Effect of 2-methoxyestradiol on mammary tumor initiation and progression.

BACKGROUND: The anti-cancer agent 2-methoxyestradiol (2-ME) has been shown to have anti-proliferative and anti-angiogenic properties. Previously, the effect of 2-ME on early- and late-stage breast cancer (BC) was investigated in vivo using a transgenic mouse model (FVB/N-Tg(MMTV-PyVT)) of spontaneous mammary carcinoma. Anti-tumor effects were observed in late-stage BC with no effect on early-stage BC. Given the contrasting results obtained from the different BC stages, we have now investigated the effect of 2-ME when administered before the appearance of palpable tumors. METHODS: Each mouse received 100 mg/kg 2-ME on day 30 after birth, twice per week for 28 days, while control mice received vehicle only. Animals were terminated on day 59. Lung and mammary tissue were obtained for immunohistochemical analysis of CD163 and CD3 expression, and histological examination was performed to analyze tumor necrosis. Additionally, blood samples were collected to measure plasma cytokine levels. RESULTS: 2-ME increased tumor mass when compared to the untreated animals (p = .0139). The pro-tumorigenic activity of 2-ME was accompanied by lower CD3+ T-cell numbers in the tumor microenvironment (TME) and high levels of the pro-inflammatory cytokine interleukin (IL)-1ß. Conversely, 2-ME-treatment resulted in fewer CD163+ cells detectable in the TME, increased levels of tumor necrosis, increased IL-10 plasma levels, and low IL-6 and IL-27 plasma levels. CONCLUSION: Taken together, these findings suggest that 2-ME promotes early-stage BC development.

Comparative RNA isolation methods from fresh ejaculated spermatozoa in Sahiwal cattle (Bos indicus) and Murrah buffalo (Bubalus bubalis) bulls for high quality and enhanced RNA yield.

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. The aim of the study was to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bulls. Ten fresh ejaculates from each Sahiwal (n = 10 bulls × 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) resulted significantly (p < .05) higher total RNA quantity (300-340 ng/µL) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm.

Inhibition of JAK1/STAT3 pathway by 2-methoxyestradiol ameliorates psoriatic features in vitro and in an imiquimod-induced psoriasis-like mouse model.

Psoriasis is characterized by hyperproliferative keratinocytes, dilated capillaries and leukocyte infiltration. 2-Methoxyestradiol (2-ME) has shown significant inhibition on proliferation, angiogenesis and inflammation. To evaluate the anti-psoriatic potential of 2-ME, psoriasis-like dermatitis was induced by topical application of imiquimod (IMQ) on the dorsal skin of C57BL/6 mice for seven consecutive days, followed by treatment of vehicle or 2-ME ointment from Day 4 on. The psoriasis area and severity index (PASI) was assessed daily. On Day 8, skin histology and spleen index were assessed. The effects of 2-ME on the proliferation, apoptosis, cell cycle, vascular endothelial growth factor A (VEGFA), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways of HaCaT cells stimulated by interleukin-17 (IL-17A) were detected, together with its effect on the proliferation, tube formation and VEGF receptor expression of human umbilical vein endothelial cells (HUVECs). We found that topical 2-ME treatment significantly improved IMQ-induced psoriasis-like dermatitis and decreased the PASI scores, the activation of STAT3 in the skin (P < 0.05), and the spleen index in mice (P < 0.01). In vitro, 2-ME inhibited the proliferation of HaCaT cells by inducing apoptosis and G2/M phase arrest (P < 0.01). Moreover, 2-ME suppressed IL-17A-induced VEGFA (2.5 µM: P < 0.05; 5 µM: P < 0.01) and phosphorylation of STAT3 by blocking p-JAK1 in HaCaT cells and prevented tube formation (P < 0.01) and proliferation by targeting VEGF receptors 1 (VEGFR1) and 2 (VEGFR2) in HUVECs. We conclude that 2-ME alleviated psoriasis in vivo and in vitro by inhibiting JAK1/STAT3 pathway and was a promising therapeutic agent for psoriasis.

ß-Mercaptoethanol in culture medium improves cryotolerance of in vitro-produced bovine embryos.

The objective of this study was to investigate the effects of adding ß-mercaptoethanol (ßME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 µM ßME for 72 h. Embryos cultured in 100 µM ßME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) µM ßME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 µM ßME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 µM ßME, in a 2 × 2 factorial design: (i) CTRL-CTRL, control IVC and control PWC; (ii) CTRL-ßME, control IVC and ßME-supplemented PWC; (iii) ßME-CTRL, ßME-supplemented IVC and control PWC; or (iv) ßME-ßME, ßME-supplemented IVC and ßME-supplemented PWC. ßME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in ßME-CTRL (84.0%) and ßME-ßME (87.5%) than in CTRL-CTRL (71.0%) and CTRL-ßME (73.1%). Hatching rates were higher in CTRL-ßME (58.1%) and ßME-ßME (63.8%) than in CTRL-CTRL (36.6%) and ßME-CTRL (42.0%). Total cell number in hatched blastocysts was higher in ßME-ßME (181.2 ± 7.4 cells) than CTRL-CTRL (139.0 ± 9.9 cells). Adding ßME to the IVC medium reduced development but increased cryotolerance, whereas adding ßME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

The effect of HIF-1α inhibition in breast cancer cells prior to doxorubicin treatment under conditions of normoxia and hypoxia.

BACKGROUND: Oxygen deprivation is a key hallmark within solid tumours that contributes to breast-tumour pathophysiology. Under these conditions, neoplastic cells activate several genes, regulated by the HIF-1 transcription factor, which alters the tumour microenvironment to promote survival - including resistance to cell death in therapeutic attempts such as doxorubicin (Dox) treatment. METHODS: We investigated HIF-1ɑ as a therapeutic target to sensitize breast cancer cells to Dox treatment. Under both normoxic (21% O2) and hypoxic (∼0.1% O2) conditions, the HIF-1 inhibitor, 2-methoxyestradiol (2-ME), was investigated as an adjuvant for its ability to alter MCF-7 cell viability, apoptosis, autophagy and molecular pathways which are often associated with increased cell survival. RESULTS: Here we observed that an inverse relationship between HIF-1ɑ and apoptosis exists and that Dox promotes autophagy under hypoxic conditions. Although adjuvant therapy with 2-ME induced an antagonistic effect in breast cancer cells, upregulated HIF-1ɑ expression in a hypoxic environment promotes treatment resistance and this was attenuated once HIF-1ɑ gene expression was silenced. CONCLUSION: Therefore, highlighting the identification of possible hypoxia-targeting therapies for breast cancer patients can be beneficial by promoting more favourable treatment responses.

Study of lead-induced neurotoxicity in cholinergic cells differentiated from bone marrow-derived mesenchymal stem cells.

The developing brain is susceptible to the neurotoxic effects of lead. Exposure to lead has main effects on the cholinergic system and causes reduction of cholinergic neuron function during brain development. Disruption of the cholinergic system by chemicals, which play important roles during brain development, causes of neurodevelopmental toxicity. Differentiation of stem cells to neural cells is recently considered a promising tool for neurodevelopmental toxicity studies. This study evaluated the toxicity of lead acetate exposure during the differentiation of bone marrow-derived mesenchyme stem cells (bone marrow stem cells, BMSCs) to CCholinergic neurons. Following institutional animal care review board approval, BMSCs were obtained from adult rats. The differentiating protocol included two stages that were pre-induction with ß-mercaptoethanol (BME) for 24 h and differentiation to cholinergic neurons with nerve growth factor (NGF) over 5 days. The cells were exposed to different lead acetate concentrations (0.1-100 µm) during three stages, including undifferentiated, pre-induction, and neuronal differentiation stages; cell viability was measured by MTT assay. Lead exposure (0.01-100 µg/ml) had no cytotoxic effect on BMSCs but could significantly reduce cell viability at 50 and 100 µm concentrations during pre-induction and neuronal differentiation stages. MAP2 and choline acetyltransferase (ChAT) protein expression were investigated by immunocytochemistry. Although cells treated with 100 µm lead concentration expressed MAP2 protein in the differentiation stages, they had no neuronal cell morphology. The ChAT expression was negative in cells treated with lead. The present study showed that differentiated neuronal BMSCs are sensitive to lead toxicity during differentiation, and it is suggested that these cells be used to study neurodevelopmental toxicity.

2-Mercaptoethanol promotes porcine oocyte maturation in vitro by maintaining autophagy homeostasis.

2-Mercaptoethanol (2-ME) is often used as an antioxidant to optimize culture systems for in vitro oocyte maturation in livestock. However, the relationship between 2-ME and autophagy has not yet been elucidated. In this study, we hypothesized that 2-ME can promote porcine oocyte maturation in vitro by maintaining autophagy homeostasis. To test this hypothesis, we explored the effects of 2-ME on the maturation of porcine oocytes exposed to an autophagy activator (rapamycin) or an autophagy inhibitor (3-methyladenine, i.e., 3-MA) in vitro. Rapamycin-induced autophagy over-activation significantly increased autophagy- and apoptosis-related gene expression, oxidative stress, apoptosis rates, abnormal mitochondrial redistribution, and significantly decreased oocyte first polar body extrusion (PBE) rates, spindle/chromosome integrity and developmental competence. 3-MA-mediated autophagy inhibition exerted similar effects on all these parameters except the expression of genes that promote autophagy and inhibit apoptosis. Importantly, 2-ME supplementation significantly attenuated the detrimental effects of rapamycin and 3-MA. Interestingly, we observed that 44 h of coincubation with rapamycin/3-MA and 2-ME restored autophagy homeostasis in vitro. In conclusion, our study confirmed that 2-ME promotes porcine oocyte maturation and embryo development in vitro by maintaining autophagy homeostasis and lays a foundation for further research on the underlying mechanism.

Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus.

BACKGROUND: In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. RESULT: The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of ß-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. CONCLUSION: The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas ß-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste.

The photodynamic reaction with IR-775 cyanine combined with 2-methoxyestradiol in ovarian (SKOV-3) and human breast adenocarcinoma (MDA MB-231) cell lines.

BACKGROUND: Photodynamic therapy (PDT) is known anticancer approach used mainly for topical skin cancer. However, it could serve as an excellent alternative to traditional anticancer therapies, such as chemotherapy or radiotherapy. AIMS: The study aimed to assess the effect of PDT, where the combination of cyanine with 2-methoxyestradiol (2-Me) was used on mammary and ovary adenocarcinoma human cell lines. MATERIALS AND METHODS: The cyanine IR-775 was used as the photosensitizer. Two human malignant adenocarcinoma cell lines - ovary and mammary adenocarcinoma (MDA MB-231 and SKOV-3) were investigated in photodynamic reaction (PDR), with the enhancement of 2-Me. PDR efficiency was evaluated by the MTT test. Photosensitizer intracellular distribution was assessed by fluorescent microscopy. Additionally, apoptotic and oxidative stress markers were investigated by immunocytochemistry staining. RESULTS AND CONCLUSIONS: It was observed that PDR enhanced by 2-Me is effective against two common but different types of cancer. The treatment decreased cells' viability by around 70%. Immunocytochemical staining of SOD2 and caspase-12 indicated apoptosis and oxidative stress induction in tested cell lines. The results suggest that the therapy could be involved in further oncological applications.

The Impact of Sodium Dodecyl Sulfate and 2-Mercaptoethanol on Antibody and Antigen Binding.

OBJECTIVE: To evaluate the effect of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) on antigen-antibody binding when incubated at 100°C, which is the pretreatment temperature required for western blots. METHODS: Serum that tested positive for hepatitis B surface antigen (HBsAg) plus loading buffer were mixed at a ratio of 4:1 and incubated in a water bath. We then detected HBsAg using double immunodiffusion and ELISA. RESULTS: The HBsAg titer was 1:512 in the control group when incubated at 37°C. Incubation with SDS at 100°C reduced the antigen titer to 1:32. The inhibitory effect on HBsAg titer reached 96.9% after incubation at 100°C with SDS and 2-ME. CONCLUSION: We detected strong inhibition of antigens in western blots via SDS and 2-ME. It is likely that false-negative results will be obtained from western blots of antigens with weak resistance to these reagents.

Cutting Edge of the Pathogenesis of Atopic Dermatitis: Sphingomyelin Deacylase, the Enzyme Involved in Its Ceramide Deficiency, Plays a Pivotal Role.

Atopic dermatitis (AD) is characterized clinically by severe dry skin and functionally by both a cutaneous barrier disruption and an impaired water-holding capacity in the stratum corneum (SC) even in the nonlesional skin. The combination of the disrupted barrier and water-holding functions in nonlesional skin is closely linked to the disease severity of AD, which suggests that the barrier abnormality as well as the water deficiency are elicited as a result of the induced dermatitis and subsequently trigger the recurrence of dermatitis. These functional abnormalities of the SC are mainly attributable to significantly decreased levels of total ceramides and the altered ceramide profile in the SC. Clinical studies using a synthetic pseudo-ceramide (pCer) that can function as a natural ceramide have indicated the superior clinical efficacy of pCer and, more importantly, have shown that the ceramide deficiency rather than changes in the ceramide profile in the SC of AD patients plays a central role in the pathogenesis of AD. Clinical studies of infants with AD have shown that the barrier disruption due to the ceramide deficiency is not inherent and is essentially dependent on postinflammatory events in those infants. Consistently, the recovery of trans-epidermal water loss after tape-stripping occurs at a significantly slower rate only at 1 day post-tape-stripping in AD skin compared with healthy control (HC) skin. This resembles the recovery pattern observed in Niemann-Pick disease, which is caused by an acid sphingomyelinase (aSMase) deficiency. Further, comparison of ceramide levels in the SC between before and after tape-stripping revealed that whereas ceramide levels in HC skin are significantly upregulated at 4 days post-tape-stripping, their ceramide levels remain substantially unchanged at 4 days post-tape-stripping. Taken together, the sum of these findings strongly suggests that an impaired homeostasis of a ceramide-generating process may be associated with these abnormalities. We have discovered a novel enzyme, sphingomyelin (SM) deacylase, which cleaves the N-acyl linkage of SM and glucosylceramide (GCer). The activity of SM deacylase is significantly increased in AD lesional epidermis as well as in the involved and uninvolved SC of AD skin, but not in the skin of patients with contact dermatitis or chronic eczema, compared with HC skin. SM deacylase competes with aSMase and ß-glucocerebrosidase (BGCase) to hydrolyze their common substrates, SM and GCer, to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine (GSP), respectively, instead of ceramide. Consistently, those reaction products (SPC and GSP) accumulate to a greater extent in the involved and uninvolved SC of AD skin compared with chronic eczema or contact dermatitis skin as well as HC skin. Successive chromatographies were used to purify SM deacylase to homogeneity with a single band of ≈43 kDa and with an enrichment of >14,000-fold. Analysis of a protein spot with SM deacylase activity separated by 2D-SDS-PAGE using MALDI-TOF MS/MS allowed its amino acid sequence to be determined and to identify it as the ß-subunit of acid ceramidase (aCDase), an enzyme consisting of α- and ß-subunits linked by amino-bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ≈56 and ≈13 kDa and the ß-subunit at ≈43 kDa, the purified SM deacylase was detectable only by the antibody to the ß-subunit at ≈43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with an apparent size of ≈40 kDa upon gel chromatography in contrast to aCDase activity with an apparent size of ≈50 kDa in untreated recombinant human aCDase. These results provide new insights into the essential role of SM deacylase as the ß-subunit aCDase that causes the ceramide deficiency in AD skin.

A Modified MTS Proliferation Assay for Suspended Cells to Avoid the Interference by Hydralazine and ß-Mercaptoethanol.

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay is one of the most commonly used tests of cell proliferation. Hydralazine has been reported to interfere with the performance of the MTS assay when used on adherent cells. This study aimed to investigate whether hydralazine interferes with the performance of the MTS assay on suspended cells. THP-1 (a monocytic leukemia cell line) cells were cultured in the presence or absence of hydralazine (0, 10, 50, 100, and 500 µM) for 2 or 24 h. Cell numbers were analyzed using the MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established by centrifuging the cells and replacing the test medium with fresh culture medium immediately before the addition of the MTS reagent. Culture of THP-1 cells with hydralazine at concentrations of 50, 100, and 500 µM for 2 h increased absorbance (p < 0.001) in the standard MTS assay, whereas both the trypan blue exclusion assay and microscopy suggested no change in cell numbers. Culture of THP-1 cells with 100 and 500 µm hydralazine for 24 h increased absorbance (p < 0.05) in the standard MTS assay; however, trypan blue exclusion and microscopy suggested a decrease in cell numbers. In a cell-free system, hydralazine (100 and 500 µM) increased absorbance in a time- and concentration-dependent manner. The modified MTS assay produced results consistent with trypan blue exclusion and microscopy using THP-1 cells. In addition, the modified MTS assay produced reliable results when K562 and Jurkat cells were incubated with hydralazine or ß-mercaptoethanol (ßME). In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and ßME when assessing suspended cells.

Differentiation of canine adipose mesenchymal stem cells into insulin-producing cells: comparison of different culture medium compositions.

The aim of this study was to differentiate canine adipose-derived mesenchymal stem cells (ADMSCs) into insulin-producing cells by using culture media with different compositions to determine the most efficient media. Stem cells isolated from the fat tissues close to the bitch uterus were distributed into 6 groups: (1) Dulbecco's modified Eagle medium (DMEM)-high glucose (HG), ß-mercaptoethanol, and nicotinamide; (2) DMEM-HG, ß-mercaptoethanol, nicotinamide, and exendin-4; (3) DMEM-HG, ß-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, and l-glutamine; (4) DMEM-HG, ß-mercaptoethanol, and nicotinamide (for the initial 8-d period), and DMEM-HG, ß-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, l-glutamine, and basic fibroblast growth factor (for the remaining 8-d period); (5) DMEM-HG and fetal bovine serum; and (6) DMEM-low glucose and fetal bovine serum (standard control group). Adipose-derived mesenchymal stem cells from groups 1 to 5 gradually became round in shape and gathered in clusters. These changes differed between the groups. In group 3, the cell clusters were apparently more in numbers and gathered as bigger aggregates. Dithizone staining showed that groups 3 and 4 were similar in terms of the mean area of each aggregate stained for insulin. However, only in group 4, the number of insulin aggregates and the total area of aggregates stained were significantly bigger than in the other groups. The mRNA expression of PDX1, BETA2, MafA, and Insulin were also confirmed in all the groups. We conclude that by manipulating the composition of the culture medium it is possible to induce canine ADMSCs into insulin-producing cells, and the 2-staged protocol that was used promoted the best differentiation.

Extracellular neutral protease from Arthrospira platensis: Production, optimization and partial characterization.

Proteases are industrially important catalysts. They belong to a complex family of enzymes that perform highly focused proteolysis functions. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. In the present study, a novel extracellular neutral protease produced from Arthrospira platensis was detected and characterized. Its proteolytic activity was strongly activated by ß-mercaptoethanol, 5,5-dithio-bis-(2-nitrobenzoic acid) and highly inhibited by Hg2+ and Zn2+ metal ions which support the fact that the studied protease belongs to the cysteine protease family. Using statistical modelling methodology, the logistic model has been selected to predict A. platensis growth-kinetic values. The optimal culture conditions for neutral protease production were found using Box-Behnken Design. The maximum experimental protease activities (159.79 U/mL) was achieved after 13 days of culture in an optimized Zarrouk medium containing 0.625 g/L NaCl, 0.625 g/L K2HPO4 and set on 9.5 initial pH. The extracellular protease of A. platensis can easily be used in the food industry for its important activity at neutral pH and its low production cost since it is a valuation of the residual culture medium after biomass recovery.

Potential alteration of tumor microenvironments by ß-mercaptoethanol.

The therapeutic effectiveness of immune checkpoint inhibitors in cancer patients is quite profound. However, it is generally accepted that further progress is curtailed by accompanying adverse events and by low cure rates linked to the tumor microenvironment. The multitudes of immune processes altered by low-molecular-weight thiols published over the past decades suggest they have potential to alter tumor microenvironment processes which could result in an increase in immune checkpoint inhibitor survival rates. Based on one of the most studied and most potent low-molecular-weight thiols, ß-mercaptoethanol (BME), it is proposed that clinical assessment be undertaken to identify any BME benefits with relevance for proliferation/differentiation of immune cells, lymphocyte exhaustion, immunogenicity of tumor antigens and inactivation of suppressor cells/factors. The BME alterations projected to be most effective are: maintenance/replacement of glutathione in lymphocytes via facilitation of cysteine uptake, inhibition of suppressor cells/soluble factors and inactivation of free-radical, reactive oxygen species.

Aquaporin3 expression and the potential role of aquaporins in motility and mitochondrial membrane potential in human spermatozoa.

Aquaporins are water selective channels which play important roles in cell volume regulation during the transmission of spermatozoa to female tract. This study investigated the expression of aquaporin3 and determined the role of aquaporins in human sperm motility and mitochondrial membrane potential (MMP). RT-PCR and flow cytometry analysis were done to investigate aquaporin3 expression levels, and immunolocalisation of aquaporin3 in the spermatozoa was detected using immunocytochemical analysis. The sperm suspension was divided into four groups of spermatozoa: (a) Spermatozoa at 0 hr, (b) spermatozoa in control group, (c) spermatozoa treated with HgCl2 (as an aquaporin inhibitor) and (d) spermatozoa treated with HgCl2 + and 2-mercaptoethanol. The sperm samples were examined in terms of sperm motility and mitochondrial membrane potential. Results confirmed aquaporin3 expression in human spermatozoa and immunocytochemistry results showed an intense immunoreactivity in whole sperm tail. After 60 min, HgCl2 showed a significant decrease in motility and MMP compared to the control group. At this time point, 2-mercaptoethanol in the HgCl2 + 2-mercaptoethanol group reversed the effects of HgCl2 as compared to the HgCl2 group. Present study showed the expression and immunolocalisation of AQP3 in human spermatozoa and the potential role of AQPs in the sperm motility and MMP.

Biochemical characterization of the endo-α-N-acetylgalactosaminidase pool of the human gut symbiont Tyzzerella nexilis.

Three large (2084-, 984-, and 2104-amino acids) endo-α-N-acetylgalactosaminidase candidate genes from the commensal human gut bacterium Tyzzerella nexilis were successfully cloned and subsequently expressed in Escherichia coli. Activity tests of the purified proteins revealed that two of the candidate genes (Tn0153 and Tn2105) were able to hydrolyze the disaccharide unit from Galß1-3GalNAc-α-pNP. The biochemical characterization revealed optimum pH conditions of 4.0 for both enzymes and temperature optima of 50 °C. The addition of 2-mercaptoethanol, Triton X-100 and urea had only minor effects on the activity of the enzymes, and the addition of imidazole and sodium dodecyl sulfate led to a significant reduction of the enzymes' activities. A mutational study identified and confirmed the role of the catalytically significant amino acids. The present study describes the first functional characterization of members of the GH101 family from this human gut symbiont.

The Small Protein CydX Is Required for Cytochrome bd Quinol Oxidase Stability and Function in Salmonella enterica Serovar Typhimurium: a Phenotypic Study.

Cytochrome bd quinol oxidases, which have a greater affinity for oxygen than heme-copper cytochrome oxidases (HCOs), promote bacterial respiration and fitness in low-oxygen environments, such as host tissues. Here, we show that, in addition to the CydA and CydB subunits, the small protein CydX is required for the assembly and function of the cytochrome bd complex in the enteric pathogen Salmonella enterica serovar Typhimurium. Mutant S Typhimurium lacking CydX showed a loss of proper heme arrangement and impaired oxidase activity comparable to that of a ΔcydABX mutant lacking all cytochrome bd subunits. Moreover, both the ΔcydX mutant and the ΔcydABX mutant showed increased sensitivity to ß-mercaptoethanol and nitric oxide (NO). Cytochrome bd-mediated protection from ß-mercaptoethanol was not a result of resistance to reducing damage but, rather, was due to cytochrome bd oxidase managing Salmonella respiration, while ß-mercaptoethanol interacted with the copper ions necessary for the HCO activity of the cytochrome bo-type quinol oxidase. Interactions between NO and hemes in cytochrome bd and cytochrome bd-dependent respiration during nitrosative stress indicated a direct role for cytochrome bd in mediating Salmonella resistance to NO. Additionally, CydX was required for S Typhimurium proliferation inside macrophages. Mutants deficient in cytochrome bd, however, showed a significant increase in resistance to antibiotics, including aminoglycosides, d-cycloserine, and ampicillin. The essential role of CydX in cytochrome bd assembly and function suggests that targeting this small protein could be a useful antimicrobial strategy, but potential drug tolerance responses should also be considered.IMPORTANCE Cytochrome bd quinol oxidases, which are found only in bacteria, govern the fitness of many facultative anaerobic pathogens by promoting respiration in low-oxygen environments and by conferring resistance to antimicrobial radicals. Thus, cytochrome bd complex assembly and activity are considered potential therapeutic targets. Here we report that the small protein CydX is required for the assembly and function of the cytochrome bd complex in S Typhimurium under stress conditions, including exposure to ß-mercaptoethanol, nitric oxide, or the phagocytic intracellular environment, demonstrating its crucial function for Salmonella fitness. However, cytochrome bd inactivation also leads to increased resistance to some antibiotics, so considerable caution should be taken when developing therapeutic strategies targeting the CydX-dependent cytochrome bd.

Chemical Reprogramming of Somatic Cells in Neural Direction: Myth or Reality?

In in vitro experiments on cultures of human multipotent stem cells from the human bonearrow and dental pulp, we studied direct reprogramming towards neuro-glial lineage cells using a cocktail of small molecules. Reprogramming by the previously published protocol (with a cocktail containing ß-mercaptoethanol, LIF, VPA, CHIR99021, and RepSox) and by the optimized protocol (VPA, RG108, А83-01, dorsomorphin, thiazovivin, CHIR99021, forskolin, and Isx9) allows obtaining cells with immunophenotypic and genetic signs of neural stem cells. However, neither the former, nor the optimized protocols allowed preparing neural progenitors capable of adequate terminal differentiation from both bone marrow-derived mesenchymal stem cells and nestin-positive neural crest-derived mesenchymal stem cells. Real-time PCR demonstrated the expression of some neurogenesis markers, but neural stem cell-specific expression pattern was not observed. The findings lead us to a conclusion that reprogramming with small molecules without additional factors modifying gene expression does not allow reproducible production of human neural stem cell-like progenitors that can be used as the source of neural tissue for the regenerative therapy.

In vitro transdifferentiation of human adipose tissue-derived stem cells to neural lineage cells - a stage-specific incidence.

The present Study investigated the intrinsic ability of adipose tissue-derived stem cells (ADSCs) and their neural transdifferentiation in a stage-specific manner. Woodbury's Chemical induction was implemented with modifications to achieve neural transdifferentiation. In Group I, ADSCs were preinduced with ß-mercaptoethanol (ß-ME) and later, with neural induction medium (NIM). In Group II, ADSCs were directly treated with NIM. In Group III, a DNA methyltransferase (DNMT) inhibitor 5-azacytidine was applied to understand whether transdifferentiation is controlled by epigenetic marks. Irrespective of the presence of (ß-ME), the differentiation protocol resulted in glial-lineage cells. Group III produced poorly -differentiated neural cells with neuron-specific enolase positivity. A neuroprogenitor stage (NPC) was identified at d 11 after induction only in Group I. In other groups, this stage was not morphologically distinct. We explored the stage-specific incidence NPC, by alternatively treating them with basic fibroblast growth factor (bFGF), and antioxidants to validate if different signalling could cause varied outcomes (Group IV). They differentiated into neurons, as defined by cell polarity and expression of specific proteins. Meanwhile, neuroprogenitors exposed to NIM (Group I) produced glial-lineage cells. Further refinement and study of the occurrence and terminal differentiation of neuroprogenitors would identify a promising source for neural tissue replacement.