Crotonylation of NAE1 Modulates Cardiac Hypertrophy via Gelsolin Neddylation.

BACKGROUND: Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8 [neural precursor cell expressed developmentally downregulated protein 8]-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy. METHODS: Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. Tandem mass tag (TMT)-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 (lysine residue at site 238) crotonylation. RESULTS: The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy. CONCLUSIONS: Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

The function and therapeutic potential of transfer RNA-derived small RNAs in cardiovascular diseases: A review.

Transfer RNA-derived small RNAs (tsRNAs) are a class of small non-coding RNA (sncRNA) molecules derived from tRNA, including tRNA derived fragments (tRFs) and tRNA halfs (tiRNAs). tsRNAs can affect cell functions by participating in gene expression regulation, translation regulation, intercellular signal transduction, and immune response. They have been shown to play an important role in various human diseases, including cardiovascular diseases (CVDs). Targeted regulation of tsRNAs expression can affect the progression of CVDs. The tsRNAs induced by pathological conditions can be detected when released into the extracellular, giving them enormous potential as disease biomarkers. Here, we review the biogenesis, degradation process and related functional mechanisms of tsRNAs, and discuss the research progress and application prospects of tsRNAs in different CVDs, to provide a new perspective on the treatment of CVDs.

[Ginsenoside Rg3 Regulates Cisplatin Resistance in Gastric Cancer by Wnt/ß-catenin Signaling Pathway].

Objective To investigate the inhibitory effect of ginsenoside Rg3 combined with cisplatin (DDP) on DDP-resistant cell line SGC-7901/DDP and their molecular mechanism.Methods SGC-7901/DDP cells were divided into four groups including a control group,a ginsenoside Rg3 (40 µg/ml) treatment group,a DDP (1.40 µg/ml) treatment group,and a drug combination treatment group.The proliferation ability of SGC-7901/DDP cells was detected by MTT,EdU,and colony formation assays.The apoptosis ability of SGC-7901/DDP cell was detected by flow cytometry and Hoechst 33342 staining.The protein levels of apoptosis-related markers were detected by Western blotting.The migration ability of SGC-7901/DDP cells was detected by wound healing and Transwell assays.The expression levels of proteins in epithelial-mesenchymal transformation (EMT) and Wnt/ß-catenin signaling pathway were determined by Western blotting and immunofluorescence staining.Results Compared with the ginsenoside Rg3 or the DDP treatment groups,the drug combination treatment group inhibited the proliferation (t=8.062,P=0.001;t=7.090,P=0.002),colony formation (t=8.062,P=0.001;t=6.144,P=0.004),and migration (t=7.424,P=0.002;t=4.317,P=0.013),and promoted the apoptosis (t=5.530,P=0.031;t=6.036,P=0.026) of SGC-7901/DDP cells.Compared with the ginsenoside Rg3 and the DDP treatment groups,the drug combination treatment group down-regulated the expression levels of EMT-associated proteins including vimentin (t=24.450,P<0.001;t=14.750,P<0.001),Snail (t=29.640,P<0.001;t=70.700,P<0.001),Slug (t=89.230,P<0.001;t=87.360,P<0.001),matrix metalloproteinase (MMP) 2 (t=84.540,P<0.001;t=67.120,P<0.001),and MMP9 (t=19.010,P<0.001;t=10.890,P<0.001),as well as those of Wnt/ß-catenin signaling pathway related proteins including Wnt (t=35.480,P<0.001;t=14.670,P<0.001),ß-catenin (t=155.800,P<0.001;t=118.100,P<0.001),C-myc (t=20.870,P<0.001;t=3.334,P=0.029),and cyclin D1 (t=5.007,P=0.008;t=8.347,P=0.001).Meanwhile,it up-regulated the expression of epithelial cells including E-cadherin (t=36.450,P<0.001;t=33.810,P<0.001) and ZO-1 (t=37.060,P<0.001;t=37.030,P<0.001).Conclusion Ginsenoside Rg3 enhanced the sensitivity of SGC-7901/DDP cells to DDP by inhibiting the activity of Wnt/ß-catenin signaling pathway.

Classification of induced malaria case in an elimination setting: investigation of transfusion-transmitted malaria cases.

BACKGROUND: Since the National Malaria Elimination Action Plan was launched in China in 2010, local malaria transmission has decreased rapidly. Zero indigenous cases were reported since 2017. However, after 2010, the proportion of imported cases in China increased from 45.7% in 2010 to 99.9% in 2016, and almost all provinces of China have reported imported cases in recent years. Prevention of the reintroduction of malaria into China is crucial for the maintenance of its malaria-free status. Hence, it is of utmost importance to correctly identify the source of malaria infections within the country. CASE INTRODUCTION AND RESPONSE: In 2016 and 2017, three laboratory-confirmed cases of malaria caused by Plasmodium falciparum were identified in patients with no previous travel history to endemic areas were reported in Jiangsu Province, China, where malaria due to P. falciparum was eliminated about 30 years ago. These were diagnosed after 41, 31 and 39 days of seeking treatment, respectively, and all of them had received blood transfusions. Further investigations indicated that two of the cases had received blood from foreign students (from Indonesia and Ghana), and the other had received blood from an individual who had worked in Equatorial Guinea. All three blood donors were traced, and found to be carrying asymptomatic P. falciparum infections by microscopic examination and PCR. Furthermore, five polymorphic microsatellite markers (C1M4, C4M62, C13M13, C14M17, and C13M63) were typed and used to link parasites from the donors with those of the transfusion-receiving patients. CONCLUSIONS: Three transfusion-transmitted malaria cases were identified in China, all of which were due to the transfusion of blood donated by individuals who had contracted malaria outside the country. These cases can provide a reference for those faced with similar challenges in malaria case identification and classification in other regions. In addition, a stricter screening policy including the use of appropriate detection methods for malaria parasites should be developed and adopted for blood donation in regions undergoing malaria elimination.

Effect of scrotal heating on sperm quality, seminal biochemical substances, and reproductive hormones in human fertile men.

At present, male contraceptive methods are only vasectomy and condoms, so it is necessary to research on male contraceptive techniques. The aim of this study is to observe the effects of scrotal heating (SH) on semen parameters, seminal l-carnitine (LC), epidermal growth factor (EGF), macrophage migration inhibitory factor (MIF), reproductive hormones and sperm chromosome numbers of adult healthy men, and to provide the experimental data for male contraception. The scrotums of 30 healthy male volunteers were exposed to the condition of 40 to 43°C SH belt warming 40 minutes each day for successive 2 days per week. The course of SH was continuous for 3 months. Computer-assisted semen analysis and hypo-osmotic swelling test, sperm DNA integrity, l-carnitine, MIF and EGF, and sperm fluorescence in situ hybridization were performed before, during, and after SH. The serum level of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were measured by chemiluminescent immunoassay. The mean parameters of sperm concentration, vitality, and normal morphological sperm were significantly decreased in groups with sperms being collected during 1, 2, and 3 months of SH when compared with those in groups of pre-SH (P < 0.01). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane functionality, levels of LC and MIF in semen, and LH, FSH, and T in serum were observed between the groups of before SH and after SH 3 months and the groups of during SH 1, 2, and 3 months (P < 0.001). The total rate of chromosome number for 13, 18, 21, X, and Y in the 3 months of SH was 13.7-fold greater (13.72%/1.69%) than before SH (P < 0.001). The constant SH can impact the semen quality, sperm DNA integrity, sperm chromosome, LC and MIF, and LH, FSH, and T in serum. Transient SH may be a new method for male contraception.

[Yam polysaccharide improves sperm viability and protects sperm DNA integrity in vitro].

OBJECTIVE: To clarify the roles of yam polysaccharide (YPS) in improving sperm viability and protecting sperm DNA integrity in vitro and provide a new approach to the treatment of oligoasthenozoospermia. METHODS: We collected samples by masturbation from 36 normal fertile males aged 27－39 years. Each sample was divided into six groups: blank control or treated with normal saline, vitamin C solution, and YPS solution at low (0.25 mg/ml), medium (1.0 mg/ml) or high concentration (5.0 mg/ml). Using eosin-Y staining, sperm hypotonic swelling (HOS) and sperm chromatin diffusion (SCD) test, we observed the effects of different concentrations of YPS on sperm viability, membrane integrity and nuclear DNA. RESULTS: After 24 and 48 hours of treatment, sperm viability was markedly reduced in the vitamin C (ï¼»28.5 ± 3.1ï¼½ and ï¼»6.5 ± 1.2ï¼½%), low-YPS (ï¼»31.3 ± 3.5ï¼½ and ï¼»6.5 ± 2.2ï¼½%), medium-YPS (ï¼»37.1 ± 3.5ï¼½ and ï¼»9.5 ± 2.8ï¼½%) and high-YPS groups (ï¼»38.3 ± 3.3ï¼½ and ï¼»9.0 ± 3.2ï¼½%) as compared with the blank control (ï¼»17.3 ± 2.1ï¼½ and ï¼»3.2 ± 1.3ï¼½%) (P <0.01) and normal saline groups (ï¼»13.4 ± 4.1ï¼½ and ï¼»3.1 ± 2.0ï¼½%) (P <0.01), and it was significantly higher in the medium- and high-YPS than in the vitamin C group (P <0.05 and P <0.01). The rate of sperm DNA fragmentation was remarkably decreased at 48 hours in the vitamin C (ï¼»30.5 ± 3.1ï¼½%), low-YPS (ï¼»29.4 ± 2.6ï¼½%), medium-YPS (ï¼»28.5 ± 2.3ï¼½%) and high-YPS groups (ï¼»27.9 ± 1.9ï¼½%) in comparison with the blank control (ï¼»41.7 ± 2.2ï¼½%) (P <0.01) and normal saline groups (ï¼»42.1 ± 3.3ï¼½%), markedly lower in the medium- and high-YPS than in the blank control, normal saline and vitamin C groups (P <0.05 or P <0.01), but with no statistically significant difference between the low-YPS and vitamin C groups (P >0.05). CONCLUSIONS: Yam polysaccharide can improve sperm viability and protect sperm DNA integrity in vitro.

Molecular mechanisms of myocardial nitroso-redox imbalance during on-pump cardiac surgery.

BACKGROUND: The mechanism responsible for left ventricular dysfunction after cardiac surgery is only partly understood. In isolated rat hearts subjected to an ischaemia-reperfusion protocol, left ventricular dysfunction was associated with uncoupling of endothelial nitric oxide synthase (NOS) activity secondary to oxidation of the NOS cofactor, tetrahydrobiopterin (BH4). Here we investigated the effect of cardiopulmonary bypass and reperfusion on myocardial nitroso-redox balance in patients undergoing cardiac surgery. METHODS: From 116 patients who underwent elective cardiac surgery on cardiopulmonary bypass, paired samples of the right atrial appendages were obtained before venous cannulation of the right atrium and after myocardial reperfusion. Superoxide production from atrial samples was measured by lucigenin (5 µmol/L) enhanced chemiluminescence and 2-hydroxyethidium (2-OHE) detection by high-performance liquid chromatography (HPLC). BH4, oxidised biopterins, GTP-cyclohydrolase 1 (GTPCH-1, the rate-limiting enzyme in BH4 synthesis), and NOS activity ((14)C L-arginine to L-citrulline conversion) were measured by HPLC. FINDINGS: Atrial superoxide production increased significantly after reperfusion (from mean 37·83 relative light units per s per mg [SE 3·71] before cannulation to 65·02 [6·01] after reperfusion, p<0·0001; n=46 samples from 23 patients) due to increased mitochondrial and NOX2 oxidase activity (by 309% and 149%; p=0·002 and p=0·0002, respectively) and uncoupling of NOS activity. Atrial content of BH4 after perfusion was reduced (by 32%, p=0·001), as was activity of GTPCH1 (50%, p<0·0001). NOS activity decreased significantly after reperfusion (60%, p=0·0005) and this reduction was not affected by BH4 supplementation (10 µM) or NOX2 inhibition ex vivo. Instead, we identified increased endothelial NOS s-glutathionylation as the main mechanism for NOS uncoupling after reperfusion. Reversing NOS s-glutathionylation with dithiothreitol (100 µmol/L) completely restored NOS activity after reperfusion (p=0·34). INTERPRETATION: Our findings suggest that NOS s-glutathionylation, rather than BH4 depletion, accounts for NOS dysfunction in patients after cardiac surgery and cardiopulmonary bypass. FUNDING: British Heart Foundation.

Scrotal heat stress causes sperm chromatin damage and cysteinyl aspartate-spicific proteinases 3 changes in fertile men.

PURPOSE: To observe changes in semen parameters, sperm DNA integrity, chromatin condensation and cysteinyl aspartate-spicific proteinases (Caspase-3) in adult healthy men after scrotal heat stress (SHS). METHODS: The scrotums of 19 healthy male volunteers were exposed to the condition of 40-43 °C SHS belt warming 40 min each day for successive 2 days per week. The course of SHS was continuously 3 months. Routine semen analysis, hypo-osmotic swelling (HOS) test, eosin Y (EY) staining sperm HOS and chromatin dispersion (HOS/SCD) test, HOS and aniline blue (HOS/AB) staining test were carried out before, during and after SHS. The activated Caspase 3 levels of spermatozoa were determined with a microtiter plate reader. RESULTS: The mean parameters of sperm concentration, motility and normal morphological sperm were significantly decreased in groups with sperm being collected during SHS 1, 2 and 3 months when compared with those in groups of pre-SHS (P < 0.01). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane and vitality, and Caspase-3 activity were observed between the groups of before SHS and after SHS 3 months and the groups of during SHS 1, 2 and 3 months (P < 0.001). Three months the SHS stopped, various parameters recovered to the level before SHS. Abnormal sperm with HOS/AB and HOS/SCD showed a negatively significant correlation with normal sperm by HOS/EY test, and WBC in semen showed a positively significant correlation with Caspase-3 activity. The percentage of abnormal sperm by using the test of HOS/SCD showed a positively significant correlation with that of HOS/AB. CONCLUSIONS: The continuously constant SHS can impact the semen quality, sperm DNA integrity, chromatin condensation and Caspase-3, and the combination of HOS plus AB test may simultaneously determine the integrity of membrane and chromatin condensation at the same spermatozoon.

CD34+ VEGFR-3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells.

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34(+) VEGFR-3(+) EPCs were isolated from mononuclear cells of human cord blood by fluorescence-activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7-10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF-C for 2 weeks, CD34(+) VEGFR-3(+) EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5'-nucleotidase, LYVE-1 and Prox-1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary-like structures in three-dimensional collagen gel and Matrigel. VEGF-C enhanced VEGFR-3 mRNA expression. After interfering with VEGFR-3 siRNA, the effects of VEGF-C were diminished. These results demonstrate that there is a population of CD34(+) VEGFR-3(+) EPCs with lymphatic potential in human cord blood. VEGF-C/VEGFR-3 signalling pathway mediates differentiation of CD34(+) VEGFR-3(+) EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood-derived CD34(+) VEGFR-3(+) EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.

Cardiomyocyte GTP cyclohydrolase 1 and tetrahydrobiopterin increase NOS1 activity and accelerate myocardial relaxation.

RATIONALE: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOS). Oral BH4 supplementation preserves cardiac function in animal models of cardiac disease; however, the mechanisms underlying these findings are not completely understood. OBJECTIVE: To study the effect of myocardial transgenic overexpression of the rate-limiting enzyme in BH4 biosynthesis, GTP cyclohydrolase 1 (GCH1), on NOS activity, myocardial function, and Ca2+ handling. METHODS AND RESULTS: GCH1overexpression significantly increased the biopterins level in left ventricular (LV) myocytes but not in the nonmyocyte component of the LV myocardium or in plasma. The ratio between BH4 and its oxidized products was lower in mGCH1-Tg, indicating that a large proportion of the myocardial biopterin pool was oxidized; nevertheless, myocardial NOS1 activity was increased in mGCH1-Tg, and superoxide release was significantly reduced. Isolated hearts and field-stimulated LV myocytes (3 Hz, 35°C) overexpressing GCH1 showed a faster relaxation and a PKA-mediated increase in the PLB Ser16 phosphorylated fraction and in the rate of decay of the [Ca2+]i transient. RyR2 S-nitrosylation and diastolic Ca2+ leak were larger in mGCH1-Tg and ICa density was lower; nevertheless the amplitude of the [Ca2+]i transient and contraction did not differ between genotypes, because of an increase in the SR fractional release of Ca2+ in mGCH1-Tg myocytes. Xanthine oxidoreductase inhibition abolished the difference in superoxide production but did not affect myocardial function in either group. By contrast, NOS1 inhibition abolished the differences in ICa density, Ser16 PLB phosphorylation, [Ca2+]i decay, and myocardial relaxation between genotypes. CONCLUSIONS: Myocardial GCH1 activity and intracellular BH4 are a limiting factor for constitutive NOS1 and SERCA2A activity in the healthy myocardium. Our findings suggest that GCH1 may be a valuable target for the treatment of LV diastolic dysfunction.

Development and application of an AllGlo probe-based qPCR assay for detecting knockdown resistance (kdr) mutations in Anopheles sinensis.

BACKGROUND: Anopheles sinensis is one of the most important malaria vectors in China and other Southeast Asian countries. High levels of resistance have been reported in this species due to the long-term use of insecticides, especially pyrethroids, for public health and agricultural purposes. Knockdown resistance (kdr) caused by a single base pair mutation in the gene encoding the sodium channel is strongly associated with pyrethroid insecticide resistance in many Anopheles mosquitoes. There are few methods currently available for detecting kdr mutations in An. sinensis. METHODS: A novel AllGlo probe-based qPCR (AllGlo-qPCR) method was developed to screen for the predominant kdr mutations in An. sinensis mosquitoes from the Jiangsu Province. The results from AllGlo-qPCR, allele-specific PCR (AS-PCR), and TaqMan-MGB probe-based qPCR (TaqMan-qPCR) were compared. A comparative analysis of the equipment required, ease of use and cost of the available methods was also performed. Finally, the AllGlo-qPCR method was used to detect the frequencies of kdr mutations from the other four provinces in central China. RESULTS: Six kdr genotypes were detected in An. sinensis from the Jiangsu Province by DNA sequencing. The AllGlo-qPCR method detected all of the kdr genotypes with a high level of accuracy (97% sensitivity and 98% specificity). AllGlo-qPCR correctly determined the kdr genotypes of 98.73% of 158 An. sinensis samples, whereas TaqMan-qPCR and AS-PCR correctly identified 96.84% and 88.61% of mutations, respectively. Furthermore, the AllGlo-qPCR method is simpler to perform, requires less equipment, and exhibits a moderate expense cost comparing with the other tested methods of kdr mutation detection. Samples collected from four of the other provinces in central China showed a high frequency of kdr mutation in An. sinensis, as detected by the established AllGlo-qPCR method. CONCLUSION: The novel AllGlo-qPCR method developed for kdr mutation detection in An. sinensis exhibits greater specificity and sensitivity than currently available methods and is more cost-effective; therefore, it represents a useful tool for entomological surveillance.

Recent advances of PIWI-interacting RNA in cardiovascular diseases.

BACKGROUND: The relationship between noncoding RNAs (ncRNAs) and human diseases has been a hot topic of research, but the study of ncRNAs in cardiovascular diseases (CVDs) is still in its infancy. PIWI-interacting RNA (piRNA), a small ncRNA that binds to the PIWI protein to maintain genome stability by silencing transposons, was widely studied in germ lines and stem cells. In recent years, piRNA has been shown to be involved in key events of multiple CVDs through various epigenetic modifications, revealing the potential value of piRNA as a new biomarker or therapeutic target. CONCLUSION: This review explores origin, degradation, function, mechanism and important role of piRNA in CVDs, and the promising therapeutic targets of piRNA were summarized. This review provide a new strategy for the treatment of CVDs and lay a theoretical foundation for future research. KEY POINTS: piRNA can be used as a potential therapeutic target and biomaker in CVDs. piRNA influences apoptosis, inflammation and angiogenesis by regulating epigenetic modificaions. Critical knowledge gaps remain in the unifying piRNA nomenclature and PIWI-independent function.

An aquaporin and an aquaglyceroporin have roles in low temperature adaptation of mosquitoes (Anopheles sinensis).

Mosquitoes (Anopheles sinensis), widely geographically distributed in Asia including China, are the primary vector of the malaria parasite Plasmodium vivax and other parasitic diseases such as Malayan filariasis. An. sinensis can survive through low winter temperatures. Aquaporin channels are found in all life forms, where they facilitate environmental adaptation by allowing rapid trans-cellular movement of water (classical aquaporins) or water and solutes such as glycerol (aquaglyceroporins). Here, we identified and characterized 2 aquaporin (AQP) homologs in An. sinensis: AsAQP2 (An. sinensis aquaglyceroporin) and AsAQP4 (An. sinensis aquaporin). When expressed in frog (Xenopus laevis) oocytes, AsAQP2 transported water, glycerol, and urea; AsAQP4 transported only water. Water permeation through AsAQP2 and AsAQP4 was inhibited by mercuric chloride. AsAQP2 expression was slightly higher in adult female mosquitoes than in males, and AsAQP4 expression was significantly higher in adult males. The 2 AsAQPs were highly expressed in Malpighian tubules and midgut. AsAQP2 and AsAQP4 expression was up-regulated by blood feeding compared with sugar feeding. At freezing point (0 °C), the AsAQP4 expression level increased and An. sinensis survival time reduced compared with those at normal temperature (26 °C). At low temperature (8 °C), the AsAQP2 and AsAQP4 expression levels decreased and survival time was significantly longer compared with those at 26 °C. These results suggest that AsAQP2 and AsAQP4 have roles in water homeostasis during blood digestion and in low temperature adaptation of A. sinensis. Together, our results show that the 2 AQPs are important for mosquito diuresis after blood feeding and when exposed to low temperatures.

Regulation of endothelial nitric-oxide synthase (NOS) S-glutathionylation by neuronal NOS: evidence of a functional interaction between myocardial constitutive NOS isoforms.

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial ß(3)-adrenergic receptor (ß(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in ß(3)-AR signaling and found that the ß(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-L-thiocitrulline. LV superoxide (O(2)(·-)) production was increased in nNOS(-/-) mice and reduced by L-N(ω)-nitroarginine methyl ester (L-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O(2)(·-), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to ß(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)(·-) production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of ß(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.

[Epithelioid hemangioma: a clinicopathologic analysis of 7 cases].

OBJECTIVE: To study the clinicopathologic features, diagnosis and differential diagnosis of epithelioid hemangioma. METHODS: The morphologic features of 7 cases of epithelioid hemangioma of skin, bone and venous vessels were studied. RESULTS: There were altogether 4 male and 3 female patients (median age = 34 years; age range from 14 to 54 years). The 3 skin cases presented as single or multiple erythematous to bluish nodules or papules, with or without itchiness. The 2 bone cases appeared as osteolytic expansile lesions on radiologic examination. The remaining 2 cases involved medium-sized venous structures and presented as small isolated nodules in soft tissue. Histologically, the lesions were characterized by the presence of exuberant endothelial proliferations with various degree of inflammatory reaction. The neoplastic endothelial cells were plump, eosinophilic and polygonal, forming vascular channels. Occasional solid sheet-like arrangement was demonstrated. Intracytoplasmic vacuoles were commonly identified, indicating formation of primary lumen. The surrounding stroma contained various number of eosinophils and lymphoplasmacytic cells. Immunohistochemical study showed that the tumor cells were positive for endothelial markers (CD31 and CD34) and negative for epithelial marker (cytokeratin). Follow-up information was available in 6 cases. The duration of follow-up ranged from 5 to 36 months (median = 14 months). There was no evidence of recurrence or distant metastasis. CONCLUSIONS: Epithelioid hemangioma is a rare benign curable lesion which can be multifocal, involving skin, soft tissue and bone. It needs to be distinguished from Kimura's disease and epithelioid hemangioendothelioma.

Male reproductive system and simulated high-altitude environment: preliminary results in rats.

This study assessed the effects of a simulated high-altitude environment on the reproductive system of prepubertal male rats and the reversibility of these effects upon return to a normal environment. Three-week-old male Wistar rats were randomly allocated to 4 groups that were exposed to different conditions: a normal environment for 6 weeks and 12 weeks, respectively, hypobaric hypoxia for 6 weeks, and hypobaric hypoxia for 6 weeks followed by a normal environment for 6 weeks. Multiple pathophysiological parameters were evaluated at the histological, endocrine, and molecular levels. Hypobaric hypoxia exposure for 6 weeks during the prepubertal phase significantly altered physiological parameters, body functions, blood indices, and reproductive potential. Six weeks after returning to a normal environment, the damaged reproductive functions partially recovered due to compensatory mechanisms. However, several changes were not reversed after returning to a normal environment for 6 weeks, including disorders of body development and metabolism, increased red blood cells, increased fasting blood glucose, abnormal blood lipid metabolism, decreased testicular and epididymis weights, abnormal reproductive hormone levels, excessive apoptosis of reproductive cells, and decreased sperm concentration. In summary, a hypobaric hypoxic environment significantly impaired the reproductive function of prepubertal male rats, and a return to normal conditions during the postpubertal phase did not fully recover these impairments.

Rapid, direct effects of statin treatment on arterial redox state and nitric oxide bioavailability in human atherosclerosis via tetrahydrobiopterin-mediated endothelial nitric oxide synthase coupling.

BACKGROUND: Treatment with statins improves clinical outcome, but the exact mechanisms of pleiotropic statin effects on vascular function in human atherosclerosis remain unclear. We examined the direct effects of atorvastatin on tetrahydrobiopterin-mediated endothelial nitric oxide (NO) synthase coupling in patients with coronary artery disease. METHODS AND RESULTS: We first examined the association of statin treatment with vascular NO bioavailability and arterial superoxide (O(2)(·-)) in 492 patients undergoing coronary artery bypass graft surgery. Then, 42 statin-naïve patients undergoing elective coronary artery bypass graft surgery were randomized to atorvastatin 40 mg/d or placebo for 3 days before surgery to examine the impact of atorvastatin on endothelial function and O(2)(·-) generation in internal mammary arteries. Finally, segments of internal mammary arteries from 26 patients were used in ex vivo experiments to evaluate the statin-dependent mechanisms regulating the vascular redox state. Statin treatment was associated with improved vascular NO bioavailability and reduced O(2)(·-) generation in internal mammary arteries. Oral atorvastatin increased vascular tetrahydrobiopterin bioavailability and reduced basal and N-nitro-l-arginine methyl ester-inhibitable O(2)(·-) in internal mammary arteries independently of low-density lipoprotein lowering. In ex vivo experiments, atorvastatin rapidly improved vascular tetrahydrobiopterin bioavailability by upregulating GTP-cyclohydrolase I gene expression and activity, resulting in improved endothelial NO synthase coupling and reduced vascular O(2)(·-). These effects were reversed by mevalonate, indicating a direct effect of vascular hydroxymethylglutaryl-coenzyme A reductase inhibition. CONCLUSIONS: This study demonstrates for the first time in humans the direct effects of statin treatment on the vascular wall, supporting the notion that this effect is independent of low-density lipoprotein lowering. Atorvastatin directly improves vascular NO bioavailability and reduces vascular O(2)(·-) through tetrahydrobiopterin-mediated endothelial NO synthase coupling. These findings provide new insights into the mechanisms mediating the beneficial vascular effects of statins in humans. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01013103.

Sperm chromosomal aneuploidy and DNA integrity of infertile men with anejaculation.

PURPOSE: To explore sperm chromosomal aneuploidy, sperm membrane and DNA integrity in infertile patients with anejaculation. METHODS: Semen samples were collected from 18 infertile men with spinal cord injury (SCI) by penile vibratory stimulation (PVS) and from 14 psychogenic anejaculation (PA) patients by percutaneous vasal sperm aspiration (PVSA). These semen samples as well as samples from 16 donors were analyzed using the hypo-osmotic swelling (HOS) test, the sperm chromatin dispersion (SCD) test and multi-color fluorescence in situ hybridization (FISH) with probes specific for chromosomes 13, 18, 21, X and Y. RESULTS: There were significant differences in the percentages of motile sperm, normal morphologic sperm and sperm DNA fragmentation between the infertile men with SCI and the control group (P < 0.05 and P < 0.01). The sperm motility was significantly greater in the PA-PVSA group than in the SCI-PVS group (P < 0.01). The number of round cells per mL of semen obtained from the 18 SCI patients by PVS was between 1 and 8 million. The rate of sperm DNA fragmentation in the SCI-PVS group was higher than that of the PA-PVSA group (P < 0.05). The aneuploidy rates for the SCI patients were 2.4-fold higher for chromosomes 13, 18 and 21 and 2.2-fold higher for chromosomes X and Y than for patients in the control group (P < 0.0001). CONCLUSIONS: The semen quality is poorer, sperm DNA fragmentation and sperm chromosomal aneuploidies are seen at a higher rate for SCI patients compared to healthy, fertile and normospermic men. Whether the difference in yield is due to increased scrotal temperature, genitourinary infection, or other reasons requires further study.

Exploring cryptic biodiversity in a world heritage site: a new pitviper (Squamata, Viperidae, Crotalinae) from Jiuzhaigou, Aba, Sichuan, China.

This study presents a comprehensive morphological comparison along with molecular phylogeny of the genus Gloydius based on five mitochondrial genes (12S, 16S, COI, cytb, and ND4). The specimens collected from Jiuzhaigou National Nature Reserve are shown to be a new species, Gloydiuslateralis sp. nov. Zhang, Shi, Jiang & Shi based on a combination of morphological and molecular accounts. G.lateralis sp. nov. differs from other congeneric species by a series of diagnostic morphological characteristics and forms a strongly supported monophyletic group. The new species is phylogenetically closely related to G.swild, another recently described species from Heishui, Aba, Sichuan.

Preoperative atorvastatin treatment in CABG patients rapidly improves vein graft redox state by inhibition of Rac1 and NADPH-oxidase activity.

BACKGROUND: Statins improve clinical outcome of patients with atherosclerosis, but their perioperative role in patients undergoing coronary artery bypass grafting (CABG) is unclear. We hypothesized that short-term treatment with atorvastatin before CABG would improve the redox state in saphenous vein grafts (SVGs), independently of low-density lipoprotein cholesterol (LDL)-lowering. METHODS AND RESULTS: In a randomized, double-blind controlled trial, 42 statin-naïve patients undergoing elective CABG received atorvastatin 40 mg/d or placebo for 3 days before surgery. Circulating inflammatory markers and malondialdehyde (MDA) were measured before and after treatment. SVG segments were used to determine vascular superoxide (O(2)(*-)) and Rac1 activation. For ex vivo studies, SVG segments from 24 patients were incubated for 6 hours with atorvastatin 0, 5, or 50 µmol/L. Oral atorvastatin reduced vascular basal and NADPH-stimulated O(2)(*-) in SVGs (P<0.05 for all versus placebo) and reduced plasma MDA (P<0.05), independently of LDL-lowering and of changes in inflammatory markers. In SVGs exposed to atorvastatin ex vivo, without exposure to LDL, basal and NADPH-stimulated O(2)(·-) were significantly reduced (P<0.01 for both concentrations versus 0 µmol/L) in association with a striking reduction in Rac1 activation and 1 membrane-bound Rac1 and p67(phox) subunit. The antioxidant effects of atorvastatin were reversed by mevalonate, implying a dependence on vascular HMG-CoA reductase inhibition. CONCLUSIONS: Short-term treatment with atorvastatin 40 mg/d before CABG improves redox state in SVGs, by inhibiting vascular Rac1-mediated activation of NADPH-oxidase. These novel findings suggest that statin therapy should be maintained or initiated in patients undergoing CABG, independently of LDL levels. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01013103.