Sectioning and Counting of Motor Neurons in the L3 to L6 Region of the Adult Mouse Spinal Cord

Histology is the study of the microscopic structure of tissues. This protocol permits the generation of frozen transverse sections of lumbar spinal cord regions L3 to L6. It enables counting of murine ventral horn lumbar motor neurons in a reproducible manner. Methods include spinal column dissection, hydraulic extrusion, and histological processing. The preparation for cryo-sectioning includes embedding lumbar spinal cord in optimal cutting temperature (OCT) medium. The correct orientation of the tissue is critical as calculating the amount of tissue to discard saved time overall. Specific details regarding section thickness and mounting are described. These requirements not only allow optimum coverage of specific regions but also ensure that no individual motor neuron was counted twice. The Nissl bodies of the motor neurons were stained using gallocyanin. The sections obtained are all of a comparable area and quality assurance is consistent. The specificity of the staining enables the scientist to identify and reliably quantify lumbar motor neurons. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Euthanasia of mouse and isolation of spinal cord. Basic Protocol 2: Hydraulic extrusion of the spinal cord. Basic Protocol 3: Identification of the lumbar region. Basic Protocol 4: Embedding cord in OCT. Basic Protocol 5: Collection of frozen sections onto slides. Basic Protocol 6: Gallocyanin staining. Basic Protocol 7: Motor neuron counting

Incorporating stakeholder perspectives into the assessment and provision of captive elephant welfare

Recent concerns over the welfare of elephants in UK zoos have implications for their future in captivity, and it is clear that improvements in welfare should be made. Evidence suggests that the knowledge of experienced stakeholders is vital to captive animal welfare assessment. However, there have been few attempts to consult with zoo personnel and other stakeholders on the assessment of elephant welfare, and much of their valuable knowledge of routine husbandry has not been captured in the published literature. As part of a research project commissioned by the Department for Environment Food and Rural Affairs, open response focus groups and workshop discussions were conducted with representatives from 15 UK elephant-holding facilities, and other experts in the welfare and behaviour of captive or free-ranging elephants. Participants described three broad categories of welfare indicators: behavioural, physical and physiological. Resources perceived to be of importance to elephants included aspects of the physical environment, such as feeding opportunities and appropriate substrate, and aspects of the social environment, including group size and relatedness. The data obtained during this study can be used to develop an elephant welfare assessment strategy, informed by the knowledge and expertise of experienced stakeholders, and for consideration of potential changes to guidelines for managing elephants in captivity. Our approach to capturing the views of those who work closely with captive species could be applied elsewhere, in order to draw upon the extensive knowledge of expert stakeholders and consider ways to improve the welfare of captive animals

Multiple independent origins of a protease inhibitor resistance mutation in salvage therapy patients

<p>Abstract</p> <p>Background</p> <p>Combination anti-viral therapies have reduced treatment failure rates by requiring multiple specific mutations to be selected on the same viral genome to impart high-level drug resistance. To determine if the common protease inhibitor resistance mutation L90M is only selected once or repeatedly on different HIV genetic backbones during the course of failed anti-viral therapies we analyzed a linked region of the viral genome during the evolution of multi-drug resistance.</p> <p>Results</p> <p>Using L90M allele specific PCR we amplified and sequenced gag-pro regions linked to very early L90M containing HIV variants prior to their emergence and detection as dominant viruses in 15 failed salvage therapy patients. The early minority L90M linked sequences were then compared to those of the later L90M viruses that came to dominate the plasma quasispecies. Using Bayesian evolutionary analysis sampling trees the emergence of L90M containing viruses was seen to take place on multiple occasion in 5 patients, only once for 2 patients and an undetermined number of time for the remaining 8 patients.</p> <p>Conclusion</p> <p>These results indicate that early L90M mutants can frequently be displaced by viruses carrying independently selected L90M mutations rather than by descendents of the earlier mutants.</p

A review of current indicators of welfare in captive elephants (Loxodonta africana and Elephas maximus)

Concerns over elephant welfare in UK zoos have implications for their future in captivity. To monitor improvements made to elephant welfare in UK zoos, non-invasive, valid and reliable indicators of welfare are needed. Using a rapid review strategy and critical appraisal tool, we aimed to appraise evidence from peer-reviewed literature on potential welfare indicators for captive elephants. Scopus, Web of Knowledge and Ovid were searched in January 2014 using terms relevant to captive elephants and welfare assessment. Inclusion and exclusion criteria were applied and remaining articles were critically appraised against a specially designed welfare indicator appraisal tool. Thirty-seven unique indicators of welfare were extracted from 30 peer-reviewed papers which met the inclusion criteria. Behavioural measures of welfare (n = 21) were more common than either physical (n = 11) or physiological (n = 5) measures. Stereotypies were the most frequently used behavioural measure, glucocorticoids were the most frequently used physiological measure and body condition scores were the most frequently used physical measure. There was most support for the following indicators of improved welfare state: reduced stereotypies, reduced glucocorticoids and improved body condition scores. Additional measures which require further validation but had strong associations with the most supported measures, and thus have potential use in welfare assessment, were: increased lying rest and positive social interactions. Further validation of the described measures is needed, but this information forms a crucial part of the knowledge required to efficiently monitor and improve the welfare of elephants in captivity

True density of soot particles: A comparison of results highlighting the influence of the organic contents

International audienc

Zr-metal adhesion on graphenic nanostructures

3 pages, 3 figures.-- PACS nrs.: 68.35.Np, 61.46.Fg, 61.46.Df.Our high resolution transmission electronic microscopy studies of multiwall carbon nanotubes show, after the growth of zirconia nanoparticles by a hydrothermal route, the presence of surface Zr, forming an atomically thin layer. Using first-principles calculations we investigate the nature of the Zr–C interaction, which is neither ionic nor covalent, and the optimal coverage for the Zr metal in a graphene flake. This preferred coverage is in agreement with that deduced from electron energy loss spectra experiments. We show also that the amount of charge transferred to the C layer saturates as the Zr coverage increases and the Zr–C bond becomes weaker.We want to acknowledge the support by the ETORTEK (NANOMAT) program of the Basque government, the Intramural Special Project (Reference No. 2006601242), the Spanish Ministerio de Ciencia y Tecnología (MCyT) of Spain (Grant No. Fis 2007-66711-C02-C01), and the European Network of Excellence NANOQUANTA (NM4-CT-2004-500198). Y.S.P. gratefully acknowledges his DIPC grant.Peer reviewe

Toxicity screening of two prevalent metal organic frameworks for therapeutic use in human lung epithelial cells

Introduction: The flexibility and tunability of metal organic frameworks (MOFs), crystal- line porous materials composed of a network of metal ions coordinated by organic ligands, confer their variety of applications as drug delivery systems or as sensing and imaging agents. However, such properties also add to the difficulty in ensuring their safe implementa- tion when interaction with biological systems is considered. Methods: In the current study, we used real-time sensorial strategies and cellular-based approaches to allow for fast and effective screening of two MOFs of prevalent use, namely, MIL-160 representative of a hydrophilic and ZIF-8 representative of a hydrophobic frame- work. The two MOFs were synthesized “in house” and exposed to human bronchial epithelial (BEAS-2B) cells, a pertinent toxicological screening model. Results: Analysis allowed evaluation and differentiation of particle-induced cellular effects as well identification of different degrees and routes of toxicity, all in a high-throughput manner. Our results show the importance of performing screening toxicity assessments before introducing MOFs to biomedical applications. Discussion: Our proposed screening assays could be extended to a wider variety of cell lines to allow for identification of any deleterious effects of MOFs, with the range of toxic mechanisms to be differentiated based on cell viability, morphology and cell–substrate interactions, respectively. Conclusion: Our analysis highlights the importance of considering the physicochemical properties of MOFs when recommending a MOF-based therapeutic option or MOFs imple- mentation in biomedical applications

Moving towards effective therapeutic strategies for Neuronal Ceroid Lipofuscinosis.

The Neuronal Ceroid Lipofuscinoses (NCLs) are a family of autosomal recessive neurodegenerative disorders that annually affect 1:100,000 live births worldwide. This family of diseases results from mutations in one of 14 different genes that share common clinical and pathological etiologies. Clinically, the diseases are subcategorized into infantile, late-infantile, juvenile and adult forms based on their age of onset. Though the disease phenotypes may vary in their age and order of presentation, all typically include progressive visual deterioration and blindness, cognitive impairment, motor deficits and seizures. Pathological hallmarks of NCLs include the accumulation of storage material or ceroid in the lysosome, progressive neuronal degeneration and massive glial activation. Advances have been made in genetic diagnosis and counseling for families. However, comprehensive treatment programs that delay or halt disease progression have been elusive. Current disease management is primarily targeted at controlling the symptoms rather than curing the disease. Recognizing the growing need for transparency and synergistic efforts to move the field forward, this review will provide an overview of the therapeutic approaches currently being pursued in preclinical and clinical trials to treat different forms of NCL as well as provide insight to novel therapeutic approaches in development for the NCLs

Development of a behavioural welfare assessment tool for routine use with captive elephants

There has been much concern in recent years about the welfare of elephants in zoos across North America and Europe. While some previous studies have assessed captive elephant welfare at a particular point in time, there has been little work to develop methods which could be used for regular, routine welfare assessment. Such assessment is important in order to track changes in welfare over time. A welfare assessment tool should be rapid, reliable, and simple to complete, without requiring specialist training and facilities; welfare assessments based on behavioural observations are well suited to this purpose. This report describes the development of a new elephant behavioural welfare assessment tool designed for routine use by elephant keepers. Tool development involved: (i) identification of behavioural indicators of welfare from the literature and from focus groups with relevant stakeholders; (ii) development of a prototype tool; (iii) testing of the tool at five UK zoological institutions, involving 29 elephants (representing 46% of the total UK captive elephant population of 63 animals); (iv) assessment of feasibility and reliability of aspects of the prototype tool; (v) assessment of the validity of each element of the tool to reflect the relevant behaviour by comparing detailed behavioural observations with data from the prototype tool; (vi) assessment of known-groups criterion validity by comparing prototype tool scores in individuals with demographics associated with better or worse welfare; (vii) development of a finalised tool which incorporated all elements of the tool which met the criteria set for validity and reliability. Elements of the tool requiring further consideration are discussed, as are considerations for appropriate application and interpretation of scores. This novel behavioural welfare assessment tool can be used by elephant-holding facilities for routine behavioural welfare monitoring, which can inform adjustments to individual welfare plans for each elephant in their collection, to help facilities further assess and improve captive elephant welfare. This study provides an example of how an evidence-based behavioural welfare assessment tool for use by animal caretakers can be developed within the constraints of zoo-based research, which could be applied to a range of captive species

Searching for Novel Biomarkers Using a Mouse Model of CLN3-Batten Disease

CLN3-Batten disease is a rare, autosomal recessive disorder involving seizures, visual, motor and cognitive decline, and premature death. The Cln3Δex7/8 mouse model recapitulates several phenotypic characteristics of the most common 1.02kb disease-associated deletion. Identification of reproducible biomarker(s) to facilitate longitudinal monitoring of disease progression and provide readouts for therapeutic response has remained elusive. One factor that has complicated the identification of suitable biomarkers in this mouse model has been that variations in animal husbandry appear to significantly influence readouts. In the current study, we cross-compared a number of biological parameters in blood from Cln3Δex7/8 mice and control, non-disease mice on the same genetic background from multiple animal facilities in an attempt to better define a surrogate marker of CLN3-Batten disease. Interestingly, we found that significant differences between Batten and non-disease mice found at one site were generally not maintained across different facilities. Our results suggest that colony variation in the Cln3Δex7/8 mouse model of CLN3-Batten disease can influence potential biomarkers of the disease