bluuebunny/crossref_metadata_2025_split huggingface dataset:
Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy
Authors: de Moraes Luciana V. , Dechavanne Sebastien , Sousa Patrícia M. , Barateiro André , Cunha Sónia F. , Nunes-Silva Sofia , Lima Flávia A. , Murillo Oscar , Marinho Claudio R. F. , Gangnard Stephane , Srivastava Anand , Braks Joanna A. , Janse Chris J. , Gamain Benoit , Franke-Fayard Blandine , Penha-Gonçalves Carlos 
Published: April 2016

ABSTRACT

Plasmodium falciparum
infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the
P. falciparum
IE membrane.
Plasmodium berghei
IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic
P. berghei
parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) fused to a
P. berghei
exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of
P. berghei-
var2CSA (
P. berghei
-VAR) or
P. berghei
wild-type IEs. Infection with 10
4
P. berghei
-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than
P. berghei
. At doses of 10
5
and 10
6
IEs,
P. berghei
-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in
P. berghei
-VAR-infected mice than in
P. berghei
-infected mice.
In vitro
assays showed that higher numbers of
P. berghei
-VAR IEs than
P. berghei
IEs adhered to placental tissue. Immunization of mice with
P. berghei
-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6–EMAP1 on
P. berghei
-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by
P. berghei
-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA.
Cellular Microbiology of Mycoplasma canis
Authors: Michaels Dina L. , Leibowitz Jeffrey A. , Azaiza Mohammed T. , Shil Pollob K. , Shama Suzanne M. , Kutish Gerald F. , Distelhorst Steven L. , Balish Mitchell F. , May Meghan A. , Brown Daniel R. 
Published: April 2016

ABSTRACT

Mycoplasma canis
can infect many mammalian hosts but is best known as a commensal or opportunistic pathogen of dogs. The unexpected presence of
M. canis
in brains of dogs with idiopathic meningoencephalitis prompted new
in vitro
studies to help fill the void of basic knowledge about the organism's candidate virulence factors, the host responses that it elicits, and its potential roles in pathogenesis. Secretion of reactive oxygen species and sialidase varied quantitatively (
P
< 0.01) among strains of
M. canis
isolated from canine brain tissue or mucosal surfaces. All strains colonized the surface of canine MDCK epithelial and DH82 histiocyte cells and murine C8-D1A astrocytes. Transit through MDCK and DH82 cells was demonstrated by gentamicin protection assays and three-dimensional immunofluorescence imaging. Strains further varied (
P
< 0.01) in the extents to which they influenced the secretion of tumor necrosis factor alpha (TNF-α) and the neuroendocrine regulatory peptide endothelin-1 by DH82 cells. Inoculation with
M. canis
also decreased major histocompatibility complex class II (MHC-II) antigen expression by DH82 cells (
P
< 0.01), while secretion of gamma interferon (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and complement factor H was unaffected. The basis for differences in the responses elicited by these strains was not obvious in their genome sequences. No acute cytopathic effects on any homogeneous cell line, or consistent patterns of
M. canis
polyvalent antigen distribution in canine meningoencephalitis case brain tissues, were apparent. Thus, while it is not likely a primary neuropathogen,
M. canis
has the capacity to influence meningoencephalitis through complex interactions within the multicellular and neurochemical
in vivo
milieu.
The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation
Authors: Wang Xiaowei , Shaw Dana K. , Sakhon Olivia S. , Snyder Greg A. , Sundberg Eric J. , Santambrogio Laura , Sutterwala Fayyaz S. , Dumler J. Stephen , Shirey Kari Ann , Perkins Darren J. , Richard Katharina , Chagas Andrezza C. , Calvo Eric , Kopecký Jan , Kotsyfakis Michail , Pedra Joao H. F. 
Published: April 2016

ABSTRACT

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent
Anaplasma phagocytophilum
. Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1β (IL-1β) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly,
Annexin a2
-deficient mice were more susceptible to
A. phagocytophilum
infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.
Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK
Authors: Eaton Daniel S. , Crosson Sean , Fiebig Aretha 
Published: April 2016

ABSTRACT

Growth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotroph
Caulobacter crescentus
can elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control of
C. crescentus
holdfast development.
C. crescentus
mutants partially or completely lacking the C-terminal substrate binding “lid” domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3- to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain of
C. crescentus
DnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control.

IMPORTANCE
Regulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control of
Caulobacter crescentus
cell adhesion.
C. crescentus
surface adhesion is mediated by an envelope-attached organelle known as the holdfast. Holdfast development is tightly controlled by HfiA, a small protein inhibitor that directly interacts with a WecG/TagA-family glycosyltransferase required for holdfast biosynthesis. We demonstrate that the C-terminal lid domain of DnaK is not essential for growth but is necessary for proper control of HfiA levels in the cell and for control of holdfast adhesin development.
Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome
Authors: Hadizadeh Nastaran , Johnson Reid C. , Marko John F. 
Published: April 2016

ABSTRACT

Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or “facilitated dissociation.” Here, we demonstrate that this effect occurs for the major
Escherichia coli
nucleoid protein Fis on isolated bacterial chromosomes. We isolated
E. coli
nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an “exchange” rate constant (
k
exch
) of ≈10
4
M
−1
s
−1
, comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed
in vitro
on chromosomes assembled
in vivo
.

IMPORTANCE
Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated
E. coli
chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in
in vitro
experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.
Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase
Authors: Donnelly M. Lauren , Fimlaid Kelly A. , Shen Aimee 
Published: April 2016

ABSTRACT

The spore-forming obligate anaerobe
Clostridium difficile
is a leading cause of antibiotic-associated diarrhea around the world. In order for
C. difficile
to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In
C. difficile
, cortex hydrolysis is necessary for DPA release, whereas in
Bacillus subtilis
, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for
C. difficile
spore germination by constructing mutations in either
spoVAC
or
dpaAB
, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively.
C. difficile
spoVAC
and
dpaAB
mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast,
B. subtilis
spoVAC
and
dpaAB
mutant spores were unstable. Although
C. difficile
spoVAC
and
dpaAB
mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly,
C. difficile
spoVAC
mutant spores were significantly more sensitive to heat treatment than
dpaAB
mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control
C. difficile
spore resistance and reveal differential requirements for these proteins among the
Firmicutes
.

IMPORTANCE
Clostridium difficile
is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for
C. difficile
to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for
C. difficile
spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.
Genomic Characterization of a Pattern D Streptococcus pyogenes
emm53
Isolate Reveals a Genetic Rationale for Invasive Skin Tropicity
Authors: Bao Yun-Juan , Liang Zhong , Mayfield Jeffrey A. , Donahue Deborah L. , Carothers Katelyn E. , Lee Shaun W. , Ploplis Victoria A. , Castellino Francis J. 
Published: April 2016

ABSTRACT

The genome of an invasive skin-tropic strain (AP53) of serotype M53 group A
Streptococcus pyogenes
(GAS) is composed of a circular chromosome of 1,860,554 bp and carries genetic markers for infection at skin locales,
viz
.,
emm
gene family pattern D and FCT type 3. Through genome-scale comparisons of AP53 with other GAS genomes, we identified 596 candidate single-nucleotide polymorphisms (SNPs) that reveal a potential genetic basis for skin tropism. The genome of AP53 differed by ∼30 point mutations from a noninvasive pattern D serotype M53 strain (Alab49), 4 of which are located in virulence genes. One pseudogene, yielding an inactive sensor kinase (CovS
−
) of the two-component transcriptional regulator CovRS, a major determinant for invasiveness, severely attenuated the expression of the secreted cysteine protease SpeB and enhanced the expression of the hyaluronic acid capsule compared to the isogenic noninvasive AP53/CovS
+
strain. The collagen-binding protein transcript
sclB
differed in the number of 5′-pentanucleotide repeats in the signal peptides of AP53 and Alab49 (9 versus 15), translating into different lengths of their signal peptides, which nonetheless maintained a full-length translatable coding frame. Furthermore, GAS strain AP53 acquired two phages that are absent in Alab49. One such phage (ΦAP53.2) contains the known virulence factor superantigen exotoxin gene tandem
speK-slaA
. Overall, we conclude that this bacterium has evolved in multiple ways, including mutational variations of regulatory genes, short-tandem-repeat polymorphisms, large-scale genomic alterations, and acquisition of phages, all of which may be involved in shaping the adaptation of GAS in specific infectious environments and contribute to its enhanced virulence.

IMPORTANCE
Infectious strains of
S. pyogenes
(GAS) are classified by their serotypes, relating to the surface M protein, the
emm
-like subfamily pattern, and their tropicity toward the nasopharynx and/or skin. It is generally agreed that M proteins from pattern D strains, which also directly bind human host plasminogen, are skin tropic. We have sequenced and characterized the genome of an invasive pattern D GAS strain (AP53) in comparison to a very similar strain (Alab49) that is noninvasive and developed a genomic rationale as to possible reasons for the skin tropicity of these two strains and the greater invasiveness of AP53.
Type IV Pilus Expression Is Upregulated in Nontypeable Haemophilus influenzae Biofilms Formed at the Temperature of the Human Nasopharynx
Authors: Mokrzan Elaine M. , Ward Michael O. , Bakaletz Lauren O. 
Published: April 2016

ABSTRACTNontypeableHaemophilus influenzae(NTHI), a commensal of the human nasopharynx (hNP), is a common cause of biofilm-associated diseases of the respiratory tract. However, NTHI biofilm biology at the average hNP temperature, i.e., 34°C, has not been well studied. Here we grew NTHI biofilms at 34°C and 37°C, to evaluate relative biofilm growth, expression, and function of the type IV pilus (Tfp), a critical adhesin important for NTHI biofilm formation. The kinetics and regulation of Tfp expression in NTHI biofilms are unclear, especially at 34°C. Tfp expression, as estimated bypilApromoter activity, was distributed throughout the biofilms, with a unique pattern that was dependent on temperature, time in culture, and position within the maturing biofilm. Tfp expression was required for the formation of the characteristic tower structures of NTHI biofilms and was significantly upregulated in NTHI biofilms formed at 34°C versus 37°C. This increase correlated with significantly greater twitching motility at 34°C than at 37°C. Treatment with antisera targeting the major subunit of Tfp (PilA) significantly inhibited NTHI biofilm formation at both temperatures, confirming the importance of this critical adhesin in biofilm formation. Additionally, treatment of preestablished biofilms with antisera against PilA significantly decreased biofilm biomass and mean thickness at both temperatures. These results demonstrated a pivotal role for Tfp in NTHI biofilm formation and stability at the temperature of the hNP, and they underscore the utility of PilA as a vaccine candidate for treatment and/or prevention of NTHI biofilm-associated diseases.IMPORTANCENTHI is an important cause of chronic respiratory tract infections, including otitis media, chronic rhinosinusitis, and exacerbations of chronic obstructive pulmonary disease and cystic fibrosis. The chronic and recurrent nature of these diseases is attributed to the presence of bacterial biofilms, which are highly resistant to antimicrobials. We characterized NTHI biofilm growth and expression of PilA, the major subunit of the Tfp, at the temperature of the hNP, which is the commensal habitat of NTHI. Our results expand the current understanding of the role of Tfp during biofilm formation and maturation at the temperature of both the hNP and the middle ear, and they strengthen support for PilA as a vaccine candidate for the prevention and treatment of NTHI biofilm-associated diseases.
Adaptor Scaffoldins: An Original Strategy for Extended Designer Cellulosomes, Inspired from Nature
Authors: Stern Johanna , Moraïs Sarah , Lamed Raphael , Bayer Edward A. 
Published: April 2016

ABSTRACT
Designer cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities—the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes—4 xylanases and 4 cellulases—thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes.

IMPORTANCE
Plant cell wall residues represent a major source of renewable biomass for the production of biofuels such as ethanol via breakdown to soluble sugars. The natural microbial degradation process, however, is inefficient for achieving cost-effective processes in the conversion of plant-derived biomass to biofuels, either from dedicated crops or human-generated cellulosic wastes. The accumulation of the latter is considered a major environmental pollutant. The development of designer cellulosome nanodevices for enhanced plant cell wall degradation thus has major impacts in the fields of environmental pollution, bioenergy production, and biotechnology in general. The findings reported in this article comprise a true breakthrough in our capacity to produce extended designer cellulosomes via synthetic biology means, thus enabling the assembly of higher-order complexes that can supersede the number of enzymes included in a single multienzyme complex.
Behaviour-change intervention in a multicentre, randomised, placebo-controlled COPD study: methodological considerations and implementation
Authors: Bourbeau Jean , Lavoie Kim L , Sedeno Maria , De Sousa Dorothy , Erzen Damijan , Hamilton Alan , Maltais François , Troosters Thierry , Leidy Nancy 
Published: April 2016

IntroductionChronic obstructive pulmonary disease is generally progressive and associated with reduced physical activity. Both pharmacological therapy and exercise training can improve exercise capacity; however, these are often not sufficient to change the amount of daily physical activity a patient undertakes. Behaviour-change self-management programmes are designed to address this, including setting motivational goals and providing social support. We present and discuss the necessary methodological considerations when integrating behaviour-change interventions into a multicentre study.Methods and analysisPHYSACTO is a 12-week phase IIIb study assessing the effects on exercise capacity and physical activity of once-daily tiotropium+olodaterol 5/5 µg with exercise training, tiotropium+olodaterol 5/5 µg without exercise training, tiotropium 5 µg or placebo, with all pharmacological interventions administered via the Respimat inhaler. Patients in all intervention arms receive a behaviour-change self-management programme to provide an optimal environment for translating improvements in exercise capacity into increases in daily physical activity. To maximise the likelihood of success, special attention is given in the programme to: (1) the Site Case Manager, with careful monitoring of programme delivery; (2) the patient, incorporating patient-evaluation/programme-evaluation measures to guide the Site Case Manager in the self-management intervention; and (3) quality assurance, to help identify and correct any problems or shortcomings in programme delivery and ensure the effectiveness of any corrective steps. This paper documents the comprehensive methods used to optimise and standardise the behaviour-change self-management programme used in the study to facilitate dialogue on the inclusion of this type of programme in multicentre studies.Ethics and disseminationThe study has been approved by the relevant Institutional Review Boards, Independent Ethics Committee and Competent Authority according to national and international regulations. The results of this study will be disseminated through relevant, peer-reviewed journals and international conference presentations.Trial registration numberNCT02085161.