白斑狗鱼StAR基因克隆及其表达分析

【目的】克隆白斑狗鱼（Esox lucius）类固醇激素合成急性调节蛋白（StAR）基因（ElStAR）并分析其组织表达差异,为开展StAR基因生物学功能研究及揭示白斑狗鱼性腺发育机制提供基础资料。【方法】根据GenBank已公布的白斑狗鱼基因组测序结果（NC_047581.1）设计特异性引物,采用RT-PCR克隆ElStAR基因cDNA序列,通过ClustalX、ExPASy、TargetP 1.1、TMHMM 2.0和SignalP 5.0等在线软件进行生物信息学分析,并采用实时荧光定量PCR进行ElStAR基因组织表达定量分析。【结果】ElStAR基因cDNA序列长度1485 bp,其开放阅读框（ORF）为864 bp,共编码287个氨基酸残基;ElStAR氨基酸序列与北极红点鲑StAR氨基酸序列的相似性最高（93.33%）,与海鳟、条纹鲈鱼、金头鲷、大菱鲆、斑马鱼、半滑舌鳎的相似性均高于75.00%,而与小家鼠的相似性最低（59.15%）;基于StAR氨基酸序列相似性构建的系统发育进化树也显示白斑狗鱼与北极红点鲑和海鳟等鲑科鱼类处于同一分支。ElStAR蛋白相对分子量为32.23 kD,理论等电点（pI）为8.98,为不稳定的亲水性蛋白;具有START结构域,无信号肽及跨膜结构,符合线粒体靶向肽的基本特征。ElStAR蛋白二级结构由α-螺旋（占40.77%）、无规则卷曲（占35.89%）、延伸链（占18.12%）和β-折叠（占5.23%）组成,其三级结构是由α-螺旋配合多个β-折叠卷曲盘旋而成。ElStAR基因在白斑狗鱼精巢、卵巢、头肾、肝脏、肌肉及脑组织中均有不同程度的表达,且在精巢中的相对表达量极显著高于在卵巢中的相对表达量（P<0.01）,呈明显的性别二态性表达模式。【结论】ElStAR基因编码蛋白结构和功能十分保守,具有典型的START结构域,对胆固醇的运输和调节起重要作用。ElStAR基因在白斑狗鱼精巢及卵巢中的表达存在极显著差异,可能在维持白斑狗鱼精巢的发育形成中发挥重要作用。     

Cloning and expression of coxsakievirus B3 viral protein-1 in E. coli

BACKGROUND: Viral protein-1 (VP1) is a major capsid protein of Coxsakievirus B3 (CVB3) that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein (VP1) was over expressed in E. coli. METHODS: The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA (cells and virus) was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors (32 a and 28 a). The recombinant vector was transferred into competent E. coli (BL-21) and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein (VP1). The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. RESULTS: Molecular techniques such as PCR which showed exact defined size of the VP1 (819 bp), restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. CONCLUSION: In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3.     

Acylated flavonol glycoside from Platanus orientalis

The ethylacetate and n-butanol fractions of ethanolic extract of Platanus orientalis leaves led to the isolation of new acylated flavonol glycoside as 3',5,7-trihydroxy-4'-methoxyflavonol 3-[O-2-O-(2,4-Dihydroxy)-E-cinnamoyl-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl (1→2)]-β-D-glucopyranoside, along with seven known compounds. All the compounds were characterized by NMR including 2D NMR techniques. The isolates were evaluated for NF-κB, nitric oxide (NO), aromatase and QR2 chemoprevention activities and some of them appeared to be modestly active.     

Prevalence and molecular characterization of bovine Cryptosporidium in Qazvin province, Iran

The aim of this study was to determine the prevalence, variability with host age, and the genotypes of species of Cryptosporidium in cattle from 15 dairy farms in Qazvin province, Iran. Fecal samples, collected from 272 cattle during May 2006 to December 2007, were characterized microscopically. Oocysts from 51 positive samples were analyzed using PCR assay of 18S SSU rRNA, restriction fragment length polymorphism (RFLP) and sequencing. We identified 72.6% of the positive samples as Cryptosporidium parvum, 17.7% as Cryptosporidium andersoni, 7.8% as Cryptosporidium bovis and 1.9% as a novel genotype of C. parvum possessing a single mutation on MboII restriction. An infection rate of 19.5% of C. parvum among 174 pre-weaned calves was significantly higher than the 3.1% among 98 post-weaned calves (P < 0.0006). This is the first report of C. bovis and the new subgenotype of C. parvum in Iranian cattle.     

Effects of plant residue management on soil properties,surface runoff,and soil loss under rainfall simulation in a semi-arid region in Iran

A 5-year field study was conducted on the effect of plant residue management on soil erosion, runoff, bulk density, penetration resistance, and organic carbon. There were three treatments: burning residues on field (BR), returning residues unto the soil surface after harvesting (TR), and removing residues from soil surface (RR) (control treatment). At the end of the 5-year treatment, a rainfall simulation at 90?mm?h^?1 was applied to each plot. After rainfall simulation started, the runoff volume and soil loss amount in samples were collected at four sampling times. The results showed that the runoff volume for BR (from 145.5?±?12.2 to 190.0?±?11.8?mL) differed significantly (p?≤?0.01) from that of RR (from 32.3?±?5.5 to 67.5?±?11.1?mL) and TR (from 10.0?±?0.7 to 16.7?±?3.3?mL). A significant difference (p?≤?0.01) was also observed between RR and TR regarding runoff volume and soil loss amount in different sampling times, except for the first sampling time (2–4?min). The runoff volume in BR and TR was 215% higher and 294% lower than that of the control (RR), respectively. As compared to the control (RR), soil loss decreased by 96.5% in TR but increased by 192% in BR. The BR increased soil bulk density and penetration resistance by 4.9% and 12.4%, whereas TR reduced them by 2.1% and 15.8%, respectively, as compared to the control (RR). The results indicated that the highest (0.35) and lowest (0.03) runoff coefficients were obtained for BR and TR treatments, respectively. It is concluded that returning plant residues to soil is the best residue management practice in decreasing soil runoff volume and controlling soil erosion in semi-arid regions.     

Bacterial susceptibility to and chemical composition of essential oils from Thymus kotschyanus and Thymus persicus

Susceptibility of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, and Pseudomonas aeroginosa to the essential oils extracted from two varieties of Thyme, i.e., Thymus kotschyanus Boiss. and Hohen. and Thymus persicus L. at preflowering and flowering stages were studied. The disk diffusion method was used to evaluate the zone of microbial growth inhibition at various concentrations of the oils. Minimal inhibitory concentration and minimal bactericidal concentration of the oils were determined and compared with each other. The oils from the above plants were found to be strongly bactericidal with that of T. kotschyanus being more effective. T. kotschyanusand T. persicus oils analyzed by gas chromatography (GC) and GC/mass spectrometry (MS) lead to identification of 33 and 26 components, respectively. The profile of the oil components from T. persicuswas similar to that of T. kotschyanus in almost all of the compounds but at different concentrations. The major components of T. kotschyanus oil before and at the flowering stages were carvacrol (35.06, 22.75%), thymol (26.60, 16.52%), gamma-terpinene (7.81, 0.34%), gamma-terpinene (4.34, 0%), borneol (2.29, 4.52%), myrcene (0.26, 12.65%), thymolquinone (0, 11.39%), nerol (0, 6.10%), and beta-caryophyllene (0, 5.54%), respectively, and those of T. persicus at the same stages were carvacrol (38.96, 27.07%), thymol (6.48, 11.86%), P-cymene (7.51, 10.16%), gamma-terpineol (0, 9.51%), nerol (15.66, 9.41%), gamma-terpinene (6.11, 6.51%), and thymol acetate (5.29, 5.30%), respectively. The contribution of oil components to its antibacterial property is discussed. High aromatic compound content of the phenol-rich oils seems to account for strong antibacterial activity.     

Forest Road Detection Using LiDAR Data

We developed a three-step classification approach for forest road extraction utilizing LiDAR data. The first step employed the IDW method to interpolate LiDAR point data （first and last pulses） to achieve DSM, DTM and DNTM layers （at 1 m resolution）. For this interpolation RMSE was 0.19 m. In the second step, the Support Vector Machine （SVM） was employed to classify the LiDAR data into two classes, road and non-road. For this classification, SVM indicated the merged distance layer with intensity data and yielded better identification of the road position. Assessments of the obtained results showed 63% correctness, 75% completeness and 52% quality of classification. In the next step, road edges were defined in the LiDAR-extracted layers, enabling accu- rate digitizing of the centerline location. More than 95% of the Li- DAR-derived road was digitized within 1.3 m to the field surveyed nor- rnal. The proposed approach can provide thorough and accurate road inventory data to support forest management.     

Morphometric analysis of the intercarpal ligaments of the equine proximal carpal bones during simulated flexion and extension of cadaver limbs

Despite many reported cases of carpal lameness associated with intercarpal ligament injuries in horses, the morphometry, movement pattern and general intrinsic biomechanics of the carpus are largely unknown. Using osteoligamentous preparation of the carpus prepared from 14 equine cadaver forelimbs (aged 9.62 ± 4.25 years), locomotory simulations of flexion and extension movements of the carpal joint were carried out to observed carpal biomechanics and, thereafter, the limbs were further dissected to obtain morphometric measurements of the medial and lateral collateral ligaments (MLC and LCL); medial and lateral palmar intercarpal ligaments (MPICL and LPICL); intercarpal ligaments between radial (Cr) and intermediate (Ci) carpal bones (Cr-Ci ICL); and intercarpal ligaments between Ci and ulnar (Cu) carpal bones (Ci-Cu ICL). The Cr, Ci, Cu and Ca are held together by a series of intercarpal ligaments and move in unison lateropalmarly during flexion, and mediodorsally during extension with a distinguishable proximo-distal sliding movement (gliding) of Cr and Ci against each other during movement. The mean length of MCL (108.82 ± 9.64 mm) was significantly longer (p = 0.042) than LCL (104.43 ± 7.65 mm). The Cr-Ci ICL has a dorsopalmar depth of 37.58 ± 4.14 mm and a midpoint width of 12.05 ± 3.09 mm and its fibres ran diagonally from the medial side of the Ci in a proximo-palmar disto-dorsal direction (i.e. palmarodistally) to the lateral side of the Cr. The specialized movement of the Cr-Ci ICL, which appeared to be further facilitated by a longer MCL suggest a biomechanical function by which carpal damage may be minimized in the equine carpus.     

Landing,consumption, and DNA barcoding of commercial seabream (Perciformes: Sparidae) in Kuwait

1. To our knowledge, this is the first inclusive assessment of the status of Sparidae fisheries in the territorial waters of Kuwait, integrating information on catch trends, consumer preferences, recreational fishing behaviour, and molecular based identification of fish species by DNA barcoding
2. Fisheries landing data were obtained from the official fisheries bulletin released by the Kuwait Central Statistical Bureau. Surveys were conducted to assess consumer preferences on seabream species, landed seabream species, and the behaviour of recreational anglers towards seabream species. DNA barcodes were generated to authenticate commercial seabream using the mitochondrial cytochrome c oxidase subunit I (COXI) gene and then compared with GenBank sequence entries; these sequences were then used to construct a neighbour-joining (NJ) phylogenetic tree.
3. Yellowfin seabream Acanthopagrus latus was the prime and most favoured commercial seabream species (45%), followed by Sparidentex hasta, which was the top-rated secondary, unassessed seabream species. Approximately 54% of households conduct recreational fishing more than twice a month, and most (77%) primarily target S. hasta and Ac. latus. Consumer demand for secondary seabreams, which were not assessed and are listed in the bulletin under the category ‘others’, was evident.
4. The NJ phylogenetic tree revealed that Arabian Gulf seabreams, including those of Kuwait, are genetically different from their counterparts inhabiting neighbouring waters.
5. The data presented here highlight the urgency to modify the existing fisheries list using accurate identification tools, such as DNA barcoding, for the sustainable and conservation-oriented management of local fisheries, which are in decline.