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Background: People with cystic fibrosis carrying two nonsense alleles lack CFTR-specific treatment. Growing evidence supports the hypothesis that nonsense mutation identity affects therapeutic response, calling for mutation-specific CF models. We describe a novel W1282X mouse model and compare it to an existing G542X mouse., Methods: The W1282X mouse was created using CRISPR/Cas9 to edit mouse Cftr. In this model, Cftr transcription was assessed using qRT-PCR and CFTR function was measured in the airway by nasal potential difference and in the intestine by short circuit current. Growth, survival, and intestinal motility were examined as well. Correction of W1282X CFTR was assessed pharmacologically and by gene-editing using a forskolin-induced swelling (FIS) assay in small intestine-derived organoids., Results: Homozygous W1282X mice demonstrate decreased Cftr mRNA, little to no CFTR function, and reduced survival, growth, and intestinal motility. W1282X organoids treated with various combinations of pharmacologic correctors display a significantly different amount of CFTR function than that of organoids from G542X mice. Successful gene editing of W1282X to wildtype sequence in intestinal organoids was achieved leading to restoration of CFTR function., Conclusions: The W1282X mouse model recapitulates common human manifestations of CF similar to other CFTR null mice. Despite the similarities between the congenic W1282X and G542X models, they differ meaningfully in their response to identical pharmacological treatments. This heterogeneity highlights the importance of studying therapeutics across genotypes., Competing Interests: Declaration of competing interest Authors report no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In Rho Q344X/+ mice, mRNA transcripts from the wild-type (Rho) and Rho Q344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHO Q344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHO Q344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both Rho Q344X/+ and Rho Q344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHO Q344X is associated with the concurrent mislocalization of wild-type rhodopsin in Rho Q344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Objective: Describe the first hybrid global simulation-based comprehensive cleft care workshop, evaluate impact on participants, and compare experiences based on in-person versus virtual attendance., Design: Cross-sectional survey-based evaluation., Setting: International comprehensive cleft care workshop., Participants: Total of 489 participants., Interventions: Three-day simulation-based hybrid comprehensive cleft care workshop., Main Outcome Measures: Participant demographic data, perceived barriers and interventions needed for global comprehensive cleft care delivery, participant workshop satisfaction, and perceived short-term impact on practice stratified by in-person versus virtual attendance., Results: The workshop included 489 participants from 5 continents. The response rate was 39.9%. Participants perceived financial factors (30.3%) the most significant barrier and improvement in training (39.8%) as the most important intervention to overcome barriers facing cleft care delivery in low to middle-income countries. All participants reported a high level of satisfaction with the workshop and a strong positive perceived short-term impact on their practice. Importantly, while this was true for both in-person and virtual attendees, in-person attendees reported a significantly higher satisfaction with the workshop (28.63 ± 3.08 vs 27.63 ± 3.93; P  = .04) and perceived impact on their clinical practice (22.37 ± 3.42 vs 21.02 ± 3.45 P  = .01)., Conclusion: Hybrid simulation-based educational comprehensive cleft care workshops are overall well received by participants and have a positive perceived impact on their clinical practices. In-person attendance is associated with significantly higher satisfaction and perceived impact on practice. Considering that financial and health constraints may limit live meeting attendance, future efforts will focus on making in-person and virtual attendance more comparable.

Osteoporosis is an increasing burden on public health as the world-wide population ages and effective therapeutics are severely needed. Two pathways with high potential for osteoporosis treatment are the retinoic acid (RA) and endocannabinoid system (ECS) signaling pathways. We sought to elucidate the roles that these pathways play in bone development and maturation. Here, we use chemical treatments to modulate the RA and ECS pathways at distinct early, intermediate, and late times bone development in zebrafish. We further assessed osteoclast activity later in zebrafish and medaka. Finally, by combining sub-optimal doses of AR and ECS modulators, we show that enhancing RA signaling or reducing the ECS promote bone formation and decrease osteoclast abundance and activity. These data demonstrate that RA signaling and the ECS can be combined as sub-optimal doses to influence bone growth and may be key targets for potential therapeutics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: Evaluate simulation-based comprehensive cleft care workshops as a reproducible model for education with sustained impact., Design: Cross-sectional survey-based evaluation., Setting: Simulation-based comprehensive cleft care workshop., Participants: Total of 180 participants., Interventions: Three-day simulation-based comprehensive cleft care workshop., Main Outcome Measures: Number of workshop participants stratified by specialty, satisfaction with the workshop, satisfaction with simulation-based workshops as educational tools, impact on cleft surgery procedural confidence, short-term impact on clinical practice, medium-term impact on clinical practice., Results: The workshop included 180 participants from 5 continents. The response rate was 54.5%, with participants reporting high satisfaction with all aspects of the workshop and with simulation-based workshops as educational tools. Participants reported a significant improvement in cleft lip (33.3 ± 5.7 vs 25.7 ± 7.6; P < .001) and palate (32.4 ± 7.1 vs 23.7 ± 6.6; P < .001) surgery procedural confidence following the simulation sessions. Participants also reported a positive short-term and medium-term impact on their clinical practices., Conclusion: Simulation-based comprehensive cleft care workshops are well received by participants, lead to improved cleft surgery procedural confidence, and have a sustained positive impact on participants' clinical practices. Future efforts should focus on evaluating and quantifying this perceived positive impact, as well reproducing these efforts in other areas of need.

Objective: It is well known that patients with oral clefts have challenges with feeding. Enteral feeding access, in the form of gastrostomy, is often utilized to supplement or replace oral intake. Although commonly performed, these procedures have reported complication rates as high as 83%. We intend to discover rates of enteral access in patients with oral clefts and report-related outcomes., Design: The Healthcare Cost Utilization Project Kids' Inpatient Database from 2000 to 2012 was analyzed using patients with oral clefts and enteral access procedures. The χ 2 test was used for univariate analyses of proportions, and linear regression was used to analyze trends. Multivariate logistic regression was used to analyze odds ratios., Results: Of the 46 617 patient admissions included, 14.6% had isolated cleft lip (CL), 51.7% cleft lip and palate (CLP), and 43.7% isolated cleft palate. The rates of enteral access in the oral cleft population increased from 3.7% in 2000 to 5.8% in 2012 ( P < .001). Increased rates were identified in patients with ( P = .019) and without ( P < .001) complex conditions. A significant increase in the rate of enteral access was seen in patients with CLP ( P < .001) and isolated cleft palate ( P < .001). No difference was seen in the isolated CL group ( P = .096). Patients with complex conditions were at a 4.4-fold increased risk and those admitted to urban, teaching hospitals were at a 4.7-fold risk of enteral access placement., Conclusions: The rates for enteral feeding access increased significantly from 2000 to 2012. The reasons for the increased incidence are unclear. Invasive enteral access procedures have been shown to have a multitude of complications. Careful patient selection should be done before placement of invasive enteral access.

Nonsense mutations are present in 10% of patients with CF, produce a premature termination codon in CFTR mRNA causing early termination of translation, and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study, we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels, demonstrates absence of CFTR function, and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly, CFTR restoration is observed in G542X intestinal organoids treated with G418, an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations., Competing Interests: The authors have declared that no competing interests exist.

Nasal stenosis is an uncommon and challenging deformity. Most common etiologies for nasal stenosis include congenital, iatrogenic, trauma, and infection. Repair techniques typically include tissue replacement with grafts or flaps with subsequent stent placement. These procedures often require general anesthesia and carry high rates of restenosis. We describe a case of a 10-year-old girl with Teebi syndrome and iatrogenic nasal stenosis who underwent successful nasal dilation with inexpensive, minimally invasive steel gauge earrings.

Deformities of the cranium in patients with nonsyndromic single-suture synostosis occur because of growth restriction at fused sutures and growth over compensation at normal sutures. Traditional surgery includes ostectomies of the synostotic suture to release these restricted areas and osteotomies to enable immediate cranial remodeling. In the process of reshaping the cranium, traditional approaches usually involve obliteration of both the normal functioning suture and the pathologic suture. The directive growth approach (DGA) is a new, simpler, more natural way to repair deformities caused by single-suture cranial synostosis. The DGA works by reversing the original deforming forces by temporarily restricting growth in areas of over compensation and forcing growth in areas of previous synostotic restriction. Most importantly, it preserves a normal functioning suture to allow for improved future cranial growth. Eighteen consecutive nonsyndromic patients with unilateral coronal synostosis were used to illustrate the efficacy of the DGA. Ten patients who underwent DGA treatment were compared with a control group of 8 patients treated with traditional frontal orbital advancement. Postoperative three-dimensional computed tomography (CT) comparison measurements were taken, including bilateral vertical and transverse orbital dimensions, lateral orbital rim to external auditory canal, and forehead measurements from the superior aspect of the orbital rim to the pituitary fossa. The traditional treatment group showed absence of the coronal sutures bilaterally on long-term CT scans. The DGA group showed normal coronal sutures on the unaffected sides. Postoperative CT measurements showed no statistical difference between the 2 techniques (P < 0.05).

Oropharyngeal stenosis (OPS) is a rare postoperative complication of adenotonsillectomy that can be a source of considerable patient distress and morbidity. Circumferential scarring of the soft palate and tonsillar pillars leads to narrowing of the oropharyngeal aperture. This case report describes the novel use of bilateral buccal myomucosal flaps for the repair of postoperative OPS in a 20-year-old woman presenting with dysphagia, odynophagia, dyspnea, and intermittent hypernasal speech. Postoperatively, the patient noted immediate improvement of her symptoms. At 1-month follow-up, she noted complete resolution of her symptoms with no dysphagia, nasal regurgitation, speaking difficulty, dyspnea, or gagging. The buccal flaps were well healed and completely intact, maintaining appropriate height of the tonsillar pillars. The buccal myomucosal flap is an effective tool for numerous palatal and oropharyngeal abnormalities and, as described in this case study, is a reliable, safe, and effective technique that can be considered for the reconstruction of postsurgical OPS.

Background: Motorcycle helmet legislation has been a contentious topic for over a half-century. Benefits of helmet use in motorcycle trauma patients are well documented. In 2012, Michigan repealed its universal motorcycle helmet law in favor of a partial helmet law. The authors describe the early clinical effects on facial injuries throughout Michigan., Methods: Retrospective data from the Michigan Trauma Quality Improvement Program trauma database were evaluated. Included were 4643 motorcycle trauma patients presenting to 29 Level I and II trauma centers throughout Michigan 3 years before and after the law repeal (2009 to 2014). Demographics, external cause of injury codes, International Classification of Diseases, Ninth Revision diagnosis codes, and injury details were gathered., Results: The proportion of unhelmeted trauma patients increased from 20 percent to 44 percent. Compared with helmeted trauma patients, unhelmeted patients were nearly twice as likely to sustain craniomaxillofacial injuries (relative risk, 1.90), including fractures (relative risk, 2.02) and soft-tissue injuries (relative risk, 1.94). Unhelmeted patients had a lower Glasgow Coma Scale score and higher Injury Severity Scores. Patients presenting after helmet law repeal were more likely to sustain craniomaxillofacial injuries (relative risk, 1.46), including fractures (relative risk, 1.28) and soft-tissue injuries (relative risk, 1.56). No significant differences were observed for age, sex, Injury Severity Score, or Glasgow Coma Scale score (p > 0.05)., Conclusions: This study highlights the significant negative impact of relaxed motorcycle helmet laws leading to an increase in craniomaxillofacial injuries. The authors urge state and national legislators to reestablish universal motorcycle helmet laws.

Background: Standard methods of cleft palate repair rely on existing palatal tissue to achieve closure. These procedures often require relaxing incisions, causing scars and growth restriction, and may result in insufficient palatal length and suboptimal positioning of the velar musculature. The Furlow double opposing Z-plasty improves palatal length and repositions the velar musculature; however, relaxing incisions may still be needed. The addition of buccal flaps to the Furlow repair obviates the need for relaxing incisions and allows the Furlow repair to be used in wide clefts., Methods: A retrospective review was performed on 505 patients; all patients were treated with the double opposing Z-plasty plus or minus buccal flap approach. Outcomes included nasal resonance, secondary speech surgery, and postoperative complications. A comparison was made between patients treated with double opposing Z-plasty alone and those treated with double opposing Z-plasty plus buccal flaps., Results: The average nasal resonance score was 1.38 and was equivalent in both the double opposing Z-plasty alone and with buccal flap groups, despite significantly more wide clefts in the buccal flap group (56 percent versus 8 percent). The secondary surgery rate for velopharyngeal insufficiency was 6.6 percent and the fistula rate was 6.1 percent. The large fistula rate (>2 mm) was 2.7 percent., Conclusions: The double opposing Z-plasty plus or minus buccal flap approach is a useful alternative to standard palate repairs. Speech outcomes were excellent, even in wider clefts, and postoperative complications were minimal. Buccal flaps allow the benefits of the Furlow repair to be applied to any size cleft, without the need for relaxing incisions., Clinical Question/level of Evidence: Therapeutic, III.

Background and Objective: Wound healing inevitably leads to scarring, which leads to functional and cosmetic defects. It is the goal of this study to investigate the immediate use of ablative fractional CO 2 lasers to reduce post-operative scarring secondary to surgical wounds., Study Design/materials and Methods: In this prospective controlled study, 20 surgical incisions were created on each of three pigs. Fifteen of the incisions were treated with an ablative fractional CO 2 laser at one of three laser settings. The remaining five incisions served as a control. Punch biopsies were taken post-operatively over time. Digital photographs were taken of each incisional scar at each time period. Blinded evaluators used a previously verified scoring system to score photographs of the incisional scars taken at the 6 month time period., Results: With regards to the comparison between the three individual laser treatment groups and the control, there were no statistically significant effects for treatment (P = 0.40), time (P = 0.48), or for the interaction of time and treatment (P = 0.57). With regards to the visual assessment tool, there were no statistically significant differences between treatments for Overall Appearance (P = 0.21) or for Total Score (P = 0.24)., Conclusions: In the limited setting of this pilot study, treatment of surgical incisions with ablative fractional CO 2 lasers does not significantly lessen scar formation. In addition, photographic analysis was not able to demonstrate a significant difference. Future studies on this topic will need a larger sample size to better answer whether a statistically significant difference may exist. Lasers Surg. Med. 49:122-128, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background and Objectives: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients., Methods: GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET)., Results: GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding., Conclusions: GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.

Reconstruction for single sutural synostosis typically involves cranial reshaping to correct for compensatory growth changes. Current remodeling techniques involve obliteration of both pathologic and normal sutures. Presented here is a case report describing a new approach to the treatment of single cranial synostosis. The concept involves excision of the offending suture and transient plating of the remaining functional sutures. Compensatory sutures are then allowed to direct the growth forces to the area of the synostosis, leading to the reversal of the compensatory shape deformity. This more natural approach leaves functioning sutures intact and allows for their active participation in the reshaping process.

Background: The tongue-lip adhesion has undergone several modifications in an attempt to reduce surgical complications and failure rates. Current techniques rely on the use of a button at the tongue base for suspension, which raises concerns about possible aspiration and interference with oral motor function and bottle-feeding. A new technique for tongue-lip adhesion is proposed that adds a tongue suspension to the standard surgical adhesion., Methods: A total of 22 patients with Pierre Robin sequence who received a tongue-lip adhesion via a tongue suspension technique were reviewed. The surgical technique differs from the standard surgical approach by the use of a suture weave across the base of the tongue instead of a standard button to suspend the tongue anteriorly., Results: Average age at the time of tongue-lip adhesion was 13.9 days, with a mean operative time of 88.8 minutes. A marked improvement in postoperative oxygenation was seen in the majority of patients. One dehiscence occurred secondary to a traumatic postoperative extubation, eventually requiring a tracheostomy for subglottic stenosis., Conclusion: A technical innovation for performing a tongue-lip adhesion using a tongue suspension in conjunction with a standard transverse adhesion of the lip is described. The advantage of the tongue-lip adhesion with suspension includes immediate postoperative extubation, as well as removal of concerns regarding button aspiration and possible interference in early developmental oral motor function and bottle-feeding. This technique is reproducible, expanding the craniofacial surgeon's armamentarium for the management of difficult airways in Pierre Robin sequence.

Reconstruction of the craniofacial skeleton has undergone a significant evolution during the past century. Initially, the use of autogenous bone grafts from various sites was the criterion standard. However, owing to donor site morbidity and lack of sufficient bone for large defects, surgeons have relied on various bone substitutes. Hydroxyapatite (HA) has served as an alternative to autogenous grafts, but questions regarding biocompatibility, risk of infection, and slow set times have hampered its acceptance. This article serves as a review of a single surgeon's experience using HA in the craniofacial skeleton. Eighteen patients receiving HA between March 2000 and November 2006 were observed. Sixteen underwent recontouring of skull-based bone defects, and 2 underwent recontouring for nasal and alveolar defects. The mean amount of HA used in each patient was 30.2 g. For large contour irregularities, the maximum thickness of HA used was 8 mm. The size of bone defects ameliorated averaged 4.8 cm(2). Complications occurred in 3 (16.7%) of 18 patients and included scalp hematoma and superficial cellulitis. In addition, 1 patient developed a facial abscess after placement along the alveolar floor, which necessitated removal. Hydroxyapatite represents a viable alternative to autogenous bone grafts when used in the correct manner. Hydroxyapatite should be used only for smaller defects or used in conjunction with absorbable plates when attempting to fill larger defects. Use of HA for nasal piriform augmentation or alveolar bone grafting should not be considered owing to problems with late infections.

Background: Velopharyngeal dysfunction has been treated with either a pharyngeal flap or sphincteroplasty with varying degrees of success. Both of these entities have their own series of problems, with sleep apnea and nasal mucous flow disruptions at the forefront. The purpose of this study was to review the senior author's (R.J.M.) experience performing the double-opposing buccal flap for palatal lengthening., Methods: All patients who were treated with double-opposing buccal flaps between October of 1994 and July of 2007 were reviewed. These patients presented with varying degrees of velopharyngeal dysfunction showing some degree of velar movement at the time of surgery. Preoperative and postoperative speech results were reviewed for comparison., Results: Twenty-seven patients underwent palatal lengthening, with an average length of follow-up of 58 months. Distal flap necrosis occurred in two patients. The level of intelligibility (65.4 percent versus 95.5 percent) and resonance (moderately hypernasal versus normal resonance) improved significantly postoperatively (p < 0.0001). Only one patient required the addition of a pharyngeal flap for persistent velopharyngeal dysfunction, and there were no postoperative issues with sleep apnea., Conclusions: The double-opposing buccal flap is an effective technique for lengthening the palate, improving speech, and decreasing the risks of postoperative sleep apnea. All patients experienced a dramatic improvement in their resonance and intelligibility. This technique appears most effective in patients with intact velar movement who demonstrate a small to moderate posterior velar gap. The double-opposing buccal flap is a useful means of treating velopharyngeal dysfunction, thus serving as an adjunct when improving pharyngeal closure.

G protein-coupled receptors (GPCRs) are seven transmembrane α-helical (7TM) integral membrane proteins that play a central role in both cell signaling and in the action of many pharmaceuticals. The crystal structures of several Family A GPCRs have shown the presence of a disulfide bond linking transmembrane helix 3 (TM3) to the second extracellular loop (ECL2), enabling ECL2 to stabilize and contribute to the ligand binding pocket. Family B GPCRs share no significant sequence identity with those in Family A but nevertheless share two conserved cysteines in topologically equivalent positions. Since there are no available crystal structures for the 7TM domain of any Family B GPCR, we used mutagenesis alongside pharmacological analysis to investigate the role of ECL2 and the conserved cysteine residues. We mutated Cys-226, at the extracellular end of TM3 of the glucagon-like peptide-1 (GLP-1) receptor, to alanine and observed a 38-fold reduction in GLP-1 potency. Interestingly, this potency loss was restored by the additional substitution of Cys-296 in ECL2 to alanine. Alongside the complete conservation of these cysteine residues in Family B GPCRs, this functional coupling suggested the presence of a disulfide bond. Further mutagenesis demonstrated that the low potency observed at the C226A mutant, compared with the C226A-C296A double mutant, was the result of the bulky nature of the released Cys-296 side chain. Since this suggested that ECL2 was in close proximity to the agonist activation pocket, an alanine scan of ECL2 was carried out which confirmed the important role of this loop in agonist-induced receptor activation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In a time of rapid change in the health care delivery system, health care professionals are challenged to fully understand their legal and ethical obligations for practice. The interface between law and ethics as it affects professional practice is complex and confusing. Three areas of legal and ethical interface and their associated history are explored: laws affecting scope of practice and the ethical obligation to provide access to care; laws affecting informed consent and the ethical obligation to respect autonomy; and laws limiting disclosure of personal identifiable information and the ethical obligation to protect privacy. A trend toward the use of statutes to protect and enforce patient rights has emerged, which has altered and limited professional practice.

Germline mutations of the fumarate hydratase (FH, fumarase) gene are found in the recessive FH deficiency syndrome and in dominantly inherited susceptibility to multiple cutaneous and uterine leiomyomatosis (MCUL). We have previously reported a number of germline FH mutations from MCUL patients. In this study, we report additional FH mutations in MCUL and FH deficiency patients. Mutations can readily be found in about 75% of MCUL cases and most cases of FH deficiency. Some of the more common FH mutations are probably derived from founding individuals. Protein-truncating FH mutations are functionally null alleles. Disease-associated missense FH changes map to highly conserved residues, mostly in or around the enzyme's active site or activation site; we predict that these mutations severely compromise enzyme function. The mutation spectra in FH deficiency and MCUL are similar, although in the latter mutations tend to occur earlier in the gene and, perhaps, are more likely to result in a truncated or absent protein. We have found that not all mutation-carrier parents of FH deficiency children have a strong predisposition to leiomyomata. We have confirmed that renal carcinoma is sometimes part of MCUL, as part of the variant hereditary leiomyomatosis and renal cancer (HLRCC) syndrome, and have shown that these cancers may have either type II papillary or collecting duct morphology. We have found no association between the type or site of FH mutation and any aspect of the MCUL phenotype. Biochemical assay for reduced FH functional activity in the germline of MCUL patients can indicate carriers of FH mutations with high sensitivity and specificity, and can detect reduced FH activity in some patients without detectable FH mutations. We conclude that MCUL is probably a genetically homogeneous tumour predisposition syndrome, primarily resulting from absent or severely reduced fumarase activity, with currently unknown functional consequences for the smooth muscle or kidney cell.

Chronically elevated luteinizing hormone (LH) induces significant pathology in the LHbetaCTP transgenic mouse model, which uses the bovine gonadotropin alpha (alpha)-subunit promoter to direct transgene expression specifically to gonadotropes in the anterior pituitary. Previously, it was shown that female LHbetaCTP mice are infertile due to anovulation, develop granulosa cell tumors, and undergo precocious puberty from elevated LH and steroid hormones that fail to completely repress the alpha-subunit promoter. This chapter will discuss recent studies that further elucidate the impact of chronically elevated LH on diverse physiological systems. Granulosa cell tumors induced by elevated LH are strain dependent and prevented when transgenics are treated with human chorionic gonadotropin (hCG) surges. A granulosa cell tumor-associated transcriptome is generated, revealing several possible gene candidates for ovarian granulosa cell tumorigenesis. Primordial follicles in LHbetaCTP transgenics become depleted and oocytes exhibit increased rates of meiotic segregation defects, although meiotic competency is acquired normally. Anovulation can be rescued in transgenics by superovulation, though pregnancy fails at midgestation due to maternal factors. Uterine receptivity defects prevent implantation of normal embryos following induction of pseuodpregnancy. Transgenics develop Cushing-like adrenocortical hyperfunction with increased corticosterone production following induction of adrenal LH receptor expression. Elevated LH acts as a tumor promoter in the gonads and the adrenal gland, when expressed in conjunction with the inhibin-alpha SV40 transgene. Finally, chronic elevated LH promotes mammary tumorigenesis. The understanding of multiple clinical pathologies--including ovarian cancer, perimenopausal reproductive aging, premature ovarian failure, polycystic ovarian syndrome, Cushing's syndrome, and breast cancer--may be enhanced through further study of this useful transgenic mouse model.

Objective: (1) To integrate clinical problem solving into freshman cell and tissue biology (CTB) and (2) to enhance understanding of diabetes using CTB principles to explain the etiology, management, and development of complications in terms of cell, tissue, and organ structure and function., Description: First-year medical students often question the need to learn detailed basic science material. Although clinical content has increased throughout basic science courses, little attempt has been made to link clinical correlations to one another or to enhance use of basic science material in clinical problem solving. The CTB course applies pertinent cell biology concepts such as cell proliferation, differentiation, migration, adhesion, and morphogenesis to tissue and organ function. Diabetes mellitus was chosen as a theme for CTB as diabetes has devastating effects on multiple tissues, and the disease has reached epidemic proportions in the United States, affecting individuals of every age and population group. Type I diabetes, presenting as ketoacidosis in a ten-year-old boy, was introduced by generalist physicians using a "grand rounds" approach. This format challenged students on the first day of medical school to diagnose a patient's problem and to explain the clinical findings in terms of anatomic, biochemical, and physiologic changes. The role of the blood/bicarbonate buffering system was the main focus of faculty-led discussion. The patient was then presented at age 25 with many diabetic complications. This stimulated discussion of the etiology of diabetes (type I versus type II), glycation, and the use of hemoglobin (Hb) A1c to monitor blood sugar control. Compliance and other aspects of diabetes management were added to the discussion. Faculty provided scientific information as necessary, and summary materials were distributed after the sessions. The interaction of a cell biologist with two generalist physicians optimized the integration of basic science with clinical problem solving. During the two semesters of CTB, the diabetes case is frequently referenced. Insulin synthesis provides the model for protein synthesis. Glycation, advanced glycation end products (AGE), and receptors (RAGE) are discussed. Other diabetes-related topics include wound healing (epithelium), basement membrane thickening (connective tissue), insulin regulation of muscle metabolism, diabetic neuropathy (neurohistology), platelet adhesiveness, glycation and HbA1c (blood), osteoporosis and Charcot joints (skeletal system), autoimmune mechanisms (cellular immunology), atherosclerosis and high blood pressure (blood vessels), diabetic nephropathy (renal), altered hepatic and gastrointestinal function, impotence (male reproductive system), and a comparison of type I and type II diabetes (endocrine system)., Discussion: Students have provided very positive feedback. The initial case enhanced interest in CTB, established clinical relevance, and has motivated learning and integration of materials from different parts of CTB and other courses. Other courses are now formally linking to the theme. For example, neuroscience will revisit diabetic neuropathy and retinopathy, physiology will relate ketoacidosis to acid-base balance, a human anatomy clinical correlation is being designed for transplantation surgeons to "cure" our diabetic patient with a renal-pancreas transplant. Uses of the case for introduction to clinical medicine, aspects of medical ethics, preventive medicine, and courses in pharmacology and pathology are contemplated.

The purpose of this study was to explore perceptions of pregnant and parenting substance-abusing women in an outpatient drug treatment program regarding provider and social support. Also identified were aspects of the rehabilitation program perceived by the women as assisting them to maintain abstinence from substance use. Data were collected through a demographic questionnaire and a tool designed by the authors based on the Social Stress Model of Substance Abuse (Lindenberg et al., 1993) and the literature of social support. The majority of the women were satisfied with their social support from family and friends. Sixty-seven percent of the women felt the support received from medical providers were not adequate. Also, the majority of the women received no information on risks of drug use and pregnancy from their medical providers. The women felt the program helped maintain abstinence by providing education, coping mechanisms, resources, 12-step programs, and spiritual guidance.

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca(2+)-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca(2+)/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca(2+)-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca(2+)-dependent and Zn(2+)-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

A heterotrimeric member of the AMP-activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an alpha2 catalytic and two non-catalytic subunits, beta2 and gamma1. The AMPK beta2 cDNA (271 amino acids (aa), molecular weight (MW)=30¿ omitted¿307, pI 6. 3) was cloned from skeletal muscle and found to share an overall identity of 70% with beta1 (270 aa, MW=30¿ omitted¿475, pI 6.0). In the liver AMPK beta1 subunit, Ser-182 is constitutively phosphorylated whereas in skeletal muscle beta2 isoform, we find that Ser-182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser-24, Ser-25 found in the beta1 are replaced by Ala-Glu in the beta2 isoform. beta2 contains seven more Ser and one less Thr residues than beta1, raising the possibility of differential post-translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively beta1 associated with alpha2, whereas extensor digitorum longus muscle alpha2 (EDL, fast twitch) associates with beta2 as well as beta1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co-immunoprecipitated with the AMPK alpha2beta2gamma1 complex.

This study describes the personal experiences of pregnancy for African-American women. Data were obtained from two group interviews with four African-American nurse-midwives who had experienced pregnancy and had extensive professional experience in the provision of health care services to pregnant African Americans. Three major themes were constructed from the interview narratives. The first concerned the experience of pregnancy as a transition experience from childhood to adulthood and from womanhood to motherhood, involving heightened senses of maturity, self-esteem, and intimacy. The second identified stresses experienced by African-American women, including the lack of material resources and emotional support. The last theme concerned the provision of effective support in pregnancy. The significance of interpersonal relationships with the pregnant women's mothers, other significant women, and their partners was described. Implications for practice included suggestions for the provision of effective emotional support from health care professionals such as attentive listening and the elimination of environmental factors that communicate lowered personal value.

Elevated levels of LH have been associated with infertility and miscarriage in women. Previously, we have reported generating a transgenic mouse model that hypersecretes LH. Female transgenics exhibit extensive pathology including enlarged, cystic, and hemorrhagic ovaries; elevated testosterone:estradiol ratios; and infertility primarily due to anovulation. Here we show that anovulation can be reversed in transgenics and that, despite development within a pathological ovary, oocytes from transgenics are remarkably healthy. Fertilized ova from transgenics are capable of normal development to term when transferred into nontransgenic pseudopregnant recipients. However, reciprocal transfers of nontransgenic embryos into transgenic recipients failed due to lack of uterine receptivity. In addition, while superovulated and mated transgenics appear to have normal early pregnancy, embryos are resorbed at midgestation due to maternal hormonal defects. Transgenic infertility can be rescued by ovariectomy with progesterone and estradiol replacement. These studies are particularly intriguing in light of data indicating an increased rate of miscarriage among women undergoing infertility treatments who are diagnosed with polycystic ovarian syndrome.

The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the alpha2 and beta2 subunits were increased between embryonic days 10 and 14, whereas expression of alpha1, beta1, and gamma1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The alpha2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the alpha1 catalytic subunit showed low expression in neuropil. The gamma1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the beta1 and beta2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either beta isoform. Preferential nuclear localization of the alpha2, beta1, and gamma1 subunits suggests new functions of the AMP-activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits alpha1 and alpha2 point to different physiological roles.

The underlying pathology of the cleft lip nasal deformity has yet to be fully realized, and cleft lip rhinoplasty continues to challenge the reconstructive surgeon. A new model is proposed, which is composed of elements that represent known anatomical structures of the nose. These structures are considered elemental to the mechanism of the primary cleft lip nasal deformity. The lobule is reduced to four arches. Five points on the skull provide foundations for these arches, which react interdependently to extrinsic forces and positional change. When certain changes are imposed on the model, predictable alterations in the configuration of the model imitate the observed deformities in the spectrum of the cleft lip nasal deformity, unilateral and bilateral, mild through severe. The model is described with illustrations, anatomic dissection, physical models, and selected clinical cases. A better understanding of the mechanisms of the cleft nasal deformities can be obtained through analysis of the model.

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA. The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immuno-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor alpha-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) non-activated STAT1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

A few years before reproductive senescence, primordial follicles are depleted from the ovary at a dramatically accelerated rate. It has been proposed that this depletion is due to transient increases in gonadotropin levels. To test this hypothesis, we used mice that produce chronically elevated levels of serum LH via expression of an LHbeta subunit transgene. Ovaries were collected from transgenic and control mice, and complete serial sections were prepared for histological examination. Each section was scanned for morphological abnormalities, and every fifth section was sampled to estimate the total number of primordial, primary, and large preantral follicles per ovary. Until 3 wk postpartum, ovaries from transgenic and control mice were morphologically similar. By 5 wk, control ovaries contained many healthy primordial, primary, and large preantral follicles as well as atretic follicles. Transgenic ovaries contained blood-filled cysts, misshapen granulosa cells, luteinized cells, and approximately 45% fewer primordial follicles than controls. By 3 mo, transgenic ovaries had about 68% fewer primordial follicles and 53% fewer primary follicles than controls. These results suggest that, in addition to having profound effects on growing follicles, chronically elevated LH levels deplete the primordial follicle pool and thus may hasten the onset of reproductive senescence.

The giant myosin-associated twitchin kinase, a member of the Ca2+-regulated protein kinase superfamily, is activated by the EF-hand protein S100A1 in a Ca2+-dependent and Zn2+-enhanced manner. We used recombinant S100A1 to further characterize the interaction between the two proteins. Zn2+ enhanced the binding of Ca2+/S100A1 to twitchin kinase fragments (Kd < 50 nM) in assays using a BIAcore biosensor by reducing the S100A1 off rate. Other Ca2+-binding proteins (S100A6, calmodulin, and the calmodulin-like domain of Ca2+-dependent protein kinase alpha) bound to the kinase but did not activate it. These results indicate that binding of Ca2+-binding proteins alone is insufficient to trigger the intramolecular rearrangement of kinase autoinhibitory contacts required for twitchin kinase activation that is specifically elicited by the S100A1 protein. Kinase fragments that contained only the autoinhibited catalytic sequence or an additional immunoglobulin-like domain had very similar properties, indicating that the tethered immunoglobulin-like domain does not modulate kinase regulation.