Impact of α-crystallin protein loss on zebrafish lens development.

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

Barrett's esophagus before and after Roux-en-Y gastric bypass for severe obesity.

INTRODUCTION: Barrett's esophagus (BE) has been associated with obesity and metabolic syndrome. Laparoscopic Roux-en-Y gastric bypass (LRYGB) may represent a surgical approach which addresses both severe obesity and BE. LRYGB diverts bile away from the gastric pouch and esophagus due to the long Roux limb, and very little acid is produced in the cardia-based gastric pouch. Furthermore, surgically induced weight loss may diminish systemic inflammation, which may contribute to metaplastic changes in the esophagus. Moreover, improved compliance with proton-pump inhibitor therapy, as a consequence of enrolling in a bariatric program, will decrease acid production further. Decreased duodeno-gastro-esophageal reflux should lead to decreased BE. In this study we examine the effect of LRYGB on regression of BE. METHODS AND PROCEDURES: We performed a review of all patients with biopsy proven BE, who underwent LRYGB at our institution. A total of 19 patients were identified. A subset of those patients was identified who had at least 1 year of clinical, endoscopic, or histological data, comprising a total of 14 patients. Seven of these patients had symptoms of gastroesophageal reflux. All 19 patients had short-segment BE. One patient had low-grade dysplasia. RESULTS: Post-LRYGB, 6 of 14 (42.9%) patients had histologic regression of BE to normal esophageal mucosa, with no evidence of ongoing BE. 13 of 14 patients (92.8%) reported compliance with continuing PPI therapy for at least the first year after surgery. Body mass index for the group of 14 patients improved from 46.6 to 30.3 kg/m2. CONCLUSIONS: We recommend LRYGB as an effective combined bariatric and anti-reflux surgical procedure for patients with severe obesity and BE. In short-term follow-up, LRYGB achieved endoscopic and histologic regression to normal mucosa in a substantial number of the patients in our series. Long-term follow-up for patients with BE according to standard surveillance protocols is still recommended.

Is Endoluminal Bariatric Therapy a New Paradigm of Treatment for Obesity?

The prevalence of obesity has steadily increased throughout recent decades, and along with it, the costs of caring for the associated comorbid conditions has increased as well. Traditional bariatric surgical procedures generally are safe and effective, but patient acceptance, the risk of minor and sometimes serious complications, costs, and insurance coverage have limited the application of these techniques to the treatment of a minority of patients. Endoluminal techniques represent newer approaches to weight loss that can be used independently or in concert with traditional medical and surgical treatments for obesity, with varying degrees of success. It is anticipated that less invasiveness will increase the appeal across a broader representation of patients, perhaps increasing the number of obese patients who choose an intervention over medical management and possibly resulting in a greater total loss of excess body weight across a population; this may reduce costs involved in treating the complications of weight-related comorbidities. Acceptance of endoluminal bariatric procedures and devices will hinge on proving safety, efficacy, and value.

The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function.

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

Development and evaluation of a decision-based simulation for assessment of team skills.

BACKGROUND: There is a need to train and evaluate a wide variety of nontechnical surgical skills. The goal of this project was to develop and evaluate a decision-based simulation to assess team skills. METHODS: The decision-based exercise used our previously validated Laparoscopic Ventral Hernia simulator and a newly developed team evaluation survey. Five teams of 3 surgical residents (N = 15) were tasked with repairing a 10 × 10-cm right upper quadrant hernia. During the simulation, independent observers (N = 6) completed a 6-item survey assessing: (1) work quality; (2) communication; and (3) team effectiveness. After the simulation, team members self-rated their performance by using the same survey. RESULTS: Survey reliability revealed a Cronbach's alpha of r = .811. Significant differences were found when we compared team members' (T) and observers' (O) ratings for communication (T = 4.33/5.00 vs O = 3.00/5.00, P < .01) and work quality (T = 4.33/5.00 vs O = 3.33/5.00, P < .05). The team with the greatest survey ratings was the only group to successfully complete the task. CONCLUSION: The team evaluation survey had good reliability and correlated with task performance on the simulator. Our current and previous work provides strong evidence that nontechnical and team related skills can be assessed without simulating a crisis situation.