Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide-rich de novo designed peptides.

Disulfide-rich peptides represent an important protein family with broad pharmacological potential. Recent advances in computational methods have made it possible to design new peptides which adopt a stable conformation de novo. Here, we describe a system to produce disulfide-rich de novo peptides using Escherichia coli as the expression host. The advantage of this system is that it enables production of uniformly 13 C- and 15 N-labeled peptides for solution nuclear magnetic resonance (NMR) studies. This expression system was used to isotopically label two previously reported de novo designed peptides, and to determine their solution structures using NMR. The ensemble of NMR structures calculated for both peptides agreed well with the design models, further confirming the accuracy of the design protocol. Collection of NMR data on the peptides under reducing conditions revealed a dependency on disulfide bonds to maintain stability. Furthermore, we performed long-time molecular dynamics (MD) simulations with tempering to assess the stability of two families of de novo designed peptides. Initial designs which exhibited a stable structure during simulations were more likely to adopt a stable structure in vitro, but attempts to utilize this method to redesign unstable peptides to fold into a stable state were unsuccessful. Further work is therefore needed to assess the utility of MD simulation techniques for de novo protein design.

Heterotopic ossification of the temporomandibular joint in juvenile idiopathic arthritis.

OBJECTIVE: To describe the clinical, laboratory, pathologic, and radiographic imaging characteristics of a series of children with juvenile idiopathic arthritis (JIA) and radiographic imaging evidence of heterotopic ossification of their temporomandibular joint (TMJ). METHODS: Children were identified through search of an administrative database of imaging results at Seattle Children's Hospital. Retrospective chart review was performed to collect data on each patient's clinical and laboratory characteristics, systemic therapies, timing and number of TMJ intraarticular corticosteroid injections (IAS), TMJ symptoms, and TMJ findings on physician examination. TMJ imaging studies for which heterotopic ossifications were reported were reviewed. Pathology specimens were reviewed for the 2 children who underwent synovial biopsy of their TMJ. RESULTS: Twelve children were identified. The average duration between onset of JIA and detection of heterotopic ossification of TMJ on an imaging study was 36 months (range 19-94). Half the children had abnormal mouth-opening for age when the calcifications were first detected. In each case, the heterotopic ossification was first detected by computed tomography scan, and in 11 of the cases they were associated with synovial pannus formation as documented on an imaging study. Two children underwent synovial biopsy, which revealed reactive parosteal osteochondromatosis in one case and findings consistent with an intraarticular rheumatoid nodule in the other. CONCLUSION: Heterotopic ossifications of the TMJ may be seen in children with JIA and are associated with particularly severe TMJ arthritis, joint destruction, and pannus formation. Pathology from these joints suggests that the heterotopic ossification may result from multiple pathological processes.

Are pediatric autoimmune diseases primarily genetic diseases?

PURPOSE OF REVIEW: Powerful new methods are allowing identification of genetic risk factors in large populations of adults with autoimmune diseases. In this review, we describe the advantages and limitations of genetic methodologies, and how these methods have been used to discover candidate genes in smaller populations of pediatric patients. We also introduce novel concepts for nontraditional modes of genetic inheritance that may be important in the pathogenesis of autoimmunity. RECENT FINDINGS: Candidate genes identified by linkage analyses and genome-wide association scans in adult populations have led to focused genetic studies in pediatric populations. Some genes are associated with subsets of both adult and pediatric patients; others appear to be age specific. Novel concepts in genetics have uncovered potential contributions of maternal compared with paternal transmission, noninherited maternal alleles that may work through maternal microchimerism, and sex-specific epigenetic mechanisms of immunoregulation. SUMMARY: Advancing methods are leading to the discovery of genes associated with childhood autoimmune diseases. However, the genetic contribution to disease risk for any one gene remains less than 30% for most diseases, suggesting that pediatric autoimmunity is not primarily genetic in a classical sense. A combinatorial approach considering the contributions of multiple genes, mode of inheritance, and environmental influences will be required to fully understand the mechanisms of pathogenesis in pediatric autoimmune disease.

Characterization of the peripheral retinopathy in X-linked and autosomal recessive Alport syndrome.

BACKGROUND: Alport syndrome is an inherited disease resulting in kidney failure, hearing loss and ocular abnormalities. Alport syndrome is however often unrecognized, and the aim of this study was to characterize the associated but rarely described peripheral retinopathy and determine whether its demonstration was diagnostically helpful. METHODS: Index cases were diagnosed with Alport syndrome on renal biopsy in themselves or a family member. Inheritance and affected status were determined using microsatellite markers at the COL4A5 and COL4A3/COL4A4 loci, respectively. Participants' eyes were dilated, and examined with direct and indirect ophthalmoscopy, and slit lamp biomicroscopy by an expert ophthalmologist who was unaware of the patients' disease status. RESULTS: Ten males and nine females with X-linked Alport syndrome and seven with autosomal recessive disease were studied. Of the 26 patients, 16 had central retinopathy (62%), and 19 patients had peripheral retinopathy (74%). The peripheral changes occurred in both males and females with X-linked and autosomal recessive Alport syndrome, and were more common when renal failure, hearing loss, lenticonus and the central changes were present, but were also noted in 3 X-linked carriers with normal renal function. CONCLUSIONS: The peripheral retinopathy occurs in X-linked and autosomal recessive Alport syndrome even when the central retinopathy is absent. Careful retinal examination and photography that includes the periphery is a safe and inexpensive method that may help in the diagnosis of Alport syndrome especially in carriers of X-linked disease.

Access to colposcopy services for high-risk Canadian women: can we do better?

BACKGROUND: Despite overall decreasing mortality from cervical cancer, selected groups of Canadian women continue to have suboptimal access to diagnostic and treatment interventions for cervical cancer. In this paper, we present an evaluation of a colposcopy program developed to improve attendance for colposcopy in a lower socio-economic and immigrant population. METHODS: All women attending the North Hamilton Community Health Centre (CHC) who required colposcopic assessment and were referred to a newly developed colposcopy program based at the CHC were evaluated. Attendance rates for consultation, follow up and treatment in women referred for colposcopy were compared retrospectively for the CHC-based colposcopy program and concurrently with the regional colposcopy clinic (RCC). RESULTS: Women referred to the CHC colposcopy program had a significant reduction in their no-show rate after the introduction of the locally based colposcopy program (17.2% vs. 1.3%, p<0.01). Comparing the same time periods, there was no significant reduction in the default rate at the RCC (2.5% vs. 3.3%, p=0.21). Despite serving a population of women who were at higher risk for non-attendance, patients at the CHC had a default rate for appointments similar to that of the RCC (1.3% vs. 3.3%, p=0.55) after the introduction of the local colposcopy program. CONCLUSIONS: Lower socio-economic status and immigrant women receiving care from a CHC-based colposcopy program had a significant decrease in their no-show rate for colposcopic evaluation after the introduction of the on-site program. Consideration must be given to locating diagnostic colposcopy programs in settings more accessible to women who require these services the most.