A homozygous ADAMTS2 nonsense mutation in a Doberman Pinscher dog with Ehlers Danlos syndrome and extreme skin fragility.

An eight-week old Doberman Pinscher was diagnosed with Ehlers Danlos syndrome based on the dog's hyper-mobile carpal, tarsal and stifle joints and abnormal skin. The skin was loose and hyper-elastic with several wounds and large atrophic scars. The dog was euthanized after a severe degloving injury from minimal trauma. A whole-genome sequence, generated with DNA from the dog's blood, contained a rare, homozygous C-to-T transition at position 2408978 on chromosome 11. This transition is predicted to alter the ADAMTS2 transcript (ADAMTS2:c.769C>T) and encode a nonsense mutation (p.Arg257Ter). Biallelic ADAMTS2 mutations have caused a type of Ehlers Danlos syndrome known as dermatosparaxis in other species.

Golden Retriever dogs with neuronal ceroid lipofuscinosis have a two-base-pair deletion and frameshift in CLN5.

We studied a recessive, progressive neurodegenerative disease occurring in Golden Retriever siblings with an onset of signs at 15 months of age. As the disease progressed these signs included ataxia, anxiety, pacing and circling, tremors, aggression, visual impairment and localized and generalized seizures. A whole genome sequence, generated with DNA from one affected dog, contained a plausibly causal homozygous mutation: CLN5:c.934_935delAG. This mutation was predicted to produce a frameshift and premature termination codon and encode a protein variant, CLN5:p.E312Vfs*6, which would lack 39 C-terminal amino acids. Eighteen DNA samples from the Golden Retriever family members were genotyped at CLN5:c.934_935delAG. Three clinically affected dogs were homozygous for the deletion allele; whereas, the clinically normal family members were either heterozygotes (n = 11) or homozygous for the reference allele (n = 4). Among archived Golden Retrievers DNA samples with incomplete clinical records that were also genotyped at the CLN5:c.934_935delAG variant, 1053 of 1062 were homozygous for the reference allele, 8 were heterozygotes and one was a deletion-allele homozygote. When contacted, the owner of this homozygote indicated that their dog had been euthanized because of a neurologic disease that progressed similarly to that of the affected Golden Retriever siblings. We have collected and stored semen from a heterozygous Golden Retriever, thereby preserving an opportunity for us or others to establish a colony of CLN5-deficient dogs.

Cascaded all-optical operations in a hybrid integrated 80-Gb/s logic circuit.

We demonstrate logic functionalities in a high-speed all-optical logic circuit based on differential Mach-Zehnder interferometers with semiconductor optical amplifiers as the nonlinear optical elements. The circuit, implemented by hybrid integration of the semiconductor optical amplifiers on a planar lightwave circuit platform fabricated in silica glass, can be flexibly configured to realize a variety of Boolean logic gates. We present both simulations and experimental demonstrations of cascaded all-optical operations for 80-Gb/s on-off keyed data.

Characterization of neuronal ceroid-lipofuscinosis in 3 cats.

Three young domestic shorthair cats were presented for necropsy with similar histories of slowly progressive visual dysfunction and neurologic deficits. Macroscopic examination of each cat revealed cerebral and cerebellar atrophy, dilated lateral ventricles, and slight brown discoloration of the gray matter. Histologically, there was bilateral loss of neurons within the limbic, motor, somatosensory, visual, and, to a lesser extent, vestibular systems with extensive astrogliosis in the affected regions of all 3 cases. Many remaining neurons and glial cells throughout the entire central nervous system were distended by pale yellow to eosinophilic, autofluorescent cytoplasmic inclusions with ultrastructural appearances typical of neuronal ceroid-lipofuscinoses (NCLs). Differences in clinical presentation and neurological lesions suggest that the 3 cats may have had different variants of NCL. Molecular genetic characterization in the 1 cat from which DNA was available did not reveal any plausible disease-causing mutations of the CLN1 (PPT1), CLN3, CLN5, CLN8, and CLN10 (CTSD) genes. Further investigations will be required to identify the mutations responsible for NCLs in cats.

Public health approach to detection of non-O157 Shiga toxin-producing Escherichia coli: summary of two outbreaks and laboratory procedures.

Routine laboratory testing may not detect non-O157 Shiga toxin-producing Escherichia coli (STEC) reliably. Active clinical, epidemiological, environmental health, and laboratory collaboration probably influence successful detection and study of non-O157 STEC infection. We summarized two outbreak investigations in which such coordinated efforts identified non-O157 STEC disease and led to effective control measures. Outbreak 1 involved illness associated with consuming unpasteurized apple cider from a local orchard. Public health personnel were notified by a local hospital; stool specimens from ill persons contained O111 STEC. Outbreak 2 involved bloody diarrhoea at a correctional facility. Public health personnel were notified by the facility infection control officer; O45 STEC was the implicated agent. These reports highlight the ability of non-O157 STEC to cause outbreaks and demonstrate that a coordinated effort by clinicians, infection-control practitioners, clinical diagnostic laboratorians, and public health personnel can lead to effective identification, investigation, and prevention of non-O157 STEC disease.

Prevalence, geographic distribution, and impact on lifespan of a dilated cardiomyopathy-associated RNA-binding motif protein 20 variant in genotyped dogs.

INTRODUCTION: The study objectives were to determine the prevalence and geographic distribution of a dilated cardiomyopathy (DCM)-associated RNA-binding motif protein 20 (RBM20) variant in canine DNA samples submitted for testing and to evaluate the influence of the genotype on cardiac phenotype and lifespan. ANIMALS: Samples from 2136 dogs including 1834 Standard Schnauzers (SSNZ), 266 Giant Schnauzers (GSNZ), and 36 dogs of other breeds. METHODS: The University of Missouri Canine Genetics Laboratory's sample-accession spreadsheet and Orthopedic Foundation for Animals' database were retrospectively reviewed for samples submitted for RBM20 genotyping from November, 2013, through May, 2018. Data analyzed included breed, date of birth, RBM20 genotype (homozygous wild-type, heterozygous variant [HET], or homozygous variant [HOM]), geographic origin of submission, pedigree, cardiac phenotype, and date of death or current age if alive. RESULTS AND DISCUSSION: The RBM20 variant was only detected in SSNZ and GSNZ. A total of 389 SSNZ were variant-positive (prevalence = 21.2%), with 361 HET (19.7%) and 28 HOM (1.5%). Of the HOM SSNZ, DCM was confirmed in 26 of 28 (92.9%), with the remainder lost to follow-up. The median lifespan of HOM SSNZ (3.06 years) was significantly shorter than that for HET (15.11 years) and wild-type (15.18 years) SSNZ. Twenty-six GSNZ were variant-positive (prevalence = 9.8%), with 23 HET (8.6%) and three HOM (1.1%). Nine GSNZ belonged to one family, including the three HOM GSNZ that all had DCM. CONCLUSIONS: The HOM genotype is associated with DCM and premature death in SSNZ and GSNZ.

Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

An information-intensive approach to the molecular pharmacology of cancer.

Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.

Long-term Treatment with Methylene Blue in a Dog with Hereditary Methemoglobinemia Caused by Cytochrome b5 Reductase Deficiency.

A juvenile male mixed breed dog was presented for lethargy, exercise intolerance, and aggression when touched on the head. Cyanosis, tachycardia, and tachypnea were observed and persisted during oxygen supplementation. Arterial blood gas analysis by co-oximetry identified an increased methemoglobin concentration (27%; normal, <2%) with normal arterial oxygen tension. The methemoglobinemia and associated clinical signs resolved after administration of methylene blue (1 mg/kg) IV, and the dog was discharged. The affected dog's whole-genome sequence contained 2 potentially causal heterozygous CYB5R3 missense mutations suggesting that cytochrome b5 reductase deficiency was responsible for the methemoglobinemia. This hypothesis was confirmed by enzyme analysis that identified cytochrome b5 reductase activity in the affected dog's erythrocytes to only approximately 6% of that in a control sample. Clinical signs recurred 11 days after discharge but normalized and the methemoglobin concentration decreased with methylene blue administration PO (1.5 mg/kg, initially daily and then every other day).

GM2 Gangliosidosis in Shiba Inu Dogs with an In-Frame Deletion in HEXB.

Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young-adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL-related variants were identified in a whole-genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole-genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3-bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin-layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3-bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole-genome sequencing can lead to the early identification of potentially disease-causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality.

Homozygous PPT1 Splice Donor Mutation in a Cane Corso Dog With Neuronal Ceroid Lipofuscinosis.

A 10-month-old spayed female Cane Corso dog was evaluated after a 2-month history of progressive blindness, ataxia, and lethargy. Neurologic examination abnormalities indicated a multifocal lesion with primarily cerebral and cerebellar signs. Clinical worsening resulted in humane euthanasia. On necropsy, there was marked astrogliosis throughout white matter tracts of the cerebrum, most prominently in the corpus callosum. In the cerebral cortex and midbrain, most neurons contained large amounts of autofluorescent storage material in the perinuclear area of the cells. Cerebellar storage material was present in the Purkinje cells, granular cell layer, and perinuclear regions of neurons in the deep nuclei. Neuronal ceroid lipofuscinosis (NCL) was diagnosed. Whole genome sequencing identified a PPT1c.124 + 1G>A splice donor mutation. This nonreference assembly allele was homozygous in the affected dog, has not previously been reported in dbSNP, and was absent from the whole genome sequences of 45 control dogs and 31 unaffected Cane Corsos. Our findings indicate a novel mutation causing the CLN1 form of NCL in a previously unreported dog breed. A canine model for CLN1 disease could provide an opportunity for therapeutic advancement, benefiting both humans and dogs with this disorder.

Increase in hormonal activation of adenylate cyclase by treatment of cultured cells with N-alkyl,N-nitrosourea.

Treatment of cultured normal rat kidney cells with the nitrosourea-containing compounds streptozotocin, chlorozotocin, or 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea resulted in a time-dependent potentiation in the ability of prostaglandin E1 and (-)-isoproterenol to elevate intracellular cAMP levels. This hormone response increased at 4 hours and reached a maximum at 15--25 hours after addition of the nitrosoureas. Basal cAMP levels were not affected. The greater response was apparently due to an increase in the GTP-dependent step in hormonal activation of adenylate cyclase, inasmuch as GTP- and GTP plus hormone-stimulated adenylate cyclase activities were enhanced twofold to threefold in crude membranes prepared from nitrosourea-treated cells. Fluoride-stimulated adenylate cyclase activity was increased only 10--25%. Nicotinamide did not prevent the elevated response, and NAD+ plus NADH levels were not appreciably altered after 42 hours; treatment with streptozotocin. The results suggest a possible involvement of cAMP in the biologic actions of nitrosoureas.

Selective toxicity induced by picolinic acid in simian virus 40-transformed cells in tissue culture.

When cultured normal and SV40-transformed normal rat kidney and BALB/3T3 cells were exposed to picolinic acid, cell proliferation ceases. Most of the normal cells remained in a quiescent G1 (G0) state and viable for prolonged periods of time. In contrast, SV40-transformed cells progressed to the S and G2 phases of the cell cycle and remained viable only up to 90 to 120 hr. Then, most of the cells began to die. However, a very small fraction of the cell population (approximately 0.01 percent) developed into variants resistant to picolinic acid. Prevention of development of variants, and therefore destruction of all transformed cells, was obtained by addition of glycerol to picolinic acid-treated cells. Untransformed cells were unaffected by the same treatment. These results suggest that differential tumor toxicity should be feasible.

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.

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.

The coding sequence of the bovine ferrochelatase gene.

The amino acid code and surrounding regions in the bovine ferrochelatase gene were amplified by a combination of reverse transcriptase PCR and vectorette PCR and sequenced. The bovine code was 86% homologous to the human ferrochelatase code but was altered at a position corresponding to the presumed human initiator codon.

Acute oral Bryostatin-1 administration improves learning deficits in the APP/PS1 transgenic mouse model of Alzheimer's disease.

BACKGROUND: Previous studies showed that Bryostatin-1, a potent PKC modulator and alphasecretase activator, can improve cognition in models of Alzheimer's disease (AD) with chronic (>10 weeks), intraperitoneal (i.p.) administration of the drug. We compared learning and spatial memory in the APPswe, PSEN1dE985Dbo (APP/PS1) mouse model of AD and studied the ability of acute intraperitoneal and oral Bryostatin-1 to reverse cognitive deficits in this model. Compared to wild-type (WT) mice, APP/PS1 mice showed significant delays in learning the location of a submerged platform in the Morris water maze. Bryostatin-1 was administered over a 2-week course prior to and during water maze testing. RESULTS: Acute i.p. Bryostatin-1 administration did not improve latency to escape but oral Bryostatin-1 significantly improved memory (measured by a reduction in latency to escape). This benefit of oral Bryostatin-1 administration was most apparent during the first 3 days of testing. These findings show that: 1) Bryostatin-1 is orally active in models of learning and memory, 2) this effect can be produced in less than 2 weeks and 3) this effect is not seen with i.p. administration. We conclude that oral Bryostatin-1 represents a novel, potent and long-acting memory enhancer with future clinical applications in the treatment of human AD.

Protein alterations in olfactory neuroblasts from Alzheimer donors.

Definitive diagnosis of Alzheimer's disease (AD) is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. To date, there are no good biological markers for a positive diagnosis of AD in the living patient. In an effort to identify biological markers useful both in the clinical and pathologic diagnosis of AD, we have investigated disease-specific protein alterations in cultured olfactory neurons. Olfactory neurons are readily accessible by biopsy, can be propagated in primary cell culture as olfactory neuroblasts (ONs), and exhibit several elements of AD brain pathophysiology making them powerful tools for the study of AD. Two-dimensional gel analysis of ON proteins from neuropsychologically evaluated AD donors revealed a set of five proteins (Mr 17-50 kD, pI 4.8-6.7) that were significantly altered in concentration when compared to cells from age-matched controls. Further characterization and microsequence analysis could lead to the identification of proteins that may have important diagnostic or therapeutic value in the treatment of AD.

Immunochemical localization of the Batten disease (CLN3) protein in retina.

PURPOSE: Batten disease, also known as juvenile ceroid-lipofuscinosis and CLN3, is an autosomal recessively inherited disorder that results in blindness due to retinal degeneration. The CLN3 gene has been identified, but the function of the protein that this gene encodes is unknown. Experiments were conducted to determine where the CLN3 protein is localized in the mouse retina. Localization should provide a clue in evaluating potential functions of this protein. METHODS: Using oligonucleotide primers based on the reported human CLN3 cDNA sequence, the mouse cDNA nucleotide sequence was determined from products of the reverse transcriptase-polymerase chain reaction and 3' rapid amplification of cDNA ends. A synthetic 20-amino-acid peptide corresponding to an internal hydrophilic region of the predicted amino acid sequence of the mouse CLN3 protein was used to immunize rabbits. The resulting antiserum was used in immunoblot analysis of mouse retina homogenates and in electron microscopic immunocytochemical labeling of mouse retina sections. RESULTS: The peptide antibody labeled a single protein band of approximately 50 kDa on immunoblots of mouse retina homogenates. No labeling was detected with homogenates from human retinas. The antibody specifically labeled mitochondria of Müller cells and inner retinal neurons. Little labeling was observed in mitochondria of the photoreceptor cells. Mitochondria of other cell types, including the retinal pigment epithelium and choroidal cells, were not labeled. CONCLUSIONS: The retinal CLN3 protein appears to be localized almost exclusively in the mitochondria, but was detected only in certain cell types. Batten disease is characterized by massive lysosomal accumulations of a small inner mitochondrial membrane protein (subunit c of ATP synthase). The mitochondrial localization of the CLN3 protein suggests that it may play a role in the normal processing of subunit c.

Protein differences in tau mutant hamsters: candidate clock proteins.

In the tau mutant hamster, the period of the circadian rhythm is shortened from about 24 h to about 22 h in heterozygotes and to about 20 h in homozygotes. Understanding the biochemical basis of the period changes in the tau mutant may elucidate the regulation of the vertebrate pacemaker. Using two-dimensional gel electrophoresis, we have found two sets of proteins that differ between the different genotypes. P33tau (about 33 kDa; pI 6.5) was found in all gels from wild type and heterozygous animals, but was absent in gels from all except one of the homozygous mutant animals. P32tau (about 32 kDa; pI 4.8) was a chain of spots, which showed a striking difference in pattern between gels from wild type animals and from mutant animals. P33tau was greatly enriched in soluble cellular fractions, whereas P32tau was found only in insoluble fractions. These differences between P33tau and P32tau were apparent in gels from both SCN and cortical tissue, suggesting that both proteins are distributed throughout the brain. These proteins should be useful as new tools to explore the biochemistry of circadian pacemakers.