Analyzing pre-symptomatic tissue to gain insights into the molecular and mechanistic origins of late-onset degenerative trinucleotide repeat disease.

How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one-two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.

Impact of postoperative complications on disease recurrence and long-term survival following oesophagogastric cancer resection.

BACKGROUND: Postoperative complications after resection of oesophagogastric carcinoma can result in considerable early morbidity and mortality. However, the long-term effects on survival are less clear. METHODS: All patients undergoing intentionally curative resection for oesophageal or gastric cancer between 2006 and 2016 were selected from an institutional database. Patients were categorized by complication severity according to the Clavien-Dindo classification (grades 0-V). Complications were defined according to an international consensus statement. The effect of leak and severe non-leak-related complications on overall survival, recurrence and disease-free survival was assessed using Kaplan-Meier analyses to evaluate differences between groups. All factors significantly associated with survival in univariable analysis were entered into a Cox multivariable regression model with stepwise elimination. RESULTS: Some 1100 patients were included, with a median age of 69 (range 28-92) years; 48·1 per cent had stage III disease and cancer recurred in 428 patients (38·9 per cent). Complications of grade III or higher occurred in 244 patients (22·2 per cent). The most common complications were pulmonary (29·9 per cent), with a 13·0 per cent incidence of pneumonia. Rates of atrial dysrhythmia and anastomotic leak were 10·0 and 9·6 per cent respectively. Patients with a grade III-IV leak did not have significantly reduced overall survival compared with those who had grade 0-I complications. However, patients with grade III-IV non-leak-related complications had reduced median overall survival (19·7 versus 42·7 months; P < 0·001) and disease-free survival (18·4 versus 36·4 months; P < 0·001). Cox regression analysis identified age, tumour stage, resection margin and grade III-IV non-leak-related complications as independent predictors of poor overall and disease-free survival. CONCLUSION: Beyond the acute postoperative period, anastomotic leak does not adversely affect survival, however, other severe postoperative complications do reduce long-term overall and disease-free survival.

Ruthenium photoredox-triggered phospholipid membrane formation.

As more methodologies for generating and manipulating biomimetic cellular systems are developed, opportunities arise for combining different methods to create more complex synthetic biological constructs. This necessitates an increasing need for tools to selectively trigger individual methodologies. Here we demonstrate ruthenium tris-bipyridine mediated photoredox triggering of the copper catalyzed alkyne azide cycloaddition reaction (CuAAC), resulting in the synthesis of biomimetic phospholipids in situ, and subsequent membrane assembly. The use of a ruthenium-copper electron transport chain to trigger phospholipid assembly opens up future opportunities for spatiotemporal synthesis of membranes.

The development and validation of a meta-tool for quality appraisal of public health evidence: Meta Quality Appraisal Tool (MetaQAT).

OBJECTIVES: Most quality appraisal tools were developed for clinical medicine and tend to be study-specific with a strong emphasis on risk of bias. In order to be more relevant to public health, an appropriate quality appraisal tool needs to be less reliant on the evidence hierarchy and consider practice applicability. Given the broad range of study designs used in public health, the objective of this study was to develop and validate a meta-tool that combines public health-focused principles of appraisal coupled with a set of design-specific companion tools. STUDY DESIGN: Several design methods were used to develop and validate the tool including literature review, synthesis, and validation with a reference standard. METHODS: A search of critical appraisal tools relevant to public health was conducted; core concepts were collated. The resulting framework was piloted during three feedback sessions with public health practitioners. Following subsequent revisions, the final meta-tool, the Meta Quality Appraisal Tool (MetaQAT), was then validated through a content analysis of appraisals conducted by two groups of experienced public health researchers (MetaQAT vs generic appraisal form). RESULTS: The MetaQAT framework consists of four domains: relevancy, reliability, validity, and applicability. In addition, a companion tool was assembled from existing critical appraisal tools to provide study design-specific guidance on validity appraisal. Content analysis showed similar methodological and generalizability concerns were raised by both groups; however, the MetaQAT appraisers commented more extensively on applicability to public health practice. CONCLUSIONS: Critical appraisal tools designed for clinical medicine have limitations for use in the context of public health. The meta-tool structure of the MetaQAT allows for rigorous appraisal, while allowing users to simultaneously appraise the multitude of study designs relevant to public health research and assess non-standard domains, such as applicability.

Multiple shape memory polymers based on laminates formed from thiol-click chemistry based polymerizations.

This investigation details the formation of polymer network trilayer laminates formed by thiol-X click chemistries, and their subsequent implementation and evaluation for quadruple shape memory behavior. Thiol-Michael addition and thiol-isocyanate-based crosslinking reactions were employed to fabricate each of the laminate's layers with independent control of the chemistry and properties of each layer and outstanding interlayer adhesion and stability. The characteristic features of step-growth thiol-X reactions, such as excellent network uniformity and narrow thermal transitions as well as their stoichiometric nature, enabled fabrication of trilayer laminates with three distinctly different glass transition temperatures grouped within a narrow range of 100 °C. Through variations in the layer thicknesses, a step-wise modulus drop as a function of temperature was achieved. This behavior allowed multi-step programming and the demonstration and quantification of quadruple shape memory performance. As is critical for this performance, the interface connecting the layers was evaluated in stoichiometric as well as off-stoichiometric systems. It was shown that the laminated structures exhibit strong interfacial binding and hardly suffer any delamination during cyclic material testing and deformation.

Colorectal cancer is reliably excluded in the frail and elderly population by minimal preparation CT.

BACKGROUND: This study aimed to retrospectively assess the accuracy of minimal preparation computed tomography (MPCT) in the detection of colorectal cancer (CRC) within the frail and elderly population and to evaluate the relevance of extra-colonic findings (ECF). METHODS: Radiology reports, clinical notes and follow-up reports from 207 patients who underwent MPCT to investigate for CRC between 2005 and 2009 were analysed. Patients were scanned following the administration of oral contrast for 48 h, without bowel preparation or colonic insufflation. MPCT results were measured against patient outcomes, with a minimum of 2 years of follow-up. RESULTS: Twelve cases of clinically relevant CRC were confirmed (5.8 %). MPCT correctly identified 11 of these lesions (sensitivity 91.6 %). Thirty-one patients had a possible CRC identified by MPCT, which was not confirmed by further examination (specificity 84.1 %). This results in a positive predictive value of 26.2 % and a negative predictive value of 99.4 %. Five of the patients with colon cancer underwent curative surgery. Sixty-eight clinically relevant ECF were confirmed, including 14 previously undiagnosed extra-colonic malignancies. ECF were considered to account for the presenting complaint in 15.0 % (31/207) of all patients. CONCLUSIONS: Minimal preparation computed tomography is an effective and reliable investigation for the exclusion of clinically relevant CRC in this population. It provides clinicians with a valuable and pragmatic alternative to colonoscopy and CT colonography when invasive examination or cathartic bowel preparation will be poorly tolerated and small polyps are of limited significance. MPCT has an advantage over purely luminal imaging in the detection of extra-colonic pathology and appears to have an equally important role in the detection of CRC.

HIV-1 co-receptor expression and epithelial immune cells of the cervix in asymptomatic women attending a genitourinary medicine clinic.

OBJECTIVES: The aim of the study was to qualitatively and semiquantitatively characterize the expression of the principal HIV co-receptors chemokine (C-C motif) receptor 5 (CCR5) and chemokine (C-X-C motif) receptor 4 (CXCR4) on susceptible CD4 T-helper cell, monocyte/macrophage and Langerhans dendritic cell populations within the cervical epithelia of asymptomatic women attending a genitourinary medicine clinic. METHODS: Of 77 asymptomatic women recruited, 35 were excluded: 21 because they were found to have bacterial vaginosis, eight because they were found to have candida and six for other reasons. Cervical cytobrush samples from 11 women with Chlamydia trachomatis infection and 31 women without any detectable genital infection were stained with fluorescently labelled antibodies specific for cell surface CCR5, CXCR4, CD4, CD3, CD1a and CD19 expression, then analysed by flow cytometry. RESULTS: CD4/CD3 T-helper cells (84%), CD1a Langerhans dendritic cells (75%) and CD4/CD14 monocytes/macrophages (59%) were detected in the samples. CCR5 and CXCR4 HIV co-receptor expression was observed on 46-86% of the above subsets. CD1a cells exhibited significantly higher CCR5 and CXCR4 positivity and median fluorescence than CD4 cells and higher CXCR4 positivity and median fluorescence than CD14 cells (P < 0.05 or less). Increased detection of CCR5 over CXCR4 was seen in CD14 cells (P < 0.05). No significant differences in CCR5 or CXCR4 expression were found in samples from asymptomatic women with or without chlamydial infection. CONCLUSIONS: Co-receptor expression confirms the potential for CD1a Langerhans cells, monocytes/macrophages and T-helper cells in the cervix as primary targets for HIV infection. Previously observed selective transmission of CCR5-tropic isolates cannot be accounted for by a lack of CXCR4-expressing CD4 cervical immune cells. We were unable to identify any specific impact of chlamydial infection on co-receptor expression in this study.

Reproductive and developmental safety of nirmatrelvir (PF-07321332), an oral SARS-CoV-2 Mpro inhibitor in animal models.

Nirmatrelvir (PF-07321332; NMV) the antiviral component of PAXLOVID™ is a potent and selective inhibitor of the SARS-CoV-2 main protease (Mpro), which plays a critical role in viral replication. PAXLOVID, comprised of nirmatrelvir and ritonavir (used as a pharmacokinetic enhancer), is an oral therapy currently in development as a therapeutic option for those infected with SARS-CoV-2 to prevent progression to severe disease, hospitalization, and death. PAXLOVID has been shown to be efficacious against hospitalization and death in two Phase 2/3 clinical studies that evaluated non hospitalized patients both with and without high risk factors for progression to severe illness. Given that males and females of reproductive age are included in the intended patient population, we assessed the potential effects of NMV up to the limit dose of 1000 mg/kg/day in ICH guideline embryo-fetal development studies in rats and rabbits, and a fertility and early embryonic development study in rats. There were no effects on male and female fertility or early embryonic development in rats, and no severe manifestations of developmental toxicity in rats or rabbits. The lack of adverse findings reported here in nonclinical species is consistent with the intended therapeutic target of NMV (a virus specific protein not present in mammalian cells), the favorable off-target selectivity profile, and lack of genetic toxicity. The results of these nonclinical studies with NMV along with existing ritonavir safety information indicate that there are no clinically relevant risks associated with PAXLOVID administration during pregnancy and in males and females of reproductive age.

Lack of direct bone effect of tofacitinib, a JAK inhibitor, in juvenile rats and evaluation of the association between offspring growth and femur length.

Bone has recently emerged as a target organ for some Janus kinase (JAK) inhibitors in adult and/or juvenile animal toxicity studies. Oral administration of tofacitinib, a JAK inhibitor, was not associated with clinical or macroscopic effects on bone growth and development in a rat juvenile animal study (JAS) with tofacitinib dosing starting on postnatal day (PND) 21. However, given that previous JAS did not include a targeted evaluation of bone, inclusive of microscopic examination, an additional rat JAS was conducted to further assess this risk. In this subsequent JAS, administration of tofacitinib from PND 7-49 or from PND 21-49 did not result in any direct effects on bone, with no histologic effects on developing bone. The only bone effect in this JAS was nonadverse shorter femur length, which was not considered to be a direct effect of tofacitinib, but rather an indicator of growth delay, as this was associated with lower body weights. There were no effects on femur length or body weight after a 2-month recovery period. To further explore the relationship between body weight and femur length, historical control data were analyzed from control rats in other JAS. This analysis clearly demonstrated that shorter femur length can occur as an indirect effect that is highly associated with lower body weight, consistent with what was observed in the JAS with tofacitinib. These analyses provide a robust and valuable data set to support the interpretation of such data in JAS, and further support the lack of direct effects of tofacitinib on bone growth and development. As with the previously conducted juvenile studies with tofacitinib, the additional JAS did not identify any special JAS-based concerns for use in pediatric patients as young as 2 years of age.

Studies on the mode of action of diphtheria toxin. III. Effect on subcellular components of protein synthesis from the tissues of intoxicated guinea pigs and rats.

The effect of diphtheria toxin on subcellular components of protein synthesis was determined. Polyribosomes prepared from intoxicated guinea pigs functioned normally in an in vitro assay system, while the activity of soluble enzymes (transferases) from toxin-treated animals was significantly reduced. At high toxin dosages, this reduction was widespread, but when levels of toxin comparable to those which might be generated in a natural infection were given, inhibition of soluble enzyme activity was found only in extracts from heart and skeletal muscle. Possible nonspecific inhibition in the assay system due to interference by free toxin or by a serum component was eliminated. Since it was possible to demonstrate reactivation of soluble enzyme activity with nicotinamide and toxin, it was suggested that diphtheria toxin acts in the intact sensitive animal in a manner analogous to its action in tissue culture or in cell-free systems. It was hypothesized that the lethal biochemical lesion of the toxin in sensitive animals was the inactivation of transferase enzymes, principally in the heart. It was also suggested that the lethal lesion induced in diphtheria-sensitive and resistant species may not be identical.

Efficient production of a high-performance dispersion strengthened, multi-principal element alloy.

Additive manufacturing currently facilitates new avenues for materials discovery that have not been fully explored. In this study we reveal how additive manufacturing can be leveraged to produce dispersion strengthened (DS), multi-principal element alloys (MPEA) without the use of traditional mechanical alloying or chemical reactions. This new processing technique employed resonant acoustic mixing to coat an equiatomic NiCoCr powder with nano-scale yttrium oxides. Then, through laser powder bed fusion (L-PBF), the coated powder was successfully consolidated into 99.9% dense parts. Microstructural analysis confirmed the successful incorporation and dispersion of nano-scale oxides throughout the build volume. Furthermore, high temperature mechanical testing of the DS alloys showed significant improvements in strength and ductility over the baseline NiCoCr. As a result, this recently discovered processing route opens a new alloy design and production path that is synergistic between additive manufacturing and dispersion strengthening, possibly enabling a new generation of high-performance alloys.

Genome-wide approaches to identify pharmacogenetic contributions to adverse drug reactions.

Adverse drug reactions (ADRs) have a major impact on patients, physicians, health care providers, regulatory agencies and pharmaceutical companies. Identifying the genetic contributions to ADR risk may lead to a better understanding of the underlying mechanisms, identification of patients at risk and a decrease in the number of events. Technological advances have made the routine monitoring and investigation of the genetic basis of ADRs during clinical trials possible. We demonstrate through simulation that genome-wide genotyping, coupled with the use of clinically matched or population controls, can yield sufficient statistical power to permit the identification of strong genetic predictors of ADR risk in a prospective manner with modest numbers of ADR cases. The results of a 500,000 single nucleotide polymorphism analysis of abacavir-associated hypersensitivity reaction suggest that the known HLA-B gene region could be identified with as few as 15 cases and 200 population controls in a sequential analysis.

Osmotic behavior and permeability of osmotically lysed mitochondria.

Experiments were carried out with water-treated isolated rat liver mitochondria (mitochondria ghosts) previously studied by Caplan and Greenawalt (Caplan, A.I., and J.W. Greenawalt. 1966. J. Cell Biol. 31:455-472) and Vasington and Greenawalt (Vasington, F., and J. Greenawalt. 1968. J. Cell Biol. 39:661-675). The ghosts have permeability properties and osmotic behavior comparable to those of isolated mitochondria. Although they have lost most of their internal contents, they must have resealed. Four properties were found which have not been previously described in systems derived from biological membranes: (a) an osmotic behavior in the virtual absence of internal components. (b) a self-arranging property in the formation of invaginations corresponding in morphology to the cristae. The results suggest that the assembly of the molecular components of the inner membrane is sufficient to specify the morphology. Hence the surface area to volume ratio of the vesicles may specify the presence or absence of cristae-like folds. (c) an increase in the permeability of the membranes to sucrose in the presence of iso-osmotic concentrations of sucrose. (d) an independence of the light transmitted by suspensions of the vesicles from the refractive index of the external medium. This observations run counter to the general previous experience with either mitochondria or liposomes.

Electrophoretic injection of a fluorescent dye into giant mitochondria and mitoplasts.

Studies of the electrical properties of giant mitochondria and mitoplasts with microelectrodes have indicated that there are no significant metabolically dependent membrane potentials. The internal location of the microelectrode has been confirmed by electrophoretically microinjecting the water-soluble dye Lucifer yellow CH into giant mitochondria or mitoplasts.

Complete genome sequence of Treponema pallidum, the syphilis spirochete.

The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.

Cost-effective airway cultures in the cystic fibrosis patient.

BACKGROUND: Cystic fibrosis (CF) patients have a high incidence of multidrug-resistant infections, rendering CF patients a treatment challenge. OBJECTIVE: To evaluate culture protocols for CF patients and develop a cost-effective culture regimen that identifies clinically relevant pathogens. STUDY DESIGN: Retrospective review. METHODS: At the time of endoscopic sinus surgery, CF patients underwent both sinus and bronchial lavage cultures. Medical records from 2002 to 2006 were reviewed. RESULTS: Twenty-four cases were identified; 12 had complete sets of cultures. Seven of 12 had sinus aerobic bacteria that were also present in bronchial culture. Anaerobic cultures from both sites were all negative (42%) or nondiagnostic (58%). Thirty-three percent of sinus fungal cultures and 91.6% of bronchial fungal cultures were positive. Sinus acid fast bacillus cultures were all negative. CONCLUSION: CF culture protocols may be streamlined by eliminating all anaerobic cultures, as well as sinus acid fast bacillus and fungal cultures for a 52% reduction in cost.

Ipratropium bromide for acute asthma exacerbations in the emergency setting: a literature review of the evidence.

Since the 1970s, when inhaled anticholinergic agents were first introduced as adjunct therapies for the immediate treatment of pediatric asthma exacerbations, several trials have shown varying degrees of benefit from their use as bronchodilators in combination with inhaled short-acting beta-adrenergic agonists and systemic corticosteroids. Although other anticholinergics exist, ipratropium bromide (IB) specifically has emerged as the overwhelming choice of pulmonologists and emergency physicians because of its limited systemic absorption from the lungs when given as an inhaled preparation. However, although the varying trials, predominantly in the emergency department setting, have typically shown a trend toward improved outcomes, none has set forth clear dosing protocol recommendations for use by practicing physicians. It is our goal in this review of the available literature on the use of IB, as an adjunct to inhaled short-acting beta-adrenergic agonists, to summarize practical, evidence-based recommendations for use in the pediatric emergency department setting for acute asthma exacerbations. We also hope to better delineate the most effective dosing regimen in those patients who might benefit most from the addition of IB and to explore proposed additional benefits it may have as a modulator of cholinergic-induced effects from high-dose beta-agonist therapy and viral triggers.

Reproductive and developmental toxicity assessment of palbociclib, a CDK4/6 inhibitor, in Sprague-Dawley rats and New Zealand White rabbits.

Palbociclib is a selective inhibitor of the cyclin-dependent kinase (CDK) 4/6, approved for the treatment of breast cancer. We assessed the potential effects of oral administration of palbociclib on reproduction and development. There were no effects on female or male fertility indices; however, in the male there was seminiferous tubule degeneration in the testes and secondary findings in the epididymides, lower testicular and epididymal weights, sperm density and motility. Palbociclib was not teratogenic in rats or rabbits; however, in the presence of maternal toxicity (lower maternal body weight gain and food consumption), low fetal body weights were observed in rats and small forepaw phalanges were noted in rabbits. There were, however, no adverse effects on the F1 generation in a pre- and post-natal developmental toxicity study in the rat.

Physically principled reflection models applied to filtered camera imaging inversions in metal walled fusion machines.

Ray-tracing techniques are applied to filtered divertor imaging, a diagnostic that has long suffered from artifacts due to the polluting effect of reflected light in metal walled fusion machines. Physically realistic surface reflections were modeled using a Cook-Torrance micro-facet bi-directional reflection distribution function applied to a high resolution mesh of the vessel geometry. In the absence of gonioreflectometer measurements, a technique was developed to fit the free parameters of the Cook-Torrance model against images of the JET in-vessel light sources. By coupling this model with high fidelity plasma fluid simulations, photo-realistic renderings of a number of tokamak plasma emission scenarios were generated. Finally, a sensitivity matrix describing the optical coupling of a JET divertor camera and the emission profile of the plasma was obtained, including full reflection effects. These matrices are used to perform inversions on measured data and shown to reduce the level of artifacts in inverted emission profiles.

An examination of protein binding and protein-facilitated uptake relating to in vitro-in vivo extrapolation.

As explained by the free drug theory, the unbound fraction of drug has long been thought to drive the efficacy of a molecule. Thus, the fraction unbound term, or fu, appears in equations for fundamental pharmacokinetic parameters such as clearance, and is used when attempting in vitro to in vivo extrapolation (IVIVE). In recent years though, it has been noted that IVIVE does not always yield accurate predictions, and that some highly protein bound ligands have more efficient uptake than can be explained by their unbound fractions. This review explores the evolution of fu terms included when implementing IVIVE, the concept of protein-facilitated uptake, and the mechanisms that have been proposed to account for facilitated uptake.