Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

Novel preclinical model for CDKL5 deficiency disorder.

Cyclin-dependent kinase-like-5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodegenerative disease characterised by early-onset epileptic seizures, low muscle tone, progressive intellectual disability and severe motor function. CDD affects ∼1 in 60,000 live births, with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD, with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput preclinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss-of-function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model for investigating CDD disease pathophysiology and allowing high-throughput screening for effective therapies. This article has an associated First Person interview with the first author of the paper.

Inhibition of Amyloid Aggregation and Toxicity with Janus Iron Oxide Nanoparticles.

Amyloid aggregation is a ubiquitous form of protein misfolding underlying the pathologies of Alzheimer's disease (AD), Parkinson's disease (PD) and type 2 diabetes (T2D), three primary forms of human amyloid diseases. While much has been learned about the origin, diagnosis and management of these neurological and metabolic disorders, no cure is currently available due in part to the dynamic and heterogeneous nature of the toxic oligomers induced by amyloid aggregation. Here we synthesized beta casein-coated iron oxide nanoparticles (ßCas IONPs) via a BPA-P(OEGA-b-DBM) block copolymer linker. Using a thioflavin T kinetic assay, transmission electron microscopy, Fourier transform infrared spectroscopy, discrete molecular dynamics simulations and cell viability assays, we examined the Janus characteristics and the inhibition potential of ßCas IONPs against the aggregation of amyloid beta (Aß), alpha synuclein (αS) and human islet amyloid polypeptide (IAPP) which are implicated in the pathologies of AD, PD and T2D. Incubation of zebrafish embryos with the amyloid proteins largely inhibited hatching and elicited reactive oxygen species, which were effectively rescued by the inhibitor. Furthermore, Aß-induced damage to mouse brain was mitigated in vivo with the inhibitor. This study revealed the potential of Janus nanoparticles as a new nanomedicine against a diverse range of amyloid diseases.

RNA-induced inflammation and migration of precursor neurons initiates neuronal circuit regeneration in zebrafish.

Tissue regeneration and functional restoration after injury are considered as stem- and progenitor-cell-driven processes. In the central nervous system, stem cell-driven repair is slow and problematic because function needs to be restored rapidly for vital tasks. In highly regenerative vertebrates, such as zebrafish, functional recovery is rapid, suggesting a capability for fast cell production and functional integration. Surprisingly, we found that migration of dormant "precursor neurons" to the injury site pioneers functional circuit regeneration after spinal cord injury and controls the subsequent stem-cell-driven repair response. Thus, the precursor neurons make do before the stem cells make new. Furthermore, RNA released from the dying or damaged cells at the site of injury acts as a signal to attract precursor neurons for repair. Taken together, our data demonstrate an unanticipated role of neuronal migration and RNA as drivers of neural repair.

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA.

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgshΔex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgshΔex5-6 zebrafish is largely dependent on interleukin-1ß and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgshΔex5-6 mutant larvae in a context-dependent manner. We expect the sgshΔex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.

Selection for CD26- and CD49A+ Cells From Pluripotent Stem Cells-Derived Islet-Like Clusters Improves Therapeutic Activity in Diabetic Mice.

Background: Cell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the ß-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells. Methods: Functional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice. Results: CD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A + cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26 + cells followed by enrichment for CD49A + cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26 - /CD49A + enriched ILC exhibited improved function over non-sorted ILC or CD49A + cells in diabetic mice and maintain prolonged blood C-peptide levels. Conclusions: Refining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.

Cellular and Molecular Characterization of the Effects of the Zebrafish Embryo Genotyper Protocol.

The Zebrafish Embryo Genotyper (ZEG) device provides a promising tool for genotyping live embryos. Although the gross morphology and survival of embryos after the use of ZEG are unaffected, the cellular and molecular effects of the ZEG protocol remain unknown. To address this, we have examined the integrity of specific tissues, and evaluated the expression of stress-responsive genes to determine the impact of the ZEG protocol. Our analyses reveal that although ZEG results in a low-level acute stress response, no long-lasting effects are evident, supporting its utilization for a variety of downstream assays.

Combined iStent® Inject Trabecular Micro-Bypass and Phacoemulsification in Australian Patients with Open-Angle Glaucoma.

PURPOSE: This retrospective audit aimed to evaluate the impact of combined iStent® Inject (iSI) and phacoemulsification on medication number in Australians with open-angle glaucoma. Secondary outcomes included intraocular pressure (IOP), best-corrected visual acuity, refraction and visual fields. PATIENTS AND METHODS: Patients with glaucoma that received combined iSI and phacoemulsification by the same surgeon between 1 February 2016 and 1 February 2018 were audited for postoperative medication number, pressures after 1 day, 1 week, 4 weeks and 6, 12, 18 and 24 months, visual acuity, refraction and visual fields. These parameters were compared to baseline levels and with those from a separate cohort of patients without glaucoma that received standalone phacoemulsification. RESULTS: Forty-one patients (63 eyes) received the combined procedure. Thirty-four patients (59 eyes) received standalone phacoemulsification. Four weeks after receiving combined iSI and phacoemulsification the mean medication number was significantly reduced by 1.3 (p < 0.001) for those on medication at baseline and by 0.5 (p = 0.002) overall. Mean IOP was significantly reduced from baseline after 6 months (-16%; p = 0.012; n = 35) and 12 months (-29%; p = 0.004; n = 16). Patients receiving standalone phacoemulsification had short-term reductions in IOP at 4 weeks (-8%; p < 0.001; n = 57) and 6 months (-16%; p < 0.001; n = 32). These patients without glaucoma had lower pressures overall compared to those with glaucoma that received the combined procedure (p = 0.019). There were no differences in final visual acuity or refractive outcomes between groups. CONCLUSION: This audit suggests that iSI and phacoemulsification are at least as effective in controlling IOP as medical therapy. It may have an important role in reducing the medication burden in Australians with cataract and glaucoma. This study is one of the first to confirm refractive stability in concomitant iSI and phacoemulsification.

Accelerated brain aging towards transcriptional inversion in a zebrafish model of the K115fs mutation of human PSEN2.

BACKGROUND: The molecular changes involved in Alzheimer's disease (AD) progression remain unclear since we cannot easily access antemortem human brains. Some non-mammalian vertebrates such as the zebrafish preserve AD-relevant transcript isoforms of the PRESENILIN genes lost from mice and rats. One example is PS2V, the alternative transcript isoform of the PSEN2 gene. PS2V is induced by hypoxia/oxidative stress and shows increased expression in late onset, sporadic AD brains. A unique, early onset familial AD mutation of PSEN2, K115fs, mimics the PS2V coding sequence suggesting that forced, early expression of PS2V-like isoforms may contribute to AD pathogenesis. Here we use zebrafish to model the K115fs mutation to investigate the effects of forced PS2V-like expression on the transcriptomes of young adult and aged adult brains. METHODS: We edited the zebrafish genome to model the K115fs mutation. To explore its effects at the molecular level, we analysed the brain transcriptome and proteome of young (6-month-old) and aged (24-month-old) wild type and heterozygous mutant female sibling zebrafish. Finally, we used gene co-expression network analysis (WGCNA) to compare molecular changes in the brains of these fish to human AD. RESULTS: Young heterozygous mutant fish show transcriptional changes suggesting accelerated brain aging and increased glucocorticoid signalling. These early changes precede a transcriptional 'inversion' that leads to glucocorticoid resistance and other likely pathological changes in aged heterozygous mutant fish. Notably, microglia-associated immune responses regulated by the ETS transcription factor family are altered in both our zebrafish mutant model and in human AD. The molecular changes we observe in aged heterozygous mutant fish occur without obvious histopathology and possibly in the absence of Aß. CONCLUSIONS: Our results suggest that forced expression of a PS2V-like isoform contributes to immune and stress responses favouring AD pathogenesis. This highlights the value of our zebrafish genetic model for exploring molecular mechanisms involved in AD pathogenesis.

Bioinspired polynorepinephrine nanoparticles as an efficient vehicle for enhanced drug delivery.

An innovative drug delivery vehicle based on polynorepinephrine (PNE) with controllable size modification, high delivery efficacy and low cytotoxicity is presented. Highly monodisperse PNE nanoparticles are fabricated by the autoxidation of norepinephrine monomers in an alkaline water/ethanol mixture via stirring at room temperature. We demonstrated the facile optimization of particle size to enhance particle stability and biocompatibility by varying solvent and monomer dosage. To demonstrate the suitability and potential application of PNE particles in cancer therapy, we show that these particles are biocompatible in vitro with HeLa cells and in vivo in zebrafish embryos. After loading the anti-cancer chemotherapy drug doxorubicin (DOX) into the PNE nanoparticles, a consistent and pH responsive drug release profile of DOX was achieved in different environmental conditions. It was found that DOX loaded PNE nanoparticles (PNE/DOX) exhibit much higher pharmaceutical cytotoxicity than free DOX on HeLa cells. Furthermore, the amount of drug released was significantly enhanced in acidic environments that mimic the pH of extracellular tumour microenvironments. Taken together, the PNE nanoparticles represent a new class of melanin particles with promising potential in drug delivery and as a therapeutic platform for cancer treatment.

Single-Molecular Heteroamyloidosis of Human Islet Amyloid Polypeptide.

Human amyloids and plaques uncovered post mortem are highly heterogeneous in structure and composition, yet literature concerning the heteroaggregation of amyloid proteins is extremely scarce. This knowledge deficiency is further exacerbated by the fact that peptide delivery is a major therapeutic strategy for targeting their full-length counterparts associated with the pathologies of a range of human diseases, including dementia and type 2 diabetes (T2D). Accordingly, here we examined the coaggregation of full-length human islet amyloid polypeptide (IAPP), a peptide associated with type 2 diabetes, with its primary and secondary amyloidogenic fragments 19-29 S20G and 8-20. Single-molecular aggregation dynamics was obtained by high-speed atomic force microscopy, augmented by transmission electron microscopy, X-ray diffraction, and super-resolution stimulated emission depletion microscopy. The coaggregation significantly prolonged the pause phase of fibril elongation, increasing its dwell time by 3-fold. Surprisingly, unidirectional elongation of mature fibrils, instead of protofilaments, was observed for the coaggregation, indicating a new form of tertiary protein aggregation unknown to existing theoretical models. Further in vivo zebrafish embryonic assay indicated improved survival and hatching, as well as decreased frequency and severity of developmental abnormalities for embryos treated with the heteroaggregates of IAPP with 19-29 S20G, but not with 8-20, compared to the control, indicating the therapeutic potential of 19-29 S20G against T2D.

Additives migrating from 3D-printed plastic induce developmental toxicity and neuro-behavioural alterations in early life zebrafish (Danio rerio).

The environmental impact of exposure to 3D-printed plastics as well as potential migration of toxic chemicals from 3D-printed plastics remains largely unexplored. In this work we applied leachates from plastics fabricated using a stereolithography (SLA) process to early developmental stages of zebrafish (Danio rerio) to investigate developmental toxicity and neurotoxicity. Migration of unpolymerized photoinitiator, 1-hydroxycyclohexyl phenyl ketone (1-HCHPK) from a plastic solid phase to aqueous media at up to 200 mg/L in the first 24 h was detected using gas chromatography-mass spectrometry. Both plastic extracts (LC50 22.25% v/v) and 1-HCHPK (LC50 60 mg/L) induced mortality and teratogenicity within 48 h of exposure. Developmental toxicity correlated with in situ generation of reactive oxygen species (ROS), an increase in lipid peroxidation and protein carbonylation markers and enhanced activity of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in embryos exposed to concentrations as low as 20% v/v for plastic extracts and 16 mg/L for 1-HCHPK. ROS-induced cellular damage led to induction of caspase-dependent apoptosis which could be pharmacologically inhibited with both antioxidant ascorbic acid and a pan-caspase inhibitor. Neuro-behavioral analysis showed that exposure to plastic leachates reduced spontaneous embryonic movement in 24-36 hpf embryos. Plastic extracts in concentrations above 20% v/v induced rapid retardation of locomotion, changes in photomotor response and habituation to photic stimuli with progressive paralysis in 120 hpf larvae. Significantly decreased acetylcholinesterase (AChE) activity with lack of any CNS-specific apoptotic phenotypes as well as lack of changes in motor neuron density, axonal growth, muscle segment integrity or presence of myoseptal defects were detected upon exposure to plastic extracts during embryogenesis. Considering implications of the results for environmental risk assessment and the growing usage of 3D-printing technologies, we speculate that some 3D-printed plastic waste may represent a significant and yet very poorly uncharacterized environmental hazard that merits further investigation on a range of aquatic and terrestrial species.

Midbrain tectal stem cells display diverse regenerative capacities in zebrafish.

How diverse adult stem and progenitor populations regenerate tissue following damage to the brain is poorly understood. In highly regenerative vertebrates, such as zebrafish, radial-glia (RG) and neuro-epithelial-like (NE) stem/progenitor cells contribute to neuronal repair after injury. However, not all RG act as neural stem/progenitor cells during homeostasis in the zebrafish brain, questioning the role of quiescent RG (qRG) post-injury. To understand the function of qRG during regeneration, we performed a stab lesion in the adult midbrain tectum to target a population of homeostatic qRG, and investigated their proliferative behaviour, differentiation potential, and Wnt/ß-catenin signalling. EdU-labelling showed a small number of proliferating qRG after injury (pRG) but that progeny are restricted to RG. However, injury promoted proliferation of NE progenitors in the internal tectal marginal zone (TMZi) resulting in amplified regenerative neurogenesis. Increased Wnt/ß-catenin signalling was detected in TMZi after injury whereas homeostatic levels of Wnt/ß-catenin signalling persisted in qRG/pRG. Attenuation of Wnt signalling suggested that the proliferative response post-injury was Wnt/ß-catenin-independent. Our results demonstrate that qRG in the tectum have restricted capability in neuronal repair, highlighting that RG have diverse functions in the zebrafish brain. Furthermore, these findings suggest that endogenous stem cell compartments compensate lost tissue by amplifying homeostatic growth.

A Modular Millifluidic Homeostatic Imaging Plate for Imaging of Larval Zebrafish.

Zebrafish larvae are suitable in vivo models for toxicological and pharmacological screens due to their transparency, small size, ex utero development, and genetic and physiological similarity to humans. Using modern imaging techniques, cells and tissues can be dynamically visualized over several days in multiple zebrafish larvae. However, precise specimen immobilization and maintenance of homeostatic conditions remain a challenge for longitudinal studies. A highly customizable mounting configuration with inbuilt means of controlling temperature and media flow would therefore be a valuable tool to facilitate long-term imaging of a large number of specimens. Using three-dimensional printing, we have developed a millifluidic, modular homeostatic imaging plate (HIP), which consists of a customizable sample insert and a temperature-controlled incubation chamber that is continuously perfused, providing an ideal environment for long-term experiments where homeostatic conditions are desired. The HIP is cheap to produce, has a standard microtiter well plate format, and can be fitted to most microscopes. We used the device to image dynamic regeneration of spinal cord neurons. The flexibility and adaptability of the HIP facilitate long-term in vivo imaging of many samples, and can be easily adapted to suit a broad range of specimens.

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia.

Tolerance to antifungal drug concentrations above the minimal inhibitory concentration (MIC) is rarely quantified, and current clinical recommendations suggest it should be ignored. Here, we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC, and find that it is distinct from susceptibility/resistance. Instead, tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population, and correlates inversely with intracellular drug accumulation. Many adjuvant drugs used in combination with fluconazole, a widely used fungistatic drug, reduce tolerance without affecting resistance. Accordingly, in an invertebrate infection model, adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with low tolerance isolates. Furthermore, isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole. Thus, tolerance correlates with, and may help predict, patient responses to fluconazole therapy.

A Whole Brain Staining, Embedding, and Clearing Pipeline for Adult Zebrafish to Visualize Cell Proliferation and Morphology in 3-Dimensions.

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish (Danio rerio) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2'-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a valuable tool to unlock new ways of understanding systemic patterns in cell proliferation in the healthy and injured brain, brain-wide cellular interactions, stem cell niche development, and changes in brain morphology.

Laryngeal snaring by ingested fishing net in a common bottlenose dolphin (Tursiops truncatus) off the Israeli shoreline.

We report an unusual snaring of the larynx in an adult, female common bottlenose dolphin (Tursiops truncatus). The dolphin was observed swimming and diving in Haifa Port, Israel, but was found dead the next day, 60 km south, on the coast. Postmortem examination revealed stranded-cordage, nylon filaments wrapped around the larynx, cutting through the soft tissue, and extending down into the forestomach, where a large mass of netting was found. The cachectic state of the dolphin and the subacute to chronic, hyper-plastic response of soft tissue surrounding the filaments lodged around the larynx, suggest a prolonged period of starvation, which led to the final weakness and wasting of the dolphin.

The ascent of cat breeds: genetic evaluations of breeds and worldwide random-bred populations.

The diaspora of the modern cat was traced with microsatellite markers from the presumed site of domestication to distant regions of the world. Genetic data were derived from over 1100 individuals, representing 17 random-bred populations from five continents and 22 breeds. The Mediterranean was reconfirmed to be the probable site of domestication. Genetic diversity has remained broad throughout the world, with distinct genetic clustering in the Mediterranean basin, Europe/America, Asia and Africa. However, Asian cats appeared to have separated early and expanded in relative isolation. Most breeds were derived from indigenous cats of their purported regions of origin. However, the Persian and Japanese bobtail were more aligned with European/American than with Mediterranean basin or Asian clusters. Three recently derived breeds were not distinct from their parental breeds of origin. Pure breeding was associated with a loss of genetic diversity; however, this loss did not correlate with breed popularity or age.

Microsurgical management of penetrating peripheral nerve injuries: pre, intra- and postoperative analysis and results.

BACKGROUND AND METHODS: Clinical and electrophysiological motor function data were compared before and after microsurgical repair of penetrating peripheral nerve injuries. Sixty-four patients totaling 74 injured nerves (25 gunshot wounds, 49 stab wounds) were treated with external and interfascicular neurolysis and/or interfascicular nerve grafts. Microsurgery was performed 2-12 months after the injury (Group 1, 33 patients,) and 12 months-60 years after the injury (Group 2, 31 patients). The postoperative clinical and electrophysiological follow-up period ranged between 1 and 5 years. RESULTS: A statistically significant improvement in muscle strength occurred after the microsurgery, compared to before repair, gunshot wounds (p < 0.001), stab wounds (p < 0.001). Intraoperative and postoperative electrophysiological analysis showed statistically significant improvement. TIMING OF SURGERY: No statistically significant difference in muscle strength occurred between the 2 groups after the surgery, each showing statistically significant improvement, Group 1 (p < 0.001), Group 2 (p < 0.001). Patients above and below age of 40 showed an improvement in muscle strength after microsurgery, (p < 0.001) and (p < 0.001), respectively. CONCLUSION: Microsurgery can progressively improve nerve function in penetrating peripheral nerve injuries and lead to significant functional improvement, even when it is delayed for more than one year after the injury.

Thoracic outlet syndrome: a multidisciplinary problem with a perspective for microsurgical management without rib resection.

BACKGROUND: Thoracic outlet syndrome (TOS) refers to a group of complex symptoms in the upper extremity caused by compression of the brachial plexus, subclavian artery and vein. Different surgical approaches were described for the management of TOS. There is, however, no "gold standard" procedure for this complicated and multidisciplinary problem. OBJECTIVES: This study evaluated the effectiveness of a microsurgical neurovascular decompression in the treatment of TOS. METHODS: 11 patients suffering from TOS (for 1.3 to 15 years after the beginning of the symptoms) were selected for a treatment of the complex symptoms of pain (diffuse or irradiated to the arm and hand), aching or paresthesia in the neck, shoulder, anterior chest, upper extremity and hand. Four of the 11 patients were suffering from signs of vascular compression. Eight patients showed slow progressive neurological deterioration (distribution of the ulnar nerve) with partial muscle atrophy. Patients underwent a microsurgical treatment using a supraclavicular approach followed by brachial plexus neurolysis, scalenectomy and release of the subclavian artery and vein without rib resection. Postoperative results were classified, using Am. J. Surg. (176: 215-218, 1998) scale (4), as good, fair and poor. RESULTS: Surgical results were studied, with a follow-up of 24 to 48 months. Prior to surgery, all patients had partial or severe limitation in physical activities. Post-operative follow-up showed that 9 (82%) of the 11 patients returned to normal everyday physical activities with a complete or significant relief of the symptoms (good results). In 2 patients (18%) the pain decreased and the use of medication was reduced (fair results). Eight of the 11 patients returned to full or partial employment. There were no cases of poor results in the study. CONCLUSION: Microsurgical neurovascular decompression of TOS without a removal of the cervical or first rib using a supraclavicular approach is an effective treatment method for a relief or complete release from symptoms and allows most patients to return to an active normal life.