JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics.
Nucleic Acids Res.
PUBLISHED: 11-14-2014
Show Abstract
Hide Abstract
The Central Carbon Metabolic Flux Database (CeCaFDB, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. It encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information gathered from hundreds of journal articles. In addition to its comprehensive literature-derived data, the CeCaFDB supports a common text search function among the data and interactive visualization of the curated flux distributions with compartmentation information based on the Cytoscape Web API, which facilitates data interpretation. The CeCaFDB offers four modules to calculate a similarity score or to perform an alignment between the flux distributions. One of the modules was built using an inter programming algorithm for flux distribution alignment that was specifically designed for this study. Based on these modules, the CeCaFDB also supports an extensive flux distribution comparison function among the curated data. The CeCaFDB is strenuously designed to address the broad demands of biochemists, metabolic engineers, systems biologists and members of the -omics community.
Related JoVE Video
Establishment and application of a multiplex PCR for rapid and simultaneous detection of six viruses in swine.
J. Virol. Methods
PUBLISHED: 08-10-2014
Show Abstract
Hide Abstract
A multiplex PCR assay was developed and evaluated subsequently for its effectiveness in simultaneously detecting mixed viral infections of swine. Specific primers were designed and used for testing the six swine viruses: three DNA viruses, including pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine circovirus type 2 (PCV2); three common RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV). This technique has shown to be highly sensitive in that the minimum detection amounts of nucleic acids from PRV, PPV, PCV2, PRRSV, CSFV, and JEV were 6.6, 96, 12.9, 10.5, 51, and 46 pg, respectively. It also was effective for detecting one or multiple viruses in the specimens, such as the lungs, spleens, lymph nodes, and tonsils collected from clinically ill pigs. The multiplex PCR method can detect simultaneously not only infection of the six viruses, but also other swine DNA and RNA viruses. Given its rapidity, specificity, and sensitivity, the multiplex PCR is a useful tool for diagnosing clinically the mixed infections of swine DNA and RNA viruses.
Related JoVE Video
Molecular evidence for Anaplasma bovis infection in wild Reeves' muntjac (Muntiacus reevesi), Southwest China.
J. Wildl. Dis.
PUBLISHED: 08-06-2014
Show Abstract
Hide Abstract
Anaplasma bovis is one of the most important tick-borne pathogens in China. In the first report of A. bovis in China, we describe infection in eight of 17 wild Reeves' muntjac (Muntiacus reevesi) from Guangxi, southwest China.
Related JoVE Video
Required enhancer-matrin-3 network interactions for a homeodomain transcription program.
Nature
PUBLISHED: 08-03-2014
Show Abstract
Hide Abstract
Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and ?-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with ?-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with ?-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/?-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.
Related JoVE Video
An electronic environment and contact direction sensitive scoring function for predicting affinities of protein-ligand complexes in Contour(®).
J. Mol. Graph. Model.
PUBLISHED: 07-28-2014
Show Abstract
Hide Abstract
Contour(®) is a computational structure-based drug design technology that grows drug-like molecules by assembling context sensitive fragments in well-defined binding pockets. The grown molecules are scored by a novel empirical scoring function developed using high-resolution crystal structures of diverse classes of protein-ligand complexes and associated experimental binding affinities. An atomic model bearing features of the valence bond and VSEPR theories embodying their molecular electronic environment has been developed for non-covalent intermolecular interactions. On the basis of atomic hybridization and polarization states, each atom is modeled by features representing electron lone pairs, p-orbitals, and polar and non-polar hydrogens. A simple formal charge model was used to differentiate between polar and non-polar atoms. The interaction energy and the desolvation contribution of the protein-ligand association energy is computed as a linear sum of pair-wise interactions and desolvation terms. The pair-wise interaction energy captures short-range positive electrostatic interactions via hydrogen bonds, electrostatic repulsion of like charges, and non-bond contacts. The desolvation energy is estimated by calculating the energy required to desolvate interaction surfaces of the protein and the ligand in the complex. The scoring function predicts binding energies of a diverse set of protein-ligand complexes used for training with a correlation coefficient of 0.61. It also performs equally well in predicting association energies of a diverse validation set of protein-ligand complexes with a correlation coefficient of 0.57, which is equivalent to or better than 12 other scoring functions tested against this set including X-Score, GOLD, and DrugScore.
Related JoVE Video
Ectopic TBX1 suppresses thymic epithelial cell differentiation and proliferation during thymus organogenesis.
Development
PUBLISHED: 07-24-2014
Show Abstract
Hide Abstract
The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.
Related JoVE Video
Population analysis of Streptococcus suis isolates from slaughtered swine by use of minimum core genome sequence typing.
J. Clin. Microbiol.
PUBLISHED: 07-23-2014
Show Abstract
Hide Abstract
Streptococcus suis, an important zoonotic pathogen, is a highly diverse species with only a subset of strains that cause disease in humans. Our previous study proposed a minimum core genome (MCG) sequence typing method and defined seven MCG groups, with MCG group 1 as the prevalent group causing human infections. In this study, we identified a set of 10 single nucleotide polymorphisms (SNPs) distributed in six genes that were used to identify the seven MCG groups. The 10 SNPs were typed for 179 S. suis isolates collected from slaughtered pigs. The most prevalent groups among the tested isolates were MCG groups 6 and 7. Most of the isolates (147/179) were genotyped as mrp negative, epf negative, sly negative, and CDS2157 positive. The 179 isolates were also typed by multilocus sequence typing (MLST) and divided into 115 sequence types (STs), 111 of which were new. The 6 serotypes (29, 11, 5, 12, 30, and 2) represented 72.3% of the serotyped isolates. Our data show that the typing assay facilitates the application of genome data to the surveillance of S. suis.
Related JoVE Video
Genome Sequence of Borrelia garinii Strain SZ, Isolated in China.
Genome Announc
PUBLISHED: 07-19-2014
Show Abstract
Hide Abstract
We announce the genome sequence of Borrelia garinii strain SZ, isolated from Dermacentor ticks collected in northeastern China. B. garinii strain SZ carries numerous plasmids, both 10 circular and 9 linear plasmids. The 902,487-bp linear chromosome (28.2% GC content) contains 820 open reading frames, 33 tRNAs, and 4 complete rRNAs. The plasmid cp32-10 contains one clustered regularly interspaced short palindromic repeat (CRISPR) with four repeats.
Related JoVE Video
Complexity of the RAR-mediated transcriptional regulatory programs.
Subcell. Biochem.
PUBLISHED: 06-26-2014
Show Abstract
Hide Abstract
In the past several decades, intensive research in this field has uncovered a surprising number of regulatory factors and their associated enzymatic properties to reveal the network of complexes that function in activation and repression of the transcriptional programs mediated by nuclear receptors (NR). These factors and their associated complexes have been extensively characterized both biochemically and functionally [34, 87, 94]. Several principles have emerged: (1) It is widely recognized that ligand-dependent cofactor complexes mediating repression and activation exhibit ligand-dependent exchange. (2) These complexes mediate modifications of chromatin structure consequent to their binding at regulatory elements, particularly at promoter and enhancer Enhancer sites. (3) The concept about the rapid exchange of coregulatory complexes at regulatory sites has been suggested [88]. Key questions in the NR field have included: (a) What are the cofactors and exchange complexes used to mediate the ligand and signaling network-dependent switches in gene regulation programs; (b) Do long non-coding RNAs (lncRNAs) serve as regulatory "factors" for ligand-dependent gene programs, and do enhancers actually regulate transcription units encoding enhancer Enhancer non-coding RNAs (eRNAs) Enhancer RNA that might have functional significance; (c) What is the relationship between DNA damage repair machinery and transcriptional machinery? (d) Do Retinoic Acid Receptors (RAR) also regulate Pol III-dependent, non-coding repeat transcriptional units in stem cells? and (e) How have new technologies such as deep sequencing altered our ability to investigate transcriptional regulatory mechanisms utilized by NRs?
Related JoVE Video
Dermacentor everestianus Hirst, 1926 (Acari: Ixodidae): phylogenetic status inferred from molecular characteristics.
Parasitol. Res.
PUBLISHED: 05-26-2014
Show Abstract
Hide Abstract
Dermacentor everestianus Hirst, 1926, is only reported in Northwestern China and Nepal. Few researches about this species have been involved, especially for molecular characteristics. The taxonomy studies of D.everestianus are mainly based on morphological features, and its taxonomic status is an ongoing controversy. To clarify the molecular characteristics and phylogenetic status of D.everestianus and other related species, the sequences of mitochondrial 16S ribosomal DNA (rDNA) and cox1 fragments were analyzed in the present study. Analysis of 16S rDNA and cox1 sequences showed 99.3-100% identity within D.everestianus individuals, with the genetic divergence among them was 0-0.0086. The interspecific distance of 16S rDNA and cox1 between D.everestianus and some other Palaearctic species including D. silvarum, D. nuttalli, and D. marginatus was much smaller than that between D.everestianus and Nearctic Dermacentor ticks (D.albipictus, D.nitens, and D.variabilis). Such relationships of these ticks were also verified in the phylogenetic analysis. Two major clades were recovered within Dermacentor spp. with more than 90% bootstrap support in the phylogenetic trees. D.everestianus together with D.silvarum, D.nuttalli, and D.marginatus were included in the clade I (Eurasia lineage). Other analyzed tick species including D.variabilis, D.nitens, and D.albipictus formed clade II, which are distributed in Nearctic realm. These indicated that the genus Dermacentor was at least composed of two lineages. Thus, further researches including additionally molecular markers on all Dermacentor species globally should be taken to precisely resolve relationships within Dermacentor.
Related JoVE Video
Does Haemaphysalis bispinosa (Acari: Ixodidae) really occur in China?
Exp. Appl. Acarol.
PUBLISHED: 05-08-2014
Show Abstract
Hide Abstract
Haemaphysalis bispinosa Neumann has been considered to exist in China, especially in the southern part of the country. However, H. bispinosa referred to in many Chinese research papers may in fact be H. longicornis, which is widely distributed in most regions of China. In order to clarify the occurrence of H. bispinosa, Haemaphysalis ticks collected from 18 of 23 provinces of China (Hebei, Henan, Hubei, Guangxi, Gansu, Yunnan, Xinjiang, Anhui, Zhejiang, Shannxi, Guizhou, Sichuan, Shanxi, Shandong, Ningxia, Fujian, Qinghai and Jiangxi) were examined based on morphological and molecular characteristics. We found no evidence of H. bispinosa being present in China. Our results indicate that all of the so called "H. bispinosa" ticks reported in China are in fact H. longicornis.
Related JoVE Video
Identification and characterization of Argonaute gene family and meiosis-enriched Argonaute during sporogenesis in maize.
J Integr Plant Biol
PUBLISHED: 04-14-2014
Show Abstract
Hide Abstract
Argonaute (AGO) proteins play a key role in regulation of gene expression through small RNA-directed RNA cleavage and translational repression, and are essential for multiple developmental processes. In the present study, 17 AGO genes of maize (Zea mays L., ZmAGOs) were identified using a Hidden Markov Model and validated by rapid amplification of cDNA ends assay. Subsequently, quantitative PCR revealed that expressions of these genes were higher in reproductive than in vegetative tissues. AGOs presented five temporal and spatial expression patterns, which were likely modulated by DNA methylation, 5'-untranslated exons and microRNA-mediated feedback loops. Intriguingly, ZmAGO18b was highly expressed in tassels during meiosis. Furthermore, in situ hybridization and immunofluorescence showed that ZmAGO18b was enriched in the tapetum and germ cells in meiotic anthers. We hypothesized that ZmAGOs are highly expressed in reproductive tissues, and that ZmAGO18b is a tapetum and germ cell-specific member of the AGO family in maize.
Related JoVE Video
Enhancer Activation Requires trans-Recruitment of a Mega Transcription Factor Complex.
Cell
PUBLISHED: 04-03-2014
Show Abstract
Hide Abstract
Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ER? at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.
Related JoVE Video
Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants.
Vet. Parasitol.
PUBLISHED: 03-08-2014
Show Abstract
Hide Abstract
An enzyme-linked immunosorbent assay (ELISA) based on a recombinant Theileria uilenbergi immunodominant protein (rTuIP) was validated for detection of antibodies in 188 positive and 198 negative reference serum samples, respectively. The cut-off value was determined at 32.7% with 95% and 90% accuracy levels by two-graphic receiver-operating characteristic (TG-ROC). The equal diagnostic sensitivity (Se) and specificity (Sp) were calculated to be 98.4%. Further validation of the repeatability with positive and negative reference samples indicated the reliable performance of the assay. Monitoring the antibody dynamics of sheep experimentally infected with Theileria luwenshuni showed the efficient detection of antibody response against the pathogen at the early infection stage and up until two months post infection. Application of this assay for detection of antibody in field sera from previous unknown Theileria endemic regions in Suizhou and Guiyang showed 17.8% and 11.6% seroprevalence, respectively, and presence of the pathogen was confirmed by identification of the 18S rRNA gene in the corresponding blood of the seropositive animals. These data support that the rTuIP ELISA could be a useful tool to study the epidemiology of theileriosis caused by T. uilenbergi and/or T. luwenshuni.
Related JoVE Video
Susceptibility of the tick Haemaphysalis qinghaiensis to isolates of the fungus Metarhizium anisopliae in China.
Exp. Appl. Acarol.
PUBLISHED: 03-05-2014
Show Abstract
Hide Abstract
Haemaphysalis qinghaiensis, a prevalent tick species in China, causes severe economic losses. In this study, we investigated the pathogenicity of six isolates of the fungus Metarhizium anisopliae to engorged female H. qinghaiensis using concentrations of 10(6), 10(7) and 10(8) conidia ml(-1). The results indicated that M.aAT08 and M.aAT13 isolates were highly virulent against the ticks. Metarhizium anisopliae has potential for biocontrol of H. qinghaiensis.
Related JoVE Video
Molecular identification of Theileria parasites of northwestern Chinese Cervidae.
Parasit Vectors
PUBLISHED: 02-27-2014
Show Abstract
Hide Abstract
Theileria and Babesia protozoan parasites are transmitted mainly by tick vectors. These parasites cause heavy economic losses to the live-stock industry, as well as affecting the health of wild animals in parasite-endemic areas. Identification of infectious agents in wild animals is not only crucial for species preservation, but also provides valuable information on parasite epidemiology. Here, we conducted a molecular surveillance study in Northwestern China to assess the prevalence of blood pathogens in cervids.
Related JoVE Video
Scanning electron microscopy of all parasitic stages of Haemaphysalis qinghaiensis Teng, 1980 (Acari: Ixodidae).
Parasitol. Res.
PUBLISHED: 02-21-2014
Show Abstract
Hide Abstract
Haemaphysalis qinghaiensis Teng (Acta Zootaxon Sin 5:144-149, 1980) is an endemic species in China. This tick species was first described based on engorged or semi-engorged specimens, and the drawings and description in words of morphological characteristics were poor. Therefore, the present study aims to redescribe morphological characteristics of all active stages of this tick species in detail by scanning electron microscopy. Additionally, a comparison between H. qinghaiensis and other sympatric Haemaphysalis species was also analyzed. Males of H. qinghaiensis can be distinguished from sympatric Haemaphysalis species by the following characters: palpi less salient laterally and curved in contour; ventrointernal setae of palpal segment II thin, number <7; the tips of palpal segment III not so strongly recurved inward to become "pincerlike" and lacking dorsal spur; dental formula 5/5; lateral grooves enclose first festoon; coxa IV with a short, broadly triangular spur; tarsi somewhat humped; and spiracular plates long comma-shaped. Females of H. qinghaiensis can be distinguished by palpi less salient laterally and curved in contour; ventrointernal setae of palpal segment II thin, number <7; segment III of palpi lacking dorsal spur; dental formula 4/4; scutum subcircula; and tarsi somewhat humped. Nymphs of H. qinghaiensis can be distinguished from those of other species by palpi less salient laterally and curved in contour; dental formula 2/2; basis capituli rectangular, with distinct dorsal cornua, without ventral cornua; and spiracular plates with short and narrow dorsal prolongation. Larvae of H. qinghaiensis can be distinguished by palpi less salient laterally and curved in contour; basis capituli rectangular, without distinct cornua.
Related JoVE Video
Comparative genomic hybridization identifies virulence differences in Streptococcus suis.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Streptococcus suis is an important zoonotic pathogen. However, identification of virulent S. suis strains is complicated because of the high diversity of the species. Here we evaluated the genetic difference among S. suis strains using comparative genomic hybridization (CGH) and virulence variation in vivo and in vitro. We showed that different clades differed in their ability to activate TLR2/6 in vitro and their capacity to induce cytokine production in vivo as well as their resistance to phagocytosis and survival in vivo. Our data showed the S. suis strains tested can be classified into three groups having differing levels of virulence: epidemic and highly virulent strains were clustered into clade Ia (epidemic and highly virulent group, E/HV group), virulent strains were clustered into clade Ib (virulent group, V group), and intermediately or weakly virulent strains were clustered into other clades (intermediately or weakly virulent group, I/WV group). Our study provided further insight into the genomic and virulence variation of S. suis.
Related JoVE Video
Enhancement of ansamitocin P-3 production in Actinosynnema pretiosum by a synergistic effect of glycerol and glucose.
J. Ind. Microbiol. Biotechnol.
PUBLISHED: 08-31-2013
Show Abstract
Hide Abstract
Ansamitocin P-3 (AP-3), a secondary metabolite produced by Actinosynnema pretiosum, is well known for its extraordinary antitumor properties and is broadly utilized in clinical research. Through this work, we found, for the first time, that the combination of glucose and glycerol as a mixed carbon source is an appropriate approach for enhancing the production of AP-3 by A. pretiosum. The amount yielded was about threefold that obtained with glucose as the sole carbon source. In order to better understand the mechanisms that channel glycerol metabolism towards AP-3 production, the activities of some key enzymes such as glucose-6-phosphate dehydrogenase, glucose-6-phosphate isomerase, phosphoglucomutase (PGM), and fructose 1,6-bisphosphatase were assessed. The results showed that glycerol affects the production of AP-3 by increasing PGM activity. Furthermore, qRT-PCR analysis revealed that transcriptional levels of structural genes asm14 and asm24, and primary genes amir5189 and amir6327 were up-regulated in medium containing glycerol.
Related JoVE Video
Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants.
Parasitol. Res.
PUBLISHED: 08-15-2013
Show Abstract
Hide Abstract
Infections with Theileria sp. may cause significant economic losses to the sheep industry. Species identification based on microscopic examination is difficult, and more suitable methods are required for the rapid detection and identification of Theileria sp, in clinical specimens. In this study, a multiplex polymerase chain reaction (mPCR) assay was developed to simultaneously identify three individual Theileria species in small ruminants. Three pairs of specific, sensitive primers were designed on the basis of the 5.8S ribosomal RNA gene (Theileria luwenshuni and Theileria ovis) and the 18S ribosomal RNA gene (Theileria uilenbergi) to generate target products of 303, 884, and 530 bp, respectively. Standard DNA for each of the three species was extracted from blood recovered from infected sheep, and a preliminary study was conducted on 56 sheep to verify the reliability of the system. Optimal PCR conditions, including primer concentration, annealing time, and the number of amplification cycles, were established. The assay sensitivity under these conditions was 10(-3) % parasitemia, and its specificity was 100 %. The results of the study suggest that mPCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of Theileria species in small ruminants.
Related JoVE Video
Pathogenic analysis of Borrelia garinii strain SZ isolated from Northeastern China.
Parasit Vectors
PUBLISHED: 05-05-2013
Show Abstract
Hide Abstract
Various genospecies of Borrelia burgdorferi sensu lato (s.l.) have been identified from patients and animals worldwide. Genospecies-related dissemination of disease has been reported. The present study aimed to elucidate the pathogenicity of infections caused by B. garinii SZ isolated in China. B. burgdorferi B31 and B. afzelii BO23 were used for comparison.
Related JoVE Video
Coevolutionary analyses of the relationships between piroplasmids and their hard tick hosts.
Ecol Evol
PUBLISHED: 04-12-2013
Show Abstract
Hide Abstract
Host-parasite coevolution is a key driver of biological diversity. To examine the evolutionary relationships between piroplasmids and their hard tick hosts, we calculated the molecular clock and conducted phylogenetic analyses of both groups. Based on our results, we conclude that the divergence time of piroplasmids (?56 Mya) is later than divergence time of their hard tick hosts (?86 Mya). From analyses of the evolution of both piroplasmid and vector lineages and their association, we know that hard ticks transmit piroplasmids with high genus specificity and low species specificity.
Related JoVE Video
An epidemiological survey of Theileria infections in small ruminants in central China.
Vet. Parasitol.
PUBLISHED: 04-08-2013
Show Abstract
Hide Abstract
Here, we conducted an epidemiological study in five regions in central China to assess the impact of theileriosis on small ruminants. PCR analysis and microscopic evaluations of blood smears to detect ovine and caprine theileriosis was conducted, in which 256 blood samples and 250 ticks were collected from sheep and goats, and tested for Theileria uilenbergi, T. luwenshuni, and T. ovis. The 18S rRNA gene sequences were deduced from positive samples and used for phylogenetic analysis. The results showed that T. luwenshuni was found most frequently in the five investigated regions and the prevalence of T. luwenshuni was found to be very high by PCR analysis. In contrast, T. uilenbergi and T. ovis infections were not detected in these regions. Phylogenetic tree analysis showed that all of the newly isolated Theileria spp. was in the same clade as T. luwenshuni. Haemaphysalis longicornis, which can transmit T. luwenshuni, was also detected in the sampled sheep and goats in these regions. Our results provide important data to increase the understanding of the epidemiology of ovine and caprine theileriosis, and will aid in the implementation of measures to control theileriosis transmission to small ruminants in central China.
Related JoVE Video
Effect of dielectric material on bipolar nanosecond pulse diffuse dielectric barrier discharge in air at atmospheric pressure.
Spectrochim Acta A Mol Biomol Spectrosc
PUBLISHED: 04-02-2013
Show Abstract
Hide Abstract
In this paper, dielectric plates made by ceramic, quartz and polytetrafluoroethylene (PTFE) respectively are employed to generate low gas temperature, diffuse dielectric barrier discharge plasma by using a needle-plate electrode configuration in air at atmospheric pressure. Both discharge images and the optical emission spectra are obtained while ceramic, quartz and PTFE are used as dielectric material. Plasma gas temperature is also calculated by comparing the experimental emission spectra with the best fitted spectra of N2 (C(3)?u?B(3)?g 1-3) and N2 (C(3)?u?B(3)?g 0-2). The effects of different pulse peak voltages and gas gap distances on the emission intensity of N2 (C(3)?u?B(3)?g, 0-0, 337.1 nm) and the plasma area on dielectric surface are investigated while ceramic, quartz and PTFE are used as dielectric material. It is found that the permittivity of dielectric material plays an important role in the discharge homogeneity, plasma gas temperature, emission spectra intensity of the discharge, etc. Dielectric with higher permittivity i.e., ceramic means brighter discharge luminosity and stronger emission spectra intensity of N2 (C(3)?u?B(3)?g, 0-0, 337.1 nm) among the three dielectric materials. However, more homogeneous, larger plasma area on dielectric surface and lower plasma gas temperature can be obtained under dielectric with lower permittivity i.e., PTFE. The emission spectra intensity and plasma gas temperature of the discharge while the dielectric plate is made by quartz are smaller than that while ceramic is used as dielectric material and bigger than that when PTFE is used as dielectric material.
Related JoVE Video
Transdifferentiation of parathyroid cells into cervical thymi promotes atypical T-cell development.
Nat Commun
PUBLISHED: 03-25-2013
Show Abstract
Hide Abstract
The thoracic thymus is the primary vertebrate organ for T-cell generation. Accessory cervical thymi have also been identified in humans and mice, and shown in mice to be independent functional organs that support T-cell development. However, their origin and functional significance remain unclear. Here we show that cervical thymi in mice have following two origins: delayed differentiation of endodermal precursors and transdifferentiation of parathyroid-fated cells. Compared with thoracic thymus, parathyroid-origin cervical thymi (pCT) express low levels of the thymic epithelial cell-specific transcription factor FOXN1. Consequently, pCT form a distinct microenvironment that supports an atypical thymocyte development pathway, generating T cells with unconventional phenotypic characteristics. Our data demonstrate a transdifferentiation origin for a subset of cervical thymi, with specific functional consequences for T-cell development.
Related JoVE Video
Infection of small ruminants and their red blood cells with Theileria annulata schizonts.
Exp. Parasitol.
PUBLISHED: 03-11-2013
Show Abstract
Hide Abstract
Theileria annulata, the causative agent of tropical theileriosis, is a protozoan parasite that also causes lymphoproliferative diseases in cattle. In vivo, parasitized cells undergo clonal expansion and infiltrate both the lymphoid and non-lymphoid tissues of the infected host. To determine whether the small ruminants and their red blood cells (RBCs) were invaded by T. annulata schizonts or not, T. annulata schizonts were used to infect bovine, ovine and caprine RBCs in vitro, and sheep and goats in vivo. The results showed that the schizonts infected bovine, ovine and caprine RBCs in vitro, but not sheep and goats, which showed only an increase in body temperature and no development of piroplasms. To our knowledge, this is the first report of infection of small ruminants and their RBCs by T. annulata schizonts.
Related JoVE Video
The in vitro efficacy of deltamethrin and alpha-cypermethrin against engorged female Haemaphysalis qinghaiensis ticks (Acari: Ixodidae).
Exp. Parasitol.
PUBLISHED: 03-06-2013
Show Abstract
Hide Abstract
Currently, the most efficient and widely used method for tick control is the application of acaricides, especially deltamethrin and alpha-cypermethrin, two pyrethroids with neurotoxic action. In this study, the in vitro efficacy of deltamethrin and alpha-cypermethrin was assessed on engorged female Haemaphysalis qinghaiensis ticks. An in vitro bioassay (adult immersion test) was carried out to determine the LC (lethal concentration) 50 and LC90 of both compounds, calculated by probit analysis. The LC50 and LC90 values of deltamethrin and alpha-cypermethrin were 5.67 (LC50) and 51.72ppm (LC90), and 166.56 (LC50) and 1366.69ppm (LC90), respectively. This study provides important information on the efficacy of deltamethrin and alpha-cypermethrin for the control of H. qinghaiensis.
Related JoVE Video
Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene.
Korean J. Parasitol.
PUBLISHED: 01-15-2013
Show Abstract
Hide Abstract
Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.
Related JoVE Video
Development of multiplex PCR assays for the identification of the 33 serotypes of Streptococcus suis.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Streptococcussuis is an important zoonotic agent causing severe diseases in pigs and humans. To date, 33 serotypes of S. suis have been identified based on antigenic differences in the capsular polysaccharide. The capsular polysaccharide synthesis (cps) locus encodes proteins/enzymes that are responsible for capsular production and variation in the capsule structures are the basis of S. suis serotyping. Multiplex and/or simplex PCR assays have been developed for 15 serotypes based on serotype-specific genes in the cps gene cluster. In this study, we developed a set of multiplex PCR (mPCR) assays to identify the 33 currently known S. suis serotypes. To identify serotype-specific genes for mPCR, the entire genomes of reference strains for the 33 serotypes were sequenced using whole genome high-throughput sequencing, and the cps gene clusters from these strains were identified and compared. We developed a set of 4 mPCR assays based on the polysaccharide polymerase gene wzy, one of the serotype-specific genes. The assays can identify all serotypes except for two pairs of serotypes: 1 and 14, and 2 and 1/2, which have no serotype-specific genes between them. The first assay identifies 12 serotypes (serotypes 1 to 10, 1/2, and 14) that are the most frequently isolated from diseased pigs and patients; the second identifies 10 serotypes (serotypes 11 to 21 except 14); the third identifies the remaining 11 serotypes (serotypes 22 to 31, and 33); and the fourth identifies a new cps cluster of S. suis discovered in this study in 16 isolates that agglutinated with antisera for serotypes 29 and 21. The multiplex PCR assays developed in this study provide a rapid and specific method for molecular serotyping of S. suis.
Related JoVE Video
Two distinct calmodulin binding sites in the third intracellular loop and carboxyl tail of angiotensin II (AT(1A)) receptor.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT(1A)), at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214-231) and carboxyl tail of the receptor (ct, 302-317). We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT(1A) holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca²?-dependent fashion. The former is a 1-12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219) for alanine in i3, and phenylalanine (F309 or F313) for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein ?? subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter G?? binding, but reduced the ability of calmodulin to compete with G??, suggesting that calmodulin and G?? have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the G?? binding to i3 and ct regions of the AT(1A) receptor strongly suggests that calmodulin plays critical roles in regulating G??-dependent signaling of the receptor.
Related JoVE Video
Molecular survey and genetic identification of Anaplasma species in goats from central and southern China.
Appl. Environ. Microbiol.
PUBLISHED: 11-04-2011
Show Abstract
Hide Abstract
Anaplasma species are obligate intracellular rickettsial pathogens that impact the health of humans and animals. Few studies have been carried out on Anaplasma infections in central and southern China. This study was conducted to determine the coinfection rates of Anaplasma ovis, A. bovis, and A. phagocytophilum from 262 field blood samples of goats in these regions. The average prevalences of single infection of A. ovis, A. bovis, and A. phagocytophilum were 15.3, 16.0, and 6.1%, respectively. Coinfection of A. ovis and A. bovis was dominant, with an infection rate of 27.1%. Coinfection of A. ovis and A. phagocytophilum was 1.9% and that of A. bovis and A. phagocytophilum was 4.2%. Three-pathogen coinfection was found in three of four investigated provinces with a prevalence between 0 and 5.3%. The accuracy of the PCR results was corroborated by sequencing. Analysis of the 16S rRNA gene sequences of A. bovis and A. phagocytophilum confirmed the presence of these pathogens at the investigated sites and indicated the possible genetic diversity of A. phagocytophilum. Field blood inoculation of experimental animals led to successful identification and observation of the morphological shapes of A. bovis in the infected monocytes of sheep. Phylogenetic study with msp4 sequences of A. ovis indicated that the A. ovis genotypes from sheep in the north differed from the genotypes of goats in the investigated sites.
Related JoVE Video
The life cycle of Hyalomma rufipes (Acari: Ixodidae) under laboratory conditions.
Exp. Appl. Acarol.
PUBLISHED: 08-02-2011
Show Abstract
Hide Abstract
Biological characteristics of Hyalomma rufipes parasitising on rabbits and sheep were compared under laboratory conditions in Gansu, China. Mature ticks could parasitize both rabbits and sheep, while immature ticks only fed on rabbits successfully. Adults sucked blood on sheep significantly longer than on rabbits (16 and 13 days, respectively). Other adult parasite characteristics fed on the two hosts were similar, including the weight of engorged adult, female daily oviposition, and the weight and amount of the egg mass laid. Those indicated that this tick species showed little host specificity between sheep and rabbits during its adult stage. In total, the life cycle of H. rufipes was completed in an average period of 179 days. The average developmental periods were 59 days for egg incubation, 3 and 21 days for immature tick prefeeding and feeding, 2, 12 and 40 days for adult prefeeding, female preoviposition and oviposition. The longer female fed for engorgement, the shorter preoviposition period of engorged female needed, although when the feeding period was less than 15 days, this relationship was not obvious. The results confirmed the correlation between the weight of the engorged female and the number of eggs laid (r = 0.909). The reproductive efficiency index (REI) and reproductive fitness index (RFI) in females was 10.63 and 7.22, respectively. Engorged nymphs moulting to females were significantly heavier (27.6 ± 0.89 mg) than those moulting to males (22.3 ± 0.52 mg). Males outnumbered females by 1.4:1.
Related JoVE Video
Detection and differentiation of Borrelia burgdorferi sensu lato in ticks collected from sheep and cattle in China.
BMC Vet. Res.
PUBLISHED: 04-29-2011
Show Abstract
Hide Abstract
Lyme disease caused by Borrelia burgdorferi sensu lato complex is an important endemic zoonosis whose distribution is closely related to the main ixodid tick vectors. In China, isolated cases of Lyme disease infection of humans have been reported in 29 provinces. Ticks, especially ixodid ticks are abundant and a wide arrange of Borrelia natural reservoirs are present. In this study, we developed a reverse line blot (RLB) to identify Borrelia spp. in ticks collected from sheep and cattle in 7 Provinces covering the main extensive livestock regions in China.
Related JoVE Video
Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi.
Parasitol. Res.
PUBLISHED: 04-27-2011
Show Abstract
Hide Abstract
A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/?l of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.
Related JoVE Video
Sequence analysis of a Torque teno canis virus isolated in China.
Virus Res.
PUBLISHED: 04-22-2011
Show Abstract
Hide Abstract
In the present study, a total of 158 fecal samples were collected from diarrheal dogs younger than 1 year old in pet clinic in China. 20 specimens (20/158, 13%) were positive for Torque teno canis virus DNA using detection PCR. One representative positive isolate designated LDL was randomly selected, cloned and sequenced. The complete genome of the LDL Chinese strain was 2799 nucleotides in length and contains three open reading frames (ORFs), which encode 576 (ORF1), 101 (ORF2), and 243 (ORF3) aa. Compared with the human and other animal TTV genomes, the genome of the LDL strain is clearly smaller and shares 95% identity with Japanese cf-TTV10 strain (AB076002). Phylogenetic analysis showed that the present Chinese Torque teno canis virus LDL strain was also closely clustered with the previous Japanese cf-TTV10 strain, and formed a different branch together with Torque teno sus viruses 1 and 2 compared with other Torque teno viruses, Torque teno mini virus, and Torque teno midi virus. Our study demonstrated that Torque teno canis virus is present in China.
Related JoVE Video
Development and evaluation of a loop-mediated isothermal amplification method for rapid detection of Anaplasma ovis.
J. Clin. Microbiol.
PUBLISHED: 04-06-2011
Show Abstract
Hide Abstract
Anaplasma ovis is an intraerythrocytic rickettsial pathogen of small ruminants. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection method in which the target DNA can be efficiently amplified with high specificity and sensitivity under isothermal conditions. In this study, a LAMP method was developed for the specific detection of A. ovis, using LAMP primers designed on the basis of the major surface protein 4 gene. LAMP was performed at 65 °C for 30 min. Its specificity was confirmed by successful amplification of several A. ovis isolates and through EcoRI restriction analysis of LAMP products. No cross-reactivity with the A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi, or the Babesia sp. Xinjiang isolate was observed. Detection using the LAMP method was compared with that using conventional PCR in 227 field samples; LAMP demonstrated a sensitivity of 95.45%. In summary, LAMP is a specific, sensitive, and rapid test for the diagnosis of A. ovis infection, with the potential to be standardized as a detection method for A. ovis in areas of endemicity.
Related JoVE Video
Enhanced Y1H assays for Arabidopsis.
Nat. Methods
PUBLISHED: 04-01-2011
Show Abstract
Hide Abstract
We present an Arabidopsis thaliana full-length transcription factor resource of 92% of root stele-expressed transcription factors and 74.5% of root-expressed transcription factors. We demonstrate its use with enhanced yeast one-hybrid (eY1H) screening for rapid, systematic mapping of plant transcription factor-promoter interactions. We identified 158 interactions with 13 stele-expressed promoters, many of which occur physically or are regulatory in planta.
Related JoVE Video
Virulence of Beauveria bassiana, Metarhizium anisopliae and Paecilomyces lilacinus to the engorged female Hyalomma anatolicum anatolicum tick (Acari: Ixodidae).
Vet. Parasitol.
PUBLISHED: 03-11-2011
Show Abstract
Hide Abstract
The tick is a common ectoparasite of livestock and humans, and is responsible for the transmission of pathogens among hosts. Direct and indirect impacts of ticks include limiting the sustainable development of the animal husbandry industry and detrimental effects on human health. Despite these negative effects, the main method of controlling ticks remains the application of chemical acaricides, which can lead to ambient pollution and the development of tick resistance to them. The biocontrol of ticks is one of the alternative control methods that has received recent research attention. The present study used Tenebrio moliter bait methods to collect 13 species of entomopathogenic fungi from different areas in China that were then tested to observe their effects on engorged female Hyalomma anatolicum anatolicum ticks. The results showed that more than half of the isolates had some pathogenic effects on the ticks; in particular, two Beauveria bassiana strains (B.bAT01, B.bAT17) and one Metarhizium anisopliae strain (M.aAT26) were highly virulent, causing up to 90% mortality. In addition, H. anatolicum anatolicum females were treated with B. bassiana B.bAT17 using different concentrations of the fungus. Results revealed that B. bassiana B.bAT17 is highly pathogenic against engorged H. anatolicum anatolicum females. This is the first report of the pathogenic effect of entomopathogenic fungi on engorged H. anatolicum anatolicum females. However, studies of the efficiency of this fungus against ticks in the field are required before it can be used for tick management in practice.
Related JoVE Video
Biological control of engorged female Haemaphysalis qinghaiensis (Acari: Ixodidae) ticks with different Chinese isolates of Beauveria bassiana.
Parasitol. Res.
PUBLISHED: 03-09-2011
Show Abstract
Hide Abstract
Seven isolates of the fungus Beauveria bassiana (B.b) were assessed for their lethality against Haemaphysalis qinghaiensis, a prevalent tick species in China. Fourteen days after exposure to the isolates B.bAT1, B.bAT5, and B.bAT7 (at 10(8) conidia mL(-1)), the mortality rate had reached 100%. The results indicated that these three B. bassiana isolates were highly virulent against the engorged female H. qinghaiensis ticks. The present study suggests that B. bassiana has potential for biocontrol applications to eradicate H. qinghaiensis.
Related JoVE Video
An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability.
Protein Expr. Purif.
PUBLISHED: 02-14-2011
Show Abstract
Hide Abstract
Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.
Related JoVE Video
Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae).
Exp. Parasitol.
PUBLISHED: 01-20-2011
Show Abstract
Hide Abstract
Owing to the need to combat the spread of chemical acaricide resistance in ticks, we evaluated the efficacy of a mixture of the entomopathogenic fungus Beauveria bassiana AT17 and acaricides for the control of Hyalomma anatolicum anatolicum in China. A mixture of B. bassiana AT17 at the concentration of 10(8)conidia/mL and the synthetic pyrethroid deltamethrin at concentrations of 2500, 250, 25, 5, 2.5, 0.5 and 0.25ppm was tested in vitro. The germination capability, vegetative growth, conidia production, and viability of B. bassiana AT17 were assessed and the efficacy of the mixture in killing engorged H. anatolicum anatolicum females was measured. High mortality rates were achieved when the entomopathogen was combined with different concentrations of deltamethrin. Neither B. bassiana AT17 nor deltamethrin alone at the same concentrations could cause the higher mortality rates seen with the combination. In addition the combination killed the ticks more rapidly than did either agent alone (3-5days more rapidly). Our results indicate that the mixture of B. bassiana AT17 and deltamethrin has potential as a new type of reagent for integrated control of H. anatolicum anatolicum.
Related JoVE Video
Thymus-associated parathyroid hormone has two cellular origins with distinct endocrine and immunological functions.
PLoS Genet.
PUBLISHED: 09-13-2010
Show Abstract
Hide Abstract
In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2?/? mutants. However, the developmental ontogeny and cellular identity of these "thymic" PTH-expressing cells is unknown. We found that the lethality of aparathyroid Gcm2?/? mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2?/? mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant "thymic PTH."
Related JoVE Video
Crystallization and preliminary crystallographic analysis of a calcineurin B-like protein 1 (CBL1) mutant from Ammopiptanthus mongolicus.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 08-06-2010
Show Abstract
Hide Abstract
Calcineurin B-like protein 1 (CBL1) is a calcium sensor in plants. It transmits the calcium signal through the downstream protein CBL-interacting protein kinase (CIPK). CBL1 and CIPK play crucial roles in the response to environmental stresses such as low K+, osmotic shock, high salt, cold and drought. Recombinant CBL1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. However, the crystal did not diffract well. A mutant prepared using the surface-entropy method and crystallized using the hanging-drop method at 298?K with PEG 2000 MME as a precipitant diffracted to 2.90?Å resolution. The crystal belonged to space group P2(1)2(1)2, with unit-cell parameters a=99.87, b=114.42, c=63.80?Å, ?=?=?=90.00° and three molecules per asymmetric unit.
Related JoVE Video
[Construction of Helicobacter pylori Lpp20-IL2 DNA vaccine and evaluation of its immunocompetence in C57BL/6 mice].
Wei Sheng Wu Xue Bao
PUBLISHED: 06-22-2010
Show Abstract
Hide Abstract
To evaluate the humoral and cellular immune responses in C57BL/6 mice induced by pcDNA3.1(+)/Lpp20-IL2 for further development of DNA vaccine against H. pylori infection.
Related JoVE Video
Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP).
Exp. Parasitol.
PUBLISHED: 04-21-2010
Show Abstract
Hide Abstract
Theileria sergenti and Theileria sinensis are closely related members of benign Theileria species found in cattle and yaks in China. They are morphologically indistinguishable. A polymerase chain reaction (PCR) targeting major piroplasm surface protein of T. sergenti and T. sinensis was developed in this study. The newly developed oligonucleotide primer set was able to specifically amplify the DNA of T. sinensis and in conjunction with primers for T. sergenti and these two species could be detected and distinguished. Specificity testing also revealed that there was no cross-reaction with the other tick-borne diseases Theileria annulata, Babesia ovata, Anaplasma marginale as well as bovine white blood cells. Phylogenetic analysis based on the MPSP gene sequences confirmed the specificity of PCR assays. The sensitivity of the methods was 0.1pg DNA for the T. sergenti PCR and 1pg DNA for T. sinensis PCR. Two hundred and thirty-six field blood samples from of cattle and yaks were collected from five different geographical regions in China where benign Theileria species have been found. T. sergenti was found in all five provinces but was absent from one county in Gansu Province. T. sinensis was only found in Gansu Province. In both counties in Gansu where the parasites co-existed, mixed infections were detected. Our results indicate that the PCR methods developed in this study are suitable for the detection and differentiation of T. sergenti and T. sinensis.
Related JoVE Video
Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis.
Parasitol. Res.
PUBLISHED: 04-08-2010
Show Abstract
Hide Abstract
The pathogenic protozoan parasite Theileria uilenbergi is one of the causative agents of theileriosis in small ruminants in China. The infection results in great economical losses in the northwest part of China. Efforts are underway to establish an enzyme-linked immunosorbent assay (ELISA) based on a T. uilenbergi immunodominant recombinantly expressed protein using different approaches in order to perform epidemiological studies in the area. In this study, we describe the possible use of the clone-9 protein for this purpose, which was identified as a potential immunogenic piroplasm protein by random sequencing of cDNA library clones followed by bioinformatic analyses. The clone-9 gene was partially recombinantly expressed and used for the development of an indirect ELISA for the detection of circulating antibodies in sera of T. uilenbergi-infected sheep. No cross-reactivity was observed in serum from animals infected with Theileria lestoquardi. The cut-off was calculated at 48.6% positivity using 25 serum samples from uninfected animals. A total of 101 field samples collected from an endemic area in China were used to evaluate the clone-9 ELISA for its use in the field.
Related JoVE Video
The B73 maize genome: complexity, diversity, and dynamics.
Patrick S Schnable, Doreen Ware, Robert S Fulton, Joshua C Stein, Fusheng Wei, Shiran Pasternak, Chengzhi Liang, Jianwei Zhang, Lucinda Fulton, Tina A Graves, Patrick Minx, Amy Denise Reily, Laura Courtney, Scott S Kruchowski, Chad Tomlinson, Cindy Strong, Kim Delehaunty, Catrina Fronick, Bill Courtney, Susan M Rock, Eddie Belter, Feiyu Du, Kyung Kim, Rachel M Abbott, Marc Cotton, Andy Levy, Pamela Marchetto, Kerri Ochoa, Stephanie M Jackson, Barbara Gillam, Weizu Chen, Le Yan, Jamey Higginbotham, Marco Cardenas, Jason Waligorski, Elizabeth Applebaum, Lindsey Phelps, Jason Falcone, Krishna Kanchi, Thynn Thane, Adam Scimone, Nay Thane, Jessica Henke, Tom Wang, Jessica Ruppert, Neha Shah, Kelsi Rotter, Jennifer Hodges, Elizabeth Ingenthron, Matt Cordes, Sara Kohlberg, Jennifer Sgro, Brandon Delgado, Kelly Mead, Asif Chinwalla, Shawn Leonard, Kevin Crouse, Kristi Collura, Dave Kudrna, Jennifer Currie, Ruifeng He, Angelina Angelova, Shanmugam Rajasekar, Teri Mueller, Rene Lomeli, Gabriel Scara, Ara Ko, Krista Delaney, Marina Wissotski, Georgina Lopez, David Campos, Michele Braidotti, Elizabeth Ashley, Wolfgang Golser, Hyeran Kim, Seunghee Lee, Jinke Lin, Zeljko Dujmic, Woojin Kim, Jayson Talag, Andrea Zuccolo, Chuanzhu Fan, Aswathy Sebastian, Melissa Kramer, Lori Spiegel, Lidia Nascimento, Theresa Zutavern, Beth Miller, Claude Ambroise, Stephanie Müller, Will Spooner, Apurva Narechania, Liya Ren, Sharon Wei, Sunita Kumari, Ben Faga, Michael J Levy, Linda McMahan, Peter Van Buren, Matthew W Vaughn, Kai Ying, Cheng-Ting Yeh, Scott J Emrich, Yi Jia, Ananth Kalyanaraman, An-Ping Hsia, W Brad Barbazuk, Regina S Baucom, Thomas P Brutnell, Nicholas C Carpita, Cristian Chaparro, Jer-Ming Chia, Jean-Marc Deragon, James C Estill, Yan Fu, Jeffrey A Jeddeloh, Yujun Han, Hyeran Lee, Pinghua Li, Damon R Lisch, Sanzhen Liu, Zhijie Liu, Dawn Holligan Nagel, Maureen C McCann, Phillip SanMiguel, Alan M Myers, Dan Nettleton, John Nguyen, Bryan W Penning, Lalit Ponnala, Kevin L Schneider, David C Schwartz, Anupma Sharma, Carol Soderlund, Nathan M Springer, Qi Sun, Hao Wang, Michael Waterman, Richard Westerman, Thomas K Wolfgruber, Lixing Yang, Yeisoo Yu, Lifang Zhang, Shiguo Zhou, Qihui Zhu, Jeffrey L Bennetzen, R Kelly Dawe, Jiming Jiang, Ning Jiang, Gernot G Presting, Susan R Wessler, Srinivas Aluru, Robert A Martienssen, Sandra W Clifton, W Richard McCombie, Rod A Wing, Richard K Wilson.
Science
PUBLISHED: 12-08-2009
Show Abstract
Hide Abstract
We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize.
Related JoVE Video
Identification of Theileria uilenbergi immunodominant protein for development of an indirect ELISA for diagnosis of ovine theileriosis.
Int. J. Parasitol.
PUBLISHED: 09-11-2009
Show Abstract
Hide Abstract
Theileriosis of small ruminants in the northwest of China is a protozoan disease that restricts the development of the livestock industry. The disease is caused by infection with Theileria uilenbergi and Theilerialuwenshuni, both of which are transmitted by ixodid Heamaphysalis ticks. The development of serological tools as a means of integrated control of the disease is an urgent and important requirement. Here we describe the identification and partial recombinant expression of a T.uilenbergi immunodominant protein (TuIP), which was identified by immunoscreening of a merozoite cDNA library. Using the recombinant TuIP (rTuIP), a novel indirect ELISA was established using 329 negative serum samples to determine the cut-off value. The internal quality control revealed satisfactory stability and repeatability of the assay. Preliminary validation using 128 positive and 48 negative reference samples demonstrated that the rTuIP ELISA is able to detect T. uilenbergi infection with high sensitivity and specificity. No cross-reactivity was found in sera from animals infected with Theileria lestoquardi, Babesia sp. China or Anaplasma ovis. Furthermore, circulating antibodies were detected in sera collected from endemic regions in China. Analyses of the antibody responses of experimentally infected animals demonstrated that tick infestation resulted in a sharply rising and stronger production of specific antibodies against TuIP while inoculation with infected blood induced an earlier production of TuIP-specific antibodies. The persistence of the TuIP-specific antibodies lasted more than 100days p.i. These data indicate the usefulness of the TuIP antigen for the development of diagnostic methods and as a potential candidate for vaccine design.
Related JoVE Video
A genome-wide characterization of microRNA genes in maize.
PLoS Genet.
PUBLISHED: 07-14-2009
Show Abstract
Hide Abstract
MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR-RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with approximately 35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.
Related JoVE Video
Differential expression of miRNAs in response to salt stress in maize roots.
Ann. Bot.
PUBLISHED: 04-02-2009
Show Abstract
Hide Abstract
Corn (Zea mays) responds to salt stress via changes in gene expression, metabolism and physiology. This adaptation is achieved through the regulation of gene expression at the transcriptional and post-transcriptional levels. MicroRNAs (miRNAs) have been found to act as key regulating factors of post-transcriptional gene expression. However, little is known about the role of miRNAs in plants responses to abiotic stresses.
Related JoVE Video
Characterization of a concealed antigen Hq05 from the hard tick Haemaphysalis qinghaiensis and its effect as a vaccine against tick infestation in sheep.
Vaccine
PUBLISHED: 03-11-2009
Show Abstract
Hide Abstract
Positive clone Hq05 of Haemaphysalis qinghaiensis (named following those cDNA clones cloned from the tick before) was obtained by differentially screening of a cDNA library of the tick with rabbit anti-tick salivary gland serum and rabbit anti-tick saliva serum. Hq05 contains an open reading frame (ORF) of 540bp that codes for 179amino acid residues with a coding capacity of 20kDa. Sequence similarity and phylogenetic analyses indicated that Hq05 was a novel gene. Expression analysis by reverse transcription-polymerase chain reaction indicated that the gene was expressed in nymphal and adult stage of H. qinghaiensis tick and its salivary glands, but not in midguts. The cDNA was expressed as glutathione S-transferase (GST)-fused protein in a procariotic system. Western blot showed that only rabbit anti-H. qinghaiensis salivary gland serum could recognize the expressed GST-Hq05 (46kDa) protein, while both rabbit negative serum and rabbit anti-H. qinghaiensis saliva serum could not react with the expressed protein. This proved the recombinant protein was a "concealed" antigen of H. qinghaiensis. Vaccination of sheep with rHq05 conferred a significant protective immunity in sheep, resulting in a 40% reduction of the amount of eggs laid by each tick and the hatching capability of eggs decreased by 37% compared to the controls. These results showed that rHq05 could be a candidate tick vaccine molecule for the control of H. qinghaiensis.
Related JoVE Video
Detection and differentiation of ovine Theileria and Babesia by reverse line blotting in China.
Parasitol. Res.
PUBLISHED: 01-13-2009
Show Abstract
Hide Abstract
A reverse line blot (RLB) assay was developed for detection and specific identification of the different ovine Theileria and Babesia parasites. In a polymerase chain reaction (PCR), the hypervariable region 4 (V4 region) of the 18S ribosomal DNA gene was amplified with a set of general primers specific for members of the genera Theileria and Babesia. Meanwhile, specific oligonucleotide probes were designed and bound on membrane. After one single-PCR amplification, the amplified fragment was hybridized against different generic and species-specific probes. It was able to detect four species, i.e., Babesia motasi (Chengde, Lintan, Ningxian, Tianzhu), Babesia sp. (Kashi), Theileria luwenshuni (Lintan, Madang, Ningxian), Theileria uilenbergi (Longde, Zhangjiachuan) as defined previously. All probes bound to their respective target sequence only; therefore, no cross-reaction was observed, resulting in clear recognition of either individual strains, species, or groups in normal positive tests. Meanwhile, no signal was observed when ovine genomic DNA and water were used as a control, demonstrating that the signals are due to the presence of parasite DNA in the samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level between10(-3)% and 10(-8)%. Finally, 117 samples from field were tested with RLB, PCR, and enzyme-linked immunosorbent assay (ELISA). The positive rate of RLB was higher than that of PCR and ELISA, and furthermore, RLB could determinate the species of piroplasms, the samples were infected with. Samples, 1,117, from five areas in Gannan Tibet Autonomous Region have been examined with RLB assay and compared with ELISA assay for corresponding samples. The results showed that the positive rate of RLB was higher than that of ELISA test obviously, and both T. luwenshuni and T. uilenbergi were widely distributed in these areas. RLB developed here could be used for differentiation of Babesia and Theileria infection and for epidemiological survey, which was difficult to achieve by classical methods. In conclusion, the RLB is a versatile technique for simultaneous detection and identification of all ovine piroplasms.
Related JoVE Video
Experimental transmission of Theileria uilenbergi infective for small ruminants by Haemaphysalis longicornis and Haemaphysalis qinghaiensis.
Parasitol. Res.
PUBLISHED: 01-13-2009
Show Abstract
Hide Abstract
The experimental transmission of a recently designated Theileria uilenbergi pathogenic for sheep and goats in Northern China is described. Haemaphysalis qinghaiensis nymphs and adults developed from larvae and nymphs engorged on sheep infected with T. uilenbergi were able to respectively transmit the latter to sheep. However, when H. longicornis ticks picked up T. uilenbergi either at larval or nymphal, only the subsequent adult could transmit the parasites to sheep, the subsequent nymph could not transmit the parasites to sheep. This experiment suggested that the T. uilenbergi could be transmitted by at least two species of Haemaphysalis sp. ticks, H. longicornis and H. qinghaiensis, and the mode of transmission is stage to stage.
Related JoVE Video
Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences.
Exp. Parasitol.
PUBLISHED: 01-06-2009
Show Abstract
Hide Abstract
The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.
Related JoVE Video
Analysis of metabolic fluxes for better understanding of mechanisms related to lipid accumulation in oleaginous yeast Trichosporon cutaneum.
Bioresour. Technol.
Show Abstract
Hide Abstract
Microbial fermentation for producing biodiesel from lignocellulosic hydrolysates is receiving increasing attention and attempts have been made to screen an oleaginous Trichosporon sp. with high lipid content and a strong tolerance to lignocellulose hydrolysates. In order to better understand mechanisms related to its lipid accumulation, metabolic flux analysis was performed under 5gL(-1) ammonium sulfate (high nitrogen) and/or 0.4gL(-1) ammonium sulfate (low nitrogen) conditions. Cell growth phase and lipid accumulation phase were shown for cells grown under low nitrogen condition. Results of flux distribution demonstrated that NADPH provided by cytosolic malic enzyme and the acetyl-CoA from cytoplasmic citrate by the ATP: citrate lyase were the two primary sources for excess lipid accumulation. Flux data also supported the fact that the citrate pyruvate cycle plays an essential role in the lipid accumulation. The flux information obtained could also motivate new design strategies for oleaginous yeasts for enhanced biodiesel production.
Related JoVE Video
Additional data for a new Theileria sp. from China based on the sequences of ribosomal RNA internal transcribed spacers.
Exp. Parasitol.
Show Abstract
Hide Abstract
Theileria sinensis was recently isolated and named as an independent Theileria species that infects cattle in China. To date, this parasite has been described based on its morphology, transmission and molecular studies, indicating that it should be classified as a distinct species. To test the validity of this taxon, the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were cloned and sequenced from three T. sinensis isolates. The complete ITS sequences were compared with those of other Theileria sp. available in GenBank. Phylogenetic analyses based on sequence data for the complete ITS sequences indicate that T. sinensis lies in a distinct clade that is separate from that of T. buffeli/orientalis and T. annulata. Sequence comparisons indicate that different T. sinensis isolates possess unique sizes of ITS1 and ITS2 as well as species-specific nucleotide sequences. This analysis provides new molecular data to support the classification of T. sinensis as a distinct species from other known Theileria spp. based on ITS sequences.
Related JoVE Video
Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China.
Exp. Appl. Acarol.
Show Abstract
Hide Abstract
Molecular phylogenetic analyses are mainly based on the small ribosomal RNA subunit (18S rRNA), internal transcribed spacer regions, and other molecular markers. We compared the phylogenetic relationships of Babesia spp. using large subunit ribosomal RNA, i.e., 28S rRNA, and the united 28S + 18S rRNA sequence fragments from 11 isolates of Babesia spp. collected in China. Due to sequence length and variability, the 28S rRNA gene contained more information than the 18S rRNA gene and could be used to elucidate the phlyogenetic relationships of B. motasi, B. major, and B. bovis. Thus, 28S rRNA is another candidate marker that can be used for the phylogenetic analysis of Babesia spp. However, the united fragment (28S + 18S) analysis provided better supported phylogenetic relationships than single genes for Babesia spp. in China.
Related JoVE Video
The life cycle of Haemaphysalis qinghaiensis (Acari: Ixodidae) ticks under laboratory conditions.
Exp. Appl. Acarol.
Show Abstract
Hide Abstract
The developmental stages in the life cycle of Haemaphysalis qinghaiensis were investigated under laboratory conditions. The larval, nymphal and adult ticks were fed on sheep at 25-27 °C, 50 % relative humidity (RH) and exposed to daylight. All free-living stages were maintained in an incubator (28 °C with 90 % RH and a 12-h photoperiod). The whole life cycle of H. qinghaiensis was completed in an average of 176 days (range 118-247 days). The average developmental periods were 34.44 days for egg incubation; 5.83, 4.20 and 33.70 days for larval pre-feeding, feeding and pre-molting; and 3.88, 5.30 and 46.50 days for nymphal pre-feeding, feeding and pre-molting. The average times for pre-feeding, feeding, pre-oviposition and oviposition of female adult ticks were 2.60, 11.40, 8.50, and 19.35 days, respectively. The results confirmed the positive correlation between the weight of the engorged female and the egg mass laid (r = 0.557, P < 0.05). The reproductive efficiency index and reproductive fitness index in females were 5.49 and 4.98, respectively. Engorged nymphs moulting to females (4.53 ± 0.16 mg) were significantly heavier (P < 0.001) than those moulting to males (3.45 ± 0.19 mg). The overall sex ratio of the adult ticks was 1:1.1 (M:F).
Related JoVE Video
Prevalence of Anaplasma phagocytophilum in ruminants, rodents and ticks in Gansu, north-western China.
J. Med. Microbiol.
Show Abstract
Hide Abstract
The zoonotic rickettsial pathogen Anaplasma phagocytophilum has a broad geographical distribution and a high degree of biological and clinical diversity. To determine the prevalence of Anaplasma phagocytophilum in the Gannan Tibetan Autonomous Prefecture of Gansu Province, north-western China, four ruminant species, one rodent and one tick species were examined for Anaplasma phagocytophilum infection. DNA from Anaplasma phagocytophilum was detected by nested PCR in blood samples from 21/49 sheep (42.9?%), 35/91 goats (38.5?%), 51/158 yaks (32.3?%) and 7/20 cattle-yaks (35.0?%), and in spleen samples from 2/12 rodents (16.7?%). For samples from tick larvae and nymphs, 105 pools were tested; one of 46 larval tick pools was positive and seven of 59 nymphal tick pools were positive. For adult ticks, 40/598 female ticks (6.7?%) and 26/528 male ticks (4.9?%) were positive. The prevalence of Anaplasma phagocytophilum in female ticks was higher than that in males, although the difference was not statistically significant (P>0.05). Sequences analysis based on the 16S rRNA gene indicated that the strains in the study area were distinct from previously reported Anaplasma phagocytophilum in other continents. These results add new information on the epidemiology of Anaplasma phagocytophilum and indicate the tick-animal cycle of anaplasmosis in the area. To the best of our knowledge, this is the first report of Anaplasma phagocytophilum infection in Gansu Province in north-western China.
Related JoVE Video
Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay.
Vet. Parasitol.
Show Abstract
Hide Abstract
The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species -T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55 min or 50 min under isothermal conditions of 61°C or 63°C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species -T. sergenti and T. sinensis, especially in endemic countries.
Related JoVE Video
[Metabolic regulation of isocitrate lyase regulator in Escherichia coli based on metabolic flux information].
Sheng Wu Gong Cheng Xue Bao
Show Abstract
Hide Abstract
Gene expression is regulated by different transcriptional regulators. The transcriptional regulator isocitrate lyase regulator (IclR) of Escherichia coli represses the expression of the aceBAK operon that codes for the glyoxylate pathway enzymes. In this study, physiological and metabolic responses of the deletion of the ic1R gene in E. coli BW25113 were investigated based on the quantification and analysis of intracellular metabolic fluxes. The knockout of the iclR gene resulted in a decrease in the growth rate, glucose uptake rate and the acetate secretion rate, but a slight increase in biomass yield. The latter could be attributed to the lowered metabolic fluxes through several CO2 generating pathways, including the redirection of 33% of isocitrate directly to succinate and malate without CO2 production as well as the reduced flux through the pentose phosphate pathway. Furthermore, although the glyoxylate shunt was activated in the iclR mutant, the flux through phosphoenolpyruvate (PEP) carboxykinase kept almost unchanged, implying an inactive PEP-glyoxylate cycle and no extra loss of carbon atoms in the mutant strain. Both the reduced glucose uptake rate and the active glyoxylate shunt were responsible for the minor decrease in acetate secretion in the ic1R knockout strain compared to that in the wild-type E. coli strain.
Related JoVE Video
Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina.
Parasitol. Int.
Show Abstract
Hide Abstract
Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and/or Babesia bigemina and is characterized by significant morbidity and mortality worldwide. This disease is widespread in most parts of China. However, it is difficult to rapidly discriminate between the B. bovis and B. bigemina species. To detect and distinguish these species, a loop-mediated isothermal amplification (LAMP) platform that targets specific sequences of the internal transcribed spacer (ITS) genes was developed. Specificity testing revealed that there was no cross-reaction with the other tick-borne parasites B. ovate, B. major, unnamed bovine Babesia, Theileria annulata, Theileria sinensis, Theileria sergenti, and Anaplasma marginale, or with bovine white blood cells. The sensitivity of the LAMP method was 0.1 pg DNA for both B. bovis and B. bigemina, which was superior to that of the classical PCR methods. This assay was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infected cattle in China. These findings indicate that the Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic.
Related JoVE Video
Structure-based design technology contour and its application to the design of renin inhibitors.
J Chem Inf Model
Show Abstract
Hide Abstract
It is well-known that the structure-based design approach has had a measurable impact on the drug discovery process in identifying novel and efficacious therapeutic agents for a variety of disease targets. The de novo design approach has inherent potential to generate novel molecules that best fit into a protein binding site when compared to all of the computational methods applied to structure-based design. In its initial attempts, this approach did not achieve much success due to technical hurdles. More recently, the algorithmic advancements in the methodologies and clever strategies developed to design drug-like molecules have improved the success rate. We describe a state-of-the-art structure-based design technology called Contour and provide details of the algorithmic enhancements we have implemented. Contour was designed to create novel drug-like molecules by assembling synthetically viable fragments in the protein binding site using a high-resolution crystal structure of the protein. The technology consists of a sophisticated growth algorithm and a novel scoring function based on a directional model. The growth algorithm generates molecules by dynamically selecting only those fragments from the fragment library that are complementary to the binding site, and assembling them by sampling the conformational space for each attached fragment. The scoring function embodying the essential elements of the binding interactions aids in the rank ordering of grown molecules and helps identify those that have high probability of exhibiting activity against the protein target of interest. The application of Contour to identify inhibitors against human renin enzyme eventually leading to the clinical candidate VTP-27,999 will be discussed here.
Related JoVE Video
A DNA barcode for Piroplasmea.
Acta Trop.
Show Abstract
Hide Abstract
Due to the difficulty in morphological identification the development of reliable molecular tools for species distinction is a priority for piroplasma. Previous studies based on 18S rRNA and other gene sequences provided a backbone for the phylogeny of piroplasma. However, it is difficult to discriminate species in a comprehensive sample. Here, the abilities of eight DNA regions including 18S rRNA, 28S rRNA, internal transcribed spacer (ITS) regions and COI genes, have been compared as candidates of DNA barcodes for piroplasma. In total, 484 sequences of piroplasma were collected from this study and GenBank. The eight proposed DNA regions were evaluated according to the criterion of Consortium for the Barcode of Life (CBOL). From this evaluation, ITS2 had 100% PCR amplification efficiency, an ideal sequence length, the largest gap between the intra- and inter-specific divergence, 98% identification efficiency at the genus level, and 92% at the species level. Thus, we propose that ITS2 is the most ideal DNA barcode based on the current database for piroplasma.
Related JoVE Video
Characterization of miRNAs in response to short-term waterlogging in three inbred lines of Zea mays.
PLoS ONE
Show Abstract
Hide Abstract
Waterlogging of plants leads to low oxygen levels (hypoxia) in the roots and causes a metabolic switch from aerobic respiration to anaerobic fermentation that results in rapid changes in gene transcription and protein synthesis. Our research seeks to characterize the microRNA-mediated gene regulatory networks associated with short-term waterlogging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many genes involved in growth, development and various biotic and abiotic stress responses. To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and their 92 predicted targets in three inbred Zea mays lines (waterlogging tolerant Hz32, mid-tolerant B73, and sensitive Mo17). Based on our studies, miR159, miR164, miR167, miR393, miR408 and miR528, which are mainly involved in root development and stress responses, were found to be key regulators in the post-transcriptional regulatory mechanisms under short-term waterlogging conditions in three inbred lines. Further, computational approaches were used to predict the stress and development related cis-regulatory elements on the promoters of these miRNAs; and a probable miRNA-mediated gene regulatory network in response to short-term waterlogging stress was constructed. The differential expression patterns of miRNAs and their targets in these three inbred lines suggest that the miRNAs are active participants in the signal transduction at the early stage of hypoxia conditions via a gene regulatory network; and crosstalk occurs between different biochemical pathways.
Related JoVE Video
Genome-wide identification and analysis of microRNA responding to long-term waterlogging in crown roots of maize seedlings.
Physiol Plant
Show Abstract
Hide Abstract
MicroRNAs (miRNAs) are critical post-transcriptional modulators of gene expression involving in plant responses to abiotic stress. However, the regulation of miRNA in the morphological response to waterlogging is poorly understood in maize. In this study, we detected miRNAs and their targets that expressed in waterlogged crown roots of maize seedlings in two inbred lines (Hz32 and Mo17) by RNA sequencing. A total of 61 mature miRNAs were found including 36 known maize (zma) miRNAs and 25 potential novel miRNA candidates. Comparison of miRNA expression in both waterlogged and control crown roots revealed 32 waterlogging-responsive miRNAs, most were consistently downregulated under waterlogging in the two inbred lines. We identified the miRNA targets through degradome sequencing. Many known miRNA targets involving in transcription regulation and reactive oxygen species elimination were found in the degradome libraries, and 17 targets of 10 newly detected miRNAs were identified as well. Moreover, the miRNA-mediated pathways that respond to waterlogging and regulate the induction of crown roots were discussed. This study is a comprehensive survey of responsive miRNAs in waterlogged maize crown roots. The results will help to understand the miRNA expression in response to waterlogging and miRNA-mediated regulation of morphological adaptation to waterlogging in maize.
Related JoVE Video
A recently identified ovine Babesia in China: serology and sero-epidemiology.
Parasitol. Int.
Show Abstract
Hide Abstract
Babesia sp. in Xinjiang, transmitted by Hyalomma, is a large Babesia that is infective for small ruminants, but it has almost no pathogenicity in healthy sheep. On the basis of the sequences of the 18S rRNA and internal transcribed spacer (ITS) genes, morphological characteristics, vector tick species and pathogenicity it was identified recently as a novel Babesia species. In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed using soluble merozoite antigens of Babesia sp. in Xinjiang (BXJMA) derived from in vitro culture. When the positive threshold was chosen as 24.65% of the specific mean antibody rate, the specificity and sensitivity were both 97.3%. There was no cross-reaction between BXJMA and positive sera from sheep infected with other Chinese ovine piroplasms or Anaplasma ovis in the ELISA and western blotting. Specific antibodies against Babesia sp. in Xinjiang could be detected 2 weeks post infection and a high level of antibodies persisted for more than 12 weeks in experimentally infected sheep. The ELISA was tested on 3857 sera collected from small ruminants in 50 prefectures of 22 provinces to evaluate the sero-epidemiology of Babesia sp. in Xinjiang infection, and the average positive rate was 31.66%. These data provide that the developed ELISA is a powerful tool for the sero-diagnosis of Babesia sp. in Xinjiang and confirm that it is a novel species.
Related JoVE Video
Evaluation of molecular methods for detection of Borrelia burgdorferi senso lato in ticks.
Diagn. Microbiol. Infect. Dis.
Show Abstract
Hide Abstract
Borrelia burgdorferi sensu lato (s. l.), the agent of Lyme disease, is distributed widely worldwide. A large number of polymerase chain reaction (PCR) methods have been developed and used for detection of B. burgdorferi s. l. However, there is a lack of a reference standard because of the genetic diversity of the B. burgdorferi s. l. complex. In this study, 4 PCR methods, based on the OspA, flagellin, rrs, and P66 genes, for detection of B. burgdorferi s. l. were evaluated by detection of genomic DNA from 3 reference genospecies and tick samples. The sensitivity of the PCR methods was analyzed using serially diluted gDNA from B. afzelii (Bo23), B. burgdorferi sensu stricto (B31), and B. garinii (PBi). The performance of the PCRs was evaluated by detection of the gDNA of 543 ticks. The results showed that the PCRs targeting the OspA gene, fla gene, rrs gene, and P66 gene detected 37 (6.8%), 74 (13.6%), 16 (2.9%), and 14 (2.6%) tick samples, respectively. The PCR targeting the fla gene was the most sensitive method for the detection of B. burgdorferi s. l.
Related JoVE Video
Development of real-time polymerase chain reaction for detection of Borrelia burgdorferi sensu lato in China.
Vector Borne Zoonotic Dis.
Show Abstract
Hide Abstract
Universal primers and probes were selected on the basis of the 16S rRNA gene sequence of Borrelia burgdorferi in GenBank®, and a real-time polymerase chain reaction (PCR) method for detection of B. burgdorferi was established. The results showed that this method could specifically detect the B31 strain (Borrelia burgdorferi sensu stricto), the BO23 strain (Borrelia afzelii), and the SZ strain (Borrelia garinii), without cross-reaction with genome DNA of Theileria (T. luwenshuni, T. uilenbergi, T. sinensis, T. annulata, T. sergenti, T. annulata), Babesia (B. bigemina, B. ovate, B. sp. (Xinjiang)), Anaplasma (A. marginale, A. ovis), Mycoplasma mycoides subsp. capri, and Chlamydia psittaci, which are the infective pathogens to yak and/or sheep. The sensitivity of this real-time PCR is 10? times greater than that of a conventional PCR. The real-time PCR was able to amplify 16S rRNA gene from as few as 22.88 fg genomic DNA of B. burgdorferi sensu lato. Tick DNAs from 369 field samples collected from Shangzhi City of Heilongjiang Province were tested, resulting in an infection rate of 42.80%, and a total of 332 genomic DNAs from the blood of 186 yaks and 146 sheep in the Gannan Tibetan Autonomous Prefecture of Gansu Province were tested, resulting in 24.19% positive rate for the yaks and 39.04% positive rate for the sheep.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.