Drosophila sperm development and intercellular cytoplasm sharing through ring canals do not require an intact fusome.

Animal germ cells communicate directly with each other during gametogenesis through intercellular bridges, often called ring canals (RCs), that form as a consequence of incomplete cytokinesis during cell division. Developing germ cells in Drosophila have an additional specialized organelle connecting the cells called the fusome. Ring canals and the fusome are required for fertility in Drosophila females, but little is known about their roles during spermatogenesis. With live imaging, we directly observe the intercellular movement of GFP and a subset of endogenous proteins through RCs during spermatogenesis, from two-cell diploid spermatogonia to clusters of 64 post-meiotic haploid spermatids, demonstrating that RCs are stable and open to intercellular traffic throughout spermatogenesis. Disruption of the fusome, a large cytoplasmic structure that extends through RCs and is important during oogenesis, had no effect on spermatogenesis or male fertility under normal conditions. Our results reveal that male germline RCs allow the sharing of cytoplasmic information that might play a role in quality control surveillance during sperm development.

Somatic insulin signaling regulates a germline starvation response in Drosophila egg chambers.

Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers.

Randomized controlled trial of early enteral fat supplement and fish oil to promote intestinal adaptation in premature infants with an enterostomy.

OBJECTIVE: To test the hypothesis that early enteral supplementing fat and fish oil decreases the duration of parenteral nutrition (PN) and increases enteral nutrition (EN) before bowel reanastomosis in premature infants with an enterostomy. STUDY DESIGN: Premature infants (<2 months old) who had an enterostomy and tolerated enteral feeding at 20 mL/kg/day were randomized to usual care (control=18) or early supplementing enteral fat supplement and fish oil (treatment=18). Intravenous lipid was decreased as enteral fat intake was increased. Daily weight, clinical and nutrition data, and weekly length and head circumference were recorded. The primary outcomes were the duration of PN and volume of EN intake, and the secondary outcomes were weight gain (g/day), ostomy output (mL/kg/d), and serum conjugated bilirubin level (mg/dL) from initiating feeding to reanastomosis. Data were analyzed by Student t test or Wilcoxon rank sum test. RESULTS: There were no differences in the duration of PN, ostomy output, and weight gain between the 2 groups before reanastomosis. However, supplemented infants received less intravenous lipid, had greater EN intake, and lower conjugated bilirubin before reanastomosis, and they also received greater total calorie, had fewer sepsis evaluations and less exposure to antibiotics and central venous catheters before reanastomosis, and had greater weight and length gain after reanastomosis (all P<.05). CONCLUSION: Early enteral feeding of a fat supplement and fish oil was associated with decreased exposure to intravenous lipid, increased EN intake, and reduced conjugated bilirubin before reanastomosis and improved weight and length gain after reanastomosis in premature infants with an enterostomy.

Early enteral fat supplement and fish oil increases fat absorption in the premature infant with an enterostomy.

OBJECTIVE: To test the hypothesis that in the premature infant with an enterostomy, early enteral supplementation with Microlipid (fat supplement) and fish oil increases enteral fat absorption and decreases the requirement for Intralipid (intravenous fat emulsion). STUDY DESIGN: Premature infants (<2 months old) with an enterostomy after surgical treatment for necrotizing enterocolitis or spontaneous intestinal perforation and tolerating enteral feeding at 20 mL/kg/day were randomized to usual care (control 18 infants) or early supplementing enteral fat and fish oil (treatment 18 infants). Intravenous fat emulsion was decreased as enteral fat intake was increased. Daily weight, ostomy output, and nutrition data were recorded. Weekly 24-hour ostomy effluent was collected until bowel reanastomosis, and fecal fat, fecal liquid, and dry feces were measured. Fat absorption (g/kg/d) was calculated by subtracting fecal fat from dietary fat. The fecal liquid and dry feces were reported as mg/g wet stool. Date were analyzed by using ANOVA and mixed-effects model. RESULTS: The interval from initial postoperative feeding to bowel reanastomosis varied from 2 to 10 weeks. The treatment group received more dietary fat and less intravenous fat emulsion and had higher enteral fat absorption, less fecal liquid, and drier feces than the control group. These effects were greater among infants with a high ostomy compared with those with a low ostomy. Enteral fat intake was significantly correlated with fat absorption. CONCLUSION: Early enteral fat supplement and fish oil increases fat absorption and decreases the requirement for intravenous fat emulsion. This approach could be used to promote bowel adaptation and reduce the use of intravenous fat emulsion in the premature infant with an enterostomy.

Mixed Oil-Based Lipid Emulsions vs Soybean Oil-Based Lipid Emulsions on Incidence and Severity of Intestinal Failure-Associated Liver Disease in a Neonatal Intensive Care Unit.

BACKGROUND: Neonates requiring long-term parenteral nutrition (PN) are at risk for developing intestinal failure-associated liver disease (IFALD). The purpose of this study was to compare the incidence and severity of IFALD in a highly surgical neonatal population receiving mixed oil-based lipid emulsions (MOLEs) vs soybean oil-based lipid emulsions (SOLEs) for long-term PN. METHODS: This retrospective cohort study evaluated patients admitted to a neonatal intensive care nursery that received PN for ≥14 days. Patients were separated into 2 cohorts; those who received SOLE and those who received MOLE. The primary outcome of this study was the occurrence of IFALD. Secondary outcomes included time to IFALD, peak bilirubin level during therapy, incidence of hypertriglyceridemia, bronchopulmonary dysplasia, and retinopathy of prematurity. RESULTS: A total of 107 patients were included in the study, IFALD occurred in 44.8% of patients receiving SOLE compared with 30% of patients receiving MOLE (relative risk, 0.67; 95% CI, 0.39-1.15). In the multivariable analysis, adjusting for the known confounders (prematurity, necrotizing enterocolitis, presence of ostomy, and duration of PN and lipids), the type of lipids was not a significant predictor for development of IFALD. Duration of PN and duration of lipids were determined to be significant risk factors for IFALD, regardless of type of lipid emulsion (odds ratio, 1.03; 95% CI, 1.01-1.05). CONCLUSIONS: Use of MOLE resulted in no significant difference in the outcomes studied when compared with SOLE. Duration of PN and duration of lipids were significant risk factors for development of IFALD.

Exploring strategies for protein trapping in Drosophila.

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes.

The Transgenic RNAi Project at Harvard Medical School: Resources and Validation.

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

Early enteral fat supplementation improves protein absorption in premature infants with an enterostomy.

BACKGROUND: Early enteral fat supplementation and fish oil (FO) stimulates post-resection intestinal adaptation in rats and increases fat absorption in premature infants with bowel resection and an enterostomy. OBJECTIVE: To test the hypothesis that early fat supplement and FO increases post-resection protein absorption, intestinal RNA, protein without decreasing intestinal arachidonic acid (AA) in premature infants with an enterostomy. METHODS: 36 premature infants (<2 months old) with an enterostomy after surgical treatment for necrotizing enterocolitis or spontaneous intestinal perforation who tolerated enteral feeding at 20 ml/kg/day were randomized to usual care (control, n = 18) or early supplementing enteral Microlipid (ML) and FO (treatment, n = 18). Intralipid was decreased as the dose of enteral fat was increased. Daily weight, ostomy output and nutritional intake were recorded. Weekly 24-hour ostomy effluent was collected to measure fecal protein. Protein absorption was calculated by subtracting fecal protein from dietary protein. Tissue samples from the functional stoma and the nonfunctional distal diverted end were collected during bowel reanastomosis to measure RNA, protein, and fatty acid (FA) profile. RESULTS: Compared to controls, the treatment group had higher protein absorption (g/kg/day) and intestinal RNA and protein (µg/mg tissue) proximal to the ostomy. The two groups had similar FA profiles except that the treatment group had higher n-3 eicosapentaenoic acid (EPA, µg/mg tissue) proximal to the ostomy. CONCLUSION: Early supplementation of enteral ML and FO to premature infants with an enterostomy increased dietary protein absorption, intestinal RNA, protein and n-3 EPA content without altering other FA content.

A regulatory network of Drosophila germline stem cell self-renewal.

Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ∼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.

Early enteral fat supplementation with microlipid® and fish oil in the treatment of two premature infants with short bowel.

The infusion of Intralipid® is a main risk factor for parenteral nutrition-associated cholestasis in infants with short bowel syndrome. Early provision of enteral fat to reduce the use of Intralipid while providing adequate fat for the growth of infants with short bowel has not been reported. We present 2 cases of premature infants with short bowel who received early supplementation of enteral Microlipid® and fish oil. This approach allowed us to discontinue Intralipid shortly after initiating feedings. The infants tolerated Microlipid/fish oil well without adverse reactions, had appropriate weight gain and ostomy output. They underwent bowel reanastomosis 3 weeks after enteral feeding began, and were discharged on full oral feedings. In case 1, the infant did not develop parenteral nutrition-associated cholestasis; in case 2, cholestasis had developed before initiating feeds, but was not aggravated by enteral fat and was improving prior to discharge.