Validation of microarray-based resequencing of 93 worldwide mitochondrial genomes.
The human mitochondrial genome consists of a multicopy, circular dsDNA molecule of 16,569 base pairs. It encodes for 13 proteins, two ribosomal genes, and 22 tRNAs that are essential in the generation of cellular ATP by oxidative phosphorylation in eukaryotic cells. Germline mutations in mitochondrial DNA (mtDNA) are an important cause of maternally inherited diseases, while somatic mtDNA mutations may play important roles in aging and cancer. mtDNA polymorphisms are also widely used in population and forensic genetics. Therefore, methods that allow the rapid, inexpensive and accurate sequencing of mtDNA are of great interest. One such method is the Affymetrix GeneChip Human Mitochondrial Resequencing Array 2.0 (MitoChip v.2.0) (Santa Clara, CA). A direct comparison of 93 worldwide mitochondrial genomes sequenced by both the MitoChip and dideoxy terminator sequencing revealed an average call rate of 99.48% and an accuracy of > or =99.98% for the MitoChip. The good performance was achieved by using in-house software for the automated analysis of additional probes on the array that cover the most common haplotypes in the hypervariable regions (HVR). Failure to call a base was associated mostly with the presence of either a run of > or =4 C bases or a sequence variant within 12 bases up- or downstream of that base. A major drawback of the MitoChip is its inability to detect insertions/deletions and its low sensitivity and specificity in the detection of heteroplasmy. However, the vast majority of haplogroup defining polymorphism in the mtDNA phylogeny could be called unambiguously and more rapidly than with conventional sequencing.


Nanocrystalline SnS2 coated onto reduced graphene oxide: demonstrating the feasibility of a non-graphitic anode with sulfide chemistry for potassium-ion batteries.
An anode material incorporating a sulfide is reported. SnS2 nanoparticles anchored onto reduced graphene oxide are produced via a chemical route and demonstrate an impressive capacity of 350 mA h g-1, exceeding the capacity of graphite. These results open the door for a new class of high capacity anode materials (based on sulfide chemistry) for potassium-ion batteries.


Pseudomonas aeruginosa MurE amide ligase: enzyme kinetics and peptide inhibitor.
The enzyme kinetics of the amide ligase MurE, a cell wall biosynthesis enzyme, from Pseudomonas aeruginosa were determined using the synthesized nucleotide substrate UDP-MurNAc-Ala-Glu (uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamate). When coupled to a competitive bio-panning technique using a M13 phage display library encoding approximately 2.7 x 10(9) random peptide permutations and the specific substrates meso-A2pm (meso-diaminopimelic acid) and ATP, a peptide inhibitor of MurE was identified. The MurEp1 dodecamer selected and synthesized inhibited MurE ATPase activity with an IC(50) value of 500 microM. The inhibition was shown to be time-dependent and was reversed by the addition of meso-A2pm or UDP-MurNAc-Ala-Glu during the pre-incubation step. Kinetic analysis defined MurEp1 as a mixed inhibitor against both substrates with K(i) values of 160 and 80 microM respectively. MurEp1 was found to interfere in meso-A2pm and UDP-MurNAc-Ala-Glu binding necessary for amide bond formation. Modelling of Ps. aeruginosa MurE and docking of MurEp1 on the Ps. aeruginosa MurE surface indicated that MurEp1 binds at the juxtaposition of both meso-A2pm- and UDP-MurNAc-Ala-Glu-binding sites in the closed conformational state of the enzyme. Identification of the MurEp1 residues involved in MurE binding and inhibition will allow the development of a novel class of inhibitors having a novel mode of action against MurE.


Stimulated scattering of electromagnetic waves carrying orbital angular momentum in quantum plasmas.
We investigate stimulated scattering instabilities of coherent circularly polarized electromagnetic (CPEM) waves carrying orbital angular momentum (OAM) in dense quantum plasmas with degenerate electrons and nondegenerate ions. For this purpose, we employ the coupled equations for the CPEM wave vector potential and the driven (by the ponderomotive force of the CPEM waves) equations for the electron and ion plasma oscillations. The electrons are significantly affected by the quantum forces (viz., the quantum statistical pressure, the quantum Bohm potential, as well as the electron exchange and electron correlations due to electron spin), which are included in the framework of the quantum hydrodynamical description of the electrons. Furthermore, our investigation of the stimulated Brillouin instability of coherent CPEM waves uses the generalized ion momentum equation that includes strong ion coupling effects. The nonlinear equations for the coupled CPEM and quantum plasma waves are then analyzed to obtain nonlinear dispersion relations which exhibit stimulated Raman, stimulated Brillouin, and modulational instabilities of CPEM waves carrying OAM. The present results are useful for understanding the origin of scattered light off low-frequency density fluctuations in high-energy density plasmas where quantum effects are eminent.


Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS).
Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells.


Enhanced targeting of invasive glioblastoma cells by peptide-functionalized gold nanorods in hydrogel-based 3D cultures.
Cancer stem cells (CSCs) are responsible for drug resistance, tumor recurrence, and metastasis in several cancer types, making their eradication a primary objective in cancer therapy. Glioblastoma Multiforme (GBM) tumors are usually composed of a highly infiltrating CSC subpopulation, which has Nestin as a putative marker. Since the majority of these infiltrating cells are able to elude conventional therapies, we have developed gold nanorods (AuNRs) functionalized with an engineered peptide capable of specific recognition and selective eradication of Nestin positive infiltrating GBM-CSCs. These AuNRs generate heat when irradiated by a near-infrared laser, and cause localized cell damage. Nanoparticle internalization assays performed with GBM-CSCs or Nestin negative cells cultured as two-dimensional (2D) monolayers or embedded in three-dimensional (3D) biodegradable-hydrogels of tunable mechanical properties, revealed that the AuNRs were mainly internalized by GBM-CSCs, and not by Nestin negative cells. The AuNRs were taken up via energy-dependent and caveolae-mediated endocytic mechanisms, and were localized inside endosomes. Photothermal treatments resulted in the selective elimination of GBM-CSCs through cell apoptosis, while Nestin negative cells remained viable. Results also indicated that GBM-CSCs embedded in hydrogels were more resistant to AuNR photothermal treatments than when cultured as 2D monolayers. In summary, the combination of our engineered AuNRs with our tunable hydrogel system has shown the potential to provide an in vitro platform for the evaluation and screening of AuNR-based cancer therapeutics, leading to a substantial advancement in the application of AuNRs for targeted GBM-CSC therapy. There is an urgent need for reliable and efficient therapies for the treatment of Glioblastoma Multiforme (GBM), which is currently an untreatable brain tumor form with a very poor patient survival rate. GBM tumors are mostly comprised of cancer stem cells (CSCs), which are responsible for tumor reoccurrence and therapy resistance. We have developed gold nanorods functionalized with an engineered peptide capable of selective recognition and eradication of GBM-CSCs via heat generation by nanorods upon NIR irradiation. An in vitro evaluation of nanorod therapeutic activities was performed in 3D synthetic-biodegradable hydrogel models with distinct biomechanical cues, and compared to 2D cultures. Results indicated that cells cultured in 3D were more resistant to photothermolysis than in 2D systems.


Allergy and immunology in Africa: Challenges and unmet needs.
The tremendous increase in allergy in the African continent cannot simply be explained by the change in public hygiene. There are many "prehygiene" communities with sewage-contaminated water supplies, helminth infestations, bare footedness, and poor housing, and still there is a high prevalence of allergic disease. Africans can be exposed to many risk factors facilitating severe asthma and wheezing, including airborne viruses, smoke, indoor dampness, cockroaches, and poor access to health care. Although the reporting on food allergy is inadequate to perform systematic reviews or meta-analyses, the available data suggest that food allergy is underdiagnosed. The rate of new HIV infections in high-prevalence settings in Africa remains unacceptably high. Although the annual number of new HIV infections in Sub-Saharan Africa has decreased lately, new HIV infections in the Middle East and North Africa region have increased; however, the current prevalence of 0.1% is still among the lowest globally. Africa is densely populated, and consanguineous mating is high in some areas of North and Sub-Saharan Africa. This allows for emergence of many autosomal recessive primary immunodeficiency diseases. There is urgent need for the establishment of primary immunodeficiency disease registries, stem cell transplantation facilities, and neonatal screening programs. To address these expanding problems and perform local cutting-edge research, Africans need to be empowered by motivated governments, dedicated funds, and compassionate scientific partnership.


Bedside endosonography and endosonography-guided fine-needle aspiration in critically ill patients: a way out of the deadlock?
Endosonography and endosonography-guided fine-needle aspiration (EUS-FNA) are now established diagnostic techniques, which are performed electively in endoscopy suites. We report here the bedside use of EUS-FNA in three critically ill patients in an intensive-care unit, with a significant impact on the outcome. A mediastinal abscess after percutaneous dilational tracheotomy was aspirated in one patient, leading to appropriate antibiotic therapy and complete recovery. A paratracheal hematoma compressing the right main bronchus was aspirated in a patient with polytrauma, relieving the pressure effects. The third patient, who had end-stage dilated cardiomyopathy and was being evaluated for cardiac transplantation, was found to have an apical lung lesion suspicious for bronchogenic carcinoma. EUS was performed to exclude mediastinal metastasis and allow simultaneous resection at the time of transplantation. Although a metastasis was excluded by EUS-FNA, the patient died while awaiting surgery. We conclude that bedside EUS-FNA is a feasible procedure, and in experienced hands it can offer an alternative in life-threatening situations.


Interfaces of Malignant and Immunologic Clonal Dynamics in Ovarian Cancer.
High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.


Contributions of identifiable neurons and neuron classes to lamprey vertebrate neurobiology.
Among the advantages offered by the lamprey brainstem and spinal cord for studies of the structure and function of the nervous system is the unique identifiability of several pairs of reticulospinal neurons in the brainstem. These neurons have been exploited in investigations of the patterns of sensory input to these cells and the patterns of their outputs to spinal neurons, but no doubt these cells could be used much more effectively in exploring their roles in descending control of the spinal cord. The variability of cell positions of neurons in the spinal cord has precluded the recognition of unique spinal neurons. However, classes of nerve cells can be readily defined and characterized within the lamprey spinal cord and this has led to progress in understanding the cellular and synaptic mechanisms of locomotor activity. In addition, both the identifiable reticulospinal cells and the various spinal nerve cell classes and their known synaptic interactions have been used to demonstrate the degree and specificity of regeneration within the lamprey nervous system. The lack of uniquely identifiable cells within the lamprey spinal cord has hampered progress in these areas, especially in gaining a full understanding of the locomotor network and how neuromodulation of the network is accomplished.


Membrane resealing: synaptotagmin VII keeps running the show.
Ca2+ influx, an immediate consequence of plasma membrane disruption, triggers a resealing mechanism involving exocytosis. Although this has been known for about a decade, a better understanding of the organelles involved and of the molecular machinery controlling membrane repair has been slower to emerge. Recent studies have changed this picture, by identifying lysosomes as exocytotic vesicles involved in membrane resealing and the Ca2+-binding protein synaptotagmin VII as a regulator of this process. New evidence reinforces the role of the C2A and C2B domains of synaptotagmin VII in plasma membrane repair, highlighting the importance of this molecule as a powerful tool for future studies.


Vitamin D analogs, a new treatment for retinoblastoma: The first Ellsworth Lecture.
This lecture honors the memory of Dr. Robert M. Ellsworth, an important figure in the development of current treatments of retinoblastoma (RB), and reviews our studies of vitamin D analogs as treatments for retinoblastoma in two experimental mouse models. We identified vitamin D receptors in retinoblastoma and examined the effectiveness and mechanism of action of these analogs. Reverse-transcriptase polymerase chain reaction (RT-PCR) amplification was used to detect vitamin D receptor mRNAs in human and mouse retinoblastomas. The effectiveness and toxicity of vitamin D(2), calcitriol, and synthetic analogs were studied in the athymic/Y-79 xenograft and transgenic mouse models of RB. Dosing was 5X/week for five weeks. Dose-response studies focused on tumor inhibition; toxicity studies investigated survival and serum calcium. The mechanism of action of vitamin D was investigated using terminal transferase dUTP labeling 3'-overhang ligation to measure apoptosis; immunohistochemistry measured p53-dependent gene expression and cell proliferation. Vitamin D receptor mRNAs were detectable in Y-79 RB cells, LH beta-Tag tumors, and human RB specimens using RT-PCR. Calcitriol inhibited cell growth in vitro. Calcitriol and vitamin D(2) inhibited in vivo growth in xenograft and transgenic models, but therapeutic levels were toxic due to hypercalcemia. Two analogs, 16,23-D(3) and 1 alpha-OH-D( 2), inhibited tumors in animal models of RB with reduced toxicity. The mechanism of action appears related to increased p53-related gene expression resulting in increased apoptosis. 16,23-D(3) and 1 alpha-OH-D(2) are effective in tumor reduction in two mouse models of RB with low toxicity. These results warrant initiating phase 1 and phase 2 clinical studies in children.


Dual Lewis Acid/Lewis Base Catalyzed Acylcyanation of Aldehydes: A Mechanistic Study.
A mechanistic investigation, which included a Hammett correlation analysis, evaluation of the effect of variation of catalyst composition, and low-temperature NMR spectroscopy studies, of the Lewis acid-Lewis base catalyzed addition of acetyl cyanide to prochiral aldehydes provides support for a reaction route that involves Lewis base activation of the acyl cyanide with formation of a potent acylating agent and cyanide ion. The cyanide ion adds to the carbonyl group of the Lewis acid activated aldehyde. O-Acylation by the acylated Lewis base to form the final cyanohydrin ester occurs prior to decomplexation from titanium. For less reactive aldehydes, the addition of cyanide is the rate-determining step, whereas, for more reactive, electron-deficient aldehydes, cyanide addition is rapid and reversible and is followed by rate-limiting acylation. The resting state of the catalyst lies outside the catalytic cycle and is believed to be a monomeric titanium complex with two alcoholate ligands, which only slowly converts into the product.


The Effectiveness and Long-Term Outcome of Conjunctivodacryocystorhinostomy With Frosted Jones Tubes.
To describe success rates and long-term outcomes of conjunctivodacryocystorhinostomy (CDCR) with frosted Jones tubes (FJT) for epiphora with proximal outflow obstruction. A retrospective chart review of all patients undergoing external and endoscopic CDCR with FJTs by one author (RAD) was performed between January 1, 2006 and November 1, 2014 at the Casey Eye Institute. Patient demographics, etiology of tearing, concurrent endonasal and eyelid procedures, and FJT size were recorded. After CDCR, follow-up time, tube size changes, tube position, and tearing status were noted. Exclusion criteria included follow up less than 6 months and/or prior CDCR. The study was IRB approved, HIPAA compliant, and adherent to the declaration of Helsinki. Forty-two eyes of 31 patients met the inclusion criteria, with the majority having epiphora from canalicular obstruction (31%) or flaccid canaliculi (31%). Average follow up was 1,088 days. Forty of 42 eyes, or 30 of 31 patients, had complete resolution of tearing after surgery. Twenty of 42 eyes required tube size changes, usually an increase in collar size (45%) and/or decrease in tube length (55%). Six of 42 FJTs were lost, one migrating outward, with an average time to loss between 61 and 1,122 days (mean 817 days). After collars larger than 4 mm became available, only one tube was lost. All epiphora resolved after repeat CDCR. The most common complication was intermittent irritation (17%) near the FJT that resolved after antibiotic-steroid drops and/or tube replacement/cleaning. CDCR with FJTs is highly effective in correcting epiphora, and well tolerated by the majority.


Cell therapy for spinal cord injury using induced pluripotent stem cells.
For the past few decades, spinal cord injury (SCI) has been believed to be an incurable traumatic condition, but with recent developments in stem cell biology, the field of regenerative medicine has gained hopeful momentum in the development of a treatment for this challenging pathology. Among the treatment candidates, transplantation of neural precursor cells has gained remarkable attention as a reasonable therapeutic intervention to replace the damaged central nervous system cells and promote functional recovery. Here, we highlight transplantation therapy techniques using induced pluripotent stem cells to treat SCI and review the recent research giving consideration to future clinical applications.


The hemophilias: progress and problems.
Known since the beginning of the first millenium, the hemophilias are among the most frequent inherited disorders of blood coagulation and definitely the most severe. In the 1970s, with the availability of concentrated preparations of the deficient coagulation factors VIII and IX and with the large-scale adoption of home treatment, hemophilia care became one of the most gratifying examples of successful secondary prevention of a chronic disease. Unfortunately, in the early 1980s It was recognized that factor concentrates prepared from plasma pooled from thousands of donors transmitted the hepatitis and the human immunodeficiency viruses. The scientific community reacted promptly to the devastation brought about by hepatitis and AIDS. The last 15 years of the second millenium have witnessed the development of methods that applied during concentrate manufacturing inactivate viruses escaping the screening procedures. The adoption of these measures has reduced dramatically the risk of transmission of bloodborne infections. The production of recombinant factors and their availability for patient treatment epitomize progress in hemophilia care through DNA technology. Methods based on the polymerase chain reaction (PCR) have unraveled an array of gene lesions associated with hemophilia, permitting improved secondary control of the disease through carrier detection in women from affected families and prenatal termination of their affected male infants. This article will review the aforementioned areas of progress and discuss unresolved problems (such as treatment of patients with antibodies, the risk of new infectious complications, and the issue of secondary tumors). Hopes and expectations for further improvement in the third millenium and particularly the prospects of hemophilia cure through gene replacement therapy will also be mentioned.


A rapid flow cytometric assay to detect CD4+ and CD8+ T-helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy.
The immune response in atopic dermatitis (AD) is thought to be driven by T-helper (Th) 2 cytokines. Using flow cytometry, higher frequencies of peripheral blood CD4+ and CD8+ T cells producing interleukin (IL)-4 and correspondingly lower frequencies of CD4+ T cells producing interferon (IFN)-gamma have been found in patients with AD compared with healthy controls. It would be of interest to know whether other Th1 and Th2 cytokines such as IL-5, IL-13 and tumour necrosis factor (TNF)-alpha are similarly skewed in patients with AD and whether this immune skewing, detected via a simple blood assay, can be correlated with other clinical measurements or treatments in AD. To use a rapid (4-h) flow cytometric assay to study a wide range of Th1 and Th2 cytokine patterns in peripheral blood lymphocytes from patients with AD, comparing them with non-atopic healthy controls. To correlate cytokine patterns with the degree of eosinophilia observed and in the case of one patient with severe disease, to observe the effect of cyclosporin therapy on peripheral blood cytokine patterns. Peripheral blood from eight patients with AD and 23 healthy controls was examined for the frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4, IL-5, IL-13, IFN-gamma and TNF-alpha using flow cytometry. Significantly higher frequencies of CD4+/IL-4+ (P < 0.005) and CD4+/IL-13+ (P < 0.0001) and lower frequencies of CD4+/IFN-gamma+ (P < 0.002) and CD8+/TNF-alpha+ (P < 0.05) T lymphocytes were found in patients with AD compared with controls. There were significant positive correlations with the increased percentages of CD4+/IL-4+ and CD4+/IL-13+ T lymphocytes and the degree of eosinophilia observed (P < 0.05, P < 0.001) and a negative correlation between the percentage of CD4+/IFN-gamma+ T lymphocytes and eosinophilia (P < 0.05). In one patient examined before and 8 days after cyclosporin therapy, 50% or greater reductions were observed in percentages of peripheral blood CD8+/IL-5+, CD8+/IL-13+, CD4+/IL-4+ and CD4+/IL-5+ T lymphocytes following cyclosporin therapy. A smaller reduction of 15% after cyclosporin therapy was found in percentages of CD4+/IL-13+ T cells. These data strongly support a Th2 predominance in the peripheral blood of AD. The results suggest that administration of cyclosporin therapy in patients with AD may help to restore the Th2 cytokine imbalance seen in these patients.


Pre-exposure rabies prophylaxis: a systematic review.
To review the safety and immunogenicity of pre-exposure rabies prophylaxis (including accelerated schedules, co-administration with other vaccines and booster doses), its cost-effectiveness and recommendations for use, particularly in high-risk settings. We searched the PubMed, Centre for Agriculture and Biosciences International, Cochrane Library and Web of Science databases for papers on pre-exposure rabies prophylaxis published between 2007 and 29 January 2016. We reviewed field data from pre-exposure prophylaxis campaigns in Peru and the Philippines. Pre-exposure rabies prophylaxis was safe and immunogenic in children and adults, also when co-administered with routine childhood vaccinations and the Japanese encephalitis vaccine. The evidence available indicates that shorter regimens and regimens involving fewer doses are safe and immunogenic and that booster intervals could be extended up to 10 years. The few studies on cost suggest that, at current vaccine and delivery costs, pre-exposure prophylaxis campaigns would not be cost-effective in most situations. Although pre-exposure prophylaxis has been advocated for high-risk populations, only Peru and the Philippines have implemented appropriate national programmes. In the future, accelerated regimens and novel vaccines could simplify delivery and increase affordability. Pre-exposure rabies prophylaxis is safe and immunogenic and should be considered: (i) where access to postexposure prophylaxis is limited or delayed; (ii) where the risk of exposure is high and may go unrecognized; and (iii) where controlling rabies in the animal reservoir is difficult. Pre-exposure prophylaxis should not distract from canine vaccination efforts, provision of postexposure prophylaxis or education to increase rabies awareness in local communities.


The interplay between structure and agency in shaping the mental health consequences of job loss.
Job loss is a discrete life event, with multiple adverse consequences for physical and mental health and implications for agency. Our research explores the consequences of job loss for retrenched workers' mental health by examining the interplay between their agency and the structures shaping their job loss experiences. We conducted two waves of in-depth, semi-structured interviews with a sample of 33 of the more than 1000 workers who lost their jobs at Mitsubishi Motors in South Australia during 2004 and 2005 as a result of industry restructuring. Interviews capturing the mental health consequences of job loss were recorded and transcribed verbatim. Thematic analysis was employed to determine the health consequences of the job loss and the impact of structural factors. Main themes that emerged from the qualitative exploration of the psychological distress of job loss included stress, changes to perceived control, loss of self-esteem, shame and loss of status, experiencing a grieving process, and financial strain. Drawing on two models of agency we identified the different ways workers employed their agency, and how their agency was enabled, but mainly constrained, when dealing with job loss consequences. Respondents' accounts support the literature on the moderating effects of economic resources such as redundancy packages. The results suggest the need for policies to put more focus on social, emotional and financial investment to mediate the structural constraints of job loss. Our study also suggests that human agency must be understood within an individual's whole of life circumstances, including structural and material constraints, and the personal or interior factors that shape these circumstances.


Racial differences in the management of unstable angina: results from the multicenter GUARANTEE registry.
Prior studies, usually conducted with the use of insurance databases, have shown differences in the use of cardiac procedures between black patients and white patients hospitalized with various types of coronary artery disease. However, few data are available in prospectively collected cohorts of patients with unstable angina or on the use of appropriate medications or interventions. We evaluated 2948 consecutive patients with unstable angina admitted to 35 hospitals across the United States in 1996, comparing nonwhite and white patients. Seventy-seven percent of patients were white, 14% were black, 4% were Hispanic, 1% were Asian, and 3% were other or unknown race. Differences were seen in coronary risk profile, with a higher incidence of hypertension and diabetes mellitus in nonwhites. Cardiac catheterization was performed less often in nonwhites compared with whites (36% vs 53%, P =.001). Even in patients meeting the criteria for appropriate catheterization in the Agency for Health Care Policy Research unstable angina guidelines, fewer nonwhites underwent catheterization (44% vs 61%, P =.001), but among these, fewer nonwhites had significant coronary stenosis (72% vs 90%, P =.001). However, among patients catheterized who had indications for revascularization, angioplasty and coronary artery bypass grafting were performed equally often in nonwhites and whites. Current guidelines would recommend more aggressive use of cardiac catheterization for nonwhite patients. However, our findings suggest that racial differences may need to be included in the diagnostic and interventional algorithms.


Understanding healing relationships in primary care.
Clinicians often have an intuitive understanding of how their relationships with patients foster healing. Yet we know little empirically about the experience of healing and how it occurs between clinicians and patients. Our purpose was to create a model that identifies how healing relationships are developed and maintained. Primary care clinicians were purposefully selected as exemplar healers. Patients were selected by these clinicians as having experienced healing relationships. In-depth interviews, designed to elicit stories of healing relationships, were conducted with patients and clinicians separately. A multidisciplinary team analyzed the interviews using an iterative process, leading to the development of case studies for each clinician-patient dyad. A comparative analysis across dyads was conducted to identify common components of healing relationships Three key processes emerged as fostering healing relationships: (1) valuing/creating a nonjudgmental emotional bond; (2) appreciating power/consciously managing clinician power in ways that would most benefit the patient; and (3) abiding/displaying a commitment to caring for patients over time. Three relational outcomes result from these processes: trust, hope, and a sense of being known. Clinician competencies that facilitate these processes are self-confidence, emotional self-management, mindfulness, and knowledge. Healing relationships have an underlying structure and lead to important patient-centered outcomes. This conceptual model of clinician-patient healing relationships may be generalizable to other kinds of healing relationships.


Dynamics of spiking neurons with electrical coupling.
We analyze the existence and stability of phase-locked states of neurons coupled electrically with gap junctions. We show that spike shape and size, along with driving current (which affects network frequency), play a large role in which phase-locked modes exist and are stable. Our theory makes predictions about biophysical models using spikes of different shapes, and we present simulations to confirm the predictions. We also analyze a large system of all-to-all coupled neurons and show that the splay-phase state can exist only for a certain range of frequencies.


Pyridin-2-yl guanidine derivatives: conformational control induced by intramolecular hydrogen-bonding interactions.
The synthesis and conformational analysis of a series of pyridin-2-yl guanidine derivatives using NMR, X-ray crystallography, and B3LYP/6-31+G** theoretical studies are reported. A remarkable difference was observed in the (1)H NMR spectra of the guanidinium salts as compared with their N,N'-di-Boc protected and neutral analogues. This difference corresponds to a 180° change in the dihedral angle between the guanidine/ium moiety and the pyridine ring in the salts as compared to the Boc-protected derivatives, a conclusion that was supported by theoretical studies, X-ray data, and NMR analysis. Moreover, our data sustain the existence of two intramolecular hydrogen-bonding systems: (i) between the pyridine N1 atom and the guanidinium protons in the salts and (ii) within the tert-butyl carbamate groups of the Boc-protected derivatives. To verify that the observed conformational control arises from these intramolecular interactions, a new series of N-Boc-N'-propyl-substituted pyridin-2-yl guanidines were also prepared and studied.


Comparative genomic analysis of ten Streptococcus pneumoniae temperate bacteriophages.
Streptococcus pneumoniae is an important human pathogen that often carries temperate bacteriophages. As part of a program to characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, we performed comparative genomic analysis of 10 temperate pneumococcal phages. All of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis clusters. All of the phage particles observed showed a Siphoviridae morphology. The only genes that are well conserved in all the genomes studied are those involved in the integration and the lysis of the host in addition to two genes, of unknown function, within the replication module. We observed that a high percentage of the open reading frames contained no similarities to any sequences catalogued in public databases; however, genes that were homologous to known phage virulence genes, including the pblB gene of Streptococcus mitis and the vapE gene of Dichelobacter nodosus, were also identified. Interestingly, bioinformatic tools showed the presence of a toxin-antitoxin system in the phage phiSpn_6, and this represents the first time that an addition system in a pneumophage has been identified. Collectively, the temperate pneumophages contain a diverse set of genes with various levels of similarity among them.


Patient-, organization-, and system-level barriers and facilitators to preventive oral health care: a convergent mixed-methods study in primary dental care.
Dental caries is the most common chronic disease of adult and childhood, a largely preventable yet widespread, costly public health problem. This study identified patient-, organization-, and system-level factors influencing routine delivery of recommended care for prevention and management of caries in primary dental care. A convergent mixed-methods design assessed six guidance-recommended behaviours to prevent and manage caries (recording risk, risk-based recall intervals, applying fluoride varnish, placing preventive fissure sealants, demonstrating oral health maintenance, taking dental x-rays). A diagnostic questionnaire assessing current practice, beliefs, and practice characteristics was sent to a random sample of 651 dentists in National Health Service (NHS) Scotland. Eight in-depth case studies comprising observation of routine dental visits and dental team member interviews were conducted. Patient feedback was collected from adult patients with recent checkups at case study practices. Key informant interviews were conducted with decision makers in policy, funding, education, and regulation. The Theoretical Domains Framework within the Behaviour Change Wheel was used to identify and describe patient-, organization-, and system-level barriers and facilitators to care. Findings were merged into a matrix describing theoretical domains salient to each behaviour. The matrix and Behaviour Change Wheel were used to prioritize behaviours for change and plan relevant intervention strategies. Theoretical domains associated with best practice were identified from the questionnaire (N-196), case studies (N = 8 practices, 29 interviews), and patient feedback (N = 19). Using the study matrix, key stakeholders identified priority behaviours (use of preventive fissure sealants among 6-12-year-olds) and strategies (audit and feedback, patient informational campaign) to improve guidance implementation. Proposed strategies were assessed as appropriate for immediate implementation and suitable for development with remaining behaviours. Specific, theoretically based, testable interventions to improve caries prevention and management were coproduced by patient-, practice-, and policy-level stakeholders. Findings emphasize duality of behavioural determinants as barriers and facilitators, patient influence on preventive care delivery, and benefits of integrating multi-level interests when planning interventions in a dynamic, resource-constrained environment. Interventions identified in this study are actively being used to support ongoing implementation initiatives including guidance, professional development, and oral health promotion.


Adoptive transfer of immunity to oral challenge with virulent salmonellae in innately susceptible BALB/c mice requires both immune serum and T cells.
The mechanisms of immunity to salmonellae conferred by immunization with live vaccines were studied by adoptive transfer using the mouse-virulent strain Salmonella typhimurium C5 and innately susceptible BALB/c (ltys) mice. This organism cannot establish a sublethal infection in naive BALB/c mice. Animals immunized 2 to 3 months earlier with the S. typhimurium SL3261 aroA live vaccine were used as donors of serum, spleen cells, and mesenteric lymph node cells for naive recipients which were challenged orally with the virulent C5 strain. Simultaneous transfer of both immune serum and immune cells was necessary for protection. Simultaneously depleting the donors of CD4+ and CD8+ T cells by administration of antisera in vivo prior to cell harvesting showed that T cells were necessary for protection. The results demonstrate that both antibody and T cells are required for recall of immunity to oral challenge with virulent salmonellae in innately susceptible mice and suggest that the ability to elicit opsonizing antibody in addition to cell-mediated immunity is important for optimal protection induced by salmonella vaccines.


Unusual behavioral profile of alprazolam in squirrel monkeys.
Alprazolam was studied for its effects on schedule-controlled behavior of squirrel monkeys. Monkeys responded by pressing a lever under fixed-ratio or two separate fixed-interval (FI) schedules. Responding was maintained by the presentation of food under all three schedules and was suppressed under one of the FI schedules by electric shock. Unlike lorazepam, alprazolam did not increase rates of either suppressed or nonsuppressed responding under the two FI schedules after acute administration. When given for 3 (at 0.03 mg/kg) or 11 (at 0.1 mg/kg) consecutive days immediately before test sessions, alprazolam also did not increase suppressed responding. Under all three schedules, alprazolam (0.03-3.0 mg/kg) produced dose-dependent decreases in response rate. The rate-decreasing effects of alprazolam were attenuated by drugs known to have benzodiazepine (BZ) antagonist action: Ro 15-1788 (0.01 and 0.3 mg/kg), Ro 15-4513 (0.01 and 0.1 mg/kg) and 3-(methoxycarbonyl)amino-beta-carboline (10.0 and 30.0 mg/kg). These latter drugs alone either had no effect on or decreased fixed-ratio responding. Alprazolam also was tested in combination with lorazepam under the two FI schedules. In general, low doses of lorazepam, equal to or less than those having little effect on response rate when given alone, increased suppressed and nonsuppressed responding when combined with low to moderate doses of alprazolam. The results show that although the rate-decreasing effects of alprazolam probably are due to actions at BZ recognition sites, alprazolam alone does not have typical BZ-like agonist effects on FI responding; it did, however, potentiate the behavioral effects of lorazepam.


A Patient-Centered PaTH to Address Diabetes: Protocol for a Study on the Impact of Obesity Counseling.
Overweight and obesity are America's number one health concern. The prevalence of obesity in the United States is greater than 36%, a rate that has doubled since 1970. As the second most preventable cause of death, obesity is a risk factor for diabetes, cardiovascular disease, stroke, and cancer, all major causes of death. Primary care clinics may be an ideal setting for weight control interventions to help manage and prevent diabetes. For this reason, the Centers for Medicare and Medicaid Services (CMS) implemented a health care procedure coding system code for intensive behavioral therapy (IBT) for obesity within primary care in 2012 to facilitate payment for addressing obesity, which was followed by broader coverage by most insurers for IBT for adults in 2013. However, the impact of this coverage on patient-centered outcomes is largely unknown. The overarching goal of this study is to understand the comparative effectiveness of obesity counseling as covered by CMS and other insurers in improving weight loss for adults either with or at increased risk for type 2 diabetes. This study leverages the novel infrastructure of the Patient-Centered Outcomes Research Institute-funded PaTH Clinical Data Research Network. The PaTH network is comprised of Geisinger Health System, Johns Hopkins University, Johns Hopkins Health System, Lewis Katz School of Medicine at Temple University, Temple Health System, Penn State College of Medicine, Penn State Milton S Hershey Medical Center, University of Pittsburgh, UPMC and UPMC Health Plan, and the University of Utah. Electronic health record (EHR) data will originate from the 6 PaTH health systems. Specifically, we will (1) evaluate the impact of broader preventive service coverage for obesity screening and counseling on weight loss, diabetes incidence, and diabetes outcomes in patients with diabetes or at increased risk for diabetes (defined by body mass index [BMI] ≥25). We will determine how the annual probability of receiving obesity and/or nutritional counseling changed pre- and postpolicy across all insurers in a cohort of patients with diabetes and at high risk for diabetes. We will (2) compare patient weight loss and diabetes-related outcomes among those who receive obesity screening and counseling with those who do not, following implementation of preventive service coverage. We will examine postpolicy impact of obesity screening and counseling in a cohort of patients with diabetes and at increased risk for diabetes. Specific outcomes to be examined include weight loss, diabetes incidence, and diabetes outcomes. Exploratory outcomes will include patient-reported outcomes. Furthermore, we will determine patient characteristics, including demographics, and practice characteristics, including provider type. Our PCORI-funded study is underway. To date, we have obtained our second data extraction from the PaTH CDRN and are performing data editing and cleaning. Next steps include analysis of early policy change. Given patients who are overweight are at highest risk for diabetes, improved weight management services could prevent diabetes and its negative health outcomes. Comparing weight and diabetes outcomes in 3 states using EHRs and claims data before and after this policy was implemented using the PaTH Network will allow important insight into policy effectiveness. DERR1-10.2196/12054.


Loss of the Synaptic Vesicle Protein SV2B results in reduced neurotransmission and altered synaptic vesicle protein expression in the retina.
The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content.


Postmeiotic transcription of X and Y chromosomal genes during spermatogenesis in the mouse.
During the meiotic prophase of spermatogenesis, the X and Y chromosomes form the heterochromatic sex body, showing little transcriptional activity. It has been suggested that transcription of the Xist gene is involved in this inactivation. After completion of the meiotic divisions, at least two Y chromosomal genes, Zfy and Sry, are transcribed in haploid spermatids. In contrast, postmeiotic transcription of X chromosomal genes has not been demonstrated. Using highly purified preparations of mouse pachytene spermatocytes, round spermatids, and cytoplasmic fragments from elongated spermatids, the present experiments show differential postmeiotic expression of the Y chromosomal genes Ubely and Sry, with highest mRNA levels in round spermatids and cytoplasmic fragments, respectively. Postmeiotic transcription of the X chromosomal gene Ube1x is indicated by an increased level of Ube1x mRNA in round spermatids and cytoplasmic fragments. The X chromosomal gene MHR6A shows a marked temporary postmeiotic expression in round spermatids. This postmeiotic activity of the X chromosome is a novel finding, which may have implications for our understanding of X chromosome inactivation during spermatogenesis and paternal genome imprinting.


Stroke Risk Factors, Genetics, and Prevention.
Stroke is a heterogeneous syndrome, and determining risk factors and treatment depends on the specific pathogenesis of stroke. Risk factors for stroke can be categorized as modifiable and nonmodifiable. Age, sex, and race/ethnicity are nonmodifiable risk factors for both ischemic and hemorrhagic stroke, while hypertension, smoking, diet, and physical inactivity are among some of the more commonly reported modifiable risk factors. More recently described risk factors and triggers of stroke include inflammatory disorders, infection, pollution, and cardiac atrial disorders independent of atrial fibrillation. Single-gene disorders may cause rare, hereditary disorders for which stroke is a primary manifestation. Recent research also suggests that common and rare genetic polymorphisms can influence risk of more common causes of stroke, due to both other risk factors and specific stroke mechanisms, such as atrial fibrillation. Genetic factors, particularly those with environmental interactions, may be more modifiable than previously recognized. Stroke prevention has generally focused on modifiable risk factors. Lifestyle and behavioral modification, such as dietary changes or smoking cessation, not only reduces stroke risk, but also reduces the risk of other cardiovascular diseases. Other prevention strategies include identifying and treating medical conditions, such as hypertension and diabetes, that increase stroke risk. Recent research into risk factors and genetics of stroke has not only identified those at risk for stroke but also identified ways to target at-risk populations for stroke prevention.


The survival outcome of patients with metastatic colorectal cancer based on the site of metastases and the impact of molecular markers and site of primary cancer on metastatic pattern.
Pattern of spread in patients with metastatic colorectal cancer (mCRC) is variable and may reflect different biology in subsets of patients. This is a retrospective study to explore the outcome of patients with mCRC based on their site of metastasis at diagnosis and to explore the association between tumor characteristics [KRAS/RAS, BRAF, mismatch repair (MMR) status, site of primary] and the site of metastasis. Patients from two Australian databases were divided into six groups based on site of metastasis at time of diagnosis of metastatic disease; lung-only, liver-only, lymph node-only or any patients with brain, bone or peritoneal metastases. Primary endpoint was overall survival (OS) of each cohort compared with the rest of the population. A Mantel-Haenszel chi-squared test used to explore the association between site of metastasis and selected tumor characteristics. Five thousand nine hundred and sixty-seven patients were included. In a univariate analysis, median OS was significantly higher when metastases were limited to lung or liver and shorter for those with brain, bone or peritoneal metastases (p < .001) in both datasets. BRAF mutation was strongly associated with peritoneal metastases (relative risk = 1.8, p < .001) with lower incidence of lung (RR = 0.3, p = .004) and liver (RR = 0.7, p = .005) limited metastases. Lung-only metastases were more frequent with KRAS/RAS mutation (RR = 1.4, p = .007). Left colon tumors were associated with bone (RR = 1.6, p < .001) and lung-only metastases (RR = 2.3, p = .001) while peritoneal spread was less frequent compared with right colon tumors (RR = 0.6, p < .001). Rectal cancer was associated with brain, bone and lung metastases (RR = 1.7; p = .002, 1.7; p < .001, 2.0; p < .001). Liver-only metastases were less frequent in deficient MMR tumors (RR = 0.7, p = .01). Survival duration with mCRC is related to the site of metastases with lung limited disease showing a more favorable survival outcome compared to other single metastatic site disease. The BRAF mutation and primary rectal cancer were associated with poor prognostic metastatic sites.


Real Objects Can Impede Conditional Reasoning but Augmented Objects Do Not.
In this study, Knauff and Johnson-Laird's (2002) visual impedance hypothesis (i.e., mental representations with irrelevant visual detail can impede reasoning) is applied to the domain of external representations and diagrammatic reasoning. We show that the use of real objects and augmented real (AR) objects can control human interpretation and reasoning about conditionals. As participants made inferences (e.g., an invalid one from "if P then Q" to "P"), they also moved objects corresponding to premises. Participants who moved real objects made more invalid inferences than those who moved AR objects and those who did not manipulate objects (there was no significant difference between the last two groups). Our results showed that real objects impeded conditional reasoning, but AR objects did not. These findings are explained by the fact that real objects may over-specify a single state that exists, while AR objects suggest multiple possibilities.


Pediatric stroke related to Lyme neuroborreliosis: Data from the Swiss NeuroPaediatric Stroke Registry and literature review.
Cerebrovascular complications of Lyme neuroborreliosis (LNB) are poorly documented in the paediatric population. We performed a retrospective analysis from prospectively registered cases of acute ischemic stroke (AIS) from the Swiss NeuroPaediatric Stroke Registry (SNPSR) from 2000 to 2015. Only cases with serologically confirmed LNB were included. In addition, a literature review on paediatric stroke cases secondary to Lyme neuroborreliosis in the same time frame was performed. 4 children out of 229 children with arterial ischemic childhood stroke and serologically confirmed LNB were identified in the SNPSR giving a global incidence of 1.7%. Median age was 9.9 years. A prior history of tick bites or erythema migrans (EM) was reported in two cases. Clinical presenting signs were suggestive of acute cerebellar/brainstem dysfunction. On imaging, three children demonstrated a stroke in the distribution of the posterior inferior cerebellar artery. The remaining fourth child had a "stroke-like" picture with scattered white matter lesions and a multifocal vasculitis with prominent basilar artery involvement. Lymphocytic pleocytosis as well as intrathecal synthesis of Borrelia burgdorferi antibodies were typical biological features. Acute intravenous third generation cephalosporins proved to be effective with rapid improvement in all patients. No child had recurrent stroke. Data from the literature concerning eight patients gave similar results, with prominent posterior circulation stroke, multifocal vasculitis and abnormal CSF as distinctive features. Lyme Neuroborreliosis accounts for a small proportion of paediatric stroke even in an endemic country. The strong predilection towards posterior cerebral circulation with clinical occurrence of brainstem signs associated with meningeal symptoms and CSF lymphocytosis are suggestive features that should rapidly point to the diagnosis. This can be confirmed by appropriate serological testing in the serum and CSF. Clinicians must be aware of this rare neurological complication of Lyme disease that demands specific antibiotic treatment.


Interactive visual steering--rapid visual prototyping of a common rail injection system.
Interactive steering with visualization has been a common goal of the visualization research community for twenty years, but it is rarely ever realized in practice. In this paper we describe a successful realization of a tightly coupled steering loop, integrating new simulation technology and interactive visual analysis in a prototyping environment for automotive industry system design. Due to increasing pressure on car manufacturers to meet new emission regulations, to improve efficiency, and to reduce noise, both simulation and visualization are pushed to their limits. Automotive system components, such as the powertrain system or the injection system have an increasing number of parameters, and new design approaches are required. It is no longer possible to optimize such a system solely based on experience or forward optimization. By coupling interactive visualization with the simulation back-end (computational steering), it is now possible to quickly prototype a new system, starting from a non-optimized initial prototype and the corresponding simulation model. The prototyping continues through the refinement of the simulation model, of the simulation parameters and through trial-and-error attempts to an optimized solution. The ability to early see the first results from a multidimensional simulation space--thousands of simulations are run for a multidimensional variety of input parameters--and to quickly go back into the simulation and request more runs in particular parameter regions of interest significantly improves the prototyping process and provides a deeper understanding of the system behavior. The excellent results which we achieved for the common rail injection system strongly suggest that our approach has a great potential of being generalized to other, similar scenarios.


Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016.
Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0-78.4) for IgM, 88.2% (95% CI: 64.4-98.0) for IgG, and 100% (95% CI: 78.4-100) for IgM/IgG, at 99.8% (95% CI: 99.2-100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0-3.0) and 0.4% (95% CI: 0-2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas.


Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome.
Andersen's syndrome is characterized by periodic paralysis, cardiac arrhythmias, and dysmorphic features. We have mapped an Andersen's locus to chromosome 17q23 near the inward rectifying potassium channel gene KCNJ2. A missense mutation in KCNJ2 (encoding D71V) was identified in the linked family. Eight additional mutations were identified in unrelated patients. Expression of two of these mutations in Xenopus oocytes revealed loss of function and a dominant negative effect in Kir2.1 current as assayed by voltage-clamp. We conclude that mutations in Kir2.1 cause Andersen's syndrome. These findings suggest that Kir2.1 plays an important role in developmental signaling in addition to its previously recognized function in controlling cell excitability in skeletal muscle and heart.


Development of a Swine Polytrauma Model in the Absence of Fluid Resuscitation.
In locations in which access to resuscitative therapy may be limited, treating polytraumatized patients present a challenge. There is a pressing need for adjuncts that can be delivered in these settings. To assess these adjuncts, a model representative of this clinical scenario is necessary. We aimed to develop a hemorrhage and polytrauma model in the absence of fluid resuscitation. This study consisted of two parts: pulmonary contusion dose-finding (n = 6) and polytrauma with evaluation of varying hemorrhage volumes (n = 6). We applied three, six, or nine nonpenetrating captive bolt-gun discharges to the dose-finding group and obtained computed tomography (CT) images. We segmented images to assess contusion volumes. We subjected the second group to tibial fracture, pulmonary contusion, and controlled hemorrhage of 20%, 30%, or 40% and observed for 3 hours or until death. We used Kaplan-Meier analysis to assess survival. We also assessed hemodynamic and metabolic parameters. Contusion volumes for three, six, and nine nonpenetrating captive bolt-gun discharges were 24 ± 28, 50 ± 31, and 63 ± 77 cm3, respectively (p = .679). Animals receiving at least six discharges suffered concomitant parenchymal laceration, whereas one of two swine subjected to three discharges had lacerations. Mortality was 100% at 12 and 115 minutes in the 40% and 30% hemorrhage groups, respectively, and 50% at 3 hours in the 20% group. This study characterizes a titratable hemorrhage and polytrauma model in the absence of fluid resuscitation. This model can be useful in evaluating resuscitative adjuncts that can be delivered in areas remote to healthcare access.


Haematopoietic development and HSC formation in vitro: promise and limitations of gastruloid models.
Haematopoietic stem cells (HSCs) are the most extensively studied adult stem cells. Yet, six decades after their first description, reproducible and translatable generation of HSC in vitro remains an unmet challenge. HSC production in vitro is confounded by the multi-stage nature of blood production during development. Specification of HSC is a late event in embryonic blood production and depends on physical and chemical cues which remain incompletely characterised. The precise molecular composition of the HSC themselves is incompletely understood, limiting approaches to track their origin in situ in the appropriate cellular, chemical and mechanical context. Embryonic material at the point of HSC emergence is limiting, highlighting the need for an in vitro model of embryonic haematopoietic development in which current knowledge gaps can be addressed and exploited to enable HSC production. Gastruloids are pluripotent stem cell-derived 3-dimensional (3D) cellular aggregates which recapitulate developmental events in gastrulation and early organogenesis with spatial and temporal precision. Gastruloids self-organise multi-tissue structures upon minimal and controlled external cues, and are amenable to live imaging, screening, scaling and physicochemical manipulation to understand and translate tissue formation. In this review, we consider the haematopoietic potential of gastruloids and review early strategies to enhance blood progenitor and HSC production. We highlight possible strategies to achieve HSC production from gastruloids, and discuss the potential of gastruloid systems in illuminating current knowledge gaps in HSC specification.


Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains.
Pulsed-fieldgel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several othercountries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.


Augmentation of the virulence of murine coxsackie-virus B-3 myocardiopathy by exercise.
Coxsackievirus B-3 myocardiopathy was induced in weanling mice by intraperitoneal and intracerebral inoculations of the Nancy strain. Acute mortality was 5.5%. The cardiomyopathy is characterized by an early phase lasting about 9 days with myocardial necrosis, associated inflammation, and healing by fibrosis and calcification involving 25 to 50% of the contractile fibers in each affected mouse. Infectious coxsackievirus may be recovered from the heart during this phase. Continuing myocardial inflammatory lesions follow during the later phase, but infectious virus is no longer present. When mice were forced to swim in a preheated pool (33 degrees C) during both phases of their myocardiopathy, virulence was strikingly augmented. Fully half of the mice died of congestive failure, the majority while swimming. Hearts were dilated, hypertrophied, and grossly necrotic. The myocardium was transformed to a completely necrotic, inflammatory, calcifying mass. At the peak of the infectious phase, myocardial replication of coxsackievirus was increased 530 times in nurslings which had been forced to swim. Myositis in hind limbs was more frequent, and inflammatory lesions in perirenal and pericardial fat were more severe in the mice which were forced to swim. When swimming was begun on the 9th day after infection, the virulence and lethality (13.8%) of infection were moderately increased.


Roots of blindsight.
The chapter reviews the historical background to demonstrations that there is residual visual function in the total absence of striate cortex (V1) in monkey and humans. The late 19th century evidence by Munk and others, as reviewed by William James, was that this was not possible in humans, and doubtful at best in monkeys. It has gradually become realized, starting in the middle of the 20th century, that even total bilateral removal of striate cortex in monkeys does not abolish all visual capacity, including spatial and pattern vision. The situation regarding unilateral or incomplete bilateral lesions in the monkey did not become clarified until Cowey's doctoral work in the 1960s, demonstrating that field defects were not absolute, that sensitivity continued to improve over several months of postoperative testing, that the size of the field defect gradually shrank, that the sensitivity was poorest at the center of the field defect, and that recovery was not spontaneous but depended on sustained practice. In human subjects with unilateral lesions from the 1970s onwards, using forced-choice methodology parallel to animal studies, a wide range of visual discriminations was demonstrated but with alterations or complete absence of acknowledged awareness by subjects (blindsight). Various varieties of skepticism are discussed and rebutted. The gap between humans and animals was diminished by the demonstration by Cowey and Stoerig that monkeys, like humans, classify responses to blind-field stimuli as being 'unseen'. Further recent degrees of closure and developments in human blindsight research are discussed.


Gene mapping in the 20th and 21st centuries: statistical methods, data analysis, and experimental design.
In the 20th century geneticists began to unravel some of the simpler aspects of the etiology of inherited diseases in humans. The theory of linkage analysis was developed and applied long before the advent of molecular biology, but only the technological advances of the second half of the 20th century made large-scale gene mapping with a dense genome-spanning set of markers a reality. More recently, the primary topic of interest has shifted from simple Mendelian diseases, for which genotypes of some gene are the cause of disease, to more complex diseases, for which genotypes of some set of genes together with environmental factors merely alter the probability that an individual gets the disease, although individual factors are typically insufficient to cause the disease outright. To this end, a great deal of dogma has evolved about the best way to skin this cat, although to date success has been minimal with any approach. We postulate that the main reason for this is a lack of attention to experimental design. Once the data have been ascertained, the most powerful statistical methods will not be able to salvage an inappropriately designed study (Andersen 1990). Each phenotype and/or population mandates its own individually tailored study design to maximize the chances of successful gene mapping. We suggest that careful consideration of the available data from real genotype-phenotype correlation studies (as opposed to oversimplified theoretically tractable models), and the practical feasibility of different ascertainment schemes dictate how one should proceed. In this review we review the theory and practice of gene mapping at the close of the 20th century, showing that most methods of linkage and linkage disequilibrium analysis are similar in a fundamental sense, with the differences being related more to study design and ascertainment than to technical details of the underlying statistical analysis. To this end, we propose a new focus in the field of statistical genetics that more explicitly highlights the primacy of study design as the means to increase power for gene mapping.


Five Years Later: Awareness Of New York City's Calorie Labels Declined, With No Changes In Calories Purchased.
To follow up on a previous study that examined how the mandated displaying of calorie information on menu boards in fast-food restaurants in New York City influenced consumers' behavior, we analyzed itemized cash register receipts and survey responses from 7,699 consumers at four fast-food chains. Using a difference-in-differences study design, we found that consumers exposed to menu labeling immediately after the mandate took effect in 2008 and at three points in 2013-14 reported seeing and using the information more often than their counterparts at fast-food restaurants without menu labeling. In each successive period of data collection, the percentage of respondents noticing and using the information declined, while remaining above the prelabeling baseline. There were no statistically significant changes over time in levels of calories or other nutrients purchased or in the frequency of visits to fast-food restaurants. Menu labeling at fast-food chain restaurants, which the Affordable Care Act requires to be implemented nationwide in 2016, remains an unproven strategy for improving the nutritional quality of consumer food choices at the population level. Additional policy efforts that go beyond labeling and possibly alter labeling to increase its impact must be considered.


ZFP36L1 negatively regulates plasmacytoid differentiation of BCL1 cells by targeting BLIMP1 mRNA.
The ZFP36/Tis11 family of zinc-finger proteins regulate cellular processes by binding to adenine uridine rich elements in the 3' untranslated regions of various mRNAs and promoting their degradation. We show here that ZFP36L1 expression is largely extinguished during the transition from B cells to plasma cells, in a reciprocal pattern to that of ZFP36 and the plasma cell transcription factor, BLIMP1. Enforced expression of ZFP36L1 in the mouse BCL1 cell line blocked cytokine-induced differentiation while shRNA-mediated knock-down enhanced differentiation. Reconstruction of regulatory networks from microarray gene expression data using the ARACNe algorithm identified candidate mRNA targets for ZFP36L1 including BLIMP1. Genes that displayed down-regulation in plasma cells were significantly over-represented (P = <0.0001) in a set of previously validated ZFP36 targets suggesting that ZFP36L1 and ZFP36 target distinct sets of mRNAs during plasmacytoid differentiation. ShRNA-mediated knock-down of ZFP36L1 in BCL1 cells led to an increase in levels of BLIMP1 mRNA and protein, but not for mRNAs of other transcription factors that regulate plasmacytoid differentiation (xbp1, irf4, bcl6). Finally, ZFP36L1 significantly reduced the activity of a BLIMP1 3' untranslated region-driven luciferase reporter. Taken together, these findings suggest that ZFP36L1 negatively regulates plasmacytoid differentiation, at least in part, by targeting the expression of BLIMP1.


A fluid percussion model of experimental brain injury in the rat.
Fluid percussion models produce brain injury by rapidly injecting fluid volumes into the cranial cavity. The authors have systematically examined the effects of varying magnitudes of fluid percussion injury in the rat on neurological, systemic physiological, and histopathological changes. Acute neurological experiments showed that fluid percussion injury in 53 rats produced either irreversible apnea and death or transient apnea (lasting 54 seconds or less) and reversible suppression of postural and nonpostural function (lasting 60 minutes or less). As the magnitude if injury increased, the mortality rate and the duration of suppression of somatomotor reflexes increased. Unlike other rat models in which concussive brain injury is produced by impact, convulsions were observed in only 13% of survivors. Transient apnea was probably not associated with a significant hypoxic insult to animals that survived. Ten rats that sustained a moderate magnitude of injury (2.9 atm) exhibited chronic locomotor deficits that persisted for 4 to 8 days. Systemic physiological experiments in 20 rats demonstrated that all levels of injury studied produced acute systemic hypertension, bradycardia, and increased plasma glucose levels. Hypertension with subsequent hypotension resulted from higher magnitudes of injury. The durations of hypertension and suppression of amplitude on electroencephalography were related to the magnitudes of injury. While low levels of injury produced no significant histopathological alterations, higher magnitudes produced subarachnoid and intraparenchymal hemorrhage and, with increasing survival, necrotic change and cavitation. These data demonstrate that fluid percussion injury in the rat reproduces many of the features of head injury observed in other models and species. Thus, this animal model could represent a useful experimental approach to studies of pathological changes similar to those seen in human head injury.


Understanding the Ureter: Challenges and Opportunities.
The ureter is possibly the least studied and most poorly understood organ of the urinary tract. The pathophysiologic basis underlying the use of α-blockers to improve ureteral stone passage or to treat ureteral stent symptoms is poorly understood. This, in part, may explain why clinical studies of medical expulsive therapy for ureteral stone passage are fraught with conflicting data. Methods to study human ureter in vivo are few and challenging. The findings of many of the ureteral studies are from observational in vitro studies and were evaluated in other animal species that may not be applicable in human beings. There are few mechanistic studies evaluating the underlying molecular pathophysiologic mechanisms of human ureter. This is critical to our understanding and treatment of stent symptoms, including the development of a patient friendly ureteral stent and for the pharmacologic modulation of ureteral activity. The following is an overview of some of the observational and mechanistic ureteral studies evaluating the pharmacologic and stent effects, including potential areas for further research.


End-of-Life Cancer Care Resource Utilisation in Rural Versus Urban Settings: A Systematic Review.
Despite the advances in End-of-life (EOL) cancer care, disparities remain in the accessibility and utilisation of EOL cancer care resources. Often explained by socio-demographic factors, geographic variation exists in the availability and provision of EOL cancer care services among EOL cancer decedents across urban versus rural settings. This systematic review aims to synthesise mortality follow-back studies on the patterns of EOL cancer care resource use for adults (>18 years) during end-of-life cancer care. Five databases were searched and data analysed using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Inclusion criteria involved; a) original research; b) quantitative studies; c) English language; d) palliative care related service use in adults (>18 years) with any malignancy excluding non-melanoma skin cancers; e) exclusive end of life focus; f) urban-rural focus. Narrative reviews and discussions were excluded. 24 studies met the inclusion criteria. End-of-life cancer care service utilisation patterns varied by rurality and treatment intent. Rurality was strongly associated with higher rates of Emergency Department (ED) visits and hospitalisations and lower rates of hospice care. The largest inequities between urban and rural health service utilisation patterns were explained by individual level factors including age, gender, proximity to service and survival time from cancer diagnosis. Rurality is an important predictor for poorer outcomes in end-of-life cancer care. Findings suggest that addressing the disparities in the urban-rural continuum is critical for efficient and equitable palliative cancer care. Further research is needed to understand barriers to service access and usage to achieve optimal EOL care for all cancer patient populations.


Inactivation of inhibitors by the receptor-destroying enzyme of influenza C virus.
The importance of the receptor-destroying enzyme of influenza C virus for inactivation of inhibitors was analysed. Using three different inhibitors (rat serum, bovine submandibulary mucin and bovine brain gangliosides) inhibition of virus infection was observed only at an inhibitor concentration that was about 100-fold higher than the maximum concentration of inhibitor that could be inactivated by the receptor-destroying enzyme of a given amount of virus. From our data and other observations we conclude that the receptor-destroying enzyme is not required to inactivate inhibitors.


Morphological changes of the left atrial appendage after catheter ablation of atrial fibrillation.
The left atrial appendage (LAA) has been proven to be the most important site of thrombus formation in patients with atrial fibrillation (AF). However, the information regarding the morphometric alteration of the LAA related to the outcome of AF ablation is still lacking. Thus, we evaluated the long-term changes of the LAA morphology in patients undergoing catheter ablation of AF using magnetic resonance angiography (MRA). Group 1 included 15 controls without any AF history. Group 2 included 40 patients with drug-refractory paroxysmal AF. They were divided into two subgroups: group 2a included 30 patients without AF recurrence after pulmonary vein (PV) ablation. Group 2b included 10 patients with late recurrence of AF. The LAA morphology before and after (20 +/- 11 months) ablation was evaluated by three-dimensional MRA. The group 2 patients had a larger baseline LAA size (including the LAA orifice, neck, and length) and less eccentric LAA orifice and neck. After the AF ablation, there was a significant reduction in the LAA size in the group 2a patients, and the morphology of the LAA neck became more eccentric during the follow-up period. In group 2b, the LAA size increased and no significant change in the eccentricity of the orifice and neck could be noted. The morphometric remodeling of the LAA in the AF patients could be reversed after a successful ablation of the AF. Progressive dilation of the LAA was noted in the patients with AF recurrence. These structural changes in the LAA may play a role in reducing the potential risk of cerebrovascular accidents.


Supplementation with a synthetic polyphenol limits oxidative stress and enhances neuronal cell viability in response to hypoxia-re-oxygenation injury.
Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured human neuronal cells exposed to experimental hypoxia-re-oxygenation (H/R) injury responded with an increased production of reactive oxygen species (ROS) and a significant decrease in intracellular ATP. Expression of genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), glucose transporter-1 (Glut-1), the oxygen-sensor neuroglobin (Nb) and Cu,Zn-superoxide dismutase (SOD1), catalase (CAT) and glutathione peroxidase-1 (Gpx-1) increased significantly in response to the insult. Enhanced expression of HO-1, SOD1 and CAT correlated with an increase in the corresponding protein activity. Despite the cellular response to bolster antioxidant capacity, apoptosis and necrosis increased following H/R injury. In contrast, ROS accumulation, the endogenous gene response and cell death was limited in neuronal cells pre-incubated with 50 or 100, but not 10 microM of the phenolic antioxidant 3,3',5,5'-tetra-t-butyl-biphenyl-4,4'-diol (BP) prior to H/R injury. These data indicate that the early endogenous gene response to H/R injury is unable to inhibit neuronal dysfunction and that increasing cellular antioxidant capacity with a synthetic polyphenol (>10 microM) is potentially neuro-protective.


Autoantibody profiling in multiple sclerosis using arrays of human protein fragments.
Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ∼38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then re-evaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.


Using a Value Chain Approach to Map the Pig Production System in Rwanda, Its Governance, and Sanitary Risks.
Rwanda has a fast growing pig production sector projected to continue expansion, due to rising local and regional demand. We undertook a value chain analysis to establish the flows of pigs and pork in Rwanda and the roles of various actors involved, and to understand governance and sanitary risks in the value chain. Cross-sectional qualitative data were collected through focus group discussions and key informant interviews with farmers, brokers, butchers, abattoir managers, and veterinarians. Data were collected on pig production methods and inputs, the source and destination of live and slaughtered pigs, value-adding infrastructures (abattoirs and processing factories), the people involved and interactions between them, governance, and challenges. Pig production in Rwanda is dominated by smallholders, mainly as a source of supplementary income and secondarily for manure. Emerging medium-sized and large pig farms were also identified, located mainly around urban areas. Live pig markets are the main mechanism allowing various actors to buy/sell pigs. Brokers have an important role in pig transactions: they are key in setting prices at markets, examining pigs for disease, organising the supply of pigs for abattoirs and for export. Only a few formal pig abattoirs were identified, which mainly supply to pork processing factories based in Kigali and/or export to customers. Local consumers rely on informal slaughtering at farm or bar/restaurant backyards, with irregular veterinary inspection. Formal abattoirs were attended by a veterinary inspector, however a lack of record keeping was noted. Sanitary risks identified were a lack of biosecurity throughout the chain and poor hygiene at slaughter places. Lingual palpation was practised in pig markets to identify cysticercosis infection, however cyst-positive pigs were not destroyed, but were sold for reduced prices in the same market or later informally sold by the owner. There are few veterinarians attending farms, with most services provided by less qualified technicians or self-treatment of pigs by farmers. Overall, this production system is characterised by a high degree of informality at all nodes, combined with the rapid growth trajectory in the sector. These findings provide a basis to plan interventions tailored to vulnerabilities identified in the Rwanda pig value chain.


Structure of equine infectious anemia virus matrix protein.
The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.


Desire to Be Underweight: Exploratory Study on a Weight Loss App Community and User Perceptions of the Impact on Disordered Eating Behaviors.
Mobile health (mHealth) apps for weight loss (weight loss apps) can be useful diet and exercise tools for individuals in need of losing weight. Most studies view weight loss app users as these types of individuals, but not all users have the same needs. In fact, users with disordered eating behaviors who desire to be underweight are also utilizing weight loss apps; however, few studies give a sense of the prevalence of these users in weight loss app communities and their perceptions of weight loss apps in relation to disordered eating behaviors. The aim of this study was to provide an analysis of users' body mass indices (BMIs) in a weight loss app community and examples of how users with underweight BMI goals perceive the impact of the app on disordered eating behaviors. We focused on two aspects of a weight loss app (DropPounds): profile data and forum posts, and we moved from a broader picture of the community to a narrower focus on users' perceptions. We analyzed profile data to better understand the goal BMIs of all users, highlighting the prevalence of users with underweight BMI goals. Then we explored how users with a desire to be underweight discussed the weight loss app's impact on disordered eating behaviors. We found three main results: (1) no user (regardless of start BMI) starts with a weight gain goal, and most users want to lose weight; (2) 6.78% (1261/18,601) of the community want to be underweight, and most identify as female; (3) users with underweight BMI goals tend to view the app as positive, especially for reducing bingeing; however, some acknowledge its role in exacerbating disordered eating behaviors. These findings are important for our understanding of the different types of users who utilize weight loss apps, the perceptions of weight loss apps related to disordered eating, and how weight loss apps may impact users with a desire to be underweight. Whereas these users had underweight goals, they often view the app as helpful in reducing disordered eating behaviors, which led to additional questions. Therefore, future research is needed.


Infrared imaging and spectral-domain optical coherence tomography findings correlate with microperimetry in acute macular neuroretinopathy: a case report.
Spectral-domain optical coherence tomography findings in a patient with acute macular neuroretinopathy, and correlation with functional defects on microperimetry, are presented. A 25-year old Caucasian woman presented with bitemporal field defects following an upper respiratory tract infection. Her visual acuity was 20/20 in both eyes and a dilated fundus examination revealed bilateral hyperpigmentary changes in the papillomacular bundle. Our patient underwent further evaluation with spectral-domain optical coherence tomography, infrared and fundus autofluorescence imaging. Functional changes were assessed by microperimetry. Infrared imaging showed the classic wedge-shaped defects and spectral-domain optical coherence tomography exhibited changes at the inner segment-outer segment junction, with a thickened outer plexiform layer overlying these areas. Fluorescein and indocyanine green angiography did not demonstrate any perfusion defects or any other abnormality. Microperimetry demonstrated focal elevation in threshold correlating with the wedge-shaped defects in both eyes. Spectral-domain optical coherence tomography findings provide new evidence of the involvement of the outer plexiform layer of the retina in acute macular neuroretinopathy.


Quantitative detection of codeine in human plasma using surface-enhanced Raman scattering via adaptation of the isotopic labelling principle.
In this study surface enhanced Raman scattering (SERS) combined with the isotopic labelling (IL) principle has been used for the quantification of codeine spiked into both water and human plasma. Multivariate statistical approaches were employed for the analysis of these SERS spectral data, particularly partial least squares regression (PLSR) which was used to generate models using the full SERS spectral data for quantification of codeine with, and without, an internal isotopic labelled standard. The PLSR models provided accurate codeine quantification in water and human plasma with high prediction accuracy (Q2). In addition, the employment of codeine-d6 as the internal standard further improved the accuracy of the model, by increasing the Q2 from 0.89 to 0.94 and decreasing the low root-mean-square error of predictions (RMSEP) from 11.36 to 8.44. Using the peak area at 1281 cm-1 assigned to C-N stretching, C-H wagging and ring breathing, the limit of detection was calculated in both water and human plasma to be 0.7 μM (209.55 ng mL-1) and 1.39 μM (416.12 ng mL-1), respectively. Due to a lack of definitive codeine vibrational assignments, density functional theory (DFT) calculations have also been used to assign the spectral bands with their corresponding vibrational modes, which were in excellent agreement with our experimental Raman and SERS findings. Thus, we have successfully demonstrated the application of SERS with isotope labelling for the absolute quantification of codeine in human plasma for the first time with a high degree of accuracy and reproducibility. The use of the IL principle which employs an isotopolog (that is to say, a molecule which is only different by the substitution of atoms by isotopes) improves quantification and reproducibility because the competition of the codeine and codeine-d6 for the metal surface used for SERS is equal and this will offset any difference in the number of particles under analysis or any fluctuations in laser fluence. It is our belief that this may open up new exciting opportunities for testing SERS in real-world samples and applications which would be an area of potential future studies.


In vitro Antioxidant and Antiproliferative Activities of Various Solvent Fractions from Clerodendrum viscosum Leaves.
Free radicals such as reactive oxygen and nitrogen species, generated in the body, play an important role in the fulfillment of various physiological functions but their imbalance in the body lead to cellular injury and various clinical disorders such as cancer, neurodegenaration, and inflammation. The objective of this study is to fight this problem, natural antioxidant from plants can be considered as possible protective agents against various diseases such as cancer which might also modify the redox microenvironment to reduce the genetic instability. This study was designed to evaluate the antioxidant and antiproliferative potential of Clerodendrum viscosum fractions against various carcinomas. In this present study, 70% methanolic extract of C. viscosum leaves have been fractionated to obtain hexane, chloroform, ethyl acetate, butanol, and water fractions, which were tested for their antioxidant and anticancer properties. It was observed that chloroform and ethyl acetate fractions showed good free radical scavenging properties as well as inhibited the proliferation of human lung cancer (A459), breast (MCF-7), and brain (U87) cells. Moreover, they arrested the cell cycle at G2/M phase of breast and brain cancer. These inhibitory effects were further confirmed by bromodeoxyuridine uptake imaging. Phytochemical investigations further indicate the presence of tannic acid, quercetin, ellagic caid, gallic acid, reserpine, and methyl gallate which might be the reason for these fractions' antioxidant and antiproliferative activities. Clerodendrum viscosum leaf chloroform and Clerodendrum viscosum leaf ethyl acetate fractions from C. viscosum showed good reactive oxygen species and reactive nitrogen species scavenging potential. Both the fractions arrested cell cycle at G2/M phase in MCF-7 and U87 cells which lead to induce apoptosis. Crude extract of Clerodendrum viscosum leaves was fractionated using different solventsAmong them, chloroform and ethyl acetate fractions exhibited excellent free radical scavenging propertiesThe same fractions inhibited the proliferation of human lung cancer (A459), breast (MCF-7), and brain (U87) cellsChloroform and ethyl acetate fractions arrested the cell cycle at G2/M phase of breast and brain cancerPhytochemical investigations further indicate the presence of several bioactive principles present in them. Abbreviations used: CVLME: Clerodendrum viscosum leaf methanolic extract; CVLH: Clerodendrum viscosum leaf hexane; CVLC: Clerodendrum viscosum leaf chloroform; CVLE: Clerodendrum viscosum leaf ethyl acetate; CVLB: Clerodendrum viscosum leaf butanol; CVLW: Clerodendrum viscosum leaf water; BrdU: Bromodeoxyuridine; WST-1: Water soluble tetrazolium salt.


Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.
Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.