Effects of poor maternal diet during gestation are detected in F2 offspring.

Poor maternal nutrition of F0 ewes impairs F1 offspring growth, with minimal differences in glucose tolerance or select metabolic circulating factors, and independent of differences in residual feed intake (RFI). To determine if poor maternal nutrition in F0 ewes alters F2 offspring growth, circulating leptin, feed efficiency, or glucose tolerance, F0 ewes (n = 46) pregnant with twins were fed 100% (control), 60% (restricted), or 140% (over) of National Research Council requirements from days 30 ±â€…0.02 of gestation until parturition. At 16 to 19 mo of age, female F1 (n = 36) offspring were bred to generate F2 offspring [CON-F2 (n = 12 ewes; 6 rams), RES-F2 (n = 7 ewes; 13 rams), or OVER-F2 (n = 13 ewes; 9 rams) corresponding to diets of the granddam (F0)]. Lamb body weights (BW) and blood samples were collected weekly from days 0 to 28 and every 14 d until day 252 of age. Circulating leptin was measured in serum at days 0, 7, 14, 56, 210, and 252. An intravenous glucose tolerance test was performed at days 133 ±â€…0.28. At days 167 ±â€…0.33, individual daily intake was recorded over a 77-d feeding period to determine RFI. Rams were euthanized at days 285 ±â€…0.93, and body morphometrics, loin eye area (LEA), back fat thickness, and organ weights were collected and bone mineral density (BMD) and length were determined in the right hind leg. During gestation, OVER-F1 ewes tended to be 8.6% smaller than CON-F1 ewes (P ≤ 0.06). F2 offspring were of similar BW from birth to day 70 (P ≥ 0.20). However, from days 84 to 252, RES-F2 offspring tended to be 7.3% smaller than CON-F2 (P ≤ 0.10). Granddam diet did not influence F2 ram body morphometrics, organ or muscle weights, LEA, adipose deposition, or leg BMD (P ≥ 0.84). RES-F2 (-0.20) and CON-F2 (-0.45) rams tended to be more feed efficient than CON-F2 ewes (0.31; P ≤ 0.08). No effects of granddam diet were observed on glucose or insulin average or baseline concentrations, area under the curve, first-phase response, or ratio (P ≥ 0.52). However, CON-F2 rams (297 mg/dL ±â€…16.5) had a greater glucose peak compared with RES-F2 rams (239 mg/dL ±â€…11.2; P = 0.05). Peak insulin concentrations were not influenced by granddam diet (P = 0.75). At d 56, RES-F2 and OVER-F2 offspring had 53.5% and 61.8% less leptin compared with CON-F2 offspring, respectively (P ≤ 0.02). These data indicate that poor maternal nutrition impacts offspring growth into the second generation with minimal impacts on offspring RFI, glucose tolerance, and circulating leptin.

A Single Dose of Ibuprofen Impacts IL-10 Response to 164-km Road Cycling in the Heat.

Purpose: The purpose was to determine the effect of a single-dose prophylactic ibuprofen use before a 164-km road cycling event in high ambient temperature on the circulating cytokine and leukocyte responses. Methods: Twenty-three men (53 ± 8 y, 172.0 ± 22.0 cm, 85.1 ± 12.8 kg, 19.6 ± 4.4% body fat) completed a 164-km self-paced recreational road cycling event in a hot, humid, sunny environment (WBGT = 29.0 ± 2.9°C) after consuming 600 mg of ibuprofen (n = 13) or a placebo (n = 10). Blood samples were obtained one to two hours before (PRE) and immediately after (POST) the event, and analyzed for concentrations of circulating cytokines interleukins (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, GM-CSF, IFN-γ, and TNF-α and leukocytes (total leukocytes, granulocytes, monocytes, and lymphocytes). Results: Event completion time was 400.2 ± 74.8 min. Concentrations of all cytokines (except IL-1ß, IL-2, IL-5, IL-12, GM-CSF, and IFN-γ) and of all leukocyte subsets increased from PRE to POST. Ibuprofen ingestion attenuated the increase in IL-10 (86% increase with Ibuprofen; 270% increase with placebo). Conclusions: Consuming 600 mg of Ibuprofen prior to a 164-km road cycling event in a hot-humid environment attenuates exercise-induced increases in the concentration of the anti-inflammatory cytokine IL-10, but does not alter the effect of the exercise event on concentrations of other circulating cytokines or leukocyte subset concentrations.

Knowledge of pet-related zoonotic diseases and pet care in Hong Kong, a heavily crowded urban setting.

BACKGROUND: With the rapid expansion of pet animal populations worldwide, pet-related zoonotic diseases are becoming an important issue in public health. Hong Kong (HK), located in southern China, is one of the most crowded urban centres in the world. The population of pets, especially exotic pets, in HK has grown significantly in recent decades, potentially elevating the risk of pet-related zoonotic diseases. However, no studies have been conducted to explore the knowledge of HK public towards pet-related zoonotic diseases and animal husbandry practices. OBJECTIVES: To evaluate the level of awareness among the HK public of pet-related zoonotic diseases and their understanding of proper animal husbandry practices. METHODS: The study was carried out in HK from June-August 2019 using both online and paper versions of a questionnaire. A total of 362 completed questionnaires (74.3% return rate) were collected and the responses analysed. RESULTS: Sixty percent of the participants were current or past pet owners or planned on becoming pet owners in the coming 2 years, irrespective of their income or size of their living space. Among the participants, pet owners (including those who planned pet ownership) had a relatively higher level of awareness of pet-related zoonotic disease. However, the overall awareness of zoonotic diseases among both pet and non-pet owners was low with a knowledge score of <50%. A similar trend was observed for knowledge about proper animal husbandry practices. CONCLUSIONS: This study showed that the HK public was generally not familiar with pet-related zoonotic diseases and proper pet care. These knowledge gaps could potentially increase the risk of disease transmission. Further studies focusing on specific pet species and on people of different social-economic backgrounds are needed to provide future direction of efforts to reduce the risk of pet-related zoonotic diseases and to enhance pet-related animal and human welfare.

Effects of Alzheimer's disease and Lewy body disease on subcortical atrophy.

BACKGROUND AND PURPOSE: Subcortical structures are affected by neurodegeneration in Alzheimer's disease (AD) and Lewy body disease (LBD). Although the co-occurrence of AD and LBD pathologies and their possible interaction have been reported, the effect of AD and LBD on subcortical structures remains unknown. The effects of AD and LBD on subcortical atrophy and their relationship with cognitive dysfunction were investigated. METHODS: The cross-sectional study recruited 42 patients with pure AD related cognitive impairment (ADCI), 30 patients with pure LBD related cognitive impairment (LBCI), 58 patients with mixed ADCI and LBCI, and 29 normal subjects. A general linear model was used to compare subcortical volume and shape amongst the groups, to investigate the independent and interaction effects of ADCI and LBCI on subcortical shape and volume, and to analyze the relationship between subcortical volume and cognitive dysfunction in each group. RESULTS: Alzheimer's disease related cognitive impairment and LBCI were independently associated with subcortical atrophies in the hippocampus and amygdala and in the hippocampus and putamen respectively, but their interaction effect was not significant. Compared to the control group, the pure LBCI group exhibited additional local atrophies in the amygdala, caudate and thalamus. Subcortical atrophies correlated differently with cognitive dysfunction according to the underlying causes of cognitive dysfunction. CONCLUSIONS: The patterns of subcortical atrophies and their correlation with cognitive dysfunction differ according to the underlying AD, LBD or concomitant AD and LBD.

Racial disparities after stoma construction in colorectal surgery.

AIM: Racial disparities are under-recognized among patients undergoing colorectal surgery. The purpose of this study was to determine the complication rates and surgical outcomes stratified by race and ethnicity among patients undergoing colorectal surgery with intestinal stoma creation. METHOD: The ACS NSQIP database from 2013 to 2016 was used. Colon, rectum and small bowel cases requiring intestinal stoma creation were selected. Both African-American and other groups of minority patients were compared with Caucasian patients using a complex multivariable analysis model. Primary outcomes of interest were complication rates, mortality and extended hospital length of stay. RESULTS: The study included 38 088 admissions. After multivariable analysis, African-American patients still had a prolonged length of hospital stay and higher complication rates. Other minorities also had a prolonged length of hospital stay and higher complication rates. CONCLUSIONS: Both African-American and other groups of minority patients requiring an ostomy suffer significantly higher postoperative complication rates and a prolonged hospital length of stay, even after comorbidity adjustment. Access to care, socioeconomic status and comorbid disease management are all important factors for minority patients who undergo colorectal surgery requiring intestinal stoma construction.

Living-donor Liver Retransplantation Using the Vessels of the Previous Allograft by Means of Intragraft Dissection.

Retransplantation with the use of a living-donor graft can be the only therapeutic option for patients with irreversible graft failure, especially in regions with limited access to deceased donors, but it can be technically demanding because of severe adhesion around the hepatic hilum and inferior vena cava. We introduce an effective and safe technique to overcome this challenge for right-lobe living-donor liver retransplantation by using the vessels of the previous right liver allograft with the use of intragraft dissection. The technique was used in 2 critically ill patients undergoing the graft failure. The operative times were 360 and 410 minutes. The recipients were discharged on days 18 and 25 with normal liver function. One postoperative complication occurred 3 months after retransplantation: biliary leakage, corrected with the use of percutaneous transhepatic biliary drainage. Both patients were alive with a functioning allograft at last follow-up of >3 years. Intragraft dissection to use the vessels of the previous right-liver allograft can be a useful technique and should be considered for right-lobe living-donor liver retransplantation.

Randomized clinical trial of preoperative skin antisepsis with chlorhexidine gluconate or povidone-iodine.

BACKGROUND: Skin antiseptic agents are used to prevent surgical-site infection (SSI); few trials have reported the superiority of any specific agent in clean-contaminated abdominal surgery. This RCT was designed to compare the effectiveness of chlorhexidine gluconate and povidone-iodine. METHODS: Consecutive patients who underwent clean-contaminated upper gastrointestinal or hepatobiliary-pancreatic open surgery between 2011 and 2014 were assigned randomly to either chlorhexidine gluconate or povidone-iodine. The primary endpoint was the occurrence of SSI within 30 days of surgery. Secondary endpoints included causative organisms and risk factors for SSI. RESULTS: A total of 534 patients were randomized; 31 (5·8 per cent) developed an SSI. There was no difference in the overall SSI rate in the chlorhexidine gluconate and povidone-iodine groups: 15 of 267 (5·6 per cent) and 16 of 267 (6·0 per cent) respectively (P = 0·853). The most common causative organism was Enterococcus faecalis. In subgroup analysis, biliary-pancreatic surgery had a higher SSI rate (26 of 127, 20·5 per cent) than upper gastrointestinal (2 of 204, 1·0 per cent) and hepatic (3 of 203, 1·5 per cent) resection. Both age (60 years and over) and type of incision were associated with the risk of SSI. CONCLUSION: No difference was detected between chlorhexidine gluconate and povidone-iodine antiseptics for prevention of SSI. Registration number: NCT01495117 (http://www.clinicaltrials.gov).

Extracellular and cellular Hsp72 differ as biomarkers in acute exercise/environmental stress and recovery.

Stress-inducible Hsp72 is a potential biomarker to track risk of exertional heat illness during exercise/environmental stress. Characterization of extracellular (eHsp72) vs cellular Hsp72 (iHsp72) responses is required to define the appropriate use of Hsp72 as a reliable biomarker. In each of four repeat visits, participants (n = 6 men, 4 trials; total n = 24): (a) passively dehydrated overnight, (b) exercised (2 h) with no fluid in a hot, humid environmental chamber, (c) rested and rehydrated (1 h), (d) maximally exercised for 0.5 h, and (e) returned after 24 h of at-home recovery and rehydration. We measured rectal temperature, hydration status (% body mass loss, urine markers, serum osmolality), and Hsp72 (ELISA, flow cytometry. eHsp72 (circulating) and iHsp72 (CD3+ PBMCs) correlated (P < 0.05) with markers of heat, exercise, and dehydration stresses. eHsp72 immediately post-exercise (>15% above baseline, P < 0.05) decreased back to baseline levels by 1 h post-exercise, but iHsp72 expression continued to rise and remained elevated 24 h post-exercise (~2.5-fold baseline, P < 0.05). These data suggest that in addition to the classic physiological biomarkers of exercise heat stress, using cellular Hsp72 as an indicator of lasting effects of stress into recovery may be most appropriate for determining long-term effects of stress on risk for exertional heat illness.

Cathelicidin suppresses lipid accumulation and hepatic steatosis by inhibition of the CD36 receptor.

BACKGROUND AND OBJECTIVES: Obesity is a global epidemic which increases the risk of the metabolic syndrome. Cathelicidin (LL-37 and mCRAMP) is an antimicrobial peptide with an unknown role in obesity. We hypothesize that cathelicidin expression correlates with obesity and modulates fat mass and hepatic steatosis. MATERIALS AND METHODS: Male C57BL/6 J mice were fed a high-fat diet. Streptozotocin was injected into mice to induce diabetes. Experimental groups were injected with cathelicidin and CD36 overexpressing lentiviruses. Human mesenteric fat adipocytes, mouse 3T3-L1 differentiated adipocytes and human HepG2 hepatocytes were used in the in vitro experiments. Cathelicidin levels in non-diabetic, prediabetic and type II diabetic patients were measured by enzyme-linked immunosorbent assay. RESULTS: Lentiviral cathelicidin overexpression reduced hepatic steatosis and decreased the fat mass of high-fat diet-treated diabetic mice. Cathelicidin overexpression reduced mesenteric fat and hepatic fatty acid translocase (CD36) expression that was reversed by lentiviral CD36 overexpression. Exposure of adipocytes and hepatocytes to cathelicidin significantly inhibited CD36 expression and reduced lipid accumulation. Serum cathelicidin protein levels were significantly increased in non-diabetic and prediabetic patients with obesity, compared with non-diabetic patients with normal body mass index (BMI) values. Prediabetic patients had lower serum cathelicidin protein levels than non-diabetic subjects. CONCLUSIONS: Cathelicidin inhibits the CD36 fat receptor and lipid accumulation in adipocytes and hepatocytes, leading to a reduction of fat mass and hepatic steatosis in vivo. Circulating cathelicidin levels are associated with increased BMI. Our results demonstrate that cathelicidin modulates the development of obesity.

Does visual fatigue from 3D displays affect autonomic regulation and heart rhythm?

Most investigations into the negative effects of viewing stereoscopic 3D content on human health have addressed 3D visual fatigue and visually induced motion sickness (VIMS). Very few, however, have looked into changes in autonomic balance and heart rhythm, which are homeostatic factors that ought to be taken into consideration when assessing the overall impact of 3D video viewing on human health. In this study, 30 participants were randomly assigned to two groups: one group watching a 2D video, (2D-group) and the other watching a 3D video (3D-group). The subjects in the 3D-group showed significantly increased heart rates (HR), indicating arousal, and an increased VLF/HF (Very Low Frequency/High Frequency) ratio (a measure of autonomic balance), compared to those in the 2D-group, indicating that autonomic balance was not stable in the 3D-group. Additionally, a more disordered heart rhythm pattern and increasing heart rate (as determined by the R-peak to R-peak (RR) interval) was observed among subjects in the 3D-group compared to subjects in the 2D-group, further indicating that 3D viewing induces lasting activation of the sympathetic nervous system and interrupts autonomic balance.

Single-incision vs straight laparoscopic segmental colectomy: a case-controlled study.

PURPOSE: Single-incision laparoscopic surgery is gaining momentum in general surgery but it is essentially unstudied for laparoscopic colectomy. The aim of our study was to compare outcomes for single-incision laparoscopic colectomy with laparoscopic-assisted colectomy. METHODS: Patients undergoing laparoscopic colectomy were prospectively entered into an institutional review board-approved database. Those that underwent single-incision laparoscopic colectomy were case matched for sex, age, disease, surgery, body mass index, previous surgeries, and surgeon with patients undergoing LAC. RESULTS: Twenty-nine single-incision laparoscopic segmental colectomies were performed for polyps (4), adenocarcinoma (12), diverticulitis (6), and Crohn's disease (7) and were case matched to laparoscopic-assisted colectomy for the same indications. Mean body mass index was 28.8 ± 3 kg/m². Operative time was longer for single-incision laparoscopic colectomy (134.4 ± 40 vs 103.8 ± 54 min; P = .0002). Four single-incision laparoscopic colectomies were converted to LAC requiring either one extra port (2) or 2 extra ports (2), and there was one conversion to laparotomy. Extraction scar length (millimeters) was similar (38 ± 6.0 vs 45 ± 6.2; P = .746). Postoperative morbidity (5/29 vs 7/29; P = .284) and length of stay (day) (3.7 ± 1.1 vs 3.9 ± 1.1; P = .445) were similar between groups. CONCLUSIONS: Single-incision laparoscopic colectomy is feasible and safe but takes more time than laparoscopic-assisted colectomy. Although results approximate those for laparoscopic-assisted colectomy, an additional learning curve is involved, and extra incisions are sometimes required. Single-incision laparoscopic colectomy requires further prospective validation so that the cost of the device can be justified by an improved clinical outcome.

Optical frequency domain imaging with a rapidly swept laser in the 815-870 nm range.

Optical frequency domain imaging (OFDI) in the 800-nm biological imaging window is demonstrated by using a novel wavelength-swept laser source. The laser output is tuned continuously from 815 to 870 nm at a 43.2-kHz repetition rate with 7-mW average power. Axial resolution of 10-mum in biological tissue and peak sensitivity of 96 dB are achieved. In vivo imaging of Xenopus laevis is demonstrated with an acquisition speed of 84 frames per second (512 axial lines per frame). This new imaging technique may prove useful in comprehensive retinal screening for medical diagnosis and contrast-agent-based imaging for biological investigations.

High-speed imaging of human retina in vivo with swept-source optical coherence tomography.

We present the first demonstration of human retinal imaging in vivo using optical frequency domain imaging (OFDI) in the 800-nm range. With 460-muW incident power on the eye, the sensitivity is 91 dB at maximum and >85 dB over 2-mm depth range. The axial resolution is 13 mum in air. We acquired images of retina at 43,200 depth profiles per second and a continuous acquisition speed of 84 frames/s (512 A-lines per frame) could be maintained over more than 2 seconds.

Ergonomics and human factors in endoscopic surgery: a comparison of manual vs telerobotic simulation systems.

BACKGROUND: Minimally invasive surgical techniques expose surgeons to a variety of occupational hazards that may promote musculoskeletal disorders. Telerobotic systems for minimally invasive surgery may help to reduce these stressors. The objective of this study was to compare manual and telerobotic endoscopic surgery in terms of postural and mental stress. METHODS: Thirteen participants with no experience as primary surgeons in endoscopic surgery performed a set of simulated surgical tasks using two different techniques--a telerobotic master--slave system and a manual endoscopic surgery system. The tasks consisted of passing a soft spherical object through a series of parallel rings, suturing along a line 5-cm long, running a 32-in ribbon, and cannulation. The Job Strain Index (JSI) and Rapid Upper Limb Assessment (RULA) were used to quantify upper extremity exposure to postural and force risk factors. Task duration was quantified in seconds. A questionnaire provided measures of the participants' intuitiveness and mental stress. RESULTS: The JSI and RULA scores for all four tasks were significantly lower for the telerobotic technique than for the manual one. Task duration was significantly longer for telerobotic than for manual tasks. Participants reported that the telerobotic technique was as intuitive as, and no more stressful than, the manual technique. CONCLUSIONS: Given identical tasks, the time to completion is longer using the telerobotic technique than its manual counterpart. For the given simulated tasks in the laboratory setting, the better scores for the upper extremity postural analysis indicate that telerobotic surgery provides a more comfortable environment for the surgeon without any additional mental stress.

Programmed cell death and survival pathways in prostate cancer cells.

Programmed cell death, or apoptosis, is a series of morphologically and biochemically related processes. The extrinsic (death receptor mediated) and intrinsic (mitochondrial-mediated) apoptotic pathways can be triggered by physiological and pharmacological substances. However, other molecular events influence the sensitivity of prostate cancer cells to apoptotic stimuli, leading to marked variations in the responsiveness of prostate cancer cell lines to individual stimuli. Modulation of apoptotic responses by over expression of anti-apoptotic proteins (NF-kappaB, IAPs and Bcl-2), or attenuation of pro-apoptotic proteins (PTEN and Bax) may be responsible for the variations in sensitivity of these cell lines to hormone and chemotherapy. The expression of anti- and pro-apoptotic proteins in some of the widely used in vitro models of prostate cancer is reviewed.

Antiandrogen-induced cell death in LNCaP human prostate cancer cells.

Antiandrogens such as Casodex (Bicalutamide) are designed to treat advance stage prostate cancer by interfering with androgen receptor-mediated cell survival and by initiating cell death. Treatment of androgen sensitive, non-metastatic LNCaP human prostate cancer cells with 0-100 microM Casodex or 0-10 ng/ml TNF-alpha induces cell death in 20-60% of the cells by 48 h in a dose-dependent manner. In cells treated with TNF-alpha, this is accompanied by the loss of mitochondrial membrane potential (DeltaPsim) and cell adhesion. In contrast, cells treated with Casodex display loss of cell adhesion, but sustained mitochondrial dehydrogenase activity. Overexpression of Bcl-2 in LNCaP cells attenuates the induction of cell death by TNF-alpha but not Casodex, suggesting that mitochondria depolarization is not required for the induction of cell death by Casodex. While both TNF-alpha and Casodex-induced release of cytochrome c in LNCaP cell is predominantely associated with the translocation and cleavage of Bax, our data also suggest that Casodex induces cell death by acting on components downstream of decline of DeltaPsim and upstream of cytochrome c release. Furthermore, while induction of both caspase-3 and caspase-8 activities are observed in TNF-alpha and Casodex-treated cells, a novel cleavage product of procaspase-8 is seen in Casodex-treated cells. Taken together, these data support the hypothesis that Casodex induces cell death by a pathway that is independent of changes in DeltaPsim and Bcl-2 actions and results in an extended lag phase of cell survival that may promote the induction of an invasive phenotype after treatment.

Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice.

Mutations at the waltzer (v) locus result in deafness and vestibular dysfunction due to degeneration of the neuroepithelium within the inner ear. Here, we use a positional cloning approach to show that waltzer encodes a novel cadherin (Cdh23), which is most closely related to the Drosophila Fat protein. A single nucleotide deletion in the v(J) allele and a single nucleotide insertion in the v allele are predicted to truncate each protein near the N-terminus and produce a functional null allele. In situ hybridization analysis showed that Cdh23 is expressed in the sensory hair cells of the inner ear, where it has been suggested to be a molecule critical for crosslinking of the stereocilia. In addition, Cdh23 is expressed in the urticulo-saccular foramen,the ductus reuniens, and Reissner's membrane, suggesting that Cdh23 may also be involved in maintaining the ionic composition of the endolymph. Finally, mutations in human CDH23 have recently been described for two loci, DFNB12 and USH1D, which cause nonsyndromic deafness, identifying waltzer as a mouse model for human hearing loss.

A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.

Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

Rapid engineering of bacterial artificial chromosomes using oligonucleotides.

A rapid method obviating the use of selectable markers to genetically manipulate large DNA inserts cloned into bacterial artificial chromosomes is described. Mutations such as single-base changes, deletions, and insertions can be recombined into a BAC by using synthetic single-stranded oligonucleotides as targeting vectors. The oligonucleotides include the mutated sequence flanked by short homology arms of 35-70 bases on either side that recombine with the BAC. In the absence of any selectable marker, modified BACs are identified by specific PCR amplification of the mutated BAC from cultures of pooled bacterial cells. Each pool represents about 10 electroporated cells from the original recombination mixture. Subsequently, individual clones containing the desired alteration are identified from the positive pools. Using this BAC modification method, we have observed a frequency of one recombinant clone per 90-260 electroporated cells. The combination of high targeting frequency and the sensitive yet selective PCR-based screening method makes BAC manipulation using oligonucleotides both rapid and simple.

The scabrous protein can act as an extracellular antagonist of notch signaling in the Drosophila wing.

Notch (N) is a receptor for signals that inhibit neural precursor specification [1-6]. As N and its ligand Delta (DI) are expressed homogeneously, other molecules may be differentially expressed or active to permit neural precursor cells to arise intermingled with nonneural cells [7,8]. During Drosophila wing development, the glycosyltransferase encoded by the gene fringe (fng) promotes N signaling in response to DI, but inhibits N signaling in response to Serrate (Ser), which encodes a ligand that is structurally similar to DI. Dorsal expression of Fng protein localizes N signaling to the dorsoventral (DV) wing margin [9-11]. The secreted protein Scabrous (Sca) is a candidate for modulation of N in neural cells. Mutations at the scabrous (sca) locus alter the locations where precursor cells form in the peripheral nervous system [12,13]. Unlike fringe, sca mutations act cell non-autonomously [12]. Here, we report that targeted misexpression of Sca during wing development inhibited N signaling, blocking expression of all N target genes. Sca reduced N activation in response to DI more than in response to Ser. Ligand-independent signaling by overexpression of N protein, or by expression of activated truncated N molecules, was not inhibited by Sca. Our results indicate that Sca can act on N to reduce its availability for paracrine and autocrine interactions with DI and Ser, and can act as an antagonist of N signaling.