In situ dynamics of Vibrio parahaemolyticus and Vibrio vulnificus in water, sediment and triploid Crassostrea virginica oysters cultivated in floating gear.

AIMS: To understand spatial-temporal distribution of Vibrio parahaemolyticus and Vibrio vulnificus in triploid Crassostrea virginica in off-bottom aquaculture. METHODS AND RESULTS: Oysters, sediments and water were seasonally collected in Georgia, USA. V. parahaemolyticus and V. vulnificus were quantified with tlh/tdh/trh and vvhA genes, respectively. No tdh/trh genes were detected. Highest concentrations of tlh gene were observed in summer sediments > oysters > water (105 /g, 104 /g, 103 /ml). VvhA concentrations were similar in sediments and oysters but never exceeded ≥3 × 101 /ml in water. Concentrations of tlh and vvhA genes correlated to temperature and turbidity, respectively, which along with their different spatial distribution indicated different environmental drivers. In oysters, ratios of the tlh and vvhA to 16S rRNA gene have increased from 0 to 10-1 and 10-2 in summer, while these ratios in water and sediments were lower by 2-3 orders of magnitude. CONCLUSIONS: Dynamics of tlh and vvhA concentrations and abundances suggested enrichment of V. parahaemolyticus and V. vulnificus by off-bottom triploid oysters in summer resulting in their abundance by far exceeding that in water. SIGNIFICANCE AND IMPACT OF THE STUDY: This first report on enrichment of Vibrio pathogens in triploid oysters with no direct contact to sediments reveals a threat to human health suggesting their monitoring in triploid off-bottom C. virginica aquaculture.

Automated next-generation profiling of genomic alterations in human cancers.

The lack of validated, distributed comprehensive genomic profiling assays for patients with cancer inhibits access to precision oncology treatment. To address this, we describe elio tissue complete, which has been FDA-cleared for examination of 505 cancer-related genes. Independent analyses of clinically and biologically relevant sequence changes across 170 clinical tumor samples using MSK-IMPACT, FoundationOne, and PCR-based methods reveals a positive percent agreement of >97%. We observe high concordance with whole-exome sequencing for evaluation of tumor mutational burden for 307 solid tumors (Pearson r = 0.95) and comparison of the elio tissue complete microsatellite instability detection approach with an independent PCR assay for 223 samples displays a positive percent agreement of 99%. Finally, evaluation of amplifications and translocations against DNA- and RNA-based approaches exhibits >98% negative percent agreement and positive percent agreement of 86% and 82%, respectively. These methods provide an approach for pan-solid tumor comprehensive genomic profiling with high analytical performance.

Chronic, programmed polypeptide delivery from an implanted, multireservoir microchip device.

Implanted drug delivery systems are being increasingly used to realize the therapeutic potential of peptides and proteins. Here we describe the controlled pulsatile release of the polypeptide leuprolide from microchip implants over 6 months in dogs. Each microchip contains an array of discrete reservoirs from which dose delivery can be controlled by telemetry.

Comparison of Continuous Versus Interrupted Chest Compressions during CPR in a Rural Community.

INTRODUCTION: Cardiopulmonary resuscitation (CPR) in patients with out-of-hospital cardiac arrest (OHCA) have interruption of manual chest compressions for airway management and breathing when performed by medical personnel trained by Advanced Cardiac Life Support (ACLS) standards. This interruption likely reduces blood flow and possibly survival. Traditional CPR (30:2 compressions to ventilations) was compared with continuous chest compressions, CCC (also termed Cardiocerebral Resuscitation, CCR) in a rural community. METHODS: A retrospective cohort analysis of three years of traditional CPR (June 2008 - May 2011) for OHCA was compared to three years of using CCC protocols (June 2011 - May 2014). Primary outcomes were survival at one and six months. RESULTS: There were 58 0HCA patients in the six year study period (June 2008 - May 2014). Forty (69%) received CPR and 18 (31%) received CCC. Two (5%) survived at least one month with CPR and eight (44%) survived at least one month with CCC (p = 0.0007). After six months, 0/40 (0%) who received CPR had survived and 6/18 (33%) who received CCC survived (p = 0.0018). For the patient found in ventricular fibrillation or tachycardia (a shockable rhythm), 0/13 (0.0%) survived one month after CPR and 7/9 (78%) survived with CCC (p < 0.01). After six months 0/13 (0.0%) survived with CPR and 6/9 (67%) survived with CCC (p < 0.05). CONCLUSIONS: For patients in a rural environment with OHCA, CCC had a more favorable outcome than traditional CPR. For the patient found in ventricular fibrillation or ventricular tachycardia, there was a profound survival benefit of CCC over CPR.

Development of Glatopa® (Glatiramer Acetate): The First FDA-Approved Generic Disease-Modifying Therapy for Relapsing Forms of Multiple Sclerosis.

The multiple sclerosis (MS) treatment landscape in the United States has changed dramatically over the past decade. While many disease-modifying therapies (DMTs) have been approved by the US Food and Drug Administration (FDA) for the treatment of relapsing forms of MS, DMT costs continue to rise. The availability of generics and biosimilars in the MS-treatment landscape is unlikely to have a major impact on clinical benefit. However, their availability will provide alternative treatment options and potentially lower costs through competition, thus increasing the affordability of and access to these drugs. In April 2015, the first generic version of the complex drug glatiramer acetate (Glatopa® 20 mg/mL) injection was approved in the United States as a fully substitutable generic for all approved indications of the 20 mg/mL branded glatiramer acetate (Copaxone®) dosage form. Despite glatiramer acetate's complex nature-being a chemically synthesized (ie, nonbiologic) mixture of peptides-the approval occurred without conducting any clinical trials. Rather, extensive structural and functional characterization was performed to demonstrate therapeutic equivalence to the innovator drug. The approval of Glatopa signifies an important milestone in the US MS-treatment landscape, with the hope that the introduction of generic DMTs and eventually biosimilar DMTs will lead to future improvements in the affordability and access of these much-needed treatments for MS.

Addition of chromosomal microarray and next generation sequencing to FISH and classical cytogenetics enhances genomic profiling of myeloid malignancies.

Comprehensive genetic profiling is increasingly important for the clinical workup of hematologic tumors, as specific alterations are now linked to diagnostic characterization, prognostic stratification and therapy selection. To characterize relevant genetic and genomic alterations in myeloid malignancies maximally, we utilized a comprehensive strategy spanning fluorescence in situ hybridization (FISH), classical karyotyping, Chromosomal Microarray (CMA) for detection of copy number variants (CNVs) and Next generation Sequencing (NGS) analysis. In our cohort of 569 patients spanning the myeloid spectrum, NGS and CMA testing frequently identified mutations and copy number changes in the majority of genes with important clinical associations, such as TP53, TET2, RUNX1, SRSF2, APC and ATM. Most importantly, NGS and CMA uncovered medically actionable aberrations in 75.6% of cases normal by FISH/cytogenetics testing. NGS identified mutations in 65.5% of samples normal by CMA, cytogenetics and FISH, whereas CNVs were detected in 10.1% cases that were normal by all other methodologies. Finally, FISH or cytogenetics, or both, were abnormal in 14.1% of cases where NGS or CMA failed to detect any changes. Multiple mutations and CNVs were found to coexist, with potential implications for patient stratification. Thus, high throughput genomic tumor profiling through targeted DNA sequencing and CNV analysis complements conventional methods and leads to more frequent detection of actionable alterations.

Demonstration of equivalence of a generic glatiramer acetate (Glatopa™).

Glatiramer acetate (GA) has been available under the brand name Copaxone® for nearly two decades. Recently, the US Food and Drug Administration (FDA) approved the first generic GA, Glatopa™, as fully substitutable for all indications for which Copaxone 20mg is approved; Glatopa also represents the first FDA-approved "AP-rated," substitutable generic for treating patients with MS. Glatiramer acetate is a complex mixture of polypeptides and, consequently, its characterization presented challenges not generally encountered in drug development. Despite its complexity, and without requiring any clinical data, approval was accomplished through an Abbreviated New Drug Application in which equivalence to Copaxone was evaluated across four criteria: starting materials and basic chemistry; structural signatures for polymerization, depolymerization, and purification; physicochemical properties; and biological and immunological properties. This article describes the rigorous overall scientific approach used to successfully establish equivalence between Glatopa and Copaxone, and presents key representative data from several of the comprehensive sets of physicochemical (structural) and biological (functional) assays that were conducted.

Contribution of Beta-HPV Infection and UV Damage to Rapid-Onset Cutaneous Squamous Cell Carcinoma during BRAF-Inhibition Therapy.

PURPOSE: BRAF-inhibition (BRAFi) therapy for advanced melanoma carries a high rate of secondary cutaneous squamous cell carcinoma (cSCC) and risk of other cancers. UV radiation and α-genus human papillomavirus (HPV) are highly associated with SCC, but a novel role for ß-genus HPV is suspected in BRAFi-cSCC. Cutaneous ß-HPV may act in concert with host and environmental factors in BRAFi-cSCC. EXPERIMENTAL DESIGN: Primary BRAFi-cSCC tissue DNA isolated from patients receiving vemurafenib or dabrafenib from two cancer centers was analyzed for the presence of cutaneous oncogenic viruses and host genetic mutations. Diagnostic specimens underwent consensus dermatopathology review. Clinical parameters for UV exposure and disease course were statistically analyzed in conjunction with histopathology. RESULTS: Twenty-nine patients contributed 69 BRAFi-cSCC lesions. BRAFi-cSCC had wart-like features (BRAFi-cSCC-WF) in 22% of specimens. During vemurafenib therapy, BRAFi-cSCC-WF arose 11.6 weeks more rapidly than conventional cSCC when controlled for gender and UV exposure (P value = 0.03). Among all BRAFi-cSCC, ß-genus HPV-17, HPV-38, HPV-111 were most frequently isolated, and novel ß-HPV genotypes were discovered (CTR, CRT-11, CRT-22). Sequencing revealed 63% of evaluated BRAFi-cSCCs harbored RAS mutations with PIK3CA, CKIT, ALK, and EGFR mutations also detected. CONCLUSIONS: We examined clinical, histopathologic, viral, and genetic parameters in BRAFi-cSCC demonstrating rapid onset; wart-like histomorphology; ß-HPV-17, HPV-38, and HPV-111 infection; UV damage; and novel ALK and CKIT mutations. Discovered ß-HPV genotypes expand the spectrum of tumor-associated viruses. These findings enhance our understanding of factors cooperating with BRAF inhibition that accelerate keratinocyte oncogenesis as well as broaden the knowledge base of multifactorial mediators of cancer in general.

The use of stratefied sampling of blend and dosage units to demonstrate adequacy of mix for powder blends.

In response to concerns expressed by applicants regarding inconsistent policies in establishing blend uniformity acceptance criteria to demonstrate adequacy of mix, the FDA Office of Generic Drugs (OGD) issued the draft document Guidance for Industry, ANDAs: Blend Uniformity Analysis (August 1999). Both generic and innovator pharmaceutical companies raised a number of concerns following the publication of this document. As a result, the Product Quality Research Institute (PQRI) Blend Uniformity Working Group (BUWG) was established in February 2000. One of the primary goals of this group was to draft a scientifically based alternative to the OGD document. The resulting recommendation addresses both FDA and industry concerns by substantially enhancing product quality assurance without increasing regulatory burden. The PQRI BUWG recommends that these blend and dosage unit uniformity requirements be administered uniformly throughout the industry. PQRI submitted the following recommendation to the FDA on December 31, 2002, providing the Agency with an alternative strategy to consider when drafting future regulatory policy to assess blend and dosage unit uniformity.

Enhanced brain connectivity in math-gifted adolescents: An fMRI study using mental rotation.

Mathematical giftedness is a form of intelligence related to enhanced mathematical reasoning that can be tested using a variety of numerical and spatial tasks. A number of neurobiological mechanisms related to exceptional mathematical reasoning ability have been postulated, including enhanced brain connectivity. We aimed to further investigate this possibility by comparing a group of mathematically gifted adolescents with an average math ability control group performing mental rotation of complex three-dimensional block figures. Functional magnetic resonance imaging (fMRI) data were collected and differences in intrahemispheric and interhemispheric connectivity between the groups were assessed using structural equation modeling (SEM). The math-gifted showed heightened intrahemispheric frontoparietal connectivity, as well as enhanced interhemispheric frontal connectivity between the dorsolateral prefrontal and premotor cortex. These enhanced connectivity patterns are consistent with previous studies linking increased activation of the frontal and parietal regions with high fluid intelligence, and may be a unique neural characteristic of the mathematically gifted brain.

Long-term stability and in vitro release of hPTH(1-34) from a multi-reservoir array.

PURPOSE: Therapeutic peptides generally exhibit poor oral bioavailability and require alternative methods of delivery. Implanted microelectromechanical systems-based multi-reservoir devices enable programmable, chronic, pulsatile peptide delivery. This report describes parathyroid hormone fragment (hPTH (1-34)) formulations suitable for delivery from a multi-reservoir array. METHODS: The stability of hPTH(1-34) lyophilizates obtained from aqueous acidic solutions was assessed by reverse phase high pressure liquid chromatography. An in vitro test device measured in vitro release kinetics. RESULTS: Novel, highly concentrated (>50 mg/mL) hPTH(1-34) solutions were dispensed as bulk samples (1-3 mg peptide) in vials and as individual doses (13-21 microg peptide) in reservoir arrays. Bulk and array samples were lyophilized and stored at 37 degrees C. Bulk lyophilizate hPTH(1-34) purity after lyophilization, after 8 weeks, and after 26 weeks exceeded 96%, 90%, and 80%, respectively. The hPTH(1-34) stored in multi-reservoir arrays exhibited similar purity over 29 weeks at 37 degrees C. Initially and over 29 weeks, over half of the peptide was consistently released from arrays into neutral, isotonic solution in less than 30 min with quantitative recoveries (>95%) within 3 h. CONCLUSIONS: Clinically relevant formulations of hPTH(1-34) for use with implantable multi-reservoir devices are achievable.

Dosage form development, in vitro release kinetics, and in vitro-in vivo correlation for leuprolide released from an implantable multi-reservoir array.

PURPOSE: Implanted multi-reservoir arrays improve dosing control relative to osmotic pumps or polymer depots. The limited reservoir volume requires concentrated formulations. This report describes the development of a stable solid phase formulation of leuprolide acetate for chronic in vivo delivery from a multi-reservoir microchip and examines the correlation between in vitro release kinetics and serum pharmacokinetics. MATERIALS AND METHODS: Concentrated formulations (>10% w/v) were prepared using small volume processing methods. Drug yield, release kinetics, and formulation stability were evaluated in vitro by HPLC. The correlation between in vitro and in vivo kinetic data was determined for a solid formulation by direct comparison of data sets and using absorption kinetics calculated from the Wagner-Nelson equation. RESULTS: High yield and the control of release kinetics by altering peptide formulation or reservoir geometry were demonstrated. Lyophilized leuprolide in a soluble solid matrix exhibited reproducible release kinetics and was stable (>95% leuprolide monomer) after 6 months at 37 degrees C. A strong correlation was found between in vitro release kinetics and in vivo absorption by direct comparison of data sets and using the Wagner-Nelson absorption (slopes of 1.01 and 0.91; R(2) 0.99). CONCLUSIONS: Reproducible releases of a stable solid leuprolide formulation from a multi-reservoir microchip were achieved in vitro. Chronic pulsatile release was subsequently performed in vivo. Comparison of in vitro and in vivo data reveals that pharmacokinetics were controlled by the rate of release from the device.