Understanding Racial, Ethnic, and Socioeconomic Differences in the Ambulatory Care Experience.

BACKGROUND AND OBJECTIVE: Racial and ethnic and socioeconomic differences in patient experience are prevalent and can negatively impact health outcomes. Our objective was to examine differences in family experience of care in the pediatric ambulatory setting. METHODS: We conducted interviews with parents of patients from different demographic groups who had received care at 1 of 3 clinics at a quaternary children's hospital. Multidisciplinary team conducted inductive and deductive thematic analysis of transcribed interviews. Sentiments and recurring themes were compared within and between racial and ethnic groups, insurance status, and language. RESULTS: Eighty parents were interviewed. Three primary themes were identified: (1) mitigation of system issues: parents' mixed experiences with staff or clinicians mitigating system issues impacted their overall perceptions of care; (2) pivotal role of personal interactions: clinicians' interactions positively influenced family-clinician relationships and offset negative experiences; (3) effective explanations: clinicians' clear and thorough explanations were crucial in enhancing parent confidence in care. As an overarching theme, discrimination and disrespect by staff undermined trust in care, affecting all aspects of experience. With the exception of explanations, a higher proportion of publicly-insured parents reported negative experiences across all themes compared to those with private insurance. Asian parents with public insurance had the highest proportion of interviews that were mainly negative in sentiment. CONCLUSIONS: Our findings offer nuanced insights into differences in the experience of ambulatory care. Insurance status emerged as an important marker of differential perceptions of care. Our study points to areas for improvement and highlights family-clinician interactions as vital to overall positive experience.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

The novel missense mutation Met48Lys in FKBP22 changes its structure and functions.

Mutations in the FKBP14 gene encoding FKBP22 (FK506 Binding Protein 22 kDa) cause kyphoscoliotic Ehlers-Danlos Syndrome (kEDS). The first clinical report showed that a lack of FKBP22 protein due to mutations causing nonsense-mediated decay of the mRNA leads to a wide spectrum of clinical phenotypes including progressive kyphoscoliosis, joint hypermobility, hypotonia, hyperelastic skin, hearing loss and aortic rupture. Our previous work showed that these phenotypic features could be correlated with the functions of FKBP22, which preferentially binds to type III, VI and X collagens, but not to type I, II or V collagens. We also showed that FKBP22 catalyzed the folding of type III collagen through its prolyl isomerase activity and acted as a molecular chaperone for type III collagen. Recently, a novel missense mutation Met48Lys in FKBP22 was identified in a patient with kEDS. In this report, we expand the list of substrates of FKBP22 and also demonstrate that the Met48Lys mutation diminishes the activities of FKBP22, indicating that pathology can arise from absence of FKBP22, or partial loss of its function.

Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum.

Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/SERPINH1) and 65-kDa FK506-binding protein (FKBP65/FKBP10), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes.

Authentication of collagen VI antibodies.

BACKGROUND: Collagen VI is a ubiquitously-expressed macromolecule that forms unique microfibrillar assemblies in the extracellular matrix. Mutations in the COL6A1, COL6A2 and COL6A3 genes result in congenital muscular dystrophy, arguing that collagen is critical for skeletal muscle development and function. Antibodies against collagen VI are important clinical and diagnostic tools in muscular dystrophy. They are used to confirm genetic findings by detecting abnormalities in the distribution, organization and overall levels of collagen VI in cells and tissues isolated from patients. METHODS: Many antibodies have been raised against tissue-purified collagen VI and individual collagen VI chains, however few have been properly validated for sensitivity and chain specificity. To address this deficiency, we compared the ability of 23 commercially-available antibodies to detect extracellular collagen VI by immunohistochemistry on frozen tissue sections. To determine chain specificity, immunoblot analyses were conducted on cell lysates isolated from cells transfected with cDNAs for each individual chain and cells expressing all three chains together. RESULTS: Our analyses identified 15 antibodies that recognized tissue collagen VI by immunohistochemistry at varying intensities and 20 that successfully detected collagen VI by immunoblotting. Three antibodies failed to recognize collagen VI by either method under the conditions tested. All chain-specific antibodies that worked by immunoblotting specifically recognized their correct chain, and no other chains. CONCLUSIONS: This series of side-by-side comparisons reveal at least two antibodies specific for each chain that work well for immunohistochemistry on frozen sections. This validation study expands the repertoire of antibodies available for muscular dystrophy studies caused by defects in collagen VI.

Exploring the evolutionary origins of overimitation: a comparison across domesticated and non-domesticated canids.

When learning from others, human children tend to faithfully copy - or 'overimitate' - the actions of a demonstrator, even when these actions are irrelevant for solving the task at hand. We investigate whether domesticated dogs (Canis familiaris) and dingoes (Canis dingo) share this tendency to overimitate in three experiments. In Experiment 1, dogs and dingoes had the opportunity to solve a puzzle after watching an ostensive demonstrator who used both a relevant action and an irrelevant action. We find clear evidence against overimitation in both species. In contrast to human children (Horner & Whiten, 2005), dogs and dingoes used the irrelevant action less often across trials, suggesting that both species were filtering out the irrelevant action as they gained experience with the puzzle (like chimpanzees; Horner & Whiten, 2005). Experiments 2 and 3 provide further evidence against overimitation, demonstrating that both species' behavior is better characterized by individual exploration than overimitation. Given that both species, particularly dogs, show human-like social learning in other contexts, these findings provide additional evidence that overimitation may be a unique aspect of human social learning. A video abstract of this article can be viewed at: https://youtu.be/g2mRniJZ7aU.

Novel compound heterozygous mutations in inositol polyphosphate phosphatase-like 1 in a family with severe opsismodysplasia.

This study aimed to identify the genetic basis of a severe skeletal lethal dysplasia. The main clinical features of two affected fetuses included short limbs with flared metaphyses, bowed radii, femora and tibiae, irregular ossification of hands and feet, and marked platyspondyly. Affected and nonaffected family members were subjected to whole-exome sequencing, followed by immunoblot analysis on amniocytes isolated from one of the affected individuals. Unique compound heterozygous variants in the inositol polyphosphate phosphatase-like 1 (INPPL1) gene encoding the SHIP2 protein were identified in both affected individuals. One variant was inherited from each unaffected parent. Both allelic variants, c.(2327-1G>C);(1150_1151delGA), are predicted to result in premature stop codons leading to nonsense-mediated mRNA decay of the mutant alleles and no production of SHIP2. The absence of SHIP2 was confirmed by immunoblot analysis of proband amniocytes. This skeletal disorder is caused by the complete absence of the SHIP2 protein. INPPL1 mutations have been reported in opsismodysplasia, an autosomal recessive skeletal dysplasias with significant delayed bone formation. Our finding highlights the critical role that INPPL1/SHIP2 plays in skeletal development.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

Hypoxia-inducible factor 3-alpha expression is associated with the stable chondrocyte phenotype.

The hypoxia-inducible factors HIF-1α and HIF-2α are important regulators of the chondrocyte phenotype but little is known about HIF-3α in cartilage. The objective of this study was to characterize HIF-3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three-dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype.

Assessing the susceptibility of transgenic mice overexpressing deer prion protein to bovine spongiform encephalopathy.

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.

The expanded collagen VI family: new chains and new questions.

Collagen VI is a component of the extracellular matrix of almost all connective tissues, including cartilage, bone, tendon, muscles and cornea, where it forms abundant and structurally unique microfibrils organized into different suprastructural assemblies. The precise role of collagen VI is not clearly defined although it is most abundant in the interstitial matrix of tissues and often found in close association with basement membranes. Three genetically distinct collagen VI chains, α1(VI), α2(VI) and α3(VI), encoded by the COL6A1. COL6A2 and COL6A3 genes, were first described more than 20 years ago. Their molecular assembly and role in congenital muscular dystrophy has been broadly characterized. In 2008, three additional collagen VI genes arrayed in tandem at a single gene locus on chromosome 3q in humans, and chromosome 9 in mice, were described. Following the naming scheme for collagens the new genes were designated COL6A4. COL6A5 and COL6A6 encoding the α4(VI), α5(VI) and α6(VI) chains, respectively. This review will focus on the current state of knowledge of the three new chains.

PHENOTYPIC VARIABILITY IN INDIVIDUALS WITH TYPE V OSTEOGENESIS IMPERFECTA WITH IDENTICAL IFITM5 MUTATIONS.

BACKGROUND: Osteogenesis imperfecta (OI) type V is a dominantly inherited skeletal dysplasia characterized by fractures and progressive deformity of long bones. In addition, patients often present with radial head dislocation, hyperplastic callus, and calcification of the forearm interosseous membrane. Recently, a specific mutation in the IFITM5 gene was found to be responsible for OI type V. This mutation, a C to T transition 14 nucleotides upstream from the endogenous start codon, creates a new start methionine that appears to be preferentially used by the translational machinery. However, the mechanism by which the lengthened protein results in a dominant type of OI is unknown. METHODS AND RESULTS: We report 7 ethnically diverse (African-American, Caucasian, Hispanic, and African) individuals with OI type V from 2 families and 2 sporadic cases. Exome sequencing failed to identify a causative mutation. Using Sanger sequencing, we found that all affected individuals in our cohort possess the c.-14 IFITM5 variant, further supporting the notion that OI type V is caused by a single, discrete mutation. Our patient cohort demonstrated inter-and intrafamilial phenotypic variability, including a father with classic OI type V whose daughter had a phenotype similar to OI type I. This clinical variability suggests that modifier genes influence the OI type V phenotype. We also confirm that the mutation creates an aberrant IFITM5 protein containing an additional 5 amino acids at the N-terminus. CONCLUSIONS: The variable clinical signs in these cases illustrate the significant variability of the OI type V phenotype caused by the c.-14 IFITM5 mutation. The affected individuals are more ethnically diverse than previously reported.

A disintegrin-like and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5) is a novel fibrillin-1-, fibrillin-2-, and heparin-binding member of the ADAMTS superfamily containing a netrin-like module.

ADAMTS-like proteins are related to ADAMTS metalloproteases by their similarity to ADAMTS ancillary domains. Here, we have characterized ADAMTSL5, a novel member of the superfamily with a unique modular organization that includes a single C-terminal netrin-like (NTR) module. Alternative splicing of ADAMTSL5 at its 5' end generates two transcripts that encode different signal peptides, but the same mature protein. These transcripts differ in their translational efficiency. Recombinant ADAMTSL5 is a secreted, N-glycosylated 60kDa glycoprotein located in the subcellular matrix, on the cell-surface, and in the medium of transfected cells. RT-PCR and western blot analysis of adult mouse tissues showed broad expression. Western blot analysis suggested proteolytic release of the NTR module in transfected cells as well as in some mouse tissues. Immunostaining during mouse organogenesis identified ADAMTSL5 in musculoskeletal tissues such as skeletal muscle, cartilage and bone, as well as in many epithelia. Affinity-chromatography demonstrated heparin-binding of ADAMTSL5 through its NTR-module. Recombinant ADAMTSL5 bound to both fibrillin-1 and fibrillin-2, and co-localized with fibrillin microfibrils in the extracellular matrix of cultured fibroblasts, but without discernible effect on microfibril assembly. ADAMTSL5 is the first family member shown to bind both fibrillin-1 and fibrillin-2. Like other ADAMTS proteins implicated in microfibril biology through identification of human and animal mutations, ADAMTSL5 could have a role in modulating microfibril functions.

A novel form of chondrocyte stress is triggered by a COMP mutation causing pseudoachondroplasia.

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH-MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH.

Long-term analysis of surgical correction of the senile upper lip.

OBJECTIVE: A quantitative comparison of immediate and long-term results of surgical correction of the senile upper lip using lip advancement and lip lift. METHODS: Retrospective review of 30 patients who underwent senile upper lip repair, including lip advancement or lip lift. Digital image analysis was used to standardize each patient's preoperative and postoperative photographs for accurate, objective comparison. RESULTS: Lip lift and lip advancement achieve significant improvement in the appearance of the senile upper lip (P < .001). This improvement is sustained during many years (mean, 5 y; P < .001). Using repeated measures analysis of variance, no significant difference was found in the operative group compared with the control group when examining age-related change. CONCLUSION: Lip advancement and lip lift can restore the senile upper lip to a more youthful and natural appearance with sustained long-term benefits.

Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.

Lemierre syndrome: a pediatric case series and review of literature.

BACKGROUND: Lemierre syndrome is a rare disease of the head and neck often affecting adolescents and young adults. Classically, infection begins in the oropharynx with thrombosis of the tonsillar veins followed by involvement of the parapharyngeal space and the internal jugular vein. Septicemia and pulmonary lesions develop as infection spreads via septic emboli. Although a rare entity in modern times, Lemierre syndrome remains a disease of considerable morbidity and potential mortality. METHODS: This was a retrospective review of 3 cases and associated literature. RESULTS: A common 1- to 2-week history of fever, sore throat, neck pain, and fatigue was observed in all patients. Patient 1 developed right facial swelling, neck tenderness, trismus, and tonsillar exudate. Patient 2 displayed right tonsillar erythema and enlargement with right neck tenderness. Patient 3 revealed bilateral tonsillar enlargement with exudate and left neck tenderness. Subsequent studies included blood cultures and computed tomography, after which empiric antibiotic therapy was started. Patient 1 underwent drainage of a right peritonsillar abscess, right pressure equalization tube placement, and ligation of the right external jugular vein. He subsequently developed subdural empyemas, cavernous sinus thrombosis, and carotid artery narrowing and required 9 weeks of antibiotic therapy. Patients 2 and 3 developed pulmonary lesions and received 6 weeks of antibiotic therapy. Timing was crucial in all cases. CONCLUSIONS: Lemierre syndrome is a rare but severe opportunistic infection with poor prognostic outcomes if left untreated. Early diagnosis and treatment is essential. Aggressive antibiotic therapy coupled with surgical intervention, when necessary, provides excellent outcomes.

Cartilage oligomeric matrix protein promotes cell attachment via two independent mechanisms involving CD47 and alphaVbeta3 integrin.

Cartilage oligomeric matrix protein (COMP) is a pentameric approximately 524 kDa multidomain extracellular matrix protein and is the fifth member of the thrombospondin family. COMP is abundantly expressed in proliferating and hypertrophic chondrocytes of the growth plate, articular cartilage, synovium, tendon, and ligament. The spatial localization of COMP highlights its importance in the phenotypes of pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), COMP disorders that are characterized by disproportionate short stature, brachydactyly, scoliosis, early-onset osteoarthritis, and joint hypermobility. In this study, the role of COMP in ligament was investigated with a series of cell attachment assays using ligament cells binding to COMP. A dose-dependent cell attachment activity was found, which was inhibited by a peptide containing the SFYVVMWK amino acid sequence derived from the globular C-terminal domain of COMP. This activity was independent of the recently described RGD-dependent attachment activity. Function-blocking antibodies to CD47 and alphaVbeta3 integrin reduced cell attachment to COMP, implicating the participation of these cell surface molecules in COMP cell binding. Immunofluorescence studies showed that cell attachment to COMP induced the formation of lamellae containing F-actin microspikes associated with fascin. We propose that COMP promotes cell attachment via two independent mechanisms involving cell surface CD47 and alphaVbeta3 integrin and that a consequence of cell attachment to COMP is the specific induction of fascin-stabilized actin microspikes.

Crude subcellular fractionation of cultured mammalian cell lines.

BACKGROUND: The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. FINDINGS: We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. CONCLUSION: We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

Minimally invasive ear reshaping with a 1450-nm diode laser using cryogen spray cooling in New Zealand white rabbits.

BACKGROUND: Otoplasty is the current standard of care for treating prominent ears, a psychologically and sometimes functionally disabling disorder. The technically demanding procedure carries many risks such as poor aesthetic outcome, need for revision surgery, and need for general anesthesia. This study investigates the use of laser irradiation combined with cryogen skin cooling and stenting to reshape cartilage in the ears of New Zealand white rabbits. METHODS: In this prospective, randomized, internally controlled animal study, the right ears of 9 rabbits were mechanically deformed with a jig and then irradiated with a 1450-nm diode laser combined with cryogen skin cooling (14 J/pulse with cryogen spray for 33 milliseconds per cycle and a 6-mm spot size). The left ear served as the control. The ears were splinted for 1, 3, or 4 weeks. The rabbits were then given a lethal dose of intravenous pentobarbital, and the splints were removed and ears examined and photographed. Light and confocal microscopy were performed on the specimens. RESULTS: Shape change was observed in all 9 treated rabbit ears, while none of the control ears (stenting alone) showed significant change. Qualitatively, reshaped ears were stiffer after 4 weeks of splinting than after 1 or 3 weeks. None of the rabbits showed evidence of skin injury nor did they show signs of postprocedural pain. Findings from histologic analysis in the treated areas showed evidence of an expanded chondrocyte population in the region of laser irradiation, along with some perichondrial thickening and some fibrosis of the deep dermis. Confocal microscopy revealed minimal cellular death at 1 week and none thereafter. CONCLUSIONS: Cartilage reshaping using laser energy can be performed safely transcutaneously using cryogen spray cooling in rabbits. This animal model has similarity to human ears with regard to skin and cartilage thickness and is a stepping stone toward developing minimally invasive laser auricle reshaping in humans.