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Regulating tension in three-dimensional culture environments

Sun, 10/25/2015 - 7:56pm

The processes of development, repair, and remodeling of virtually all tissues and organs, are dependent upon mechanical signals including external loading, cell-generated tension, and tissue stiffness. Over the past few decades, much has been learned about mechanotransduction pathways in specialized two-dimensional culture systems; however, it has also become clear that cells behave very differently in two- and three-dimensional (3D) environments. Three-dimensional in vitro models bring the ability to simulate the in vivo matrix environment and the complexity of cell-matrix interactions together. In this review, we describe the role of tension in regulating cell behavior in three-dimensional collagen and fibrin matrices with a focus on the effective use of global boundary conditions to modulate the tension generated by populations of cells acting in concert. The ability to control and measure the tension in these 3D culture systems has the potential to increase our understanding of mechanobiology and facilitate development of new ways to treat diseased tissues and to direct cell fate in regenerative medicine and tissue engineering applications.

RNA recognition by the Caenorhabditis elegans oocyte maturation determinant OMA-1

Sun, 10/25/2015 - 7:56pm

Maternally supplied mRNAs encode proteins that pattern early embryos in many species. In the nematode Caenorhabditis elegans, a suite of RNA-binding proteins regulates expression of maternal mRNAs during oogenesis, the oocyte to embryo transition, and early embryogenesis. To understand how these RNA-binding proteins contribute to development, it is necessary to determine how they select specific mRNA targets for regulation. OMA-1 and OMA-2 are redundant proteins required for oocyte maturation--an essential part of meiosis that prepares oocytes for fertilization. Both proteins have CCCH type tandem zinc finger RNA-binding domains. Here, we define the RNA binding specificity of OMA-1 and demonstrate that OMA-1/2 are required to repress the expression of a glp-1 3'-UTR reporter in developing oocytes. OMA-1 binds with high affinity to a conserved region of the glp-1 3'-UTR previously shown to interact with POS-1 and GLD-1, RNA-binding proteins required for glp-1 reporter repression in the posterior of fertilized embryos. Our results reveal that OMA-1 is a sequence-specific RNA-binding protein required to repress expression of maternal transcripts during oogenesis and suggest that interplay between OMA-1 and other factors for overlapping binding sites helps to coordinate the transition from oocyte to embryo.

Hematocrit alters VerifyNow P2Y12 assay results independently of intrinsic platelet reactivity and clopidogrel responsiveness

Sun, 10/25/2015 - 7:56pm

BACKGROUND: The VerifyNow P2Y12 assay assesses the adequacy of clopidogrel therapy by measuring ADP-induced platelet activation in whole blood. Low hematocrit is associated with high clopidogrel on-treatment platelet reactivity (HTPR) defined by this assay.

OBJECTIVES: To characterize the effect of hematocrit on VerifyNow values and determine if it is due to hematocrit-dependent changes in intrinsic platelet reactivity or an in vitro assay phenomenon.

PATIENTS/METHODS: Adenosine diphosphate-induced platelet activation was measured using the VerifyNow P2Y12 assay, whole blood impedance and light transmission platelet aggregometry (LTA) before and after clopidogrel loading in 113 patients undergoing elective cardiac catheterization. Iso-TRAP-induced platelet activation was additionally measured using the VerifyNow device. Multivariate modeling employing clinical and laboratory variables was used to investigate the association between hematocrit and VerifyNow values.

RESULTS: VerifyNow P2Y12 reaction units (PRU) and iso-TRAP Base units before and after clopidogrel loading, but not their relative change, exhibited strong negative correlation with hematocrit (P < /= 0.0005 for both). While hematocrit remained a strong predictor of post-clopidogrel PRU (P = 0.001) in multivariate modeling, it was independent of post-clopidogrel ADP-induced platelet reactivity as measured by LTA (P = 0.001). Correcting for the effects of hematocrit resulted in a 15-39% reduction in the prevalence of HTPR defined by thresholds of 208-236 PRU.

CONCLUSIONS: The effect of hematocrit on VerifyNow PRU values is an in vitro phenomenon that is independent of intrinsic change in ADP-induced platelet reactivity and clopidogrel responsiveness. Correcting for hematocrit when using this assay may more accurately identify patients with HTPR that may benefit from alternative antiplatelet therapy.

Memory CD4 T cell-mediated immunity against influenza A virus: more than a little helpful

Sun, 10/25/2015 - 7:56pm

Recent observations have uncovered multiple pathways whereby CD4 T cells can contribute to protective immune responses against microbial threats. Incorporating the generation of memory CD4 T cells into vaccine strategies thus presents an attractive approach toward improving immunity against several important human pathogens, especially those against which antibody responses alone are inadequate to confer long-term immunity. Here, we review how memory CD4 T cells provide protection against influenza viruses. We discuss the complexities of protective memory CD4 T cell responses observed in animal models and the potential challenges of translating these observations into the clinic. Specifically, we concentrate on how better understanding of organ-specific heterogeneity of responding cells and defining multiple correlates of protection might improve vaccine-generated memory CD4 T cells to better protect against seasonal, and more importantly, pandemic influenza.

Efficacy and safety of natalizumab in Crohn's disease patients treated at 6 Boston academic hospitals

Sun, 10/25/2015 - 7:56pm

BACKGROUND: Despite trials demonstrating its efficacy, many physicians harbor concerns regarding the use of natalizumab in the treatment of patients with refractory Crohn's disease (CD). The purpose of this study was to perform a descriptive analysis of a series of CD patients not currently enrolled in a clinical trial.

METHODS: A retrospective case review of patients treated with natalizumab at 6 sites in Massachusetts: Boston Medical Center, Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Lahey Clinic, Massachusetts General Hospital, and UMass Medical Center.

RESULTS: Data on 69 CD patients on natalizumab were collected. At the start of treatment, patients' disease duration was 12 years. A high proportion of patients were women (68%), presented with perianal disease (65%) and upper gastrointestinal tract involvement (14%). Prior nonbiologic therapies were steroids (96%), thiopurines (94%), antibiotics (74%), methotrexate (58%), and at least two anti-tumor necrosis factor agent failures (81%). Sixty-nine percent (44 of 64 patients) with available medical evaluation had a partial or complete clinical response. Loss of response was 13% after an average of 1 year of treatment. Adverse events were infusion reactions, headaches, fever, and infections. No case of progressive multifocal leukoencephalopathy was observed.

CONCLUSIONS: In our clinical experience outside the context of a clinical trial, natalizumab is largely reserved for CD patients with extensive ileocolonic disease who have failed conventional immunosuppressants and of at least 2 anti-tumor necrosis factor agents. This drug is, however, well tolerated and offers significant clinical improvement for more than a year in one-third of these difficult-to-treat CD patients.

Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers

Sun, 10/25/2015 - 7:56pm

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered > 250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

A systematic review of pharmacological pain management in multiple sclerosis

Sun, 10/25/2015 - 7:56pm

BACKGROUND: Both chronic and acute pain have been cited as the most common symptoms amongst patients with multiple sclerosis (MS), with recent prevalence estimates as high as 83 %. The evidence for spasticity and trigeminal neuralgia pharmacological treatments in MS has been systematically reviewed, but no equivalent reviews have been published concerning MS pain unrelated to these two conditions.

OBJECTIVE: Our objective was to systematically review pain management strategies for the reduction of non-spastic and non-trigeminal neuralgic pain in MS patients.

DATA SOURCES: Experimental studies published after 1965 were chosen for review by searching electronic databases (e.g. PubMed, Cumulative Index to Nursing and Allied Health Literature, Science Citation Index Expanded, Conference Proceedings Citation Index-Science, and and bibliographies/citations of previously published reviews.

STUDY SELECTION: Studies were included if all participants were adults clinically diagnosed with MS, study sample was not restricted to participants with spasticity or trigeminal neuralgia, and participant-reported pain was a primary or secondary outcome measured with a validated tool.

STUDY APPRAISAL AND SYNTHESIS METHODS: Records were screened and methodological qualities of included studies were assessed independently by two reviewers under the supervision of another reviewer using the principles recommended in the Cochrane Handbook for Systematic Review of Interventions and the levels of evidence espoused by the American Academy of Neurology.

RESULTS: Fifteen studies met the inclusion and exclusion criteria for review; interventions included antidepressants, anticonvulsants, dextromethorphan/quinidine, cannabinoids, and opioids/opioid antagonists. The pooled effect size for anticonvulsants (4 studies, 78 participants) was -1.88 (95 % CI: -3.13 to -0.64). The pooled effect size for cannabinoids (3 studies, 565 participants) was 0.08 (95 % CI: -0.74 to 0.89). Overall, only four trials reported Class 1 evidence. For these trials, dizziness was the most commonly reported adverse event, followed by nausea and somnolence.

LIMITATIONS: The relatively small number of trials in MS patients with chronic pain precludes specific recommendations for treatment strategies. The review did not reveal any studies of drug combinations.

CONCLUSIONS: More trials with rigorous design and reporting are needed to determine effective treatments for specific pain types presenting in people living with MS.

Characterization and optimization of the glucan particle-based vaccine platform

Sun, 10/25/2015 - 7:56pm

Glucan particles (GPs) are hollow porous Saccharomyces cerevisiae cell walls that are treated so that they are composed primarily of beta-1,3-d-glucans. Our previous studies showed that GPs can serve as an effective vaccine platform. Here, we characterize CD4(+) T-cell and antibody responses in immunized mice as a function of antigen (ovalbumin) encapsulation, antigen dose, particle numbers, time, immunization schedule, and trapping methods. Although we found that GPs served as an effective adjuvant when admixed with free antigens for IgG1 antibody production, stronger CD4(+) T-cell and IgG2c antibody responses were stimulated when antigens were encapsulated inside GPs, suggesting that the GP platform acts as both an adjuvant and a delivery system. Vigorous T-cell and antibody responses were stimulated even at submicrogram antigen doses, as long as the number of GPs was kept at 5 x 10(7) particles per immunization. One prime and one boost were sufficient to elicit robust immune responses. In addition, strong antigen-specific antibody and T-cell responses prevailed up to 20 months following the last immunization, including those of gamma interferon (IFN-gamma), interleukin 17A (IL-17A), and dual IFN-gamma/IL-17A-secreting CD4(+) T cells. Finally, robust immune responses were observed using generally recognized as safe (GRAS) materials (alginate and calcium, with or without chitosan) to trap antigens within GPs. Thus, these studies demonstrate that antigens encapsulated into GPs make an effective vaccine platform that combines adjuvanticity and antigen delivery to elicit strong durable immune responses at relatively low antigen doses using translationally relevant formulations.

QTc-prolongation in posterior circulation stroke

Sun, 10/25/2015 - 7:56pm

BACKGROUND AND PURPOSE: To evaluate the relationship between infarct location and QTc-prolongation in patients with posterior circulation strokes.

METHODS: Admission electrocardiograms (ECG) of 131 patients among a prospective sample of 407 consecutive adult patients in the New England Medical Center Posterior Circulation Registry were retrospectively analyzed. The QT interval (ms) was measured and corrected using Bazett's formula (QTcBazett) as well as linear regression functions (QTcLinear). QTcBazett > 440 ms and QTcLinear > /= 450 ms for men ( > /=460 ms for women) were considered prolonged. Multivariable linear and logistic regression analyses were used to identify independent predictors of the QTc.

RESULTS: Overall, 34% of patients had a prolonged QTcBazett and 7% had a prolonged QTcLinear noted on the admission ECG. There was a significant association between temporal lobe infarction and QTcBazett and QTcLinear (p < 0.001 for both) in multivariable linear regression analyses adjusting for demographics, ECG parameters, and preadmission medication use. In multivariable logistic regression analysis, temporal lobe infarction emerged as an independent predictor of prolonged QTcBazett (p = 0.009) and QTcLinear (p = 0.008), respectively. Sensitivity analyses excluding patients with transient ischemic attack yielded similar results. Exploratory analyses indicated that patients with temporal lobe infarction had worse functional 30-day outcomes in multivariable logistic regression (p = 0.022). However, there was no significant association between QTc and 30-day functional outcome.

CONCLUSIONS: QTc-prolongation is common after posterior circulation stroke and associated with temporal lobe infarction. Prospective studies are needed to confirm these preliminary findings and to examine potential long-term consequences.

Investigating the effect of characteristic x-rays in cadmium zinc telluride detectors under breast computerized tomography operating conditions

Sun, 10/25/2015 - 7:56pm

A number of research groups have been investigating the use of dedicated breast computerized tomography (CT). Preliminary results have been encouraging, suggesting an improved visualization of masses on breast CT as compared to conventional mammography. Nonetheless, there are many challenges to overcome before breast CT can become a routine clinical reality. One potential improvement over current breast CT prototypes would be the use of photon counting detectors with cadmium zinc telluride (CZT) (or CdTe) semiconductor material. These detectors can operate at room temperature and provide high detection efficiency and the capability of multi-energy imaging; however, one factor in particular that limits image quality is the emission of characteristic x-rays. In this study, the degradative effects of characteristic x-rays are examined when using a CZT detector under breast CT operating conditions. Monte Carlo simulation software was used to evaluate the effect of characteristic x-rays and the detector element size on spatial and spectral resolution for a CZT detector used under breast CT operating conditions. In particular, lower kVp spectra and thinner CZT thicknesses were studied than that typically used with CZT based conventional CT detectors. In addition, the effect of characteristic x-rays on the accuracy of material decomposition in spectral CT imaging was explored. It was observed that when imaging with 50-60 kVp spectra, the x-ray transmission through CZT was very low for all detector thicknesses studied (0.5-3.0 mm), thus retaining dose efficiency. As expected, characteristic x-ray escape from the detector element of x-ray interaction increased with decreasing detector element size, approaching a 50% escape fraction for a 100 mum size detector element. The detector point spread function was observed to have only minor degradation with detector element size greater than 200 mum and lower kV settings. Characteristic x-rays produced increasing distortion in the spectral response with decreasing detector element size. If not corrected for, this caused a large bias in estimating tissue density parameters for material decomposition. It was also observed that degradation of the spectral response due to characteristic x-rays caused worsening precision in the estimation of tissue density parameters. It was observed that characteristic x-rays do cause some degradation in the spatial and spectral resolution of thin CZT detectors operating under breast CT conditions. These degradations should be manageable with careful selection of the detector element size. Even with the observed spectral distortion from characteristic x-rays, it is still possible to correctly estimate tissue parameters for material decomposition using spectral CT if accurate modeling is used.

DNA vaccine prime followed by boost with live attenuated virus significantly improves antigen-specific T cell responses against human cytomegalovirus

Sun, 10/25/2015 - 7:56pm

As a leading cause of congenital infection and a major threat to immunocompromised individuals, human cytomegalovirus (HCMV) is a major global public health concern. Effective HCMV vaccines would need to induce potent and balanced humoral and cellular immune responses. In this pilot study, immunogenicity studies were conducted in mice to examine HCMV antigen-specific antibody and T cell responses when a heterologous prime-boost immunization strategy was tested. DNA vaccines expressing either targets of protective antibody responses (gB and gM/gN) or well characterized T cell immunogens (pp65, pp150, and IE1) were used as the priming immunization while the live attenuated HCMV vaccine Towne strain was used as the boost, which may act like an inactivated vaccine due to the inability of HCMV to replicate in a mouse host. Our data indicate that while DNA vaccines were effective in priming HCMV-specific antibody responses, the final titers of gB- or gM-specific antibodies were not much different from those elicited by using multiple immunizations of HCMV alone. In contrast, DNA priming significantly enhanced T cell responses against gB, pp65, and IE1 as measured by IFN-gamma. However, HCMV alone was not effective in eliciting strong T cell immune responses when used in a mouse host. Our data indicate that the complexity of antigen composition from a large virus, such as HCMV, may affect the profile of immune responses when viral vaccines are used as a boost.

Apoptosis induced by the fungal pathogen gliotoxin requires a triple phosphorylation of Bim by JNK

Sun, 10/25/2015 - 7:56pm

We previously reported that gliotoxin (GT), the major virulence factor of the mold Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients, induces apoptosis in a Bak-dependent manner. The signaling pathway leading to Bak activation and subsequent mitochondrial outer membrane permeabilization (MOMP) is elusive. Here, we show that GT and the supernatant of A. fumigatus (but not its GT-defective mutant) activate the JNK pathway and require a co-operative JNK-mediated BimEL phosphorylation at three sites (S100, T112 and S114) to induce apoptosis in mouse fibroblasts, human bronchial and mouse alveolar epithelial cells. Cells (i) treated with the JNK inhibitor SP600125, (ii) deleted or knocked down for JNK1/2 or Bim or (iii) carrying the BimEL triple phosphomutant S100A/T112A/S114A instead of wild-type BimEL are similarly resistant to GT-induced apoptosis. Triple-phosphorylated BimEL is more stable, redistributes from a cytoskeletal to a membrane fraction, better interacts with Bcl-2 and Bcl-xL and more effectively activates Bak than the unphosphorylated mutant. These data indicate that JNK-mediated BimEL phosphorylation at S100, T112 and S114 constitutes a novel regulatory mechanism to activate Bim in response to apoptotic stimuli.

Evolving concepts of gliogenesis: a look way back and ahead to the next 25 years

Sun, 10/25/2015 - 7:56pm

Glial cells are present in all organisms with a CNS and, with increasing brain complexity, glial cells have undergone substantive increases in cell number, diversity, and functions. Invertebrates, such as Drosophila, possess glial subtypes with similarity to mammalian astrocytes in their basic morphology and function, representing fertile ground for unraveling fundamental aspects of glial biology. Although glial subtypes in simple organisms may be relatively homogenous, emerging evidence suggests the possibility that mammalian astrocytes might be highly diversified to match the needs of local neuronal subtypes. In this Perspective, we review classic and new roles identified for astrocytes and oligodendrocytes by recent studies. We propose that delineating genetic and developmental programs across species will be essential to understand the core functions of glia that allow enhanced neuronal function and to achieve new insights into glial roles in higher-order brain function and neurological disease.

Heart rate is a prognostic risk factor for myocardial infarction: a post hoc analysis in the PERFORM (Prevention of cerebrovascular and cardiovascular Events of ischemic origin with teRutroban in patients with a history oF ischemic strOke or tRansient isc

Sun, 10/25/2015 - 7:56pm

BACKGROUND: Elevated resting heart rate is known to be detrimental to morbidity and mortality in cardiovascular disease, though its effect in patients with ischemic stroke is unclear. We analyzed the effect of baseline resting heart rate on myocardial infarction (MI) in patients with a recent noncardioembolic cerebral ischemic event participating in PERFORM.

METHODS: We compared fatal or nonfatal MI using adjusted Cox proportional hazards models for PERFORM patients with baseline heart rate < 70 bpm (n=8178) or > /=70 bpm (n=10,802). In addition, heart rate was analyzed as a continuous variable. Other cerebrovascular and cardiovascular outcomes were also explored.

RESULTS: Heart rate > /=70 bpm was associated with increased relative risk for fatal or nonfatal MI (HR 1.32, 95% CI 1.03-1.69, P=0.029). For every 5-bpm increase in heart rate, there was an increase in relative risk for fatal and nonfatal MI (11.3%, P=0.0002). Heart rate > /=70 bpm was also associated with increased relative risk for a composite of fatal or nonfatal ischemic stroke, fatal or nonfatal MI, or other vascular death (excluding hemorrhagic death) (P < 0001); vascular death (P < 0001); all-cause mortality (P < 0001); and fatal or nonfatal stroke (P=0.04). For every 5-bpm increase in heart rate, there were increases in relative risk for fatal or nonfatal ischemic stroke, fatal or nonfatal MI, or other vascular death (4.7%, P < 0.0001), vascular death (11.0%, P < 0.0001), all-cause mortality (8.0%, P < 0.0001), and fatal and nonfatal stroke (2.4%, P=0.057).

CONCLUSION: Elevated heart rate > /=70 bpm places patients with a noncardioembolic cerebral ischemic event at increased risk for MI.

Cutting edge: TLR signaling licenses IRAK1 for rapid activation of the NLRP3 inflammasome

Sun, 10/25/2015 - 7:55pm

Activation of the NLRP3 inflammasome by diverse stimuli requires a priming signal from TLRs and an activation signal from purinergic receptors or pore-forming toxins. In this study, we demonstrate, through detailed analysis of NLRP3 activation in macrophages deficient in key downstream TLR signaling molecules, that MyD88 is required for an immediate early phase, whereas Toll/IL-1R domain-containing adapter inducing IFN-beta is required for a subsequent intermediate phase of posttranslational NLRP3 activation. Both IL-1R-associated kinase (IRAK) 1 and IRAK4 are critical for rapid activation of NLRP3 through the MyD88 pathway, but only IRAK1 is partially required in the Toll/IL-1R domain-containing adapter inducing IFN-beta pathway. IRAK1 and IRAK4 are also required for rapid activation of NLRP3 by Listeria monocytogenes, as deletion of IRAK1 or IRAK4 led to defective inflammasome activation. These findings define the pathways that lead to rapid NLRP3 activation and identify IRAK1 as a critical mediator of a transcription-independent,inflammasome-dependent early warning response to pathogenic infection.

Assessing the need for improved access to rheumatology care: a survey of Massachusetts community health center medical directors

Sun, 10/25/2015 - 7:55pm

OBJECTIVE: Access to rheumatology care can expedite diagnosis and treatment of rheumatic diseases and reduce disparities. We surveyed community health center (CHC) medical directors to evaluate rheumatology care in underserved areas and potential strategies for improvement.

METHODS: We identified 77 Massachusetts CHCs that provide adult medical services and sent a 40-item survey to their physician medical directors. Survey questions assessed the centers' prevalence of rheumatic diseases, prescribing practices of immunosuppressive medications, and possible interventions to improve care. We compared CHC characteristics and rheumatology-specific items and then stratified our data by the response to whether improved access to rheumatology care was needed. Qualitative data were analyzed thematically.

RESULTS: Thirty-six CHC physician medical directors returned surveys (47% response rate). Fifty-five percent indicated a need for better access to rheumatology care. Eighty-six percent of CHC physicians would not start a patient with rheumatoid arthritis on a disease-modifying antirheumatic drug; 94% would not start a patient with systemic lupus erythematosus on an immunosuppressant. When we compared CHCs that reported needing better access to rheumatology care to those that did not, the former described a significantly greater percentage of patients with private insurance or Medicaid who required outside rheumatology referrals (P < 0.05). Language differences and insurance status were highlighted as barriers to obtaining rheumatology care. Sixteen directors (57%) ranked the patient navigator-a layperson to assist with care coordination-as their first-choice intervention.

CONCLUSIONS: Community health center medical directors expressed a need for better access to rheumatology services. A patient navigator for rheumatic diseases was proposed to help improve care and reduce health disparities.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis

Sun, 10/25/2015 - 7:55pm

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position or gene--we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Cost-effectiveness of helicopter versus ground emergency medical services for trauma scene transport in the United States

Sun, 10/25/2015 - 7:55pm

STUDY OBJECTIVE: We determine the minimum mortality reduction that helicopter emergency medical services (EMS) should provide relative to ground EMS for the scene transport of trauma victims to offset higher costs, inherent transport risks, and inevitable overtriage of patients with minor injury.

METHODS: We developed a decision-analytic model to compare the costs and outcomes of helicopter versus ground EMS transport to a trauma center from a societal perspective during a patient's lifetime. We determined the mortality reduction needed to make helicopter transport cost less than $100,000 and $50,000 per quality-adjusted life-year gained compared with ground EMS. Model inputs were derived from the National Study on the Costs and Outcomes of Trauma, National Trauma Data Bank, Medicare reimbursements, and literature. We assessed robustness with probabilistic sensitivity analyses.

RESULTS: Helicopter EMS must provide a minimum of a 15% relative risk reduction in mortality (1.3 lives saved/100 patients with the mean characteristics of the National Study on the Costs and Outcomes of Trauma cohort) to cost less than $100,000 per quality-adjusted life-year gained and a reduction of at least 30% (3.3 lives saved/100 patients) to cost less than $50,000 per quality-adjusted life-year. Helicopter EMS becomes more cost-effective with significant reductions in patients with minor injury who are triaged to air transport or if long-term disability outcomes are improved.

CONCLUSION: Helicopter EMS needs to provide at least a 15% mortality reduction or a measurable improvement in long-term disability to compare favorably with other interventions considered cost-effective. Given current evidence, it is not clear that helicopter EMS achieves this mortality or disability reduction. Reducing overtriage of patients with minor injury to helicopter EMS would improve its cost-effectiveness. Inc. All rights reserved.

Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays

Sun, 10/25/2015 - 7:55pm

BACKGROUND: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.

METHODOLOGY: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.

FINDINGS AND CONCLUSIONS: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

Oncofetal protein IMP3: a new diagnostic biomarker for laryngeal carcinoma

Sun, 10/25/2015 - 7:55pm

An accurate diagnosis of laryngeal squamous cell carcinoma (LSCC) is essential for patient management. The diagnosis of LSCC, especially in superficial biopsies, can present a diagnostic challenge for pathologists. The ability to diagnose LSCC would be greatly improved by the detection of a tumor-associated antigen. IMP3 is an oncofetal protein associated with aggressive and advanced tumors and is specifically expressed in malignant tumors but not found in benign tissues. The aim of this study was to determine the expression and diagnostic value of IMP3 in LSCC to determine whether it can serve as a diagnostic biomarker. A total of 238 cases (laryngectomy, n = 121; biopsy, n = 117) consisting of 11 laryngeal carcinoma in situ/severe dysplasia and 227 invasive LSCC were examined by immunohistochemistry for IMP3 expression. IMP3 showed strong cytoplasmic staining in 217 (92%) of 238 LSCCs regardless of histologic grade. In addition, 58 (89%) of 65 small biopsies ( < /=5 mm in greatest dimension) containing a minute amount of carcinoma were positive for IMP3. In contrast to malignant tumors, IMP3 expression was not found in any of the adjacent benign squamous epithelium (0/118 cases; 0%), mild or moderate dysplasia (0/139 cases; 0%), or pseudoepitheliomatous hyperplasia (0/99 cases; 0%). In summary, we are the first to describe that IMP3 is a highly sensitive and specific biomarker for LSCC. The expression of IMP3 in LSCC can be used as a positive biomarker to increase the level of confidence in establishing a definitive diagnosis of a malignancy in laryngeal biopsy.