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1.  Australian Cattle Dogs with Neuronal Ceroid Lipofuscinosis are Homozygous for a CLN5 Nonsense Mutation Previously Identified in Border Collies 
Background
Neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease, has been diagnosed in young adult Australian Cattle Dogs.
Objective
Characterize the Australian Cattle Dog form of NCL and determine its molecular genetic cause.
Animals
Tissues from 4 Australian Cattle Dogs with NCL‐like signs and buccal swabs from both parents of a fifth affected breed member. Archived DNA samples from 712 individual dogs were genotyped.
Methods
Tissues were examined by fluorescence, electron, and immunohistochemical microscopy. A whole‐genome sequence was generated for 1 affected dog. A TaqMan allelic discrimination assay was used for genotyping.
Results
The accumulation of autofluorescent cytoplasmic storage material with characteristic ultrastructure in tissues from the 4 affected dogs supported a diagnosis of NCL. The whole‐genome sequence contained a homozygous nonsense mutation: CLN5:c.619C>T. All 4 DNA samples from clinically affected dogs tested homozygous for the variant allele. Both parents of the fifth affected dog were heterozygotes. Archived DNA samples from 346 Australian Cattle Dogs, 188 Border Collies, and 177 dogs of other breeds were homozygous for the reference allele. One archived Australian Cattle Dog sample was from a heterozygote.
Conclusions and Clinical Importance
The homozygous CLN5 nonsense is almost certainly causal because the same mutation previously had been reported to cause a similar form of NCL in Border Collies. Identification of the molecular genetic cause of Australian Cattle Dog NCL will allow the use of DNA tests to confirm the diagnosis of NCL in this breed.
doi:10.1111/jvim.13971
PMCID: PMC5084771  PMID: 27203721
Autofluorescence; Lysosomal storage disease; Molecular genetics; Whole‐genome sequence
2.  Degenerative Encephalopathy in Nova Scotia Duck Tolling Retrievers Presenting with a Rapid Eye Movement Sleep Behavior Disorder 
Background
Neurodegenerative diseases are a heterogeneous group of disorders characterized by loss of neurons and are commonly associated with a genetic mutation.
Hypothesis/Objectives
To characterize the clinical and histopathological features of a novel degenerative neurological disease affecting the brain of young adult Nova Scotia Duck Tolling Retrievers (NSDTRs).
Animals
Nine, young adult, related NSDTRs were evaluated for neurological dysfunction and rapid eye movement sleep behavior disorder.
Methods
Case series review.
Results
Clinical signs of neurological dysfunction began between 2 months and 5 years of age and were progressive in nature. They were characterized by episodes of marked movements during sleep, increased anxiety, noise phobia, and gait abnormalities. Magnetic resonance imaging documented symmetrical, progressively increasing, T2‐weighted image intensity, predominantly within the caudate nuclei, consistent with necrosis secondary to gray matter degeneration. Abnormalities were not detected on clinicopathological analysis of blood and cerebrospinal fluid, infectious disease screening or urine metabolite screening in most cases. Postmortem examination of brain tissue identified symmetrical malacia of the caudate nuclei and axonal dystrophy within the brainstem and spinal cord. Genealogical analysis supports an autosomal recessive mode of inheritance.
Conclusions and Clinical Importance
A degenerative encephalopathy was identified in young adult NSDTRs consistent with a hereditary disease. The prognosis is guarded due to the progressive nature of the disease, which is minimally responsive to empirical treatment.
doi:10.1111/jvim.14575
PMCID: PMC5032881  PMID: 27717189
Axonopathy; Basal nuclei; Behavioral changes; Familial
3.  Clinical Characteristics and Outcome in Dogs with Splenic Marginal Zone Lymphoma 
Background
Splenic marginal zone lymphoma (MZL) is a form of indolent B-cell lymphoma that is not well characterized in dogs.
Hypothesis/Objectives
The purpose of this study was to describe clinical characteristics and outcome in dogs with splenic MZL confirmed by histopathology, immunophenotyping, and molecular clonality assessment. We hypothesized that affected dogs would have prolonged survival time with splenectomy alone.
Animals
Thirty-four dogs were included. Twenty-nine dogs were diagnosed after splenectomy, and 5 dogs were diagnosed at necropsy.
Methods
Pathology records were searched for dogs with histologically confirmed splenic MZL. Clinical and outcome data were retrospectively collected by medical record review, and prognostic factors were evaluated. Histopathology was reviewed by a board-certified pathologist, and tissue sections were subjected to immunophenotyping and molecular clonality assessment by PCR.
Results
Immunohistochemistry confirmed a B-cell phenotype for all dogs. Molecular clonality assessment was performed in 33 of 34 dogs, of which 24 had clonal rearrangement of immunoglobulin (Ig) loci, 3 had pseudoclonal rearrangement, and 6 had polyclonal rearrangement. The overall median survival time (MST) for the 29 dogs that underwent splenectomy was 383 days. The MST for 14 of 29 asymptomatic dogs that underwent splenectomy for MZL was 1,153 days as compared to 309 days for 15/29 dogs with clinical signs referable to splenic MZL (P = .018). Lymph node involvement, hemoabdomen, anemia, chemotherapy, and concurrent malignancy did not affect survival outcome.
Conclusions and Clinical Importance
Dogs diagnosed with splenic MZL can have prolonged survival with splenectomy alone, without the use of adjuvant chemotherapy. Asymptomatic dogs may have a better survival outcome.
doi:10.1111/jvim.12116
PMCID: PMC5012422  PMID: 23734665
Canine; Indolent lymphoma; Neoplasia
4.  Degenerative Encephalopathy in Nova Scotia Duck Tolling Retrievers Presenting with a Rapid Eye Movement Sleep Behavior Disorder 
Background
Neurodegenerative diseases are a heterogeneous group of disorders characterized by loss of neurons and are commonly associated with a genetic mutation.
Hypothesis/Objectives
To characterize the clinical and histopathological features of a novel degenerative neurological disease affecting the brain of young adult Nova Scotia Duck Tolling Retrievers (NSDTRs).
Animals
Nine, young adult, related NSDTRs were evaluated for neurological dysfunction and rapid eye movement sleep behavior disorder.
Methods
Case series review.
Results
Clinical signs of neurological dysfunction began between 2 months and 5 years of age and were progressive in nature. They were characterized by episodes of marked movements during sleep, increased anxiety, noise phobia, and gait abnormalities. Magnetic resonance imaging documented symmetrical, progressively increasing, T2‐weighted image intensity, predominantly within the caudate nuclei, consistent with necrosis secondary to gray matter degeneration. Abnormalities were not detected on clinicopathological analysis of blood and cerebrospinal fluid, infectious disease screening or urine metabolite screening in most cases. Postmortem examination of brain tissue identified symmetrical malacia of the caudate nuclei and axonal dystrophy within the brainstem and spinal cord. Genealogical analysis supports an autosomal recessive mode of inheritance.
Conclusions and Clinical Importance
A degenerative encephalopathy was identified in young adult NSDTRs consistent with a hereditary disease. The prognosis is guarded due to the progressive nature of the disease, which is minimally responsive to empirical treatment.
doi:10.1111/jvim.14575
PMCID: PMC5032881  PMID: 27717189
Axonopathy; Basal nuclei; Behavioral changes; Familial
5.  The missing large impact craters on Ceres 
Nature Communications  2016;7:12257.
Asteroids provide fundamental clues to the formation and evolution of planetesimals. Collisional models based on the depletion of the primordial main belt of asteroids predict 10–15 craters >400 km should have formed on Ceres, the largest object between Mars and Jupiter, over the last 4.55 Gyr. Likewise, an extrapolation from the asteroid Vesta would require at least 6–7 such basins. However, Ceres' surface appears devoid of impact craters >∼280 km. Here, we show a significant depletion of cerean craters down to 100–150 km in diameter. The overall scarcity of recognizable large craters is incompatible with collisional models, even in the case of a late implantation of Ceres in the main belt, a possibility raised by the presence of ammoniated phyllosilicates. Our results indicate that a significant population of large craters has been obliterated, implying that long-wavelength topography viscously relaxed or that Ceres experienced protracted widespread resurfacing.
Studying craters on atmosphere-less bodies can unlock information about planetesimal histories. Here, Marchi et al. present results from the NASA Dawn mission to Ceres showing that craters >100–150 km in size are largely absent, and find that Ceres' internal evolution is responsible for their absence.
doi:10.1038/ncomms12257
PMCID: PMC4963536  PMID: 27459197
6.  Identification of Candidate Genes for Prostate Cancer Risk-SNPs Utilizing a Normal Prostate Tissue eQTL Dataset 
Nature communications  2015;6:8653.
Multiple studies have identified loci associated with risk of developing prostate cancer but the associated genes are not well studied. Here we create a normal prostate tissue-specific eQTL dataset and then apply this dataset to a set of previously identified prostate cancer (PrCa) risk-SNPs in an effort to identify candidate target genes. The eQTL dataset was constructed by genotyping and RNA sequencing of 471 samples. We focus on identifying eQTLs for 146 PrCa risk-SNPs, including all SNPs in linkage disequilibrium (LD) with each risk-SNP, resulting in 100 unique risk-intervals. We further focus only on cis-acting associations where the transcript is located within 2Mb (+/-1Mb) of the risk-SNP interval. Of all SNP - gene combinations tested, 41.7% of SNPs demonstrate a significant eQTL signal after adjustment for sample histology and 14 expression principal component covariates. Of the 100 PrCa risk-intervals, 51 (51%) have a significant eQTL signal and these are associated with 88 genes. This study provides a rich resource to study biological mechanisms underlying genetic risk to prostate cancer.
doi:10.1038/ncomms9653
PMCID: PMC4663677  PMID: 26611117
7.  A Homozygous RAB3GAP1:c.743delC Mutation in Rottweilers with Neuronal Vacuolation and Spinocerebellar Degeneration 
Background
A variety of presumed hereditary, neurologic diseases have been reported in young Rottweilers. Overlapping ages of onset and clinical signs have made antemortem diagnosis difficult. One of these diseases, neuronal vacuolation and spinocerebellar degeneration (NVSD) shares clinical and histological features with polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV), a recently described hereditary disease in Black Russian Terriers (BRTs). Dogs with POANV harbor mutations in RAB3GAP1 which codes for a protein involved in membrane trafficking.
Hypothesis
Rottweilers with NVSD will be homozygous for the RAB3GAP1:c.743delC allele associated with POANV in BRTs.
Animals
Eight Rottweilers with NVSD confirmed at necropsy, 128 Rottweilers without early onset neurologic signs, and 468 randomly selected dogs from 169 other breeds.
Methods
Retrospective case–control study. Dogs were genotyped for the RAB3GAP1:c.743delC allele with an allelic discrimination assay.
Results
All 8 NVSD‐affected dogs were homozygous for the RAB3GAP1:c.743delC allele while the 128 NVSD‐free Rottweilers were either homozygous for the reference allele (n = 105) or heterozygous (n = 23) and the 468 genotyped dogs from other breeds were all homozygous for the reference allele.
Conclusions and Clinical Importance
The RAB3GAP1:c.743delC mutation is associated with a similar phenotype in Rottweilers and BRTs. Identification of the mutation permits a DNA test that can aid in the diagnosis of NVSD and identify carriers of the trait so that breeders can avoid producing affected dogs. Disruption of membrane trafficking could explain the neuronal vacuolation seen in NVSD and other spongiform encephalopathies.
doi:10.1111/jvim.13921
PMCID: PMC4913561  PMID: 26968732
Canine; Molecular genetics; Peripheral nervous system disorders; Rab GTPase; Spongiform encephalopathies; Warburg micro syndrome
8.  A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study 
Leukemia  2016;30(4):929-936.
In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.
doi:10.1038/leu.2015.313
PMCID: PMC4832072  PMID: 26639181
9.  Molecular Profiling Reveals Prognostically Significant Subtypes of Canine Lymphoma 
Veterinary pathology  2012;50(4):693-703.
We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.
doi:10.1177/0300985812465325
PMCID: PMC4683027  PMID: 23125145
canine; lymphoma subclassification; gene expression profiling; prognostication
10.  Identification of candidate genes for prostate cancer-risk SNPs utilizing a normal prostate tissue eQTL data set 
Nature Communications  2015;6:8653.
Multiple studies have identified loci associated with the risk of developing prostate cancer but the associated genes are not well studied. Here we create a normal prostate tissue-specific eQTL data set and apply this data set to previously identified prostate cancer (PrCa)-risk SNPs in an effort to identify candidate target genes. The eQTL data set is constructed by the genotyping and RNA sequencing of 471 samples. We focus on 146 PrCa-risk SNPs, including all SNPs in linkage disequilibrium with each risk SNP, resulting in 100 unique risk intervals. We analyse cis-acting associations where the transcript is located within 2 Mb (±1 Mb) of the risk SNP interval. Of all SNP–gene combinations tested, 41.7% of SNPs demonstrate a significant eQTL signal after adjustment for sample histology and 14 expression principal component covariates. Of the 100 PrCa-risk intervals, 51 have a significant eQTL signal and these are associated with 88 genes. This study provides a rich resource to study biological mechanisms underlying genetic risk to PrCa.
Single nucleotide polymorphisms—SNPs—have been identified for prostate cancer but whether these SNPs alter the expression of genes is largely unknown. In this study, the authors search for genes located within 2 Mb of the SNPs and identify SNPs that influence gene expression, so called expression quantitative trait loci.
doi:10.1038/ncomms9653
PMCID: PMC4663677  PMID: 26611117
11.  Protective Efficacy and Pharmacokinetics of Human/Mouse Chimeric Anti-Stx1 and Anti-Stx2 Antibodies in Mice 
In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.
doi:10.1128/CVI.00022-15
PMCID: PMC4375342  PMID: 25716230
12.  Helmet-Cam: tool for assessing miners’ respirable dust exposure 
Mining engineering  2013;65(9):78-84.
Video technology coupled with datalogging exposure monitors have been used to evaluate worker exposure to different types of contaminants. However, previous application of this technology used a stationary video camera to record the worker’s activity while the worker wore some type of contaminant monitor. These techniques are not applicable to mobile workers in the mining industry because of their need to move around the operation while performing their duties. The Helmet-Cam is a recently developed exposure assessment tool that integrates a person-wearable video recorder with a datalogging dust monitor. These are worn by the miner in a backpack, safety belt or safety vest to identify areas or job tasks of elevated exposure. After a miner performs his or her job while wearing the unit, the video and dust exposure data files are downloaded to a computer and then merged together through a NIOSH-developed computer software program called Enhanced Video Analysis of Dust Exposure (EVADE). By providing synchronized playback of the merged video footage and dust exposure data, the EVADE software allows for the assessment and identification of key work areas and processes, as well as work tasks that significantly impact a worker’s personal respirable dust exposure. The Helmet-Cam technology has been tested at a number of metal/nonmetal mining operations and has proven to be a valuable assessment tool. Mining companies wishing to use this technique can purchase a commercially available video camera and an instantaneous dust monitor to obtain the necessary data, and the NIOSH-developed EVADE software will be available for download at no cost on the NIOSH website.
PMCID: PMC4570878  PMID: 26380529
13.  Enhanced Virulence of the Escherichia coli O157:H7 Spinach-Associated Outbreak Strain in Two Animal Models Is Associated with Higher Levels of Stx2 Production after Induction with Ciprofloxacin 
Infection and Immunity  2014;82(12):4968-4977.
Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains both stx2a and stx2c. We hypothesized that the enhanced virulence of K3995 reflects the combination of stx2 alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of the stx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a+ Stx2a+) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995 stx2a::cat were not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a+). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.
doi:10.1128/IAI.02361-14
PMCID: PMC4249278  PMID: 25225244
14.  Severely Fibrotic Pancreases from Young Patients with Chronic Pancreatitis: Evidence for a Ductal Origin of Islet Neogenesis 
Acta diabetologica  2011;50(5):807-814.
While it is known that islet cell mass increases considerably after birth, general uncertainty surrounds the source of new beta cells in humans. Chronic pancreatitis (CP) presents a natural injury model for studying postnatal beta-cell regeneration in the human pancreas. In this report, we present histological evidence from human CP pancreases to support the theory that islet neogenesis can occur from ductal precursor cells after birth. Three young patients (ages 16, 12, and 28 years) underwent total pancreatectomy for the management of CP followed by islet isolation and autologous transplantation to prevent or minimize post-surgical diabetes. In all cases, the pancreases had extensive fibrosis, a rock-like consistency, and calcifications in the ducts. During islet isolations, we observed the unusual release of islets with many ductal fragments. In histopathological evaluation of these pancreases, solid cords of cells sometimes formed islet like structures intraductally or extending from ductal structures. Immunofluorescence staining for chromogranin, insulin, proinsulin, PDX1, glucagon and cytokeratins confirmed these structures to be composed of chromogranin-positive endocrine cells which included both β-cells and α-cells. Labeling for Ki67 to demonstrate mitotic activity showed frequent labeling of duct epithelial cells and of some periductal cells. Using insulin and wide-spectrum cytokeratin double-immunofluorescent labeling, we found insulin positive cells to be present within the ductal lumens, among the cytokeratin positive ductal epithelium, and extending from the ductal epithelium into surrounding connective tissues, providing evidence for a ductal origin of islet neogenesis.
doi:10.1007/s00592-011-0306-9
PMCID: PMC4124082  PMID: 21773756
islet neogenesis; chronic pancreatitis; islet precursor cell; beta cell; islet progenitor cell
15.  Oral Intoxication of Mice with Shiga Toxin Type 2a (Stx2a) and Protection by Anti-Stx2a Monoclonal Antibody 11E10 
Infection and Immunity  2014;82(3):1213-1221.
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB5 toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD50) of 2.9 μg, but no morbidity occurred after oral intoxication with up to 157 μg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.
doi:10.1128/IAI.01264-13
PMCID: PMC3957995  PMID: 24379294
16.  DNA Testing in Neurologic Diseases 
DNA testing is available for a growing number of hereditary diseases in neurology and other specialties. In addition to guiding breeding decisions, DNA tests are important tools in the diagnosis of diseases, particularly in conditions for which clinical signs are relatively nonspecific. DNA testing also can provide valuable insight into the risk of hereditary disease when decisions about treating comorbidities are being made. Advances in technology and bioinformatics will make broad screening for potential disease‐causing mutations available soon. As DNA tests come into more common use, it is critical that clinicians understand the proper application and interpretation of these test results.
doi:10.1111/jvim.12383
PMCID: PMC4857950  PMID: 24962505
Genetic mapping; Genetic markers; Genetics; Neurology
17.  A Homozygous KCNJ10 Mutation in Jack Russell Terriers and Related Breeds with Spinocerebellar Ataxia with Myokymia, Seizures, or Both 
Background
Juvenile‐onset spinocerebellar ataxia has been recognized in Jack Russell Terriers and related Russell group terriers (RGTs) for over 40 years. Ataxia occurs with varying combinations of myokymia, seizures, and other signs of neurologic disease. More than 1 form of the disease has been suspected.
Hypothesis/Objectives
The objective was to identify the mutation causing the spinocerebellar ataxia associated with myokymia, seizures, or both and distinguish the phenotype from other ataxias in the RGTs.
Animals
DNA samples from 16 RGTs with spinocerebellar ataxia beginning from 2 to 12 months of age, 640 control RGTs, and 383 dogs from 144 other breeds along with the medical records of affected dogs were studied.
Methods
This case‐control study compared the frequencies of a KCNJ10 allele in RGTs with spinocerebellar ataxia versus control RGTs. This allele was identified in a whole‐genome sequence of a single RGT with spinocerebellar ataxia and myokymia by comparison to whole‐genome sequences from 81 other canids that were normal or had other diseases.
Results
A missense mutation in the gene coding for the inwardly rectifying potassium channel Kir4.1 (KCNJ10:c.627C>G) was significantly (P < .001) associated with the disease. Dogs homozygous for the mutant allele all had spinocerebellar ataxia with varying combinations of myokymia and seizures.
Conclusions and Clinical Importance
Identification of the KCNJ10 mutation in dogs with spinocerebellar ataxia with myokymia, seizures, or both clarifies the multiple forms of ataxia seen in these breeds and provides a DNA test to identify carriers.
doi:10.1111/jvim.12355
PMCID: PMC4238845  PMID: 24708069
Epilepsy; Kir4.1; Peripheral nerve hyperexcitability; Potassium channel
18.  Breed Distribution of SOD1 Alleles Previously Associated with Canine Degenerative Myelopathy 
Background
Previous reports associated 2 mutant SOD1 alleles (SOD1:c.118A and SOD1:c.52T) with degenerative myelopathy in 6 canine breeds. The distribution of these alleles in other breeds has not been reported.
Objective
To describe the distribution of SOD1:c.118A and SOD1:c.52T in 222 breeds.
Animals
DNA from 33,747 dogs was genotyped at SOD1:c.118,SOD1:c.52, or both. Spinal cord sections from 249 of these dogs were examined.
Methods
Retrospective analysis of 35,359 previously determined genotypes at SOD1:c.118G>A or SOD1:c.52A>T and prospective survey to update the clinical status of a subset of dogs from which samples were obtained with a relatively low ascertainment bias.
Results
The SOD1:c.118A allele was found in cross‐bred dogs and in 124 different canine breeds whereas the SOD1:c.52T allele was only found in Bernese Mountain Dogs. Most of the dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A homozygotes, but 8 dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A/G heterozygotes and had no other sequence variants in their SOD1 amino acid coding regions. The updated clinical conditions of dogs from which samples were obtained with a relatively low ascertainment bias suggest that SOD1:c.118A homozygotes are at a much higher risk of developing degenerative myelopathy than are SOD1:c.118A/G heterozygotes.
Conclusions and Clinical Importance
We conclude that the SOD1:c.118A allele is widespread and common among privately owned dogs whereas the SOD1:c.52T allele is rare and appears to be limited to Bernese Mountain Dogs. We also conclude that breeding to avoid the production of SOD1:c.118A homozygotes is a rational strategy.
doi:10.1111/jvim.12317
PMCID: PMC4238831  PMID: 24524809
Amyotrophic lateral sclerosis; Cytoplasmic aggregates; DNA test; Spinal cord
19.  Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia 
Journal of Medical Microbiology  2012;61(Pt 10):1380-1392.
Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ΔAmes strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.
doi:10.1099/jmm.0.045393-0
PMCID: PMC3541767  PMID: 22767539
20.  Surgical Protocol Involving the Infusion of Paramagnetic Microparticles for Preferential Incorporation within Porcine Islets 
Transplantation proceedings  2010;42(10):4209-4212.
Introduction
Despite significant advances, widespread applicability of islet cell transplantation (ICT) remains elusive. Refinement of current islet isolation protocols may improve ICT outcomes. Islet purification by magnetic separation (MS) has shown early promise. However, surgical protocols must be optimized to maximize the incorporation of paramagnetic microparticles (MP) within a greater number of islets. The objective of this study is to explore the impact of MP concentration and infusion method on optimizing MP incorporation within islets.
Methods
Five porcine pancreata were procured from donors following cardiac death. Splenic lobes were isolated and infused with varying concentrations of MP (8, 16 and 32 × 108 MP/L of cold preservation solution) and using one of two delivery techniques (hanging bag versus hand-syringe). Following procurement and infusion, pancreata were stored at 0–4°C during transportation (< 1 hour), fixed in 10% buffered formalin and examined by standard MRI and histopathology.
Results
T2*-weighted MRI illustrated homogeneous distribution of MP in all experimental splenic lobes. In addition, histologic analysis confirmed that MP were primarily located within the microvasculature of islets (82–85%), with few MP present in acinar tissue (15–18%), with an average of 5–7 MP per islet (within a 5 μm thick section). The highest MP incorporation was achieved at a concentration of 16 × 108 MP/L using the hand-syringe technique.
Conclusion
This preliminary study suggests that optimization of a surgical protocol, MP concentrations and applied infusion pressures may enable more uniform distribution of MP in the porcine pancreas and better control of MP incorporation within islets. These results may have implications in maximizing the efficacy of islet purification by MS.
doi:10.1016/j.transproceed.2010.09.138
PMCID: PMC3035915  PMID: 21168666
21.  Speriolin is a novel human and mouse sperm centrosome protein 
Human Reproduction (Oxford, England)  2010;25(8):1884-1894.
BACKGROUND
Oocytes in humans, mice and other mammals lack identifiable centrioles. The proximal centriole brought in by the fertilizing sperm in humans and most other mammals appears to gives rise to the centrioles at the spindle poles in the zygote, and is believed to indicate that centrioles are inherited through the paternal lineage. However, both the proximal and distal sperm centrioles degenerate in mice and other rodents. A bipolar mitotic spindle nucleates from multiple centrosome-like structures in the mouse zygote and centrioles are not seen until the blastocyst stage, suggesting that centrioles are inherited through the maternal lineage in mice. We previously identified speriolin as a spermatogenic cell-specific binding partner of Cdc20 that co-localizes with pericentrin in mouse spermatocytes and is present in the centrosome in round spermatids.
METHODS
The nature and localization of speriolin in mouse and human sperm and the fate of speriolin following fertilization in the mouse were determined using immunofluorescence microscopy, immunoelectron microscopy and western blotting.
RESULTS
Speriolin surrounds the intact proximal centriole in human sperm, but is localized at the periphery of the disordered distal centriole in mouse sperm. Human speriolin contains an internal 163-amino acid region not present in mouse that may contribute to localization differences. Speriolin is carried into the mouse oocyte during fertilization and remains associated with the decondensing sperm head in zygotes. The speriolin spot appears to undergo duplication or splitting during the first interphase and is detectable in 2-cell embryos.
CONCLUSIONS
Speriolin is a novel centrosomal protein present in the connecting piece region of mouse and human sperm that is transmitted to the mouse zygote and can be detected throughout the first mitotic division.
doi:10.1093/humrep/deq138
PMCID: PMC2907228  PMID: 20542897
centriole; flagellum; fertilization; paternal inheritance; zygote
22.  Rapid Quantitative Assessment of the Pig Pancreas Biopsy Predicts Islet Yield 
Transplantation proceedings  2010;42(6):2036-2039.
Background
The cost of islet procurement from donor pigs is increased by the use of organs that produce low yields. We developed an assessment system using dithizone-stained pig pancreas biopsies to enable the preselection of donor organs.
Methods
Pig pancreas biopsy slices were soaked in dithizone solution. The islets were evaluated before islet isolation by converting the islet counts (IC) to islet equivalents (IE), then determining the IE/cm2, IE/IC, % >150 μm islets, and % >200 μm islets. These parameters were evaluated in 3 different areas of pancreas (duodenal, splenic, and connecting lobe; n = 42 each). Stepwise multivariate linear regression analysis was performed to assess for correlations with islet yield and decide which area of the pancreas had the most predictive value. To identify other predictors, including donor and islet isolation variables, we performed binary logistic regression analysis with significant variables from the univariate analysis (n = 67). For this analysis, the pigs were categorized into high (n = 23) and low (n = 44) yield groups.
Results
Stepwise multivariate linear regression analysis revealed that IE/cm2 of the splenic lobe significantly predicted islet yield. Binary logistic regression analysis indicated that the IE/mm2 of the splenic lobe was the only parameter that significantly correlated with successful pig islet isolations (P = .01; odds ratio 3.605). Variables associated with donor and islet isolation, such as age, gender, ischemic time, or enzyme lot, were not significantly correlated with islet yield.
Conclusion
Our study suggests that islet distribution of splenic lobe biopsies can be a reliable predictor of islet yield from pig pancreata.
doi:10.1016/j.transproceed.2010.05.113
PMCID: PMC2922853  PMID: 20692401
23.  Persufflation Improves Pancreas Preservation When Compared With the Two-Layer Method 
Transplantation proceedings  2010;42(6):2016-2019.
Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the high dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in lower islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from non–heart-beating donors and preserved by either TLM or persufflation for 24 hours and fixed. Biopsies were collected from several regions of the pancreas, sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries due to gas perfusion and significantly less autolysis/cell death than regions not exposed to persufflation or tissues exposed to TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.
doi:10.1016/j.transproceed.2010.05.092
PMCID: PMC2956134  PMID: 20692396
25.  Schwann cell invasion of the conus medullaris: case report 
European Spine Journal  2002;12(3):328-331.
Abstract.
As Schwann cells possess regenerative capabilities there is intense interest concerning their role in central nervous system (CNS) regeneration. We report on a case of an intramedullary schwannoma involving the conus medullaris and spinal cord above it. We discuss the possible origin of these cells and the mechanisms by which these cells may invade the CNS. We offer imaging and discuss experimental studies to support our hypothesis. This case concerns a 48-year-old man, who presented with a 6-month history of bilateral lower extremity weakness. Magnetic resonance imaging (MRI) revealed an intramedullary tumour extending from the conus to T11. At operation, following laminectomy and durotomy, a schwannoma was dissected free from the conus. Total gross resection of tumour was achieved. The patient made an uneventful and full recovery. This case shows that Schwann cells can invade the CNS. Manipulation of the transitional zone astrocytic barrier may offer a potential avenue for Schwann cells to enter the CNS in pathological states.
doi:10.1007/s00586-002-0484-9
PMCID: PMC3615487  PMID: 12800008
CNS regeneration Intramedullary schwannoma Transitional zone

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