Planctomonas psychrotolerans sp. nov., isolated from rhizosphere soil of Suaeda salsa.

A psychrotolerant actinobacterium, designated strain J5903T, was isolated from an alkaline soil sample from the rhizosphere of Suaeda salsa collected in desertification land surrounding Jiuliancheng Nur in Hebei Province, PR China. Cells of the isolate were Gram-stain-positive, aerobic, non-motile and non-spore-forming cocci. Strain J5903T grew optimally at 20‒25 °C, at pH 7.0‒7.5 and with <1 % (w/v) NaCl. The cell-wall peptidoglycan type was B2γ with d-2,4-diaminobutyric acid and l-2,4-diaminobutyric acid as diagnostic amino acids. The muramyl residue was acetyl type. The menaquinones were MK-11, MK-12, MK-10 and MK-13. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid. The major whole-cell fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was 69.1 mol%. It shared the highest average nucleotide identity and digital DNA-DNA hybridization values with Planctomonas deserti 13S1-3T. Phylogenies based on genome sequence showed that strain J5903T and P. deserti 13S1-3T formed a robust cluster with high bootstrap support. Strain J5903T shared typical chemotaxonomic characteristics with P. deserti 13S1-3T. Combining the polyphasic taxonomic evidence, strain J5903T represents a novel species of the genus Planctomonas, for which the name Planctomonas psychrotolerans sp. nov. is proposed. The type strain is J5903T (=DSM 101894T=CGMCC 1.15523T).

Phytoactinopolyspora mesophila sp. nov., isolated from a saline-alkaline soil.

A novel moderately halophilic, filamentous actinobacterium, designated as XMNu-373T, was isolated from a saline-alkaline soil sample collected from the Mongolia Plateau, Dongwu County, Inner Mongolia Autonomous Region, PR China. The isolate grew optimally at 28‒37 °C, pH 7.0‒8.0 and with 2-5 % (w/v) NaCl. The substrate mycelia fragmented into rod-like elements, and the white aerial mycelia formed spore chains at maturity. The predominant menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, three unidentified phosphoglycolipids, an unidentified aminophospholipid, two phosphatidylinositol mannosides, four unidentified phospholipids, phosphatidylglycerol and two unidentified lipids. The major cellular fatty acids were iso-C16 : 0, anteiso-C17 : 0 and anteiso-C15 : 0. The genomic DNA G+C content was 66.2 mol%. It shared high 16S rRNA gene sequence similarities to Phytoactinopolyspora halotolerans YIM 96448T (96.1 %) and Phytoactinopolyspora endophytica EGI 60009T (96.0 %). Phylogenetic trees based on 16S rRNA gene sequences revealed that strain XMNu-373T resided in the clade of family Jiangellaceae, and it formed a monophyletic branch distinct from four other recognized type species in the subclade of the genus Phytoactinopolyspora. On the basis of polyphasic taxonomic evidence, strain XMNu-373T represents a novel species of the genus Phytoactinopolyspora, for which the name Phytoactinopolyspora mesophila sp. nov. is proposed. The type strain is XMNu-373T (=JCM 33740T=CGMCC 4.7654T).

Mutation analysis of FBN1 gene in two Chinese families with congenital ectopia lentis in northern China.

AIM: To summarize the phenotypes and identify the underlying genetic cause of the fibrillin-1 (FBN1) gene responsible for congenital ectopia lentis (EL) in two Chinese families in northern China. METHODS: A detailed family history and clinical data from all participants were collected by clinical examination. The candidate genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing. Haplotyping was used to confirm the mutation sequence. Real-time PCR was used to determine the FBN1 messenger ribonucleic acid (mRNA) levels in patients with EL and in unaffected family members. RESULTS: The probands and other patients in the two families were affected with congenital isolated EL. A heterozygous FBN1 mutation in exon 21 (c.2420_IVS20-8 delTCTGAAACAinsCGAAAG) was identified in FAMILY-1. A heterozygous FBN1 mutation in exon 14 (c.1633C>T, p.R545C) was identified in FAMILY-2. Each mutation co-segregated with the affected individuals in the family and did not exist in unaffected family members and 200 unrelated normal controls. CONCLUSION: The insertion-deletion mutation (c.2420 IVS20-8delTCTGAAACA insCGAAAG) in the FBN1 gene is first identified in isolated EL. The mutation (c.1633C>T) in the FBN1 gene was a known mutation in EL patient. The variable phenotypes among the patients expand the phenotypic spectrum of EL in a different ethnic background.

Blockade of the forward Na+ /Ca2+ exchanger suppresses the growth of glioblastoma cells through Ca2+ -mediated cell death.

BACKGROUND AND PURPOSE: The Na+ /Ca2+ exchanger (NCX) working in either forward or reverse mode participates in maintaining intracellular Ca2+ ([Ca2+ ]i ) homeostasis, which is essential for determining cell fate. Previously, numerous blockers targeting reverse or forward NCX have been developed and studied in ischaemic tissue injury but barely examined in glioblastoma for the purpose of anti-tumour therapy. We assessed the effect of NCX blockers on glioblastoma growth and whether NCX can become a therapeutic target. EXPERIMENTAL APPROACH: Patch-clamp recording, Ca2+ imaging, flow cytometry, and Western blot were used to study the effects of specific and non-specific NCX blockers on cultured glioblastoma cells. In vivo bioluminescent imaging was used to measure effects on grafted glioblastoma. KEY RESULTS: Selectively blocking the reverse NCX with SEA0400, SN-6, and YM-244769 did not affect tumour cell viability. Blocking the forward NCX with bepridil, CB-DMB, or KB-R7943 elevated [Ca2+ ]i and killed glioblastoma cells. Bepridil and CB-DMB caused Ca2+ -dependent cell cycle arrest together with apoptosis, which were all attenuated by a Ca2+ chelator BAPTA-AM. Systemic administration of bepridil inhibited growth of brain-grafted glioblastoma. Bepridil did not appear to have a cytotoxic effect on human astrocytes, which have higher functional expression of NCX than glioblastoma cells. CONCLUSIONS AND IMPLICATIONS: Low expression of the NCX makes glioblastoma cells sensitive to disturbance of [Ca2+ ]i . Interventions designed to block the forward NCX can cause Ca2+ -mediated injury to glioblastoma thus having therapeutic potential. Bepridil could be a lead compound for developing new anti-tumour drugs.

Neuronal and Endothelial Nitric Oxide Synthases in the Paraventricular Nucleus Modulate Sympathetic Overdrive in Insulin-Resistant Rats.

A central mechanism participates in sympathetic overdrive during insulin resistance (IR). Nitric oxide synthase (NOS) and nitric oxide (NO) modulate sympathetic nerve activity (SNA) in the paraventricular nucleus (PVN), which influences the autonomic regulation of cardiovascular responses. The aim of this study was to explore whether the NO system in the PVN is involved in the modulation of SNA in fructose-induced IR rats. Control rats received ordinary drinking water, whereas IR rats received 12.5% fructose-containing drinking water for 12 wks to induce IR. Basal SNA was assessed based on the changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in response to chemicals administered to the PVN. We found an increased plasma norepinephrine level but significantly reduced NO content and neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression levels in the PVN of IR rats compared to Control rats. No difference in inducible NOS (iNOS) protein expression was observed between the two groups. In anesthetized rats, the microinjection of sodium nitroprusside (SNP), an NO donor, or Nω-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, into the PVN significantly decreased and increased basal SNA, respectively, in both normal and IR rats, but these responses to SNP and L-NAME in IR rats were smaller than those in normal rats. The administration of selective inhibitors of nNOS or eNOS, but not iNOS, to the PVN significantly increased basal SNA in both groups, but these responses were also smaller in IR rats. Moreover, IR rats exhibited reduced nNOS and eNOS activity in the PVN. In conclusion, these data indicate that the decreased protein expression and activity levels of nNOS and eNOS in the PVN lead to a reduction in the NO content in the PVN, thereby contributing to a subsequent enhancement in sympathoexcitation during IR.

Activation of muscarinic receptors protects against retinal neurons damage and optic nerve degeneration in vitro and in vivo models.

AIMS: Muscarinic acetylcholine receptor agonist pilocarpine reduces intraocular pressure (IOP) of glaucoma mainly by stimulating ciliary muscle contraction and then increasing aqueous outflow. It is of our great interest to know whether pilocarpine has the additional properties of retinal neuroprotection independent of IOP lowering in vitro and in vivo models. METHODS: In rat primary retinal cultures, cell viability was measured using an MTT assay and the trypan blue exclusion method, respectively. Retinal ganglion cells (RGCs) were identified by immunofluorescence and quantified by flow cytometry. For the in vivo study, the retinal damage after retinal ischemia/reperfusion injury in rats was evaluated by histopathological study using hematoxylin and eosin staining, transmission electron microscopy, and immunohistochemical study on cleaved caspase-3, caspase-3, and ChAT. RESULTS: Pretreatment of pilocarpine attenuated glutamate-induced neurotoxicity of primary retinal neurons in a dose-dependent manner. Protection of pilocarpine in both retinal neurons and RGCs was largely abolished by the nonselective muscarinic receptor antagonist atropine and the M1-selective muscarinic receptor antagonist pirenzepine. After ischemia/reperfusion injury in retina, the inner retinal degeneration occurred including ganglion cell layer thinning and neuron lost, and the optic nerve underwent vacuolar changes. These degenerative changes were significantly lessened by topical application of 2% pilocarpine. In addition, the protective effect of pilocarpine on the ischemic rat retina was favorably reflected by downregulating the expression of activated apoptosis marker cleaved caspase-3 and caspase-3 and upregulating the expression of cholinergic cell marker ChAT. CONCLUSIONS: Taken together, this highlights pilocarpine through the activation of muscarinic receptors appear to afford significant protection against retinal neurons damage and optic nerve degeneration at clinically relevant concentrations. These data also further support muscarinic receptors as potential therapeutic neuroprotective targets in glaucoma.

Establishment and application of hepatitis B virus persistent replication model in IFNAR(-/-) mouse.

The type I interferon and IFNAR play an important role in hepatitis B virus (HBV) infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more insights into the role of type I interferon and type I interferon receptor (IFNAR) in HBV infection, we established an HBV persistent replication IFNAR knockout (IFNAR(-/-)) mouse model and preliminarily applied this model. At first, the progeny of IFNAR(-/-) mouse was reproduced. Then hydrodynamic injection with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR(-/-) mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir (ETV) on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline (NS) treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days after the ETV treatment [P=0.035, P=0.00, P=0.149 and P=0.084, IFNAR knockout (KO) control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR(-/-) and C57BL/6 mice. This result suggests that HBV suppression during ETV treatments doesn't depend on type I interferon and IFNAR. Collectively, persistent HBV replication IFNAR(-/-) mouse model that we established is a useful and convenient tool to detect the function of the type I interferon and IFNAR in HBV infection and anti-HBV treatments.

[Assessment of mitochondrial toxicity induced by zidovudine and adefovir dipivoxil in rats].

OBJECTIVE: To explore the mitochondrial toxicities induced by zidovudine (AZT) and adefovir dipivoxil (ADV) antiviral drugs using a rat model system. METHODS: Twelve healthy Sprague-Dawley rats were randomly divided into three equal groups and treated by oral gavage with zidovudine (125 mg/kg/day), adefovir (40 mg/kg/day), or saline (equal volume) for 28 days. The rats' body weights were measured once a week, and blood was collected every two weeks for blood and biochemical tests. All animals were sacrificed at the end of treatment, and liver, kidney, skeletal muscle, and cardiac muscle were collected by necropsy. Mitochondria were isolated from the respective tissue samples, and the activities of respiratory chain complexes were measured. DNA was purified from each sample and the mitochondrial DNA (mtDNA) content was monitored by quantitative real time PCR. Mitochondrial morphology was analyzed under electron microscope. RESULTS: No significant adverse effects, including body weight loss, abnormal blood or biochemistry, were observed in rats treated with AZT or ADV. The activities of mitochondrial cytochrome c oxidase in liver and cardiac muscle were slightly decreased in rats treated with AZT (liver: 9.44+/-3.09 vs. 17.8+/-12.38, P?=?0.21; cardiac muscle: 32.74+/-5.52 vs. 24.74+/-20.59, P?=?0.28; kidney: 4.42+/-1.53 vs. 14.45+/-13.75, P?=?0.18; skeletal muscle: 33.75+/-8.74 vs. 40.04+/-2.49, P?=?0.45). The mtDNA content was significantly decreased in cardiac muscle of AZT-treated rats (cardiac muscle: 0.15+/-0.13 vs. 0.32+/-0.42, P?=?0.85). The morphology of mitochondria in liver, kidney, skeletal muscle, and cardiac muscle was significantly altered in the AZT-treated rats and included disappearance of the outer membrane, severely damaged structure, and swollen or completely absent cristae. No obvious effects were noted in the ADV- or saline-treated rats. CONCLUSION: Significant adverse effects related to mitochondrial toxicity were observed in rats treated with AZT. The slightly decreased mtDNA content in ADV-treated rats may suggest that this antiviral drug can also cause mitochondrial toxic effects.

Expression of neuroglobin in ocular hypertension induced acute hypoxic-ischemic retinal injury in rats.

AIM: To investigate the expression of neuroglobin (Ngb) in the retina of rats with ocular hypertension induced acute retinal hypoxic-ischemic injury. METHODS: Seventy Wistar rats were divided into 7 groups randomly. The experimental model was induced by elevation of intraocular pressure via anterior chamber canula insertion in the left eyes and the fellow eyes were preserved as normal controls. The retinal tissues were taken at 1, 5, 10, 15, 20, 30 and 60 minutes after hypoxic-ischemia injury. Protein was extracted, and then analyzed by Western-blot method. SPSS was used for statistical analysis. RESULTS: The time-depended expressions of Ngb were observed. The level of Ngb increased rapidly at 1 minute after ischemia and reached to the peak at 5 minutes, which had significant difference from that of control group (P<0.05). It kept in high level during 5-15 minutes (P<0.05), then decreased after 20 minutes till 60 minutes. There were no significant differences between experimental and control group in the latter period (P>0.05). CONCLUSION: The expression of Ngb in retinal tissue increased rapidly after hypoxic-ischemic injury in rats, suggesting that Ngb may play an important role in the process of acute retinal hypoxic-ischemic injury.

[Establishment and application of a loop-mediated isothermal amplification method for rapid diagnosis of Vibrio cholerae].

OBJECTIVE: To establish a loop-mediated isothermal amplification (LAMP) method for rapid diagnosis of Vibrio cholerae. METHODS: Based on the ompW nucleic sequence of Vibrio cholerae, a pair of primers was designed for LAMP. The reaction conditions were optimized, and the specificity, sensitivity, and practicability of LAMP were tested using 47 bacterial strains and simulated contaminated sites. RESULTS: The results of viable bacterium count showed that LAMP was capable of detecting Vibrio cholerae at a level as low as 1.6x10(2) cfu/ml. The minimal detectable concentration was 1.6+10(3) cfu/ml for simulated contaminated samples such as feces and seawater, and 1.6+10(4) cfu/ml for contaminated milk. All the 21 strains of Vibrio cholerae yielded positive results in LAMP, and the 26 strains of other bacteria all showed negative results, with a detection specificity of 100%. CONCLUSION: The established LAMP method has high specificity and sensitivity for detecting Vibrio cholerae and is applicable in field monitoring and epidemiological study of Vibrio cholerae.

Quantitative determination of a novel enantiomeric tropane analog, (-)-satropane, in biological fluids using liquid chromatography/tandem mass spectrometry.

A sensitive, accurate and precise liquid chromatography-tandem mass spectrometry method was developed for the determination of (-)-satropane (3alpha-paramethyl-benzenesulfonyloxy-6beta-acetoxy-tropane) in rabbit aqueous humor. Since (-)-satropane may be absorbed from the aqueous humour with resultant systemic side effects, the LC-MS/MS method was also evaluated for its applicability in analyzing plasma samples containing this compound. (-)-Satropane and phentolamine (the internal standard, represented as IS) were detected by multiple reaction monitoring using the transitions m/z 354-182 and 282-212, respectively. The calibration curve was linear over the ranges 2-500 and 5-1000 ng/mL, and the values of the lower limit of quantification were 2 and 5 ng/mL for the microdialysis dialysate and rat plasma samples, respectively. The intra-day and inter-day precision and accuracy were better than 8.6 and 6.00%, respectively, in both matrices investigated. The absolute recovery of the plasma samples was more than 76.30%. The average matrix effects of (-)-satropane were 91.72 and 83.05% in the microdialysis dialysate and plasma samples, respectively. The validated method was successfully applied to analyze (-)-satropane in microdialysis dialysate and rat plasma samples, and this assay has been used to quantify (-)-satropane in the pharmacokinetic and toxicokinetic studies in our laboratory.

Preparation and evaluation of a novel delayed-onset sustained-release system of propranolol hydrochloride.

The objective of this work was to prepare and evaluate a new delayed-onset sustained-release system, comprising a sustained-release core tablet with hydroxypropyl methylcellulose as polymer matrix and an ethylcellulose/Eudragit L coating capable of delaying the drug release. The sustained core containing propranolol hydrochloride as the model drug was prepared by granulate tableting and the polymer coating was applied in a computer-controlled coating pan. The dissolution tests demonstrated that the in-vitro drug release was pH-dependent with sufficient gastric resistance, and the lag time (t(10%)) could be controlled by adjusting the coating level. Three dosage forms including commercial tablet, sustained-release tablet and the delayed-onset sustained-release tablet were administrated to six beagle dogs and the plasma levels of propranolol hydrochloride were measured with high-performance liquid chromatography. The delayed-onset sustained-release tablet had a lag time of 3.0 h in-vitro and 3.5 h in-vivo, and a t(max) of 7.0 h. The relative bioavailability for delayed-onset sustained-release tablet was 96.98% compared with commercial tablets. The results indicate that the new propranolol delayed-onset sustained-release system could achieve a relatively constant drug release followed by a programmed lag time, and this may provide a promising drug delivery form for chronopharmacotherapy of certain cardiovascular diseases.

Synergistically enhanced transdermal permeation and topical analgesia of tetracaine gel containing menthol and ethanol in experimental and clinical studies.

The aim of this study is to observe the synergistically enhanced percutaneous penetration and skin analgesia of tetracaine gel containing menthol and ethanol through experimental and clinical studies. Four anesthetic gels containing 4% tetracaine in carbomer vehicle named T-gel (containing no menthol or ethanol), 5%M/T-gel (containing 5% menthol), 70%E/T-gel (containing 70% ethanol, an optimal concentration for antiseptic), and 5%M+70%E/T-gel (containing both 5% menthol and 70% ethanol), respectively, were fabricated. The in vitro mouse skin permeation was investigated using a Franz diffusion cell. The mouse skin morphology was examined by a scanning electron microscope. The in vivo skin analgesic effect in mice was evaluated using the von Frey tests. To determine the efficacy of tetracaine gels for managing the pain in human volunteers, a paralleled, double-blinded, placebo-controlled, randomized controlled trial design combined with verbal pain scores (VPS) was performed. The combination of menthol and ethanol (5%M+70%E/T-gel) conferred significantly higher tetracaine diffusion across full-thickness mouse skin than 5%M/T-gel, 70%E/T-gel, and T-gel. The ultra structure changes of mouse skin stratum corneum treated with 5%M+70%E/T-gel were more marked compared with those of any other tetracaine gel. von Frey tests in mice showed a synergistically enhanced effect of menthol and ethanol on the analgesia of tetracaine gel. The mean VPS were significantly lower for volunteers treated with 5%M+70%E/T-gel than those receiving other gels or the EMLA cream. 5%M+70%E/T-gel possessed the shortest anesthesia onset time, the longest anesthesia duration and the strongest anesthesia efficacy. Seventy percent ethanol in 5%M+70%E/T-gel not only improved the analgesic efficacy of the tetracaine gel through synergistically enhanced percutaneous permeation with menthol but also served as an antiseptic agent keeping drug application site from infection. 5%M+70%E/T-gel is a potential topical anesthesia preparation for clinical use.

[Design of mechanical structure dynamic characteristics and development of verification system].

OBJECTIVE: To present an effective method for structural dynamic characteristics design, required design and test means for space products' development. METHOD: Computational modal analysis method was used to evaluate natural frequencies and modes of the products in the beginning of design, and test modal analysis method was used to identify the dynamic parameters of products in the test period. RESULT: The proposed computational modal analysis system is capable of making static and dynamic analysis, as well as natural frequencies and modes analysis of the structure, and this system could be applied in mechanical structure dynamic characteristics design. The proposed test modal analysis system could acquire real time force and acceleration signals, and obtain the basic modal parameters by processing the sampled data. This system could be used to modify mechanical products' dynamic characteristics and decrease vibration amplitude. CONCLUSION: The computational and test results of two test models demonstrated that the developed computational and test modal system is effective. The method can be used for design of structural dynamic characteristics of space products.

[Development of a shock response spectrum test facility].

OBJECTIVE: To develop a shock response spectrum test facility for testing space products in the pyrotechnic environments. METHOD: The natural frequencies of the resonant plate, a key part of the facility, were calculated with finite element method. The dynamic response of the whole system was simulated when the machine was excited by a real shock generated by a dropping hammer. A shock response spectrum test facility was developed under the guide of the simulation results. RESULT: The resonant plate drop machine could simulate pyrotechnic shock by adjusting the force, the natural frequencies of the plate and the damping materials above and under the plate. CONCLUSION: The agreement between the measurement and the simulation indicates that the computer simulation results are reliable. It is feasible to use the resonant plate drop machine to simulate pyrotechnic environment.

[Development of a measurement and control system of human centrifuge].

OBJECTIVE: To develop measurement and control system for human centrifuge to simulate arbitrary hypergravity curves. METHOD: The human centrifuge is controlled by the upper and lower level computers. The measurement and control computer is as upper, and the real-time controller as lower. RESULT: The system performance could satisfy the requirement of the human centrifuge. CONCLUSION: The measurement and control system can achieve a high control precision. It is safe, reliable and easy to operate.