Publications by authors named "Eric J Schmidt"

23 Publications

  • Page 1 of 1

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Proc Natl Acad Sci U S A 2021 Jun;118(23)

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605;

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.
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http://dx.doi.org/10.1073/pnas.2024605118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201830PMC
June 2021

Gene Expression in Meniscal Tears at the Time of Arthroscopic Partial Meniscectomy Predicts the Progression of Osteoarthritis Within 6 Years of Surgery.

Orthop J Sports Med 2020 Aug 14;8(8):2325967120936275. Epub 2020 Aug 14.

Department of Orthopaedic Surgery, Washington University School of Medicine, St Louis, Missouri, USA.

Background: While knees with meniscal tears are associated with a heightened risk of developing osteoarthritis (OA), it is difficult to predict which patients are at the greatest risk for OA. Gene signatures in menisci that are resected during arthroscopic partial meniscectomy (APM) may provide insight into the risk of OA progression.

Hypothesis: Meniscal gene signatures at the time of APM will predict radiographic OA progression.

Study Design: Case series; Level of evidence, 4.

Methods: Meniscal fragments were collected from 38 patients without OA during clinically indicated APM of the medial meniscus. The expression of 28 candidate genes with known roles in cartilage homeostasis, OA, extracellular matrix degradation, and obesity was assessed by quantitative real-time polymerase chain reaction. Weightbearing radiographs obtained before surgery and at final follow-up were graded by a musculoskeletal radiologist using the Kellgren-Lawrence classification of OA. The association of meniscal gene expression at baseline with the progression of radiographic OA was determined.

Results: Gene expression and baseline and follow-up radiographic data were available from 31 patients (81.6%) at a mean follow-up of 6.2 ± 1.3 years. Patients without OA progression had significantly higher expression of 7 genes: (5.1-fold; = .002), (2.9-fold; = .016), (3.7-fold; = .032), (4.5-fold; = .008), (6.2-fold; = .010), (5.2-fold; = .004), and (46-fold; = .008).

Conclusion: Gene expression in the meniscus at the time of APM may be associated with the risk for progression of OA after surgery. Elevated expression of the aforementioned genes may reflect a chondroprotective response. Stratifying the risk for OA progression after APM could facilitate targeted interventions to delay or prevent the development of OA. Further studies in a larger cohort with an extended follow-up, and inclusion of additional genes, are warranted to better characterize this association.
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http://dx.doi.org/10.1177/2325967120936275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446268PMC
August 2020

Duration of symptoms prior to partial meniscectomy is not associated with the expression of osteoarthritis genes in the injured meniscus.

J Orthop Res 2020 06 13;38(6):1268-1278. Epub 2020 Jan 13.

Department of Orthopedic Surgery, Musculoskeletal Research Center, Washington University School of Medicine, St. Louis, Missouri.

While there is emerging data on how duration of symptoms prior to surgery relates to outcomes of patients undergoing arthroscopic partial meniscectomy, little is known about how duration of symptoms relates to the biology of the knee in these patients. The purpose of this study was to test the hypothesis that duration of symptoms prior to arthroscopic partial meniscectomy is associated with expression of osteoarthritis (OA)-related genes in the meniscus. We collected resected meniscus from patients (N = 76) undergoing clinically indicated arthroscopic partial meniscectomy from knees without advanced degenerative changes. RNA from 64 patients was analyzed for 28 candidate OA transcripts by real-time polymerase chain reaction (PCR). RNA was also probed for identification of novel genes by RNA microarray in 12 patients followed by validation of selected candidates by real-time PCR. The association of gene expression with duration of symptoms prior to surgery was tested. Additional screening was performed with known OA genetic risk alleles assembled from published literature and with gene transcripts differentially expressed between non-OA and OA cartilage and menisci. Our data revealed that duration of symptoms did not predict expression of OA genes in the meniscus, other than limited association with CXCL3, BMP2, and HLA-DQA1. Microarray identified new genes and pathways with unknown role(s) in meniscus injury and OA and validation of a subset of genes by real-time PCR showed expression pattern highly concordant with the microarray data. While duration of symptoms prior to arthroscopic partial meniscectomy does not significantly alter the expression of OA related genes, the association with novel genes and pathways deserves further investigation.
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http://dx.doi.org/10.1002/jor.24574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225063PMC
June 2020

Nonlinear gene expression-phenotype relationships contribute to variation and clefting in the A/WySn mouse.

Dev Dyn 2019 12 14;248(12):1232-1242. Epub 2019 Sep 14.

Department of Cell Biology and Anatomy, Alberta Children's Hospital Research Institute and McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, Alberta, Canada.

Background: Cleft lip and palate is one of the most common human birth defects, but the underlying etiology is poorly understood. The A/WySn mouse is a spontaneously occurring model of multigenic clefting in which 20% to 30% of individuals develop an orofacial cleft. Recent work has shown altered methylation at a specific retrotransposon insertion downstream of the Wnt9b locus in clefting animals, which results in decreased Wnt9b expression.

Results: Using a newly developed protocol that allows us to measure morphology, gene expression, and DNA methylation in the same embryo, we relate gene expression in an individual embryo directly to its three-dimensional morphology for the first time. We find that methylation at the retrotransposon relates to Wnt9b expression and morphology. IAP methylation relates to shape of the nasal process in a manner consistent with clefting. Embryos with low IAP methylation exhibit increased among-individual variance in facial shape.

Conclusions: Methylation and gene expression relate nonlinearly to nasal process morphology. Individuals at one end of a continuum of phenotypic states display a clinical phenotype and increased phenotypic variation. Variable penetrance and expressivity in this model is likely determined both by among-individual variation in methylation and changes in phenotypic robustness along the underlying liability distribution for orofacial clefting.
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http://dx.doi.org/10.1002/dvdy.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210921PMC
December 2019

Contact Lens Versus Non-Contact Lens-Related Corneal Ulcers at an Academic Center.

Eye Contact Lens 2019 Sep;45(5):301-305

Department of Ophthalmology (L.B., S.T., B.E., E.J.S., R.P., C.H., and S.L.E.), Saint Louis University Eye Institute, St. Louis, MO; Department of Ophthalmology (H.H.), Doheny Eye Institute, Los Angeles, CA; and Department of Ophthalmology (H.H.), David Geffen School of Medicine at UCLA, Los Angeles, CA.

Purpose: To compare the infectious contact lens-related corneal ulcer (CLRU) and non-CLRU cases at Saint Louis University.

Methods: Retrospective review of corneal ulcer cases identified by search of the ophthalmology and microbiology department databases between 1999 and 2016.

Results: Six hundred seventy-seven cases of corneal ulcers were identified, of which 46% were CLRU. CLRU cases were seen more commonly in younger patients (P<0.001) and women (P=0.03) than non-CLRU cases. Many of the infections were vision-threatening as defined by central/paracentral location (73% CLRU and 71% non-CLRU [P=0.60]) and large size of ulcer >2 mm in 36% CLRU and 51% non-CLRU (P=0.002). Causative pathogen in cultured CLRU was predominately Pseudomonas species (44%, P<0.001 vs. the non-CLRU group), other gram-negative (6%), gram-positive (33%), fungi (13%), and Acanthamoeba (5%). Comparatively, cultured non-CLRU was predominately gram-positive (64%, P<0.001 vs. the CLRU group), gram-negative (26%), and fungi (11%). The combined oxacillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus isolates were 35% and 34%, respectively. Despite the progressive increase in the number of corneal ulcers seen, the annual trend for any one particular organism for either CLRU cases or non-CLRU cases did not change significantly.

Conclusions: Most of the cases were non-CLRU. CLRU was disproportionately associated with Pseudomonas species and non-CLRU with Staphylococcal species. Fungal infections were predominately caused by filamentous organisms in both groups. Acanthamoeba keratitis was exclusively associated with CL use.
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http://dx.doi.org/10.1097/ICL.0000000000000568DOI Listing
September 2019

An In Vitro Model of Charcot-Marie-Tooth Disease Type 4B2 Provides Insight Into the Roles of MTMR13 and MTMR2 in Schwann Cell Myelination.

ASN Neuro 2018 Jan-Dec;10:1759091418803282

1 Department of Neurology, Jungers Center for Neurosciences Research, Oregon Health & Science University, Portland, OR, USA.

Charcot-Marie-Tooth Disorder Type 4B (CMT4B) is a demyelinating peripheral neuropathy caused by mutations in myotubularin-related (MTMR) proteins 2, 13, or 5 (CMT4B1/2/3), which regulate phosphoinositide turnover and endosomal trafficking. Although mouse models of CMT4B2 exist, an in vitro model would make possible pharmacological and reverse genetic experiments needed to clarify the role of MTMR13 in myelination. We have generated such a model using Schwann cell-dorsal root ganglion (SC-DRG) explants from Mtmr13 mice. Myelin sheaths in mutant cultures contain outfoldings highly reminiscent of those observed in the nerves of Mtmr13 mice and CMT4B2 patients. Mtmr13 SC-DRG explants also contain reduced Mtmr2, further supporting a role of Mtmr13 in stabilizing Mtmr2. Elevated PI(3,5)P has been implicated as a cause of myelin outfoldings in Mtmr2 models. In contrast, the role of elevated PI3P or PI(3,5)P in promoting outfoldings in Mtmr13 models is unclear. We found that over-expression of MTMR2 in Mtmr13 SC-DRGs moderately reduced the prevalence of myelin outfoldings. Thus, a manipulation predicted to lower PI3P and PI(3,5)P partially suppressed the phenotype caused by Mtmr13 deficiency. We also explored the relationship between CMT4B2-like myelin outfoldings and kinases that produce PI3P and PI(3,5)P by analyzing nerve pathology in mice lacking both Mtmr13 and one of two specific PI 3-kinases. Intriguingly, the loss of vacuolar protein sorting 34 or PI3K-C2β in Mtmr13 mice had no impact on the prevalence of myelin outfoldings. In aggregate, our findings suggest that the MTMR13 scaffold protein likely has critical functions other than stabilizing MTMR2 to achieve an adequate level of PI 3-phosphatase activity.
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http://dx.doi.org/10.1177/1759091418803282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6236487PMC
July 2019

Laboratory Results, Epidemiologic Features, and Outcome Analyses of Microbial Keratitis: A 15-Year Review From St. Louis.

Am J Ophthalmol 2019 02 9;198:54-62. Epub 2018 Oct 9.

Department of Ophthalmology, Saint Louis University School of Medicine, St Louis, Missouri, USA.

Purpose: To evaluate the laboratory results and prognostic factors of poor clinical outcomes in microbial keratitis cases over 15 years at Saint Louis University.

Design: Retrospective cohort and trend study.

Methods: Microbiological and clinical information from culture-positive cases seen at Saint Louis University from 1999 to 2013 were reviewed retrospectively. Statistical analyses were used to determine microbiological and antibiotic susceptibility trends. Prognostic factors of poor clinical outcome from the literature were used to create multivariate regression models to describe our cohort.

Results: Gram-positive organisms predominated (48%), followed by gram-negative organisms (34%) and fungi (16%). The most commonly isolated organism was Pseudomonas aeruginosa (21%). Oxacillin-resistant rates of Staphylococcus aureus and coagulase-negative staphylococci were 45% and 43%, respectively. Only the proportion of Pseudomonas changed significantly over time (P = .02). The only antibiotic found to lose efficacy over time was gentamicin for gram-positive organisms (P = .005). Multivariate logistic regression analyses revealed that major complications were associated with large ulcers (P < .006), fungal cases (P < .001), and comorbid ophthalmic conditions (P < .001). Poor healing was associated with large ulcers (P < .001) and fungal cases (P < .001). Lastly, poor visual outcome was associated with large ulcers (P < .01) and age ≥ 60 years (P < .02).

Conclusions: In the St Louis area, oxacillin-resistant organisms, Pseudomonas aeruginosa, and fungi are commonly recovered from microbial keratitis cases with a disproportionally high incidence. Hence, empiric antibiotic choice should reflect these trends. Special care needs to be taken for patients with large ulcers and fungal infections, as well as elderly patients with comorbid ophthalmic conditions, as these patients have worse clinical outcomes.
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http://dx.doi.org/10.1016/j.ajo.2018.09.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349514PMC
February 2019

Evidence for Genetic Contribution to Variation in Posttraumatic Osteoarthritis in Mice.

Arthritis Rheumatol 2019 03 25;71(3):370-381. Epub 2019 Jan 25.

Washington University in St. Louis, St. Louis, Missouri.

Objective: Recombinant inbred mouse strains generated from an LG/J and SM/J intercross offer a unique resource to study complex genetic traits such as osteoarthritis (OA). We undertook this study to determine the susceptibility of 14 strains to various phenotypes characteristic of posttraumatic OA. We hypothesized that phenotypic variability is associated with genetic variability.

Methods: Ten-week-old male mice underwent surgical destabilization of the medial meniscus (DMM) to induce posttraumatic OA. Mice were killed 8 weeks after surgery, and knee joints were processed for histology to score cartilage degeneration and synovitis. Micro-computed tomography was used to analyze trabecular bone parameters including subchondral bone plate thickness and synovial ectopic calcifications. Gene expression in the knees was assessed using a QuantiGene Plex assay.

Results: Broad-sense heritability ranged from 0.18 to 0.58, which suggested that the responses to surgery were moderately heritable. The LGXSM-33, LGXSM-5, LGXSM-46, and SM/J strains were highly susceptible to OA, while the LGXSM-131b, LGXSM-163, LGXSM-35, LGXSM-128a, LGXSM-6, and LG/J strains were relatively OA resistant. This study was the first to accomplish measurement of genetic correlations of phenotypes that are characteristic of posttraumatic OA. Cartilage degeneration was significantly positively associated with synovitis (r = 0.83-0.92), and subchondral bone plate thickness was negatively correlated with ectopic calcifications (r = -0.59). Moreover, we showed that 40 of the 78 genes tested were significantly correlated with various OA phenotypes. However, unlike the OA phenotypes, there was no evidence for genetic variation in differences in gene expression levels between DMM-operated and sham-operated knees.

Conclusion: For these mouse strains, various characteristics of posttraumatic OA varied with genetic composition, which demonstrated a genetic basis for susceptibility to posttraumatic OA. The heritability of posttraumatic OA was established. Phenotypes exhibited various degrees of correlations; cartilage degeneration was positively correlated with synovitis, but not with the formation of ectopic calcifications. Further investigation of the genome regions that contain genes implicated in OA, as well as further investigation of gene expression data, will be useful for studying mechanisms of OA and identifying therapeutic targets.
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http://dx.doi.org/10.1002/art.40730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393186PMC
March 2019

Post-Traumatic Osteoarthritis in Mice Following Mechanical Injury to the Synovial Joint.

Sci Rep 2017 03 27;7:45223. Epub 2017 Mar 27.

Department of Orthopaedic Surgery, Musculoskeletal Research Center, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, MO, USA.

We investigated the spectrum of lesions characteristic of post-traumatic osteoarthritis (PTOA) across the knee joint in response to mechanical injury. We hypothesized that alteration in knee joint stability in mice reproduces molecular and structural features of PTOA that would suggest potential therapeutic targets in humans. The right knees of eight-week old male mice from two recombinant inbred lines (LGXSM-6 and LGXSM-33) were subjected to axial tibial compression. Three separate loading magnitudes were applied: 6N, 9N, and 12N. Left knees served as non-loaded controls. Mice were sacrificed at 5, 9, 14, 28, and 56 days post-loading and whole knee joint changes were assessed by histology, immunostaining, micro-CT, and magnetic resonance imaging. We observed that tibial compression disrupted joint stability by rupturing the anterior cruciate ligament (except for 6N) and instigated a cascade of temporal and topographical features of PTOA. These features included cartilage extracellular matrix loss without proteoglycan replacement, chondrocyte apoptosis at day 5, synovitis present at day 14, osteophytes, ectopic calcification, and meniscus pathology. These findings provide a plausible model and a whole-joint approach for how joint injury in humans leads to PTOA. Chondrocyte apoptosis, synovitis, and ectopic calcification appear to be targets for potential therapeutic intervention.
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http://dx.doi.org/10.1038/srep45223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366938PMC
March 2017

Corneal Toxicity Associated With Aquarium Coral Palytoxin.

Am J Ophthalmol 2017 Feb 25;174:119-125. Epub 2016 Oct 25.

Departments of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

Purpose: To report a series of patients who developed corneal toxicity after exposure to aquarium coral palytoxin.

Design: Multicenter retrospective case series.

Methods: Retrospective review.

Results: Seven patients presented with corneal findings ranging from superficial punctate epitheliopathy to bilateral corneal melt with subsequent perforation. Among those with mild corneal findings, resolution was achieved with topical steroids and lubrication, whereas some patients who developed progressive corneal melt required therapeutic penetrating keratoplasty. The history in all patients revealed exposure to aquarium zoanthid corals shortly before disease onset. A review of the literature revealed that there are few prior reports of coral-associated corneal toxicity and that some species of coral secrete a substance known as palytoxin, a potent vasoconstrictor that inhibits the membranous sodium-potassium ATPase pump across cell types and can cause rapid death if inhaled or ingested.

Conclusions: This is the largest case series to date demonstrating patients with aquarium coral palytoxin-associated corneal toxicity, and is the first to provide details of related histopathologic findings. Similar to other forms of toxic keratoconjunctivitis, a detailed history and careful clinical assessment are required, as well as timely removal of the offending agent from the patients' ocular milieu and environment. Mild ocular surface and corneal disease may be treated effectively with aggressive topical steroid therapy and lubrication. Given the potential severity of ocular as well as systemic adverse effects, there should be increased awareness of this entity among eye care professionals, aquarium enthusiasts, and the general public.
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http://dx.doi.org/10.1016/j.ajo.2016.10.007DOI Listing
February 2017

Genetic loci that regulate ectopic calcification in response to knee trauma in LG/J by SM/J advanced intercross mice.

J Orthop Res 2015 Oct 19;33(10):1412-23. Epub 2015 Jun 19.

Department of Orthopaedic Surgery, Musculoskeletal Research Center, Washington University School of Medicine at Barnes-Jewish Hospital, 425 S. Euclid Avenue MS 8233, St. Louis, Missouri, 63110.

This study reports on genetic susceptibility to ectopic calcification in the LG/J and SM/J advanced intercross mice. Using 347 mice in 98 full-sibships, destabilization of medial meniscus (DMM) was performed to induce joint injury. We found that joint destabilization instigated ectopic calcifications as detected and quantified by micro-CT. We performed quantitative trait locus (QTL) analysis to map ectopic calcification phenotypes to discrete genomic locations. To validate the functional significance of the selected QTL candidate genes, we compared mRNA expression between parental LG/J and SM/J inbred strains. Overall, we detected 20 QTLs affecting synovial and meniscus calcification phenotypes with 11 QTLs linked to synovial calcification. Functional and bioinformatic analyses of single nucleotide polymorphism (SNP) identified functional classifications relevant to angiogenesis (Myo1e, Kif26b, Nprl3, Stab2, Fam105b), bone metabolism/calcification (Tle3, Tgfb2, Lipc, Nfe2l1, Ank, Fam105b), arthritis (Stab2, Tbx21, Map4k4, Hoxb9, Larp6, Col1a2, Adam10, Timp3, Nfe2l1, Trpm3), and ankylosing-spondylitis (Ank, Pon1, Il1r2, Tbkbp1) indicating that ectopic calcification involves multiple mechanisms. Furthermore, the expression of 11 out of 78 candidate genes was significantly different between LG/J and SM/J. Correlation analysis showed that Aff3, Fam81a, Syn3, and Ank were correlated with synovial calcification. Taken together, our findings of multiple genetic loci suggest the involvement of multiple genes contributing to ectopic calcification.
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http://dx.doi.org/10.1002/jor.22944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025301PMC
October 2015

Anisometropia in children with neurofibromatosis type 1 and unilateral optic glioma.

J AAPOS 2014 Dec 1;18(6):623. Epub 2014 Nov 1.

Saint Louis University Department of Ophthalmology.

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http://dx.doi.org/10.1016/j.jaapos.2014.07.161DOI Listing
December 2014

Molecular insight into the association between cartilage regeneration and ear wound healing in genetic mouse models: targeting new genes in regeneration.

G3 (Bethesda) 2013 Nov 6;3(11):1881-91. Epub 2013 Nov 6.

Department of Orthopaedic Surgery, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110.

Tissue regeneration is a complex trait with few genetic models available. Mouse strains LG/J and MRL are exceptional healers. Using recombinant inbred strains from a large (LG/J, healer) and small (SM/J, nonhealer) intercross, we have previously shown a positive genetic correlation between ear wound healing, knee cartilage regeneration, and protection from osteoarthritis. We hypothesize that a common set of genes operates in tissue healing and articular cartilage regeneration. Taking advantage of archived histological sections from recombinant inbred strains, we analyzed expression of candidate genes through branched-chain DNA technology directly from tissue lysates. We determined broad-sense heritability of candidates, Pearson correlation of candidates with healing phenotypes, and Ward minimum variance cluster analysis for strains. A bioinformatic assessment of allelic polymorphisms within and near candidate genes was also performed. The expression of several candidates was significantly heritable among strains. Although several genes correlated with both ear wound healing and cartilage healing at a marginal level, the expression of four genes representing DNA repair (Xrcc2, Pcna) and Wnt signaling (Axin2, Wnt16) pathways was significantly positively correlated with both phenotypes. Cluster analysis accurately classified healers and nonhealers for seven out of eight strains based on gene expression. Specific sequence differences between LG/J and SM/J were identified as potential causal polymorphisms. Our study suggests a common genetic basis between tissue healing and osteoarthritis susceptibility. Mapping genetic variations causing differences in diverse healing responses in multiple tissues may reveal generic healing processes in pursuit of new therapeutic targets designed to induce or enhance regeneration and, potentially, protection from osteoarthritis.
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http://dx.doi.org/10.1534/g3.113.007302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815053PMC
November 2013

The CMT4B disease-causing phosphatases Mtmr2 and Mtmr13 localize to the Schwann cell cytoplasm and endomembrane compartments, where they depend upon each other to achieve wild-type levels of protein expression.

Hum Mol Genet 2013 Apr 7;22(8):1493-506. Epub 2013 Jan 7.

The Jungers Center for Neurosciences Research, Department of Neurology, Oregon Health & Science University, Portland, OR 97239, USA.

The demyelinating peripheral neuropathy Charcot-Marie-Tooth type 4B (CMT4B) is characterized by axonal degeneration and myelin outfoldings. CMT4B results from mutations in either myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2), phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P2, lipids which regulate endo-lysosomal membrane traffic. The catalytically active MTMR2 and catalytically inactive MTMR13 physically associate, although the significance of this association is not well understood. Here we show that Mtmr13 loss leads to axonal degeneration in sciatic nerves of older mice. In addition, CMT4B2-like myelin outfoldings are present in Mtmr13(-/-) nerves at postnatal day 3. Thus, Mtmr13(-/-) mice show both the initial dysmyelination and later degenerative pathology of CMT4B2. Given the key role of PI 3-kinase-Akt signaling in myelination, we investigated the state of the pathway in nerves of CMT4B models. We found that Akt activation is unaltered in Mtmr13(-/-) and Mtmr2(-/-) mice. Mtmr2 and Mtmr13 are found within the Schwann cell cytoplasm, where the proteins are partially localized to punctate compartments, suggesting that Mtmr2-Mtmr13 may dephosphorylate their substrates on specific intracellular compartments. Mtmr2-Mtmr13 substrates play essential roles in endo-lysosomal membrane traffic. However, endosomes and lysosomes of Mtmr13(-/-) and Mtmr2(-/-) Schwann cells are morphologically indistinguishable from those of controls, indicating that loss of these proteins does not cause wholesale dysregulation of the endo-lysosomal system. Notably, Mtmr2 and Mtmr13 depend upon each other to achieve wild-type levels of protein expression. Mtmr2 stabilizes Mtmr13 on membranes, indicating that the Mtmr13 pseudophosphatase is regulated by its catalytically active binding partner.
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http://dx.doi.org/10.1093/hmg/dds562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605828PMC
April 2013

Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling.

Mol Biol Cell 2012 Jul 30;23(14):2723-40. Epub 2012 May 30.

Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0380, USA.

Cells rely on the coordinated regulation of lipid phosphoinositides and Rab GTPases to define membrane compartment fates along distinct trafficking routes. The family of disease-related myotubularin (MTM) phosphoinositide phosphatases includes catalytically inactive members, or pseudophosphatases, with poorly understood functions. We found that Drosophila MTM pseudophosphatase Sbf coordinates both phosphatidylinositol 3-phosphate (PI(3)P) turnover and Rab21 GTPase activation in an endosomal pathway that controls macrophage remodeling. Sbf dynamically interacts with class II phosphatidylinositol 3-kinase and stably recruits Mtm to promote turnover of a PI(3)P subpool essential for endosomal trafficking. Sbf also functions as a guanine nucleotide exchange factor that promotes Rab21 GTPase activation associated with PI(3)P endosomes. Of importance, Sbf, Mtm, and Rab21 function together, along with Rab11-mediated endosomal trafficking, to control macrophage protrusion formation. This identifies Sbf as a critical coordinator of PI(3)P and Rab21 regulation, which specifies an endosomal pathway and cortical control.
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http://dx.doi.org/10.1091/mbc.E12-05-0375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395661PMC
July 2012

Epigenetic integration of the developing brain and face.

Dev Dyn 2011 Oct 7;240(10):2233-44. Epub 2011 Sep 7.

McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, Alberta, Canada.

The integration of the brain and face and to what extent this relationship constrains or enables evolutionary change in the craniofacial complex is an issue of long-standing interest in vertebrate evolution. To investigate brain-face integration, we studied the covariation between the forebrain and midface at gestational days 10-10.5 in four strains of laboratory mice. We found that phenotypic variation in the forebrain is highly correlated with that of the face during face formation such that variation in the size of the forebrain correlates with the degree of prognathism and orientation of the facial prominences. This suggests strongly that the integration of the brain and face is relevant to the etiology of midfacial malformations such as orofacial clefts. This axis of integration also has important implications for the evolutionary developmental biology of the mammalian craniofacial complex.
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http://dx.doi.org/10.1002/dvdy.22729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246636PMC
October 2011

Estimating cell count and distribution in labeled histological samples using incremental cell search.

Int J Biomed Imaging 2011 24;2011:874702. Epub 2011 May 24.

Department of Computer Science, Faculty of Science, Memorial University of Newfoundland, St John's, NL, Canada A1B 3X5.

Cell proliferation is critical to the outgrowth of biological structures including the face and limbs. This cellular process has traditionally been studied via sequential histological sampling of these tissues. The length and tedium of traditional sampling is a major impediment to analyzing the large datasets required to accurately model cellular processes. Computerized cell localization and quantification is critical for high-throughput morphometric analysis of developing embryonic tissues. We have developed the Incremental Cell Search (ICS), a novel software tool that expedites the analysis of relationships between morphological outgrowth and cell proliferation in embryonic tissues. Based on an estimated average cell size and stain color, ICS rapidly indicates the approximate location and amount of cells in histological images of labeled embryonic tissue and provides estimates of cell counts in regions with saturated fluorescence and blurred cell boundaries. This capacity opens the door to high-throughput 3D and 4D quantitative analyses of developmental patterns.
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http://dx.doi.org/10.1155/2011/874702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124674PMC
July 2011

Engaging physicians in risk factor reduction.

Popul Health Manag 2010 Oct;13(5):255-61

OptumHealth Care Solutions , Scottsdale, AZ 85260, USA.

OptumHealth tested the feasibility of physician-directed population management in 3 primary care practices and with 546 continuously insured patients who exhibited claims markers for coronary artery disease, diabetes, and/or hypertension. During the intervention portion of the study, we asked physicians to improve the following health measurements: blood pressure, body mass index, cholesterol, hemoglobin A1c, and smoking status. We offered a modest pay-for-outcomes incentive for each risk factor improvement achieved. Additionally, on an eligible subset of these patients, we asked physicians to actively refer to population management programs those patients they determined could benefit from nurse or health coach interventions, advising us as to which components of their treatment plan they wished us to address. The 6-month intervention period exhibited a 10-fold improvement in the trend rate of risk factor management success when compared to the prior 6-month period for the same patients. A net of 96 distinct risk factor improvements were achieved by the 546 patients during the intervention period, whereas 9 net risk factor improvements occurred in the comparison period. This difference in improvement trends was statistically significant at P < 0.01. Of the 546 study participants, a subset of 187 members was eligible for participation in OptumHealth care management programs. Physicians identified 80 of these 187 eligible members as appropriate targets for program intervention. Representing ourselves as "calling on behalf" of the physician practices, we established contact with 50 referred members; 43 members (86%) actively enrolled in our programs. This enrollment rate is 2 to 3 times the rate of enrollment through our standard program outreach methods. We conclude that physician-directed population management with aligned incentives offers promise as a method of achieving important health and wellness goals.
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http://dx.doi.org/10.1089/pop.2009.0072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128448PMC
October 2010

Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development.

BMC Dev Biol 2010 Feb 17;10:18. Epub 2010 Feb 17.

Department of Cell Biology & Anatomy, The McCaig Bone and Joint Institute, and the Alberta Children's Hospital Institute for Child and Maternal Health, University of Calgary, Calgary, AB, Canada.

Background: Growing demand for three dimensional (3D) digital images of embryos for purposes of phenotypic assessment drives implementation of new histological and imaging techniques. Among these micro-computed tomography (microCT) has recently been utilized as an effective and practical method for generating images at resolutions permitting 3D quantitative analysis of gross morphological attributes of developing tissues and organs in embryonic mice. However, histological processing in preparation for microCT scanning induces changes in organ size and shape. Establishing normative expectations for experimentally induced changes in size and shape will be an important feature of 3D microCT-based phenotypic assessments, especially if quantifying differences in the values of those parameters between comparison sets of developing embryos is a primary aim. Toward that end, we assessed the nature and degree of morphological artifacts attending microCT scanning following use of common fixatives, using a two dimensional (2D) landmark geometric morphometric approach to track the accumulation of distortions affecting the embryonic head from the native, uterine state through to fixation and subsequent scanning.

Results: Bouin's fixation reduced average centroid sizes of embryonic mouse crania by approximately 30% and substantially altered the morphometric shape, as measured by the shift in Procrustes distance, from the unfixed state, after the data were normalized for naturally occurring shape variation. Subsequent microCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes. Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%. Changes in craniofacial shape progressively increased post-fixation.

Conclusions: The degree to which artifacts are introduced in the generation of random craniofacial shape variation relates to the degree of specimen dehydration during the initial fixation. Fixation methods that better maintain original craniofacial dimensions at reduced levels of dehydration and tissue shrinkage lead to the progressive accumulation of random shape variation during handling and data acquisition. In general, to the degree that embryonic organ size and shape factor into microCT-based phenotypic assessments, procedurally induced artifacts associated with fixation and scanning will influence results. Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.
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http://dx.doi.org/10.1186/1471-213X-10-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836989PMC
February 2010

Alveolar macrophages are necessary for the systemic inflammation of acute alveolar hypoxia.

J Appl Physiol (1985) 2007 Oct 26;103(4):1386-94. Epub 2007 Jul 26.

Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA.

Alveolar hypoxia (Fi(O(2)) 0.10) rapidly produces inflammation in the microcirculation of skeletal muscle, brain, and mesentery of rats. Dissociation between tissue Po(2) values and inflammation, plus the observation that plasma from hypoxic rats activates mast cells and elicits inflammation in normoxic tissues, suggest that the response to hypoxia is initiated when mast cells are activated by an agent released from a distant site and carried by the circulation. These experiments tested the hypothesis that this agent originates in alveolar macrophages (AM). Male rats were depleted of AM by tracheal instillation of clodronate-containing liposomes. Four days after treatment, AM recovered by bronchoalveolar lavage were <10% of control. Control rats received buffer-containing liposomes. As expected, alveolar hypoxia (Fi(O(2)) 0.10) in control rats increased leukocyte-endothelial adherence, produced degranulation of perivascular mast cells, and increased fluorescent albumin extravasation in the cremaster microcirculation. None of these effects was seen when AM-depleted rats were exposed to hypoxia. Plasma obtained from control rats after 5 min of breathing 10% O(2) elicited inflammation when applied to normoxic cremasters. In contrast, normoxic cremasters did not develop inflammation after application of plasma from hypoxic AM-depleted rats. Supernatant from AM cultured in 10% O(2) produced increased leukocyte-endothelial adherence, vasoconstriction, and albumin extravasation when applied to normoxic cremasters. Normoxic AM supernatant did not produce any of these responses. The effects of hypoxic supernatant were attenuated by pretreatment of the cremaster with the mast cell stabilizer cromolyn. These data support the hypothesis that AM are the source of the agent that initiates hypoxia-induced systemic inflammation by activating mast cells.
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http://dx.doi.org/10.1152/japplphysiol.00312.2007DOI Listing
October 2007

A new time-scale for ray-finned fish evolution.

Proc Biol Sci 2007 Feb;274(1609):489-98

Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637, USA.

The Actinopterygii (ray-finned fishes) is the largest and most diverse vertebrate group, but little is agreed about the timing of its early evolution. Estimates using mitochondrial genomic data suggest that the major actinopterygian clades are much older than divergence dates implied by fossils. Here, the timing of the evolutionary origins of these clades is reinvestigated using morphological, and nuclear and mitochondrial genetic data. Results indicate that existing fossil-based estimates of the age of the crown-group Neopterygii, including the teleosts, Lepisosteus (gar) and Amia (bowfin), are at least 40 Myr too young. We present new palaeontological evidence that the neopterygian crown radiation is a Palaeozoic event, and demonstrate that conflicts between molecular and morphological data for the age of the Neopterygii result, in part, from missing fossil data. Although our molecular data also provide an older age estimate for the teleost crown, this range extension remains unsupported by the fossil evidence. Nuclear data from all relevant clades are used to demonstrate that the actinopterygian whole-genome duplication event is teleost-specific. While the date estimate of this event overlaps the probable range of the teleost stem group, a correlation between the genome duplication and the large-scale pattern of actinopterygian phylogeny remains elusive.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1766393PMC
http://dx.doi.org/10.1098/rspb.2006.3749DOI Listing
February 2007

Role of the renin-angiotensin system in the systemic microvascular inflammation of alveolar hypoxia.

Am J Physiol Heart Circ Physiol 2007 May 5;292(5):H2285-94. Epub 2007 Jan 5.

Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.

Alveolar hypoxia (AH) induces widespread systemic inflammation. Previous studies have shown dissociation between microvascular Po(2) and inflammation. Furthermore, plasma from AH rats (PAHR) induces mast cell (MC) activation, inflammation, and vasoconstriction in normoxic cremasters, while plasma from normoxic rats does not produce these responses. These results suggest that inflammation of AH is triggered by a blood-carried agent. This study investigated the involvement of the renin-angiotensin system (RAS) in the inflammation of AH. Both an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin II (ANG II) receptor blocker (ANG II RB) inhibited the leukocyte-endothelial adherence produced by AH, as well as the inflammation produced by PAHR in normoxic rat cremasters. MC stabilization with cromolyn blocked the effects of PAHR but not those of topical ANG II on normoxic cremasters, suggesting ANG II generation via MC activation by PAHR. This was supported by the observation that ACE inhibition and ANG II RB blocked the leukocyte-endothelial adherence produced by the MC secretagogue compound 48/80. These results suggest that the intermediary agent contained in PAHR activates MC and stimulates the RAS, leading to inflammation, and imply an RAS role in AH-induced inflammation.
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http://dx.doi.org/10.1152/ajpheart.00981.2006DOI Listing
May 2007
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