Search

Objective: GM2 gangliosidosis is usually fatal by 5 years of age in its 2 major subtypes, Tay-Sachs and Sandhoff disease. First reported in 1881, GM2 gangliosidosis has no effective treatment today, and children succumb to the disease after a protracted neurodegenerative course and semi-vegetative state. This study seeks to further develop adeno-associated virus (AAV) gene therapy for human translation., Methods: Cats with Sandhoff disease were treated by intracranial injection of vectors expressing feline β-N-acetylhexosaminidase, the enzyme deficient in GM2 gangliosidosis., Results: Hexosaminidase activity throughout the brain and spinal cord was above normal after treatment, with highest activities at the injection sites (thalamus and deep cerebellar nuclei). Ganglioside storage was reduced throughout the brain and spinal cord, with near complete clearance in many regions. While untreated cats with Sandhoff disease lived for 4.4 ± 0.6 months, AAV-treated cats lived to 19.1 ± 8.6 months, and 3 of 9 cats lived >21 months. Correction of the central nervous system was so effective that significant increases in lifespan led to the emergence of otherwise subclinical peripheral disease, including megacolon, enlarged stomach and urinary bladder, soft tissue spinal cord compression, and patellar luxation. Throughout the gastrointestinal tract, neurons of the myenteric and submucosal plexuses developed profound pathology, demonstrating that the enteric nervous system was inadequately treated., Interpretation: The vector formulation in the current study effectively treats neuropathology in feline Sandhoff disease, but whole-body targeting will be an important consideration in next-generation approaches. ANN NEUROL 2023;94:969-986., (© 2023 American Neurological Association.)

Rabies is the deadliest viral infection known, with no reliable treatment, and although it is entirely preventable, rabies continues to kill more than 60,000 people every year, mostly children in countries where dog rabies is endemic. America is only 1 generation away from the time when rabies killed more than 10,000 animals and 50 Americans every year, but 3 to 5 Americans continue to die annually from rabies. Distressingly, > 50,000 Americans undergo rabies prevention therapy every year after exposure to potentially rabid animals. While enormous progress has been made, more must be done to defeat this ancient but persistent, fatal zoonosis. In the US, lack of public awareness and ambivalence are the greatest dangers imposed by rabies, resulting in unnecessary exposures, anxiety, and risk. Veterinarians have a special role in informing and reassuring the public about prevention and protection from rabies. This summary of current facts and future advances about rabies will assist veterinarians in informing their clients about the disease.

Background: Panic disorder is a debilitating anxiety disorder that has a serious impact on adolescents' social and academic functioning and general wellbeing. Panic disorder is experienced by around 1 to 3% of the adolescent population. The aim of this study was to examine adolescents' experiences of having panic disorder., Methods: Semi-structured interviews were conducted with eight adolescents with a primary diagnosis of panic disorder. Interpretative Phenomenological Analysis was used to gain an understanding of adolescents' lived experience of panic disorder., Results: Two superordinate themes were identified: (1) Drowning in sensations, and (2) An unacceptable self. The findings show that adolescents experience panic disorder as extremely overwhelming and unpleasant, with debilitating feelings of drowning in sensations. Adolescents' experiences largely fit with the cognitive model of panic, in which catastrophic misinterpretation of bodily sensations is associated with anxiety, avoidance, and safety behaviours, creating a vicious cycle. Attempts to avoid or prevent the attacks appear to inadvertently make them worse. Social worries, feeling broadly misunderstood, and unhelpful responses from others, contributed to feelings of being different or abnormal and were connected to a negative self-concept. Negative social interactions with teachers and peers in the school environment were particularly damaging., Conclusions: These findings offer new insight into these adolescents' lived experience of panic disorder and highlight the need for adolescents to access timely, evidence-based treatment, as well as the need for increased awareness and understanding of panic disorder in schools., (© 2022. The Author(s).)

Anxiety disorders are common in adolescence but outcomes for adolescents are unclear and we do not know what factors moderate treatment outcome for this age group. We conducted meta-analyses to establish the effectiveness of psychological therapies for adolescent anxiety disorders in (i) reducing anxiety disorder symptoms, and (ii) remission from the primary anxiety disorder, compared with controls, and examine potential moderators of treatment effects. The protocol was registered with PROSPERO (CRD42018091744). Electronic databases (Web of Science, MEDLINE, Psycinfo, EMBASE) were searched from January 1990 to December 2019. 2511 articles were reviewed, those meeting strict criteria were included. Random effects meta-analyses were conducted. Analyses of symptom severity outcomes comprised sixteen studies (CBT k = 15, non-CBT k = 1; n = 766 adolescents), and analyses of diagnostic remission outcomes comprised nine (CBT k = 9; n = 563 adolescents). Post-treatment, those receiving treatment were significantly more likely to experience reduced symptom severity (SMD = 0.454, 95% CI 0.22-0.69) and remission from the primary anxiety disorder than controls (RR = 7.94, 95% CI 3.19-12.7) (36% treatment vs. 9% controls in remission). None of the moderators analysed were statistically significant. Psychological therapies targeting anxiety disorders in adolescents are more effective than controls. However, with only just over a third in remission post-treatment, there is a clear need to develop more effective treatments for adolescents, evaluated through high-quality randomised controlled trials incorporating active controls and follow-up data., (© 2021. The Author(s).)

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the β-subunit of β-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.

Background: Panic disorder is experienced by around 1% of adolescents and has a significant impact on social and academic functioning. Preliminary evidence supports the effectiveness of panic disorder-specific treatment in adolescents with panic disorder; however, panic disorder may be overlooked in adolescents due to overlapping symptoms with other anxiety disorders and other difficulties being more noticeable to others. The aim of this study was to establish what training National Health Service (NHS) Child and Adolescent Mental Health Services (CAMHS) clinicians have received in psychological therapies and panic disorder and how they identify and treat panic disorder in adolescents., Method: CAMHS clinicians from a range of professions (n = 427), who were delivering psychological treatments to children and adolescents with anxiety disorders, participated. They completed a cross-sectional, online survey, including a vignette describing an adolescent with panic disorder, and were asked to identify the main diagnosis or presenting problem., Results: Less than half the clinicians (48.6%) identified panic disorder or panic symptoms as the main presenting problem from the vignette. The majority of clinicians suggested CBT would be their treatment approach. However, few identified an evidence-based treatment protocol for working with young people with panic disorder. Almost half the sample had received no training in cognitive behaviour therapy (CBT), and around a fifth had received no training in delivering psychological treatments., Conclusions: Only half of CAMHS clinicians identified panic disorder from a vignette and although CBT treatments are widely offered, only a minority of adolescents with panic disorder are receiving treatments developed for and evaluated with young people with panic disorder. There is a vital need for clinician training, the use of tools that aid identification and the implementation of evidence-based treatments within CAMHS., Key Practitioner Message: Panic disorder affects around 1% of adolescents, adversely impacting social, academic and long-term life functioning. There is preliminary evidence for the effectiveness of a panic disorder-specific treatment of panic disorder in adolescents. Clinicians struggle to identify panic disorder or suitable treatment protocols for treating adolescents, although most would use CBT as the treatment approach. There is a vital need for clinician training, tools that aid identification of young people with panic disorder and evidence-based treatments that can be delivered in routine clinical settings., (© 2020 The Authors. Child and Adolescent Mental Health published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health.)

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment., (Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.)

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity., Competing Interests: On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

This paper reports the development of an analytical model, with supporting experimental data, which quite accurately describes the key features of CO 2 laser ablation of fused silica glass. The quantitative model of nonexplosive, evaporative material removal is shown to match the experimental data very well, to the extent that it can be used as a tool for ablative measurements of absorption coefficient and vaporization energy. The experimental results indicated that a minimum of 12  MJ kg -1 is required to fully vaporize fused quartz initially held at room temperature, which is in good agreement with the prediction of the model supplied with input data available in the literature. An optimal window for the machining of fused quartz was revealed in terms of pulse duration 20-80 μs and CO 2 laser wavelength optimized for maximum absorption coefficient. Material removal rates of 0.33 μm per J cm -2 allow for a high-precision depth control with modest laser stability. The model may also be used as a parameter selection guide for CO 2 laser ablation of fused silica or other materials of similar thermophysical properties.

Background: The English NHS Bowel Cancer Screening Programme biennially invites individuals aged 60-74 to participate in screening. The booklet, 'Bowel Cancer Screening: The Facts' accompanies this invitation. Its primary aim is to inform potential participants about the aims, advantages and disadvantages of colorectal cancer screening., Objective: To provide detailed commentary on how individuals process the information contained within 'The Facts' booklet., Design, Setting and Participants: This study comprised of 18 interviews with individuals aged 45-60 and used a 'think-aloud' paradigm in which participants read aloud the booklet. Participant utterances (verbal statements made in response to researcher-led prompts) were transcribed and analysed using a combination of content and thematic analysis., Results: A total of 776 coded utterances were analysed (mean = 43.1 per person; range = 8-95). While overall comprehension was satisfactory, several problem areas were identified such as the use of complex unfamiliar terminology and the presentation of numerical information. Specific sections such as colonoscopy risk information evoked negative emotional responses. Participants made several suggestions for ways in which comprehension might be improved., Conclusion: Public perceptions of the NHS Bowel Cancer Screening Programme information materials indicated that specific aspects of the booklet were difficult to process. These materials may be an appropriate target to improve public understanding of the aims, benefits and disadvantages of colorectal cancer screening. These findings will contribute to a broader NIHR-funded project that aims to design a supplementary 'gist-based' information leaflet suitable for low literacy populations., (© 2013 The Authors Health Expectations Published by John Wiley & Sons Ltd.)

Sandhoff disease (SD) is a fatal neurodegenerative disease caused by a mutation in the enzyme β-N-acetylhexosaminidase. Children with infantile onset SD develop seizures, loss of motor tone and swallowing problems, eventually reaching a vegetative state with death typically by 4years of age. Other symptoms include vertebral gibbus and cardiac abnormalities strikingly similar to those of the mucopolysaccharidoses. Isolated fibroblasts from SD patients have impaired catabolism of glycosaminoglycans (GAGs). To evaluate mucopolysaccharidosis-like features of the feline SD model, we utilized radiography, MRI, echocardiography, histopathology and GAG quantification of both central nervous system and peripheral tissues/fluids. The feline SD model exhibits cardiac valvular and structural abnormalities, skeletal changes and spinal cord compression that are consistent with accumulation of GAGs, but are much less prominent than the severe neurologic disease that defines the humane endpoint (4.5±0.5months). Sixteen weeks after intracranial AAV gene therapy, GAG storage was cleared in the SD cat cerebral cortex and liver, but not in the heart, lung, skeletal muscle, kidney, spleen, pancreas, small intestine, skin, or urine. GAG storage worsens with time and therefore may become a significant source of pathology in humans whose lives are substantially lengthened by gene therapy or other novel treatments for the primary, neurologic disease., (Published by Elsevier Inc.)

Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients., (© The Author(s) 2015.)

Sandhoff disease (SD) is caused by deficiency of N-acetyl-β-hexosaminidase (Hex) resulting in pathological accumulation of GM2 ganglioside in lysosomes of the central nervous system (CNS) and progressive neurodegeneration. Currently, there is no treatment for SD, which often results in death by the age of five years. Adeno-associated virus (AAV) gene therapy achieved global CNS Hex restoration and widespread normalization of storage in the SD mouse model. Using a similar treatment approach, we sought to translate the outcome in mice to the feline SD model as an important step toward human clinical trials. Sixteen weeks after four intracranial injections of AAVrh8 vectors, Hex activity was restored to above normal levels throughout the entire CNS and in cerebrospinal fluid, despite a humoral immune response to the vector. In accordance with significant normalization of a secondary lysosomal biomarker, ganglioside storage was substantially improved, but not completely cleared. At the study endpoint, 5-month-old AAV-treated SD cats had preserved neurological function and gait compared with untreated animals (humane endpoint, 4.4±0.6 months) demonstrating clinical benefit from AAV treatment. Translation of widespread biochemical disease correction from the mouse to the feline SD model provides optimism for treatment of the larger human CNS with minimal modification of approach.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme β-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were largely normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated SD patients and those in future clinical trials., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Progressive debilitating neurological defects characterize feline G(M1) gangliosidosis, a lysosomal storage disease caused by deficiency of lysosomal β-galactosidase. No effective therapy exists for affected children, who often die before age 5 years. An adeno-associated viral vector carrying the therapeutic gene was injected bilaterally into two brain targets (thalamus and deep cerebellar nuclei) of a feline model of G(M1) gangliosidosis. Gene therapy normalized β-galactosidase activity and storage throughout the brain and spinal cord. The mean survival of 12 treated G(M1) animals was >38 months, compared to 8 months for untreated animals. Seven of the eight treated animals remaining alive demonstrated normalization of disease, with abrogation of many symptoms including gait deficits and postural imbalance. Sustained correction of the G(M1) gangliosidosis disease phenotype after limited intracranial targeting by gene therapy in a large animal model suggests that this approach may be useful for treating the human version of this lysosomal storage disorder.

Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of β-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and β-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.

Wavelength-locking by a volume holographic grating (VHG) is reported for a diode laser bar with 49 single mode emitters, fitted with a dual-axis collimation phase-plate for smile elimination and excellent beam pointing correction. The much-improved VHG feedback with the ultra-collimated array beam gives 100% wavelength locking at 975 nm over a 17°C temperature range and external cavity lengths up to 110 mm. This enables a folded cavity configuration to provide a fully-locked array with wavelength selection into 200 pm channels over an 8 nm band, suitable for multi-bar dense wavelength-combining.

We report a new technique for the rapid fabrication of microstripe cylindrical and toroidal mirrors with a high ratio (>10) of the two principal radii of curvature (RoC(1)/RoC(2)), and demonstrate their effectiveness as mode-selecting resonator mirrors for high-power planar waveguide lasers. In this process, the larger radius of curvature (RoC(1)) is determined by the planar or cylindrical shape of the fused silica substrate selected for laser processing, whilst the other (RoC(2)) is produced by controlled CO(2) laser-induced vaporization of the glass. The narrow stripe mirror aperture is achieved by applying a set of partially overlapped laser scans, with the incident laser power, the number of laser scans, and their spacing being used to control the curvature produced by laser evaporation. In this work, a 1 mm diameter laser spot is used to produce grooves of cylindrical/toroidal shape with 240 μm width and 16 mm length. After high reflectance coating, these grooves are found to provide excellent mode selectivity as resonator mirrors for a 150 μm core Yb:YAG planar waveguide laser, producing high brightness output at more than 300 W. The results show clearly that the laser-generated microstripe mirrors can improve the optical performance of high-power planar waveguide lasers when applied in a low-loss mode-selective resonator configuration.

We report the demonstration of a high-power single-side-pumped Yb:YAG planar waveguide amplifier seeded by an Yb:KYW femtosecond laser. Five passes through the amplifier yielded 700-fs pulses with average powers of 50 W at 1030 nm. A numerical simulation of the amplifier implied values for the laser transition saturation intensity, the small-signal intensity gain coefficient and the gain bandwidth of 10.0 kW cm(-2), 1.6 cm(-1), and 3.7 nm respectively, and identified gain-narrowing as the dominant pulse-shaping mechanism.

Objective: To determine the earliest day of gestation at which relaxin could be detected in pregnant queens by use of a commercially available point-of-care test designed for use in dogs, and to calculate sensitivity and specificity of the test for pregnancy detection on any specified day of gestation., Design: Evaluation study., Animals: 162 female cats (24 queens from a breeding colony, 128 stray and feral queens undergoing ovariohysterectomy, and 10 ovariohysterectomized cats)., Procedures: 24 queens were monitored for pregnancy. Blood samples were collected daily and tested for relaxin until 2 consecutive positive test results were obtained. The earliest day of pregnancy detection was estimated by counting backward from the day of parturition to the day of the first positive test. The uteri, ovaries, and any fetuses of 128 stray and feral queens undergoing ovariohysterectomy were examined grossly, and gestational day in pregnant queens was determined on the basis of fetal crown-rump length. Blood samples from these queens and from 10 cats ovariohysterectomized prior to the study were collected for relaxin testing., Results: Pregnancy was detected by use of the relaxin test kit as early as gestational day 20; sensitivity of the test was 100% on and after gestational day 29. False-positive results were detected in 3 queens, 2 of which had large (approx 2×3-cm) ovarian cysts, resulting in a specificity of 95.9%., Conclusions and Clinical Relevance: A commercially available relaxin test kit designed for use in dogs can be used to reliably detect pregnancy in cats.

A theory based healthy eating leaflet was evaluated against an existing publicly available standard leaflet. The intervention leaflet was designed to encourage healthy eating in 18-30 year olds and was developed by modifying an existing British Nutrition Foundation leaflet. The intervention leaflet targeted attitudes and self-efficacy. Participants (n = 104) were randomly assigned either to the intervention, Foundation or a local food leaflet control condition. Cognitions were measured pre-intervention, immediately after reading the corresponding leaflet, and once again at two weeks follow-up. Critically, intentions to eat healthily were significantly greater at follow-up in the Intervention group compared to the other two groups, with the former leaflet also being perceived as more persuasive. The Intervention group also showed evidence of healthier eating at two weeks compared to the other two groups. Collectively the results illustrate the utility of a targeted theory-based approach., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Zona pellucida (ZP) glycoproteins play a central role in sperm-oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs., (Copyright 2010 Elsevier B.V. All rights reserved.)

We describe a promising approach to the processing of micro-optical components, where CO(2) laser irradiation in raster scan is used to generate localized surface melting of binary or multilevel structures on silica, fabricated by conventional reactive-ion etching. The technique is shown to provide well-controlled local smoothing of step features by viscous flow under surface tension forces, relaxing the scale length of etch steps controllably between 1 and 30 microm. Uniform treatment of extended areas is obtained by raster scanning with a power stabilized, Gaussian beam profile in the 0.5 to 1 mm diameter range. For step heights of 1 microm or less, the laser-induced relaxation is symmetric, giving softening of just the upper and lower corners at a threshold power of 4.7 W, extending to symmetric long scale relaxation at 7.9 W, with the upper limit set by the onset of significant vaporization. Some asymmetry of the relaxation is observed for 3 microm high steps. Also, undercut steps or troughs produced by photolithography and etching of a deep 64 level multistep surface are found to have a polarization-dependent distortion after laser smoothing. The laser reflow process may be useful for improving the diffraction efficiency by suppressing high orders in binary diffractive optical elements, or for converting multilevel etched structures in fused silica into smoothed refractive surfaces in, for example, custom microlens arrays.

A single optical component for a diode laser bar combines fast-axis smile and lens error correction with slow-axis collimation. Produced by laser-machining/polishing, it provides 0.9 mm focal length, 200 microm pitch slow-axis collimation on the same surface that corrects fast-axis errors. Custom fabrication enables fill-factor optimization for the 49 single-mode beams and gives parallel collimation with rms pointing errors of 3% and 6% of the far-field divergence for the fast- and slow-axis array respectively. Sub-micron pitch mismatch between the slow-axis lens and emitter arrays, and beam pointing changes by thermal expansion of the laser bar are detected.

GM2 gangliosidosis is a fatal, progressive neuronopathic lysosomal storage disease resulting from a deficiency of beta-N-acetylhexosaminidase (EC 3.2.1.52) activity. GM2 gangliosidosis occurs with varying degrees of severity in humans and in a variety of animals, including cats. In the current research, European Burmese cats presented with clinical neurological signs and histopathological features typical of a lysosomal storage disease. Thin layer chromatography revealed substantial storage of GM2 ganglioside in brain tissue of affected cats, and assays with a synthetic fluorogenic substrate confirmed the absence of hexosaminidase activity. When the hexosaminidase beta-subunit cDNA was sequenced from affected cats, a 91 base pair deletion constituting the entirety of exon 12 was documented. Subsequent sequencing of introns 11 and 12 revealed a 15 base pair deletion at the 3' end of intron 11 that included the preferred splice acceptor site, generating two minor transcripts from cryptic splice acceptor sites in affected Burmese cats. In the cerebral cortex of affected cats, hexosaminidase beta-subunit mRNA levels were approximately 1.5 times higher than normal (P<0.001), while beta-subunit protein levels were substantially reduced on Western blots.

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.

To evaluate neural stem/progenitor cell (NPC) transplantation therapy in cat models of neurodegenerative diseases, we have isolated, expanded and characterized feline NPCs (fNPCs) from normal fetal cat brain. Feline NPCs responsive to both human epidermal growth factor (hEGF) and human fibroblast growth factor 2 (hFGF2) proliferated as neurospheres, which were able to differentiate to neurons and glial cells. The analysis of growth factors indicated that both hEGF and hFGF2 were required for proliferation of fNPCs. In contrast to the effect on human NPCs, human leukemia inhibitory factor (hLIF) enhanced differentiation of fNPCs. Expanded fNPCs were injected into the brains of normal adult cats. Immunohistochemical analysis showed that the majority of transplanted cells were located adjacent to the injection site and some fNPCs differentiated into neurons. The survival of transplanted fNPCs over time was monitored using non-invasive bioluminescent imaging technology. This study provided the first evidence of allotransplantation of fNPCs into feline CNS. Cats have heterogeneous genetic backgrounds and possess neurological diseases that closely resemble analogous human diseases. The characterization of fNPCs and exploration of non-invasive bioluminescent imaging to track transplanted cells in this study will allow evaluation of NPC transplantation therapy using feline models of human neurological diseases.