Nanopore analysis of cis-diols in fruits.

Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need of complex sample pretreatment, direct and rapid analysis of fruits in a hand-held device has never been previously reported. Nanopore, a versatile single molecule sensor, can be specially engineered to perform this task. A hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore modified with a sole phenylboronic acid (PBA) adapter is prepared. This engineered MspA accurately recognizes 1,2-diphenols, alditols, α-hydroxy acids and saccharides in prune, grape, lemon, different varieties of kiwifruits and commercial juice products. Assisted with a custom machine learning program, an accuracy of 99.3% is reported and the sample pretreatment is significantly simplified. Enantiomers such as DL-malic acids can also be directly identified, enabling sensing of synthetic food additives. Though demonstrated with fruits, these results suggest wide applications of nanopore in food and drug administration uses.

Unambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore.

Natural proteins are composed of 20 proteinogenic amino acids and their post-translational modifications (PTMs). However, due to the lack of a suitable nanopore sensor that can simultaneously discriminate between all 20 amino acids and their PTMs, direct sequencing of protein with nanopores has not yet been realized. Here, we present an engineered hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a sole Ni2+ modification. It enables full discrimination of all 20 proteinogenic amino acids and 4 representative modified amino acids, Nω,N'ω-dimethyl-arginine (Me-R), O-acetyl-threonine (Ac-T), N4-(ß-N-acetyl-D-glucosaminyl)-asparagine (GlcNAc-N) and O-phosphoserine (P-S). Assisted by machine learning, an accuracy of 98.6% was achieved. Amino acid supplement tablets and peptidase-digested amino acids from peptides were also analyzed using this strategy. This capacity for simultaneous discrimination of all 20 proteinogenic amino acids and their PTMs suggests the potential to achieve protein sequencing using this nanopore-based strategy.

Simultaneous Identification of Major Thyroid Hormones by a Nickel Immobilized Biological Nanopore.

Thyroid hormones (THs) are a variety of iodine-containing hormones that demonstrate critical physiological impacts on cellular activities. The assessment of thyroid function and the diagnosis of thyroid disorders require accurate measurement of TH levels. However, largely due to their structural similarities, the simultaneous discrimination of different THs is challenging. Nanopores, single-molecule sensors with a high resolution, are suitable for this task. In this paper, a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a single nickel ion immobilized to the pore constriction has enabled simultaneous identification of five representative THs including l-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2). To automate event classification and avoid human bias, a machine learning algorithm was also developed, reporting an accuracy of 99.0%. This sensing strategy is also applied in the analysis of TH in a real human serum environment, suggesting its potential use in a clinical diagnosis.

Discrimination of Disaccharide Isomers of Different Glycosidic Linkages Using a Modified MspA Nanopore.

Disaccharides are composed of two monosaccharide subunits joined by a glycosidic linkage in an α or ß configuration. Different combinations of isomeric monosaccharide subunits and different glycosidic linkages result in different isomeric disaccharide products. Thus, direct discrimination of these disaccharide isomers from a mixture is extremely difficult. In this paper, a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore conjugated with a phenylboronic acid (PBA) adapter was applied for disaccharide sensing, with which three most widely known disaccharides in nature, including sucrose, lactose and maltose, were clearly discriminated. Besides, all six isomeric α-D-glucopyranosyl-D-fructoses, differing only in their glycosidic linkages, were also well resolved. Assisted by a custom machine learning algorithm, a 0.99 discrimination accuracy is achieved. Nanopore discrimination of disaccharide isomers with different glycosidic linkages, which has never been previously demonstrated, is inspiring for nanopore saccharide sequencing. This sensing capacity was also applied in direct identification of isomaltulose additives in a commercial sucrose-free yogurt, from which isomaltulose, lactose and L-lactic acid were simultaneously detected.

Probiotic Escherichia coli Nissle 1917 protect chicks from damage caused by Salmonella enterica serovar Enteritidis colonization.

As a foodborne pathogen of global importance, Salmonella enterica serovar Enteritidis (S. Enteritidis) is a threat to public health that is mainly spread by poultry products. Intestinal Enterobacteriaceae can inhibit the colonization of S. Enteritidis and are regarded as a potential antibiotic substitute. We investigated, in chicks, the anti-S. Enteritidis effects of Escherichia coli (E. coli) Nissle 1917, the most well-known probiotic member of Enterobacteriaceae. Eighty 1-d-old healthy female AA broilers were randomly divided into 4 groups, with 20 in each group, namely the negative control (group P), the E. coli Nissle 1917-treated group (group N), the S. Enteritidis-infected group (group S) and the E. coli Nissle 1917-treated and S. Enteritidis-infected group (group NS). From d 5 to 7, chicks in groups N and NS were orally gavaged once a day with E. coli Nissle 1917 and in groups P and S were administered the same volume of sterile PBS. At d 8, the chicks in groups S and NS were orally gavaged with S. Enteritidis and in groups P and N were administered the same volume of sterile PBS. Sampling was conducted 24 h after challenge. Results showed that gavage of E. coli Nissle 1917 reduced the spleen index, Salmonella loads, and inflammation (P < 0.05). It improved intestinal morphology and intestinal barrier function (P < 0.05). S. Enteritidis infection significantly reduced mRNA expression of angiotensin-converting enzyme 2 (ACE2) and solute carrier family 6-member 19 (SLC6A19) in the cecum and the content of Gly, Ser, Gln, and Trp in the serum (P < 0.05). Pretreatment with E. coli Nissle 1917 yielded mRNA expression of ACE2 and SLC6A19 in the cecum and levels of Gly, Ser, Gln, and Trp in the serum similar to that of uninfected chicks (P < 0.05). Additionally, E. coli Nissle 1917 altered cecum microbiota composition and enriched the abundance of E. coli, Lactobacillales, and Lachnospiraceae. These findings reveal that the probiotic E. coli Nissle 1917 reduced S. Enteritidis infection and shows enormous potential as an alternative to antibiotics.

Supplemental dietary genistein improves the laying performance and antioxidant capacity of Hy-Line brown hens during the late laying period.

The present study evaluated the effects of 3 supplemental levels of dietary genistein ingested during the late laying period (66-73 wk) of laying hens. A total of 384 Hy-Line brown hens (66 wk old) were randomly divided into 4 groups (6 replicates of 16 hens in each group), the basal diet group (CON), and groups for the basal diet supplemented with 80, 120, and 400 mg/kg of genistein, G1, G2, and G3, respectively. The results of the present study showed an increased laying rate in groups G2 and G3 (linear, P < 0.01), and decreased feed-egg ratios (linear, P < 0.05) and broken egg rate (P < 0.01) in all genistein-treated groups compared with the CON group. Moreover, the G2 group showed an increase in eggshell strength and ratio (linear, P < 0.05), whereas all genistein-treated groups saw a decrease in the L* value (linear, P < 0.01) and an increase in the a* value (linear, P < 0.05) compared with the CON group. Additionally, all genistein-treated groups had an increase in the total antioxidant capacity of plasma (linear, P < 0.05), along with reduced plasma, ovarian, and yolk malondialdehyde levels (linear, P < 0.05), compared with the CON group. The G2 group had an increase in both the superoxide dismutase activity of plasma (P < 0.01) and the total antioxidant capacity of the ovaries (linear, P < 0.05), compared with the CON group. The G3 group had an increase in both the glutathione peroxidase concentration of plasma (linear, P < 0.05) and the total antioxidant capacity of the ovaries (linear, P < 0.01), compared with the CON group. The transcript levels of nuclear factor erythroid 2-related factor 2, superoxide dismutase 1, and catalase were increased in all of the genistein-treated groups (P < 0.05) compared with the CON group, whereas heme oxygenase 1 and glutamate-cysteine ligase modifier subunit were increased only in the G2 group (P < 0.05). In conclusion, supplementation with 120 mg/kg dietary genistein had the best effect on improving the laying rate, eggshell quality, and antioxidant capacity in Hy-Line brown hens during the late laying period.

Correction to 'Cytotoxicity studies of Fe3O4 nanoparticles in chicken macrophage cells'.

[This corrects the article DOI: 10.1098/rsos.191561.].

Cytotoxicity studies of Fe3O4 nanoparticles in chicken macrophage cells.

Magnetic Fe3O4 nanoparticles (Fe3O4-NPs) have been widely investigated for their biomedical applications. The main purpose of this study was to evaluate the cytotoxic effects of different sizes of Fe3O4-NPs in chicken macrophage cells (HD11). Experimental groups based on three sizes of Fe3O4-NPs (60, 120 and 250 nm) were created, and the Fe3O4-NPs were added to the cells at different doses according to the experimental group. The cell activity, oxidative index (malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS)), apoptosis and pro-inflammatory cytokine secretion level were detected to analyse the cytotoxic effects of Fe3O4-NPs of different sizes in HD11 cells. The results revealed that the cell viability of the 60 nm Fe3O4-NPs group was lower than those of the 120 and 250 nm groups when the same concentration of Fe3O4-NPs was added. No significant difference in MDA was observed among the three Fe3O4-NP groups. The SOD level and ROS production of the 60 nm group were significantly greater than those of the 120 and 250 nm groups. Furthermore, the highest levels of apoptosis and pro-inflammatory cytokine secretion were caused by the 60 nm Fe3O4-NPs. In conclusion, the smaller Fe3O4-NPs produced stronger cytotoxicity in chicken macrophage cells, and the cytotoxic effects may be related to the oxidative stress and apoptosis induced by increased ROS production as well as the increased expression of pro-inflammatory cytokines.

Salmonella Interacts With Autophagy to Offense or Defense.

Autophagy is an important component of the innate immune system in mammals. Low levels of basic autophagy are sustained in normal cells, to help with the clearance of aging organelles and misfolded proteins, thus maintaining their structural and functional stability. However, when cells are faced with challenges, such as starvation or pathogenic infection, their level of autophagy increases significantly. Salmonella is a facultative intracellular pathogen, which imposes an economic burden on the poultry farming industry and human public health. Previous studies have shown that Salmonella can induce the autophagy of cells following invasion, which to a certain extent helps to protect the cells from bacterial colonization. This review summarizes the latest research in the field of Salmonella-induced autophagy, including: (i) the autophagy induction and escape mechanisms employed by Salmonella during the infection of host cells; (ii) the effect of autophagy on intracellular Salmonella; (iii) the important autophagy adaptors that recognize intracellular Salmonella in host cells; and (iv) the effect of autophagy-modulating drugs on Salmonella infection.

Effects of dietary N-carbamylglutamate supplementation on growth performance, tissue development and blood parameters of yellow-feather broilers.

The effects of N-carbamylglutamate (NCG) on the growth performance, tissue development, and blood parameters of broilers are unknown. In this study, 2 linked experiments were conducted to investigate the effects of 4 graded dietary levels and 3 dietary stages of NCG in a Chinese indigenous yellow-feather broiler breed during 2 growth phases: 1 to 18 d and 19 to 36 d. The dietary levels of NCG were 0.05%, 0.10%, 0.15%, and 0.20%, and dietary stages were designed to add NCG during the starter stage or grower stage or throughout the experimental period. At the age of 18 d, graded doses of NCG from 0.05 to 0.20% in the diet produced quadratic (P < 0.05) positive responses in body weight, width of intermuscular fat cingulum, liver weight, serum blood urea nitrogen, and serum low-density lipoprotein as well as linear (P < 0.05) positive responses in albumin serum concentration. The average feed per gain and mortality were unaffected by dietary NCG levels. Among 3 dietary treatments, only NCG dietary treatments throughout the experimental period improved the body weight and daily weight gain linearly (P < 0.05). The daily weight gain under the 3 dietary treatments used indicated that the most fitting dose is 0.1% NCG among the 4 dietary levels of NCG (P < 0.05). At this dose, muscle weight increased, whereas subcutaneous adipose as well as the serum contents of uric acid, triglyceride, and albumin decreased. Considering the growth performance and tissue development under the conditions used in this study, the best-fit model for NCG requirements of Chinese yellow-feather broilers was estimated from regression analysis to be 0.09 to 0.12% dietary NCG treatments during the grower stage. The modified blood parameters indicated that NCG dietary effects on broiler growth may be accompanied by modified homeostasis of arginine metabolism, lipid deposition, protein synthesis, and immune response.

Fe3O4 Nanoparticles Attenuated Salmonella Infection in Chicken Liver Through Reactive Oxygen and Autophagy via PI3K/Akt/mTOR Signaling.

Recently nanomaterials have received substantial attention in biotechnology areas for their innovative properties in physical and chemical function. One of the most arrestive properties of nanomaterials that has been reported is their bacteriostatic activity. Our previous research found that Fe3O4 magnetic nanoparticles (Fe3O4-NPs) could effectively reduce the viability of intracellular Salmonella Enteritidis in chicken cells. There is an essential need to explore whether the bacteriostatic activity of Fe3O4-NPs is available in vivo. As an extension of this research, we conducted the present study to investigate the potential effect of Fe3O4-NPs used for S. Enteritidis control in chickens and to extensively investigate the underlying mechanisms in the process. The overall study included the evaluation of pathological sections, antioxidant status, inflammation, and the autophagy status of chicken liver, including the signaling pathway involved in the process. Results indicated that Fe3O4-NPs pretreatment can effectively inhibit the invasion of S. Enteritidis in chicken liver. Fe3O4-NPs pretreatment significantly increased reactive oxygen species (ROS) generation in chickens, including antioxidant enzyme activities. S. Enteritidis infection significantly increased the protein expression of the autophagy marker LC3. Additionally, the inflammation response and pathological changes caused by S. Enteritidis infection were alleviated by Fe3O4-NPs pretreatment. Phosphorylated mTOR was significantly increased in S. Enteritidis infected chickens, but showed no difference in chickens pretreated with Fe3O4-NPs. In summary, the results demonstrated that ROS and autophagy were involved in the inhibition of S. Enteritidis in chickens by Fe3O4-NPs pretreatment. The redox balance and inflammation response appeared normal in the process, as did the expression of the PI3K/Akt/mTOR signaling pathways. Taken together, our research demonstrate that the bacteriostatic activity of Fe3O4-NPs in chickens is avaliable and safe, which can be an alternative to antibiotics for bacterial inhibition in poultry industry.

Iron oxide nanozyme suppresses intracellular Salmonella Enteritidis growth and alleviates infection in vivo.

Rational:Salmonella Enteritidis (S. Enteritidis) is a globally significant zoonotic foodborne pathogen which has led to large numbers of deaths in humans and caused economic losses in animal husbandry. S. Enteritidis invades host cells and survives within the cells, causing resistance to antibiotic treatment. Effective methods of elimination and eradication of intracellular S. Enteritidis are still very limited. Here we evaluated whether a new intracellular antibacterial strategy using iron oxide nanozymes (IONzymes) exerted highly antibacterial efficacy via its intrinsic peroxidase-like activity in vitro and in vivo.Methods: The antibacterial activities of IONzymes against planktonic S. Enteritidis, intracellular S. Enteritidis in Leghorn Male Hepatoma-derived cells (LMH), and liver from specific pathogen free (SPF) chicks were investigated by spread-plate colony count method and cell viability assay. Changes in levels of microtubule-associated protein light chain 3 (LC3), a widely used marker for autophagosomes, were analyzed by immunoblotting, immunofluorescence, and electron microscopy. Reactive oxygen species (ROS) production was also assessed in vitro. High-throughput RNA sequencing was used to investigate the effects of IONzymes on liver transcriptome of S. Enteritidis-infected chicks. Results: We demonstrated that IONzymes had high biocompatibility with cultured LMH cells and chickens, which significantly inhibited intracellular S. Enteritidis survival in vitro and in vivo. In addition, co-localization of IONzymes with S. Enteritidis were observed in autophagic vacuoles of LMH cells and liver of chickens infected by S. Enteritidis, indicating that IONzymes mediated antibacterial reaction of S. Enteritidis with autophagic pathway. We found ROS level was significantly increased in infected LMH cells treated with IONzymes, which might enhance the autophagic elimination of intracellular S. Enteritidis. Moreover, orally administered IONzymes decreased S. Enteritidis organ invasion of the liver and prevented pathological lesions in a chicken-infection model. Non-target transcriptomic profiling also discovered IONzymes could change hepatic oxidation-reduction and autophagy related gene expressions in the S. Enteritidis infected chickens. Conclusion: These data suggest that IONzymes can increase ROS levels to promote the antibacterial effects of acid autophagic vacuoles, and thus suppress the establishment and survival of invading intracellular S. Enteritidis. As a result, IONzymes may be a novel alternative to current antibiotics for the control of intractable S. Enteritidis infections.

Comprehensive evaluation of the role of soy and isoflavone supplementation in humans and animals over the past two decades.

Soy and soy-based foods are considered healthy, particularly in many Asia-Pacific countries, where soy products have long been consumed. Soy and soy-related products have been found to help prevent the occurrence of cardiovascular diseases and certain types of cancer, such as breast and prostate cancer. These products can also have antioxidative effects that alleviate hot flashes during menopause and bone loss. These biological and therapeutic functions are primarily due to the isoflavones derived from soy, whose structure is similar to the structure of 17-ß-oestradiol. Despite the many health benefits for humans and animals, the application of isoflavones remains controversial because of their anti-oestrogenic properties. We focused on general information regarding isoflavones, as well as their structure, function, and application. We summarized evidence showing that dietary or supplemental isoflavones exert protective effects on the health of humans and animals. Based on the literature, we conclude that soy foods and isoflavones may be effective and safe; however, more high-quality trials are needed to fully substantiate their potential use.

Synthesis and biological evaluation of novel hydrogen sulfide releasing nicotinic acid derivatives.

Twelve novel hybrids of slowly releasing hydrogen sulfide donor ADT-OH combined with nicotinic acid were synthesized. All of their structures had been confirmed by 1H NMR, 13C NMR and MS spectra. The target compounds were evaluated for their neuroprotective effects on hippocampal neuron HT22 cells against glutamate-induced injury at the concentrations of 1-100µM with MTT assay, and their toxicity on HT22 cells untreated by glutamine at the concentration of 100µM. The active compound was further investigated for its effect on ischemic infarct volume by intraperitoneal injection at 3h after ischemia in mice models of permanent middle cerebral artery occlusion (pMCAO). The results showed that all the compounds significantly protected HT22 cells from glutamate-induced damage at most of the experimental concentrations, and had no or little neurotoxicity on normal HT22 cells at the high concentration. More importantly, compound A6 significantly reduced infarct volume in the pMCAO model. These results suggested that compound A6 may be promising for further evaluation for the intervention of cerebral ischemic injury.

Hydrogen Sulfide Attenuates Tissue Plasminogen Activator-Induced Cerebral Hemorrhage Following Experimental Stroke.

Tissue plasminogen activator (tPA), the only approved drug for the treatment of ischemic stroke, increases the risk of cerebral hemorrhage. Here, we investigated whether the newly identified gaso-transmitter hydrogen sulfide (H2S), when used in combination with tPA, reduced the hemorrhagic transformation following stroke. In a mouse model of middle cerebral artery occlusion (MCAO), intravenous injection of tPA enhanced cerebral hemorrhage, which was significantly attenuated by the co-administration of two structurally unrelated H2S donors, ADT-OH and NaHS. By assessing extravasation of Evans blue into the ischemic hemisphere as well as brain edema following MCAO, we further showed that a tPA-exacerbated BBB disruption was significantly ameliorated by the co-administration of ADT-OH. In the mouse MCAO model, tPA upregulated Akt activation, vascular endothelial growth factor (VEGF) expression, and metalloproteinase 9 (MMP9) activity in the ischemic brain, which was remarkably attenuated by ADT-OH. In the in vitro glucose-oxygen deprivation (OGD) model, ADT-OH markedly attenuated tPA-enhanced Akt activation and VEGF expression in brain microvascular endothelial cells. Finally, ADT-OH improved functional outcomes in mice subjected to MCAO and tPA infusion. In conclusion, H2S donors reduced tPA-induced cerebral hemorrhage by possibly inhibiting the Akt-VEGF-MMP9 cascade. Administration of H2S donors has potential as a novel modality to improve the safety of tPA following stroke.

The 5-Lipoxygenase Inhibitor Zileuton Confers Neuroprotection against Glutamate Oxidative Damage by Inhibiting Ferroptosis.

5-Lipoxygenase (5-LOX) inhibitors have been shown to be protective in several neurodegenerative disease models; however, the underlying mechanisms remain unclear. We investigated whether 5-LOX inhibitor zileuton conferred direct neuroprotection against glutamate oxidative toxicity by inhibiting ferroptosis, a newly identified iron-dependent programmed cell death. Treatment of HT22 mouse neuronal cell line with glutamate resulted in significant cell death, which was inhibited by zileuton in a dose-dependent manner. Consistently, zileuton decreased glutamate-induced production of reactive oxygen species but did not restore glutamate-induced depletion of glutathione. Moreover, the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-fmk) neither prevented HT22 cell death induced by glutamate nor affected zileuton protection against glutamate oxidative toxicity, suggesting that zileuton did not confer neuroprotection by inhibiting caspase-dependent apoptosis. Interestingly, glutamate-induced HT22 cell death was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. Moreover, zileuton protected HT22 neuronal cells from erastin-induced ferroptosis. However, we did not observe synergic protective effects of zileuton and ferrostatin-1 on glutamate-induced cell death. These results suggested that both the 5-LOX inhibitor zileuton and the ferropotosis inhibitor ferrostatin-1 acted through the same cascade to protect against glutamate oxidative toxicity. In conclusion, our results suggested that zileuton protected neurons from glutamate-induced oxidative stress at least in part by inhibiting ferroptosis.

Sphingosine kinase 1 mediates neuroinflammation following cerebral ischemia.

Sphingosine kinases (Sphks) are the rate-limiting kinases in the generation of sphingosine-1-phosphate, which is a well-established intracellular pro-survival lipid mediator. Sphk2 has been reported to be protective following experimental stroke. We investigated the role of Sphk1 in cerebral ischemia using a mouse middle cerebral artery occlusion (MCAO) model and an in vitro glucose-oxygen deprivation (OGD) model. Sphk expression and activity were assessed in the ischemic brain with quantitative PCR (qPCR), Western blot, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Pharmacological and gene knockdown approaches were utilized to investigate the effects of Sphk1 on stroke outcomes. The expression of Sphk1 but not that of Sphk2 was rapidly induced in the cortical penumbra over 96h after MCAO, and the microglia were one of the major cellular sources of Sphk1 induction. Consistently, Sphk activity was enhanced in the cortical penumbra. In contrast to the protective role of Sphk2, pharmacological inhibition and cortical knockdown of Sphk1 reduced infarction at 24 and 96h after reperfusion. Additionally, the Sphk1 inhibitor improved the neurological deficits at 96h after reperfusion. Mechanistically, Sphk1 inhibition and knockdown significantly attenuated MCAO-induced expression of inflammatory mediators in the cortical penumbra. Moreover, using a conditioned medium transfer approach, we demonstrated that OGD-treated neurons induced the expression of Sphk1 and pro-inflammatory mediators in primary microglia, and the microglial induction of pro-inflammatory mediators by ischemic neurons was blunted by Sphk1 inhibition. Taken together, our results indicate that Sphk1 plays an essential role in mediating post-stroke neuroinflammation.

Hydrogen sulfide protects blood-brain barrier integrity following cerebral ischemia.

By using two structurally unrelated hydrogen sulfide (H2 S) donors 5-(4-methoxyphenyl) -3H-1, 2-dithiole-3-thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H2 S protected blood-brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H2 S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post-ischemic inflammation-induced Matrix Metalloproteimase-9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H2 S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H2 S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro-inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL-1ß while enhanced expression of anti-inflammatory markers arginase 1 and IL-10 in the ischemic brain. Accordingly, ADT attenuated ischemia-induced expression and activity of MMP9. Moreover, ADT reduced NOX-4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa-B (NF-κB) in the ischemic brain. In conclusion, H2 S donors protected BBB integrity following experimental stroke possibly by acting through NF-κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4-derived free radicals. To determine H2 S effects on blood-brain barrier (BBB) disruption following stroke, we used two structurally unrelated H2 S donors ADT and NaHS. Both ADT and NaHS remarkably protected BBB integrity following experimental stroke. The slow-releasing donor ADT also reduced post-ischemic inflammation-induced expression and activity of MMP9 and NOX4 in the ischemic brain possibly by inhibiting NF-κB activation.

Differential mechanisms underlying neuroprotection of hydrogen sulfide donors against oxidative stress.

This study investigated whether slow-releasing organic hydrogen sulfide donors act through the same mechanisms as those of inorganic donors to protect neurons from oxidative stress. By inducing oxidative stress in a neuronal cell line HT22 with glutamate, we investigated the protective mechanisms of the organic donors: ADT-OH [5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione], the most widely used moiety for synthesizing slow-releasing hydrogen sulfide donors, and ADT, a methyl derivative of ADT-OH. The organic donors were more potent than the inorganic donor sodium hydrogensulfide (NaHS) in protecting HT22 cells against glutamate toxicity. Consistent with previous publications, NaHS partially restored glutamate-depleted glutathione (GSH) levels, protected HT22 from direct free radical damage induced by hydrogen peroxide (H2O2), and NaHS protection was abolished by a KATP channel blocker glibenclamide. However, neither ADT nor ADT-OH enhanced glutamate-depleted GSH levels or protected HT22 from H2O2-induced oxidative stress. Glibenclamide, which abolished NaHS neuroprotection against oxidative stress, did not block ADT and ADT-OH neuroprotection against glutamate-induced oxidative stress. Unexpectedly, we found that glutamate induced AMPK activation and that compound C, a well-established AMPK inhibitor, remarkably protected HT22 from glutamate-induced oxidative stress, suggesting that AMPK activation contributed to oxidative glutamate toxicity. Interestingly, all hydrogen sulfide donors, including NaHS, remarkably attenuated glutamate-induced AMPK activation. However, under oxidative glutamate toxicity, compound C only increased the viability of HT22 cells treated with NaHS, but did not further increase ADT and ADT-OH neuroprotection. Thus, suppressing AMPK activation likely contributed to ADT and ADT-OH neuroprotection. In conclusion, hydrogen sulfide donors acted through differential mechanisms to confer neuroprotection against oxidative toxicity and suppressing AMPK activation was a possible mechanism underlying neuroprotection of organic hydrogen sulfide donors against oxidative toxicity.

Effects of repeated injection of kisspeptin-10 on the initiation of egg-laying in juvenile quail.

The kisspeptin (Kp) signaling pathway plays an essential role in the onset of reproduction in mammals. To investigate the effects of Kp on the initiation of egg laying in birds, juvenile female quail were given daily intraperitoneal injections of 300µl saline (control, Con), or 10nmol (low dosage, L) or 100nmol (high dosage, H) kisspeptin-10 (Kp-10) dissolved in 300µl saline for 3 weeks. The ratio of egg laying of quail in the L and H groups was notably increased compared to that of the Con group (P<0.01), which paralleled earlier ovarian growth and increases in circulating estrogen (E(2)) concentrations. In the hypothalamus, gonadotropin-releasing hormone-I (GnRH-I) mRNA expression was markedly up-regulated, whereas the level of gonadotropin-inhibiting hormone mRNA was down-regulated by high-dose Kp-10 (P<0.05). In the pituitary gland, expression of GnRH receptor type II, but not type I mRNA was significantly up-regulated by high-dose Kp-10 administration (P<0.05). Moreover, compared with the Con group, follicle-stimulating hormone (FSH) gene expression in the pituitary was significantly decreased in the L and H groups (P<0.05), whereas luteinizing hormone (LH) mRNA expression was significantly increased in the H, but not the L group (P<0.05). These results indicate that repeated peripheral Kp-10 injections can advance the sexual maturation of female quail by regulating the activities of the hypothalamus-pituitary-gonadal axis.