CRISPR/Cas9-Mediated Genome Editing in Zebrafish.

The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.

Fishing for answers to hemostatic and thrombotic disease: Genome editing in zebrafish.

Over the past two decades, the teleost vertebrate Danio rerio (zebrafish) has emerged as a model for hemostasis and thrombosis. At genomic and functional levels, there is a high degree of conservation of the hemostatic system with that of mammals. Numerous features of the fish model offer unique advantages for investigating hemostasis and thrombosis. These include high fecundity, rapid and external development, optical transparency, and extensive functional homology with mammalian hemostasis and thrombosis. Zebrafish are particularly suited to genome-wide mutagenesis experiments for the study of modifier genes. They are also amenable to whole-organism small-molecule screens, a feature that is exceptionally relevant to hemostasis and thrombosis. Zebrafish coagulation factor knockouts that are in utero or neonatal lethal in mammals survive into adulthood before succumbing to hemorrhage or thrombosis, enabling studies not possible in mammals. In this illustrated review, we outline how zebrafish have been employed for the study of hemostasis and thrombosis using modern genome editing techniques, coagulation assays in larvae, and in vivo evaluation of patient-specific variants to infer causality and demonstrate pathogenicity. Zebrafish hemostasis and thrombosis models will continue to serve as a clinically directed basic research tool and powerful alternative to mammals for the development of new diagnostic markers and novel therapeutics for coagulation disorders through high-throughput genetic and small-molecule studies.

Acitretin mitigates uroporphyrin-induced bone defects in congenital erythropoietic porphyria models.

Congenital erythropoietic porphyria (CEP) is a rare genetic disorder leading to accumulation of uro/coproporphyrin-I in tissues due to inhibition of uroporphyrinogen-III synthase. Clinical manifestations of CEP include bone fragility, severe photosensitivity and photomutilation. Currently there is no specific treatment for CEP, except bone marrow transplantation, and there is an unmet need for treating this orphan disease. Fluorescent porphyrins cause protein aggregation, which led us to hypothesize that uroporphyrin-I accumulation leads to protein aggregation and CEP-related bone phenotype. We developed a zebrafish model that phenocopies features of CEP. As in human patients, uroporphyrin-I accumulated in the bones of zebrafish, leading to impaired bone development. Furthermore, in an osteoblast-like cell line, uroporphyrin-I decreased mineralization, aggregated bone matrix proteins, activated endoplasmic reticulum stress and disrupted autophagy. Using high-throughput drug screening, we identified acitretin, a second-generation retinoid, and showed that it reduced uroporphyrin-I accumulation and its deleterious effects on bones. Our findings provide a new CEP experimental model and a potential repurposed therapeutic.

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish.

The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete deficiency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafish are able to tolerate severe hemostatic deficiencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafish prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fibrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafish and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafish are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.

How frequently are patients weighed in hospital ? Results from a five-year cross-sectional audit of clinical practice in nine hospitals.

BACKGROUND AND AIMS: Knowledge of a person's weight is important for identifying malnutrition; occupational safety reasons; medication dosing and evaluating effectiveness of medical nutrition therapy. However, weighing of patients in hospitals is known to be problematic and suboptimal. METHODS: Five annual cross-sectional audits of patients admitted to nine hospitals were conducted to determine the frequency that patients were weighed (i) on admission and (ii) during admission. Characteristics such as age, length of stay, the presence of cognitive impairment, culturally and linguistically diverse (CALD) background, mobility status and single room isolation were also recorded. RESULTS: The frequency of weighing patients on admission was only 20.3%. Approximately 62.4% of patients had been weighed at least once during their admission. Individuals who were admitted to rehabilitation wards or those with independent mobility had significantly higher odds of being weighed during the admission, in addition to those with a longer length of stay (Odds Ratios 5.98 (95% CI: 2.51-10.3); 2.34 (95% CI: 1.60-3.4); and 1.05 (95% CI: 1.03-1.07) respectively, all p < 0.001). Differences between ward types were also evident with rehabilitation, paediatric, renal and mental health wards exhibiting a higher incidence of weighing patients during their admission. CONCLUSIONS: The practice of weighing patients in this health district was suboptimal and places patients at high risk of a deterioration in their clinical condition. Strategies to ensure all patients are weighed including those who have a short hospital stay and those with poor mobility is required.

Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis.

In humans, coagulation factor V (FV) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of FV in zebrafish through targeted mutagenesis and apply the model to the study of human F5 variants. CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f5 -/- mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood. Human F5 variants of uncertain significance from patients with FV deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual FV function, with minimal activity being required to restore hemostasis in response to laser-induced endothelial injury. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology, although limitations exist. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities.

Evaluation of the impact of a post-hospital discharge Transitional Aged Care Service on frailty, malnutrition and functional ability.

AIM: To investigate the relationship between nutritional status, functional ability and frailty in older adults participating in a 12-week Transitional Aged Care Service program. METHODS: A retrospective analysis of a clinical cohort of older adults aged 65+ years after hospital discharge. At entry into the program and at completion, nutritional status was measured using the Mini Nutritional Assessment (MNA), frailty status was measured using the Groningen Frailty Indicator and functional ability was measured using the Modified Barthel Index (MBI). Demographic data were obtained from electronic medical records. RESULTS: Baseline data were available for 115 participants (mean age = 81.7 (SD =7.9) years; 20.9% classified as malnourished and 89.6% as frail). A positive association was found between nutritional status and frailty (r = 0.298; P = 0.001), and frailty and functional ability (r = 0.204; P = 0.029). Multiple regression analysis, accounting for the cofounders of baseline MNA, MBI, age, gender, length of hospital stay and living situation, found that nutritional status and functional ability were able to indicate the presence of frailty on admission to the program (P = 0.002, P = 0.007, respectively). In those program completers (n = 79), significant improvements were found in nutritional status, frailty and functional ability (P < 0.0005). CONCLUSIONS: Nutrition status, frailty and functional ability are closely and positively related, and should therefore be considered simultaneously in rehabilitation for older adults. A post-hospital transitional program with a multidisciplinary approach significantly improved all three outcomes, suggesting its value in enabling frail older people to remain independent for as long as possible.

A Journey to Improved Inpatient Glycemic Control by Redesigning Meal Delivery and Insulin Administration.

PURPOSE: The purpose of this quality improvement project was to redesign a hospital meal delivery process in order to shorten the time between blood glucose monitoring and corresponding insulin administration and improve glycemic control. DESCRIPTION: This process change redesigned the workflow of the dietary and nursing departments. Modifications included nursing, rather than dietary, delivering meal trays to patients receiving insulin. Dietary marked the appropriate meal trays and phoned each unit prior to arrival on the unit. The process change was trialed on 2 acute care units prior to implementation hospital wide. Elapsed time between blood glucose monitoring and insulin administration was analyzed before and after process change as well as evaluation of glucometrics: percentage of patients with blood glucose between 70 and 180 mg/dL (percent perfect), blood glucose greater than 300 mg/dL (extreme hyperglycemia), and blood glucose less than 70 mg/dL (hypoglycemia). RESULTS: Percent perfect glucose results improved from 45% to 53%, extreme hyperglycemia (blood glucose >300 mg/dL) fell from 11.7% to 5%. Hypoglycemia demonstrated a downward trend line, demonstrating that with improving glycemic control hypoglycemia rates did not increase. Percentage of patients receiving meal insulin within 30 minutes of blood glucose check increased from 35% to 73%. CONCLUSION: In the hospital, numerous obstacles were present that interfered with on-time meal insulin delivery. Establishing a meal delivery process with the nurse performing the premeal blood glucose check, delivering the meal, and administering the insulin improves overall blood glucose control. IMPLICATIONS: Nurse-led process improvement of blood glucose monitoring, meal tray delivery, and insulin administration does lead to improved glycemic control for the inpatient population.

Validating workplace performance assessments in health sciences students: a case study from speech pathology.

Valid assessment of health science students' ability to perform in the real world of workplace practice is critical for promoting quality learning and ultimately certifying students as fit to enter the world of professional practice. Current practice in performance assessment in the health sciences field has been hampered by multiple issues regarding assessment content and process. Evidence for the validity of scores derived from assessment tools are usually evaluated against traditional validity categories with reliability evidence privileged over validity, resulting in the paradoxical effect of compromising the assessment validity and learning processes the assessments seek to promote. Furthermore, the dominant statistical approaches used to validate scores from these assessments fall under the umbrella of classical test theory approaches. This paper reports on the successful national development and validation of measures derived from an assessment of Australian speech pathology students' performance in the workplace. Validation of these measures considered each of Messick's interrelated validity evidence categories and included using evidence generated through Rasch analyses to support score interpretation and related action. This research demonstrated that it is possible to develop an assessment of real, complex, work based performance of speech pathology students, that generates valid measures without compromising the learning processes the assessment seeks to promote. The process described provides a model for other health professional education programs to trial.

What Latino Puerto Ricans and non-Latinos say when they talk about Alzheimer's disease.

BACKGROUND: To discover whether Latino Puerto Rican and non-Latino communities differ in the words they use to talk about Alzheimer's disease (AD). METHODS: Four groups of 30 persons per group defined by self-identified ethnicity and caregiver status: Latino Puerto Ricans and non-Latino Whites, who were either caregivers or non-caregivers completed free-listing exercises to identify the words they use when they describe AD causes, symptoms, caregiving, and research risks and benefits. RESULTS: Both Latino Puerto Ricans and non-Latino Whites recognize AD as a disease of memory loss and other cognitive problems. Although both groups used the term "sadness" to describe AD, non-Latino Whites did not feature emotional, behavioral, or psychological problems as among the causes of AD. Although all the groups' descriptions of a person who lives with and cares for a person with AD shared the word "loving," Latino Puerto Ricans focused on a good spouse who exercises intelligence, patience, and attention on behalf of the person with AD and did not use the term "caregiver." In contrast, non-Latino Whites typically used the term "caregiver." Both groups' lists shared words that describe research as presenting harms to an AD patient and requiring a commitment of time. Latino Puerto Ricans' lists suggested an understanding of research benefits akin to clinical care. CONCLUSIONS: Notable differences exist in how Latino Puerto Ricans and non-Latino Whites talk about AD and AD research. Clinicians, clinical investigators, and patient educators need to consider these differences when they conduct clinical care and research and design outreach and educational materials.

Desolvation barrier effects are a likely contributor to the remarkable diversity in the folding rates of small proteins.

The variation in folding rate among single-domain natural proteins is tremendous, but common models with explicit representations of the protein chain are either demonstrably insufficient or unclear as to their capability for rationalizing the experimental diversity in folding rates. In view of the critical role of water exclusion in cooperative folding, we apply native-centric, coarse-grained chain modeling with elementary desolvation barriers to investigate solvation effects on folding rates. For a set of 13 proteins, folding rates simulated with desolvation barriers cover approximately 4.6 orders of magnitude, spanning a range essentially identical to that observed experimentally. In contrast, folding rates simulated without desolvation barriers cover only approximately 2.2 orders of magnitude. Following a Hammond-like trend, the folding transition-state ensemble (TSE) of a protein model with desolvation barriers generally has a higher average number of native contacts and is structurally more specific, that is, less diffused, than the TSE of the corresponding model without desolvation barriers. Folding is generally significantly slower in models with desolvation barriers because of their higher overall macroscopic folding barriers as well as slower conformational diffusion speeds in the TSE that are approximately 1/50 times those in models without desolvation barriers. Nonetheless, the average root-mean-square deviation between the TSE and the native conformation is often similar in the two modeling approaches, a finding suggestive of a more robust structural requirement for the folding rate-limiting step. The increased folding rate diversity in models with desolvation barriers originates from the tendency of these microscopic barriers to cause more heightening of the overall macroscopic folding free-energy barriers for proteins with more nonlocal native contacts than those with fewer such contacts. Thus, the enhancement of folding cooperativity by solvation effects is seen as positively correlated with a protein's native topological complexity.

Growing length scale in gravity-driven dense granular flow.

We report simulations of a two-dimensional, dense, bidisperse system of inelastic hard disks falling down a vertical tube under the influence of gravity. We examine the approach to jamming as the average flow of particles down the tube is slowed by making the outlet narrower. Defining coarse-grained velocity and stress fields, we study two-point temporal and spatial correlation functions of these fields in a region of the tube where the time-averaged velocity is spatially uniform. We find that fluctuations in both velocity and stress become increasingly correlated as the system approaches jamming. We extract a growing length scale and time scale from these correlations.

Preference-based quality of life in patients with Alzheimer's disease.

BACKGROUND: This study was developed to evaluate the feasibility, reliability, and validity of use of patients' ratings of health preference measures as outcomes for cost-effectiveness analyses in persons with very mild to moderate Alzheimer's disease (AD). METHODS: Patients and caregivers completed ratings of the EuroQol-5D system (EQ-5D) and the Health Utilities Index Mark 2 (HUI2) and instruments that assess cognition, mood, insight, AD-specific and generic health-related quality of life (QOL) and activities of daily living. RESULTS: Patients' HUI2 scores were reliable. EQ-5D scores were somewhat less so. Patients rated their utility high, with overall EQ-5D and HUI2 scores greater than 0.8. Neither the EQ-5D nor the HUI2 scores had a relationship with severity of cognitive impairment. Both the EQ-5D and the HUI2 had expected relationships with patient-rated measures of QOL, function, mood, and specific subscales we hypothesized should be associated with patient preferences. Patient insight into functional and cognitive impairments had little association with their health preference scores. CONCLUSIONS: AD patients' scores on the EQ-5D and the HUI2 have many of the characteristics of valid preference measures. However, the proportions of persons who do not perceive any disability, the lack of association with the caregivers' ratings of activities of daily living, limited associations with insight, and no association with their Mini-Mental State Exam scores suggest that patients' reports of disability might reflect legitimate self-perceptions of mood and function that are associated with comorbidities rather than with AD.

Caregivers' assessments of preference-based quality of life in Alzheimer's disease.

BACKGROUND: This study was designed to evaluate the feasibility, reliability, and validity of use of caregivers' ratings of two health preference measures as outcomes for cost-effectiveness analyses in persons with very mild to moderate Alzheimer's disease (AD). METHODS: Caregivers completed ratings of preference for AD patients' health by use of the EuroQol-5D system (EQ-5D) and the Health Utilities Index Mark 2 (HUI2). They also rated patients' cognition, mood, burden, AD-specific and generic health-related quality of life (QOL), and activities of daily living. RESULTS: Caregivers' HUI2 scores were reliable. Neither the caregiver ratings of the patients' health by use of the EQ-5D nor the HUI2 had a relationship with severity of cognitive impairment. Both the EQ-5D and the HUI2 had expected relationships with caregivers' assessments of patients' function, AD-specific QOL, and physical and mental health and selected subscales of the measures of AD-specific QOL and overall health. In addition, caregiver scores showed relationships with patient self-rated function, mood, and physical health but not AD-specific QOL. Caregiver burden was associated with caregivers' scores. CONCLUSIONS: Caregiver-completed ratings of preference for patients' health made by use of the EQ-5D and the HUI2 have many of the characteristics of valid preference measures. However, the lack of association with patient Mini-Mental Status Exam scores and patient self-rated AD-specific QOL and the associations with caregiver subjective burden might present limitations to their use as proxy measures for cost-effectiveness analyses.

Stress and large-scale spatial structures in dense, driven granular flows.

We study the appearance of large-scale dynamical heterogeneities in a simplified model of a driven, dissipative granular system. Simulations of steady-state gravity-driven flows of inelastically colliding hard disks show the formation of large-scale linear structures of particles with a high collision frequency. These chains can be shown to carry much of the collisional stress in the system due to a dynamical correlation that develops between the momentum transfer and time between collisions in these frequently colliding particles. The lifetime of these dynamical stress heterogeneities is seen to grow as the flow velocity decreases toward jamming, leading to slowly decaying stress correlations reminiscent of the slow dynamics observed in supercooled liquids.

Secondary structures in long compact polymers.

Compact polymers are self-avoiding random walks that visit every site on a lattice. This polymer model is used widely for studying statistical problems inspired by protein folding. One difficulty with using compact polymers to perform numerical calculations is generating a sufficiently large number of randomly sampled configurations. We present a Monte Carlo algorithm that uniformly samples compact polymer configurations in an efficient manner, allowing investigations of chains much longer than previously studied. Chain configurations generated by the algorithm are used to compute statistics of secondary structures in compact polymers. We determine the fraction of monomers participating in secondary structures, and show that it is self-averaging in the long-chain limit and strictly less than 1. Comparison with results for lattice models of open polymer chains shows that compact chains are significantly more likely to form secondary structure.