bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stable transformation and reverse genetic analysis of Penium margaritaceum: a platform for studies of charophyte green algae, the immediate ancestors of land plants
Authors: Sørensen Iben , Fei Zhangjun , Andreas Amanda , Willats William G. T. , Domozych David S. , Rose Jocelyn K. C. 
Published: November 2013

SummaryThe charophyte green algae (CGA, Streptophyta, Viridiplantae) occupy a key phylogenetic position as the immediate ancestors of land plants but, paradoxically, are less well‐studied than the other major plant lineages. This is particularly true in the context of functional genomic studies, where the lack of an efficient protocol for their stable genetic transformation has been a major obstacle. Observations of extant CGA species suggest the existence of some of the evolutionary adaptations that had to occur for land colonization; however, to date, there has been no robust experimental platform to address this genetically. We present a protocol for high‐throughput Agrobacterium tumefaciens‐mediated transformation of Penium margaritaceum, a unicellular CGA species. The versatility of Penium as a model for studying various aspects of plant cell biology and development was illustrated through non‐invasive visualization of protein localization and dynamics in living cells. In addition, the utility of RNA interference (RNAi) for reverse genetic studies was demonstrated by targeting genes associated with cell wall modification (pectin methylesterase) and biosynthesis (cellulose synthase). This provided evidence supporting current models of cell wall assembly and inter‐polymer interactions that were based on studies of land plants, but in this case using direct observation in vivo. This new functional genomics platform has broad potential applications, including studies of plant organismal biology and the evolutionary innovations required for transition from aquatic to terrestrial habitats.
The potential for manipulating RNA with pentatricopeptide repeat proteins
Authors: Yagi Yusuke , Nakamura Takahiro , Small Ian 
Published: November 2013

SummaryThe pentatricopeptide repeat (PPR) protein family, which is particularly prevalent in plants, includes many sequence‐specific RNA‐binding proteins involved in all aspects of organelle RNA metabolism, including RNA stability, processing, editing and translation. PPR proteins consist of a tandem array of 2‐30 PPR motifs, each of which aligns to one nucleotide in the RNA target. The amino acid side chains at two or three specific positions in each motif confer nucleotide specificity in a predictable and programmable manner. Thus, PPR proteins appear to provide an extremely promising opportunity to create custom RNA‐binding proteins with tailored specificity. We summarize recent progress in understanding RNA recognition by PPR proteins, with a particular focus on potential applications of PPR‐based tools for manipulating RNA, and on the challenges that remain to be overcome before these tools may be routinely used by the scientific community.
Dynamic rotating‐shield brachytherapy
Authors: Liu Yunlong , Flynn Ryan T. , Kim Yusung , Yang Wenjun , Wu Xiaodong 
Published: November 2013

Purpose:To present dynamic rotating shield brachytherapy (D‐RSBT), a novel form of high‐dose‐rate brachytherapy (HDR‐BT) with electronic brachytherapy source, where the radiation shield is capable of changing emission angles during the radiation delivery process.Methods:A D‐RSBT system uses two layers of independently rotating tungsten alloy shields, each with a 180° azimuthal emission angle. The D‐RSBT planning is separated into two stages: anchor plan optimization and optimal sequencing. In the anchor plan optimization, anchor plans are generated by maximizing theD90 for the high‐risk clinical‐tumor‐volume (HR‐CTV) assuming a fixed azimuthal emission angle of 11.25°. In the optimal sequencing, treatment plans that most closely approximate the anchor plans under the delivery‐time constraint will be efficiently computed. Treatment plans for five cervical cancer patients were generated for D‐RSBT, single‐shield RSBT (S‐RSBT), and 192Ir‐based intracavitary brachytherapy with supplementary interstitial brachytherapy (IS + ICBT) assuming five treatment fractions. External beam radiotherapy doses of 45 Gy in 25 fractions of 1.8 Gy each were accounted for. The high‐risk clinical target volume (HR‐CTV) doses were escalated such that the D2cc of the rectum, sigmoid colon, or bladder reached its tolerance equivalent dose in 2 Gy fractions (EQD2 with α/β = 3 Gy) of 75 Gy, 75 Gy, or 90 Gy, respectively.Results:For the patients considered, IS + ICBT had an average total dwell time of 5.7 minutes/fraction (min/fx) assuming a 10 Ci192 Ir source, and the average HR‐CTV D90 was 78.9 Gy. In order to match the HR‐CTV D90 of IS + ICBT, D‐RSBT required an average of 10.1 min/fx more delivery time, and S‐RSBT required 6.7 min/fx more. If an additional 20 min/fx of delivery time is allowed beyond that of the IS + ICBT case, D‐RSBT and S‐RSBT increased the HR‐CTV D90 above IS + ICBT by an average of 16.3 Gy and 9.1 Gy, respectively.Conclusions:For cervical cancer patients, D‐RSBT can boost HR‐CTVD90 over IS + ICBT and S‐RSBT without violating the tolerance doses to the bladder, rectum, or sigmoid. The D90 improvements from D‐RSBT depend on the patient, the delivery time budget, and the applicator structure.
Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in Clinical Isolates by Multiplex PCR
Authors: Nakanaga Kazue , Sekizuka Tsuyoshi , Fukano Hanako , Sakakibara Yumi , Takeuchi Fumihiko , Wada Shinpei , Ishii Norihisa , Makino Masahiko , Kuroda Makoto , Hoshino Yoshihiko 
Published: November 2013

ABSTRACT

The rapidly growing mycobacterium
M. abscessus

sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures.
M. abscessus
sensu lato

can be divided into at least three subspecies:
M. abscessus
subsp.
abscessus
,
M. abscessus
subsp.
massiliense
, and
M. abscessus
subsp.
bolletii
. Clinical isolates of rapidly growing mycobacteria were previously identified as
M. abscessus
by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as
M. abscessus
subsp.
massiliense
using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish
M. abscessus
subsp.
massiliense
from
M. abscessus
subsp.
abscessus
, a multiplex PCR assay, was developed using the whole-genome sequence of
M. abscessus
subsp.
massiliense
JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of
M. abscessus
subsp.
massiliense
from samples previously identified as
M. abscessus
. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays.
Clinical Comparison of Simplexa Universal Direct and BD GeneOhm Tests for Detection of Toxigenic Clostridium difficile in Stool Samples
Authors: Nolte Frederick S. , Ribeiro-Nesbitt Danielle G. 
Published: November 2013

ABSTRACT

We compared the performance characteristics of the Simplexa universal direct (Focus Diagnostics, Cypress, CA) and BD GeneOhm (BD Diagnostics/GeneOhm Sciences, San Diego, CA) tests for detection of toxigenic
Clostridium difficile
in 459 stool samples (9.4% positive). The observed agreement for the results of the two tests with 452 samples with valid test results was 98.2% (kappa, 0.9;
P
value of 0.73 by the McNemar test). When samples with discordant or invalid results were retested, the agreement increased to 100%.
Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States
Authors: Chen Qi , Li Ganwu , Stasko Judith , Thomas Joseph T. , Stensland Wendy R. , Pillatzki Angela E. , Gauger Phillip C. , Schwartz Kent J. , Madson Darin , Yoon Kyoung-Jin , Stevenson Gregory W. , Burrough Eric R. , Harmon Karen M. , Main Rodger G. , Zhang Jianqiang 
Published: November 2013

ABSTRACTPorcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 102to 2 × 10550% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.
Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes
Authors: Salganik Maxim , Aydemir Fikret , Nam Hyun-Joo , McKenna Robert , Agbandje-McKenna Mavis , Muzyczka Nicholas 
Published: November 2013

ABSTRACT

A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>10
7
-fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3- to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome.
A Targeted Mutation within the Feline Leukemia Virus (FeLV) Envelope Protein Immunosuppressive Domain To Improve a Canarypox Virus-Vectored FeLV Vaccine
Authors: Schlecht-Louf Géraldine , Mangeney Marianne , El-Garch Hanane , Lacombe Valérie , Poulet Hervé , Heidmann Thierry 
Published: November 2013

ABSTRACTWe previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagationin vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the “mechanical” function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressivein vivoand that this immunosuppressive activity can be “switched off” by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressivein vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.
Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components
Authors: Zivanovic Yvan , Confalonieri Fabrice , Ponchon Luc , Lurz Rudi , Chami Mohamed , Flayhan Ali , Renouard Madalena , Huet Alexis , Decottignies Paulette , Davidson Alan R. , Breyton Cécile , Boulanger Pascale 
Published: November 2013

ABSTRACT

Bacteriophage T5 represents a large family of lytic
Siphoviridae
infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.
Viral MicroRNAs Targeting Virus Genes Promote Virus Infection in Shrimp
In Vivo
Authors: He Yaodong , Yang Kai , Zhang Xiaobo 
Published: November 2013

ABSTRACT

Viral microRNAs (miRNAs), most of which are characterized in cell lines, have been found to play important roles in the virus life cycle to avoid attack by the host immune system or to keep virus in the latency state. Viral miRNAs targeting virus genes can inhibit virus infection. In this study,
in vivo
findings in
Marsupenaeus japonicus
shrimp revealed that the viral miRNAs could target virus genes and further promote the virus infection. The results showed that white spot syndrome virus (WSSV)-encoded miRNAs WSSV-miR-66 and WSSV-miR-68 were transcribed at the early stage of WSSV infection. When the expression of WSSV-miR-66 and WSSV-miR-68 was silenced with sequence-specific anti-miRNA oligonucleotides (AMOs), the number of copies of WSSV and the WSSV-infected shrimp mortality were significantly decreased, indicating that the two viral miRNAs had a great effect on virus infection. It was revealed that the WSSV
wsv094
and
wsv177
genes were the targets of WSSV-miR-66 and that the
wsv248
and
wsv309
genes were the targets of WSSV-miR-68. The data demonstrate that the four target genes play negative roles in the WSSV infection. The targeting of the four virus genes by WSSV-miR-66 and WSSV-miR-68 led to the promotion of virus infection. Therefore, our
in vivo
findings show a novel aspect of viral miRNAs in virus-host interactions.
Exceptionally Potent and Broadly Cross-Reactive, Bispecific Multivalent HIV-1 Inhibitors Based on Single Human CD4 and Antibody Domains
Authors: Chen Weizao , Feng Yang , Prabakaran Ponraj , Ying Tianlei , Wang Yanping , Sun Jianping , Macedo Camila D. S. , Zhu Zhongyu , He Yuxian , Polonis Victoria R. , Dimitrov Dimiter S. 
Published: November 2013

ABSTRACT
Soluble forms of the human immunodeficiency virus type 1 (HIV-1) primary receptor CD4 (soluble CD4 [sCD4]) have been extensively characterized for a quarter of a century as promising HIV-1 inhibitors, but they have not been clinically successful. By combining a protein cavity-filling strategy and the power of library technology, we identified an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties, including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor, an antibody domain (m36.4) that targets the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested, with potencies about 10-, 50-, and 200-fold higher than those of the broadly neutralizing antibody VRC01, the U.S. FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity and a lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be useful for HIV-1 prevention and therapy, including eradication of the virus.
Development of Virus-Specific CD4
+
and CD8
+
Regulatory T Cells Induced by Human Herpesvirus 6 Infection
Authors: Wang Fang , Chi Jing , Peng Guangyong , Zhou Feng , Wang Jinfeng , Li Lingyun , Feng Dongju , Xie Fangyi , Gu Bin , Qin Jian , Chen Yun , Yao Kun 
Published: November 2013

ABSTRACT

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus. The mechanisms by which HHV-6 establishes latency and immunosuppression in its host are not well understood. Here we characterized HHV-6-specific T cells in peripheral blood mononuclear cells (PBMCs) from HHV-6-infected donors. Our results showed that HHV-6 infection could induce both CD4
+
and CD8
+
HHV-6-specific regulatory T (Treg) cells. These HHV-6-specific Treg cells had potent suppressive activity and expressed high levels of Treg-associated molecules CD25, FoxP3, and GITR. Both CD4
+
and CD8
+
Treg cells secreted gamma interferon (IFN-γ) and interleukin-10 (IL-10) but little or no IL-2, IL-4, or transforming growth factor β (TGF-β). Furthermore, HHV-6-specifc Treg cells not only could suppress naive and HHV-6-specific CD4
+
effector T cell immune responses but also could impair dendritic cell (DC) maturation and functions. In addition, the suppressive effects mediated by HHV-6-specific Treg cells were mainly through a cell-to-cell contact-dependent mechanism but not through the identified cytokines. These results suggest that HHV-6 may utilize the induction of Treg cells as a strategy to escape antivirus immune responses and maintain the latency and immunosuppression in infected hosts.
Autophagy and the Effects of Its Inhibition on Varicella-Zoster Virus Glycoprotein Biosynthesis and Infectivity
Authors: Buckingham Erin M. , Carpenter John E. , Jackson Wallen , Grose Charles 
Published: November 2013

ABSTRACT

Autophagy and the effects of its inhibition or induction were investigated during the entire infectious cycle of varicella-zoster virus (VZV), a human herpesvirus. As a baseline, we first enumerated the number of autophagosomes per cell after VZV infection compared with the number after induction of autophagy following serum starvation or treatment with tunicamycin or trehalose. Punctum induction by VZV was similar in degree to punctum induction by trehalose in uninfected cells. Treatment of infected cells with the autophagy inhibitor 3-methyladenine (3-MA) markedly reduced the viral titer, as determined by assays measuring both cell-free virus and infectious foci (
P
< 0.0001). We next examined a virion-enriched band purified by density gradient sedimentation and observed that treatment with 3-MA decreased the amount of VZV gE, while treatment with trehalose increased the amount of gE in the same band. Because VZV gE is the most abundant glycoprotein, we selected gE as a representative viral glycoprotein. To further investigate the role of autophagy in VZV glycoprotein biosynthesis as well as confirm the results obtained with 3-MA inhibition, we transfected cells with ATG5 small interfering RNA to block autophagosome formation. VZV-induced syncytium formation was markedly reduced by ATG5 knockdown (
P
< 0.0001). Further, we found that both expression and glycan processing of VZV gE were decreased after ATG5 knockdown, while expression of the nonglycosylated IE62 tegument protein was unchanged. Taken together, our cumulative results not only documented abundant autophagy within VZV-infected cells throughout the infectious cycle but also demonstrated that VZV-induced autophagy facilitated VZV glycoprotein biosynthesis and processing.