bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread
Authors: Ohshima Kazusato , Yamaguchi Yuka , Hirota Ryo , Hamamoto Tamaki , Tomimura Kenta , Tan Zhongyang , Sano Teruo , Azuhata Fumio , Walsh John A. , Fletcher John , Chen Jishuang , Gera Abed , Gibbs Adrian
Published: August 2015

Turnip mosaic virus (TuMV), a species of the genus Potyvirus, occurs worldwide. Seventy-six isolates of TuMV were collected from around the world, mostly from Brassica and Raphanus crops, but also from several non-brassica species. Host tests grouped the isolates into one or other of two pathotypes; Brassica (B) and Brassica–Raphanus (BR). The nucleotide sequences of the first protein (P1) and coat protein (CP) genes of the isolates were determined. One-tenth of the isolates were found to have anomalous and variable phylogenetic relationships as a result of recombination. The 5′-terminal 300 nt of the P1 gene of many isolates was also variable and phylogenetically anomalous, whereas the 380 nt 3′ terminus of the CP gene was mostly conserved. Trees calculated from the remaining informative parts of the two genes of the non-recombinant sequences by neighbour-joining, maximum-likelihood and maximum-parsimony methods were closely similar, and so these parts of the sequences were concatenated and trees calculated from the resulting 1150 nt. The isolates fell into four consistent groups; only the relationships of these groups with one another and with the outgroup differed. The ‘basal-B’ cluster of eight B-pathotype isolates was most variable, was not monophyletic, and came from both brassicas and non-brassicas from southwest and central Eurasia. Closest to it, and forming a monophyletic subgroup of it in most trees, and similarly variable, was the ‘basal-BR’ group of eight BR pathotype Eurasian isolates. The third and least variable group, the ‘Asian-BR’ group, was of 22 BR-pathotype isolates, all from brassicas, mostly Raphanus, and all from east Asia mostly Japan. The fourth group of 36 isolates, the ‘world-B’ group, was from all continents, most were isolated from brassicas and most were of the B-pathotype. The simplest of several possible interpretations of the trees is that TuMV originated, like its brassica hosts, in Europe and spread to the other parts of the world, and that the BR pathotype has recently evolved in east Asia.
Detection of neutralizing antibodies to hepatitis C virus using a biliary cell infection model
Authors: Bichr Saadia , Rende-Fournier Rosanna , Vona Giovanna , Yamamoto Ana-Maria , Depla Erik , Maertens Geert , Bréchot Christian
Published: August 2015

The identification and characterization of neutralizing anti-hepatitis C virus (HCV) antibodies may have a major impact on understanding HCV pathogenesis. However, to date, their detection has only been based on the inhibition of either the E2 envelope protein or HCV virions binding to different target cells. The permissivity of primary biliary cells for HCV infection has been demonstrated previously. In the present report, infection of biliary cells was demonstrated further by combining PCR and immunohistochemical detection of the HCV core protein. This study demonstrates, using both serum and purified IgG, the presence of neutralizing anti-HCV antibodies in the serum of patients showing long-term response to antiviral therapy. Overall, the usefulness of the primary biliary cell infection model to investigate anti-HCV neutralization is shown.
Mapping of tissue tropism determinants in coxsackievirus genomes
Authors: Harvala Heli , Kalimo Hannu , Dahllund Leif , Santti Juhana , Hughes Pamela , Hyypiä Timo , Stanway Glyn
Published: August 2015

Genomic regions responsible for the different tissue tropisms of coxsackievirus A9 (CAV9) and coxsackievirus B3 (CBV3) in newborn mice were investigated using recombinant viruses. Infectious cDNA clones of CAV9, a virus known to infect striated muscle, and CBV3, affecting the central nervous system, pancreas, liver, brown fat and striated muscle, were used to generate chimeric viruses. In situ hybridization analysis of different tissues from mice infected with the recombinant viruses, constructed by exchanging the 5′ non-coding region (5′NCR), structural and non-structural genes, demonstrated that the pancreo- and liver tropism map predominantly to CBV3 sequences within the capsid genes, evidently due to receptor recognition. Although the major neurotropism determinant in the CBV3 genome was in the capsid region, viruses containing the CAV9 capsid were also able to initiate infection in the central nervous system provided they contained the CBV3 5′NCR. The presence of the 5′NCR of CAV9 clearly enhanced muscle tissue tropism.
Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: age-dependent paralysis and a mucosal route of infection
Authors: Crotty Shane , Hix Laura , Sigal Luis J. , Andino Raul
Published: August 2015

We constructed a poliovirus receptor (PVR) transgenic mouse line carrying a PVRδ cDNA driven by a β-actin promoter. We refer to this model as the cPVR mouse. The cPVR mice express Pvr in a variety of tissues (including small intestines, brain, spinal cord, muscle, blood and liver) and are susceptible to infection after intraperitoneal, intracerebral or intramuscular inoculation of poliovirus. After intraperitoneal inoculation, poliovirus replication is observed in cPVR muscle, brain, spinal cord and, notably, small intestine. The cPVR mice exhibit a striking age-dependent paralysis after intramuscular infection, with 2-week-old mice being 10000-fold more susceptible to paralytic disease than adult mice. The cPVR mice are also susceptible to paralysis following intranasal infection with poliovirus. After intranasal infection, virus replication is observed in the olfactory bulb, cerebrum, brain stem and spinal cord, suggesting that intranasal infection of cPVR mice is a model for bulbar paralysis. Intranasally infected mice frequently display unusual neurological behaviours. The PVR transgenic mouse reported here provides the first available model for a mucosal route of infection with poliovirus.
Sequence analysis of Potato leafroll virus isolates reveals genetic stability, major evolutionary events and differential selection pressure between overlapping reading frame products
Authors: Guyader Sébastien , Ducray Danièle Giblot
Published: August 2015

In order to investigate the genetic diversity ofPotato leafroll virus(PLRV), seven new complete genomic sequences of isolates collected worldwide were compared with the five sequences available in GenBank. Then, a restricted polymorphic region of the genome was chosen to further analyse new sequences. The sequences of PLRV open reading frames (ORFs) 3 and 4 were also compared with those of two other poleroviruses and the non-synonymous to synonymous substitution ratio distribution was analysed in overlapping and non-overlapping regions of the genome using maximum-likelihood models. Results confirmed that PLRV sequences from around the world are very closely related and showed that the region encoding protein P0 allowed the detection of three groups of isolates. When compared to other poleroviruses, PLRV was the most conserved in both ORFs 3 and 4. However, the results suggest that important events, such as deletion, mutation at a stop codon and intraspecific homologous recombination events, have occurred during the evolution of PLRV. Finally, it was shown that the translation products of ORFs 0 and 3 are significantly more conserved than those of the overlapping ORFs 1 and 4, respectively. All together, the results allow the proposal of new hypotheses to explain the apparent genetic stability of PLRV and its evolution.
Vaccination of pigs with a DNA construct expressing an influenza virus M2–nucleoprotein fusion protein exacerbates disease after challenge with influenza A virus
Authors: Heinen Paul P. , Rijsewijk Frans A. , de Boer-Luijtze Els A. , Bianchi André T. J.
Published: August 2015

In mice, vaccines inducing antibodies to the extracellular domain of the M2 protein (M2e) can confer protection to influenza A virus infection. Unlike the surface glycoproteins, haemagglutinin and neuraminidase, this domain of M2 is highly conserved and is therefore a potential broad-spectrum immunogen. In this study, the protection conferred by vaccines inducing antibodies to M2e was evaluated in a challenge model for swine influenza in pigs. A protein resulting from the fusion between M2e and the hepatitis B virus core protein (M2eHBc), with or without adjuvant, was evaluated. In addition, a DNA construct expressing a fusion protein between M2e and influenza virus nucleoprotein (M2eNP) was evaluated to see if the broad-spectrum protection conferred by antibodies could be further enhanced by T helper cells and cytotoxic T cells. All vaccines induced an antibody response against M2e, and the M2eNP DNA vaccine additionally induced an influenza virus-specific lymphoproliferation response. However, after challenge with a swine influenza virus (H1N1), no protection was observed in the vaccinated groups compared with the non-vaccinated control group. On the contrary, vaccinated pigs showed more severe clinical signs than the control pigs. The M2eNP DNA-vaccinated pigs showed the most severe clinical signs and three out of six pigs died on days 1 and 2 post-challenge. These results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease. Thus, clinical signs after infection should be observed closely in further studies using M2e as an immunogen and caution should be exercised in using M2e in humans.
Infection of SCID mice with Montana Myotis leukoencephalitis virus as a model for flavivirus encephalitis
Authors: Charlier Nathalie , Leyssen Pieter , Paeshuyse Jan , Drosten Christian , Schmitz Herbert , Van Lommel Alfons , De Clercq Erik , Neyts Johan
Published: August 2015

We have established a convenient animal model for flavivirus encephalitis using MontanaMyotisleukoencephalitis virus (MMLV), a bat flavivirus. This virus has the same genomic organization, and contains the same conserved motifs in genes that encode potential antiviral targets, as flaviviruses that cause disease in man (N. Charlieret al., accompanying paper), and has a similar particle size (approximately 40 nm). MMLV replicates well in Vero cells and appears to be equally as sensitive as yellow fever virus and dengue fever virus to a selection of experimental antiviral agents. Cells infected with MMLV show dilation of the endoplasmic reticulum, a characteristic of flavivirus infection. Intraperitoneal, intranasal or direct intracerebral inoculation of SCID mice with MMLV resulted in encephalitis ultimately leading to death, whereas immunocompetent mice were refractory to either intranasal or intraperitoneal infection with MMLV. Viral RNA and/or antigens were detected in the brain and serum of MMLV-infected SCID mice, but not in any other organ examined: MMLV was detected in the olfactory lobes, the cerebral cortex, the limbic structures, the midbrain, cerebellum and medulla oblongata. Infection was confined to neurons. Treatment with the interferon-α/β inducer poly(I)·poly(C) protected SCID mice against MMLV-induced morbidity and mortality, and this protection correlated with a reduction in infectious virus titre and viral RNA load. This validates the MMLV model for use in antiviral drug studies. The MMLV SCID model may, therefore, be attractive for the study of chemoprophylactic or chemotherapeutic strategies against flavivirus infections causing encephalitis.
Serial passage of foot-and-mouth disease virus in sheep reveals declining levels of viraemia over time
Authors: Hughes Gareth J. , Mioulet Valerie , Haydon Daniel T. , Kitching R. Paul , Donaldson Alex I. , Woolhouse Mark E. J.
Published: August 2015

If an infectious agent is to maintain itself within a closed population by means of an unbroken serial chain of infections, it must maintain the level of infectiousness of individuals through time, or termination of the transmission chain is inevitable. One possible cause of diminution in infectiousness along serial chains of transmission may be that individuals are unable to amplify and transmit comparable levels of the infectious agent. Here, the results are reported of a novel experiment designed specifically to assess the effects of serial passage of foot-and-mouth disease virus (FMDV) in experimental groups of sheep. A virus isolate taken from an epidemic of foot-and-mouth disease (FMD) characterized by rapid fade-out of infection was passed serially through four groups of sheep housed in an isolation unit. Although it was not possible to measure individual infectiousness directly, blood virus load from infected individuals was quantified using a real-time PCR assay and used as an underlying indicator of the level of infection. The results of this assay concurred well with those of the traditional tissue-culture assay and were shown to be highly repeatable. The level of peak viraemia was shown to fall significantly with the time of infection and with passage group, both in terms of the group mean and regression analysis of individual values, suggesting that this isolate of FMDV may, under certain conditions, be unable to maintain itself indefinitely in susceptible sheep populations. The results of these experiments are discussed in terms of the epidemiology of FMD in sheep.
A divalent antibody format is required for neutralization of human cytomegalovirus via antigenic domain 2 on glycoprotein B
Authors: Lantto Johan , Fletcher Jean M. , Ohlin Mats
Published: August 2015

Glycoprotein B (gB) of human cytomegalovirus (HCMV) is the dominating protein in the envelope of this virus and gives rise to virus-neutralizing antibodies in most infected individuals. We have previously isolated a neutralizing human antibody specific for antigenic domain 2 (AD-2) on gB, a poorly immunogenic epitope, which nevertheless is capable of eliciting potent neutralizing antibodies. In order to define parameters important for the neutralization of HCMV via gB, we have investigated the virus-neutralizing capacity and the kinetics of the interaction with AD-2 of the monomeric and dimeric forms of a single chain variable fragment (scFv) corresponding to this antibody. We demonstrate here that neutralization of HCMV via AD-2 on gB can be mediated by dimeric scFv, while monomeric fragments cannot mediate neutralization of the virus, despite a slow dissociation from the intact glycoprotein. This finding is discussed in the context of possible mechanisms for antibody-mediated virus neutralization.
Polydnavirus replication: the EP1 segment of the parasitoid wasp Cotesia congregata is amplified within a larger precursor molecule
Authors: Pasquier-Barre F. , Dupuy C. , Huguet E. , Monteiro F. , Moreau A. , Poirié M. , Drezen J.-M.
Published: August 2015

Polydnaviruses are unique viruses: they are essential for successful parasitism by tens of thousands of species of parasitoid wasps. These viruses are obligatorily associated with the wasps and are injected into the host during oviposition. Molecular analyses have shown that each virus sequence in the segmented polydnavirus genome is present in the wasp DNA in two forms: a circular form found in the virus particles and an integrated form found in the wasp chromosomes. Recent studies performed on polydnaviruses from braconid wasps suggested that the circular forms were excised from the chromosome. The different forms of the EP1 circle of Cotesia congregata polydnavirus during the pupal–adult development of the parasitoid wasp were analysed. Unexpectedly, an off-size fragment formerly used to diagnose the integration of the EP1 sequence into wasp genomic DNA was found to be amplified in female wasps undergoing virus replication. The EP1 sequence is amplified within a larger molecule comprising at least two virus segments. The amplified molecule is different from the EP1 chromosomally integrated form and is not encapsidated into virus particles. These findings shed light on a new step towards EP1 circle production: the amplification of virus sequences preceding individual circle excision.
Adenovirus E1-transformed cells grow despite the continuous presence of transcriptionally active p53
Authors: Löber Christian , Lenz-Stöppler Claudia , Dobbelstein Matthias
Published: August 2015

The E1 region of adenovirus (Ad) type 5 is capable of transforming cells. According to current concepts, the Ad E1B 55 kDa (E1B 55K) protein enables transformed cells to grow by constantly binding and inactivating the p53 tumour suppressor protein. To test this model, the transcriptional activity of p53 was determined in Ad E1-transformed cells. Surprisingly, it was found that a p53-responsive promoter is highly active in Ad E1-transformed cells and further activated only 3- to 4-fold (compared to 200-fold in p53
−/− cells) by exogenously expressed p53 or p53mt24–28, a p53 mutant that is transcriptionally active but unable to bind the E1B 55K. On the other hand, the transient overexpression of E1B 55K led to a strong downregulation of a p53-responsive promoter relative to its baseline activity in Ad E1-transformed cells but not in p53
−/− cells. COS-7 cells, transformed by simian virus 40 (SV40), also showed constitutive p53 activity, whereas HeLa cells, transformed with oncogenic human papillomavirus, did not. Upon stable transfection, Ad E1-transformed cells but not p53
−/− cells gave rise to colonies that expressed exogenous p53 or p53mt24–28 but, nonetheless, grew at near-wild-type rates. It is proposed that E1B 55K or the SV40 tumour antigen are saturated by the p53 protein, which accumulates in virus-transformed cells, leaving a proportion of active p53 molecules. The transformation of cells by the Ad E1 genes confers permissiveness for active p53, conceivably by inactivating the relevant products of p53 target genes that would otherwise prevent cell growth. Thus, Ad-transformed cells contain and tolerate active p53.
Occupancy and mechanism in antibody-mediated neutralization of animal viruses
Authors: Klasse P. J. , Sattentau Q. J.
Published: August 2015

Neutralization of virus infectivity by antibodies is an important component of immunity to several virus infections. Here, the immunochemical basis for the action of neutralizing antibodies, and what role their induction of conformational changes in the antigen might play, is reviewed. Theories of the mechanisms by which antibodies neutralize virus infectivityin vitroare also presented. The theoretical and empirical foundation of the hypothesis that viruses are neutralized by a single antibody per virion is critically reviewed. The relationship between antibody occupancy on virions and the mechanism of neutralization is explored. Examples of neutralization mediated through antibody interference with virus attachment and entry are discussed and test implications of refined theories of neutralization by antibody coating of virions are formulated.
Hepatitis C virus core protein represses the p21 promoter through inhibition of a TGF-β pathway
Authors: Lee Mi Nam , Jung Eun Young , Kwun Hyun Jin , Jun Hong Ki , Yu Dae-Yeul , Choi Yung Hyun , Jang Kyung Lib
Published: August 2015

The increased proliferation rate of hepatocytes is one of the major risk factors for the development of hepatocellular carcinoma. In this study, we investigated the mechanism by which hepatitis C virus (HCV) core protein represses transcription of the universal cyclin-dependent kinase inhibitor p21 gene in murine fibroblast NIH 3T3 cells. From the transient reporter assays of p21 promoter, we found that the TGF-β-responsive element (TβRE) located between −83 and −74 of the p21 promoter is responsible for the effect. The TGF-β-induced p21 promoter activity was specifically decreased by HCV core protein and in the presence of the inhibitory Smad7 the repression effect was almost completely abolished. Furthermore, HCV core protein stimulated the growth rate of NIH 3T3 cells and could overcome growth arrest by TGF-β but not by butyrate, suggesting that HCV core protein stimulates cell cycle progression by repressing p21 transcription through a TGF-β pathway.
Human immunodeficiency virus type 1 Vif binds the viral protease by interaction with its N-terminal region
Authors: Baraz Lea , Hutoran Marina , Blumenzweig Immanuel , Katzenellenbogen Mark , Friedler Assaf , Gilon Chaim , Steinitz Michael , Kotler Moshe
Published: August 2015

The vif gene, one of the six auxiliary genes of human immunodeficiency virus (HIV), is essential for virus propagation in peripheral blood lymphocytes and macrophages and in certain T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV type 1 (HIV-1) Gag–Pol polyproteins expressed in bacterial cells, as well as the protease-mediated cleavage of synthetic peptides in vitro. Peptides derived from the aa 78–98 region in the Vif molecule specifically inhibit and bind the HIV-1 protease in vitro and arrest the production of infectious viruses in HIV-1-infected cells. This study demonstrates that (i) purified recombinant Vif protein and HIV-1 but not avian sarcoma leukaemia virus protease specifically bind each other and (ii) the interaction between these two proteins takes place at the N terminus of the protease (aa 1–9) and the central part of Vif (aa 78–98). The data presented in this report suggest a model in which Vif interacts with the dimerization sites of the viral protease.
PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B
Authors: Bartosch Birke , Weiss Robin A. , Takeuchi Yasuhiro
Published: August 2015

Two pig endogenous retroviruses (PERV), PERV-A and -B, productively infect human cells and are therefore considered to constitute a potential risk in pig-to-human xenotransplantation. A PCR-based cloning technique to isolate infectious PERV proviruses was established. Overlapping 3′ half and 5′ halves of PERV proviral genomes were amplified using DNA extracted from human 293 cells infected with PERV-A or -B. These clones were fused at a unique restriction site in the overlapping region and tested for their infectivity. Representative constructs possessed the same infectious properties as their parent isolates. We also developed a polyclonal anti-PERV serum by using recombinant PERV capsid protein derived from one of the infectious constructs as immunogen and established an immunocytological method for detection and titration of PERV infection. This detection method proved to be more sensitive than the current method of choice (transfer of MLV-lacZ vectors) for infectivity assessment of PERV. These findings should be considered for future characterization of PERV isolates.
Susceptibility of sensory neurons to apoptosis following infection by bovine herpesvirus type 1
Authors: Delhon Gustavo A. , González Marcelo J. , Murcia Pablo R.
Published: August 2015

Like other members of the alpha subfamily of herpesviruses, bovine herpesvirus type 1 (BHV-1) establishes latent infections in sensory neurons. BHV-1 induces apoptosis in lymphoid cells in vivo and in epithelial cell lines, but the ability of BHV-1 to induce apoptosis in sensory neurons remains unknown. In this report, the susceptibility of rabbit ganglionic neurons to infection by BHV-1 was examined in vitro and in vivo. Following infection of cultured neurons with BHV-1, hallmarks of apoptosis such as chromatin condensation, DNA fragmentation and membrane blebbing were detected. The appearance of these changes was preceded by active viral DNA replication as determined by in situ hybridization. When viral DNA replication was blocked by treatment of cultures with an inhibitor of eukaryotic DNA polymerases, apoptosis but not virus attachment to neurons or bICP0 gene expression was completely prevented. Taken together, these results demonstrate that sensory neurons are not intrinsically resistant to BHV-1-induced apoptosis and that viral DNA replication plays a role in triggering the apoptotic programme. Infection of rabbits with BHV-1 resulted in pathological changes in the trigeminal ganglia (TG) which included mononuclear cell infiltration and neuronophagia. Morphological evidence of apoptosis was not detected in neurons, even in cells with advanced cytophatology. Furthermore, whereas DNA fragmentation was common in infiltrating cells, it was very rare and sporadic in neurons. Therefore, mechanisms in the TG should exist to prevent neuronal apoptosis upon BHV-1 infection.
The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome
Authors: Collins Christopher M. , Medveczky Maria M. , Lund Troy , Medveczky Peter G.
Published: August 2015

The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8). Both belong to the gamma-2 herpesvirus subgroup. The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA). Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F′ replicon-based E. coli vector. Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit. T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome. Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA. Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers. These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells. Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence. Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E. coli.
No association between human parvovirus B19 and testicular germ cell cancer
Authors: Tolfvenstam T. , Papadogiannakis N. , Andersen A. , Akre O.
Published: August 2015

The incidence of testicular germ cell cancer, which is the most common cancer among young male adults, is increasing. The aetiology remains unknown, although a virus has been proposed. A previous study has shown a high prevalence of human parvovirus B19 (B19) DNA in the testes of patients with testicular germ cell tumours (85%) and suggested that B19 may play a role in tumour development. To address this question of causality, seroreactivity to B19 was studied among cases (n=80) and controls (n=241) using serum samples drawn before the onset of disease, in addition to an elucidation of the frequency of virus DNA in a retrospectively collected 2-year testicular carcinoma series. No association was found between B19 seropositivity and the risk of testicular cancer (odds ratio=1·03; 95% confidence interval=0·60–1·77) nor was there any dose-response relation (P for trend=0·53). This study did, however, confirm the observation that B19 DNA can be detected in testicular carcinoma tissue, as 4 of 24 cases were found to be positive, while no B19 DNA could be detected in the control cases. It is speculated that this finding may be due to susceptibility of the carcinoma cells to B19 virus owing to high-level expression of the viral receptor glycosphingolipid (Gb4) and possible other putative cellular factors resulting in a localized persistence initiated after the development of cancer.
‘Streptomyces nanchangensis’, a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters
Authors: Sun Yuhui , Zhou Xiufen , Liu Jun , Bao Kai , Zhang Guiming , Tu Guoquan , Kieser Tobias , Deng Zixin
Published: August 2015

Several independent gene clusters containing varying lengths of type I polyketide synthase genes were isolated from ‘Streptomyces nanchangensis’ NS3226, a producer of nanchangmycin and meilingmycin. The former is a polyether compound similar to dianemycin and the latter is a macrolide compound similar to milbemycin, which shares the same macrolide ring as avermectin but has different side groups. Clusters A–H spanned about 133, 132, 104, 174, 122, 54, 37 and 59 kb, respectively. Two systems were developed for functional analysis of the gene clusters by gene disruption or replacement. (1) Streptomyces phage ϕC31 and its derived vectors can infect and lysogenize this strain. (2) pSET152, an Escherichia coli plasmid with ϕC31 attP site, and pHZ1358, a Streptomyces–Escherichia coli shuttle cosmid vector, both carrying oriT from RP4, can be mobilized from E. coli into NS3226 by conjugation. pHZ1358 was shown to be generally useful for generating mutant strains by gene disruption and replacement in NS3226 as well as in several other Streptomyces strains. A region in cluster A (∼133 kb) seemed to be involved in nanchangmycin production because replacement of several DNA fragments in this region by an apramycin resistance gene [aac3(IV)] gave rise to nanchangmycin non-producing mutants.
Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this paper are AJ224870, AJ276618, AJ277778, AJ295338 and AJ295339.
Authors: Borovok Ilya , Kreisberg-Zakarin Rachel , Yanko Michaela , Schreiber Rachel , Myslovati Margarita , Aslund Fredrik , Holmgren Arne , Cohen Gerald , Aharonowitz Yair
Published: August 2015

Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genusStreptomyces. One gene,nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of ∼108 kDa. The activity of this protein depended on the presence of 5′-deoxyadenosylcobalamine (coenzyme B12), establishing it as a class II RNR. TheStreptomyces clavuligerus nrdJgene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa. Molecular phylogenetic analysis showed that theS. clavuligerusclass II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes,nrdAandnrdB, were initially identified in theStreptomyces coelicolorgenome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that thenrdABgenes were present in differentStreptomycesspp. TheS. coelicolor nrdABgenes were cloned and expressed inEscherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that theS. clavuligerusclass Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class IInrdJtranscripts was approximately constant throughout the exponential phase of vegetative growth (3–5×105copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class IanrdABtranscripts was some 10- to 20-fold less than that ofnrdJin the early-exponential growth phase (2·8×104copies), and decreased markedly at the mid-exponential (4×103copies) and late-exponential phases (1·1×103copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.
In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion
Authors: Dieterich C , Schandar M , Noll M , Johannes F.-J , Brunner H , Graeve T , Rupp S
Published: August 2015

The individual and synergistic contributions of two transcription factors,EFG1andCPH1, have been characterized with regard to adhesion to, and invasion of, human epithelia byCandida albicans. For this purpose twoin vitroreconstructed tissue models were developed. A multi-layered model of human epidermis was used to simulate superficial infections of the skin, whereas a reconstructed human intestinal model was used to mimic the first steps of systemic infections. It was shown thatC. albicansdeleted for both transcription factorsCPH1andEFG1, in contrast to the congenic clinical isolate Sc5314, was neither able to adhere to, nor to penetrate, either of the model systems. A strain deleted forEFG1alone showed significant reduction in adhesion and was not able to penetrate through the stratum corneum. However, strains deleted forCPH1showed phenotypes paralleling the phenotypes of the clinical isolate Sc5314. Using different types of multi-layered human tissues reconstructedin vitrothe individual contributions of Efg1p and Cph1p to two important virulence factors ofC. albicans, namely adhesion and invasion, could be defined.
The metIC operon involved in methionine biosynthesis in Bacillus subtilis is controlled by transcription antitermination
Authors: Auger Sandrine , Yuen W. H , Danchin Antoine , Martin-Verstraete Isabelle
Published: August 2015

There are two major pathways for methionine biosynthesis in micro-organisms. Little is known about these pathways inBacillus subtilis. The authors assigned a function to themetI(formerlyyjcI) andmetC(formerlyyjcJ) genes ofB. subtilisby complementingEscherichia coli metBandmetCmutants, analysing the phenotype ofB. subtilis metIandmetCmutants, and carrying out enzyme activity assays. These genes encode polypeptides belonging to the cystathionine γ-synthase family of proteins. Interestingly, the MetI protein has both cystathionine γ-synthase andO-acetylhomoserine thiolyase activities, whereas the MetC protein is a cystathionine β-lyase. InB. subtilis, the transsulfuration and the thiolation pathways are functionalin vivo. Due to its dual activity, the MetI protein participates in both pathways. ThemetIandmetCgenes form an operon, the expression of which is subject to sulfur-dependent regulation. When the sulfur source is sulfate or cysteine the transcription of this operon is high. Conversely, when the sulfur source is methionine its transcription is low. An S-box sequence, which is located upstream of themetIgene, is involved in the regulation of themetICoperon. Northern blot experiments demonstrated the existence of two transcripts: a small transcript corresponding to the premature transcription termination at the terminator present in the S-box and a large one corresponding to transcription of the completemetICoperon. When methionine levels were limiting, the amount of the full-length transcript increased. These results substantiate a model of regulation by transcription antitermination.
Bacterial deposition in a parallel plate and a stagnation point flow chamber: microbial adhesion mechanisms depend on the mass transport conditions
Authors: Bakker Dewi P , Busscher Henk J , van der Mei Henny C
Published: August 2015

Deposition onto glass in a parallel plate (PP) and in a stagnation point (SP) flow chamber of Marinobacter hydrocarbonoclasticus, Psychrobacter sp. and Halomonas pacifica, suspended in artificial seawater, was compared in order to determine the influence of methodology on bacterial adhesion mechanisms. The three strains had different cell surface hydrophobicities, with water contact angles on bacterial lawns ranging from 18 to 85 degrees. Bacterial zeta potentials in artificial seawater were essentially zero. The three strains showed different adhesion kinetics and the hydrophilic bacterium H. pacifica had the greatest affinity for hydrophilic glass. On average, initial deposition rates were two- to threefold higher in the SP than in the PP flow chamber, possibly due to the convective fluid flow toward the substratum surface in the SP flow chamber causing more intimate contact between a substratum and a bacterial cell surface than the gentle collisions in the PP flow chamber. The ratios between the experimental deposition rates and theoretically calculated deposition rates based on mass transport equations not only differed among the strains, but were also different for the two flow chambers, indicating different mechanisms under the two modes of mass transport. The efficiencies of deposition were higher in the SP flow chamber than in the PP flow chamber: 62±4 and 114±28% respectively. Experiments in the SP flow chamber were more reproducible than those in the PP flow chamber, with standard deviations over triplicate runs of 8% in the SP and 23% in the PP flow chamber. This is probably due to better-controlled convective mass transport in the SP flow chamber, as compared with the diffusion-controlled mass transport in the PP flow chamber. In conclusion, this study shows that bacterial adhesion mechanisms depend on the prevailing mass transport conditions in the experimental set-up used, which makes it essential in the design of experiments that a methodology is chosen with mass transport conditions resembling the problem under investigation.
The Rhizobium leguminosarum bv. viciae VF39 γ-aminobutyrate (GABA) aminotransferase gene (gabT) is induced by GABA and highly expressed in bacteroids
The GenBank accession number for the sequence determined in this work is AF335502.
Authors: Prell Jürgen , Boesten Bert , Poole Philip , Priefer Ursula B
Published: August 2015

A Rhizobium leguminosarum bv. viciae VF39 gene (gabT) encoding a γ-aminobutyrate (GABA) aminotransferase was identified, cloned and characterized. This gene is thought to be involved in GABA metabolism via the GABA shunt pathway, a theoretical bypass of the 2-oxoglutarate dehydrogenase complex. Mutants in gabT are still able to grow on GABA as a sole carbon and nitrogen source. 2-Oxoglutarate-dependent GABA aminotransferase activity is absent in these mutants, while pyruvate-dependent activity remains unaffected. This indicates that at least two enzymes with different substrate specifities are involved in the GABA metabolism of R. leguminosarum bv. viciae VF39. The gabT promoter was cloned into a newly constructed, stable promoter-probe vector pJP2, suitable for the study of transcriptional GUS fusions in free-living bacteria and during symbiosis. Under free-living conditions the gabT promoter is induced by GABA and repressed by succinate. Transcriptional regulation is mediated by GabR in a repressor-like manner. During symbiosis with the pea host plant gabT is induced and highly expressed in the symbiotic zone. Nodules induced by gabT mutants, however, are still effective in nitrogen fixation.
Rhodococcus lactis sp. nov., an actinobacterium isolated from sludge of a dairy waste treatment plant
Authors: Singh Pradip Kumar , Kumari Annu , Chawla Niharika , Pinnaka Anil Kumar , Korpole Suresh
Published: August 2015

A Gram-stain-positive, non-motile and aerobic bacterium, designated strain DW151BT, was isolated from a sludge sample of a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis of strain DW151BT placed it within the genus Rhodococcus. It displayed significant similarity with recognized species of the genus: Rhodococcus pyridinivorans PDB9T (98.8 %), Rhodococcus gordoniae W 4937T (98.6 %), Rhodococcus rhodochrous DSM 43241T (98.5 %) and Rhodococcus artemisiae YIM 65754T (97.5 %). However, strain DW151BT differed from phylogenetically closely related species in various phenotypic properties. The cellular polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as major lipids, MK-8(H2) was the major menaquinone and meso-diaminopimelic acid was the cell-wall peptidoglycan. The fatty acid profile consisted of C16 : 0, C18 : 1
cis9 and C16 : 1
cis9 as main components. The presence of C16 : 0 and diphosphatidylglycerol as major fatty acid and polar lipid, respectively, was in accordance with chemotaxonomic markers of the genus Rhodococcus. The DNA G+C content of strain DW151BT was 69.9 mol%, a value within the limits reported for the members of this genus. Furthermore, strain DW151BT showed low similarity at the whole genome level in DNA–DNA hybridization experiments with phylogenetically closely related strains. Considering the low similarity at the genome level and differences in phenotypic properties, strain DW151BT is considered to represent a novel species of the genus Rhodococcus, for which the name Rhodococcus lactis sp. nov. is proposed. The type strain is DW151BT ( = MTCC 12279T = DSM 45625T).
OEFinder: A user interface to identify and visualize ordering effects in single-cell RNA-seq data
Authors: Leng Ning , Choi Jeea , Chu Li-Fang , Thomson James A. , Kendziorski Christina , Stewart Ron
Published: August 2015

A recent paper identified an artifact in multiple single-cell RNA-seq (scRNA-seq) data sets generated by the Fluidigm C1 platform. Specifically, Leng*et al. showed significantly increased gene expression in cells captured from sites with small or large plate output IDs. We refer to this artifact as an ordering effect (OE). Including OE genes in downstream analyses could lead to biased results. To address this problem, we developed a statistical method and software called OEFinder to identify a sorted list of OE genes. OEFinder is available as an R package along with user-friendly graphical interface implementations that allows users to check for potential artifacts in scRNA-seq data generated by the Fluidigm C1 platform.

Availability and Implementation

OEFinder is freely available athttps://github.com/lengning/OEFinder

Contact

rstewart@morgridge.org
Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting
Authors: Mavor David , Barlow Kyle A. , Thompson Samuel , Barad Benjamin A. , Bonny Alain R. , Cario Clinton L. , Gaskins Garrett , Liu Zairan , Deming Laura , Axen Seth D. , Caceres Elena , Chen Weilin , Cuesta Adolfo , Gate Rachel , Green Evan M. , Hulce Kaitlin R. , Ji Weiyue , Kenner Lillian R. , Mensa Bruk , Morinishi Leanna S. , Moss Steven M. , Mravic Marco , Muir Ryan K. , Niekamp Stefan , Nnadi Chimno I. , Palovcak Eugene , Poss Erin M. , Ross Tyler D. , Salcedo Eugenia , See Stephanie , Subramaniam Meena , Wong Allison W. , Li Jennifer , Thorn Kurt S. , Conchúir Shane Ó. , Roscoe Benjamin P. , Chow Eric D. , DeRisi Joseph L. , Kortemme Tanja , Bolon Daniel N. , Fraser James S.
Published: August 2015

Ubiquitination is an essential post-translational regulatory process that can control protein stability, localization, and activity. Ubiquitin is essential for eukaryotic life and is highly conserved, varying in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies inS. cerevisiaeindicate that ubiquitin is highly tolerant to single amino acid mutations. To resolve this paradox, we hypothesized that the set of tolerated substitutions would be reduced when the cultures are not grown in rich media conditions and that chemically induced physiologic perturbations might unmask constraints on the ubiquitin sequence. To test this hypothesis, a class of first year UCSF graduate students employed a deep mutational scanning procedure to determine the fitness landscape of a library of all possible single amino acid mutations of ubiquitin in the presence of one of five small molecule perturbations: MG132, Dithiothreitol (DTT), Hydroxyurea (HU), Caffeine, and DMSO. Our data reveal that the number of tolerated substitutions is greatly reduced by DTT, HU, or Caffeine, and that these perturbations uncover “shared sensitized positions” localized to areas around the hydrophobic patch and to the C-terminus. We also show perturbation specific effects including the sensitization of His68 in HU and tolerance to mutation at Lys63 in DTT. Taken together, our data suggest that chemical stress reduces buffering effects in the ubiquitin proteasome system, revealing previously hidden fitness defects. By expanding the set of chemical perturbations assayed, potentially by other classroom-based experiences, we will be able to further address the apparent dichotomy between the extreme sequence conservation and the experimentally observed mutational tolerance of ubiquitin. Finally, this study demonstrates the realized potential of a project lab-based interdisciplinary graduate curriculum.
Children, old-age support and pension in rural China
Authors: Zhang Chuanchuan
Published: August 2015

Purpose

– The purpose of this paper is to examine the substitute between social old-age insurance and adult children in providing old-age support, and estimate the effects of China’s old rural pension program (ORPP) on sex ratio.

Design/methodology/approach

– Using data from China’s 2005 inter-census population survey and China Health and Retirement Longitudinal Study, this paper estimates the effects of children on rural parents’ take-up of pension program and the effects of having access to pension on old parents’ choice of resource for future old-age support. The effects of China’s ORPP on sex ratio are estimated using difference-in-differences identification strategy.

Findings

– Peasants having sons are less likely to participate in pension program and each additional son and daughter significantly decreases their likelihood of participation. Moreover, the effect of sons is much larger than that of daughters. Peasants having access to pension are less likely to rely on their children for old-age support. The implementation of the ORPP moderately decreased sex ratio.

Social implications

– Implementing pension program in China’s rural area will probably affect rural people’s fertility behavior and thus be helpful in correcting sex ratio bias.

Originality/value

– This paper first estimates the effects of having access to pension on old parents’ choices of providers of old-age support, and estimates the causal effect of rural pension on sex ratio using standard DID strategy.
Irrigation investment in China: trends, correlates and impacts
Authors: He Fei , Shi Yaojiang , Luo Renfu , Zhang Linxiu , Johnson Natalie , Rozelle Scott
Published: August 2015

Purpose

– The purpose of this paper is to describe trends in irrigation investment in China’s rural villages in the 2000s, identify the types of villages in which investment occurred and examine whether this investment had an impact on agricultural land.

Design/methodology/approach

– This study makes use of longitudinal survey data from a nearly nationally representative sample of 101 villages spread across five provinces. The outcome variables are cultivated area, sown area and effectively irrigated area, and ordinary least squares regression and fixed effects models are used for the analysis.

Findings

– In spite of sustained investments into irrigation from 1998 to 2011, there has been almost no impact on agricultural land. Cultivated area and sown area have fallen across all five sample provinces while effectively irrigated area remains largely unchanged. The authors also show that there is no relationship between investment into irrigation and agricultural land. Irrigation facilities also have the lowest rate of rural resident satisfaction of any of the major public services provided.

Research limitations/implications

– More research is needed to understand the impact of irrigation investment on crop yields and water savings.

Practical implications

– Policymakers may need to rethink the current pattern of investment into irrigation.

Originality/value

– This is the first study to quantitatively model the impact of investment into irrigation at the village level all over China.