bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies
Authors: Andersen Kasper Krogh , Strokappe Nika M. , Hultberg Anna , Truusalu Kai , Smidt Imbi , Mikelsaar Raik-Hiio , Mikelsaar Marika , Verrips Theo , Hammarström Lennart , Marcotte Harold 
Published: November 2015

ABSTRACT

Clostridium difficile
is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of
C. difficile
are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered
Lactobacillus
strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB
in vitro
were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in
Lactobacillus paracasei
BL23, they were able to neutralize the cytotoxic effect of the toxin in an
in vitro
cell-based assay. Prophylactic treatment with a combination of two strains of engineered
L. paracasei
BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA
−
TcdB
+
strain of
C. difficile
(
P
< 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with
C. difficile
for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.
Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae
Authors: Morgan Sarah J. , French Emily L. , Thomson Joshua J. , Seaborn Craig P. , Shively Christian A. , Krukonis Eric S. 
Published: November 2015

ABSTRACT

TcpP and ToxR coordinately regulate transcription of
toxT
, the master regulator of numerous virulence factors in
Vibrio cholerae
. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of
toxT
activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ∼50%
toxT
activation capacity. TcpP-C218S was also TcpH independent, since deletion of
tcpH
did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression.

IMPORTANCE
The
Vibrio cholerae
transcription factor TcpP, in conjunction with ToxR, regulates transcription of
toxT
, the master regulator of numerous virulence factors in
Vibrio cholerae
. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability.
The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC
Authors: Stohl Elizabeth A. , Lenz Jonathan D. , Dillard Joseph P. , Seifert H. Steven 
Published: November 2015

ABSTRACTKey steps in bacterial cell division are the synthesis and subsequent hydrolysis of septal peptidoglycan (PG), which allow efficient separation of daughter cells. Extensive studies in the Gram-negative, rod-shaped bacteriumEscherichia colihave revealed that this hydrolysis is highly regulated spatially and temporally.Neisseria gonorrhoeaeis an obligate Gram-negative, diplococcal pathogen and is the only causative agent of the sexually transmitted infection gonorrhea. We investigated how cell separation proceeds in this diplococcal organism. We demonstrated that deletion of thenlpDgene in strain FA1090 leads to poor growth and to an altered colony and cell morphology. An isopropyl-beta-d-galactopyranoside (IPTG)-regulatednlpDcomplemented construct can restore these defects only when IPTG is supplied in the growth medium. Thin-section transmission electron microscopy (TEM) revealed that thenlpDmutant strain grew in large clumps containing live and dead bacteria, which was consistent with deficient cell separation. Biochemical analyses of purified NlpD protein showed that it was able to bind purified PG. Finally, we showed that, although NlpD has no hydrolase activity itself, NlpD potentiates the hydrolytic activity of AmiC. These results indicate thatN. gonorrhoeaeNlpD is required for proper cell growth and division through its interactions with the amidase AmiC.IMPORTANCEN. gonorrhoeaeis the sole causative agent of the sexually transmitted infection gonorrhea. The incidence of antibiotic-resistant gonococcal infections has risen sharply in recent years, andN. gonorrhoeaehas been classified as a “superbug” by the CDC. Since there is a dearth of new antibiotics to combat gonococcal infections, elucidating the essential cellular process ofN. gonorrhoeaemay point to new targets for antimicrobial therapies. Cell division and separation is one such essential process. We identified and characterized the gonococcalnlpDgene and showed that it is essential for cell separation. In contrast to other pathogenic bacteria, the gonococcal system is streamlined and does not appear to have any redundancies.
Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti
Authors: Schäper Simon , Krol Elizaveta , Skotnicka Dorota , Kaever Volkhard , Hilker Rolf , Søgaard-Andersen Lotte , Becker Anke 
Published: November 2015

ABSTRACT

Sinorhizobium meliloti
undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in
S. meliloti
Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of
N
-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the
S. meliloti
genes
pleD
,
SMb20523
,
SMb20447
,
SMc01464
, and
SMc03178
encoding putative DGCs and of
SMb21517
encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an
S. meliloti
strain that did not produce detectable levels of cdG (cdG
0
) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The
SMc01790-SMc01796
locus, homologous to the
Agrobacterium tumefaciens
uppABCDEF
cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility.

IMPORTANCE
We present the first systematic genome-wide investigation of the role of 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an
S. meliloti
strain unable to produce cdG (cdG
0
) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of
S. meliloti
and uncovered a single-domain PilZ protein as regulator of motility.
A systematic review of stroke recognition instruments in hospital and prehospital settings
Authors: Rudd Matthew , Buck Deborah , Ford Gary A , Price Christopher I 
Published: November 2015

BackgroundWe undertook a systematic review of all published stroke identification instruments to describe their performance characteristics when used prospectively in any clinical setting.MethodsA search strategy was applied to Medline and Embase for material published prior to 10 August 2015. Two authors independently screened titles, and abstracts as necessary. Data including clinical setting, reported sensitivity, specificity, positive predictive value, negative predictive value were extracted independently by two reviewers.Results5622 references were screened by title and or abstract. 18 papers and 3 conference abstracts were included after full text review. 7 instruments were identified; Face Arm Speech Test (FAST), Recognition of Stroke in the Emergency Room (ROSIER), Los Angeles Prehospital Stroke Screen (LAPSS), Melbourne Ambulance Stroke Scale (MASS), Ontario Prehospital Stroke Screening tool (OPSS), Medic Prehospital Assessment for Code Stroke (MedPACS) and Cincinnati Prehospital Stroke Scale (CPSS). Cohorts varied between 50 and 1225 individuals, with 17.5% to 92% subsequently receiving a stroke diagnosis. Sensitivity and specificity for the same instrument varied across clinical settings. Studies varied in terms of quality, scoring 13–31/36 points using modified Standards for the Reporting of Diagnostic accuracy studies checklist. There was considerable variation in the detail reported about patient demographics, characteristics of false-negative patients and service context. Prevalence of instrument detectable stroke varied between cohorts and over time. CPSS and the similar FAST test generally report the highest level of sensitivity, with more complex instruments such as LAPSS reporting higher specificity at the cost of lower detection rates.ConclusionsAvailable data do not allow a strong recommendation to be made about the superiority of a stroke recognition instrument. Choice of instrument depends on intended purpose, and the consequences of a false-negative or false-positive result.
Hyperthermia Selectively Targets Human Papillomavirus in Cervical Tumors via p53-Dependent Apoptosis
Authors: Oei Arlene L. , van Leeuwen Caspar M. , ten Cate Rosemarie , Rodermond Hans M. , Buist Marrije R. , Stalpers Lukas J.A. , Crezee Johannes , Kok H. Petra , Medema Jan Paul , Franken Nicolaas A.P. 
Published: November 2015

Abstract
Human papillomavirus (HPV) is associated with cervical cancer, the third most common cancer in women. The high-risk HPV types 16 and 18 are found in over 70% of cervical cancers and produce the oncoprotein, early protein 6 (E6), which binds to p53 and mediates its ubiquitination and degradation. Targeting E6 has been shown to be a promising treatment option to eliminate HPV-positive tumor cells. In addition, combined hyperthermia with radiation is a very effective treatment strategy for cervical cancer. In this study, we examined the effect of hyperthermia on HPV-positive cells using cervical cancer cell lines infected with HPV 16 and 18, in vivo tumor models, and ex vivo–treated patient biopsies. Strikingly, we demonstrate that a clinically relevant hyperthermia temperature of 42°C for 1 hour resulted in E6 degradation, thereby preventing the formation of the E6–p53 complex and enabling p53-dependent apoptosis and G2-phase arrest. Moreover, hyperthermia combined with p53 depletion restored both the cell-cycle distribution and apoptosis to control levels. Collectively, our findings provide new insights into the treatment of HPV-positive cervical cancer and suggest that hyperthermia therapy could improve patient outcomes. Cancer Res; 75(23); 5120–9. ©2015 AACR.
M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment
Authors: Van Overmeire Eva , Stijlemans Benoît , Heymann Felix , Keirsse Jiri , Morias Yannick , Elkrim Yvon , Brys Lea , Abels Chloé , Lahmar Qods , Ergen Can , Vereecke Lars , Tacke Frank , De Baetselier Patrick , Van Ginderachter Jo A. , Laoui Damya 
Published: November 2015

Abstract
Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-IIhi and MHC-IIlo tumor-associated macrophage (TAM) subpopulations that originate from Ly6Chi monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6Chi monocytic precursors. M-CSFR signaling blockade shifted the MHC-IIlo/MHC-IIhi TAM balance in favor of the latter as observed by the preferential differentiation of Ly6Chi monocytes into MHC-IIhi TAMs. In addition, the genetic and functional signatures of MHC-IIlo TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-IIlo TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-IIhi phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade. Cancer Res; 76(1); 35–42. ©2015 AACR.
Antibody-Dependent Phagocytosis of Tumor Cells by Macrophages: A Potent Effector Mechanism of Monoclonal Antibody Therapy of Cancer
Authors: Gül Nuray , van Egmond Marjolein 
Published: November 2015

Abstract
Nowadays, it is impossible to imagine modern cancer treatment without targeted therapies, such as mAbs, that bind to tumor-associated antigens. Subsequently, mAbs can use a wide range of effector functions that mostly engage the immune system. mAbs can bridge immune effector cells with tumor cells, which can result in antibody-dependent cytotoxicity. Increasing evidence, however, identified macrophages as prominent effector cells and induction of antibody-dependent cell phagocytosis as one of the primary mechanisms of action mediated by mAbs. Macrophages are extremely effective in eliminating tumor cells from the circulation. Several immunosuppressive mechanisms may, however, hamper their function, particularly in solid malignancies. In this review, we discuss the evolving insight of macrophages as effector cells in mAb therapy and address novel (co)therapeutic strategies that may be used to fully unleash their cytotoxic capacity for the treatment of cancer. Cancer Res; 75(23); 5008–13. ©2015 AACR.
Decreased Serum Thrombospondin-1 Levels in Pancreatic Cancer Patients Up to 24 Months Prior to Clinical Diagnosis: Association with Diabetes Mellitus
Authors: Jenkinson Claire , Elliott Victoria L. , Evans Anthony , Oldfield Lucy , Jenkins Rosalind E. , O'Brien Darragh P. , Apostolidou Sophia , Gentry-Maharaj Aleksandra , Fourkala Evangelia-O , Jacobs Ian J. , Menon Usha , Cox Trevor , Campbell Fiona , Pereira Stephen P. , Tuveson David A. , Park B. Kevin , Greenhalf William , Sutton Robert , Timms John F. , Neoptolemos John P. , Costello Eithne 
Published: November 2015

Abstract
Purpose: Identification of serum biomarkers enabling earlier diagnosis of pancreatic ductal adenocarcinoma (PDAC) could improve outcome. Serum protein profiles in patients with preclinical disease and at diagnosis were investigated.
Experimental Design: Serum from cases up to 4 years prior to PDAC diagnosis and controls (UKCTOCS, n = 174) were studied, alongside samples from patients diagnosed with PDAC, chronic pancreatitis, benign biliary disease, type 2 diabetes mellitus, and healthy subjects (n = 298). Isobaric tags for relative and absolute quantification (iTRAQ) enabled comparisons of pooled serum from a test set (n = 150). Validation was undertaken using multiple reaction monitoring (MRM) and/or Western blotting in all 472 human samples and samples from a KPC mouse model.
Results: iTRAQ identified thrombospondin-1 (TSP-1) as reduced preclinically and in diagnosed samples. MRM confirmed significant reduction in levels of TSP-1 up to 24 months prior to diagnosis. A combination of TSP-1 and CA19-9 gave an AUC of 0.86, significantly outperforming both markers alone (0.69 and 0.77, respectively; P &lt; 0.01). TSP-1 was also decreased in PDAC patients compared with healthy controls (P &lt; 0.05) and patients with benign biliary obstruction (P &lt; 0.01). Low levels of TSP-1 correlated with poorer survival, preclinically (P &lt; 0.05) and at clinical diagnosis (P &lt; 0.02). In PDAC patients, reduced TSP-1 levels were more frequently observed in those with confirmed diabetes mellitus (P &lt; 0.01). Significantly lower levels were also observed in PDAC patients with diabetes compared with individuals with type 2 diabetes mellitus (P = 0.01).
Conclusions: Circulating TSP-1 levels decrease up to 24 months prior to diagnosis of PDAC and significantly enhance the diagnostic performance of CA19-9. The influence of diabetes mellitus on biomarker behavior should be considered in future studies. Clin Cancer Res; 22(7); 1734–43. ©2015 AACR.
Promoter Methylation Analysis Reveals That KCNA5 Ion Channel Silencing Supports Ewing Sarcoma Cell Proliferation
Authors: Ryland Katherine E. , Hawkins Allegra G. , Weisenberger Daniel J. , Punj Vasu , Borinstein Scott C. , Laird Peter W. , Martens Jeffrey R. , Lawlor Elizabeth R. 
Published: November 2015

Abstract
Polycomb proteins are essential regulators of gene expression in stem cells and development. They function to reversibly repress gene transcription via posttranslational modification of histones and chromatin compaction. In many human cancers, genes that are repressed by polycomb in stem cells are subject to more stable silencing via DNA methylation of promoter CpG islands. Ewing sarcoma is an aggressive bone and soft-tissue tumor that is characterized by overexpression of polycomb proteins. This study investigates the DNA methylation status of polycomb target gene promoters in Ewing sarcoma tumors and cell lines and observes that the promoters of differentiation genes are frequent targets of CpG-island DNA methylation. In addition, the promoters of ion channel genes are highly differentially methylated in Ewing sarcoma compared with nonmalignant adult tissues. Ion channels regulate a variety of biologic processes, including proliferation, and dysfunction of these channels contributes to tumor pathogenesis. In particular, reduced expression of the voltage-gated Kv1.5 channel has been implicated in tumor progression. These data show that DNA methylation of the KCNA5 promoter contributes to stable epigenetic silencing of the Kv1.5 channel. This epigenetic repression is reversed by exposure to the DNA methylation inhibitor decitabine, which inhibits Ewing sarcoma cell proliferation through mechanisms that include restoration of the Kv1.5 channel function.
Implications: This study demonstrates that promoters of ion channels are aberrantly methylated in Ewing sarcoma and that epigenetic silencing of KCNA5 contributes to tumor cell proliferation, thus providing further evidence of the importance of ion channel dysregulation to tumorigenesis. Mol Cancer Res; 14(1); 26–34. ©2015 AACR.
Sex Differences in Dabigatran Use, Safety, And Effectiveness In a Population-Based Cohort of Patients With Atrial Fibrillation
Authors: Avgil Tsadok Meytal , Jackevicius Cynthia A. , Rahme Elham , Humphries Karin H. , Pilote Louise 
Published: November 2015

Background—
Sex differences were observed with regard to warfarin treatment in patients with atrial fibrillation, with women having a higher risk of stroke compared with men. We aimed to compare sex differences in use, safety, and effectiveness of dabigatran.

Methods and Results—

We conducted a population-based cohort study of patients with atrial fibrillation using administrative data in Quebec, Canada, 1999 to 2013. Men and women who filled a prescription for dabigatran (110 and 150 mg bid) were compared with matched warfarin users with respect to their rates of stroke, bleeding, and myocardial infarction events, using propensity score analysis. The cohort comprised 31 786 women (50.4%) and 31 324 men (49.6%). Women had a higher baseline stroke risk and lower baseline bleeding risk compared with men. Women filled more prescriptions for the lower dabigatran dose compared with men (adjusted OR, 1.35; 95% confidence interval, 1.24–1.48). In multivariable analyses adjusted for propensity scores, dabigatran use was associated with a lower risk of bleeding compared with warfarin in men (
P
for interaction=0.008). Dabigatran was associated with a trend toward lower risk of stroke in women treated with the 150-mg dose (HR, 0.79; 95% confidence interval, 0.56–1.04), but was not associated with a difference in the risk of myocardial infarction compared with warfarin in either sex.

Conclusions—
In real-life practice, women are more frequently treated with low-dose dabigatran, yet a trend toward lower stroke rates in women taking high-dose dabigatran was observed. Men benefit from lower bleeding rates with dabigatran compared with warfarin.