Of mice, cats, and men: is human breast cancer a zoonosis?

Mouse mammary tumor virus (MMTV), a member of the betaretroviridae, is the most common cause of breast cancer (BC) in mice. MMTV is transmitted in mice both in the germline as endogenous proviruses and exogenously as infectious virions. Here, we review a variety of evidence accumulated for six decades that has suggested that a human homologue of MMTV may exist. The findings include recent studies from several independent laboratories that have detected sequences very closely related to MMTV in DNA isolated from human BC tumors. Other laboratories, however, have failed to detect the MMTV-related sequences in human DNA samples, and conclusive evidence for a human mammary tumor virus has been elusive. We also reviewed additional studies, suggesting that betaretroviruses are present in a much wider range of species than previously known, including rodents, felines, and primates. The observation that a subset of cats may be infected with a close homologue of MMTV may be of epidemiological significance for human BC. Cats may become infected by MMTV from mice, and in turn may transmit the virus to humans, possibly after selection for variants with an expanded host range.     

超声表观背散射参数成像定征松质骨结构的可行性研究

骨矿密度的降低与骨微结构的退化都会导致骨强度的下降,从而引发骨质疏松.松质骨的超声背散射信号中包含骨微结构信息.本文提出用超声背散射系数(BC)和平均积分背散射系数(AIB)成像来定征松质骨微结构,并对骨样本感兴趣区域(ROI)内的BC 和AIB与μ-CT获得的骨结构参数做线性相关分析.研究结果表明,超声背散射参数的平均值与骨小梁厚度(r=0.898)、骨小梁间距(r= -0.916)有较强的相关性.说明利用背散射参数图像来反映松质骨微结构的特征是可行的.     

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes

Bacterial artificial chromosome - fluorescence in situ hybridization (BAC-FISH) and cyclingprimed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect CPRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of     

In-Fusion克隆技术构建HBVX基因真核表达质粒

目的以血清中乙型肝炎病毒（HBV）DNA 为模板,克隆构建HBV X 蛋白质的真核表达载体pcDNA-HBx.方法：设计扩增HBV X 基因的In-Fusion PCR 引物,采用高保真DNA 聚合酶分两段扩增HBV X 基因序列;采用In-Fusion 克隆技术,将两段X 基因扩增片段-步克隆入经Hind Ⅲ 和Xba I 双酶切的pcDNA3.0 真核表达载体,以构建重组真核表达载体pcDNA-HBX ;采用Hind Ⅲ 和Xba I 双酶切和DNA 序列测序筛选鉴定重组载体;pcDNAHBx转染HepG2 细胞,Western blot 检测pcDNA-HBx 转染细胞的HBx 蛋白质表达.结果：In-Fusion PCR 引物分两段扩增获得全长HBx 基因序列;In-Fusion 获得克隆的pcDNA-HBx 经双酶切筛选得到阳性重组质粒,测序分析证实插入序列正确;转染pcDNA-HBX 的HepG2 细胞,Western blot 证实表达HBx 蛋白质.结论：以血清HBV DNA 为模板克隆HBV X 基因,构建了表达HBV HBx 蛋白质的真核表达载体,为HBx 蛋白质的生物学功能研究提供支持.     

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.     

生物质谱作为SNP分型检测方法的研究

利用改良的 Miller法从人血中提取人类基因组 DNA。采用聚合酶链式反应 ( Polymerase chain reac-tion,PCR) ,对含有单核苷酸多态性 ( SNP)点的 DNA片段进行扩增 ,然后用 CIP和 Eox I去除掉 PCR体系中剩余的脱氧核糖核苷三磷酸 ( d NTP)和引物 ,最后用单碱基延伸反应获得 SNP位点处碱基类型。用基质辅助激光解吸电离 -飞行时间质谱 ( MALDI-TOFMS)或电喷雾电离 -四极杆飞行时间质谱 ( ESI-Qq TOFMS)检测延伸产物与未延伸引物间的分子量差异 ,确定该点处碱基。对不同的生物质谱检方法以及生物质谱检测方法与其它检测方法的优缺点进行了分析比较     

Fragile X syndrome,the Fragile X related proteins,and animal models

The Fragile X syndrome (FraX), which is characterized among other physical and neurologic impairments by mental retardation, is caused by the absence of the product of the FMR1 gene. The Fragile X Mental Retardation Protein (FMRP) is a member of a novel family of RNA-binding proteins. The latter includes two other proteins highly homologous with FMRP: the fragile X related proteins 1 and 2 (FXRP1 and FXRP2). Characterization of FXRPs, including their interaction with FMRP, will provide critical information about the mechanisms of action of FMRP and the role of this group of proteins in FMRP-deficient conditions such as FraX. Genetic manipulations of FMRP and the FXRPs should also provide valuable tools for investigating pathophysiology and gene therapies in FraX. The present review summarizes the strategies used for identifying the FXRPs, their chromosomal localization, molecular structure, and tissue distribution. It also reviews interactions between different members of this family of RNA-binding proteins. Animal models, both knockout and transgenic, of FMRP and the FXRPs are discussed. Phenotypic features of the FMR1 knockout mouse, the FMR1 transgenic rescue mouse, and other novel strategies for manipulating and delivering FMRP and FXRPs to the brain and other tissues are described.     

Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20–50 and 1–2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA–DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1–2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.     

Assembly sequences planning for simultaneous engineering applications

This paper describes a Generative Assembly Process Planner (GAPP) used to generate and evaluate assembly sequence alternatives which can be used in simultaneous engineering applications. Given a graph model of the product to be assembled, optimal assembly sequences are automatically generated and evaluated in a single process. Geometric feasibility and accessibility constraints on assembly operations which help prune the search space are presented. Assembly-related criteria which guide the search to an optimal solution are described. These include: number of reorientations, concurrent execution of assembly tasks, grouping of similar tasks and stability criteria. The relative weights of various criteria can be varied interactively resulting in different optimal assembly sequences. The ability of GAPP to generate and evaluate assembly alternatives when various criteria are enabled or disabled or their relative importance is changed makes it an effective tool for simultaneous engineering/manufacturing applications. Examples are included to demonstrate GAPP's use and potential for assessing assembly, disassembly, repair and maintenance procedures.     

Fluorescence in situ hybridization of three oncogenes on a leiomyoma

Using fluorescence in situ hybridization technique, expression of three oncogenes, C-myc, RARa, and cyclin-D was tested on a uterine leiomyoma. C-myc and RARa were amplified in approximately 30% and 90% of the cells, respectively. Numerous small signals of C-myc were indicative of the presence of double minutes. Amplification of RARa is being reported for the first time in a leiomyoma. Cyclin-D was normal in diploid cells while it was highly amplified in polyploid cells. Low levels of amplified C-myc and cyclin-D cells seem to be the reason for this tumor to be benign, while RARa could not be effective without the association of some other gene such as PML. Information presented here are significant toward developing new curative strategies such as gene-specific drugs and molecular manipulation to stop the activity of cancer gene. Further study may elucidate that how fibroids grow and maintain their rare benign nature.     

Molecular biomarkers: multiple roles in radiotherapy

Preoperative chemoradiation therapy (CRT) is becoming the standard treatment for patients with locally advanced rectal cancer. However, individual differences in response to treatment range from a complete response to complete resistance. Predicting the tumor response to radiotherapy may improve the efficacy of radiotherapy. This review mainly summarizes recent studies about the molecular biomarkers that can predict the response to radiotherapy in rectal cancer. These studies have indicated that the molecular markers involved in the response to radiotherapy mainly include genes related to radiosensitivity, cancer stem cell-related markers, non-coding RNAs (ncRNAs), single-nucleotide polymorphisms (SNPs) and gene methylation, and other factors including carcinoembryonic antigen (CEA) level, anemia, lymphocytes, and signaling pathways. Many of these identified markers are mainly associated with DNA repair, apoptosis, and cell cycle, but some involve unknown cell mechanisms. We speculate that predictors of radiotherapy response may involve combinations of multiple molecular biomarkers that may be useful for the development of individualized therapy for rectal cancer patients.     

Microflora associated with primary endodontic infections: Correlations among sem evaluation,clinical features,and radiographic findings

The aim of the present study was to characterize, by means of SEM, primary endodontic infections and to correlate with clinical and radiographic findings. Twelve (12) human extracted teeth (19 roots) presenting primary endodontic infection were examined. SEM qualitative observations of bacterial and defense cells, their features and distribution within the root canal lumen and root dentine were recorded for association with clinical and radiographic tabled data. Although a direct correlation between biofilm composition and clinical/radiographic findings was not established, structural organization and distribution of the biofilm, as well as the characteristics of host response, could be easily related to those features. Bacterial biofilm was predominant at the apical third. Symptomatic apical periodontitis was related to presence of bacterial biofilm all thirds. Defense cells could be seen in the apical third of some samples. These cells were present in all thirds in some of the cases with open cavities. The correlations performed in this study allowed a better understanding of the picture of primary endodontic infection, host response and relevant clinical features. The combined use of scanning electron microscopy with clinical and radiographic evaluation has the potential to overcome some limits of the current knowledge related to pulpal and periapical diseases, providing important insights for improving treatment strategies. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells

The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both nonencapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.     

Complete genome sequence of two strawberry vein banding virus isolates from China

It was rarely reported about strawberry vein banding virus (SVBV) genome sequence in China and most countries worldwide. In this work, we determined the complete genome sequences of two SVBV isolates in China, designated SVBV-AH and SVBV-BJ, that were obtained from naturally infected strawberry samples from Anhui province and Beijing city of China, respectively. The complete genomes of SVBV-AH and SVBV-BJ were 7,862 nucleotides (nts) and 7,863 nts long, respectively, and both constituted with seven genes typical of the caulimoviruses. Alignment of complete nucleotide sequences showed that SVBV-AH and SVBV-BJ shared a significant nucleotide sequence identity of 97.7% of each other and had 85.7% and 86.0% sequence identity related to SVBV from the United States (SVBV-US), respectively. Phylogenetic trees, based on the alignment of complete nucleotide sequences and amino acid sequences of Coat Protein (CP), both showed that SVBV-AH and SVBV-BJ clustered into one branch with all the other SVBV isolates, and other species of caulimoviruses clustered into another tree branch. It illustrated that all the SVBV isolates had an extremely high relationship but had a distant relationship with other species of caulimoviruses. We further confirmed that SVBV-AH infectious clone could cause similar symptoms to SVBVinfected in strawberry under natural conditions. Taken together, our study provided valuable information to elucidate the origin and dissemination of SVBV Chinese isolates, meanwhile providing the necessary vector for studying the gene functions of strawberry.     

Cloning and characterization of 66 kDa streptavidin-binding peptides (SBP) of <i>Pisum sativum</i> L. embryo specific to var. Alaska

The aim of the current research was to clone and to characterize the partial 66 kDa streptavidin-binding peptide (SBP) found in the germinated embryos of Pisum sativum L. var. Alaska. The pea (P. sativum var. Alaska) embryos possess prominent 66 kDa SBPs that gradually disappeared after few hours of germination in germinated embryos, but not in the cotyledons. The total RNA was isolated from embryos of P. sativum but could not be isolated from the cotyledons. The partial nucleotides sequences of 66 kDa SBPs of embryonic stalk (P. sativum var. Alaska) were cloned and identified using pMOSBlue vector. 66 kDa (SBP) gene from the embryos of P. sativum var. Alaska possesses 327 bp having an open reading frame (ORF) region in a part of the gene that encoded for 108 amino acids. Alignment showed similarity among 66 kDa SBPs P. sativum var. Alaska, with P. sativum seed biotinylated protein (SBP65) and P. sativum sbp65a mRNA with DNA distance matrix between 0.0094 to 1.2676. MALDI-TOF mass spectrometry analysis of 66 kDa (SBP) proteins showed it had similar short peptides to 19 proteins found in different organisms, especially Convicilin precursor, and the seed biotinylated protein in P. sativum. The alignment results of both nucleotide sequences and amino acid residues either from cloning or MALDI-TOF-MS showed differences with related species, especially P. sativum. No mRNA was found in the cotyledons during seeds germination, which means no metabolic activities and this part may act only as food reservoirs for growing newly embryos.     

FEMTOLITER MICROARRAY WELLS FOR ULTRASENSITIVE DNA/mRNA DETECTION

ABSTRACT

DNA/mRNA analysis and genetic mutation detection are important in biomedical sciences, clinical diagnosis, and environmental monitoring. In this study, a femtoliter microwell array system has been developed for ultrasensitive DNA/mRNA detection using molecular beacon (MB) DNA probes. The microwell array system, with laser-induced fluorescence imaging, can be used to achieve a detection limit of 9 Rhodamin 6G molecules in 28?fL (28?×?10^?15?L) wells in this array. Hybridization kinetics of the MBs and their complementary DNA targets has been monitored with a concentration detection limit of 3.0?nM. As few as 50 target DNA copies can be monitored in each well. The specificity of MB and the effect of DNA target concentration on the MB hybridization kinetics have been investigated in femtoliter wells. Analysis of specific rat γ-actin mRNA sequence amplified by polymerase chain reaction (PCR) and simultaneous measurement of multiple analytes have also been studied with this microwell array system. As the volume in each well is extremely small, this array system will be highly useful for single cell gene profiling and for multiple gene determination in disease diagnosis.     

Design and operation of a portable scanner for high performance microchip capillary array electrophoresis

We have developed a compact, laser-induced fluorescence detection scanner, the multichannel capillary array electrophoresis portable scanner (McCAEPs) as a platform for electrophoretic detection and control of high-throughput, integrated microfluidic devices for genetic and other analyses. The instrument contains a confocal optical system with a rotary objective for detecting four different fluorescence signals, a pneumatic system consisting of two pressure/vacuum pumps and 28 individual addressable solenoid valves for control of on-chip microvalves and micropumps, four Polymerase Chain Reaction (PCR) temperature control systems, and four high voltage power supplies for electrophoresis. The detection limit of the instrument is ~20 pM for on-chip capillary electrophoresis of fluorescein dyes. To demonstrate the system performance for forensic short tandem repeat (STR) analysis, two experiments were conducted: (i) electrophoretic separation and detection of STR samples on a 96-lane microfabricated capillary array electrophoresis microchip. Fully resolved PowerPlex(?) 16 STR profiles amplified from 1 ng of 9947A female standard DNA were successfully obtained; (ii) nine-plex STR amplification, sample injection, separation, and fluorescence detection of 100-copy 9948 male standard DNA in a single integrated PCR- capillary electrophoresis microchip. These results demonstrate that the McCAEPs can be used as a versatile control and detection instrument that operates integrated microfluidic devices for high-performance forensic human identification.