DL-dithiothreitol Research Articles

Cellular Output and Physicochemical Properties of the Membrane-Derived Vesicles Depend on Chemical Stimulants.

Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N-ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.

Green separation of galena from molybdenite by flotation using DL-dithiothreitol as a depressant

Developing an environment-friendly depressant to separate galena from molybdenite by flotation remains greatly challenging. Herein, the nontoxic DL-dithiothreitol (DLD) was used as a galena depressant in a molybdenite–galena flotation system. Flotation experiments showed that a recovery difference of 88.45 % was achieved in a binary mixed-mineral system with a DLD dosage of 40.00 mg/L at pH 8.00, indicating that DLD significantly reduced the floatability of galena while barely altering that of molybdenite. The adsorption mechanism of DLD on the surfaces of galena and molybdenite were investigated by various characterization techniques. Results showed that DLD can coordinate with unsaturated lead ions on the galena surface through its sulfhydryl groups. DLD stably adsorbed onto the galena surface because of the presence of chemical bonding (Pb–S), which significantly changed the surface potential of galena. Molybdenite cannot chemisorb DLD because only saturated sulfur atoms were exposed on its cleavage surface. Hence, the selective adsorption of DLD enhanced the hydrophilicity of the galena surface without affecting the hydrophobicity of the molybdenite surface. Evidently, DLD was a high-performance and promising industrial application of galena depressant. This study provided new insights into the separation of molybdenite and galena, thereby facilitates the sustainable development of the mining industry.

Grafting zwitterionic brushes from the surface of an epoxy-based transparent hydrogel for antifouling performance

Non-specific biofilm formation (biofouling) commonly occurs to the surface of biomedical devices, which causes infection to the human tissues and function loss after implantation. To enhance the antifouling properties on the bioinert hydrogel-based biomaterials, a novel surface grafting approach was developed using surface radical chain-transfer reaction mediated by DL-dithiothreitol (DTT), rather than catalyzed by cytotoxic metal ions. Zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) brushes were grafted on the surface of poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (PHG) to obtain PHG-graft-PMPC (PHG-g-PMPC) hydrogel, which were shown to have tunable surface hydrophilicity while maintaining high water content and transparency. Elemental composition analysis and micromorphology demonstrated the success of surface grafting. Protein adhesion assays were carried out, showing the reduction of bovine serum albumin, lactoferrin, and lysozyme adhesion by ∼90%, 80%, and 70%, respectively, compared to the pristine hydrogels. Significant resistance of bacterial attachment was observed on the surface-modified hydrogels using gram-negative Escherichia. coli and gram-positive Staphylococcus aureus, respectively. The PHG-g-PMPC hydrogel is potentially feasible in various biomedical applications, especially for preventing surface biofouling of ophthalmic implants and devices. Furthermore, this de novo approach provides a universal platform for surface functionalization via thiol-epoxy click chemistry and surface radical chain-transfer reaction.

22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a Hyaluronic Acid Vinyl Ester Hydrogel.

Three-dimensional (3D) bioinspired hydrogels have played an important role in tissue engineering, owing to their advantage of excellent biocompatibility. Here, the two-photon polymerization (TPP) of a 3D hydrogel with high precision has been investigated, using the precursor with hyaluronic acid vinyl ester (HAVE) as the biocompatibility hydrogel monomer, 3,3'-((((1E,1'E)-(2-oxocyclopentane-1,3-diylidene) bis(methanylylidene)) bis(4,1-phenylene)) bis(methylazanediyl))dipropanoate as the water-soluble initiator, and dl-dithiothreitol (DTT) as the click-chemistry cross-linker. The TPP properties of the HAVE precursors have been comprehensively investigated by adjusting the solubility and the formulation of the photoresist. The feature line width of 22 nm has been obtained at a processing laser threshold of 3.67 mW, and the 3D hydrogel scaffold structures have been fabricated. Furthermore, the average value of Young's modulus is 94 kPa for the 3D hydrogel, and cell biocompatibility has been demonstrated. This study would provide high potential for achieving a 3D hydrogel scaffold with highly precise configuration in tissue engineering and biomedicine.

Synthesis and Biodegradability of Tartaric Acid-Based Poly(ester-thioether)s via Thiol-Ene Click Polymerization.

Using scandium triflate [Sc(OTf)3] as a catalyst, chemoselective esterification of tartaric acids by 3-butene-1-ol was performed, and we produced three dialkene monomers: l-di(3-butenyl) tartrate (BTA), d-BTA, and meso-BTA. Thiol-ene polyaddition of these dialkenyl tartrates and dithiols including 1,2-ethanedithiol (ED), ethylene bis(thioglycolate) (EBTG), and d,l-dithiothreitol (DTT) proceeded in toluene at 70 °C under nitrogen to give tartrate-containing poly(ester-thioether)s (Mn, (4.2-9.0) × 103; molecular weight distribution (Mw/Mn), 1.6-2.5). In differential scanning calorimetry, the poly(ester-thioether)s showed single Tgs between -25 and -8 °C. In biochemical oxygen demand (BOD) tests using activated sludge, poly(l-BTA-alt-EBTG) and poly(l-BTA-alt-ED) showed 32 and 8% biodegradability, which is comparable to that of similar l-malate-containing poly(ester-thioether)s (23 and 13% biodegradation, respectively). Notably, we observed enantio and diastereo effects on biodegradation because poly(l-BTA-alt-EBTG), poly(d-BTA-alt-EBTG), and poly(meso-BTA-alt- EBTG) showed different degradation behaviors during the biodegradation test (BOD/theoretical oxygen demand (TOD) values after 28 days, 32, 70, and 43%, respectively). Our findings provide insights into the design of biomass-based biodegradable polymers containing chiral centers.

Inhibition of estrogen sulfation by Xian-Ling-Gu-Bao capsule

Xian-Ling-Gu-Bao capsule (XLGB) is a widely prescribed traditional Chinese medicine used for the treatment of osteoporosis. However, it significantly elevates levels of serum estrogens. Here we aimed to assess the dominant contributors of sulfotransferase (SULT) enzymes to the sulfation of estrogens and identify the effective inhibitors of this pathway in XLGB. First, estrone, 17β-estradiol, and estriol underwent sulfation in human liver S9 extracts. Phenotyping reactions and enzyme kinetics assays revealed that SULT1A1, 1A2, 1A3, 1C4, 1E1, and 2A1 all participated in estrogen sulfation, with SULT1E1 and 1A1 as the most important contributors. The incubation system for these two active enzymes were optimized with Tris-HCl buffer, DL-Dithiothreitol (DTT), MgCl2, adenosine 3'-phosphate 5'-phosphosulfate (PAPS), protein concentration, and incubation time. Then, 29 compounds in XLGB were selected to investigate their inhibitory effects and mechanisms against SULT1E1 and 1A1 through kinetic modelling. Moreover, in silico molecular docking was used to validate the obtained results. And finally, the prenylated flavonoids (isobavachin, neobavaisoflavone, etc.) from Psoralea corylifolia L., prenylated flavanols (icariside II) from Epimedium brevicornu Maxim., tanshinones (dihydrotanshinone, tanshinone II-A,) from Salvia miltiorrhiza Bge., and others (corylifol A, corylin) were identified as the most potent inhibitors of estrogen sulfation. Taken together, these findings provide insights into the understanding regioselectivity of estrogen sulfation and identify the effective components of XLGB responsible for the promotion of estrogen levels.

Effect of clinical application of anti-CD38 and anti-CD47 monoclonal antibodies on blood group detection and transfusion therapy and treatment

BackgroundTo investigate the effect of anti-CD38 monoclonal antibodies (mAb) (daratumumab, DARA) and anti-CD47 mAb combined with azacytidine on blood transfusion compatibility tests, transfusion effects in the treatment of multiple myeloma or acute myeloid leukemia and the corresponding management strategy. Materials and methodsAmong the 19 patients who were treated with DARA and anti-CD47 mAb, 4 patients with cross matching incompatibility were selected. The ABO blood group, the Rh blood group, irregular antibody screening and direct antiglobulin test (DAT) and cross matching testing were performed before and after the application of mAbs using serological methods. Then, irregular antibody screening and microcolumn gel cross matching tests were performed with donor and recipient erythrocytes and serum treated with DL-dithiothreitol (DTT) and Immucor kit, respectively. The transfusion effect was monitored. Results21.05% (4/19) patients had mismatched cross-matching results after mAb treatment. The agglutination intensity of irregular antibody screening tests (3 + ∼ 4 +) after anti-CD47 mAb was higher than that (1 + ∼ 2 +) after DARA. In the DARA group, treating RBCs with 0.2 mol L−1 DTT eliminated the DARA interference with antibody screening. In the anti-CD47 mAb group, the antibody screening, cross-matching test and DAT had been strongly interfered, and using Immucor kit eliminated the interference with antibody screening testing. There was no difference in the transfusion effect. ConclusionThe application of mAb drugs led to incompatibility of cross matching tests, and the transfusion effect was not affected.

Pasting Properties of Various Waxy Rice Flours: Effect of α-Amylase Activity, Protein, and Amylopectin

Waxy rice has a long history of being cultivated and consumed in China. In this study, the effect of different factors including α-amylase activity, protein, and amylopectin structure on the pasting properties of four waxy rice varieties were investigated. Rice flours were divided into four groups (Vietnam indica (VI), Jiangxi indica (JI), Anhui japonica (AJ), and Dongbei japonica (DJ) group) and treated with AgNO3 solution, DL-dithiothreitol (DTT), or protease (n = 3). Results suggested that both α-amylase activity and protein significantly decrease the pasting viscosity of waxy rice flours. Chain length distribution of amylopectin as measured by high performance ion exchange chromatography (HPAEC-PAD) showed that starch with a higher ratio of short chain leads to a higher pasting viscosity. X-Ray diffractograms showed that the crystal type of all the four varieties of rice starches were characteristic A-type. Relative crystallinity of each rice starch was further calculated, and higher crystallization resulted in a higher viscosity. Our study would provide a fundamental knowledge of the relationship between different factors and waxy starch pasting properties, as well as be a reference for controlling the quality of waxy rice starch-based food products.

Synthesis and Biodegradability of Poly(ester-urethane)s via the Thiol-Michael Polyaddition of Dianhydro Sugar-Based Diacrylates

We prepared dianhydro sugar-based diacrylate monomers, d-2,5-di-O-(urethane ethylene acrylate)-1,4:3,6-dianhydroglucitol (AOIIS) and -1,4:3,6-dianhydromannitol (AOIIM), via the respective reaction of 1,4:3,6-dianhydroglucitol (isosorbide (IS)) and 1,4:3,6-dianhydromannitol (isomannide (IM)) with 2-isocyanatoethyl acrylate (AOI). The thiol-Michael polyaddition of the two dianhydro sugar-based diacrylate monomers with several dithiols (ethylene bis(thioglycolate)) (EBTG); 3,6-dioxa-1,8-octanedithiol (DODT); 1,2-ethanedithiol (EDT); dl-dithiothreitol (DTT); and d-2,5-di-O-(2-mercaptoacetate)-1,4:3,6-dianhydromannitol (MAIM) proceeded in N,N-dimethylformamide (40 °C) using triethylamine as the catalyst to give poly(ester-urethane)s with the expected structures (Mn, 8.4 × 103 to 164.6 × 103; molecular dispersity index [Mw/Mn], 1.32–2.47). All of the poly(ester-urethane)s had single glass-transition temperature (Tg) values between −32 and 36 °C. In biodegradation tests using activated sludge, poly(AOIIS-alt-DODT) and poly(AOIIS-alt-DTT) had little biodegradability, but poly(AOIIS-alt-EBTG), poly(AOIIS-alt-EDT), and poly(AOIIS-alt-MAIM) showed 9, 8, and 32% of biodegradation, respectively, after 28 days. Using a conditioned sludge activated by a model compound of poly(ester-urethane), the biochemical oxygen demand (BOD)/total oxygen demand (TOD) value increased to 59% because the number of organisms able to degrade poly(AOIIM-alt-EBTG) was increased in the conditioned sludge relative to the natural sludge. Poly(AOIIS-alt-EBTG) also showed 36% of biodegradation in the activated sludge, indicating diastereo effects on biodegradability.

Oxidative stress in early metabolic syndrome impairs cardiac RyR2 and SERCA2a activity and modifies the interplay of these proteins during Ca2+ waves

We investigated how oxidative stress (OS) alters Ca2+ handling in ventricular myocytes in early metabolic syndrome (MetS) in sucrose-fed rats. The effects of N-acetyl cysteine (NAC) or dl-Dithiothreitol (DTT) on systolic Ca2+ transients (SCaTs), diastolic Ca2+ sparks (CaS) and Ca2+ waves (CaW), recorded by confocal techniques, and L-type Ca2+ current (ICa), assessed by whole-cell patch clamp, were evaluated in MetS and Control cells. MetS myocytes exhibited decreased SCaTs and CaS frequency but unaffected CaW propagation. In Control cells, NAC/DTT reduced RyR2/SERCA2a activity blunting SCaTs, CaS frequency and CaW propagation, suggesting that basal ROS optimised Ca2+ signalling by maintaining RyR2/SERCA2a function and that these proteins facilitate CaW propagation. Conversely, NAC/DTT in MetS recovered RyR2/SERCA2a function, improving SCaTs and CaS frequency, but unexpectedly decreasing CaW propagation. We hypothesised that OS decreases RyR2/SERCA2a activity at early MetS, and while decreased SERCA2a favours CaW propagation, diminished RyR2 restrains it.

Determination of selected water-soluble vitamins (thiamine, riboflavin, nicotinamide and pyridoxine) from a food matrix using hydrophilic interaction liquid chromatography coupled with mass spectroscopy

Water-soluble vitamins are essential dietary components with a multitude of important functions that require quantification from food sources to characterise the nutritional status of food. In this study, we have developed a hydrophilic interaction chromatography (HILIC) based method coupled to single-quadrupole mass spectrometry (MS) for the analysis of selected water-soluble vitamins. Due to their involvement in energy release from macronutrients, the quantification of thiamine (B1), riboflavin (B2), nicotinamide (B3) and pyridoxine (B6) offers significant value in food analysis.A commercially available vegetable soup was selected as the food matrix for this study and utilised to develop an efficient extraction procedure for the vitamins of interest. Vitamins were extracted using meta-phosphoric acid coupled with a reducing agent, DL-dithiothreitol (DTT) to produce the parent compound. The extracted vitamins were then analysed using an LC-MS system with electrospray – atmospheric pressure ionization (ES-API) source, operated in positive single ion monitoring (SIM) mode. The MS provided good linearity within the investigated range from 5 to 400 ng/mL with coefficient of determination (r2) ranging from 0.98 to 0.99. Retention times (0.65–9.04 min) were reproducible and no coelution between vitamins was observed. Limit of detection (LOD) varied from 2.4 to 9.0 ng/mL and limit of quantification (LOQ) was from 8 to 30 ng/mL, comparable to previously published studies. The extraction method provided good intra-day (%CV 1.56–6.56) and inter-day precision (%CV 8.07–10.97). Standard injections were used as part of quality control measures and provided excellent reproducibility (%CV 0.9–3.4). The overall runtime of this method was 19 min, including column reconditioning. Using this method, the quantity of thiamine (67 ± 7 ng/g), riboflavin (423 ± 39 ng/g), nicotinamide (856 ± 77 ng/g) and pyridoxine (133 ± 11 ng/g) was determined from a complex food matrix.In conclusion, we have developed a rapid and reliable, HILIC-single quad MS method utilising SIM for the low-level quantification of four B vitamins in a vegetable soup matrix in under 20 min. This method has shown excellent linearity, intra- and inter-day reproducibility and is directly applicable to other plant-based food matrices.

Conformationally Sensitive Proximity Between the TM3-4 Loop and Hairpin Loop 2 of the Glutamate Transporter EAAT2 Revealed by Paired-Cysteine Mutagenesis.

Excitatory amino acid transporters (EAATs) serve to maintain extracellular neurotransmitter concentrations below neurotoxic levels by transporting glutamate from the synaptic cleft into apposed glia and neurons. Although the crystal structures of the archaeal EAAT homologue from Pyrococcus horikoshii, GltPh, and the human glutamate transporter, EAAT1cryst, have been resolved, the transport mechanism of the transmembrane 3-4 (TM3-4) loop and its structural rearrangement during transport have remained poorly understood. In order to explore the spatial position and function of the TM3-4 loop in the transport cycle, we engineered a pair of cysteine residues between the TM3-4 loop and hairpin loop 2 (HP2) in cysteine-less EAAT2 (CL-EAAT2). We observed that the oxidative cross-linking reagent Cu(II)(1,10-phenanthroline)3 (CuPh) had a significant inhibitory effect on transport in the disubstituted A167C/G437C mutant, whereas dl-dithiothreitol (DTT) reversed the effect of cross-linking A167C/G437C on transport activity, as assayed by d-[3H]-aspartate uptake. Furthermore, we found that the effect of CuPh in this mutant was due to the formation of disulfide bonds in the transporter molecule. Moreover, dl-threo-β-benzyloxyaspartic acid (TBOA) attenuated, while l-glutamate or KCl enhanced, the CuPh-mediated inhibitory effect in the A167C/G437C mutant, suggesting that the A167C and G437C cysteines were farther apart in the outward-facing configuration and closer in the inward-facing configuration. Taken together, our findings provide evidence that the TM3-4 loop and HP2 change spatial proximity during the transport cycle.

Mucoadhesive Nanoparticles for Drug Delivery to the Anterior Eye.

While the use of topical drops for the delivery of drugs to the anterior of the eye is well accepted, it is far from efficient with as little as 5% of the drug instilled on the eye actually reaching the target tissue. The ability to prolong the residence time on the eye is desirable. Based on the acceptability of 2-hydroxyethyl methacrylate based polymers in contact lens applications, the current work focuses on the development of a poly(2-hydroxyethyl methacrylate (HEMA)) nanoparticle system. The particles were modified to allow for degradation and to permit mucoadhesion. Size and morphological analysis of the final polymer products showed that nano-sized, spherical particles were produced. FTIR spectra demonstrated that the nanoparticles comprised poly(HEMA) and that 3-(acrylamido)phenylboronic acid (3AAPBA), as a mucoadhesive, was successfully incorporated. Degradation of nanoparticles containing N,N′-bis(acryloyl)cystamine (BAC) after incubation with DL-dithiothreitol (DTT) was confirmed by a decrease in turbidity and through transmission electron microscopy (TEM). Nanoparticle mucoadhesion was shown through an in-vitro zeta potential analysis.

Pseudorabies virus infection inhibits stress granules formation via dephosphorylating eIF2α

Pseudorabies virus (PRV) is one of the most notorious pathogens in the global pig industry. During infection, viruses may evolve various strategies, such as modulating stress granules (SGs) formation, to create an optimal surroundings for viral replication. However, the interplay between PRV infection and SGs formation remains largely unknown. Here we showed that PRV infection markedly blocked SGs formation induced by sodium arsenate (AS) and DL-Dithiothreitol (DTT). Accordantly, the phosphorylation of eIF2α was markedly inhibited in PRV-infected cells, although two eIF2α kinases double-stranded RNA-activated protein kinase (PKR) and PKR-like ER kinase (PERK) were activated during PRV infection. Furthermore, we also found that the dephosphorylation of eIF2α occurred at the early stage of virus infection but without the elevated production of GADD34 and PP1. Moreover, inhibition of PP1 activity by salubrinal could counteract PRV-mediated eIF2α dephosphorylation partially and inhibit virus replication. Our results revealed that, on the one hand, PRV infection activated eIF2α kinases PKR (latter inhibited) and PERK, and on the other hand, PRV encoded-functions dephosphorylated eIF2α and inhibited SGs formation to facilitate virus replication.

Maturation Mechanism of Nitrile Hydratase From Streptomyces canus CGMCC 13662 and Its Structural Character

Nitrile hydratases have received significant interest both in the large-scale industrial production of acrylamide and nicotinamide, and the remediation of environmental contamination with nitrile-containing pollutants. Almost all known nitrile hydratases include an α-subunit (AnhA) and β-subunit (AnhB), and a specific activator protein is crucial for their maturation and catalytic activity. Many studies exist on nitrile hydratase characteristics and applications, but few have reported their metal insertion and post-translational maturation mechanism. In this study, we investigated the cobalt insertion and maturation mechanism of nitrile hydratase from Streptomyces canus CGMCC 13662 (ScNHase) bearing three subunits (AnhD, AnhE, and AnhA). ScNHase subunits were purified, and the cobalt content and nitrile hydratase activity of the ScNHase subunits were detected. We discovered that cobalt could insert into the cobalt-free AnhA of ScNHase in the absence of activator protein under reduction agent DL-dithiothreitol (DTT) environment. AnhD not only performed the function of AnhB of NHase, but also acted as a metal ion chaperone and self-subunit swapping chaperone, while AnhE did not act as similar performance. A cobalt direct-insertion under reduction condition coordinated self-subunit swapping mechanism is responsible for ScNHase post-translational maturation. Molecular docking of ScNHase and substrates suggested that the substrate specificity of ScNHase was correlated with its structure. ScNHase had a weak hydrophobic interaction with IAN through protein–ligand interaction analysis and, therefore, had no affinity with indole-3-acetonitrile (IAN). The post-translational maturation mechanism and structure characteristics of ScNHase could help guide research on the environmental remediation of nitrile-containing waste contamination and three-subunit nitrile hydratase.

Stimuli-responsive dual drugs-conjugated polydopamine nanoparticles for the combination photothermal-cocktail chemotherapy

We developed one-pot aqueous copolymerization of two dopamine prodrugs to prepare dual drugs-conjugated polydopamine nanoparticles (PDOXCBs), which integrated near infrared (NIR)-mediated photothermal effect with cocktail chemotherapy into one copolymer nanoparticle. Upon a mild NIR irradiation (808 nm, 1 W/cm2, 10 min), PDOXCBs gradually heated aqueous solution over 12.8–13.9 °C, which accordingly enhanced in vitro dual doxorubicin (DOX) and chlorambucil (CB) drug-release with assistance of the other stimuli of pH 5.0 and 10 mmol/L d,l-dithiothreitol (DTT). The combination photothermal-cocktail chemotherapy (PTT−CCT) treatment based on PDOXCB27 plus NIR irradiation gave a highly lowered half maximal inhibitory concentration (IC50) of 2.23 μg/mL and a combination index of 0.36, displaying a superior synergistic effect between PTT and CCT in vitro.

L-Carnitine improves in vitro maturation of feline oocytes and subsequent development of embryos generated by parthenogenesis

The objective of the current study was to assessment the effect of L-Carnitine as a stimulant of lipid catabolism in vitro maturation (IVM) and in vitro culture (IVC) media on IVM rate (nuclear maturation) and embryonic development. A total of 495 oocytes were assigned into two groups; the control group, oocytes matured in TCM-199 medium; L-Carnitine group oocytes matured in TCM-199 medium supplemented with L-Carnitine (0.5mg/ml). After that 250 oocytes were stained with Hoechst 33342 then with Nile Red dye. The nuclear maturation and oocyte lipid content were determined under a fluorescence microscope. The other matured oocytes were incubated for cell activation by Thimerosal and DL- Dithiothreitol (DDT). Potential zygotes were incubated in vitro culture 1 and 2, the stained embryos were counted and evaluated for morula and blastocyst stages. The Metaphase II rates were 16.2% and 42.1% in the control group and L-carnitine group, respectively. Overall, 9.3% and 14.9% of the oocytes reached at least the MI stage in the control group and L-carnitine group respectively. The addition of L-carnitine in IVM media resulted in a significant decrease in the amount of lipid in cat oocyte. Moreover, the incubation of matured oocytes of the L-carnitine group in IVC1 lead to significant increase in cleaved cells 29.4% in comparison with the control group 15.4%. The incubation of cleaved cell of the L-carnitine group in IVC2 lead to decrease significantly in degenerated cells 12.5 % compared to the control group 42.9% while caused an increase significantly in morula 50% compared to the control group 33.3% and blastocyst 37.5% comparison with the control group 23.8%. In conclusion, the use of L-Carnitine as a supplement in IVM and IVC media decrease lipid content of cat oocyte and subsequent enhance the nuclear maturation, cell activation of cat's embryo, and embryonic development.

Poly(ether-carbonate) based hydrogel with tunable mechanical strength and enhanced bioactivity prepared by Michael addition

Poly (ether-ester) based hydrogels have been broadly used as degradable biomaterials in drug delivery systems and tissue engineering. However, they were synthesized with uncontrollable mechanical properties and inert to most of the biological activities, such as cell adhesion and stem cell differentiation. Herein, we synthesized a water-soluble copolymer with poly (trimethylene carbonate-co-2-methyl,2-methylacrylate-bimethylene carbonate) (PTMAc) and poly(ethylene glycol)(PEG) blocks (PCE), in which the PTMAc segments provided free active double bonds and can be further conjugated with thiol-ended peptides to improve its cytocompatibility. Meanwhile, the extra double bonds were crosslinked by Michael addition, forming hydrogels with precisely controlled mechanical strength. The chemical structure and molecular weight of the synthesized PCE were characterized by 1H NMR and gel permeation chromatography (GPC). The composition of the PCEs was controlled by changing the feed ratios of 2-methyl,2-methylacrylate-bimethylene carbonate (Ac) comonomer (fAc). It was noted that when the ratio of comonomers to the macroinitiator PEG ([M]0/[I]0 = 18) and the ratio of DL-Dithiothreitol (DTT) to Ac ([thiol]/[Ac] = 1/1) were fixed, the storage moduli of PCE hydrogels increased from 2 kPa to 10 kPa and the gelation time decreased with increasing fAc. The pendant double bonds were easily conjugated with CRGD to promote the neurite sprouting and axonal extension from the dorsal root ganglion (DRG) neurons. Moreover, the weaker the hydrogel matrix (G′ = 0.2 kPa) was, the lower the axon extension length was, but the axon diameter became thicker. An increase in modulus is beneficial to axon extension (5 kPa), while a high modulus (10 kPa) is unfavorable. These results demonstrated that this novel poly(ether-carbonate) based hydrogel can be introduced as a suitable scaffold for fast and effective nerve regeneration.

Icariin protects vascular endothelial cells from oxidative stress through inhibiting endoplasmic reticulum stress

ObjectiveTo investigate the protective effect and underlying mechanism(s) of icariin (ICA) in preventing hydrogen peroxide (H2O2)-induced vascular endothelial cell injury via endoplasmic reticulum stress (ERS). MethodsTo study the effects of ICA on H2O2-induced damage, we used the cell counting kit-8 assay to detect cell viability and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay to determine cell adhesion and apoptosis, respectively. Spectrophotometry and enzyme-linked immunosorbent assay were used to measure the expression levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Subsequently, glucose-regulated protein 78 (GRP78), activating transcription factor-4 (ATF4) and eukaryotic initiation factor-2α (eIF2α) were detected using Western blotting. ResultsIn human umbilical vein endothelial cells, different concentrations of ICA exhibited multiple effects, including reduced H2O2 damage, improved cell viability and adhesion, reduced cell apoptosis and increased SOD and GSH-Px activity. Among the ICA concentrations used, only the H2O2 + 100 μmol/L ICA group had significant differences compared to the H2O2 group. ERS activators H2O2 and dl-dithiothreitol (DTT) significantly increased GRP78, ATF4 and eIF2α expressions, decreased cell activity and reduced SOD and GSH-Px activity. In contrast, the H2O2 + 100 μmol/L ICA and H2O2 + 100 μmol/L ICA + DTT groups had significant inhibitory effects on the expressions of GRP78, ATF4 and eIF2α proteins, showing enhanced cell viability and SOD and GSH-Px activity. ConclusionThe results showed the dose-dependent effects of ICA against H2O2-induced injury in vascular endothelial cells. The inhibition of GRP78, ATF4 and eIF2α protein expressions in the ERS, and the subsequent alleviation of oxidative stress damage, might be the molecular mechanism.

Pullulan reduces the non-specific amplification of loop-mediated isothermal amplification (LAMP).

Loop-mediated isothermal amplification (LAMP) has been developed as a versatile method for nucleic acid analysis in many applications. However, non-specific LAMP leading to false-positive outcomes has been observed frequently. To solve this problem, we selected six molecules as the additives for evaluating their effects on the improvement of the LAMP specificity. Experimental results show that bovine serum albumin (BSA) and DL-dithiothreitol (DTT) have negative effects on the LAMP specificity; dimethyl sulfoxide (DMSO), tetramethylene sulfoxide (TMSO), and glycerol could inhibit the non-specific LAMP moderately. Surprisingly, pullulan shows an ability to inhibit the non-specific amplification of LAMP significantly without affecting the sample amplification of LAMP, and this inhibitory effect is concentration dependent. Thus, pullulan could be considered as the most promising additive to improve the amplification specificity in the LAMP-based detection and analysis of nucleic acids.