bluuebunny/crossref_metadata_2025_split huggingface dataset:
The human Nav1.5 F1486 deletion associated with long QT syndrome leads to impaired sodium channel inactivation and reduced lidocaine sensitivity
Authors: Song Weihua , Xiao Yucheng , Chen Hanying , Ashpole Nicole M. , Piekarz Andrew D. , Ma Peilin , Hudmon Andy , Cummins Theodore R. , Shou Weinian 
Published: July 2012

Key points
We investigated how the F1486 deletion LQT3 mutation impairs the functional properties of the human cardiac voltage‐gated sodium channel (hNav1.5) and alters action potential firing.
Voltage‐clamp recordings from HEK 293 cells and cardiomyocytes expressing recombinant channels demonstrated that the F1486del mutation reduces peak current density but also impairs inactivation and increases late current density.
Current‐clamp recordings from cardiomyocytes indicated that the increase in late current density would result in prolonged action potential duration and this was confirmed using computer simulations.
The deletion of F1486 abolished the ability of lidocaine to stabilize the inactivated state and eliminated the high‐affinity binding of lidocaine to inactivated channels.
Our data show that the hNav1.5‐F1486del mutation has complex functional consequences and indicate that knowledge of the specific molecular defect is critical when developing potential treatments for individuals with prolonged QT intervals.
Abstract  The deletion of phenylalanine 1486 (F1486del) in the human cardiac voltage‐gated sodium channel (hNav1.5) is associated with fatal long QT (LQT) syndrome. In this study we determined how F1486del impairs the functional properties of hNav1.5 and alters action potential firing in heterologous expression systems (human embryonic kidney (HEK) 293 cells) and their native cardiomyocyte background. Cells expressing hNav1.5‐F1486del exhibited a loss‐of‐function alteration, reflected by an 80% reduction of peak current density, and several gain‐of‐function alterations, including reduced channel inactivation, enlarged window current, substantial augmentation of persistent late sodium current and an increase in ramp current. We also observed substantial action potential duration (APD) prolongation and prominent early afterdepolarizations (EADs) in neonatal cardiomyocytes expressing the F1486del channels, as well as in computer simulations of myocyte activity. In addition, lidocaine sensitivity was dramatically reduced, which probably contributed to the poor therapeutic outcome observed in the patient carrying the hNav1.5‐F1486del mutation. Therefore, despite the significant reduction in peak current density, the F1486del mutation also leads to substantial gain‐of‐function alterations that are sufficient to cause APD prolongation and EADs, the predominant characteristic of LQTs. These data demonstrate that hNav1.5 mutations can have complex functional consequences and highlight the importance of identifying the specific molecular defect when evaluating potential treatments for individuals with prolonged QT intervals.
Engineering Design at Harvey Mudd College: Innovation Institutionalized, Lessons Learned
Authors: Dym Clive L. , Gilkeson M. Mack , Phillips J. Richard 
Published: July 2012

Abstract
This paper outlines the development of Harvey Mudd College’s Engineering program, describes its resulting form, and articulates how some truly innovative ideas in engineering design education, now widely accepted as “best practices,” were developed and implemented. The paper also describes the lessons learned from Harvey Mudd College’s Engineering curriculum, as well as some of the efforts made to disseminate these lessons.
Anti-Hepatitis C Virus Activity and Toxicity of Type III Phosphatidylinositol-4-Kinase Beta Inhibitors
Authors: LaMarche M. J. , Borawski J. , Bose A. , Capacci-Daniel C. , Colvin R. , Dennehy M. , Ding J. , Dobler M. , Drumm J. , Gaither L. A. , Gao J. , Jiang X. , Lin K. , McKeever U. , Puyang X. , Raman P. , Thohan S. , Tommasi R. , Wagner K. , Xiong X. , Zabawa T. , Zhu S. , Wiedmann B. 
Published: July 2012

ABSTRACT

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus
in vitro
. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive
in vitro
profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.
Identification and Partial Characterization of Extracellular Aspartic Protease Genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384
Authors: Reid Vernita J. , Theron Louwrens W. , du Toit Maret , Divol Benoit 
Published: July 2012

ABSTRACTThe extracellular acid proteases of non-Saccharomyceswine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene fromMetschnikowia pulcherrimaIWBT Y1123, namedMpAPr1, and the other gene fromCandida apicolaIWBT Y1384, namedCaAPr1.In silicoanalysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression ofMpAPr1inSaccharomyces cerevisiaeYHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. TheMpAPr1gene was found to be present in 12 otherM. pulcherrimastrains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence.
Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System
Authors: Farhat Maha , Moletta-Denat Marina , Frï¿½re Jacques , Onillon Sï¿½verine , Trouilhï¿½ Marie-Cï¿½cile , Robine Enric 
Published: July 2012

ABSTRACT

Legionella
species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of
Legionella
spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of
Legionella
and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic
Legionella
species remained after the heat shock and chemical treatments (
Legionella pneumophila
and
Legionella anisa
, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (
Thecamoebae
sp.,
Vannella
sp., and
Hartmanella vermiformis
) and after the first heat shock treatment, but only
H. vermiformis
remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for
Legionella
, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective
Legionella
disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating
Legionella
in hot water networks requires better study of bacterial and eukaryal species associated with
Legionella
in biofilms.
Requirement of Signal Peptidase ComC and Thiol-Disulfide Oxidoreductase DsbA for Optimal Cell Surface Display of Pseudopilin ComGC in Staphylococcus aureus
Authors: van der Kooi-Pol Magdalena M. , Reilman Ewoud , Sibbald Mark J. J. B. , Veenstra-Kyuchukova Yanka K. , Kouwen Thijs R. H. M. , Buist Girbe , van Dijl Jan Maarten 
Published: July 2012

ABSTRACT

Staphylococcus aureus
is an important Gram-positive bacterial pathogen producing many secreted and cell surface-localized virulence factors. Here we report that the staphylococcal thiol-disulfide oxidoreductase DsbA is essential for stable biogenesis of the ComGC pseudopilin. The signal peptidase ComC is indispensable for ComGC maturation and optimal cell surface exposure.
Protein A-Specific Monoclonal Antibodies and Prevention of Staphylococcus aureus Disease in Mice
Authors: Kim Hwan Keun , Emolo Carla , DeDent Andrea C. , Falugi Fabiana , Missiakas Dominique M. , Schneewind Olaf 
Published: July 2012

ABSTRACTStaphylococcus aureusis a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistantS. aureus[MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope ofS. aureusis decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of VH3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpAKKAA), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the VH3+Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpAKKAAthat, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpAKKAAMAbs promoted opsonophagocytic killing of MRSA in mouse and human blood, provided protection from abscess formation, and stimulated pathogen-specific immune responses in a mouse model of staphylococcal disease. Thus, SpAKKAAMAbs may be useful for the prevention and therapy of staphylococcal disease in humans.
Innate Immune Gene Polymorphisms in Tuberculosis
Authors: Azad Abul K. , Sadee Wolfgang , Schlesinger Larry S. 
Published: July 2012

ABSTRACTTuberculosis (TB) is a leading cause worldwide of human mortality attributable to a single infectious agent. Recent studies targeting candidate genes and “case-control” association have revealed numerous polymorphisms implicated in host susceptibility to TB. Here, we review current progress in the understanding of causative polymorphisms in host innate immune genes associated with TB pathogenesis. We discuss genes encoding several types of proteins: macrophage receptors, such as the mannose receptor (MR, CD206), dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209), Dectin-1, Toll-like receptors (TLRs), complement receptor 3 (CR3, CD11b/CD18), nucleotide oligomerization domain 1 (NOD1) and NOD2, CD14, P2X7, and the vitamin D nuclear receptor (VDR); soluble C-type lectins, such as surfactant protein-A (SP-A), SP-D, and mannose-binding lectin (MBL); phagocyte cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), IL-6, IL-10, IL-12, and IL-18; chemokines, such as IL-8, monocyte chemoattractant protein 1 (MCP-1), RANTES, and CXCL10; and other important innate immune molecules, such as inducible nitric oxide synthase (iNOS) and solute carrier protein 11A1 (SLC11A1). Polymorphisms in these genes have been variably associated with susceptibility to TB among different populations. This apparent variability is probably accounted for by evolutionary selection pressure as a result of long-term host-pathogen interactions in certain regions or populations and, in part, by lack of proper study design and limited knowledge of molecular and functional effects of the implicated genetic variants. Finally, we discuss genomic technologies that hold promise for resolving questions regarding the evolutionary paths of the human genome, functional effects of polymorphisms, and corollary impacts of adaptation on human health, ultimately leading to novel approaches to controlling TB.
The Amino Acid Sequence of Neisseria lactamica PorB Surface-Exposed Loops Influences Toll-Like Receptor 2-Dependent Cell Activation
Authors: Toussi Deana N. , Carraway Margaretha , Wetzler Lee M. , Lewis Lisa A. , Liu Xiuping , Massari Paola 
Published: July 2012

ABSTRACT

Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal
Neisseria lactamica
organisms and purified
N. lactamica
PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than
N. meningitidis
organisms and
N. meningitidis
PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2
in vitro
but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between
N. lactamica
PorB and
N. meningitidis
PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical “TLR2-binding signature” within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB
in vitro
, a chimeric
N. meningitidis
strain expressing
N. lactamica
PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type
N. meningitidis
, suggesting a role for PorB in induction of host cell activation by whole bacteria.
Evaluation of the Trimeric Autotransporter Ata as a Vaccine Candidate against Acinetobacter baumannii Infections
Authors: Bentancor Leticia V. , Routray Abhisek , Bozkurt-Guzel Cagla , Camacho-Peiro Ana , Pier Gerald B. , Maira-Litrán Tomás 
Published: July 2012

ABSTRACT

Acinetobacter baumannii
is a multidrug-resistant (MDR) nosocomial pathogen for which immunotherapeutic alternatives are needed. We previously identified a surface autotransporter of
A. baumannii
, Ata, that bound to various extracellular matrix/basal membrane proteins and was required for full virulence, biofilm formation, and the adhesion of
A. baumannii
to collagen type IV. We show here that Ata binding to collagen type IV was inhibited by antibodies to Ata. In addition, in the presence of complement and polymorphonuclear cells (PMNs), antibodies to Ata were highly opsonic against
A. baumannii
ATCC 17978 and showed low to moderate killing activity against four heterologous
A. baumannii
strains, whereas in the absence of PMNs, antibody to Ata efficiently promoted complement-dependent bactericidal killing of all of the tested
A. baumannii
isolates. Using a pneumonia model of infection in both immunocompetent and immunocompromised mice, we found that, compared to normal rabbit sera, antisera to Ata significantly reduced the levels of
A. baumannii
ATCC 17978 and two MDR strains in the lungs of infected mice. The ability of Ata to engender anti-adhesive, bactericidal, opsonophagocytic, and protective antibodies validates its potential use as an antigenic target against MDR
A. baumannii
infections.
Basis for the Essentiality of H-NS Family Members in Pseudomonas aeruginosa
Authors: Castang Sandra , Dove Simon L. 
Published: July 2012

ABSTRACTMembers of the histone-like nucleoid-structuring (H-NS) family of proteins have been shown to play important roles in silencing gene expression and in nucleoid compaction. InPseudomonas aeruginosa, the two H-NS family members MvaT and MvaU are thought to bind the same AT-rich regions of the chromosome and function coordinately to control a common set of genes. Here we present evidence that the loss of both MvaT and MvaU cannot be tolerated because it results in the production of Pf4 phage that superinfect and kill cells or inhibit their growth. Using a ClpXP-based protein depletion system in combination with transposon mutagenesis, we identify mutants ofP. aeruginosathat can tolerate the depletion of MvaT in an ΔmvaUmutant background. Many of these mutants contain insertions in genes encoding components, assembly factors, or regulators of type IV pili or contain insertions in genes of the prophage Pf4. We demonstrate that cells that no longer produce type IV pili or that no longer produce the replicative form of the Pf4 genome can tolerate the loss of both MvaT and MvaU. Furthermore, we show that the loss of both MvaT and MvaU results in an increase in expression of Pf4 genes and that cells that cannot produce type IV pili are resistant to infection by Pf4 phage. Our findings suggest that type IV pili are the receptors for Pf4 phage and that the essential activities of MvaT and MvaU are to repress the expression of Pf4 genes.
Identification and Characterization of the Functional Toxboxes in the Vibrio cholerae Cholera Toxin Promoter
Authors: Dittmer Jennifer B. , Withey Jeffrey H. 
Published: July 2012

ABSTRACT

Following the consumption of contaminated food or water by a human host, the
Vibrio cholerae
bacterium produces virulence factors, including cholera toxin (CT), which directly causes voluminous diarrhea, producing cholera. A complex regulatory network controls virulence gene expression and responds to various environmental signals and transcription factors. Ultimately, ToxT, a member of the AraC/XylS transcription regulator family, is responsible for activating the transcription of the virulence genes. ToxT-regulated promoters all contain one or more copies of the toxbox, a 13-bp DNA sequence which ToxT recognizes. Nucleotides 2 through 7 of the toxbox sequence are well conserved and contain an invariant tract of four consecutive T nucleotides, whereas the remainder of the toxbox sequence is not highly conserved other than being A/T rich. The binding of ToxT to toxboxes is required to activate the transcription of virulence genes, and toxboxes in several virulence gene promoters have been characterized. However, the toxboxes required for the activation of transcription from the cholera toxin promoter P
ctxAB
have not been identified. P
ctxAB
contains a series of heptad repeats (GATTTTT), each of which matches the 5′ end of the toxbox consensus sequence and is a potential binding site for ToxT. Using site-directed mutagenesis and high-resolution copper-phenanthroline footprinting, we have identified the functional toxboxes required for the ToxT activation of P
ctxAB
. Our findings suggest that ToxT binds to only two toxboxes within P
ctxAB
, despite the presence of several other potential ToxT binding sites within the promoter. Both toxboxes are essential for DNA binding and the full activation of
ctxAB
transcription.
Virulence or Niche Factors: What's in a Name?
Authors: Hill Colin 
Published: July 2012

ABSTRACT

The increasing interest in the human microbiota raises some interesting questions about the terminology we use to describe some of the structures and strategies employed by commensal and pathogenic microbes to compete in these complex biological ecosystems. For example, all microbes arriving in the alimentary tract face the task of surviving passage through the stomach, coping with bile, interacting with the immune system, competing with the established microbiota, and obtaining sufficient nutrients to gain a foothold in this hostile environment. It is not surprising then that many gastrointestinal microbes (both pathogens and commensals) use similar strategies to overcome the challenges associated with this particular biological niche. Given that many of these structures and strategies were discovered and characterized in pathogens and because they often play important roles in establishing and maintaining an infection, they have often been characterized as virulence factors. It would be misleading to describe the same strategies and structures found in harmless commensals as “virulence factors,” since they represent a
sine qua non
for life in the gastrointestinal tract. It may be time to reconsider and refer to them as “niche factors,” both in terms of providing scientific accuracy but also in light of the growing interest in using gut microbes as probiotics, where the distinction between virulence factors and niche factors is likely to be very important from a regulatory perspective.
Evidence for Cyclic Di-GMP-Mediated Signaling in Bacillus subtilis
Authors: Chen Yun , Chai Yunrong , Guo Jian-hua , Losick Richard 
Published: July 2012

ABSTRACTCyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of c-di-GMP signaling have chiefly focused on Gram-negative bacteria. Here, we investigated c-di-GMP signaling in the Gram-positive bacteriumBacillus subtilisby constructing deletion mutations in genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative c-di-GMP-degrading phosphodiesterase, YuxH, and a putative c-di-GMP receptor, YpfA, had strong influences on motility and that these effects depended on sequences similar to canonical EAL and RxxxR—D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and thatyuxHis under the negative control of the master regulator Spo0A∼P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the downregulation ofyuxHby Spo0A∼P. We also present evidence that YpfA has a mild influence on biofilm formation.In toto, our results demonstrate the existence of a functional c-di-GMP signaling system inB. subtilisthat directly inhibits motility and directly or indirectly influences biofilm formation.