-throughput screening (HTS) of ODC inhibitors, an ODC enzyme assay using supramolecular tandem assay has been introduced. Nevertheless, there has been no study utilising the ODC tandem assay for HTS, possibly due to its intolerability to dimethyl sulfoxide (DMSO), a common amphipathic solvent used for drug libraries. Here we report a DMSO-tolerant ODC tandem assay in which DMSO-dependent fluorescence quenching becomes negligible by separating enzyme reaction and putrescine detection. Furthermore, we optimised human cell-line-based mass production of ODC for HTS. Our newly developed assay can be a crucial first step in discovering more effective ODC modulators than DFMO.

Antifreeze proteins are biologically active substances which protect living organisms against freezing injuries. The effect of a synthetic antifreeze protein carboxylated poly l -lysine (CPLL) in the extender was evaluated in the presence of a conventional cryoprotective agent, dimethyl sulfoxide (Me2SO), for freezing rabbit sperm cells. The experiment was conducted according to 2 × 3 factorial design including two Me2SO (5 or 8%) and three CPLL (0, 0.5 or 1%) concentrations. CPLL supplementation improved post-thaw live and live-acrosome intact sperm rates (P 0.05) and live-membrane intact (P>0.05) sperm rates. The most striking effect of CPLL supplementation was seen on the DNA integrity where it reduced DNA fragmentation of sperm cells significantly by interacting Me2SO (P

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Under stress conditions, luteinizing hormone (LH)-mediated ovulation is inhibited, resulting in insufficient oocyte production and excretion during follicular development. When the body is stressed, a large amount of corticosterone (CORT) is generated, which will lead to a disorder of the body’s endocrine system and damage to the body. Our previous work showed that CORT can block follicular development in mice. Since LH acts through binding with the luteinizing hormone receptor (Lhcgr), the present study aimed to investigate whether and how corticosterone (CORT) influences Lhcgr expression in mouse ovarian granulosa cells (GCs). For this purpose, three-week-old ICR female mice were injected intraperitoneally with pregnant mare serum gonadotropin (PMSG). In addition, the treatment group was injected with CORT (1 mg/mouse) at intervals of 8 h and the control group was injected with the same volume of methyl sulfoxide (DMSO). GCs were collected at 24 h, 48 h, and 55 h after PMSG injection. For in vitro experiments, the mouse GCs obtained from healthy follicles were treated with CORT alone, or together with inhibitors against the glucocorticoid receptor (Nr3c1). The results showed that the CORT caused a downregulation of Lhcgr expression in GCs, which was accompanied by impaired cell viability. Moreover, the effect of the CORT was mediated by binding to its receptor (Nr3c1) in GCs. Further investigation revealed that Nr3c1 might regulate the transcription of Lhcgr through inhibiting the expression of Lhcgr transcription factors, including AP1 and Creb. Taken together, our findings suggested a possible mechanism of CORT-induced anovulation involving the inhibition of Lhcgr expression in GCs by the CORT–Nr3c1–AP1/Creb axis.

Here we delineate simple and tunable hydrophobically driven chiral functional assemblies of diacetylene cored pseudopeptides. These amino acid appended, rigid core dialkynes constitute promising chiral supramolecular building blocks for materials properties engineering. The chiral appended amino acid elements allow for simple tuning of solubility and interaction properties as well as governing chirality, while the central dialkyne core can impart hydrophobically driven assembly and Aggregation Induced Emission (AIE) properties. The self-assembly of these rod-like dialkynes can be regulated by tuning the solvent environment, with for example self-assembly into vesicles in acetonitrile and into helical organization with AIE in a H 2 O/DMSO mixture. Of additional high interest, these supramolecular materials, themselves devoid of liquid crystal (LC) properties, can induce chirality into non-chiral LC matrices with high helical twisting power.

Daphnoretin extracted from the stem and roots of Wikstroemia indica (L.) C.A. Mey has been shown to possess antiviral and antitumor activities. Herein, we hypothesized that daphnoretin might induce megakaryocytic differentiation, thereby inhibiting the proliferation of cells and serving as a differentiation therapy agent for chronic myeloid leukemia (CML). Daphnoretin-treated K562 and HEL cells were examined for growth inhibition, cell morphology, and megakaryocyte-specific markers. Potential mechanisms of megakaryocytic differentiation of daphnoretin-treated K562 cells were evaluated. The results showed that daphnoretin inhibited the growth of K562 and HEL cells in a dose- and time-dependent manner. Flow cytometry analyses revealed that daphnoretin treatment slightly increased the proportion of sub-G1 and polyploid cells compared to that of dimethyl sulfoxide (DMSO)-treated control cells. Morphological examination showed that daphnoretin-treated K562 and HEL cells exhibited enlarged contours and multinucleation as megakaryocytic characteristics compared to DMSO-treated control cells. Daphnoretin treatment also dramatically enhanced the expression of megakaryocytic markers CD61 and CD41. Under optimal megakaryocytic differentiation conditions, daphnoretin increased the phosphorylation of STAT3 but not STAT5. In summary, daphnoretin inhibited cell growth and induced megakaryocytic differentiation in K562 and HEL cells. The efficacy of daphnoretin in vivo and in patients with CML may need further investigations for validation.

The electrode fabrication stage is a crucial step in the design of supercapacitors. The latter involves the binder generally for adhesive purposes. The binder is electrochemically dormant and has weak interactions, leading to isolating the active material and conductive additive and then compromising the electrochemical performance. Designing binder-free electrodes is a practical way to improve the electrochemical performance of supercapacitors. However, most of the methods developed for the fabrication of binder-free LDH electrodes do not accommodate LDH materials prepared via the co-precipitation or ions exchange routes. Herein, we developed a novel method to fabricate binder-free LDH electrodes which accommodates LDH materials from other synthesis routes. The induced impacts of various physical parameters such as the temperature and time applied during the fabrication process on the crystalline domain and electrochemical performances of all the binder-free LDH electrodes were studied. The electrochemical analysis showed that the electrode prepared at 200 °C-1 h exhibited the best electrochemical performance compared to its counterparts. A specific capacitance of 3050.95 Fg−1 at 10 mVs−1 was achieved by it, while its Rct value was 0.68 Ω. Moreover, it retained 97% of capacitance after 5000 cycles at 120 mVs−1. The XRD and FTIR studies demonstrated that its excellent electrochemical performance was due to its crystalline domain which had held an important amount of water than other electrodes. The as-developed method proved to be reliable and advantageous due to its simplicity and cost-effectiveness.

Highly persistent perfluorooctane sulfonate (PFOS) is an industrial fluorinated organic chemical with significant bioaccumulation and biomagnification properties. The purpose of this study was to determine the toxic effects of sublethal PFOS on the aquatic invertebrate organism, narrow-clawed crayfish [Astacus leptodactylus Eschscholtz, 1823]. The 96 h LC50 value was determined as 48.81 mg/L (34.19-63.68 mg/L) with probit analysis. The sublethal experimental design was formed into four groups solvent control (DMSO, dimethyl sulphoxide), non-treated control group, and 1/10 (5 mg/L) and 1/100 (0.5 mg/L) of 96 h LC50 of PFOS, and crayfish were exposed for 48 h, 7 d, and 21 d under laboratory conditions. Total haemocyte counts (THCs) decreased, while the haemolymph total antioxidant status (TAS) values increased (p < 0.05) after exposure to 0.5 and 5 mg/L PFOS for 48 h, 7 d, and 21 d. Haemolymph total oxidative stress (TOS) levels significantly increased at 5 mg/L PFOS concentration (p < 0.05). Catalase (CAT) activities increased at both concentrations after 48 h and 7 d and then returned to control levels after 21 d; whereas superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities did not change in muscle tissue (p > 0.05). GPX and CAT activities decreased, but SOD activity increased in hepatopancreas tissue (p < 0.05). SOD activity at both concentrations and CAT activity at 5 mg/L PFOS exposure decreased in gill tissue, while GPX activity increased at both concentrations of 48 h and 7 d and returned to control values on day 21 of exposure. Histopathological alterations were detected in hepatopancreas and gill tissues. Lamellar deformations, epithelial hyperplasia, and haemocytic infiltrations were observed in the gill tissues, whereas tubular degeneration, tubule loss, necrosis, and lesions in the hepatopancreas tissues were the major recorded alterations. As a result, the sublethal concentrations of PFOS have toxic effects on crayfish and histologically cause tissue damage. Our findings also support a better understanding of the early toxicological effects of PFOS in freshwater ecosystems. Also, it could be concluded that A. leptodactylus is a reliable model for examining histopathological alterations and differences in enzyme activities together with the haemolymph findings in toxicology studies amid aquatic species.

Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined size (i.e., ~20 μL) that were subsequently cooled by depositing on an aluminum alloy block placed in liquid nitrogen. Mathematical modeling was performed to compute the heat transfer and rate of cooling. The minimum CPA concentration needed for vitrification was determined for various CPAs (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide) and combinations thereof, while effects of droplet size and carrier solution were also identified. Sperm vitrification was eventually done using a glycerol/propylene glycol (1/1) mixture at a final concentration of 45% in buffered saline supplemented with 3% albumin and polyvinylpyrrolidon, while warming was done in standard diluent supplemented with 100 mM sucrose. The sperm concentration was found to greatly affect sperm membrane integrity after vitrification-and-warming, i.e., was found to be 21 ± 12% for 10 × 106 sperm mL-1 and 54 ± 8% for 1 × 106 sperm mL-1. However, an almost complete loss of sperm motility was observed. In conclusion, successful sperm vitrification requires establishing the narrow balance between droplet size, sperm concentration, CPA type and concentration, and exposure time.

A facile and environmentally-friendly strategy for increasing antioxidant activity is a crucial issue for value-added lignin and lignin-carbohydrate complex (LCC) as alternative antioxidants. However, the antioxidant activities of lignin and LCC by the traditional solid-liquid extraction (SLE) methods were restricted by the relatively lower solubility induced from high molecular weight (Mw), and the less functional groups including, phenolic hydroxyl and carboxyl. To improve the antioxidantion of lignin and LCC, lithium chloride/dimethyl sulfoxide (LiCl/DMSO) solvent fractionation (LDSF) was conducted to increase the functional groups and reduce Mw, in which LiCl/DMSO acted triple roles as solvent, acid, and metal chloride catalyst for the depolymerization reaction synchronously. The β-O-4′ linkages were cleaved to release the phenolic hydroxyl, resulting in decreasing Mw; the hydroxyl of the side-chain of lignin was oxidized into carboxyl. Thus, the lignin (LD-RL) and LCC (LD-LCC) samples from LDSF had a higher syringyl (S)/guaiacyl (G) ratio, phenolic hydroxyl, and carboxyl contents, but less Mw than control groups from SLE. Consequently, they presented more excellent scavenging rates toward DPPH and ABTS radicals, up to 90%. This work provided panoramic perspectives and basics of the green and convenient approach to isolate and modify lignin and LCC for great antioxidantion with LDSF.

Germplasm preservation of livestock or endangered animals and expansion of germline stem cells are important. The purpose of this study is to investigate whether supplementation of trehalose to the freezing medium (FM) reduces tissular damage and improves the quality of testicular cells in the cryopreserved bovine testicular tissues. We herein established an optimized protocol for the cryopreservation of bovine testicular tissues, and the isolation as well as culture of bovine germ cells containing spermatogonial stem cells (SSCs) from these tissues. The results showed that FM containing 10% dimethyl sulfoxide (Me2SO/DMSO), 10% knockout serum replacement (KSR) and 20% trehalose (FM5) combined with the uncontrolled slow freezing (USF) procedures has the optimized cryoprotective effect on bovine testicular tissues. The FM5 + USF protocol reduced the cell apoptosis, maintained high cell viability, supported the structural integrity and seminiferous epithelial cohesion similar to that in the fresh tissues. Viable germ cells containing SSCs were effectively isolated from these tissues and they maintained germline marker expressions in the co-testicular cells and co-mouse embryonic fibroblasts (MEF) feeder culture systems respectively, during the short-term culture. Additionally, upregulated transcriptions of spermatogenic differentiation marker C-KIT and meiotic marker SYCP3 were detected in these cells after retinoic acid-induced differentiation. Together, FM5 + USF is suitable for the cryopreservation of bovine testicular tissues, with benefits of reducing the apoptosis, maintaining the cell viability, supporting the testicular structure integrity, and sustaining the survival and differentiation potential of bovine germ cells containing SSCs.

Background: Preparation of the purified and high molecular weight DNA is considered as a first step in tissue molecular analysis. Optimization of the conditions at which samples are received as duration, temperature and preservative agents used is important to avoid affecting the quantity and quality of the extracted DNA. Aim of the work: comparing the efficiency of two DNA preservatives agents [ethanol 95% and salt-saturated dimethyl sulfoxide (SSDMSO)] on the extracted DNA from soft tissues and specifying which of the two studied organs (kidney and heart) gives better DNS yield after preservation at room temperature and at -20oC. Materials and methods: The study was done on 19 autopsy cases. 300 mg of each selected soft tissues (kidney and heart) were preserved as follow: In 2 ml sterile water (Control group), at -20°C (Freezing group), Ethanol 95% (Ethanol group) and in SSDMSO (SSDMSO group). After DNA extraction, DNA quantity was measured using NanoDrop method while DNA quality was measured using agarose gel electrophoresis. Results: All preservatives could retain DNA up to the two months. SSDMSO gave the highest DNA concentration followed by Ethanol 95%. The superiority to heart tissues in SSDMSO preservative over kidney tissues in both Ethanol 95% and SSDMSO preservatives up to two months. Conclusion: It is concluded that SSDMSO is a successful preservative agent, and it is superior to ethanol 95% for preservation of DNA in soft tissues up to two months at room temperature. Heart tissue is less susceptible to degradation and hence more suitable for DNA fingerprinting than kidney tissue. Recommendations: It is recommended to use SSDMSO as preservative for soft tissue collected for DNA analysis and to choose the heart for sampling.

BACKGROUND: Natural products and their derivatives, particularly secondary metabolites, have been recognized for many years as an important source of therapeutic agents. In this context, pentacyclic triterpene acids including betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA) are highly valuable triterpenic acids because of their wide range of biological activities. AIM: Therefore, the aim of our study was to investigate any potential effect of BA, UA, and OA on human melanoma WM-266-4 cells’ proliferation activity. METHODS: BA, UA, and OA have been prepared in dimethyl sulfoxide in concentration range from 0.002 to 200 μM separately or in selected combination (UA+OA ratio 1:1 or 3.5:1), while cells in cell culture medium served as controls. The rapid colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was used to measure proliferation activity of the metastatic melanoma cell line WM-266-4 after being exposed to selected concentrations of BA, UA, OA, or UA+OA and during different time periods. Student’s t-test was used for single statistical comparisons. Data were analyzed using IBM SPSS 25.0 (IBM Corp., Armonk, NY). To account for multiple comparisons bias, p < 0.001 was considered statistically significant. RESULTS: Our results showed decreased cell proliferation activity after 4 h of incubation of WM-266-4 cells with BA, UA, OA, and UA+OA. The highest inhibitory effect was noted when cells were incubated with selected triterpenic acids and both combinations of UA+OA during the incubation period of 48 h. When compared to control cells, concentration of 2 μM was the lowest concentration of BA that showed a significant decrease of the cells’ proliferation activity regardless the incubation period (4 h, 24 h, and 48 h) (p < 0.001). CONCLUSION: Our encouraging results could be a good starting point for further studies on possible use of BA, UA, and OA in prevention and treatment of metastatic melanoma.

Development of genomic preservation technologies for canids, especially for seasonally breeding species like the grey wolf (Canis lupus), is needed in advance of growing species conservation concerns. Here, we evaluated the efficacy of two cryopreservation protocols – needle immersion vitrification (NIV) and slow freezing (SF) on grey wolf (n = 7) testicular tissue morphology. NIV samples were equilibrated in a 7.5% v/v dimethyl sulfoxide (DMSO or Me2SO) + 7.5% ethylene glycol (EG) solution in minimum essential medium with 20% FBS for 10 min at 4 °C, then exposed to 15% DMSO + 15% EG + 0.5 M sucrose for 10 min at 4 °C before plunging into liquid nitrogen. For slow freezing, we assessed two cryoprotectant (CPA) strategies, DMSO, 15% v/v alone (SF-D) or 7.5% EG + 7.5% DMSO (SF-ED). Following thawing, there were no significant differences in seminiferous tubule area among treatment groups, although all cryopreserved tissues displayed reduced tubule size compared with fresh controls and increased apoptosis, the latter reaching significance for SF-D treated tissues. Slow freezing improved maintenance of testis architecture, with minimal detachment of seminiferous tubule basement membranes post-thaw. Spermatogonia densities were reduced in NIV tissues compared with fresh, with no differences in spermatocyte, spermatid, or Sertoli cell counts, or germ cell marker DDX4+ cell densities among groups. In sum, we conclude that slow freezing better maintained morphology of cryopreserved testicular tissues compared with needle vitrification with 15% each DMSO and EG and 0.5 M sucrose, and that DMSO + EG combination SF supports cell viability. This represents a first step in the development of male gonadal tissue preservation strategies for the grey wolf.

We tested the efficacy of lactated Ringer's solution with 3% trehalose and 5% dextran 40 (LR-3T-5D) as a vehicle solution for cryopreservation using human adipose-derived mesenchymal stromal cells (hADSCs) with dimethyl sulfoxide (Me2SO). We also tested the effect of the Me2SO concentration in the cryopreservation solution, and the effect of washing with lactated Ringer's solution with 3% trehalose (LR-3T) and replacement with LR-3T or LR-3T-5D. LR-3T-5D was more effective for cell viability, viable cell recovery ratio, annexin V-positive ratio, and colony-forming capacity as a vehicle solution for cryopreservation with 10% Me2SO than LR. The additive effects as cryoprotectants of trehalose and dextran 40 were confirmed to be dose dependent. The cell viability, cell proliferation ability, cell differentiation ability, and the ratio of cell surface positive/negative markers of hADSCs were well maintained after cryopreservation with LR-3T-5D containing 10% Me2SO in liquid nitrogen or in a −80 °C freezer. The cell viability and the proliferation curve in LR-3T-5D with 5% Me2SO were comparable to those with 10% Me2SO. LR-3T-5D was superior to LR-3T as a replacement solution in terms of viability and annexin V positivity. Our data showed that LR-3T-5D is effective as a vehicle solution for cryopreservation. Reducing Me2SO concentration to 5%, and washing and replacement with fresh LR-3T and LR-3T-5D after thawing, are feasible approaches to maintain cryopreservation efficacy.

The aim of this study was to establish a method for the cryopreservation of spermatogonia of the yellowtail (Seriola quinqueradiata), which is the most commonly farmed fish in Japan. Testicular cells were prepared by enzymatic dissociation of testicular fragments containing an abundance of type A spermatogonia and were added to cryomedium containing dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, or propylene glycol at concentrations of 0.5–2.5 M. The cells were then frozen and stored in liquid nitrogen for 3 days. After thawing, their survival and transplantability were evaluated. Testicular cells were most successfully cryopreserved in 1.0 M DMSO as indicated by survival of 34% of cells. Furthermore, in situ hybridization using the yellowtail vasa probe showed that these recovered cells contained a similar proportion of germ cells to fresh testicular cells before freezing. Transplantation of the recovered cells into the peritoneal cavities of allogeneic larvae resulted in 94% of surviving recipients having donor-derived germ cells in their gonads after 28 days. Sperm were then collected from seven randomly selected recipients once they reached 2 years of age and used to fertilize wild-type eggs, which led to an average of 26% of the first filial (F1) offspring being derived from donor fish, as confirmed through the use of microsatellite markers. Thus, we successfully cryopreserved yellowtail spermatogonia and produced functional sperm via intraperitoneal transplantation into allogeneic recipients.

Background: Pneumonia is an acute or chronic inflammatory disorder of the lungs, affecting the mucosal areas of the lung. It can be caused by bacteria, viruses or fungi. In some cases, it may be caused by physical or chemical irritants. Kolaviron, a natural bioflavonoid extract from Garcinia kola seeds, has been shown to possess anti-inflammatory properties in Flu-like conditions which are associated with cough. There has been a paucity of information on the likelihood of the effectiveness of kolaviron against pneumonia infections. Objective: To evaluate the antibacterial and anti-inflammatory effects of kolaviron on albino Wistar rats induced with pneumonia using Klebsiella pneumonia. Materials and Methods: Powdered Garcinia kola seeds were extracted with n-hexane and 100% methanol as solvents by using Soxhlet extractor. A standard method was used to obtain kolaviron from the seed extracts. A total of 24 albino Wistar rats were randomly divided into six groups A to F, each comprising four rats. The rats were allowed to acclimatize for 1 hour in very cold environments using ice packs. A standardized 1.0 x10 -5 mg/ml culture suspension was intranasally inoculated to the rats for 10 days to induce pneumonia-like symptoms. Thereafter, the kolaviron was administered to the rats such that a 500mg/kg kolaviron extract was given once daily to groups A (male rats) and B (female rats). Groups C (male rats) and D (female rats) received 250mg/kg of kolaviron extract once daily, while group E rats were given 0.5 ml of dimethyl sulfoxide (DMSO) once daily, which served as the negative control. The rats in Group F received 2.86 mg/kg of ofloxacin once daily and served as the positive control. All the treatments were done for a period of 5 days. Then 10 days after the treatments, the animals were sacrificed and the lungs were harvested for hydrostatic lung test and histopathological examination. An overnight broth culture of Klebsiella pneumonia was streaked in sterile molten nutrient agar maintained at 37°C for 24hrs. Later, a stock of 500mg/ml of kolaviron was prepared in DMSO. Two-fold dilutions were performed to obtain the following concentrations of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.565% with the stock. The anti-Klebsiella pneumonia activity of the kolaviron extract was determined using agar well diffusion methods and incubation was done at 37 o C for 24 hrs. Student t-test and Oneway Analysis of variance (ANOVA) were used for comparison of mean differences between and among the groups. Results and Discussion: The sensitivity of Klebsiella pneumonia to kolaviron was concentration- dependent. There was an increase in anti-Klebsiella pneumonia activity with a decrease in kolaviron concentration. Kolaviron (KV), at 500mg/kg concentration, was efficacious and showed significant anti-inflammatory effects (P Conclusion: Kolaviron can be used as an agent in the treatment of pneumonia as it possesses anti- inflammatory and anti-Klebsiella pneumonia activities.

Background Betulin, a natural pentacyclic triterpene with the lupane structure that is present in significant amounts in the outer bark of birch, is known for its broad array of biological and pharmacological properties. Betulin has attracted attention as a potential, natural-origin antimicrobial substance. The literature describes it as selectively toxic to neoplastic cells but safe for normal cells. The research aim was to evaluate the basal cytotoxicity of betulin towards fish (BF-2) and murine (NIH/3T3) fibroblasts. We used four colorimetric tests that provide a preliminary evaluation of possible mechanisms of the cytotoxicity of a compound to assess the degree of the toxicity of betulin after 24, 48 and 72 h of incubation with cells: the MTT assay (mitochondrial activity assessment), the NRU assay (lysosomal membrane integrity assessment), the LDH assay (cellular membrane integrity assessment) and the SRB assay (total cellular protein content determination). Results The results revealed an exceptionally high sensitivity of mitochondria to the effect of betulin, with the other endpoints being less sensitive. Although murine fibroblasts were more vulnerable to the toxic effect of betulin than fish fibroblasts, the betulin CC50 values for both cell lines were comparable with analogous IC50 values determined by other researchers in studies involving cancerous cells. Conclusions The results indicate the need to verify the claim about the selective toxicity of betulin towards malignant cells and to conduct safety/toxicity tests before any potential therapeutic use of betulin in veterinary medicine.

Busulfan (BSU) is a chemotherapeutic drug that can cause subfertility or sterility in males. We investigated the effects of adipose tissue-derived mesenchymal stem cells (AT-MSC) conditioned medium (CM) (AT-MSC-CM) on histopathological and molecular characteristics of mouse testes exposed to BSU using stereology. We used adult male mice divided randomly into five groups: control, Dulbecco's modified Eagle's medium (DMEM), dimethyl sulfoxide (DMSO), BSU, and BSU + CM. Thirty-five days following BSU injection, sperm and testis tissues were harvested for stereological and molecular studies. The BSU group exhibited significantly reduced testis volume, interstitium and tubules compared to the other groups, although the volume of the testis remained unchanged for BSU and CM groups. The number of testis cells was reduced in the BSU group compared to the other groups. The CM group exhibited a significantly increased number of testis cells compared to the BSU group. Sperm count and motility, and length density of seminiferous tubules were increased in CM group compared to the BSU group. AT-MSC-CM exhibited ameliorative effects on histopathologic changes of mouse testes exposed to BSU.