Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

A theoretical analysis of coherent cross-peaks in polarization selective 2DIR for detection of cross-α fibrils.

The coupled amide-I vibrational modes in peptide systems such as fibrillar aggregates can often provide a wealth of structural information, although the associated spectra can be difficult to interpret. Using exciton scattering calculations, we characterized the polarization selective 2DIR peak patterns for cross-α peptide fibrils, a challenging system given the similarity between the monomeric and fibrillar structures, and interpret the results in light of recently collected 2D data on the cross-α peptide phenol soluble modulin α3. We find that stacking of α-helices into fibrils couples the bright modes across helical subunits, generating three new Bloch-like extended excitonic states that we designate A⊥, E∥, and E⊥. Coherent superpositions of these states in broadband 2DIR simulations lead to characteristic signals that are sensitive to fibril length and match the experimental 2DIR spectra.

Cross-α/ß polymorphism of PSMα3 fibrils.

The formation of ordered cross-ß amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α-forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-ß polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Hypertrophy of the sublime tubercle in elbow ulnar collateral ligament injuries: a case series of baseball pitchers undergoing ulnar collateral ligament reconstruction with short-term follow-up.

Background: The ulnar collateral ligament (UCL) of the elbow is subject to repetitive stress in overhead throwing athletes. This can lead to morphologic changes at the bony attachments of the UCL and hypertrophy of the sublime tubercle. The purpose of this case series is to describe the surgical details and clinical outcomes of a series of competitive baseball pitchers with hypertrophic sublime tubercles who underwent UCL reconstruction (UCLR). Methods: All baseball pitchers who were treated for UCL injuries with significant hypertrophy of the sublime tubercle on preoperative imaging were included in the series. Clinical history, preoperative imaging, intraoperative findings during UCLR, and postoperative outcomes measured with the Conway scale were described. Results: Ten players (average age of 22.9 years [range 13-39]) were included (average follow-up 20.4 months [range 3-38 months]). Five patients also had symptoms of ulnar nerve compression, with 4 requiring transposition at the time of ULCR and 1 at 3 months postoperatively. Bony hypertrophy of the sublime tubercle was confirmed intraoperatively in all cases and excised before UCLR with the docking technique. Of the 7 patients with at least 12-month follow-up postoperatively, 6 had excellent outcomes, and 1 had a fair outcome. Conclusion: Although UCLR in the setting of hypertrophic sublime tubercle can be more complex than typical UCLR, excellent outcomes are achievable with preoperative recognition and surgical planning.

Acute Herpes Simplex Esophagitis in an Immunocompetent Adult with Eosinophilic Esophagitis.

Herpes simplex esophagitis (HSE) represents a rare entity in immunocompetent patients and has infrequently been described in association with eosinophilic esophagitis (EoE). Causality in this rare association remains a debated topic. We present a case of HSE occurring in an immunocompetent adult with EoE and review the literature in support of EoE induced mucosal disruption predisposing to increased risk of HSE.

The exploration of aza-quinolines as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors with low brain exposure.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

Quadriceps strength changes across the continuum of care in adolescent male and female athletes with anterior cruciate ligament injury and reconstruction.

OBJECTIVES: To examine the quadriceps strength (QUADS) on the surgical (SURG) and non-surgical (Non-SURG) limbs in adolescent male and female athletes at pre-operative (PRE), 12 weeks post-operative (12WK), and return to sport (RTS) time points following ACL injury and reconstruction. DESIGN: Prospective cohort study design. SETTING: Clinical Research Laboratory. PARTICIPANTS: 66 adolescent athletes. MAIN OUTCOME MEASURES: Isokinetic QUADS of the SURG and Non-SURG limbs at the PRE, 12WK, and RTS time points were assessed and compared between each time point. RESULTS: Both male and female participants had significantly lower 12 WK QUADS in the SURG limb than the PRE QUADS, but the RTS QUADS was significantly greater than the 12WK QUAD (p < 0.05). However, only female participants had greater RTS QUADS as compared to the PRE QUADS (p < 0.001). For the Non-SURG limb, only male participants had a significant improvement over time (PRE vs RTS; p < 0.001). CONCLUSION: Adolescent males and females differ in their QUADS recovery across the continuum of care following ACLR. Clinicians should consider this pattern of recovery when treating adolescent males and females.

Resolution Enhancement in Wide-Field IR Imaging and Time-Domain Spectroscopy Using Dielectric Microspheres.

Wide-field imaging through dielectric microspheres has emerged in recent years as a simple and effective approach for generating super-resolution images at visible wavelengths. We present, to our knowledge, the first demonstration that dielectric microspheres can be used in a wide-field infrared (IR) microscope to enhance the far field resolution. We have observed a substantial improvement in resolution and magnification when images are collected through polystyrene microspheres. In addition, we demonstrate that spectroscopic imaging with a pulse-shaper based femtosecond mid-IR laser system is possible through the dielectric microspheres, which is a promising first step toward applying this technique to ultrafast IR imaging methods such as pump-probe and 2DIR microscopy.

Author Correction: CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Does liquid-liquid phase separation drive peptide folding?

Proline-arginine (PR) dipeptide repeats have been shown to undergo liquid-liquid phase separation and are an example of a growing number of intrinsically disordered proteins that can assemble into membraneless organelles. These structures have been posited as nucleation sites for pathogenic protein aggregation. As such, a better understanding of the effects that the increased local concentration and volumetric crowding within droplets have on peptide secondary structure is necessary. Herein we use Fourier transform infrared (FTIR) and two-dimensional infrared (2DIR) spectroscopy to show that formation of droplets by PR20 accompanies changes in the amide-I spectra consistent with folding into poly-proline helical structures.

CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer.

Triple-negative breast cancer (TNBC), which lacks estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression, is closely related to basal-like breast cancer. Previously, we and others report that cyclin E/cyclin-dependent kinase 2 (CDK2) phosphorylates enhancer of zeste homolog 2 (EZH2) at T416 (pT416-EZH2). Here, we show that transgenic expression of phospho-mimicking EZH2 mutant EZH2T416D in mammary glands leads to tumors with TNBC phenotype. Coexpression of EZH2T416D in mammary epithelia of HER2/Neu transgenic mice reprograms HER2-driven luminal tumors into basal-like tumors. Pharmacological inhibition of CDK2 or EZH2 allows re-expression of ERα and converts TNBC to luminal ERα-positive, rendering TNBC cells targetable by tamoxifen. Furthermore, the combination of either CDK2 or EZH2 inhibitor with tamoxifen effectively suppresses tumor growth and markedly improves the survival of the mice bearing TNBC tumors, suggesting that the mechanism-based combination therapy may be an alternative approach to treat TNBC.

Computer Assisted Cobb Angle Measurements: A novel algorithm.

BACKGROUND: The standard for evaluating scoliosis is PA radiographs using Cobb angle to measure curve magnitude. Newer PACS systems allow easier Cobb angle calculations, but have not improved inter/intra observer precision of measurement. Cobb angle and its progression are important to determine treatment; therefore, angle variability is not optimal. This study seeks to demonstrate that a performance equivalent to that achieved in the manual method is possible using a novel computer algorithm with limited user input. The authors compared Cobb angles from predetermined spinal levels in the average attending score versus the computer assisted approach. METHODS: Retrospective analysis of PA radiographs from 58 patients previously evaluated for scoliosis was collected. Predesignated spinal levels (e.g., T2-T10) were assigned for different curves and calculated by Cobb method. Four spine surgeons evaluated these Cobb angles. Their average scores were measured and compared to formulated values using the novel computer-based algorithm. Literature reports inter-observer reliability is 6.3-7.2degrees. Limits of accuracy were set at 5 degrees of average orthopedic surgeons' score. RESULTS: The computer-based algorithm calculated Cobb angles within 5 degrees of orthopedic surgeons' average with a standard deviation of 3.2 degrees. This result was based on a 95% confidence interval with p values <0.001. The computer algorithm was plotted against average angle determined by the surgeons, with individual determinations and linear regression (r2 =0.90). The average difference between surgeons' measures and computer algorithm was 0.4 degrees(SD= 3.2degrees, n=79). There was a tendency for the computer algorithm program to overestimate the angle at larger angles, but difference was small with r2 = 0.09. CONCLUSIONS: Our study showed the novel computer based algorithm was an efficient and reliable method to assess scoliotic curvature in the coronal plane with the possibility of expediting clinic visits, ensuring reliability of calculation and decreasing patient exposure to radiation. Level of Evidence: III.

The public health value of vaccines beyond efficacy: methods, measures and outcomes.

BACKGROUND: Assessments of vaccine efficacy and safety capture only the minimum information needed for regulatory approval, rather than the full public health value of vaccines. Vaccine efficacy provides a measure of proportionate disease reduction, is usually limited to etiologically confirmed disease, and focuses on the direct protection of the vaccinated individual. Herein, we propose a broader scope of methods, measures and outcomes to evaluate the effectiveness and public health impact to be considered for evidence-informed policymaking in both pre- and post-licensure stages. DISCUSSION: Pre-licensure: Regulatory concerns dictate an individually randomised clinical trial. However, some circumstances (such as the West African Ebola epidemic) may require novel designs that could be considered valid for licensure by regulatory agencies. In addition, protocol-defined analytic plans for these studies should include clinical as well as etiologically confirmed endpoints (e.g. all cause hospitalisations, pneumonias, acute gastroenteritis and others as appropriate to the vaccine target), and should include vaccine-preventable disease incidence and 'number needed to vaccinate' as outcomes. Post-licensure: There is a central role for phase IV cluster randomised clinical trials that allows for estimation of population-level vaccine impact, including indirect, total and overall effects. Dynamic models should be prioritised over static models as the constant force of infection assumed in static models will usually underestimate the effectiveness and cost-effectiveness of the immunisation programme by underestimating indirect effects. The economic impact of vaccinations should incorporate health and non-health benefits of vaccination in both the vaccinated and unvaccinated populations, thus allowing for estimation of the net social value of vaccination. CONCLUSIONS: The full benefits of vaccination reach beyond direct prevention of etiologically confirmed disease and often extend across the life course of a vaccinated person, prevent outcomes in the wider community, stabilise health systems, promote health equity, and benefit local and national economies. The degree to which vaccinations provide broad public health benefits is stronger than for other preventive and curative interventions.

The Development and Validation of a Subjective Assessment Tool for the Hip in the Athletic Population.

BACKGROUND: No validated functional assessments are available that are designed specifically to evaluate the performance and function of the athletic hip. Subsections of some validated outcome assessments address recreation, but a full assessment dedicated to athletic hip function does not exist. Current hip scoring systems may not be sensitive to subtle changes in performance and function in an athletic, younger population. HYPOTHESIS: The patient-athlete subjective scoring system developed in this study will be validated, reliable, and responsive in the evaluation of hip function in the athlete. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Based on the results of a pilot questionnaire administered to 18 athletic individuals, a final 10-item questionnaire was developed. Two hundred fifty competitive athletes from multiple sports completed the final questionnaire and 3 previously validated hip outcome assessments. Each athlete was self-assigned to 1 of 3 injury categories: (1) playing without hip/groin trouble; (2) playing, but with hip/groin trouble; and (3) not playing due to hip/groin trouble. The injury categories contained 196, 40, and 14 athletes, respectively. Correlations between the assessment scores and injury categories were measured. Responsiveness testing was performed in an additional group of 24 injured athletes, and their scores before and after intervention were compared. RESULTS: The Kerlan-Jobe Orthopaedic Clinic (KJOC) Athletic Hip Score showed high correlation with the modified Harris Hip Score, the Nonarthritic Hip Score, and the International Hip Outcome Tool. The new score stratified athletes by injury category, demonstrated responsiveness and accuracy, and varied appropriately with improvements in injury category after treatment of injuries. CONCLUSION: The new KJOC Athletic Hip Score is valid, reliable, and responsive for evaluation of the hip in an athletic population. The results support its use for the functional assessment of the hip in future studies.

Open Navicular Dislocation With Midfoot Dissociation in a 45-Year-Old Man.

Traumatic dislocation of an intact tarsal navicular is an uncommon injury. In this article, we report a case of open medial navicular dislocation in a 45-year-old man. The injury was managed with open reduction and stabilization with Kirschner wires within the acute period. Ten months after injury, the patient developed avascular necrosis of the navicular and exhibited progressive collapse of the medial midfoot. He underwent naviculocuneiform arthrodesis 1 year after the index surgery. Two years after fusion, he was pain-free and ambulating independently. Successful treatment of midfoot fractures and dislocations requires an intimate understanding of anatomy, biomechanics, and both short- and long-term sequelae of injury.

Return to Golfing Activity After Joint Arthroplasty.

BACKGROUND: Many patients who are considering total joint arthroplasty, including hip, knee, and shoulder replacement, are concerned with their likelihood of returning to golf postoperatively as well as the effect that surgery will have on their game. PURPOSE: To review the existing literature on patients who have undergone major joint arthroplasty (hip, knee, and shoulder), to examine the effects of surgery on performance in golf, and to provide surgeon recommendations as related to participation in golf after surgery. A brief review of the history and biomechanics of the golf swing is also provided. STUDY DESIGN: Systematic review. METHODS: We performed a systematic review of the literature in the online Medline database, evaluating articles that contained the terms "golf" and "arthroplasty." Additionally, a web-based search evaluating clinical practice recommendations after joint arthroplasty was performed. The research was reviewed, and objective and anecdotal guidelines were formulated. RESULTS: Total joint arthroplasty provided an improvement in pain during golfing activity, and most patients were able to return to sport with variable improvements in sport-specific outcomes. CONCLUSION: In counseling patients regarding the return to golf after joint arthroplasty, it is our opinion, on the basis of our experience and those reported from others in the literature, that golfers undergoing total hip, knee, and shoulder arthroplasty can safely return to sport.

Urinary incontinence persisting after childbirth: extent, delivery history, and effects in a 12-year longitudinal cohort study.

OBJECTIVE: To investigate the extent of persistent urinary incontinence (UI) 12 years after birth, and association with delivery-mode history and other factors. DESIGN: Twelve-year longitudinal cohort study. SETTING: Maternity units in Aberdeen, Birmingham, and Dunedin. POPULATION: Women who returned questionnaires 3 months and 12 years after index birth. METHODS: Data on all births over a period of 12 months were obtained from the units and then women were contacted by post. MAIN OUTCOME MEASURE: Persistent UI reported at 12 years, with one or more previous contact. RESULTS: Of 7879 women recruited at 3 months, 3763 (48%) responded at 12 years, with 2944 also having responded at 6 years; non-responders had similar obstetric characteristics. The prevalence of persistent UI was 37.9% (1429/3763). Among those who had reported UI at 3 months, 76.4% reported it at 12 years. Women with persistent UI had lower SF12 quality of life scores. Compared with having only spontaneous vaginal deliveries (SVDs), women who delivered exclusively by caesarean section were less likely to have persistent UI (odds ratio, OR 0.42, 95% CI 0.33-0.54). This was not the case in women who had a combination of caesarean section and SVD births (OR 1.01, 95% CI 0.78-1.30). Older age at first birth, greater parity, and overweight/obesity were associated with persistent UI. Of 54 index primiparae with UI before pregnancy, 46 (85.2%) had persistent UI. CONCLUSIONS: This study, demonstrating that UI persists to 12 years in about three-quarters of women, and that risk was only reduced with caesarean section if women had no other delivery mode, has practice implications. TWEETABLE ABSTRACT: A longitudinal study of 3763 women showed a prevalence of persistent UI 12 years after birth of 37.9%.

Big Data Challenges in Climate Science.

The knowledge we gain from research in climate science depends on the generation, dissemination, and analysis of high-quality data. This work comprises technical practice as well as social practice, both of which are distinguished by their massive scale and global reach. As a result, the amount of data involved in climate research is growing at an unprecedented rate. Climate model intercomparison (CMIP) experiments, the integration of observational data and climate reanalysis data with climate model outputs, as seen in the Obs4MIPs, Ana4MIPs, and CREATE-IP activities, and the collaborative work of the Intergovernmental Panel on Climate Change (IPCC) provide examples of the types of activities that increasingly require an improved cyberinfrastructure for dealing with large amounts of critical scientific data. This paper provides an overview of some of climate science's big data problems and the technical solutions being developed to advance data publication, climate analytics as a service, and interoperability within the Earth System Grid Federation (ESGF), the primary cyberinfrastructure currently supporting global climate research activities.

Genetic and Morphological Characterization of Freshwater Shrimps (Caridina africana Kingsley, 1882) Reveals the Presence of Alien Shrimps in the Cape Floristic Region, South Africa.

Morphological identification and molecular data (mtDNA COI) were used to resolve the taxonomic identity of a non-native freshwater shrimp in the Cape Floristic Region (CFR) of South Africa and to evaluate levels of genetic diversity and differentiation in the species' core natural distribution. The species was morphologically and genetically identified as Caridina africana Kingsley, 1882, whose main natural distribution is in the KwaZulu-Natal (KZN) Province, more than 1200 km from the point of new discovery. Subsequently, sequence data from natural populations occurring in seven rivers throughout KZN showed the presence of nuclear copies of the mtDNA COI gene (NUMTs) in 46 out of 140 individuals. Upon removal of sequences containing NUMTs, levels of genetic diversity were low in the alien population (possibly as a consequence of a bottleneck event), while varying levels of genetic diversity and differentiation were found in natural populations, indicating habitat heterogeneity, fragmentation and restricted gene flow between rivers. Following the present study, the alien shrimp has survived the Western Cape's winter and dispersed into a nearby tributary of the Eerste River System, hence posing an additional potential threat to endangered endemics. Understanding the biology of this alien species will aid detection and eradication procedures.

Preliminary trauma radiographs misrepresent pubic diastasis injuries.

The goal of this study was to evaluate the role of portable primary trauma survey radiographs in the evaluation and management of anteroposterior (AP) compression pelvic injuries. A retrospective analysis was conducted at a level I academic trauma center. Twenty-seven adults with AP compressive class pelvic ring injuries who received both portable pelvic radiographs and pelvic computed tomography (CT) imaging in an unbound pelvic state were included. Three orthopedic surgeons performed independent measurements of diastasis on portable pelvic radiographs and coronal pelvic CT reconstructions. Measurement techniques were standardized among observers and were repeated after 8 weeks to assess intraobserver reliability. Nonoperative vs operative treatments were correlated with the initial magnitude of pelvic injury on CT and portable radiographic images. Independent measurements of diastasis on both radiographs and CT scans showed excellent intraobserver reliability (average correlation coefficient, 0.986) and interobserver reliability (average correlation coefficient, 0.979). Compared with diastasis measurements on CT scans, portable pelvic radiographs overestimated diastasis by an average of 49%, or 12.6 mm (P<.0001; 95% confidence interval, 9.6-15.6). Portable pelvic films were less precise than standard pelvic radiographs in measuring the size of femoral head controls (R(2)=0.919 vs 0.759; P=.004). In 12 of the 27 patients evaluated, radiographic indications for operative pelvic fixation were met by portable radiographs but not CT scans, and 11 of these patients ultimately underwent operative fixation. Portable AP pelvic radiographs may distort and exaggerate pelvic bony injuries, especially those involving anterior pelvic structures. Surgeons should use caution when making management decisions based on preliminary portable pelvic radiographs.