SHROOM3 is downstream of the planar cell polarity pathway and loss-of-function results in congenital heart defects.

Congenital heart disease (CHD) is the most common birth defect, and the leading cause of death due to birth defects, yet causative molecular mechanisms remain mostly unknown. We previously implicated a novel CHD candidate gene, SHROOM3, in a patient with CHD. Using a Shroom3 gene trap knockout mouse (Shroom3gt/gt) we demonstrate that SHROOM3 is downstream of the noncanonical Wnt planar cell polarity signaling pathway (PCP) and loss-of-function causes cardiac defects. We demonstrate Shroom3 expression within cardiomyocytes of the ventricles and interventricular septum from E10.5 onward, as well as within cardiac neural crest cells and second heart field cells that populate the cardiac outflow tract. We demonstrate that Shroom3gt/gt mice exhibit variable penetrance of a spectrum of CHDs that include ventricular septal defects, double outlet right ventricle, and thin left ventricular myocardium. This CHD spectrum phenocopies what is observed with disrupted PCP. We show that during cardiac development SHROOM3 interacts physically and genetically with, and is downstream of, key PCP signaling component Dishevelled 2. Within Shroom3gt/gt hearts we demonstrate disrupted terminal PCP components, actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation and morphology. Together, these data demonstrate SHROOM3 functions during cardiac development as an actomyosin cytoskeleton effector downstream of PCP signaling, revealing SHROOM3's novel role in cardiac development and CHD.

Hyperphagia: current concepts and future directions proceedings of the 2nd international conference on hyperphagia.

OBJECTIVE: Hyperphagia is a central feature of inherited disorders (e.g., Prader-Willi Syndrome) in which obesity is a primary phenotypic component. Hyperphagia may also contribute to obesity as observed in the general population, thus raising the potential importance of common underlying mechanisms and treatments. Substantial gaps in understanding the molecular basis of inherited hyperphagia syndromes are present as are a lack of mechanistic of mechanistic targets that can serve as a basis for pharmacologic and behavioral treatments. DESIGN AND METHODS: International conference with 28 experts, including scientists and caregivers, providing presentations, panel discussions, and debates. RESULTS: The reviewed collective research and clinical experience provides a critical body of new and novel information on hyperphagia at levels ranging from molecular to population. Gaps in understanding and tools needed for additional research were identified. CONCLUSIONS: This report documents the full scope of important topics reviewed at a comprehensive international meeting devoted to the topic of hyperphagia and identifies key areas for future funding and research.

The effects of particle size on measurement of airway hyperresponsiveness to methacholine.

BACKGROUND: The effect of particle size on methacholine provocation concentration causing a decrease in forced expiratory volume of 1 second (FEV1) of 20% (PC20) is debatable. OBJECTIVE: To evaluate the functional effects of 3 different particle size nebulizers on methacholine PC20. METHODS: Participants were randomly assigned to have 3 methacholine challenges on 3 separate days. Nebulizer mass median aerodynamic diameter (MMAD) was provided by manufacturers. The Wright nebulizer (MMAD, 1.0 µm), Aeroneb (MMAD, 3 µm), and Aeroneb (MMAD, 5 µm) were calibrated, and the nebulizer outputs were calculated to administer 0.26 mL of methacholine over 120, 112, and 83 seconds, respectively. After each inhalation, spirometry was performed and the test was terminated when the PC20 was achieved. RESULTS: Eight nonsmoking patients with mild asthma (4 male and 4 female) completed the study. The mean (SD) age was 25 (13.9) years, and the mean (SD) baseline FEV1 was 88% (11.3%). Patients using the Aeroneb (MMAD, 5 µm) nebulizer had the lowest PC20 (bronchoconstricted at lowest methacholine concentration), with a PC20 geometric mean of 0.62 mg/mL compared with patients using the Aeroneb (MMAD, 3.0 µm), who had a PC20 of 1.76 mg/mL, and patients using the Wright nebulizer (MMAD, 1.0 µm), who had a PC20 of 6.32 mg/mL. There was a significant difference in PC20 across all particle sizes (P < .001). The pairwise differences revealed a P < .001 between 3 µm and 1 µm and between 5 µm and 1 µm and a P = .008 between 5 µm and 3 µm. CONCLUSION: Our results reveal a variability in methacholine PC20 using 3 different nebulizers, despite adjusting the nebulizers' outputs. Our results are consistent with the previous reports, which recommended using larger particle size nebulizers in the assessment of airway hyperresponsiveness in asthma. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00529477.

Comparison of changes in lung function measured by plethymography and IOS after bronchoprovocation.

AIM: Lung function tests are essential for the diagnosis and management of bronchial asthma. Impulse oscillation (IOS) system is an alternative way to measure lung mechanics for some patients. We investigated the relative sensitivities of IOS, body plethysmography and spirometry in detecting allergen- and methacholine-induced bronchoconstriction. METHOD: Twenty-two subjects had single allergen inhalation and 8 subjects had 3 methacholine challenges. The tests were stopped when FEV1 fell by 20%. Lung function was measured using IOS (R5, R20, R5-R20, X5, AX, fres), plethysmography (sRaw, sGaw, FRC, lung volumes) and spirometry (FEV1, FVC, PEF, FEF50%) during inhalation challenges, and expressed as percent change from pre-challenge baseline. RESULTS: All subjects were non-smoking adults with mild allergic asthma. Following allergen challenges, the most sensitive IOS index was R5-R20 and the most sensitive plethysmography and spirometry measurements were sRaw, sGaw and FEF50%. Following methacholine challenge the most sensitive IOS index was AX, the most sensitive plethysmography measurement was sRaw. Overall, IOS (R5-R20, AX, X5Hz) proved to be more sensitive than plethysmography and spirometry measurements following allergen-induced and methacholine-induced bronchoconstriction. CONCLUSION: Our result shows that IOS is more sensitive than other lung function tests following allergen and methacholine challenge. In addition, IOS can act as an alternative measurement technique of airway resistance and obstruction in patients where manoeuvres involved in plethysmography and spirometry prove difficult to perform.

Frameshift events associated with the lysyl-tRNA and the rare arginine codon, AGA, in Escherichia coli: a case study involving the human Relaxin 2 protein.

Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in E. coli BL21 (DE3), several forms of the protein were made. One species had the expected molecular weight (19,084 Da). A second species observed had a molecular weight of 21,244 Da. A third minor species had a molecular weight of 17,118 Da. These aberrant molecular weights can be explained as follows. First, a sequence CGA-AAA-AAG-AGA, containing the rare arginine codons CGA and AGA was the site of the +1 frameshift that generated the 21,244 Da species. Since there was a limited supply of this arginyl-tRNA, the peptidyl-tRNA moved +1 nucleotide to occupy the codon and resumed protein synthesis. Second, a -1 frameshift associated with 'slippery A' sequence XXA-AAA-AAG accounted for 10% of the product with a mass of 17,118 Da. Presumably, the shift to -1 also occurred because there was a paucity of the arginyl-tRNAArgucu. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species.

Enoxaparin thromboprophylaxis in gastric bypass patients: extended duration, dose stratification, and antifactor Xa activity.

BACKGROUND: Morbidly obese patients undergoing gastric bypass surgery are at risk for postoperative venous thromboembolism. Evidence-based recommendations regarding the dosing and duration of thromboprophylaxis are lacking for morbidly obese surgical patients. The aims of this study were to evaluate the safety and efficacy of an extended duration, body mass index (BMI)-stratified enoxaparin thromboprophylaxis regimen in patients undergoing Roux-en-Y gastric bypass and to determine the resultant antifactor Xa (AFXa) activity in morbidly obese surgical patients. METHODS: In this prospective open trial, 223 patients (75% female, mean BMI 50.4 kg/m2) undergoing Roux-en-Y gastric bypass were assigned to receive enoxaparin 40 mg (BMI 50 kg/m2), n = 99) every 12 hours during hospitalization and once daily for 10 days after discharge. The AFXa levels were monitored serially, and dose adjustments were made for results outside the target prophylactic range (.2-.4 IU/mL +/- 10%) after the third dose. The safety and efficacy outcomes were major bleeding and venous thromboembolism. RESULTS: Roux-en-Y gastric bypass was performed laparoscopically in 208 subjects (93%). The duration of surgery averaged 99.5 +/- 31 minutes, and the median length of hospitalization was 3 days. Target prophylactic AFXa concentration was achieved by 74% of patients after the third enoxaparin dose; none reached the full anticoagulation concentration. One patient developed nonfatal venous thromboembolism (.45%). Four patients required transfusion (1.79%). Bleeding was not associated with a high AFXa concentration. CONCLUSION: This BMI-stratified, extended enoxaparin dosing regimen provided well-tolerated, effective prophylaxis against venous thromboembolism in patients undergoing gastric bypass surgery.

Intraperitoneal aerosolization of bupivacaine is a safe and effective method in controlling postoperative pain in laparoscopic Roux-en-Y gastric bypass.

INTRODUCTION: Obesity is a worldwide problem and has grown in severity in the last few decades making bariatric surgery and, in particular, laparoscopic banding and Roux-en-Y gastric bypass efficacious and cost-effective procedures. The laparoscopic approach has been shown to offer significant healthcare benefits, of particular interests are reports of decreased postoperative pain resulting in a shorter hospital stay and an earlier return to normal activity. However, many patients still experience significant pain, including shoulder tip pain, that require strong analgesia including opiates during their early recovery period. The aims of this study were to establish the safe use of the aerosolization technique in bariatric surgery and to investigate the possible benefits in reducing postoperative pain. METHODS: In this study, fifty patients undergoing laparoscopic gastric bypass were recruited and divided into two groups; control (n = 25) and therapeutic (n = 25). The control group received intraperitoneal aerosolization of 10 mL of 0.9% normal saline while the therapeutic group received 10 mL of 0.5% bupivacaine. All the patients had standard preoperative, intraoperative, and postoperative care. Pain scores were carried out by the nursing staff in recovery and 6 h, 12 h and 24 h postoperatively using a standard 0-10 pain scoring scale. In addition, opiate consumption via patient-controlled analgesia (PCA) was recorded. RESULTS: Aerosolized bupivacaine reduced postoperative pain in comparison to normal saline (p < 0.05). However, PCA usage showed no statistically significant change from the control group. CONCLUSION: The aims of this study were achieved and we were able to establish the safe use of the aerosolization technique in bariatric surgery and its benefits in reducing postoperative pain.

A bicistronic expression system for bacterial production of authentic human interleukin-18.

Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co-expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degrees C. An alternative bicistronic expression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production.

Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli.

In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E. coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E. coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNA(CGG) levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E. coli, allowing for efficient and accurate translation of heterologous gene products.

Rapid expression of recombinant proteins in modified CHO cells using the baculovirus system.

Baculovirus containing the mammalianCMV promoter, in place of the insect polyhedronpromoter (BacMam), has been used to transientlytransfect COS, CHO and CHOE1a (CHO cells expressing theE1a transcriptional activator). Using this system forthe expression of a cellular adhesion factor (SAF-3) Fcfusion protein in CHOE1a, we found that levels ofexpression were highest with a MOI of 100, 20mM sodiumbutyrate, at 34 degrees C. Production increased furtherif the cells were resuspended in fresh medium, about3 x 10(6) cells ml(-1), prior to addition of the virus. These conditions were used to express 3 secretedproteins, SAF-3-Fc, CD40-hexa his and Asp 2-Fc, and, at2 to 6 days post infection, protein levels ranged from4 ug ml(-1) to 25 ug ml(-1). Based on these results, theBacMam system represents a viable technique forproducing protein at ug ml(-1) levels in a relatively shortperiod of time.