Identification of Evolutionarily Conserved VSX2 Enhancers in Retinal Development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. Recently, we found that distinct modules within a murine SE regulate gene expression of master regulatory transcription factor Vsx2 in a developmental stage- and cell-type specific manner. Vsx2 is expressed in retinal progenitor cells as well as differentiated bipolar neurons and Müller glia. Mutations in VSX2 in humans and mice lead to microphthalmia due to a defect in retinal progenitor cell proliferation. Deletion of a single module within the Vsx2 SE leads to microphthalmia. Deletion of a separate module within the SE leads to a complete loss of bipolar neurons, yet the remainder of the retina develops normally. Furthermore, the Vsx2 SE is evolutionarily conserved in vertebrates, suggesting that these modules are important for retinal development across species. In the present study, we examine the ability of these modules to drive retinal development between species. By inserting the human build of one Vsx2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the implications of these SE modules in a model of human development, we generated human retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module leads to a complete loss of ON cone bipolar neurons. This prototypical SE serves as a model for uncoupling developmental stage- and cell-type specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding.

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.

Human Organoids for the Study of Retinal Development and Disease.

Recent advances in stem cell engineering have led to an explosion in the use of organoids as model systems for studies in multiple biological disciplines. Together with breakthroughs in genome engineering and the various omics, organoid technology is making possible studies of human biology that were not previously feasible. For vision science, retinal organoids derived from human stem cells allow differentiating and mature human retinal cells to be studied in unprecedented detail. In this review, we examine the technologies employed to generate retinal organoids and how organoids are revolutionizing the fields of developmental and cellular biology as they pertain to the retina. Furthermore, we explore retinal organoids from a clinical standpoint, offering a new platform with which to study retinal diseases and degeneration, test prospective drugs and therapeutic strategies, and promote personalized medicine. Finally, we discuss the range of possibilities that organoids may bring to future retinal research and consider their ethical implications.

Programmed switch in the mitochondrial degradation pathways during human retinal ganglion cell differentiation from stem cells is critical for RGC survival.

Retinal ganglion cell (RGC) degeneration is the root cause for vision loss in glaucoma as well as in other forms of optic neuropathy. A variety of studies have implicated abnormal mitochondrial quality control (MQC) as contributing to RGC damage and degeneration in optic neuropathies. The ability to differentiate human pluripotent stem cells (hPSCs) into RGCs provides an opportunity to study RGC MQC in great detail. Degradation of damaged mitochondria is a critical step of MQC, and here we have used hPSC-derived RGCs (hRGCs) to analyze how altered mitochondrial degradation pathways in hRGCs affect their survival. Using pharmacological methods, we have investigated the role of the proteasomal and endo-lysosomal pathways in degrading damaged mitochondria in hRGCs and their precursor stem cells. We found that upon mitochondrial damage induced by the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), hRGCs more efficiently degraded mitochondria than did their precursor stem cells. We further identified that for degrading damaged mitochondria, stem cells predominantly use the ubiquitine-proteasome system (UPS) while hRGCs use the endo-lysosomal pathway. UPS inhibition causes apoptosis and cell death in stem cells, while hRGC viability is dependent on the endo-lysosomal pathway but not on the UPS pathway. These findings suggest that manipulation of the endo-lysosomal pathway could be therapeutically relevant for RGC protection in treating optic neuropathies associated with mitophagy defects. Endo-lysosome dependent cell survival is also conserved in other human neurons as we found that differentiated human cerebral cortical neurons also degenerated upon endo-lysosomal inhibition but not with proteasome inhibition.

21†Daily dynamic discharge - a whole system solution to ED crowding.

INTRODUCTION: ED crowding is associated with increased mortality, poor staff and patient experience, an increased inpatient length of stay and poor compliance with the four-hour emergency access standard.1 Where crowding is caused by exit block, the focus needs to be on whole system patient management, reducing the temporal mismatch between admissions and discharges since at times of peak demand hospitals may become gridlocked until patients are discharged.In an attempt to tackle exit block, the Scottish Government Unscheduled Care Team have implemented the Daily Dynamic Discharge (DDD) approach, which aims to increase the number of inpatient discharges by 12 pm, thus enabling more timeous flow through the ED. METHODS: A series of meetings were held between the Unscheduled Care Team and the clinical and managerial staff of Dumfries and Galloway Royal Infirmary over a two-week period to train staff on implementing the elements of the Daily Dynamic Discharge approach. These included holding a daily whiteboard meeting with input from the multidisciplinary team, early determination of an Estimated Date of Discharge (EDD) for each patient, and conducting 'golden hour' ward rounds whereby the highest acuity patients were seen first followed by those who were expected to be discharged that day, thus increasing the number of discharges by 12 pm. RESULTS: Over a twelve-week period the average number of weekly discharges increased from 26.5 to 30.2, i.e., an average increase of 3.7 discharges per week. Average length of stay dropped from 6.8 days to 6.2 days, a saving of 0.6 days.The median discharge time was 32 min earlier once DDD had been implemented. Previously, a third (33%) of patients were discharged before 4 pm; after implementation, this rose to 44%. DISCUSSION: Emergency Department activity, and particularly crowding, is the barometer for the rest of the hospital, and the only way to guarantee that patients who require admission, get into the right bed, and in a timely way, is to ensure that the downstream wards discharge sufficient numbers early in the day to accommodate admissions from the ED.The DDD approach has been shown to be effective in increasing the number of discharges by 12 pm, smoothing the admission/discharge profile, and is now being adopted in other hospitals throughout Scotland. REFERENCE: Richardson DB. Increase in patient mortality at 10 days associated with emergency department overcrowding. Med J Aust2006;184(5):213-216.

How Patients and Nurses Experience an Open Versus an Enclosed Nursing Station on an Inpatient Psychiatric Unit.

The inpatient environment is a critical space for nurses and patients in psychiatric settings. In this article, we describe nurses' and patients' perceptions of the inpatient environment both before the removal of a Plexiglas enclosure around a nurses' station and after its removal. Nurses had mixed feelings about the enclosure, reporting that it provided for confidentiality and a concentrated work space but also acknowledged the challenge of the barrier for communication with their patients. Patients unanimously preferred the nurses' station without the barrier, reporting increased feelings of freedom, safety, and connection with the nurses after its removal. It is important to consider the implications of environmental decisions in inpatient settings in order to promote a healthy workplace and healing environment for all community members.

Determinants of nutrition appointment attendance among male veterans attending Veterans Health Administration clinics.

OBJECTIVE: To identify determinants of nutrition appointment attendance among male veterans attending outpatient Veterans Health Administration clinics. METHODS: Sequential use of qualitative and quantitative methods. Individual, semi-structured interviews and a mail survey were used to identify factors associated with outpatient nutrition appointment attendance. RESULTS: Qualitative analysis of 17 interviews revealed 6 themes reflecting rationales for missed appointments: travel difficulty, forgetting the appointment, competing demands, scheduling difficulty, knowledge not new or useful, and lack of provider support. Analysis of 349 returned surveys indicated past attendance history, health status, and participation in the referral and scheduling process correlated to appointment attendance (P < .05). Regression analysis substantiated the importance of social support (P < .05). CONCLUSIONS AND IMPLICATIONS: Veterans Health Administration patients should participate in the referral and scheduling process. Social support, perceived health status, and past attendance history are important considerations for patient and provider to address.

Noise-immune cavity-enhanced optical heterodyne detection of HO2 in the near-infrared range.

Accurate measurements of the absolute concentrations of radical species present in the atmosphere are invaluable for better understanding atmospheric processes and their impact on Earth systems. One of the most interesting species is HO(2), the hydroperoxyl radical, whose atmospheric daytime levels are on the order of 10 ppt and whose observation therefore requires very sensitive detection techniques. In this work, we demonstrate the first steps toward the application of external-cavity diode-laser-based noise-immune cavity-enhanced optical heterodyne molecular spectroscopy (NICE-OHMS) to the detection of the HO(2) radical in the near-infrared range. Measurements of stable species and of HO(2) were made in a laboratory setting, and the possibilities of extending the sensitivity of the technique to atmospheric conditions are discussed.

A methodology to establish a database to study gene environment interactions for childhood asthma.

BACKGROUND: Gene-environment interactions are likely to explain some of the heterogeneity in childhood asthma. Here, we describe the methodology and experiences in establishing a database for childhood asthma designed to study gene-environment interactions (PAGES--Paediatric Asthma Gene Environment Study). METHODS: Children with asthma and under the care of a respiratory paediatrician are being recruited from 15 hospitals between 2008 and 2011. An asthma questionnaire is completed and returned by post. At a routine clinic visit saliva is collected for DNA extraction. Detailed phenotyping in a proportion of children includes spirometry, bronchodilator response (BDR), skin prick reactivity, exhaled nitric oxide and salivary cotinine. Dietary and quality of life questionnaires are completed. Data are entered onto a purpose-built database. RESULTS: To date 1045 children have been invited to participate and data collected in 501 (48%). The mean age (SD) of participants is 8.6 (3.9) years, 57% male. DNA has been collected in 436 children. Spirometry has been obtained in 172 children, mean % predicted (SD) FEV1 97% (15) and median (IQR) BDR is 5% (2, 9). There were differences in age, socioeconomic status, severity and %FEV1 between the different centres (p≤0.024). Reasons for non-participation included parents not having time to take part, children not attending clinics and, in a small proportion, refusal to take part. CONCLUSIONS: It is feasible to establish a national database to study gene-environment interactions within an asthmatic paediatric population; there are barriers to participation and some different characteristics in individuals recruited from different centres. Recruitment to our study continues and is anticipated to extend current understanding of asthma heterogeneity.

Second primary tumors in children and young adults in the North of England (1968-99).

BACKGROUND: This study describes the risk of second malignancy in patients diagnosed with cancer under the age of 25 years, registered on the Northern Region Young Person's Malignant Disease Registry. PROCEDURE: Incidence rates were calculated to describe the occurrence of second malignancies, rate ratios were estimated to compare rates between subgroups. Standardized incidence ratios (SIRs) were calculated for comparison with a reference population. RESULTS: There were 4,072 children and young adults diagnosed with a first malignancy from 1968 to 1999, of whom 68 had a second malignancy (including basal cell carcinomas and meningiomas). The incidence rate of second malignancy is 1.7 per 1,000 survivor person-years (95% CI: 1.4, 2.2), reflecting a four-fold increased risk of malignancy compared with the general population. The rate of second malignancy was non-significantly higher for those diagnosed during young adulthood rather than childhood (RR = 1.2, 95% CI: 0.7, 2.0), significantly higher in females than males (RR = 1.8, 95% CI: 1.1, 3.0) and significantly lower for those diagnosed in more recent years (RR = 0.4, 95% CI: 0.2, 0.8). In contrast, the SIRs indicated that children were at substantial increased risk; whilst males and females, and those diagnosed in earlier and later time periods, were at equivalent risks. CONCLUSIONS: There is evidence of a sustained increased risk of second malignancy in those treated for primary cancer, especially those diagnosed in childhood; with no evidence that this risk is reducing.