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.     

Trauma in Rural Areas

Conservative management is important in most children's fractures. Open surgical methods may be necessary in a few specific injuries such as fractures of the lateral humeral condyle, fractures of the radial neck with severe displacement, and some fractures of the medial epicondyle.

Volkmann's ischemic contracture requires prompt surgical treatment if conservative measures fail. Other rare bone injuries in children, such as fracture of the femoral neck and osteochondral fracture within the knee joint, may require open operation.

Nonetheless, except for these uncommon situations, nature is on the side of the judicious physician who aids rather than impedes it.     

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.     

Pleiotropic syndrome of dehydrated hereditary stomatocytosis, pseudohyperkalemia, and perinatal edema maps to 16q23-q24

Dehydrated hereditary stomatocytosis (DHS) is a rare genetic disorder of red cell permeability to cations, leading to a well-compensated hemolytic anemia. DHS was shown previously to be associated in some families with a particular form of perinatal edema, which resolves in the weeks following birth and, in addition, with pseudohyperkalemia in one kindred. The latter condition was hitherto regarded as the separate entity, "familial pseudohyperkalemia." DHS and familial pseudohyperkalemia are thought to stem from the same gene, mapping to 16q23-q24. This study screened 8 French and 2 American families with DHS. DHS appeared to be part of a pleiotropic syndrome in some families: DHS + perinatal edema, DHS + pseudohyperkalemia, or DHS + perinatal edema + pseudohyperkalemia. If adequately attended to, the perinatal edema resolved spontaneously after birth. Logistic regression showed that increased mean corpuscular volume and mean corpuscular hemoglobin concentration were the parameters best related to DHS. In patients in whom cation fluxes were investigated, the temperature dependence of the monovalent cation leak exhibited comparable curves. Specific recombination events consistently suggested that the responsible gene lies between markers D16S402 and D16S3037 (16q23-q24). The 95% confidence limits (Z(max) >/= 3.02) spanned almost the complete 9-cM interval between these 2 markers.     

Platelet activation,aggregation, and life span in calves implanted with axial flow ventricular assist devices

Background. A variety of rotary blood pumps are under development worldwide to serve as chronic ventricular assist devices (VADs). Historically VADs have been associated with thrombotic and thromboembolic complications, yet the ability to evaluate the thrombotic process in preclinical device testing has been limited.

Methods. We have developed and applied flow cytometric assays for activated platelets, platelet microaggregates, and platelet life span and consumption to calves implanted with an axial flow VAD and calves undergoing a sham surgical procedure.

Results. Surgical sham calves had significant increases in circulating activated platelets (p < 0.05) that resolved within 17 days, and no increases in circulating platelet microaggregates. Calves with uneventful VAD implant periods had early transient elevations in platelet microaggregates and prolonged elevations in activated platelets that did not recover to preoperative values during the study. Daily platelet consumption in VAD implanted calves was increased by 20% ± 3%. Calves with thrombotic deposition within the VAD and elevated thromboembolism observed at autopsy experienced increases in circulating activated platelets and microaggregates at the end of the implant period when VAD flow decreased.

Conclusions. This study demonstrates the ability of flow cytometry-based platelet assays to differentiate VAD implant operations from VAD support, and suggests differences that exist between uneventful VAD support and support with complications. These techniques should have value in evaluating other cardiovascular devices undergoing preclinical testing and provide insight into the temporal impact of these devices on the hemostatic system.     

Arthroscopic repair of medium to large full-thickness rotator cuff tears: outcome at 2- to 6-year follow-up

Medium-term follow-up of arthroscopic rotator cuff repair was evaluated in 48 consecutive arthroscopic repairs of medium to large rotator cuff tears. Mean rotator cuff tear size was 2.4 cm (range, 2-4 cm), mean age at surgery was 57.6 years (range, 38-80 years), and mean follow-up was 39 months (range, 24-66 months). Mean University of California at Los Angeles End-Result Scores were 17.2 preoperatively and 33.7 postoperatively (P <.001). American Shoulder and Elbow Surgeons mean scores were 42.2 and 94.9, respectively. There were no complications requiring reoperation. There were 35 excellent, 11 good, 2 fair, and no poor results; however, 1 patient had clinical evidence of a failed repair. Forty-four of 45 patients (47/48 repairs) were satisfied with their results.     

Gait after unilateral total knee arthroplasty: Frontal plane analysis

After unilateral total knee arthroplasty (TKA), osteoarthritis (OA) in the non‐operated knee often progresses. The altered gait mechanics exhibited by patients after TKA increase the loading on the non‐operated knee and predispose it to disease progression. Therefore, our objective was to examine the potentially detrimental changes in frontal plane kinetics and kinematics during walking in patients who underwent unilateral TKA. Thirty‐one subjects 6 months after TKA, 24 subjects 1 year after unilateral TKA, and 20 control subjects were recruited. All subjects underwent 3D gait analysis. In the TKA groups, the non‐operated knee had a higher adduction angle and higher dynamic loading, knee adduction moment and impulse, compared to the operated knee. This increased loading may be an underlying reason for OA progression in the non‐operated knee. Measures of loading in the control knee did not differ from that of the non‐operated knee in the TKA group, but the TKA group walked with shorter step length. While the non‐operated knee loading was not different from controls, there may be greater risk of cumulative loading in the non‐operated knee of the TKA group given the shorter step length. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:647–652, 2011