bluuebunny/crossref_metadata_2025 huggingface dataset:
Large-Scale Analysis of the Prevalence and Geographic Distribution of HIV-1 Non-B Variants in the United States
Authors: Pyne Michael T. , Hackett John , Holzmayer Vera , Hillyard David R.
Published: June 2013

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) has significant implications for diagnosis, vaccine development, and clinical management of patients. Although HIV-1 subtype B is predominant in the United States, factors such as global travel, immigration, and military deployment have the potential to increase the proportion of non-subtype B infections. Limited data are available on the prevalence and distribution of non-B HIV-1 strains in the United States. We sought to retrospectively examine the prevalence, geographic distribution, diversity, and temporal trends of HIV-1 non-B infections in samples obtained by ARUP Laboratories, a national reference laboratory, from all regions of the United States. HIV-1
pol
sequences from 24,386 specimens collected from 46 states between 2004 and September 2011 for drug resistance genotyping were analyzed using the REGA HIV-1 Subtyping Tool, version 2.0. Sequences refractory to subtype determination or reported as non-subtype B by this tool were analyzed by PHYLIP version 3.5 and Simplot version 3.5.1. Non-subtype B strains accounted for 3.27% (798/24,386) of specimens. The 798 non-B specimens were received from 37 states and included 5 subtypes, 23 different circulating recombinant forms (CRFs), and 39 unique recombinant forms (URFs). The non-subtype B prevalence varied from 0% in 2004 (0/54) to 4.12% in 2011 (201/4,884). This large-scale analysis reveals that the diversity of HIV-1 in the United States is high, with multiple subtypes, CRFs, and URFs circulating. Moreover, the geographic distribution of non-B variants is widespread. Data from HIV-1 drug resistance testing have the potential to significantly enhance the surveillance of HIV-1 variants in the United States.
Atypical Strain of Toxoplasma gondii Causing Fatal Reactivation after Hematopoietic Stem Cell Transplantion in a Patient with an Underlying Immunological Deficiency
Authors: Štajner Tijana , Vasiljević Zorica , Vujić Dragana , Marković Marija , Ristić Goran , Mićić Dragan , Pašić Srdjan , Ivović Vladimir , Ajzenberg Daniel , Djurković-Djaković Olgica
Published: June 2013

In immunocompromized patients, including hematopoietic stem cell transplant (HSCT) recipients, life-threatening toxoplasmosis may result from reactivation of previous infection. We report a case of severe disseminated toxoplasmosis that developed early after allogeneic HSCT for T-cell lymphoblastic leukemia/lymphoma in a 15-year-old
Toxoplasma gondii
-seropositive boy with Nijmegen breakage syndrome, a rare genetic DNA repair disorder associated with immunodeficiency. The donor was the patient's HLA-identical brother. Prophylaxis with cotrimoxazole was discontinued a day before the HSCT procedure. Signs of lung infection appeared as early as day 14 post-HSCT. The presence of tachyzoite-like structures on Giemsa-stained bronchoalveolar lavage (BAL) fluid smears suggested toxoplasmosis. Real-time PCR targeted at the
T. gondii
AF146527 gene revealed extremely high parasite burdens in both blood and BAL fluid. Although immediate introduction of specific treatment resulted in a marked reduction of the parasite load and transient clinical improvement, the patient deteriorated and died of multiple organ failure on day 39 post-HSCT. Direct genotyping of
T. gondii
DNA from blood and BAL fluid with the PCR-restriction fragment length polymorphism method revealed type II alleles with
SAG1
,
SAG2
, and
GRA6
markers but alleles of both type I and type II with
GRA7
. Additional analysis with 15 microsatellite markers showed that the
T. gondii
DNA was atypical and genetically divergent from that of the clonal type I, II, and III strains. This is the first report of increased clinical severity of toxoplasmosis associated with an atypical strain in the setting of immunosuppression, which emphasizes the need to diagnose and monitor toxoplasmosis by quantitative molecular methods in cases of reactivation risk.
Phylogenetic Analyses of the VP4 and VP7 Genes of Porcine Group A Rotaviruses in São Paulo State, Brazil: First Identification of G5P[23] in Piglets
Authors: Tonietti Paloma O. , Hora Aline S. , Silva Fernanda D. F. , Ruiz Vera L. A. , Gregori Fabio
Published: June 2013

This study determined the group A rotavirus occurrence in pig farms from 7 different cities in São Paulo State, Brazil. Out of 143 samples, 70 tested positive. Sequence analyses of 37 strains indicated that the strains had the G3, G5, G9, and P[6], P[13]/P[22]-like, and P[23] genotypes.
Epidemiological Cutoff Values for Fluconazole, Itraconazole, Posaconazole, and Voriconazole for Six Candida Species as Determined by the Colorimetric Sensititre YeastOne Method
Authors: Cantón Emilia , Pemán Javier , Iñiguez Carmen , Hervás David , Lopez-Hontangas Jose L. , Pina-Vaz Cidalia , Camarena Juan J. , Campos-Herrero Isolina , García-García Inmaculada , García-Tapia Ana M. , Guna Remedios , Merino Paloma , Pérez del Molino Luisa , Rubio Carmen , Suárez Anabel ,   FUNGEMYCA Study Group
Published: June 2013

In the absence of clinical breakpoints (CBP), epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of susceptibility tests. Sensititre YeastOne (SYO) is a widely used method to determine susceptibility of
Candida
spp. to antifungal agents. The CLSI CBP have been established, but not for the SYO method. The ECVs for four azoles, obtained using MIC distributions determined by the SYO method, were calculated via five methods (three statistical methods and based on the MIC
50
and modal MIC). Respectively, the median ECVs (in mg/liter) of the five methods for fluconazole, itraconazole, posaconazole, and voriconazole (in parentheses: the percentage of isolates inhibited by MICs equal to or less than the ECVs; the number of isolates tested) were as follows: 2 (94.4%; 944), 0.5 (96.7%; 942), 0.25 (97.6%; 673), and 0.06 (96.7%; 849) for
Candida albicans
; 4 (86.1%; 642), 0.5 (99.4%; 642), 0.12 (93.9%; 392), and 0.06 (86.9%; 559) for
C. parapsilosis
; 8 (94.9%; 175), 1 (93.7%; 175), 2 (93.6%; 125), and 0.25 (90.4%; 167) for
C. tropicalis
; 128 (98.6%; 212), 4 (95.8%; 212), 4 (96.0%; 173), and 2 (98.5; 205) for
C. glabrata
; 256 (100%; 53), 1 (98.1%; 53), 1 (100%; 33), and 1 (97.9%; 48) for
C. krusei
; 4 (89.2%; 93), 0.5 (100%; 93), 0.25 (100%; 33), and 0.06 (87.7%; 73) for
C. orthopsilosis
. All methods included ≥94% of isolates and yielded similar ECVs (within 1 dilution). These ECVs would be suitable for monitoring emergence of isolates with reduced susceptibility by using the SYO method.
Osteomyelitis of the Patella Caused by Legionella anisa
Authors: Sanchez Martha Cristina , Sebti Rani , Hassoun Patrice , Mannion Ciaran , Goy Andre H. , Feldman Tatyana , Mato Anthony , Hong Tao
Published: June 2013

A 51-year-old man with a history of stage IV angioimmunoblastic T-cell lymphoma was diagnosed with osteomyelitis of the patella.
Legionella anisa
was identified by 16S rRNA gene sequencing and culture. The patient had pneumonia 2 months prior to this osteomyelitis episode.
L. anisa
was retrospectively detected in his lung tissue by 16S rRNA gene sequencing and was considered the source of the
L. anisa
that caused his patella osteomyelitis.
Structure and Receptor Binding Specificity of Hemagglutinin H13 from Avian Influenza A Virus H13N6
Authors: Lu Xishan , Qi Jianxun , Shi Yi , Wang Ming , Smith David F. , Heimburg-Molinaro Jamie , Zhang Yanfang , Paulson James C. , Xiao Haixia , Gao George F.
Published: June 2013

Interspecies transmission (host switching/jumping) of influenza viruses is a key scientific question that must be addressed. In addition to the vigorous research on highly pathogenic avian influenza viruses (HPAIVs), studies of the mechanism of interspecies transmission of low-pathogenic avian influenza viruses (LPAIVs) could also provide insights into host tropism and virulence evolution. Influenza A viruses harboring hemagglutinin (HA) H13 (e.g., H13N6) are LPAIVs. In this study, soluble H13 HA glycoprotein was purified, and its receptor binding activity was characterized. The results revealed that H13 exclusively binds the avian α2-3-linked sialic acid receptor; no binding to the mammalian α2-6-linked sialic acid receptor was detected. Furthermore, the molecular basis of the H13 receptor binding specificity was revealed by comparative analysis of the crystal structures of both receptor-bound H13 and H5 HAs, which might be contributed by the hydrophobic residue V186. Work with an H13N186 mutant confirmed the importance of V186 in the receptor binding specificity of H13 HA, which shows that the mutant protein reduced the binding of an avian receptor analog but increased the binding of a human receptor analog. Detailed structural analysis also demonstrated that the conserved binding sites of the recently well-studied broadly neutralizing human monoclonal antibodies targeting the HA2 domain are found in H13. Our results expand our understanding of virulence evolution, receptor binding preference, and species tropism of the LPAIVs and HPAIVs.
Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells
Authors: Villordo Sergio M. , Gamarnik Andrea V.
Published: June 2013

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3′ untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this
cis
-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.
Quantitative and Qualitative Differences in the T Cell Response to HIV in Uninfected Ugandans Exposed or Unexposed to HIV-Infected Partners
Authors: Pala Pietro , Serwanga Jennifer , Watera Christine , Ritchie Adam J. , Moodie Zoe , Wang Maggie , Goonetilleke Nilu , Birabwa Ester , Hughes Peter , Senkaali David , Nakiboneka Ritah , Grosskurth Heiner , Haynes Bart , McMichael Andrew , Kaleebu Pontiano ,   Center for HIV/AIDS Vaccine Immunology
Published: June 2013

HIV-exposed and yet persistently uninfected individuals have been an intriguing, repeated observation in multiple studies, but uncertainty persists on the significance and implications of this in devising protective strategies against HIV. We carried out a cross-sectional analysis of exposed uninfected partners in a Ugandan cohort of heterosexual serodiscordant couples (37.5% antiretroviral therapy naive) comparing their T cell responses to HIV peptides with those of unexposed uninfected individuals. We used an objective definition of exposure and inclusion criteria, blinded
ex vivo
and cultured gamma interferon (IFN-γ) enzyme-linked immunospot assays, and multiparameter flow cytometry and intracellular cytokine staining to investigate the features of the HIV-specific response in exposed versus unexposed uninfected individuals. A response rate to HIV was detectable in unexposed uninfected (5.7%, 95% confidence interval [CI] = 3.3 to 8.1%) and, at a significantly higher level (12.5%, 95% CI = 9.7 to 15.4%,
P
= 0.0004), in exposed uninfected individuals. The response rate to Gag was significantly higher in exposed uninfected (10/50 [20.%]) compared to unexposed uninfected (1/35 [2.9%]) individuals (
P
= 0.0004). The magnitude of responses was also greater in exposed uninfected individuals but not statistically significant. The average number of peptide pools recognized was significantly higher in exposed uninfected subjects than in unexposed uninfected subjects (1.21 versus 0.47;
P
= 0.0106). The proportion of multifunctional responses was different in the two groups, with a higher proportion of single cytokine responses, mostly IFN-γ, in unexposed uninfected individuals compared to exposed uninfected individuals. Our findings demonstrate both quantitative and qualitative differences in T cell reactivity to HIV between HESN (HIV exposed seronegative) and HUSN (HIV unexposed seronegative) subject groups but do not discriminate as to whether they represent markers of exposure or of protection against HIV infection.
B Cell Infection and Activation by Rabies Virus-Based Vaccines
Authors: Lytle Andrew G. , Norton James E. , Dorfmeier Corin L. , Shen Shixue , McGettigan James P.
Published: June 2013

Replication-deficient rabies viruses (RABV) are promising rabies postexposure vaccines due to their prompt and potent stimulation of protective virus neutralizing antibody titers, which are produced in mice by both T-dependent and T-independent mechanisms. To promote such early and robust B cell stimulation, we hypothesized that live RABV-based vaccines directly infect B cells, thereby activating a large pool of antigen-presenting cells (APCs) capable of providing early priming and costimulation to CD4+T cells. In this report, we show that live RABV-based vaccine vectors efficiently infect naive primary murine and human B cellsex vivo. Infection of B cells resulted in the significant upregulation of early markers of B cell activation and antigen presentation, including CD69, major histocompatibility complex class II (MHC-II), and CD40 in murine B cells or HLA-DR and CD40 in human B cells compared to mock-infected cells or cells treated with an inactivated RABV-based vaccine. Furthermore, primary B cells infected with a live RABV expressing ovalbumin were able to prime and stimulate naive CD4+OT-II T cells to proliferate and to secrete interleukin-2 (IL-2), demonstrating a functional consequence of B cell infection and activation by live RABV-based vaccine vectors. We propose that this direct B cell stimulation by live RABV-based vaccines is a potential mechanism underlying their induction of early protective T cell-dependent B cell responses, and that designing live RABV-based vaccines to infect and activate B cells represents a promising strategy to develop a single-dose postexposure rabies vaccine where the generation of early protective antibody titers is critical.
Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation
Authors: Li Jing , Wang Xin , Diaz Jason , Tsang Sabrina H. , Buck Christopher B. , You Jianxin
Published: June 2013

Clonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers.
Utility of the Pentax-AWS in performing tracheal intubation while wearing chemical, biological, radiation and nuclear personal protective equipment: a randomised crossover trial using a manikin
Authors: Shin Dong Hyuk , Choi Pil Cho , Na Ji Ung , Cho Jun Hwi , Han Sang Kuk
Published: June 2013

Introduction

Following a chemical, biological, radiation and nuclear (CBRN) incident, prompt establishment of an advanced airway is required for patients with respiratory failure within the warm zone, while wearing personal protective equipment (PPE). Previous studies reported that intubation attempts were prolonged, and incidence of esophageal intubation was increased with conventional Macintosh laryngoscope (McL), while wearing CBRN-PPE. Pentax-AWS (AWS), a recently introduced portable video laryngoscope, was compared with the McL to test its utility for tracheal intubation while wearing CBRN-PPE.

Methods

31 participants performed unsuited and suited intubations on an advanced life support simulator. The sequence of intubating devices and PPE wearing were randomised. Time to complete tracheal intubation (primary end point), time to see the vocal cords, overall success rate, percentage of glottic opening, dental compression and ease of intubation were measured.

Results

Suited intubations required significantly longer time to complete intubation than unsuited intubations, in both McL and AWS (22.2 vs 26.4 s, 14.2 vs 18.2 s, respectively). However, suited AWS intubations required shorter time to complete tracheal intubation than unsuited McL intubations (18.2 vs 22.2 s). In secondary outcomes, moreover, suited intubations using the AWS compared favourably with unsuited intubations using the McL.

Conclusions

Although the CBRN-PPE adversely affected time required to complete tracheal intubation with the AWS, suited intubations using the AWS were even superior to unsuited intubations using the McL. The AWS should be a promising device to perform tracheal intubation while wearing the CBRN-PPE.
Regiospecific, one-pot, and pseudo-five-component synthesis of 6,6′-(arylmethylene)bis(2-(tert-butyl)4-methylphenol) antioxidants using highly sulfonated multi-walled carbon nanotubes under solvent-free conditions
Authors: Fareghi-Alamdari Reza , Golestanzadeh Mohsen , Agend Farima , Zekri Negar
Published: June 2013

This is the first report of an innovative, one-pot, pseudo-five-component, and solvent-free synthesis of 6,6′-(arylmethylene)bis(2-(tert-butyl)4-methylphenol) antioxidants from p-cresol, methyl tert-butyl ether, and aldehydes in the presence of sulfonated multi-walled carbon nanotubes (MWCNTs-SO3H) as heterogeneous, robust, and reusable catalysts under solvent-free conditions. MWCNTs-SO3H was prepared and characterized by some microscopic and spectroscopic techniques including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, dispersibility in different solvent, and thermogravimetric analysis (one functionality every approximately five carbon atoms). The acidity of the catalyst was measured by acid–base titration (1.80 mmol g−1). This reaction proceeds smoothly to give the products in good yields. The catalyst was reused several times without efficient loss of its activity for the preparation of 6,6′-(arylmethylene)bis(2-(tert-butyl)4-methylphenol) antioxidants. In addition, high yields of the products and solvent-free and nontoxicity of the catalyst are other worthwhile advantages of the present method.
BOUNDING THE GREYBODY FACTORS FOR NON-ROTATING BLACK HOLES
Authors: NGAMPITIPAN TRITOS , BOONSERM PETARPA
Published: June 2013

Semiclassical black holes emit radiation called Hawking radiation. Such radiation, as seen by an asymptotic observer far outside the black hole, differs from the original radiation near the horizon of the black hole by a redshift factor and the so-called "greybody factor." In this paper, we concentrate on the greybody factor; various bounds for the greybody factors of non-rotaging black holes are obtained, concentrating primarily on charged Reissner–Nordström (RN) and RN–de Sitter black holes. These bounds can be derived using a 2 × 2 transfer matrix formalism. It is found that the charges of black holes act as efficient barriers. Furthermore, adding extra dimensions to spacetime can shield Hawking radiation. Finally, it is also found that the cosmological constant can increase the emission rate of Hawking radiation.
ANISOTROPY OF COSMIC ACCELERATION
Authors: ZHAO WEN , WU PUXUN , ZHANG YANG
Published: June 2013

In this paper, we study the anisotropy of cosmic acceleration by dividing the Union2 Type Ia supernova (SNIa) dataset into 12 subsets according to their positions in Galactic coordinate system. In each region, we derive the deceleration parameter q0 as the diagnostic to quantify the anisotropy level in the corresponding direction, and construct q0 anisotropic maps by combining these q0 values. In addition to the monopole component, we find the significant dipole effect in the q0-maps with the amplitude [Formula: see text], which deviates from zero at more than 2-σ level. The direction of the best-fit dipole is (θ = 108.8°, ϕ = 187.0°) in Galactic system. Interesting enough, we find the direction of this dipole is nearly perpendicular to the CMB kinematic dipole, and the angle between them is 95.7°. The perpendicular relation is anomalous at the 1-in-10 level.
BARRIERS AND BENEFITS TO LEARNING IN NATURAL ENVIRONMENTS: TOWARDS A RECONCEPTUALISATION OF THE POSSIBILITIES FOR CHANGE
Authors: DILLON JUSTIN
Published: June 2013

Policies designed to increase public engagement with biodiversity advocate increased education across a range of educational contexts. Evidence of the benefits of learning in natural environments (LINE) continues to be amassed. LINE affords direct benefits as diverse as educational, health and psychological and indirect benefits ranging from social to financial. Research into the value of LINE has failed to address the full range of benefits. Instead, there has been a narrow focus on easily measurable outcomes and a desire to seek answers to simplistic questions such as "does LINE raise standards more than learning in the classroom?" An attempt is made to outline the full range of benefits which are available to all school students. The outcomes include: benefits to individual participants (knowledge and understanding; skills; attitudes and behaviours; health and well-being; self-efficacy and self-worth); benefits to teachers, schools and the wider community, and benefits to the natural environment sector.

Several barriers exist to the effective delivery of LINE. These barriers can be grouped into those that challenge the natural environment sector and those that challenge schools. The challenges facing the sector include a lack of a coordinated effective approach to working with schools at a local level. The challenges facing schools include those frequently mentioned such as the risk of accidents, cost and curriculum pressures. However, another set of challenges exists, at local, institutional and personal levels. These challenges include teachers' confidence, self-efficacy and their access to training in using natural environments close to the school and further afield.
EVALUATING NANOTECHNOLOGIES FOR THE DEVELOPMENT OF NATIONAL AGRICULTURE INDUSTRY
Authors: GERDSRI PISEK , KOCAOGLU DUNDAR F.
Published: June 2013

This research demonstrates a systematic approach to evaluate nanotechnologies. A case study of applying nanotechnologies to the development of Thailand's agriculture industry is given as an example. A hierarchical decision model is built and qualified expert opinions are used as measurements. There are four levels in the hierarchy: mission, objectives, technological goals and research strategies. Three panels are formed based on their background and expertise in order to minimize and balance any possible biases among the members. The research results provide decision makers with a ranking of technological goals and research strategies in terms of their support for specific goals. The outcome of this research should help technology policy makers to evaluate technologies and research strategies on the basis of their contributions to the national mission.
ON REPEATED-ROOT CONSTACYCLIC CODES OF LENGTH 4ps
Authors: Dinh Hai Q.
Published: June 2013

For any odd prime p, a classification of all constacyclic codes of length 4ps over 𝔽pm is obtained, which establishes the algebraic structure in term of specified polynomial generators of such codes. Among other results, all self-dual and LCD cyclic and negacylic codes of length 4ps are obtained. As an example, all constacyclic codes of length 36 over 𝔽27 and 𝔽81 are listed.
Matrix metalloproteinase 13 is a new contributor to skeletal muscle regeneration and critical for myoblast migration
Authors: Lei Hanqin , Leong Dephne , Smith Lucas R. , Barton Elisabeth R.
Published: June 2013

Efficient skeletal muscle repair and regeneration require coordinated remodeling of the extracellular matrix (ECM). Previous reports have indicated that matrix metalloproteinases (MMPs) play the pivotal role in ECM remodeling during muscle regeneration. The goal of the current study was to determine if the interstitial collagenase MMP-13 was involved in the muscle repair process. Using intramuscular cardiotoxin injections to induce acute muscle injury, we found that MMP-13 expression and activity transiently increased during the regeneration process. In addition, in muscles from mdx mice, which exhibit chronic injury, MMP-13 expression and protein levels were elevated. In differentiating C2C12 cells, a murine myoblast cell line, Mmp13 expression was most pronounced after myoblast fusion and during myotube formation. Using pharmacological inhibition of MMP-13 to test whether MMP-13 activity is necessary for the proliferation, differentiation, migration, and fusion of C2C12 cells, we found a dramatic blockade of myoblast migration, as well as a delay in differentiation. In contrast, C2C12 cells with stable overexpression of MMP-13 showed enhanced migration, without affecting myoblast maturation. Taken together, these results support a primary role for MMP-13 in myoblast migration that leads to secondary effects on differentiation.
The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium
Authors: Dou Ying , Goodchild Samuel J. , Velde Robert Vander , Wu Yue , Fedida David
Published: June 2013

The human ether-a-go-go related (hERG) potassium channel has unusual functional characteristics in that the rates of channel activation and deactivation are much slower than inactivation, which is attributed to specific structural elements within the NH2 terminus and the S1–S4 voltage-sensing domains (VSD). Although the charged residues in the VSD have been extensively modified and mutated as a result, the role and importance of specific hydrophobic residues in the S4 has been much less explored in studies of hERG gating. We found that charged, but not neutral or hydrophobic, amino acid substitution of isoleucine 521 at the outer end of the S4 transmembrane domain resulted in channels activating at much more negative voltages associated with a marked hyperpolarization of the conductance-voltage ( G-V) relationship. The contributions of different physicochemical properties to this effect were probed by chemical modification of channels substituted with cysteine at position I521. When positively charged reagents including tetramethyl-rhodamine-5-maleimide (TMRM), 1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)oxazol-2-yl] pyridinium methane-sulfonate (PyMPO), [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET), and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) were bound to the cysteine, I521C channels activated at more negative membrane potentials. To examine the contributions to hERG gating of other residues at the outer end of S4 (520–528), we performed a cysteine scan combined with MTSET modification. Only L520C, along with I521C, shows a substantial hyperpolarizing shift of the G-V relationship upon MTSET modification. The data indicate that the neutral, hydrophobic residue I521 at the extracellular end of S4 is critical for stabilizing the closed conformation of the hERG channel relative to the open state and by comparison with Shaker supports the alignment of hERG I521 with Shaker L361.
Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis
Authors: Roy Jeremy W. , Hill Eric , Ruan Ye Chun , Vedovelli Luca , Păunescu Teodor G. , Brown Dennis , Breton Sylvie
Published: June 2013

Clear cells express the vacuolar proton-pumping H+-ATPase (V-ATPase) and acidify the lumen of the epididymis, a process that is essential for male fertility. The renin-angiotensin-aldosterone system (RAAS) regulates fluid and electrolyte balance in the epididymis, and a previous study showed binding of aldosterone exclusively to epididymal clear cells (Hinton BT, Keefer DA. Steroid Biochem 23: 231–233, 1985). We examined here the role of aldosterone in the regulation of V-ATPase in the epididymis. RT-PCR showed expression of the mineralocorticoid receptor [MR; nuclear receptor subfamily 3, group C member 2 (NR3C2)] and 11-β-dehydrogenase isozyme 2 (HSD11β2) mRNAs specifically in clear cells, isolated by fluorescence-activated cell sorting from B1-enhanced green fluorescent protein (EGFP) mice. Tail vein injection of adult rats with aldosterone, 1,2-dioctanoyl-sn-glycerol (DOG), or 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) induced V-ATPase apical membrane accumulation and extension of V-ATPase-labeled microvilli in clear cells in the caput epididymis but not in the cauda. V-ATPase activity was measured in EGFP-expressing clear cells using the intracellular pH (pHi)-sensing dye seminaphthorhodafluor-5F-5-(and 6)-carboxylic acid, acetoxymethyl ester acetate (SNARF-5F). Aldosterone induced a rapid increase in the rate of Na+- and bicarbonate-independent pHi recovery following an NH4Cl-induced acid load in clear cells isolated from the caput but not the cauda. This effect was abolished by concanamycin A, spironolactone, and chelerythrine but not myristoylated-protein kinase inhibitor (mPKI) or mifepristone. Thus aldosterone increases V-ATPase-dependent proton secretion in clear cells in the caput epididymis via MR/NR3C2 and PKC activation. This study, therefore, identifies aldosterone as an active member of the RAAS for the regulation of luminal acidification in the proximal epididymis.
Exercise prevents Western diet-associated erectile dysfunction and coronary artery endothelial dysfunction: response to acute apocynin and sepiapterin treatment
Authors: La Favor Justin D. , Anderson Ethan J. , Dawkins Jillian T. , Hickner Robert C. , Wingard Christopher J.
Published: June 2013

The aim of this study was to investigate aerobic exercise training as a means to prevent erectile dysfunction (ED) and coronary artery disease (CAD) development associated with inactivity and diet-induced obesity. Male Sprague-Dawley rats were fed a Western diet (WD) or a control diet (CD) for 12 wk. Subgroups within each diet remained sedentary (Sed) or participated in aerobic interval treadmill running throughout the dietary intervention. Erectile function was evaluated under anesthesia by measuring the mean arterial pressure and intracavernosal pressure in response to electrical field stimulation of the cavernosal nerve, in the absence or presence of either apocynin, an NADPH oxidase inhibitor, or sepiapterin, a tetrahydrobiopterin precursor. Coronary artery endothelial function (CAEF) was evaluated ex vivo with cumulative doses of ACh applied to preconstricted segments of the left anterior descending coronary artery. CAEF was assessed in the absence or presence of apocynin or sepiapterin. Erectile function ( P < 0.0001) and CAEF ( P < 0.001) were attenuated in WD-Sed. Exercise preserved erectile function ( P < 0.0001) and CAEF ( P < 0.05) within the WD. Erectile function ( P < 0.01) and CAEF ( P < 0.05) were augmented by apocynin only in WD-Sed, while sepiapterin ( P < 0.05) only augmented erectile function in WD-Sed. These data demonstrate that a chronic WD induces impairment in erectile function and CAEF that are commonly partially reversible by apocynin, whereas sepiapterin treatment exerted differential functional effects between the two vascular beds. Furthermore, exercise training may be a practical means of preventing diet-induced ED and CAD development.
Functional role of left PMd and left M1 during preparation and execution of left hand movements in older adults
Authors: Fujiyama Hakuei , Hinder Mark R. , Summers Jeffery J.
Published: June 2013

A disruptive transcranial magnetic stimulation (TMS) approach was used to determine whether the increased frontal activation and reduced hemispheric laterality brain activation patterns observed in older adults during motor tasks play a functional role. Young and older adults abducted their left index finger as soon as possible after a visual imperative signal presented 500 ms after a warning signal. TMS was applied to the dorsal premotor (PMd) or primary motor (M1) cortex in the left or right hemisphere at seven times during response preparation and execution. Both groups exhibited faster reaction times in their left hand after stimulation of the left PMd (i.e., ipsilateral to the responding hand) relative to trials with no TMS, indicating a functional role of the left PMd in the regulation of impulse control. This result also suggests that the function of the left PMd appears to be unaffected by the healthy aging process. Right M1 TMS resulted in a response time delay in both groups. Only for older adults did left M1 stimulation delay responses, suggesting the involvement of ipsilateral motor pathways in the preparation of motor actions in older adults.
Optogenetics through windows on the brain in the nonhuman primate
Authors: Ruiz Octavio , Lustig Brian R. , Nassi Jonathan J. , Cetin Ali , Reynolds John H. , Albright Thomas D. , Callaway Edward M. , Stoner Gene R. , Roe Anna W.
Published: June 2013

Optogenetics combines optics and genetics to control neuronal activity with cell-type specificity and millisecond temporal precision. Its use in model organisms such as rodents, Drosophila, and Caenorhabditis elegans is now well-established. However, application of this technology in nonhuman primates (NHPs) has been slow to develop. One key challenge has been the delivery of viruses and light to the brain through the thick dura mater of NHPs, which can only be penetrated with large-diameter devices that damage the brain. The opacity of the NHP dura prevents visualization of the underlying cortex, limiting the spatial precision of virus injections, electrophysiological recordings, and photostimulation. Here, we describe a new optogenetics approach in which the native dura is replaced with an optically transparent artificial dura. This artificial dura can be penetrated with fine glass micropipettes, enabling precisely targeted injections of virus into brain tissue with minimal damage to cortex. The expression of optogenetic agents can be monitored visually over time. Most critically, this optical window permits targeted, noninvasive photostimulation and concomitant measurements of neuronal activity via intrinsic signal imaging and electrophysiological recordings. We present results from both anesthetized-paralyzed (optical imaging) and awake-behaving NHPs (electrophysiology). The improvements over current methods made possible by the artificial dura should enable the widespread use of optogenetic tools in NHP research, a key step toward the development of therapies for neuropsychiatric and neurological diseases in humans.
State dependence of adaptation of force output following movement observation
Authors: Wanda Paul A. , Li Gang , Thoroughman Kurt A.
Published: June 2013

Humans readily learn to move through direct physical practice and by watching the movements of others. Some researchers have proposed that action observation can inform subsequent changes in control through the acquisition of a neural representation of the novel dynamics, but to date learning following observation has been described by kinematic metrics. Here we designed an experiment to consider the specificity of adaptation to novel dynamic perturbations at the level of force generation. We measured changes in temporal patterns of force output following either the performance or observation of movements perturbed by either position- or velocity-dependent dynamic environments to 1) establish whether previously described observational motor learning effects were attributable to changes in predictive limb control and 2) determine whether such adaptation reflected a learned dependence on limb states appropriate to the haptic environment. We found that subjects who observed perturbed movements produced significant compensatory changes in their lateral force output, despite never directly experiencing force perturbations firsthand while performing the motor task. The time series of observers' adapted force outputs suggested that the state dependence of observed dynamics shapes adaptation. We conclude that the brain can transform observation of kinematics into state-dependent adaptation of reach dynamics.
Investigation of the SV40 – Human Cancer Association: Look for the Full Signature of the Virus
Authors: Shah Keerti V. , Rollison Dana E. M.
Published: June 2013

In the controversy about the association of simian virus 40 with human cancers, the greatest problem is the ascertainment of SV40 exposure. This difficulty would be resolved if one were to look for all components of SV40 infection. How does SV 40 circulate in the human community? Do cancer patients with SV40‐positive tumors have serological correlates of SV 40 infection and of SV40‐induced cancer? SV40 association with a cancer should be studied in the context of the known risk factors for that cancer. The tumor cell‐virus relationship should be characterized with respect to viral integration and viral localization to the tumor cell. Specimens should be masked and the assays should include panels of specimens to estimate analytic sensitivity and specificity. In view of the rarity of some of the tumors reported to be associated with SV40, a multi‐institutional investigation initiated and coordinated by the NIH would be most effective.
A Proteomic Approach for the Discovery of Early Detection Markers of Hepatocellular Carcinoma
Authors: Steel Laura F. , Mattu Taj S. , Mehta Anand , Hebestreit Holger , Dwek Raymond , Evans Alison A. , London W. Thomas , Block Timothy
Published: June 2013

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post‐translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to “cherry pick” or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.
Pharmacogenomic Biomarkers
Authors: Kirkwood Sandra C. , Hockett Richard D.
Published: June 2013

Pharmacogenomic biomarkers hold great promise for the future of medicine and have been touted as a means to personalize prescriptions. Genetic biomarkers for disease susceptibility including both Mendelian and complex disease promise to result in improved understanding of the pathophysiology of disease, identification of new potential therapeutic targets, and improved molecular classification of disease. However essential to fulfilling the promise of individualized therapeutic intervention is the identification of drug activity biomarkers that stratify individuals based on likely response to a particular therapeutic, both positive response, efficacy, and negative response, development of side effect or toxicity. Prior to the widespread clinical application of a genetic biomarker multiple scientific studies must be completed to identify the genetic variants and delineate their functional significance in the pathophysiology of a carefully defined phenotype. The applicability of the genetic biomarker in the human population must then be verified through both retrospective studies utilizing stored or clinical trial samples, and through clinical trials prospectively stratifying patients based on the biomarker. The risk conferred by the polymorphism and the applicability in the general population must be clearly understood. Thus, the development and widespread application of a pharmacogenomic biomarker is an involved process and for most disease states we are just at the beginning of the journey towards individualized therapy and improved clinical outcome.
QT Interval Prolongation as a Biomarker for Torsades de Pointes and Sudden Death in Drug Development
Authors: Sides Gregory D.
Published: June 2013

Prolongation of the QT interval on the surface 12‐lead electrocardiogram is widely accepted as a biomarker for the potential of a drug to produce torsades de pointes and/or sudden death. Detection of drug‐induced prolongation of the QT interval in animals and man is frequently confounded by extrinsic and intrinsic factors that limit the ability to detect a true drug effect. In particular drugs that increase heart rate show an apparent increase in QT interval that confounds assessment of a true drug effect on cardiac ventricular repolarization. The basis for the use of the QT interval as a biomarker will be examined.
Nanometer‐Scale Pores: Potential Applications for Analyte Detection and DNA Characterization
Authors: Kasianowicz John J.
Published: June 2013

Several classes of transmembrane protein ion channels function in vivo as sensitive and selective detection elements for analytes. Recent studies on single channels reconstituted into planar lipid bilayer membranes suggest that nanometer‐scale pores can be used to detect, quantitate and characterize a wide range of analytes that includes small ions and single stranded DNA. We briefly review here these studies and identify leaps in technology that, if realized, might lead to innovations for the early detection of cancer.
Soluble Inter‐Cellular Adhesion Molecule‐1 in Urban Asian North Indians: Relationships with Anthropometric and Metabolic Covariates
Authors: Sethi Astha , Misra Anoop , Pandey Ravindra Mohan , Luthra Kalpana , Devi J. Rama , Sharma Rekha , Khanna Nidhi , Dwivedi Manjari , Vikram Naval Kishore
Published: June 2013

Background: High prevalence of diabetes, obesity, and dyslipidemias in people belonging to poor socio‐economic strata in urban slums of northern India has been recorded recently. To assess whether this population has high levels of soluble intercellular adhesion molecule‐1 (sICAM‐1), a cytokine involved in the pathogenesis of atherosclerosis, we investigated subjects belonging to poor socio‐economic strata in urban slums and compared them to healthy control subjects from non‐slum urban areas of New Delhi.

Design: Cross‐sectional study.

Methods: Subjects from a previously carried out cross‐sectional study, Delhi Urban Slum Project (DUSP) were divided into two groups: Group‐1 (n = 56) included subjects dwelling in slum area, having at least one risk factor (hypertension, hyperglycemia, hypertriglyceridemia and hypercholesterolemia), while group‐2 (n = 60) consisted of subjects without any risk factor dwelling in the slum area. A third group (n = 29) of non‐obese subjects without any risk factor living in non‐slum urban area was included for comparison. Measurements included; body mass index (BMI), waist‐hip ratio (W‐HR), four skinfolds, percentage body fat, fasting plasma glucose (FPG), serum lipids, and serum levels of sICAM‐1.

Results: Though statistically not significant, mean level of sICAM‐1 was higher in group‐1 (718.5 ± 232.8 ng/ml) as compared to the other groups. Of note, 35% of subjects in group‐1 (p < 0.05 as compared to other two groups), and 25.3% of all subjects had levels of sICAM‐1 in uppermost quartile (>850 ng/ml). Partial correlation coefficients (R) of sICAM‐1 levels with various parameters adjusted for age were statistically significant for BMI (R = 0.27, p < 0.05) in group‐1; W‐HR (R = 0.26, p < 0.05) and BMI (R = 0.19, p < 0.05) for group‐2; and FPG (R = 0.17, p < 0.05) for all the subjects considered together. For females, the levels of sICAM‐1 were significantly higher in the following: BMI ≥ 25 kg/m2 (p = 0.04) and FPG >7 mmol/l (p < 0.05). Multiple linear regression analysis suggests that an increment in BMI by one kg/m2 would correspond to an increase in the levels of sICAM‐1 by 8.5 units controlling for the influence of age and W‐HR in the pooled data of all subjects.

Conclusions: High percentage of subjects had levels of sICAM‐1 in the upper quartile in the study, particularly those dwelling in the slum area and having coronary risk factor (s). The levels of sICAM‐1 strongly correlated to the anthropometric and metabolic parameters, particularly in females. These observations are of potential importance for the pathogenesis of atherosclerosis in this population, though further studies are needed to predict those prone to the complications of atherosclerosis, based on sICAM‐1 levels, as has been observed in other ethnic groups.