bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered development in GABA co‐release shapes glycinergic synaptic currents in cultured spinal slices of the SOD1G93A mouse model of amyotrophic lateral sclerosis
Authors: Medelin Manuela , Rancic Vladimir , Cellot Giada , Laishram Jummi , Veeraraghavan Priyadharishini , Rossi Chiara , Muzio Luca , Sivilotti Lucia , Ballerini Laura 
Published: April 2016

Key points
Increased environmental risk factors in conjunction with genetic susceptibility have been proposed with respect to the remarkable variations in mortality in amyotrophic lateral sclerosis (ALS).
In vitro models allow the investigation of the genetically modified counter‐regulator of motoneuron toxicity and may help in addressing ALS therapy.
Spinal organotypic slice cultures from a mutant form of human superoxide dismutase 1 (SOD1G93A) mouse model of ALS allow the detection of altered glycinergic inhibition in spinal microcircuits.
This altered inhibition improved spinal cord excitability, affecting motor outputs in early SOD1G93A pathogenesis.
AbstractAmyotrophic lateral sclerosis (ALS) is a fatal, adult‐onset neurological disease characterized by a progressive degeneration of motoneurons (MNs). In a previous study, we developed organotypic spinal cultures from an ALS mouse model expressing a mutant form of human superoxide dismutase 1 (SOD1G93A). We reported the presence of a significant synaptic rearrangement expressed by these embryonic cultured networks, which may lead to the altered development of spinal synaptic signalling, which is potentially linked to the adult disease phenotype. Recent studies on the same ALS mouse model reported a selective loss of glycinergic innervation in cultured MNs, suggestive of a contribution of synaptic inhibition to MN dysfunction and degeneration. In the present study, we further exploit organotypic cultures from wild‐type and SOD1G93A mice to investigate the development of glycine‐receptor‐mediated synaptic currents recorded from the interneurons of the premotor ventral circuits. We performed single cell electrophysiology, immunocytochemistry and confocal microscopy and suggest that GABA co‐release may speed the decay of glycine responses altering both temporal precision and signal integration in SOD1G93A developing networks at the postsynaptic site. Our hypothesis is supported by the finding of an increased MN bursting activity in immature SOD1G93A spinal cords and by immunofluorescence microscopy detection of a longer persistence of GABA in SOD1G93A glycinergic terminals in cultured and ex vivo spinal slices.
Clinical Impact of Laboratory Implementation of Verigene BC-GN Microarray-Based Assay for Detection of Gram-Negative Bacteria in Positive Blood Cultures
Authors: Walker Tamar , Dumadag Sandrea , Lee Christine Jiyoun , Lee Seung Heon , Bender Jeffrey M. , Cupo Abbott Jennifer , She Rosemary C. 
Published: April 2016

ABSTRACT

Gram-negative bacteremia is highly fatal, and hospitalizations due to sepsis have been increasing worldwide. Molecular tests that supplement Gram stain results from positive blood cultures provide specific organism information to potentially guide therapy, but more clinical data on their real-world impact are still needed. We retrospectively reviewed cases of Gram-negative bacteremia in hospitalized patients over a 6-month period before (
n =
98) and over a 6-month period after (
n =
97) the implementation of a microarray-based early identification and resistance marker detection system (Verigene BC-GN; Nanosphere) while antimicrobial stewardship practices remained constant. Patient demographics, time to organism identification, time to effective antimicrobial therapy, and other key clinical parameters were compared. The two groups did not differ statistically with regard to comorbid conditions, sources of bacteremia, or numbers of intensive care unit (ICU) admissions, active use of immunosuppressive therapy, neutropenia, or bacteremia due to multidrug-resistant organisms. The BC-GN panel yielded an identification in 87% of Gram-negative cultures and was accurate in 95/97 (98%) of the cases compared to results using conventional culture. Organism identifications were achieved more quickly post-microarray implementation (mean, 10.9 h versus 37.9 h;
P
< 0.001). Length of ICU stay, 30-day mortality, and mortality associated with multidrug-resistant organisms were significantly lower in the postintervention group (
P
< 0.05). More rapid implementation of effective therapy was statistically significant for postintervention cases of extended-spectrum beta-lactamase-producing organisms (
P
= 0.049) but not overall (
P
= 0.12). The Verigene BC-GN assay is a valuable addition for the early identification of Gram-negative organisms that cause bloodstream infections and can significantly impact patient care, particularly when resistance markers are detected.
Evaluation of Various Culture Media for Detection of Rapidly Growing Mycobacteria from Patients with Cystic Fibrosis
Authors: Preece Clair L. , Wichelhaus Thomas A. , Perry Audrey , Jones Amanda L. , Cummings Stephen P. , Perry John D. , Hogardt Michael 
Published: April 2016

ABSTRACTIsolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly growing species that colonize the lungs of patients with CF. Extended incubation onBurkholderia cepaciaselective agar (BCSA) has been recommended as an expedient culture method for the isolation of rapidly growing NTM in this setting. The aim of this study was to assess five selective media designed for the isolation ofBurkholderia cepaciacomplex, along with two media designed for the isolation of mycobacteria (rapidly growing mycobacteria [RGM] medium and Middlebrook 7H11 agar), for their abilities to isolate NTM. All seven media were challenged with 147 isolates of rapidly growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for the isolation ofB. cepaciacomplex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium, compared with only 7 samples using the most selective brand of BCSA (P= 0.023). RGM medium offers a superior option, compared to other selective agars, for the isolation of rapidly growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly growing NTM in all submitted sputum samples from patients with CF.
RNA-Free and Ribonucleoprotein-Associated Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal Domain of Host RNA Polymerase II
Authors: Martínez-Alonso Mónica , Hengrung Narin , Fodor Ervin 
Published: April 2016

ABSTRACT
Influenza viruses subvert the transcriptional machinery of their hosts to synthesize their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap snatching, the virus steals short 5′ capped RNA fragments from host capped RNAs and uses them to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that the influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5-phosphorylated form of the Pol II CTD, both in the presence and in the absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation.

IMPORTANCE
Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health, as well as represent a large economic burden to health care systems globally. Existing vaccines are not always effective, as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed, it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for the development of antivirals.
HIV-1 Protease, Reverse Transcriptase, and Integrase Variation
Authors: Rhee Soo-Yon , Sankaran Kris , Varghese Vici , Winters Mark A. , Hurt Christopher B. , Eron Joseph J. , Parkin Neil , Holmes Susan P. , Holodniy Mark , Shafer Robert W. 
Published: April 2016

ABSTRACTHIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS.IMPORTANCEMost antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.
Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells
Authors: Zhou Yunying , Zhang Qishu , Gao Ge , Zhang Xiaoli , Liu Yafei , Yuan Shoudao , Wang Xiaowei , Chen Jason J. 
Published: April 2016

ABSTRACT

The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G
1
cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our studies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G
1
checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregulation of WDHD1 reduced E7-induced G
1
checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications.

IMPORTANCE
The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated in E7-expressing cells, was found to play an important role in E7-induced G
1
checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications.
Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36
Authors: Avey Denis , Tepper Sarah , Pifer Benjamin , Bahga Amritpal , Williams Hunter , Gillen Joseph , Li Wenwei , Ogden Sarah , Zhu Fanxiu 
Published: April 2016

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions.

IMPORTANCE
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis.
Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases
Authors: Lutz Lindy M. , Pace Chandler R. , Arnold Michelle M. 
Published: April 2016

ABSTRACT

The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1.
In vitro
binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity.

IMPORTANCE
The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.
Impact of Ammonium on Syntrophic Organohalide-Respiring and Fermenting Microbial Communities
Authors: Delgado Anca G. , Fajardo-Williams Devyn , Kegerreis Kylie L. , Parameswaran Prathap , Krajmalnik-Brown Rosa 
Published: April 2016

Contamination with ammonium and chlorinated solvents has been reported in numerous subsurface environments, and these chemicals bring significant challenges for
in situ
bioremediation.
Dehalococcoides mccartyi
is able to reduce the chlorinated solvent trichloroethene to the nontoxic end product ethene. Fermentative bacteria are of central importance for organohalide respiration and bioremediation to provide
D. mccartyi
with H
2
, their electron donor, acetate, their carbon source, and other micronutrients. In this study, we found that high concentrations of ammonium negatively correlated with rates of trichloroethene reductive dehalogenation and fermentation. However, detoxification of trichloroethene to nontoxic ethene occurred even at ammonium concentrations typical of those found in animal waste (up to 2 g liter
−1
NH
4
+
-N). To date, hundreds of subsurface environments have been bioremediated through the unique metabolic capability of
D. mccartyi
. These findings extend our knowledge of
D. mccartyi
and provide insight for bioremediation of sites contaminated with chlorinated solvents and ammonium.