Genomic Characterization of Novel Circular ssDNA Viruses from Insectivorous Bats in Southern Brazil

Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.     

Hox genes: the instructors working at motor pools

Motor neurons are assigned unique subidentities preceding their axon navigation. This ensures proper innervation of muscle targets and is accompanied by a stereotypical clustering of motor neuron cell bodies into "motor pools" within the spinal cord. However, the mechanisms that drive motor neuron diversification have been poorly understood. A new study by Dasen et al. (2005) in this issue of Cell shows that a network of Hox genes is responsible for instructing motor pool development.     

Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells

Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only ∼100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-β-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.     

Predation and disturbance interact to shape prey species diversity

Though predation, productivity (nutrient richness), spatial heterogeneity, and disturbance regimes are known to influence species diversity, interactions between these factors remain largely unknown. Predation has been shown to interact with productivity and with spatial heterogeneity, but few experimental studies have focused on how predation and disturbance interact to influence prey diversity. We used theory and experiments to investigate how these factors influence diversification of Pseudomonas fluorescens by manipulating both predation (presence or absence of Bdellovibrio bacteriovorus) and disturbance (frequency and intensity of disturbance). Our results show that in a homogeneous environment, predation is essential to promote prey species diversity. However, in most but not all treatments, elevated diversity was transitory, implying that the effect of predation on diversity was strongly influenced by disturbance. Both our experimental and theoretical results suggest that disturbance interacts with predation by modifying the interplay of resource and apparent competition among prey.     

Loss of cortical function in mice after decapitation, cervical dislocation, potassium chloride injection, and CO2 inhalation

Electroencephalograms (EEG) and visual evoked potentials (VEP) in mice were recorded to evaluate loss of cortical function during the first 30 s after euthanasia by various methods. Tracheal cannulae (for positive-pressure ventilation, PPV) and cortical surface electrodes were placed in mice anesthetized with inhaled halothane. Succinylcholine was used to block spontaneous breathing in the mice, which then underwent continuous EEG recording. Photic stimuli (1 Hz) were presented to produce VEPs superimposed on the EEG. Anesthesia was discontinued immediately before euthanasia. Compared with that obtained before euthanasia, EEG activity during the 30-s study period immediately after euthanasia was significantly decreased after cervical dislocation (at 5 to 10 s), 100% PPV-CO2 (at 10 to 15 s), decapitation (at 15 to 20 s), and cardiac arrest due to KCl injection (at 20 to 25 s) but not after administration of 70% PPV-CO2. Similarly, these euthanasia methods also reduced VEP amplitude, although 100% PPV-CO2 treatment affected VEP amplitude more than it did EEG activity. Thus, 100% PPV-CO2 treatment significantly decreased VEP beginning 5 to 10 s after administration, with near abolition of VEP by 30 s. VEP amplitude was significantly reduced at 5 to 10 s after cervical dislocation and at 10 to 15 s after decapitation but not after either KCl or 70% PPV-CO2 administration. The data demonstrate that 100% PPV-CO2, decapitation, and cervical dislocation lead to rapid disruption of cortical function as measured by 2 different methods. In comparison, 70% PPV-CO2 and cardiac arrest due to intracardiac KCl injection had less rapid effects on cortical function.     

Clonal Expansion of Normal-Appearing Human Hepatocytes during Chronic Hepatitis B Virus Infection

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.Transient hepatitis B virus (HBV) infections, which generally last <6 months, do not cause cirrhosis and cause only minor increases in the risk of hepatocellular carcinoma (HCC) (3, 46). Chronic infections, typically lifelong, can cause cirrhosis and HCC (3). Of the ∼350 million HBV carriers now alive, ca. 60 million will die prematurely of cirrhosis and/or HCC. Cirrhosis, which usually develops late in infection, is a significant risk factor for HCC. Early reports stated that most HCCs occur on a background of cirrhosis. However, later studies suggested that as many as 50% of HCCs may occur in noncirrhotic liver (4), that is, in patients in whom the progression of liver disease still appears rather mild. Thus, liver damage that appears severe by histologic examination is not a prerequisite for HCC.Interestingly, during chronic HBV infections there is, in the face of persistent viremia, a decline over time in the fraction of infected hepatocytes, from 100% to as little as a few percent (5, 12-14, 16, 17, 22, 23, 27, 34, 37, 38). Along with HCC, this is perhaps the most surprising and unexplained outcome of chronic infection. The timing of this decline has not been systematically studied, but it is presumably gradual, occurring over years or decades, and dependent on persistent, albeit low-level, killing of infected hepatocytes by antiviral cytotoxic T lymphocytes (CTLs) (20). It is believed that the liver is largely a closed, self-renewing population. Such a population might be expected to evolve under any strong or persistent selective pressure. In HBV-infected patients, the earliest and most persistent selective pressure is immune killing of infected hepatocytes, which should initially constitute the entire hepatocyte population. Persistent killing of HBV-infected hepatocytes could lead to clonal expansion of mutant or epigenetically altered hepatocytes that had lost the ability to support infection and that were not, therefore, targeted by antiviral CTLs.Such a selective pressure may explain why foci of altered hepatocytes (FAH) and HCC are typically virus negative (1, 6, 11, 26, 29, 31, 35, 40, 41, 44). Normal or preneoplastic hepatocytes (e.g., in FAH) that have evaded the host immune response should undergo clonal expansion, because their death rate is lower than that of surrounding hepatocytes, even if they do not have a higher growth rate. Indeed, clonal expansion of hepatocytes has been detected, in the absence of cirrhosis, in woodchucks chronically infected with woodchuck hepatitis virus (WHV) (19) and in chimpanzees chronically infected with HBV (21). The presence of discrete foci of normal-appearing but virus-negative hepatocytes in chronically infected woodchuck livers (39) suggested, but did not prove, that normal-appearing hepatocytes that had lost the ability to support virus replication might clonally expand.The purpose of the present study was, therefore, to determine if normal-appearing hepatocytes undergo clonal expansion. To address this issue, we focused on noncirrhotic livers, because hepatocyte appearance and organization in many cirrhotic nodules are often considered to indicate premalignancy (7, 24, 25, 44), and this, together with the cellular environment in the cirrhotic liver, may explain why as many as 50% of cirrhotic nodules have been found to be made up of clonally expanded hepatocytes (2, 18, 24, 25, 28, 44). In older HBV patients, cirrhosis, the result of cumulative scarring due to ongoing tissue injury, presumably produces an evolutionary pressure on the hepatocyte population due to restricted blood flow and altered hepatic architecture.Clonal expansion was detected by assaying for integrated HBV DNA by inverse PCR (19, 21). Because integration occurs at random sites in host DNA, each integration event provides a unique genetic marker for the cell in which it occurred, and for any daughter cells. Thus, the clonal expansion of these tagged hepatocytes can be measured by determining how many times a given virus-cell DNA junction is repeated in a liver fragment. Analysis of fragments of nontumorous liver from noncirrhotic HCC patients revealed that at least 1% of hepatocytes are present as clones of >1,000 cells. Examination of 5-μm-thick sections of paraffin-embedded livers from the same patients revealed that clonally expanded hepatocytes were present in liver sections lacking preneoplastic lesions or changes. Therefore, normal-appearing hepatocytes must have undergone clonal expansion. Although clonal expansion was detected by analysis of integrated HBV DNA, the expansion did not appear to be due to the site of integration of the viral DNA into host DNA.These results are consistent with the hypothesis that immune selection and the later emergence of liver cirrhosis, with altered lobular organization and restricted blood flow, may constitute the two major selective pressures on the hepatocyte population that culminate in hepatocellular carcinoma. More-direct proof of the role, if any, of immune selection in hepatocyte evolution and HCC will require, first of all, an assay with a greater ability to detect clonally expanded hepatocytes. The present approach is limited by a number of factors, including a need for integration near a particular restriction endonuclease cleavage site in host DNA and for conservation of particular viral sequences so that the integrated DNA can be amplified using the PCR primers chosen. These issues may explain why the fraction of clonally expanded hepatocytes reported here is much less than that suggested by histologic data showing that more than 50% of hepatocytes appear negative for virus replication in long-term carriers. Further dissection of this issue will also require localization and determination of the virologic status of hepatocyte clones present in tissue sections.     

Genomewide linkage scan for split-hand/foot malformation with long-bone deficiency in a large Arab family identifies two novel susceptibility loci on chromosomes 1q42.2-q43 and 6q14.1

Split-hand/foot malformation with long-bone deficiency (SHFLD) is a rare, severe limb deformity characterized by tibia aplasia with or without split-hand/split-foot deformity. Identification of genetic susceptibility loci for SHFLD has been unsuccessful because of its rare incidence, variable phenotypic expression and associated anomalies, and uncertain inheritance pattern. SHFLD is usually inherited as an autosomal dominant trait with reduced penetrance, although recessive inheritance has also been postulated. We conducted a genomewide linkage analysis, using a 10K SNP array in a large consanguineous family (UR078) from the United Arab Emirates (UAE) who had disease transmission consistent with an autosomal dominant inheritance pattern. The study identified two novel SHFLD susceptibility loci at 1q42.2-q43 (nonparametric linkage [NPL] 9.8, P=.000065) and 6q14.1 (NPL 7.12, P=.000897). These results were also supported by multipoint parametric linkage analysis. Maximum multipoint LOD scores of 3.20 and 3.78 were detected for genomic locations 1q42.2-43 and 6q14.1, respectively, with the use of an autosomal dominant mode of inheritance with reduced penetrance. Haplotype analysis with informative crossovers enabled mapping of the SHFLD loci to a region of approximately 18.38 cM (8.4 Mb) between single-nucleotide polymorphisms rs1124110 and rs535043 on 1q42.2-q43 and to a region of approximately 1.96 cM (4.1 Mb) between rs623155 and rs1547251 on 6q14.1. The study identified two novel loci for the SHFLD phenotype in this UAE family.