bluuebunny/crossref_metadata_2025 huggingface dataset:
The Interface of Mechanics and Nociception in Joint Pathophysiology: Insights From the Facet and Temporomandibular Joints
Authors: Sperry Megan M. , Ita Meagan E. , Kartha Sonia , Zhang Sijia , Yu Ya-Hsin , Winkelstein Beth
Published: January 2017

Chronic joint pain is a widespread problem that frequently occurs with aging and trauma. Pain occurs most often in synovial joints, the body's load bearing joints. The mechanical and molecular mechanisms contributing to synovial joint pain are reviewed using two examples, the cervical spinal facet joints and the temporomandibular joint (TMJ). Although much work has focused on the macroscale mechanics of joints in health and disease, the combined influence of tissue mechanics, molecular processes, and nociception in joint pain has only recently become a focus. Trauma and repeated loading can induce structural and biochemical changes in joints, altering their microenvironment and modifying the biomechanics of their constitutive tissues, which themselves are innervated. Peripheral pain sensors can become activated in response to changes in the joint microenvironment and relay pain signals to the spinal cord and brain where pain is processed and perceived. In some cases, pain circuitry is permanently changed, which may be a potential mechanism for sustained joint pain. However, it is most likely that alterations in both the joint microenvironment and the central nervous system (CNS) contribute to chronic pain. As such, the challenge of treating joint pain and degeneration is temporally and spatially complicated. This review summarizes anatomy, physiology, and pathophysiology of these joints and the sensory pain relays. Pain pathways are postulated to be sensitized by many factors, including degeneration and biochemical priming, with effects on thresholds for mechanical injury and/or dysfunction. Initiators of joint pain are discussed in the context of clinical challenges including the diagnosis and treatment of pain.
Applications of clustering model to bimodal distributions for dielectric breakdown
Authors: Wu Ernest Y. , Bolam Ronald , Filippi Ronald , Stathis James H. , Li Baozhen , Kim Andrew
Published: January 2017

In this work, the authors report the extensive time-to-breakdown (TBD) data collected from back end of line/middle of the line and metal insulator metal capacitor dielectrics that exhibit not only non-Poisson area scaling but also multiple modal (or bimodal) breakdown characteristics. They develop a new bimodal modeling approach of the distributed competition process in conjunction with the time-dependent clustering model to extract the breakdown parameters and the characteristic breakdown time and slope (t63 and β) from this seemingly intractable breakdown data. While the number of parameters increases as a result of an increase in data complexity, these extracted parameters are consistent with Poisson area-scaling of fundamental weakest-link characteristics, and can thus be used for reliability projection. As a result, our modeling approach involving clustering model provides an important solution for technology qualification and dielectric-integrity assessment.
Resource tracking within and across continents in long-distance bird migrants
Authors: Thorup Kasper , Tøttrup Anders P. , Willemoes Mikkel , Klaassen Raymond H. G. , Strandberg Roine , Vega Marta Lomas , Dasari Hari P. , Araújo Miguel B. , Wikelski Martin , Rahbek Carsten
Published: January 2017

Migrant birds track seasonally available resources on a continental scale across the annual cycle.
The burning question
Authors: Cornwall Warren
Published: January 2017

Moves to designate wood as a carbon-neutral fuel have alarmed environmentalists and divided scientists.
Microstructural proliferation in human cortex is coupled with the development of face processing
Authors: Gomez Jesse , Barnett Michael A. , Natu Vaidehi , Mezer Aviv , Palomero-Gallagher Nicola , Weiner Kevin S. , Amunts Katrin , Zilles Karl , Grill-Spector Kalanit
Published: January 2017

Brain structure and function mature together

Our ability to recognize faces improves from infancy to adulthood. This improvement depends on specific face-selective regions in the visual system. Gomez
et al.
tested face memory and place recognition in children and adults while scanning relevant brain regions. Anatomical changes co-occurred with functional changes in the brain. Some regions in the high-level visual cortex showed profound developmental maturation, whereas others were stable. Thus, improvements in face recognition involve an interplay between structural and functional changes in the brain.

Science
, this issue p.
68
Permanent human occupation of the central Tibetan Plateau in the early Holocene
Authors: Meyer M. C. , Aldenderfer M. S. , Wang Z. , Hoffmann D. L. , Dahl J. A. , Degering D. , Haas W. R. , Schlütz F.
Published: January 2017

The peopling of Tibet

The date of the first permanent human occupation of the high Tibetan Plateau has been estimated at about 3600 years ago, when agriculture became established. Meyer
et al.
used several dating techniques to analyze sediments at a high-altitude site (4270 m) where human handprints and footprints have been found. Their analysis indicates occupation of the plateau 7400 years ago and possibly earlier. These dates are consistent with the genetic history of Tibetans and suggest that a permanent preagricultural peopling of the plateau was enabled by the wetter regional climate at that time.

Science
, this issue p.
64
Robotically controlled microprey to resolve initial attack modes preceding phagocytosis
Authors: Schuerle Simone , Vizcarra Ima Avalos , Moeller Jens , Sakar Mahmut Selman , Özkale Berna , Lindo André Machado , Mushtaq Fajer , Schoen Ingmar , Pané Salvador , Vogel Viola , Nelson Bradley J.
Published: January 2017

The behavior of phagocytes to capture intruders is tracked using remotely rotated and translated nanoparticles.
The Oral β-Lactamase SYN-004 (Ribaxamase) Degrades Ceftriaxone Excreted into the Intestine in Phase 2a Clinical Studies
Authors: Kokai-Kun John F. , Roberts Tracey , Coughlin Olivia , Sicard Eric , Rufiange Marianne , Fedorak Richard , Carter Christian , Adams Marijke H. , Longstreth James , Whalen Heidi , Sliman Joseph
Published: January 2017

SYN-004 (ribaxamase) is a β-lactamase designed to be orally administered concurrently with intravenous β-lactam antibiotics, including most penicillins and cephalosporins. Ribaxamase's anticipated mechanism of action is to degrade excess β-lactam antibiotic that is excreted into the small intestine. This enzymatic inactivation of excreted antibiotic is expected to protect the gut microbiome from disruption and thus prevent undesirable side effects, including secondary infections such as
Clostridium difficile
infections, as well as other antibiotic-associated diarrheas. In phase 1 clinical studies, ribaxamase was well tolerated compared to a placebo group and displayed negligible systemic absorption. The two phase 2a clinical studies described here were performed to confirm the mechanism of action of ribaxamase, degradation of β-lactam antibiotics in the human intestine, and were therefore conducted in subjects with functioning ileostomies to allow serial sampling of their intestinal chyme. Ribaxamase fully degraded ceftriaxone to below the level of quantitation in the intestines of all subjects in both studies. Coadministration of oral ribaxamase with intravenous ceftriaxone was also well tolerated, and the plasma pharmacokinetics of ceftriaxone were unchanged by ribaxamase administration. Since ribaxamase is formulated as a pH-dependent, delayed-release formulation, the activity of ribaxamase in the presence of the proton pump inhibitor esomeprazole was examined in the second study; coadministration of these drugs did not adversely affect ribaxamase's ability to degrade ceftriaxone excreted into the intestine. These studies have confirmed the
in vivo
mechanism of action of ribaxamase, degradation of β-lactam antibiotics in the human intestine (registered at ClinicalTrials.gov under NCT02419001 and NCT02473640).
Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence
Authors: Rogers E J , Kim U J , Ambulos N P , Lovett P S
Published: January 2017

Genes encoding chloramphenicol acetyltransferase in gram-positive bacteria are induced by chloramphenicol. Induction reflects an ability of the drug to stall a ribosome at a specific site in cat leader mRNA. Ribosome stalling at this site alters downstream RNA secondary structure, thereby unmasking the ribosome-binding site for the cat coding sequence. Here, we show that ribosome stalling in the cat-86 leader is a function of leader codons 2 through 5 and that stalling requires these codons to be presented in the correct reading frame. Codons 2 through 5 specify Val-Lys-Thr-Asp. Insertion of a second copy of the stall sequence 5' to the authentic stall sequence diminished cat-86 induction fivefold. Thus, the stall sequence can function in ribosome stalling when the stall sequence is displaced from the downstream RNA secondary structure. We suggest that the stall sequence may function in cat induction at two levels. First, the tetrapeptide specified by the stall sequence likely plays an active role in the induction strategy, on the basis of previously reported genetic suppression studies (W. W. Mulbry, N. P. Ambulos, Jr., and P.S. Lovett, J. Bacteriol. 171:5322-5324, 1989). Second, we show that embedded within the stall sequence of cat leaders is a region which is complementary to a sequence internal in 16S rRNA of Bacillus subtilis. This complementarity may guide a ribosome to the proper position on leader mRNA or potentiate the stalling event, or both. The region of complementarity is absent from Escherichia coli 16S rRNA, and cat genes induce poorly, or not at all, in E. coli.
Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies
Authors: Bayer M H , Keck W , Bayer M E
Published: January 2017

We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.
Nucleotide sequence and transcriptional products of the csg locus of Myxococcus xanthus
Authors: Hagen T J , Shimkets L J
Published: January 2017

The csg locus of Myxococcus xanthus appears to control the production of an intercellular signal that is essential for development. The complete nucleotide sequence of a clone containing the csg locus was determined by the dideoxy-chain termination method. Pattern recognition analyses of the DNA sequence revealed the presence of two protein-coding regions that are convergently oriented and separated by only 8 nucleotides. Tn5 lac insertions into this clone detected two transcriptional units that are transcribed in a convergent fashion and whose expression increases during development. The two genes represented by these protein-coding regions and transcriptional units have been designated csgA and fprA. Northern (RNA) blot analyses detected an 800-nucleotide RNA specific to the csgA gene and a 900-nucleotide RNA specific to the fprA gene. Our results, along with mutational studies, identify csgA as the gene involved in cell communication. The function of the fprA gene is described in an accompanying paper (L. J. Shimkets, J. Bacteriol. 172:24-30, 1990).
Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium
Authors: Boominathan K , Dass S B , Randall T A , Kelley R L , Reddy C A
Published: January 2017

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.
Autogenous control is not sufficient to ensure steady-state growth rate-dependent regulation of the S10 ribosomal protein operon of Escherichia coli
Authors: Lindahl L , Zengel J M
Published: January 2017

The regulation of the S10 ribosomal protein operon of Escherichia coli was studied by using a lambda prophage containing the beginning of the S10 operon (including the promoter, leader, and first one and one-half structural genes) fused to lacZ. The synthesis of the lacZ fusion protein encoded by the phage showed the expected inhibition during oversynthesis of ribosomal protein L4, the autogenous regulatory protein of the S10 operon. Moreover, the fusion gene responded to a nutritional shift-up in the same way that genuine ribosomal protein genes did. However, the gene did not exhibit the expected growth rate-dependent regulation during steady-state growth. Thus, the genetic information carried on the prophage is sufficient for L4-mediated autogenous control and a normal nutritional shift-up response but is not sufficient for steady-state growth rate-dependent control. These results suggest that, at least for the 11-gene S10 ribosomal protein operon, additional regulatory processes are required to coordinate the synthesis of ribosomal proteins with cell growth rate and, furthermore, that sequences downstream of the proximal one and one-half genes of the operon are involved in this control.
Characterization of the C operon transcript of bacteriophage Mu
Authors: Stoddard S F , Howe M M
Published: January 2017

Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.
Detection of conjugal transfer systems in oral, black-pigmented Bacteroides spp
Authors: Guiney D G , Bouic K
Published: January 2017

Oral, black-pigmented Bacteroides spp. are important pathogens in oral anaerobic infections and dental disease. We detected conjugation systems in isolates of Bacteroides denticola and Bacteroides intermedius that transferred tetracycline resistance (Tetr) and penicillin resistance to Bacteroides buccae and to Bacteroides fragilis, an intestinal Bacteroides species. A cloned Tetr gene from B. fragilis hybridized to the transferable Tetr locus in the oral strains, indicating that genetic exchange occurs between these two groups of anaerobes.
Identification of Escherichia coli and Shigella Species from Whole-Genome Sequences
Authors: Chattaway Marie A. , Schaefer Ulf , Tewolde Rediat , Dallman Timothy J. , Jenkins Claire
Published: January 2017

Escherichia coli
and
Shigella
species are closely related and genetically constitute the same species. Differentiating between these two pathogens and accurately identifying the four species of
Shigella
are therefore challenging. The organism-specific bioinformatics whole-genome sequencing (WGS) typing pipelines at Public Health England are dependent on the initial identification of the bacterial species by use of a kmer-based approach. Of the 1,982
Escherichia coli
and
Shigella
sp. isolates analyzed in this study, 1,957 (98.4%) had concordant results by both traditional biochemistry and serology (TB&S) and the kmer identification (ID) derived from the WGS data. Of the 25 mismatches identified, 10 were enteroinvasive
E. coli
isolates that were misidentified as
Shigella flexneri
or
S. boydii
by the kmer ID, and 8 were
S. flexneri
isolates misidentified by TB&S as
S. boydii
due to nonfunctional
S. flexneri
O antigen biosynthesis genes. Analysis of the population structure based on multilocus sequence typing (MLST) data derived from the WGS data showed that the remaining discrepant results belonged to clonal complex 288 (CC288), comprising both
S. boydii
and
S. dysenteriae
strains. Mismatches between the TB&S and kmer ID results were explained by the close phylogenetic relationship between the two species and were resolved with reference to the MLST data.
Shigella
can be differentiated from
E. coli
and accurately identified to the species level by use of kmer comparisons and MLST. Analysis of the WGS data provided explanations for the discordant results between TB&S and WGS data, revealed the true phylogenetic relationships between different species of
Shigella
, and identified emerging pathoadapted lineages.
Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR
Authors: Kembou Tsofack Japhette Esther , Zamostiano Rachel , Watted Salsabeel , Berkowitz Asaf , Rosenbluth Ezra , Mishra Nischay , Briese Thomas , Lipkin W. Ian , Kabuusu Richard M. , Ferguson Hugh , del Pozo Jorge , Eldar Avi , Bacharach Eran
Published: January 2017

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.
Evaluation of the GenoType MTBDR
sl
Version 2.0 Assay for Second-Line Drug Resistance Detection of Mycobacterium tuberculosis Isolates in South Africa
Authors: Gardee Y. , Dreyer A. W. , Koornhof H. J. , Omar S. V. , da Silva P. , Bhyat Z. , Ismail N. A.
Published: January 2017

Early detection of resistance to second-line antituberculosis drugs is important for the management of multidrug-resistant tuberculosis (MDR-TB). The GenoType MTBDR
sl
version 2.0 (VER 2.0) line probe assay has been redesigned for molecular detection of resistance-conferring mutations of fluoroquinolones (FLQ) (
gyrA
and
gyrB
genes) and second-line injectable drugs (SLID) (
rrs
and
eis
genes). The study evaluated the diagnostic performance of the GenoType MTBDR
sl
VER 2.0 assay for the detection of second-line drug resistance compared with phenotypic drug susceptibility testing (DST), using the Bactec MGIT 960 system on
Mycobacterium tuberculosis
complex isolates from South Africa. A total of 268 repository isolates collected between 2012 and 2014, which were rifampin monoresistant or MDR based on DST, were selected. MTBDR
sl
VER 2.0 testing was performed on these isolates and the results analyzed. The MTBDR
sl
VER 2.0 sensitivity and specificity indices for culture isolates were the following: FLQ, 100% (95% confidence interval [CI] 95.8 to 100%) and 98.9% (95% CI, 96.1 to 99.9%); SLID, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.7 to 99.7%). The sensitivity and specificity observed for individual SLID were the following: amikacin, 93.8% (95% CI, 79.2 to 99.2%) and 98.5% (95% CI, 95.5 to 99.7%); kanamycin, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.5 to 99.7%); and capreomycin, 86.2% (95% CI, 68.3 to 96.1%) and 95.9% (95% CI, 92.2 to 98.2%). An interoperator reproducibility of 100% and an overall interlaboratory performance of 93% to 96% were found. The overall improvement in sensitivity and specificity with excellent reproducibility makes the GenoType MTBDR
sl
VER 2.0 a highly suitable tool for rapid screening of clinical isolates for second-line drug resistance for use in high-burden TB/HIV settings.
Evaluation of the Parasight Platform for Malaria Diagnosis
Authors: Eshel Yochay , Houri-Yafin Arnon , Benkuzari Hagai , Lezmy Natalie , Soni Mamta , Charles Malini , Swaminathan Jayanthi , Solomon Hilda , Sampathkumar Pavithra , Premji Zul , Mbithi Caroline , Nneka Zaitun , Onsongo Simon , Maina Daniel , Levy-Schreier Sarah , Cohen Caitlin Lee , Gluck Dan , Pollak Joseph Joel , Salpeter Seth J.
Published: January 2017

The World Health Organization estimates that nearly 500 million malaria tests are performed annually. While microscopy and rapid diagnostic tests (RDTs) are the main diagnostic approaches, no single method is inexpensive, rapid, and highly accurate. Two recent studies from our group have demonstrated a prototype computer vision platform that meets those needs. Here we present the results from two clinical studies on the commercially available version of this technology, the Sight Diagnostics Parasight platform, which provides malaria diagnosis, species identification, and parasite quantification. We conducted a multisite trial in Chennai, India (Apollo Hospital [
n
= 205]), and Nairobi, Kenya (Aga Khan University Hospital [
n
= 263]), in which we compared the device to microscopy, RDTs, and PCR. For identification of malaria, the device performed similarly well in both contexts (sensitivity of 99% and specificity of 100% at the Indian site and sensitivity of 99.3% and specificity of 98.9% at the Kenyan site, compared to PCR). For species identification, the device correctly identified 100% of samples with
Plasmodium vivax
and 100% of samples with
Plasmodium falciparum
in India and 100% of samples with
P. vivax
and 96.1% of samples with
P. falciparum
in Kenya, compared to PCR. Lastly, comparisons of the device parasite counts with those of trained microscopists produced average Pearson correlation coefficients of 0.84 at the Indian site and 0.85 at the Kenyan site.
Optical Screening for Rapid Antimicrobial Susceptibility Testing and for Observation of Phenotypic Diversity among Strains of the Genetically Clonal Species Bacillus anthracis
Authors: McLaughlin Heather P. , Gargis Amy S. , Michel Pierre , Sue David , Weigel Linda M.
Published: January 2017

During high-impact events involving
Bacillus anthracis
, such as the Amerithrax incident of 2001 or the anthrax outbreaks in Russia and Sweden in 2016, critical decisions to reduce morbidity and mortality include rapid selection and distribution of effective antimicrobial agents for treatment and postexposure prophylaxis. Detection of antimicrobial resistance currently relies on a conventional broth microdilution method that requires a 16- to 20-h incubation time for
B. anthracis
. Advances in high-resolution optical screening offer a new technology to more rapidly evaluate antimicrobial susceptibility and to simultaneously assess the growth characteristics of an isolate. Herein, we describe a new method developed and evaluated as a rapid antimicrobial susceptibility test for
B. anthracis
. This method is based on automated digital time-lapse microscopy to observe the growth and morphological effects of relevant antibiotics with an optical screening instrument, the oCelloScope.
B. anthracis
strains were monitored over time in the presence or absence of penicillin, ciprofloxacin, or doxycycline. Susceptibility to each antibiotic was determined in ≤4 h, 75 to 80% less than the time required for conventional methods. Time-lapse video imaging compiled from the optical screening images revealed unexpected differences in growth characteristics among strains of
B. anthracis
, which is considered to be a clonal organism. This technology provides a new approach for rapidly detecting phenotypic antimicrobial resistance and for documenting growth attributes that may be beneficial in the further characterization of individual strains.
Copy Number Heterogeneity of JC Virus Standards
Authors: Greninger Alexander L. , Bateman Allen C. , Atienza Ederlyn E. , Wendt Sharon , Makhsous Negar , Jerome Keith R. , Cook Linda
Published: January 2017

Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to obtain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and the Exact v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T antigen region. Intriguingly, several of the JCV standards sequenced in this study with large T antigen deletions were cultured in cell lines immortalized using simian virus 40 (SV40) T antigen, suggesting the possibility of transcomplementation in cell culture. Using a cutoff 5% allele fraction for junctional reads, 7 different rearrangements were present in the JC virus sequences present in the WHO standard across multiple library preparations and sequencing runs. Neither the copy number differences nor the rearrangements were observed in a clinical sample with a high copy number of JCV or a plasmid control. These results were also confirmed by the quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR), and Sanger sequencing of multiple rearrangements. In summary, targeting different regions of the same international standard can result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology.
Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain
Authors: Naito Tadasuke , Mori Kotaro , Ushirogawa Hiroshi , Takizawa Naoki , Nobusawa Eri , Odagiri Takato , Tashiro Masato , Ohniwa Ryosuke L. , Nagata Kyosuke , Saito Mineki
Published: January 2017

Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.

IMPORTANCE
Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.
Matrix Metalloproteinase Activity in Infections by an Encephalitic Virus, Mouse Adenovirus Type 1
Authors: Ashley Shanna L. , Pretto Carla D. , Stier Matthew T. , Kadiyala Padma , Castro-Jorge Luiza , Hsu Tien-Huei , Doherty Robert , Carnahan Kelly E. , Castro Maria G. , Lowenstein Pedro R. , Spindler Katherine R.
Published: January 2017

Mouse adenovirus type 1 (MAV-1) infection causes encephalitis in susceptible strains of mice and alters the permeability of infected brains to small molecules, which indicates disruption of the blood-brain barrier (BBB). Under pathological conditions, matrix metalloproteinases (MMPs) can disrupt the BBB through their proteolytic activity on basement membrane and tight junction proteins. We examined whether MAV-1 infection alters MMP activity
in vivo
and
in vitro
. Infected MAV-1-susceptible SJL mice had higher MMP2 and MMP9 activity in brains, measured by gelatin zymography, than mock-infected mice. Infected MAV-1-resistant BALB/c mice had MMP activity levels equivalent to those in mock infection. Primary SJL mouse brain endothelial cells (a target of MAV-1
in vivo
) infected
ex vivo
with MAV-1 had no difference in activities of secreted MMP2 and MMP9 from mock cells. We show for the first time that astrocytes and microglia are also infected
in vivo
by MAV-1. Infected mixed primary cultures of astrocytes and microglia had higher levels of MMP2 and MMP9 activity than mock-infected cells. These results indicate that increased MMP activity in the brains of MAV-1-infected susceptible mice may be due to MMP activity produced by endothelial cells, astrocytes, and microglia, which in turn may contribute to BBB disruption and encephalitis in susceptible mice.

IMPORTANCE
RNA and DNA viruses can cause encephalitis; in some cases, this is accompanied by MMP-mediated disruption of the BBB. Activated MMPs degrade extracellular matrix and cleave tight-junction proteins and cytokines, modulating their functions. MAV-1 infection of susceptible mice is a tractable small-animal model for encephalitis, and the virus causes disruption of the BBB. We showed that MAV-1 infection increases enzymatic activity of two key MMPs known to be secreted and activated in neuroinflammation, MMP2 and MMP9, in brains of susceptible mice. MAV-1 infects endothelial cells, astrocytes, and microglia, cell types in the neurovascular unit that can secrete MMPs.
Ex vivo
MAV-1 infection of these cell types caused higher MMP activity than mock infection, suggesting that they may contribute to the higher MMP activity seen
in vivo
. To our knowledge, this provides the first evidence of an encephalitic DNA virus in its natural host causing increased MMP activity in brains.
A Point Mutation in the N-Terminal Amphipathic Helix α
0
in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding
Authors: Yan Yu , He Ying , Boson Bertrand , Wang Xuesong , Cosset François-Loïc , Zhong Jin
Published: January 2017

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α
0
), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α
0
of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α
0
, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α
0
and aid understanding of the role of NS3 in HCV virion morphogenesis.

IMPORTANCE
HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α
0
domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α
0
in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.
Headache outcomes in children undergoing foramen magnum decompression for Chiari I malformation
Authors: Raza-Knight Saba , Mankad Kshitij , Prabhakar Prab , Thompson Dominic
Published: January 2017

Objective

A common symptom of Chiari I malformation (CIM) is headache, which is diagnosed using non-validated criteria from the International Headache Society (IHS). CIM-associated headaches should resolve following neurosurgical treatment of the malformation by foramen magnum decompression (FMD). We aimed to validate the IHS criteria and determine (1) the efficacy of FMD in treating headache and (2) whether duraplasty confers an advantage over simple bony decompression in the treatment of this symptom.

Methods

A retrospective review of CIM cases treated with FMD at Great Ormond Street Hospital from 1989 to 2014 was carried out. Clinical headache characteristics were compared against IHS criteria and correlated with outcome following FMD.

Results

Headache was a presenting symptom in 57/102 (55.9%) of patients. Of these, 42/57 (73.7%) could be classified as Chiari I-type headache. Following FMD, 42/57 (73.7%) of all presenting headaches showed a sustained improvement (>3 months) compared with 32/39 (82.1%) of Chiari I-type headaches. Duraplasty led to a sustained improvement in headache in 32/38 (84.2%) cases compared with 9/16 (56.3%) cases treated with bone-only decompression.

Conclusions

Our data suggest that 80% of headaches classified as Chiari I-type will show a sustained improvement following FMD, and, as such, the IHS criteria are clinically useful in evaluating symptoms attributable to CIM. For all headaches associated with CIM, duraplasty may confer a benefit in terms of long-term improvement, compared with bone-only decompression.
‘There were more wires than him’: the potential for wireless patient monitoring in neonatal intensive care
Authors: Bonner Oliver , Beardsall Kathryn , Crilly Nathan , Lasenby Joan
Published: January 2017

Background

The neonatal intensive care unit (NICU) can be one of the most stressful hospital environments. Alongside providing intensive clinical care, it is important that parents have the opportunity for regular physical contact with their babies because the neonatal period is critical for parent–child bonding. At present, monitoring technology in the NICU requires multiple wired sensors to track each baby's vital signs. This study describes the experiences that parents and nurses have with the current monitoring methods, and reports on their responses to the concept of a wireless monitoring system.

Methods

Semistructured interviews were conducted with six parents, each of whom had babies on the unit, and seven nurses who cared for those babies. The interviews initially focused on the participants’ experiences of the current wired system and then on their responses to the concept of a wireless system. The transcripts were analysed using a general inductive approach to identify relevant themes.

Results

Participants reported on physical and psychological barriers to parental care, the ways in which the current system obstructed the efficient delivery of clinical care and the perceived benefits and risks of a wireless system. The parents and nurses identified that the wires impeded baby–parent bonding; physically and psychologically. While a wireless system was viewed as potentially enabling greater interaction, staff and parents highlighted potential concerns, including the size, weight and battery life of any new device.

Conclusions

The many wires required to safely monitor babies within the NICU creates a negative environment for parents at a critical developmental period, in terms of physical and psychological interactions. Nurses also experience challenges with the existing system, which could negatively impact the clinical care delivery. Developing a wireless system could overcome these barriers, but there remain challenges in designing a device suitable for this unique environment.
Health hackathons: theatre or substance? A survey assessment of outcomes from healthcare-focused hackathons in three countries
Authors: Olson Kristian R , Walsh Madeline , Garg Priya , Steel Alexis , Mehta Sahil , Data Santorino , Petersen Rebecca , Guarino Anthony J , Bailey Elizabeth , Bangsberg David R
Published: January 2017

Background

Healthcare-focused hackathons are 48-hour platforms intended to accelerate novel medical technology. However, debate exists about how much they contribute to medical technology innovation. The Consortium for Affordable Medical Technologies (CAMTech) has developed a three-pronged model to maximise their effectiveness. To gauge the success of this model, we examined follow-up outcomes.

Methods

Outcomes of 12 hackathons from 2012 to 2015 in India, Uganda and the USA were measured using emailed surveys. To minimise response bias, non-responding teams were coded as having made no progress.

Results

331 individuals provided information on 196 of 356 projects (55.1% response rate), with no difference in responses from teams participating in different countries (Cramer's V=0.09, p=0.17). 30.3% of projects had made progress after a mean of 12.2 months. 88 (24.7%) teams had initiated pilot testing, with 42 (11.8%) piloting with care providers and 24 (6.7%) with patients. Overall, 97 teams (8.1 per hackathon) drafted business plans, 22 (1.8 per hackathon) had filed patents on their innovations and 15 (1.3 per hackathon) had formed new companies. Teams raised US$64.08 million in funding (average US$5.34 million per hackathon; median award size of $1800). In addition, 108 teams (30.3%) reported at least one member working on additional technologies with people they met at a hackathon. Individual confidence to address medical technology challenges was significantly increased after attending (t(1282)=192.77, p 0.001).

Conclusion

CAMTech healthcare hackathons lead to consistent output with respect to medical technology innovation, including clinical trials, business plan development, securing investment capital/funding and new company formation.
Acupuncture and PC6 stimulation for the prevention of postoperative nausea and vomiting in patients undergoing elective laparoscopic resection of colorectal cancer: a study protocol for a three-arm randomised pilot trial
Authors: Kim Kun Hyung , Kim Dae Hun , Bae Ji Min , Son Gyung Mo , Kim Kyung Hee , Hong Seung Pyo , Yang Gi Young , Kim Hee Young
Published: January 2017

Introduction

This study aims to assess the feasibility of acupuncture and a Pericardium 6 (PC6) wristband as an add-on intervention of antiemetic medication for the prevention of postoperative nausea and vomiting (PONV) in patients undergoing elective laparoscopic colorectal cancer resection.

Methods and analysis

A total of 60 participants who are scheduled to undergo elective laparoscopic resection of colorectal cancer will be recruited. An enhanced recovery after surgery protocol using standardised antiemetic medication will be provided for all participants. Participants will be equally randomised into acupuncture plus PC6 wristband (Acupuncture), PC6 wristband alone (Wristband), or no acupuncture or wristband (Control) groups using computer-generated random numbers concealed in opaque, sealed, sequentially numbered envelopes. For the acupuncture combined with PC6 wristband group, the embedded auricular acupuncture technique for preoperative anxiolysis and up to three sessions of acupuncture treatments with manual and electrical stimulation within 48 hours after surgery will be provided by qualified Korean medicine doctors. The PC6 wristband will be applied in the Acupuncture and Wristband groups, beginning 1 hour before surgery and lasting 48 hours postoperatively. The primary outcome will be the number of participants who experience moderate or severe nausea, defined as nausea at least 4 out of 10 on a severity numeric rating scale or vomiting at 24 hours after surgery. Secondary outcomes, including symptom severity, participant global assessments and satisfaction, quality of life, physiological recovery, use of medication and length of hospital stay, will be assessed. Adverse events and postoperative complications will be measured for 1 month after surgery.

Ethics and dissemination

All participants will provide written informed consent. The study has been approved by the institutional review board (IRB). This pilot trial will inform a full-scale randomised trial of acupuncture combined with PC6 stimulation for the prevention of PONV in patients undergoing elective laparoscopic colorectal cancer surgery.

Trial registration number

NCT02509143.
Double-stranded RNA upregulates the expression of inflammatory mediators in human aortic valve cells through the TLR3-TRIF-noncanonical NF-κB pathway
Authors: Zhan Qiong , Song Rui , Li Fei , Ao Lihua , Zeng Qingchun , Xu Dingli , Fullerton David A. , Meng Xianzhong
Published: January 2017

Calcific aortic valve disease is a chronic inflammatory condition, and the inflammatory responses of aortic valve interstitial cells (AVICs) play a critical role in the disease progression. Double-stranded RNA (dsRNA) released from damaged or stressed cells is proinflammatory and may contribute to the mechanism of chronic inflammation observed in diseased aortic valves. The objective of this study is to determine the effect of dsRNA on AVIC inflammatory responses and the underlying mechanism. AVICs from normal human aortic valves were stimulated with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Poly(I:C) increased the production of IL-6, IL-8, monocyte chemoattractant protein-1, and ICAM-1. Poly(I:C) also induced robust activation of ERK1/2 and NF-κB. Knockdown of Toll-like receptor 3 (TLR3) or Toll-IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF) suppressed ERK1/2 and NF-κB p65 phosphorylation and reduced inflammatory mediator production induced by poly(I:C). Inhibition of NF-κB, not ERK1/2, reduced inflammatory mediator production in AVICs exposed to poly(I:C). Interestingly, inhibition of NF-κB by prevention of p50 migration failed to suppress inflammatory mediator production. NF-κB p65 intranuclear translocation induced by the TLR4 agonist was reduced by inhibition of p50 migration; however, poly(I:C)-induced p65 translocation was not, although the p65/p50 heterodimer is present in AVICs. Poly(I:C) upregulates the production of multiple inflammatory mediators through the TLR3-TRIF-NF-κB pathway in human AVICs. The NF-κB activated by dsRNA appears not to be the canonical p65/p50 heterodimers.