Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance.     

Absence of Correlation Between the Spontaneously Hypertensive Rat's Exaggerated Preference for Sweet and Alcohol Drinking Solutions

We tested the hypothesis that the exaggerated preference of the spontaneously hypertensive rat of the Okamoto strain (SHR) for alcohol-containing drinking solutions is due to its exaggerated preference for the purported sweet subtaste of alcohol. To do, this we examined in SHR (and Brown Norway (BN) controls) whether preferences for alcohol and glucose-drinking solutions were correlated. No significant correlation was found between alcohol and glucose preference in either the SHR or BN. We conclude therefore that the exaggerated alcohol preference of the SHR is not due to an exaggerated preference for the purported sweet subtaste of alcohol.     

The Role of Community Health Centers in Providing Behavioral Health Care

The prevalence of behavioral health problems is higher for low-income individuals, yet this population is less likely to receive behavioral health treatment. Community health centers have their advantages as behavioral health-care providers because they serve a majority low-income population and are located in medically underserved areas. Their role in providing behavioral health care is expected to expand under health reform as they are expected to double their patient capacity, and due to increased insurance coverage for individuals with behavioral health problems. However, the ability of community health centers to provide behavioral health care is compromised by provider shortages and funding shortfalls.     

PRKDC mutations in a SCID patient with profound neurological abnormalities

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded byPRKDC) functions in DNA non-homologous end-joining (NHEJ), themajor DNA double strand break (DSB) rejoining pathway. NHEJ also functions duringlymphocyte development, joining V(D)J recombination intermediates during antigenreceptor gene assembly. Here, we describe a patient with compound heterozygousmutations in PRKDC, low DNA-PKcs expression, barely detectableDNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system,we found that one of the PRKDC mutations inactivated DNA-PKcs, whilethe other resulted in dramatically diminished but detectable residual function. Thepatient suffered SCID with reduced or absent T and B cells, as predicted fromPRKDC-deficient animal models. Unexpectedly, the patient was alsodysmorphic; showed severe growth failure, microcephaly, and seizures; and hadprofound, globally impaired neurological function. MRI scans revealedmicrocephaly-associated cortical and hippocampal dysplasia and progressive atrophyover 2 years of life. These neurological features were markedly more severe thanthose observed in patients with deficiencies in other NHEJ proteins. Although loss ofDNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronaldevelopment, our findings demonstrate a stringent requirement for DNA-PKcs duringhuman neuronal development and suggest that high DNA-PK protein expression isrequired to sustain efficient pre- and postnatal neurogenesis.     

An Evaluation of a Direct Instructions Flashcard System on the Acquisition and Generalization of Numerals, Shapes, and Colors for Preschool-Aged Students with Developmental Delays

The purpose of this study was to evaluate the effectiveness of the DI Flashcard system for teaching preliminary mathematic skills to three preschool students. The participants attended a self-contained special education preschool. All three participants’ eligibility category was “developmentally delayed”. A concurrent multiple baseline design across three sets (colors, shapes, and numerals) was use to evaluate the effectiveness of the DI flashcard system. The results indicated that all three participants showed an increase in their performance when DI flashcards were in effect. However, the amount of improvement varied for each participant. The importance of employing evidence-based procedures to teach skills to preschool students with developmental delays was outlined.     

Mechanical Thrombectomy for Acute Ischemic Stroke: A Case Study Using the Penumbra Stroke System

Over recent years, there have been dramatic advances in the ability to treat an acute ischemic stroke. Thrombolysis with intravenous recombinant tissue plasminogen activator can now be performed up to 4.5 hr after stroke onset in certain circumstances, and new mechanical thrombectomy devices allow intervention up to and beyond 8 hr in some instances. The Penumbra Stroke System (Penumbra Inc., Alameda, CA), one of these mechanical thrombectomy devices, uses a series of catheters to aspirate thrombus from occluded cerebral arteries and thus restore flow to the adjacent ischemic brain.This case study highlights the clinical and procedural issues involved in the treatment and postprocedural care of a patient with an acute ischemic stroke treated with the Penumbra Stroke System.     

Increased Mortality in a Colony of Zebra Finches Exposed to Continuous Light

Over a 1-mo period, increased morbidity and mortality occurred in a flock of zebra finches (Taeniopygia guttata). Complete postmortem examination was performed on 6 of the affected birds, 4 of which subsequently were diagnosed with the avian gastric yeast previously known as megabacteriosis (Macrorhabdus ornithogaster). The remaining 2 birds were diagnosed with a cloacal abscess and with large bowel perforation and peritonitis. All the birds had been prophylactically treated with amphotericin B for megabacteria 2 mo previously. An environmental assessment revealed that the light cycle had been altered, and the birds were being exposed to constant light. With correction of the light cycle, the health of the birds improved dramatically. The remaining birds were treated again with amphotericin B, and baseline mortality returned to normal. The birds in this report show several similarities to previous reports of sleep deprivation syndrome in mammals.Over a 1-mo period (from late April to late May 2011), a marked increase in morbidity and mortality was noted in a flock of zebra finches (Taeniopygia guttata) at our facility (University of Washington). These birds were involved in a project studying the neural mechanisms of vocal learning. The finches were obtained from Magnolia Bird Farm (Anaheim, CA). In March 2011, a shipment of 50 male birds arrived and was moved into a new space with 2 rooms. One room housed breeding birds in cages containing breeding pairs and offspring; the other room, into which these new arrivals were placed, housed adult male birds in cages of 4 to 9 birds.Birds in the room containing the adult nonbreeders were affected exclusively. Clinical signs consisted of puffed and ruffled feathers, lethargy, increased respiratory effort, and feather loss. These signs were noted in birds that had not undergone experimental manipulation. Several of the birds that died or were euthanized after becoming sick and lethargic were necropsied. Findings on postmortem examination for the majority of cases included emaciated body condition, necrotizing enteritis and colitis and, in individual cases, included bowel perforation and peritonitis and severe pyogranulomatous abscessation. Megabacteriosis (avian gastric yeast; Macrorhabdus ornithogaster) was diagnosed histologically in 4 of the 6 birds evaluated. A light meter was placed in the housing room; results showed that the dark period of the light cycle was absent. The light cycle was corrected, and morbidity and mortality decreased dramatically.