Cloning and developmental expression of the Xenopus Nkx6 genes

The evolutionarily conserved Nkx6 family transcription factors play important roles in the patterning of the central nervous system (CNS) and pancreas in vertebrates. In this study, we describe the cloning and expression patterns of the three Nkx6 family

Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis

Even though it generates healthy adults, nuclear transfer in mammals remains an inefficient process. Mainly attributed to abnormal reprograming of the donor chromatin, this inefficiency may also be caused at least partly by a specific effect of the clonin

Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .

Cloning,Expression and Sequence Analysis of A Luciferase Gene from the Chinese Firefly Pyrocoelia pygidialis

从一种来自中国日行性萤火虫(云南窗萤)发光器官mRNA中克隆、测序并表达了有功能的荧光素酶.云南窗萤荧光素酶的cDNA序列有1647个碱基,编码548个氨基酸残基.从推测得到的氨基酸序列的比对分析得出:云南窗萤的荧光素酶与来自Lampyris noctiluca,L.turkestanicus和Nyctophila cf.caucasica三种萤火虫的荧光素酶有97.8%的序列一致性.从推测得出的氨基酸序列进行系统发育分析,其结果表明:云南窗萤和Lampyris+Nyctophila聚在一起,与同属的发光强夜行性的萤火虫不形成的单系.云南窗萤荧光素酶在大肠杆菌中表达的条带大约70kDa,并且在有荧光素存在时发出黄绿色荧光.对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点.云南窗萤和窗萤属的其他3种萤火虫的荧光素酶卡目比,有13个不同氨基酸位点,位于模拟分子结构的表面.对于这些多肽环、不刚氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异.

Aquatic ecology monitoring: water quality, fish, fish catch, sanitation and disease vectors: monitoring no. 10 and reservoir baseline, 23rd – 30th April 2012

The results reported on were from a monitoring survey No. 10 undertaken between 23 rd and 29th April 2012 during construction period of the Bujagali Hydropower Project (BHPP). Two pre-construction, baseline surveys in April 2000 and April 2006 were conducted and so far, during construction phase of the project, nine monitoring surveys have been undertaken i.e. in September 2007, April 2008, April 2009, October 2009, April 2010, September 2010, April 2011, September 2011and the present one, in April 2012. Since 2009 biannual monitoring surveys have been conducted at an upstream and a downstream transect of the BHPP with emphasis on the following aspects: water quality determinants biology and ecology of fishes and food webs fish stock and fish catch including economic aspects of catch and sanitation/vector studies (bilharzias and river blindness) During this survey, baseline assessment of the above mentioned studies was conducted in the reservoir behind the dam, including studies on algae, zooplankton and benthic macroinvertebrates which had been restrained since April 2008. The findings of baseline assessment of the reservoir are also contained in this report and are compared with those obtained from Transect 1(Upstream) and Transect 2 (Downstream).

Aquatic ecology monitoring, water quality, fish, fish catch, sanitation and disease vectors: monitoring no. 11, 4th – 9th September 2012

This monitoring survey No. 11 undertaken between 4th and 9th September 2012 is the second one to be conducted after completion of construction of Bujagali Hydropower Dam. Two pre-construction baseline surveys in April 2000 and April 2006 were conducted and during construction phase, eight monitoring surveys (September 2007, April 2008, April 2009, October 2009, April 2010, September 2010, April 2011, September 2011) were conducted.

Aquatic ecology monitoring: water quality, fish, fish catch, sanitation and disease vectors: monitoring No.7, 4-7th September 2010

The results reported on were from a monitoring survey No.7 undertaken between 4 th and 7th September 2010 during construction period of the Bujagali Hydropower Project (BHPP). Two pre-construction, baseline surveys in April 2000 and April 2006 were conducted and so far, during construction phase of the project, six monitoring surveys have been undertaken i.e. in September 2007, April 2008, April 2009, October 2009, April 2010 and the present one, in September 2010. Since 2009 biannual monitoring surveys have been conducted at an upstream and a downstream transect of the BHPP with emphasis on the following aspects: I. water quality determinants 2. biology and ecology of fishes and food webs 3. fish stock and fish catch including economic aspects of catch and 4. sanitation/vector studies (bilharzias and river blindness)

Aquatic ecology monitoring: water quality, fish, fish catch, sanitation and disease vectors: monitoring no.12, 25th-30th April 2013

Bujagali hydropower dam construction is now completed and a reservoir behind the dam has been created, extending all the way up to Kalange-Makwanzi, an upstream transects. During the 10th monitoring survey-April 2012, a third transect was established in the mid of the reservoir where it runs up to 30 m deep and sampled similarly as at the two original sampling transects, Kalange-Makwanzi and Buyala-Kikubamutwe for comparative purposes. This monitoring survey No. 12 undertaken between 25th and 30th April 2013 is the third one to be conducted after completion of construction of Bujagali Hydropower Dam. Two pre-construction baseline surveys in April 2000 and April 2006 were conducted and during construction phase, eight monitoring surveys (September 2007, April 2008, April 2009, October 2009, April 2010, September 2010, April 2011, September 2011) were conducted. Since 2009 biannual monitoring surveys have been conducted at an upstream and a downstream transect of the BHPP with emphasis on the following aspects: water quality determinants, biology and ecology of fishes and food webs, fish stock and fish catch including economic aspects of catch and sanitation/vector studies (bilharzias and river blindness). In the post-construction monitoring surveys, the assessments of algae, zooplankton and benthic macro-invertebrates which had been restrained since April 2008 were also included.

Evaluation of indigenous molluscicides in water against schistosomiasis vectors

Seven varieties of indigenous Phytolacca dodecwulra L'Herrit (Phytolaccaceae) were field-tried for molluscicidal potency. Varieties (U96) and (U95) collected from Kabarole and Kabale respectively were the most potent with LD90 equal to 2.54 and 6.46 mg.t-· respectively. Water bodies ranging between 4,770 and 347,510 Iitres in Kibimba rice fields were treated with up to 50mg.t-· Snails kills were monitored every three months and 92 - 100% mortality rates were realized. HPLC fingerprints revealed the two P. dodecandra varieties to contain highest concentration of the active principle, oleanoglycotoxin- A or lemmatoxin - A.

XMolecular Cloning and Sequence Analyses of cDNAs Encoding Seven C2type Lectin2like Protein Subunits from Daboia russellii siamensis

按照Promega 公司的mRNA 提取试剂盒操作手册, 从圆斑蝰蛇( Daboia russellii siamensis ) 的毒腺中提 取mRNA ; 利用RT2PCR 的方法进行体外扩增, 获得C - 型凝集素蛋白的基因, 克隆到pMD182T 载体中。随机挑 选14 个阳性克隆进行核酸测序, 获得7 个编码不同蛇毒C - 型凝集素样蛋白亚基的cDNA , 分别命名为DRS2L1 、 DRS2L2 、DRS2L3 、DRS2L4 、DRS2L5 、DRS2L6 和DRS2L7 。由基因序列推导出的氨基酸序列表明, 克隆到的7 个蛇 毒C - 型凝集素样蛋白的亚基中均有糖识别结构域存在。BLAST 分析显示, 仅有DRS2L1 的蛋白序列与目前已知 的蛇毒C - 型凝集素样蛋白的α亚基相似。序列同源性比较并结合半胱氨酸位点分析, 推测DRS2L1 和DRS2L2 可能分别是圆斑蝰蛇毒Ⅹ因子激活剂的轻链LC2 和LC1 。DRS2L3 和DRS2L4 可能是高分子量的蛇毒C - 型凝集 素样蛋白的β亚基, 而DRS2L5 和DRS2L6 可能是低分子量的蛇毒C - 型凝集素样蛋白的β亚基。DRS2L7 可能是 类似于血小板膜糖蛋白Ib 结合蛋白的β亚基。

Ultrasonic imaging of 3D displacement vectors using a simulated 2D array and beamsteering.

Most quasi-static ultrasound elastography methods image only the axial strain, derived from displacements measured in the direction of ultrasound propagation. In other directions, the beam lacks high resolution phase information and displacement estimation is therefore less precise. However, these estimates can be improved by steering the ultrasound beam through multiple angles and combining displacements measured along the different beam directions. Previously, beamsteering has only considered the 2D case to improve the lateral displacement estimates. In this paper, we extend this to 3D using a simulated 2D array to steer both laterally and elevationally in order to estimate the full 3D displacement vector over a volume. The method is tested on simulated and phantom data using a simulated 6-10MHz array, and the precision of displacement estimation is measured with and without beamsteering. In simulations, we found a statistically significant improvement in the precision of lateral and elevational displacement estimates: lateral precision 35.69μm unsteered, 3.70μm steered; elevational precision 38.67μm unsteered, 3.64μm steered. Similar results were found in the phantom data: lateral precision 26.51μm unsteered, 5.78μm steered; elevational precision 28.92μm unsteered, 11.87μm steered. We conclude that volumetric 3D beamsteering improves the precision of lateral and elevational displacement estimates.

Ultrasonic imaging of 3D displacement vectors using a simulated 2D array and beamsteering

Most quasi-static ultrasound elastography methods image only the axial strain, derived from displacements measured in the direction of ultrasound propagation. In other directions, the beam lacks high resolution phase information and displacement estimation is therefore less precise. However, these estimates can be improved by steering the ultrasound beam through multiple angles and combining displacements measured along the different beam directions. Previously, beamsteering has only considered the 2D case to improve the lateral displacement estimates. In this paper, we extend this to 3D using a simulated 2D array to steer both laterally and elevationally in order to estimate the full 3D displacement vector over a volume. The method is tested on simulated and phantom data using a simulated 6-10 MHz array, and the precision of displacement estimation is measured with and without beamsteering. In simulations, we found a statistically significant improvement in the precision of lateral and elevational displacement estimates: lateral precision 35.69 μm unsteered, 3.70 μm steered; elevational precision 38.67 μm unsteered, 3.64 μm steered. Similar results were found in the phantom data: lateral precision 26.51 μm unsteered, 5.78 μm steered; elevational precision 28.92 μm unsteered, 11.87 μm steered. We conclude that volumetric 3D beamsteering improves the precision of lateral and elevational displacement estimates. © 2012 Elsevier B.V. All rights reserved.

Cloning, expression and subcellular distribution of a Rana grylio virus late gene encoding ERV1 homologue

An essential for respiration and viability (ERV1) homologue, 88R, was cloned and characterized from Rana grylio virus (RGV). Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed a highly conserved motif shared by all ERV1 family proteins: Cys-X-X-Cys. RT-PCR and western blot analysis revealed that 88R begins to transcribe and translate at 6 h postinfection (p.i.) and remains detectable at 48 h p.i. during RGV infection course. Furthermore, using drug inhibition analysis by a de novo protein synthesis inhibitor and a viral DNA replication inhibitor, RGV 88R was classified as a late (L) viral gene during the in vitro infection. 88R-EGFP fusion protein was observed in both the cytoplasm and nucleus of pEGFP-N3-88R transfected EPC cells. Although result of immunofluorescence is similar, 88R protein was not detected in viromatrix. Moreover, function of RGV 88R on virus replication were evaluated by RNAi assay. Nevertheless, effect of knockdown of RGV 88R expression on virus replication was not detected in cultured fish cell lines. Collectively, current data indicate that RGV 88R was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.

Molecular cloning and characterization analysis of immunoglobulin M heavy chain gene in European eel (Anguilla anguilla)

In this study, the immunoglobulin M heavy chain gene of European eel (Anguilla anguilla) was cloned and analyzed. The full-length cDNA of the IgM heavy chain gene (GenBank accession no. EF062515) has 2089 nucleotides encoding a putative protein of 581 amino acids. The IgM heavy chain was composed of leader peptide (L), variable domain (VH), CH1, CH2. Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 in A. anguilla-H1 and A. anguilla-H2. The deduced amino acid sequence of the European eel IgM heavy chain constant region shared similarities to that of the Ladyfish (Elops saurus). Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus carpio), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. The highest level of IgM gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the apparently healthy European eels. (C) 2008 Elsevier B.V. All rights reserved.

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.