Emergence of amoxicillin resistance and identification of novel mutations of the pbp1A gene in Helicobacter pylori in Vietnam.

BACKGROUND: Amoxicillin-resistant Helicobacter pylori (H. pylori) strains seem to have increased over time in Vietnam. This threatens the effectiveness of H. pylori eradication therapies with this antibiotic. This study aimed to investigate the prevalence of primary resistance of H. pylori to amoxicillin and to assess its association with pbp1A point mutations in Vietnamese patients. MATERIALS AND METHODS: Naive patients who presented with dyspepsia undergoing upper gastrointestinal endoscopy were recruited. Rapid urease tests and PCR assays were used to diagnose H. pylori infection. Amoxicillin susceptibility was examined by E-tests. Molecular detection of the mutant pbp1A gene conferring amoxicillin resistance was carried out by real-time PCR followed by direct sequencing of the PCR products. Phylogenetic analyses were performed using the Tamura-Nei genetic distance model and the neighbor-joining tree building method. RESULTS: There were 308 patients (46.1% men and 53.9% women, p = 0.190) with H. pylori infection. The mean age of the patients was 40.5 ± 11.4 years, ranging from 18 to 74 years old. The E-test was used to determine the susceptibility to amoxicillin (minimum inhibitory concentration (MIC) ≤ 0.125 µg/ml) in 101 isolates, among which the rate of primarily resistant strains to amoxicillin was 25.7%. Then, 270 sequences of pbp1A gene fragments were analysed. There were 77 amino acid substitution positions investigated, spanning amino acids 310-596, with the proportion varying from 0.4 to 100%. Seven amino acid changes were significantly different between amoxicillin-sensitive (AmoxS) and amoxicillin-resistant (AmoxR) samples, including Phe366 to Leu (p <  0.001), Ser414 to Arg (p <  0.001), Glu/Asn464-465 (p = 0.009), Val469 to Met (p = 0.021), Phe473 to Val (p <  0.001), Asp479 to Glu (p = 0.044), and Ser/Ala/Gly595-596 (p = 0.001). Phylogenetic analyses suggested that other molecular mechanisms might contribute to amoxicillin resistance in H. pylori in addition to the alterations in PBP1A. CONCLUSIONS: We reported the emergence of amoxicillin-resistant Helicobacter pylori strains in Vietnam and new mutations statistically associated with this antimicrobial resistance. Additional studies are necessary to identify the mechanisms contributing to this resistance in Vietnam.

Three new species of the genus Miridiba Reitter, 1902 (Scarabaeidae: Melolonthinae) from Vietnam along with a new country record.

Three new species of the genus Miridiba Reitter, 1902 from Vietnam are described and illustrated: Miridiba apicespinosa P.V. Pham & Keith, new species, M. ngoclinhensis P.V. Pham & Keith, new species, and M. quangnamensis P.V. Pham & Keith, new species. Miridiba bannaensis Gao & Fang, 2018 is reported for the first time from Vietnam. Additionally, an updated checklist and a key to all Vietnamese species are provided.

Cardioprotective Actions of the Annexin-A1 N-Terminal Peptide, Ac2-26, Against Myocardial Infarction.

The anti-inflammatory, pro-resolving annexin-A1 protein acts as an endogenous brake against exaggerated cardiac necrosis, inflammation, and fibrosis following myocardial infarction (MI) in vivo. Little is known, however, regarding the cardioprotective actions of the N-terminal-derived peptide of annexin A1, Ac2-26, particularly beyond its anti-necrotic actions in the first few hours after an ischemic insult. In this study, we tested the hypothesis that exogenous Ac2-26 limits cardiac injury in vitro and in vivo. Firstly, we demonstrated that Ac2-26 limits cardiomyocyte death both in vitro and in mice subjected to ischemia-reperfusion (I-R) injury in vivo (Ac2-26, 1 mg/kg, i.v. just prior to post-ischemic reperfusion). Further, Ac2-26 (1 mg/kg i.v.) reduced cardiac inflammation (after 48 h reperfusion), as well as both cardiac fibrosis and apoptosis (after 7-days reperfusion). Lastly, we investigated whether Ac2-26 preserved cardiac function after MI. Ac2-26 (1 mg/kg/day s.c., osmotic pump) delayed early cardiac dysfunction 1 week post MI, but elicited no further improvement 4 weeks after MI. Taken together, our data demonstrate the first evidence that Ac2-26 not only preserves cardiomyocyte survival in vitro, but also offers cardioprotection beyond the first few hours after an ischemic insult in vivo. Annexin-A1 mimetics thus represent a potential new therapy to improve cardiac outcomes after MI.

Small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice.

Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI.

The HNO donor Angeli's salt offers potential haemodynamic advantages over NO or dobutamine in ischaemia-reperfusion injury in the rat heart ex vivo.

Available inotropic pharmacotherapy for acute heart failure (HF) remains largely ineffective at ameliorating marked impairments in contractile function. Nitroxyl (HNO), the redox sibling of NO•, has recently attracted interest as a therapeutic approach for acute HF. We now compare the impact of ischaemia-reperfusion (I-R) injury on acute haemodynamic responsiveness of the HNO donor, Angeli's salt (AS), to that of NO and dobutamine. Dose-response curves to bolus doses of AS, diethylamine NONOate (DEA/NO, both 0.001-µmol) and dobutamine (0.1-100 nmol) were performed in rat isolated hearts, following I-R or normoxic perfusion. An additional 10µmol dose of Angeli's salt was included, to permit roughly equivalent inotropic responses to dobutamine. Changes in cardiac contraction, heart rate and coronary flow (CF) were determined. Although AS and DEA/NO elicited comparable dose-dependent increases in CF in normoxic hearts, only AS vasodilation was preserved after I-R. AS and dobutamine elicited dose-dependent inotropic responses in normoxic hearts and I-R blunted inotropic responses to both. Dobutamine however increased heart rate, which was exacerbated by I-R; this was not evident with AS. Further, AS infusion during reperfusion (1µM), in a separate cohort of rat hearts, improved recovery of cardiac contractility, with lower incidence of I-R-induced ventricular fibrillation. In conclusion, these observations suggest that HNO offers haemodynamic advantages over NO following I-R. Although I-R suppresses inotropy to both agents, residual contractile responses to AS following I-R is likely free of concomitant pro-arrhythmic events. HNO donors may thus offer haemodynamic advantages over existing pharmacotherapy in acute HF.

Chronic administration of the nitroxyl donor 1-nitrosocyclo hexyl acetate limits left ventricular diastolic dysfunction in a mouse model of diabetes mellitus in vivo.

BACKGROUND: Nitroxyl (HNO), a redox congener of nitric oxide (NO·), is a novel regulator of cardiovascular function, combining concomitant positive inotropic, lusitropic, and vasodilator properties. Moreover, HNO exhibits myocardial antihypertrophic and superoxide-suppressing actions. Despite these favorable actions, the impact of chronic HNO administration has yet to be reported in the context of cardiomyopathy. Diabetic cardiomyopathy is characterized by early diastolic dysfunction and adverse left ventricular (LV) structural remodeling, with LV superoxide generation playing a major causal role. We tested the hypothesis that the HNO donor 1-nitrosocyclohexylacetate (1-NCA) limits cardiomyocyte hypertrophy and LV diastolic dysfunction in a mouse model of diabetes mellitus in vivo. METHODS AND RESULTS: Diabetes mellitus was induced in male FVB/N mice using streptozotocin. After 4 weeks, diabetic and nondiabetic mice were allocated to 1-NCA therapy (83 mg/kg per day IP) or vehicle and followed up for a further 4 weeks. Diabetes mellitus-induced LV diastolic dysfunction was evident on echocardiography-derived E and A wave velocities, E:A ratio, deceleration, and isovolumic relaxation times; LV systolic function was preserved. Increased LV cardiomyocyte size, hypertrophic and profibrotic gene expression, and upregulation of LV superoxide were also evident. These characteristics of diabetic cardiomyopathy were largely prevented by 1-NCA treatment. Selectivity of 1-NCA as an HNO donor was demonstrated by sensitivity of acute 1-NCA to l-cysteine but not to hydroxocobalamin in the normal rat heart ex vivo. CONCLUSIONS: Our studies provide the first evidence that HNO donors may represent a promising strategy for treatment of diabetic cardiomyopathy and implies therapeutic efficacy in settings of chronic heart failure.

The concomitant coronary vasodilator and positive inotropic actions of the nitroxyl donor Angeli's salt in the intact rat heart: contribution of soluble guanylyl cyclase-dependent and -independent mechanisms.

BACKGROUND AND PURPOSE: The NO redox sibling nitroxyl (HNO) elicits soluble guanylyl cyclase (sGC)-dependent vasodilatation. HNO has high reactivity with thiols, which is attributed with HNO-enhanced left ventricular (LV) function. Here, we tested the hypothesis that the concomitant vasodilatation and inotropic actions induced by a HNO donor, Angeli's salt (sodium trioxodinitrate), were sGC-dependent and sGC-independent respectively. EXPERIMENTAL APPROACH: Haemodynamic responses to Angeli's salt (10 pmol-10 µmol), alone and in the presence of scavengers of HNO (L-cysteine, 4 mM) or of NO [hydroxocobalamin (HXC), 100 µM] or a selective inhibitor of sGC [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10 µM], a CGRP receptor antagonist (CGRP8-37 , 0.1 µM) or a blocker of voltage-dependent potassium channels [4-aminopyridine (4-AP), 1 mM] were determined in isolated hearts from male rats. KEY RESULTS: Angeli's salt elicited concomitant, dose-dependent increases in coronary flow and LV systolic and diastolic function. Both L-cysteine and ODQ shifted (but did not abolish) the dose-response curve of each of these effects to the right, implying contributions from HNO and sGC in both the vasodilator and inotropic actions. In contrast, neither HXC, CGRP8-37 nor 4-AP affected these actions. CONCLUSIONS AND IMPLICATIONS: Both vasodilator and inotropic actions of the HNO donor Angeli's salt were mediated in part by sGC-dependent mechanisms, representing the first evidence that sGC contributes to the inotropic and lusitropic action of HNO in the intact heart. Thus, HNO acutely enhances LV contraction and relaxation, while concomitantly unloading the heart, potentially beneficial actions in failing hearts.

HNO/cGMP-dependent antihypertrophic actions of isopropylamine-NONOate in neonatal rat cardiomyocytes: potential therapeutic advantages of HNO over NO.

Nitroxyl (HNO) is a redox congener of NO. We now directly compare the antihypertrophic efficacy of HNO and NO donors in neonatal rat cardiomyocytes and compare their contributing mechanisms of actions in this setting. Isopropylamine-NONOate (IPA-NO) elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by l-cysteine (HNO scavenger), Rp-8-pCTP-cGMPS (cGMP-dependent protein kinase inhibitor), and 1-H-(1,2,4)-oxodiazolo-quinxaline-1-one [ODQ; to target soluble guanylyl cyclase (sGC)] but were unaffected by carboxy-PTIO (NO scavenger) or CGRP8-37 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an l-cysteine-sensitive effect) and stimulated sGC activity threefold, without detectable NO release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO donor diethylamine-NONOate (DEA-NO) reproduced these IPA-NO actions but was sensitive to carboxy-PTIO rather than l-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor was attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to pressure overload in the intact heart. Together these data strongly suggest that IPA-NO protection against cardiomyocyte hypertrophy is independent of both NO and CGRP but rather utilizes novel HNO activation of cGMP signaling. Thus HNO acutely limits hypertrophy independently of NO, even under conditions of elevated superoxide. Development of longer-acting HNO donors may thus represent an attractive new strategy for the treatment of cardiac hypertrophy, as stand-alone and/or add-on therapy to standard care.

Enhanced extravasation, stability and in vivo cardiac gene silencing via in situ siRNA-albumin conjugation.

A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3'-amine, 2'-O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA ("activated siRNA"); this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA-albumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS-PAGE analysis. In Sprague-Dawley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 ± 18.2 min) compared to unactivated siRNA (5.1 ± 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 ± 4.1% of that in vehicle-treated animals (mean ± SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA-albumin constructs are therefore capable of extravasation to the myocardium resulting in silencing in this otherwise silencing-resistant organ.

Specific binding and mineralization of calcified surfaces by small peptides.

Several small (<25aa) peptides have been designed based on the sequence of the dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)(n) repeats, and though similar repeated sequences-(NTT)(n), (DTT)(n), (ETT)(n), (NSS)(n), (ESS)(n), (DAA)(n), (ASS)(n), and (NAA)(n)-also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)(n) peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed.

Beta-D-Allose inhibits fruiting body formation and sporulation in Myxococcus xanthus.

Myxococcus xanthus, a gram-negative soil bacterium, responds to amino acid starvation by entering a process of multicellular development which culminates in the assembly of spore-filled fruiting bodies. Previous studies utilizing developmental inhibitors (such as methionine, lysine, or threonine) have revealed important clues about the mechanisms involved in fruiting body formation. We used Biolog phenotype microarrays to screen 384 chemicals for complete inhibition of fruiting body development in M. xanthus. Here, we report the identification of a novel inhibitor of fruiting body formation and sporulation, beta-d-allose. beta-d-Allose, a rare sugar, is a member of the aldohexose family and a C3 epimer of glucose. Our studies show that beta-d-allose does not affect cell growth, viability, agglutination, or motility. However, beta-galactosidase reporters demonstrate that genes activated between 4 and 14 h of development show significantly lower expression levels in the presence of beta-d-allose. Furthermore, inhibition of fruiting body formation occurs only when beta-d-allose is added to submerged cultures before 12 h of development. In competition studies, high concentrations of galactose and xylose antagonize the nonfruiting response to beta-d-allose, while glucose is capable of partial antagonism. Finally, a magellan-4 transposon mutagenesis screen identified glcK, a putative glucokinase gene, required for beta-d-allose-mediated inhibition of fruiting body formation. Subsequent glucokinase activity assays of the glcK mutant further supported the role of this protein in glucose phosphorylation.

Characterization of the acute cardiovascular effects of intravenously administered insulin-like growth factor-I in conscious Sprague-Dawley rats.

1. Insulin-like growth factor (IGF)-I has acute effects on cardiovascular function, including a well-characterized vasodilator response in isolated arteries. In addition to increasing the release of nitric oxide, IGF-I also has effects on a variety of other signalling pathways that affect vascular tone, in particular interactions with the sympathetic nervous system and the renin-angiotensin-aldosterone system. We sought to characterize the effects of intravenous IGF-I on blood pressure and on responses to noradrenaline (NA), angiotensin II, acetylcholine and dobutamine. 2. Administration of IGF-I administration caused small decreases in mean arterial pressure (5.4 +/- 1.5%) and responsiveness to the prazosin-sensitive vasoconstrictor effects of NA (a 2.1 +/- 0.6-fold increase in ED(50); n = 40; P < 0.01) and both effects were maximal at 200 microg/kg IGF-I. In addition, IGF-I significantly increased pulse pressure increases induced by low doses of dobutamine (from an increase in pulse pressure of 9.9 +/- 1.2 to 13.4 +/- 1.9 mmHg; n = 39; P < 0.05). Administration of IGF-I had no significant effect on responses to AngII or ACh. 3. Intravenous administration of IGF-I receptor antisense oligonucleotides (400 microg/kg) abolished the effects of IGF-I on NA-induced vasoconstriction (n = 11; P < 0.05), whereas administration of a mismatch oligonucleotide did not. 4. These data indicate that the maximal effects of exogenously administered IGF-I include modest direct vasodilation and inhibition of constrictor responses to NA and an increase in the effect of dobutamine on pulse pressure. The magnitude of these effects was less than what previous in vitro studies and those performed in anaesthetized animals may have indicated likely. 5. The modest magnitude of the dilator effects of IGF-I observed in conscious rats in vivo in the present study suggests that IGF-I is unlikely to be a major player in regulating vascular tone in normotensive animals.

Intravenous insulin-like growth factor-I receptor antisense treatment reduces angiotensin receptor expression and function in spontaneously hypertensive rats.

The present study investigated the effects of a functional deficit in insulin-like growth factor-I signaling via chronic intravenous administration of insulin-like growth factor-I (IGF-I) receptor antisense in the conscious spontaneously hypertensive rat cardiovascular system. Insulin-like growth factor-I receptor (IGF-IR) antisense, but not full mismatch treatment, decreased IGF-IR expression in both conductance and resistance blood vessels. Aortic IGF-IR density was reduced by 67.4 +/- 6.0% in antisense-treated spontaneously hypertensive rat (SHR) compared with untreated animals, whereas mismatch treatment had no effect (analysis of variance, n = 3, P < 0.01). Aortic and tail artery angiotensin II type 1 receptor expression was significantly reduced by IGF-IR antisense treatment, whereas angiotensin II type 2 receptor expression was unaffected by administration of antisense and mismatch oligonucleotides. IGF-I receptor antisense treatment caused a significant decrease in pressor responses to angiotensin II in comparison with full-length mismatch treatment (E(max) was reduced to 65 +/- 7 mm Hg compared with 99 +/- 6 mm Hg, p < 0.05). Likewise, a reduction in pressor responses to noradrenaline was observed in hypertensive rats treated with IGF-IR antisense compared with full mismatch-treated rats (E(max) was reduced to 60 +/- 6 mm Hg compared with 108 +/- 5 mm Hg, p < 0.01). There was no clear antisense effect on resting blood pressure and no effect at on aortic medial thickness. These results suggest that although the proliferative and vasodilator effects of IGF-I are impaired in SHR, the effects on angiotensin receptor expression remain profound.

Saw palmetto is an indirectly acting sympathomimetic in the rat-isolated prostate gland.

BACKGROUND: To investigate whether saw palmetto that inhibits alpha1-adrenoceptor binding in vitro affects contractility of the rat prostate gland. METHODS: The effects of a commercially available saw palmetto extract were examined on the contractility of rat-isolated prostate glands. The extract was tested in the presence and absence of phentolamine, prazosin, yohimbine, propranolol, hexamethonium, cocaine, desipramine, nifedipine, guanethidine, atropine, and alpha,beta-methylene ATP to evaluate the mechanism of action. Isolated preparations of rat vas deferens and bladder were used for comparison. RESULTS: Unexpectedly, saw palmetto extract caused contractions of the rat prostate gland that could be attenuated by prazosin, phentolamine, nifedipine, guanethidine, cocaine, and desipramine but not by any of the other pharmacological tools. Similar contractile effects were observed in rat-isolated vas deferens preparations but not in rat-isolated bladder preparations. CONCLUSIONS: In the rat prostate gland, saw palmetto extract causes indirect alpha1-adrenoceptor-mediated contractions via the release of noradrenaline from sympathetic neurons.