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Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Clinical and pathological heterogeneity is common in patients with frontotemporal lobar degeneration (FTLD) pathology. This investigated clinical or imaging characteristics that differentiate FTLD-TDP from FTLD-tau, FTLD-TDP subtypes from each other, or pathological stages of FTLD-TDP. Initial clinical, neuropsychological and neuroimaging characteristics were compared between pathologically defined FTLD-tau and FTLD-TDP groups. Voxel-based morphometry analyses contrasted grey matter atrophy patterns. Twenty-six FTLD-TDP, 28 FTLD-tau and 78 controls were included. Amyotrophic lateral sclerosis features, when present, were highly specific FTLD-TDP, which displayed greater cortical and subcortical atrophy than FTLD-tau. FTLD-TDP-43 type B had significantly shorter survival than type A. Type A patients were more cognitively impaired than type B, and basal ganglia atrophy appeared to distinguish type A from type B. Age at onset and survival duration were comparable between stages II and IV. In conclusion, Amyotrophic lateral sclerosis features may be useful in distinguishing FTLD-TDP from FTLD-tau. TDP-43 type A and B appear to present with distinct profiles. The relationship between clinical features and pathological staging in FTLD-TDP-43 is complex, and TDP-43 subtyping may have more clinical utility.

Changes in social behavior are recognized as potential symptoms of behavioral-variant frontotemporal dementia (bvFTD) and semantic dementia (SD), yet objective ways to assess these behaviors in natural social situations are lacking. This study takes a truly social (or second-person) approach and examines changes in real-world social behavior in different dementia syndromes, by analyzing non-scripted social interactions in bvFTD patients (n = 20) and SD patients (n = 20), compared to patients with Alzheimer's disease (AD) (n = 20). Video recordings of 10-min conversations between patients and behavioral neurologists were analyzed for the presence of socially engaging (e.g., nodding, smiling, gesturing) and disengaging behavior (e.g., avoiding eye contact, self-grooming, interrupting). Results demonstrated disease-specific profiles, with bvFTD patients showing less nodding and more looking away than AD, and SD patients showing more gesturing than AD. A principal components analysis revealed the presence of four unobserved components, showing atypical disengaging patterns of behavior. Whole-brain voxel-based morphometry analyses revealed distinct neurobiological bases for each of these components, with the brain regions identified previously associated with behavior selection, abstract mentalization and processing of multi-sensory and socially-relevant information, in mediating socially engaging and disengaging behavior. This study demonstrates the utility of systematic behavioral observation of social interactions in the differential diagnosis of dementia.

Objectives Approximately 30% of Alzheimer's disease (AD) patients are misdiagnosed due to overlapping and evolving clinical features. In particular, the distinction of AD from behavioural variant frontotemporal dementia (bvFTD) can be challenging. Measures of visuospatial ability, which rely on parietal lobe function, show promise as markers of AD as the parietal lobe is preferentially affected early in the disease course. We hypothesise that traditional measures of visuospatial function may help distinguish AD from bvFTD. Materials & methods The Addenbrooke's Cognitive Examination (ACE) visuospatial subtask, Rey-Osterrieth Complex Figure (RCF) task, and subtests of the visual object and space perception battery (VOSP) were used to examine visuospatial abilities in 55 AD patients, 51 bvFTD patients, and 54 healthy Controls. A subgroup analysis was performed in patients with Pittsburgh Compound B positron emission tomography (PiB-PET) data. Results Relative to Controls, AD and bvFTD patients were impaired on almost all visuospatial tasks. Significantly worse performance was observed in AD relative to bvFTD patients on drawing tasks (ACE pentagons/loops copy, cube copy, and all RCF scores) and tasks of spatial orientation (VOSP cube analysis), when controlling for disease severity. Conclusions Visuospatial measures demonstrate limited ability to distinguish between AD and bvFTD unless disease severity is taken into consideration. Controlling for disease severity reveals a disproportionate visuospatial impairment in AD compared to bvFTD. Development of targeted measures of visuospatial function is required to improve differential diagnosis of these syndromes.

This work entails characterizing the flammability of the refrigerant R-32 (CH2F2) by both experimental measurements and modeling. Burning velocities Su were measured using a constant-volume spherical-flame method for R-32/O2/N2 mixtures with O2/N2 ratios ranging from 21% (synthetic air) to 40%, pressures of (1 to 3) bar, and equivalence ratios ϕ of (0.8 to 1.3). Based on a critical assessment of available data, and extended by our own calculations, a detailed chemical kinetic model was developed and key reactions determined using reaction path and sensitivity analyses. Initiation and combustion were identified as distinct kinetic regimes and burning velocities were found to be controlled by two primary reactions: unimolecular decomposition of CH2F2 → CHF + HF and the subsequent reaction, CHF + O2 → CHFO + O, the latter reaction initiating the radical chain propagating and branching by producing O atoms. Sensitive rate constants in the kinetic model were critically adjusted within their uncertainties and current knowledge bounds to best fit the experimental burning velocities. We found that rate constants in the model could be adjusted to match a given experimental Su for specific conditions (O2 loading, P, T, ϕ). This, however, then fixes predicted burning velocities for other all conditions within (3 to 4)% if physically realistic rate parameters are maintained. Thus, the entire set of experimental data is fit, not just to particular conditions. Relative random uncertainties in the experimental Su measurements were (4 to 6)%, but assumptions made for thermal radiation lost by the burned gas in the spherical-flame experiments add an additional systematic uncertainty. Systematic differences between the limiting cases of adiabatic (no thermal radiation lost) and optically-thin (all thermal radiation lost) varied significantly with conditions and ranged from (4 to 30)% at high to low velocities, respectively, translating into uncertainties of (2 to 15)% considering the average of two limiting cases. Comparison of experimental and kinetically modeled Su values suggests that the burned gas tends towards the optically-thin limit at the lowest pressures and fuel loadings and toward the adiabatic limit at the highest pressures and loadings. We tested and found support for this conclusion with a detailed analysis as a function of all the conditions (T, P, % O2, ϕ). This behavior appears to transition from optically-thin to adiabatic as the density of the initial fuel increases, which results in increased CO2 in the burned gas and thus increased absorption of the thermal radiation (consistent with the Beer-Lambert Law). The validated detailed model based on evaluated kinetics is shown to accurately predict burning velocities for R-32 O2/N2 mixtures over a wide range of conditions and provides a reliable basis for extrapolation to other conditions.

Measurements of flame propagation velocities and extinction states in counterflow provide a valuable source of flame data that contain information about fundamental combustion physics. The approach to properly account for stretch effects in counterflow flame measurements through non-intrusive laser-based local velocity characterization was advanced in the mid-80s by Law and coworkers at atmospheric conditions with simple fuels. Subsequently, several research groups have extended the measurements to elevated pressures and complex fuels. However, counterflow flame data at subatmospheric pressures are limited. In the present study, a method is introduced for measuring laminar flame speeds and extinction strain rates in subatmospheric counterflow flames. A numerical study was performed to assess the dynamics of tracer particles used to facilitate measurements. It was found that the particle phase dynamics used in particle velocimetry measurements are not always representative of the underlying gas phase motion due to thermophoresis and insufficient drag, especially at low pressures. A numerical scheme was implemented whereby the computed particle phases were used for proper comparison with measurements and, based on the computed results, to infer the corresponding values of the gas phase. The method was applied to premixed methane/air and non-premixed methane–nitrogen/oxygen flames at pressures as low as 0.1 atm. Complimentary flame structure simulations were carried out which show that the kinetics of formyl radical prompt dissociation strongly impact the computed subatmospheric flames and may influence the validation of unimolecular and bimolecular reactions rate constants when tested against laminar flame data.

Laminar flame speeds of methyl formate/air mixtures were measured at sub-atmospheric pressures for which limited data exist. The experiments were carried out in the counterflow configuration at an unburned mixture temperature of 333 K. The flow velocities were measured using particle image velocimetry. Particle phase slip correction was applied to low-pressure data sets for which the density disparity between the flow tracers and the gaseous phase is notable. The data were modeled using two recently developed kinetic models of methyl formate oxidation, and significant disagreements were realized at all pressures especially under fuel-rich conditions. Additionally, the computed species profiles of CO and CO2 in the burner-stabilized flame configuration using the two models were found to differ significantly. Reaction path analysis revealed that the kinetics of CH2OCHO that is produced directly from the fuel affects the overall reactivity, and the attendant rate constants differ between the two models. The variation of laminar flame speed with pressure revealed also a different behavior between experiments and simulations. Further insight into the sources causing the observed discrepancies were investigated and it was determined that reactions involving formyl radical, methanol, and formaldehyde could also be responsible for the reduction in reactivity specifically under fuel-rich conditions.

Two new tricyclic β-aminoacrylate derivatives (2e and 3e) have been found to be inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with Ki 0.037 and 0.15 μM respectively. 1H and 13C NMR spectroscopic data show that these compounds undergo ready cis-trans isomerisation at room temperature in polar solvents. In silico docking studies indicate that for both molecules there is neither conformation nor double bond configuration which bind preferentially to PfDHODH. This flexibility is favourable for inhibitors of this channel that require extensive positioning to reach their binding site.

The effect of flowfield geometry on flame propagation and extinction of atmospheric CH4/N2/air flames was studied in the counterflow configuration. Laminar flame speeds and extinction strain rates for lean premixed and non-premixed flames were measured in axisymmetric burners producing either uniform or non-uniform axial velocity exit profiles. Particle image velocimetry was used to characterize the two-dimensional flowfield between the burners. The experiments were modeled with a one-dimensional code and a detailed C2 hydrocarbon kinetic model. Laminar flame speeds were found to be insensitive to the burner exit velocity profile shape but extinction measurements were strongly affected. In non-uniform flows, two-dimensional flow field measurements revealed significant radial dependence of flow quantities and high-speed video captured off-center initiation of extinction. Thus, centerline measurements did not represent the extinction state properly, given the direct contradiction to one-dimensional modeling assumptions, and the data was deemed unreliable for kinetic model validation. Flames in uniform flows were found to exhibit minimal radial dependence with extinction initiating at near-centerline locations.

Behavioural variant frontotemporal dementia (bvFTD) is a complex and heterogeneous disorder with as yet unidentified unifying pathophysiological mechanism. There is emerging evidence that hypothalamic dysfunction, manifesting as disturbances in sleep and metabolism, is an integral component of neurodegeneration in bvFTD. Although sleep and metabolic disturbances and the behavioural abnormalities of bvFTD may appear disparate on the surface, there may be a common underlying hormonal mechanism involving orexin. Orexin is a hypothalamic neurotransmitter directly responsible for control of sleep and metabolism in healthy individuals and is implicated in many abnormal behaviours commonly seen in bvFTD - such as impulsive behaviour, hedonistic reinforcement and binge-consumption of ethanol. Further characterising orexin's role in pathophysiology of bvFTD could lead to a new paradigm for understanding this disease and may provide a new direction towards effective management and treatment.

Background There is a gap in the systematic description and investigation of functional disability in corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP). Additionally, the relations between disability, apraxia, cognitive and behavioural changes are not well understood in atypical parkinsonian syndromes. Methods Fifty patients were included in this study (CBS = 18; PSP = 11), including a subgroup of primary progressive aphasia-nonfluent variant (PPA-nfv = 21) who were used as a control group given the clinic-pathological overlap. Functional disability (basic and instrumental activities of daily living), general cognition and behavioural changes were evaluated at baseline, with a subgroup of patients being reassessed after 16 months. Results The corticobasal syndrome group had the most marked disability in basic activities in comparison to progressive supranuclear palsy and primary progressive aphasia-nonfluent variant. Longitudinal decline was marked for all three groups. In a linear regression examining factors behind functional disability in CBS and PSP, memory dysfunction emerged as the main factor (48.5%), followed by apraxia (14.9%) and atypical parkinsonian symptoms (9.6%). Conclusions Memory dysfunction is the most important factor in functional disability in CBS and PSP, which has to be taken into consideration in disease management, prognosis and planning of services to fully address patients' and families' needs.

An investigation was conducted in order to determine the viability of using two-point probability distribution functions to predict the material properties of two-phase heterogeneous materials. The material of interest in this investigation was Titanium Diboride/Alumina ceramic (TiB2 + Al2O3). In TiB2 + Al2O3 ceramics, it has been noted that the connectivity of the TiB2 second phase plays a major role in the mechanical properties of the TiB2 + Al2O3 ceramic, spall strength in particular. Two-point distribution functions were obtained for the four types of microstructures of TiB2 + Al2O3 and were compared to experimental results that have been previously reported. The two-point distribution functions showed very good correlation to connectivity and spall strength results.

The study of quantum phase transitions has brought to light novel metallic states in a number of narrow-band metals. Can a particularly high density of states be realised in geometrically frustrated systems close to a quantum critical point? The 3d heavy-fermion spinel LiV2O4 may be an example of this scenario. In the related, iso-structural and iso-electronic ( 1.5 e - per V) metal CuV2S4, a series of electronically induced phase transitions below 100 K reduce the geometrical frustration present at high temperature, T. The unusually high heat capacity ( C / T ∼ 60 mJ / mol K 2 ) even in the ambient pressure-ordered state nurtures the expectation that removing the transitions in CuV2S4 will reveal a strongly correlated state similar to that of LiV2O4. To test this idea, we have examined the high-pressure resistivity, ρ , of CuV2S4 up to about 14 GPa. A low-T maximum in ρ ( T ) has been found to be strongly p-dependent, and may shift to zero temperature at around 16.5 GPa.

Six years ago, a survey of Australian trainees in neurology highlighted a number of differences in the training offered by the various positions. There has been a subsequent increase in trainee numbers. This survey aimed to re-examine the workload and exposure provided by individual positions and to compare training in Australia and New Zealand. A questionnaire was circulated in 2012 to all advanced trainees in core adult neurology positions in Australia and New Zealand, looking at ward work, outpatient clinics, neurophysiology exposure, and on-call commitments. The response rate was 85.7%. There was a 48.7% increase in the number of core training positions in Australia, but an average increase in inpatient workload of 56%. General neurology clinic numbers were unchanged while specialist clinic exposure had risen from 1.0 to 1.8 clinics/week. In some cases, exposure to neurophysiology had fallen. The requirement for out-of-hours on-call had fallen. There were no major differences between positions in Australia and New Zealand. There have been significant improvements in advanced training in adult neurology in the 5 years between 2007 and 2012; numbers of trainees have increased, on-call commitments have fallen and exposure to specialist clinics has risen. However, inpatient workload has increased significantly, accompanied by a slight reduction in exposure to training in neurophysiology in some cases. Overall, the changes are encouraging but more work is still needed to ensure that individual positions meet the training needs of trainees.

Positive family history has been reported in a high proportion of frontotemporal dementia (FTD) patients, but a genetic cause is documented in a minority of cases. To date, very few studies have examined this apparent discord by systematically assessing the strength of family history in FTD. The present study aimed to characterise the heritability of FTD, compared to Alzheimer’s disease (AD) and controls, using the modified Goldman score. Patients with a diagnosis of FTD or AD were recruited from an FTD research clinic. Detailed clinical and neuropsychological assessments were performed to establish the diagnoses according to accepted diagnostic criteria. FTD patients were further classified into established FTD phenotypes, including behavioural variant (bvFTD) and FTD associated with motor neuron disease (FTD-MND). Comparison was made with age and sex-matched control subjects. Family history of neurodegenerative disease was obtained from patients, their carers, and other family members for collateral history. Strength of family history was graded prospectively using the modified Goldman score. In total, 308 participants (122 FTD, 98 AD, 88 control) were included in the study. Family history of neurodegenerative disease did not differ between groups (FTD 39.3%, AD 42.4%, controls 29.5%, p = 0.167), but the “strength” of family history did differ. Specifically, 17.2% of FTD patients reported a strong family history (modified Goldman score 1–3) compared to 2.3% of controls (p

Lymphoma can affect virtually any organ system including the peripheral nervous system. Neuropathy associated with lymphoma may be secondary to direct invasion of lymphomatous cells (neurolymphomatosis), immune mechanisms, or as an adverse effect of chemotherapy or radiotherapy treatment. Establishing an accurate diagnosis is vital in order to initiate appropriate therapy, particularly in the case of neurolymphomatosis. Often determining the underlying pathological process is challenging as imaging and neurophysiological findings may be nonspecific. A 76-year-old man with diffuse large B-cell lymphoma presented with progressive asymmetrical muscle wasting, sensory loss and mild quadriparesis. Neurophysiology was consistent with a distal demyelinating sensorimotor neuropathy. Only minor enhancement of the right C8 nerve root was identified on MRI. In contrast, a whole body 18 F-fluorodeoxyglucose (FDG) positron emission tomography(PET)-CT scan showed avid linear FDG uptake in the brachial plexus bilaterally as well as multiple other sites, consistent with neurolymphomatosis. There was also evidence of active lymphoma in other organs including the liver and pleura. On subsequent scans the FDG PET-CT scan was the most reliable means to determine treatment response. The neurophysiological features of lymphomatous infiltration of the peripheral nervous system were recently described. Features of demyelination were frequently observed and a third of pathologically proven neurolymphomatosis cases fulfilled the European Federation of Neurological Societies/Peripheral Nerve Society electrodiagnostic criteria for definite chronic inflammatory demyelinating polyneuropathy. This case highlights the sensitivity of FDG PET-CT scan in establishing a diagnosis of neurolymphomatosis because MRI and cerebrospinal fluid analysis are often unhelpful or non-diagnostic. This case also emphasises that neurolymphomatosis should be considered in demyelinating neuropathies associated with lymphoma even when there are minimal or no MRI changes.

Motor neuron disease (MND) and frontotemporal dementia (FTD) share clinical, genetic, and pathological features. Executive and behavioural disturbances are now recognised in MND, but non-executive cognitive impairments such as language dysfunction – a characteristic clinical feature of FTD – have not been explored to our knowledge. The present study investigated language in MND using tools specifically designed for use in different FTD phenotypes, including semantic dementia (SD) and progressive non-fluent aphasia (PNFA). Specifically, semantic deficits were assessed and compared to SD, whereas ability to interpret grammar was compared to PNFA. The study was performed in two parts. In part A, the Sydney language battery was used to assess aspects of semantic knowledge such as object naming, and results were compared to SD. In part B, interpretation of grammar was assessed using the test of reception of grammar (TROG), and results were compared to PNFA. Voxel-based morphometry was used to correlate patterns of cortical atrophy with performance on language measures. In total, 84 participants were included in part A (17 MND, 19 FTD-MND, 22 SD, and 26 controls) and 71 participants in part B (18 MND, 16 FTD-MND, 28 PNFA, and 9 controls). Part A demonstrated semantic deficits in MND and FTD-MND. For example, MND and FTD-MND were impaired on object naming (MND 25 ± 2.9, FTD-MND 17.1 ± 6.6, SD 5.4 ± 3.9, and controls 27 ± 1.9; p

Comprehensive genomic analyses of cancers have provided important insights into tumour evolution and revealed that tumours are frequently heterogeneous entities composed of multiple genetically and phenotypically distinct subclones. Intratumour heterogeneity confounds clinical decision-making, and contributes to the emergence of drug resistant tumour subclones. An important source of genetic heterogeneity in tumours is genomic instability, which is observed in a large proportion of tumours. It is therefore important to understand instability mechanisms and their associated genomic lesions. Our work has highlighted the importance of replication stress as a driver of chromosomal instability. Chromosomally unstable cancer cells display structural chromosome aberrations that lead to missegregation during cell division, thereby contributing to both numerical and structural karyotypic heterogeneity. We also identified three novel suppressors of replication stress and chromosomal instability encoded on chromosome 18q, which is recurrently lost in chromosomally unstable colorectal cancer. We are also examining the role of whole genome-doubling in the tolerance of aneuploidy and chromosomal instability, as well as its role in the rapid evolution of the unstable cancer genome. Underscoring the importance of mechanisms generating heterogeneity in disease progression, a genome-doubling event was independently predictive of poorer relapse free survival in early stage colorectal cancers in two independent patient cohorts. Disclosure: The author has declared no conflicts of interest.

A 42 year old man developed peri-orbital headache and pain on eye movements followed by progressive left monocular blindness. Five days later he developed various visual hallucinations which were both unformed and formed. The patient retained insight and had no symptoms suggestive of psychiatric illness. The hallucinations settled after several days as his visual deterioration reached its nadir. The patient had recently received four weekly injections of grass and olive tree extracts for the treatment of allergic rhinitis as part of an immune desensitisation protocol. Right visual acuity was normal with reduced colour saturation, though acuity was perception of hand movements only on the left. Fundoscopy revealed bilateral optic disc swelling but the remainder of the neurological examination was normal. Magnetic imaging revealed marked oedema and enhancement of the left optic nerve, in association with punctate T2 and FLAIR hyperintensities throughout the subcortical white matter bilaterally. Investigations for systemic vasculitis, antiphospholipid antibody syndrome, sarcoidosis and neuromyelitis optica (aquaporin-4) were negative. Given the clinical and radiological features a diagnosis of acute disseminated encephalomyelitis (ADEM) was made. To our knowledge this is the first reported case of Charles Bonnet syndrome in ADEM. The potential relationship between the illness and recent immune desensitisation therapy is discussed with reference to the literature.

Objective: To determine the clinical and neurophysiological factors on presentation that predict a response to intravenous immunoglobulin (IVIG) in patients presenting with lower motor neuronopathy. Methods: Medical records of 28 patients treated with IVIG for lower motor neuronopathy were studied. Baseline clinical, neurophysiological and laboratory parameters were compared between responders and non-responders to IVIG. Results: There were 15 responders and 13 non-responders. Responders were younger (41.73 vs 52.46 years) and more likely to have antiganglioside antibodies (46.15% vs 0%). Fasciculations were identified in responders and non-responders, but were confined to clinically affected muscles in responders. 53.33% of responders and 15.38% of non-responders met criteria for conduction block. Spontaneous discharges and fibrillations on electromyography were less common in responders than nonresponders, though fasciculations were equally present in both groups. Electromyographic discharges were only seen in responders in conjunction with conduction block. Conclusion: No single clinical, laboratory or conventional neurophysiological characteristic accurately predicts response to IVIG in all patients. Further study and application of new techniques are required. Younger patients who present with a lower motor neuronopathy without widespread fasciculations or spontaneous discharges on electromyography may respond to treatment with IVIG, whether or not conduction block and anti-GM1 antibodies are demonstrated.

The role of the adrenals in the development of the lipemic characteristic of the BHE strain of rat was studied. Male weanling rats were either adrenalectomized (ADX) or left intact and fed either a 65% starch or 65% sucrose diet. The rats were killed at 150 days of age and serum glucose, insulin, triglycerides and cholesterol plus hepatic lipids and enzymes (glucose 6-phosphate dehydrogenase, malic enzyme and glycerophosphate dehydrogenase) were determined. Adrenalectomy did not affect the sucrose-induced rise in serum glucose and insulin levels nor did it have any marked effect on serum lipid levels. Growth and hepatic metabolism also were unaffected by adrenalectomy although diet differences were observed. Sucrose-feeding the ADX rats resulted in higher enzyme levels, higher blood lipids, greater weight gains and greater feed efficiencies than starch feeding. In spite of the importance of the adrenal hormones in the regulation of fuel utilization, we concluded that these hormones do not play an active role in the development of the lipemic tendencies by the BHE strain of rat.

Axotomy induces profound morphological changes in the neuronal cell body 2,a,1°. Neurons whose axons lie within the central nervous system of higher vertebrates (intrinsic neurons) show little or no functional regenerative capability 4,8. However, many mammalian neurons whose axons lie outside the CNS, e.g., sensory and motor neuronsS, 15 and some intrinsic CNS neurons of lower vertebrates, such as teleosts and amphibia, can regenerate functional axons and re-establish normal synaptic fieldsT,9,11. This recuperative response of the perikaryon to axotomy provides a model for the study of regeneration as well as synaptogenesis and other events thought to underlie neuronal growth, recognition and plasticity. Regeneration of functional retinotectal fibers following optic nerve crush in goldfish is associated with a number of quantifiable biochemical changes in the retina, including increased nucleoside uptake and phosphorylation 1. There is also an enhanced labeling of cytoplasmic poly(A)-containing RNA 1 and of the microtubule protein tubulin 6. The enhanced radioisotopic labeling of goldfish retinal tubulin following optic nerve crush could be attributed either to its increased synthesis or decreased degradation. Since the former explanation could reflect regulation at the transcriptional or translational level and axotomy had resulted in enhanced labeling of poly(A)containing RNA, we investigated whether retinal poly(A)-RNA obtained following optic nerve crush was enriched in specific messenger RNAs, particularly that for tubulin. Translation of retinal poly(A)-containing RNA in a heterologous, cell-free protein synthesizing system, followed by SDS gel electrophoresis of the products, provided the necessary tools to detect the proposed changes in RNA populations. Cytoplasmic poly(A)-containing RNA preparations were isolated from both normal and from post-crush retinas, then incubated in a wheat germ protein synthesizing system. The protein labeling patterns from 4, 10 and 15 day post-crush incubations and their controls are shown in Fig. 1. A distinct increase in radioautographic density in the tubulin region of the gel was apparent in proteins translated

Nucleoside 5′-triphosphates in biological samples can be quantitated accurately to 0.1 pmol using a thin-layer chromatography (TLC)-coupled bioluminescence procedure. 32 P-Labeled nucleotides of known specific activities are added to a cell extract as internal standards and the mixture is separated by two-dimensional TLC. Isolated nucleotides are located on the TLC by autoradiography, eluted, and measured in a two-step reaction using nucleoside 5′-diphosphate kinase to catalyze an equimolar phosphorylation of ADP; the mass of resulting ATP is then determined in the firefly luciferin-luciferase reaction. This method provides a 10-fold increase in sensitivity as compared to previously described techniques for nucleotide measurement.