Abnormalities in whisking behaviour are associated with lesions in brain stem nuclei in a mouse model of amyotrophic lateral sclerosis

The transgenic SOD1G93A mouse is a model of human amyotrophic lateral sclerosis (ALS) and recapitulates many of the pathological hallmarks observed in humans, including motor neuron degeneration in the brain and the spinal cord. In mice, neurodegeneration particularly impacts on the facial nuclei in the brainstem. Motor neurons innervating the whisker pad muscles originate in the facial nucleus of the brain stem, with contractions of these muscles giving rise to “whisking” one of the fastest movements performed by mammals. A longitudinal study was conducted on SOD1G93A mice and wild-type litter mate controls, comparing: (i) whisker movements using high-speed video recordings and automated whisker tracking, and (ii) facial nucleus degeneration using MRI. Results indicate that while whisking still occurs in SOD1G93A mice and is relatively resistant to neurodegeneration, there are significant disruptions to certain whisking behaviours, which correlate with facial nuclei lesions, and may be as a result of specific facial muscle degeneration. We propose that measures of mouse whisker movement could potentially be used in tandem with measures of limb dysfunction as biomarkers of disease onset and progression in ALS mice and offers a novel method for testing the efficacy of novel therapeutic compounds

A method for isolating and culturing placental cells from failed early equine pregnancies

Early pregnancy loss occurs in 6–10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14–65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in AmnioChrome™ Plus. Genomic DNA of a suitable quality for molecular assays was also isolated from 29/30 of these cases. This method will enable future investigations determining pathologies causing EPL

Correlated Cryo Super‐Resolution Light and Cryo‐Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2

Super‐resolution light microscopy (SRM) enables imaging of biomolecules within cells with nanometer precision. Cryo‐fixation by vitrification offers optimal structure preservation of biological specimens and permits sequential cryo electron microscopy (cryoEM) on the same sample, but is rarely used for SRM due to various technical challenges and the lack of fluorophores developed for vitrified conditions. Here, a protocol to perform correlated cryoSRM and cryoEM on intact mammalian cells using fluorescent proteins and commercially available equipment is described. After cell culture and sample preparation by plunge‐freezing, cryoSRM is performed using the reversibly photoswitchable fluorescent protein rsEGFP2. Next, a super‐resolved image is reconstructed to guide cryoEM imaging to the feature of interest. Finally, the cryoSRM and cryoEM images are correlated to combine information from both imaging modalities. Using this protocol, a localization precision of 30 nm for cryoSRM is routinely achieved. No impediments to successive cryoEM imaging are detected, and the protocol is compatible with a variety of cryoEM techniques. When the optical set‐up and analysis pipeline is established, the total duration of the protocol for experienced cryoEM users is 3 days, not including cell culture. Microscopic imaging and technolog

ESTIMATING GENOME-WIDE COPY NUMBER USING ALLELE SPECIFIC MIXTURE MODELS

Genomic changes such as copy number alterations are thought to be one of the major underlying causes of human phenotypic variation among normal and disease subjects [23,11,25,26,5,4,7,18]. These include chromosomal regions with so-called copy number alterations: instead of the expected two copies, a section of the chromosome for a particular individual may have zero copies (homozygous deletion), one copy (hemizygous deletions), or more than two copies (amplifications). The canonical example is Down syndrome which is caused by an extra copy of chromosome 21. Identification of such abnormalities in smaller regions has been of great interest, because it is believed to be an underlying cause of cancer. More than one decade ago comparative genomic hybridization (CGH)technology was developed to detect copy number changes in a high-throughput fashion. However, this technology only provides a 10 MB resolution which limits the ability to detect copy number alterations spanning small regions. It is widely believed that a copy number alteration as small as one base can have significant downstream effects, thus microarray manufacturers have developed technologies that provide much higher resolution. Unfortunately, strong probe effects and variation introduced by sample preparation procedures have made single-point copy number estimates too imprecise to be useful. CGH arrays use a two-color hybridization, usually comparing a sample of interest to a reference sample, which to some degree removes the probe effect. However, the resolution is not nearly high enough to provide single-point copy number estimates. Various groups have proposed statistical procedures that pool data from neighboring locations to successfully improve precision. However, these procedure need to average across relatively large regions to work effectively thus greatly reducing the resolution. Recently, regression-type models that account for probe-effect have been proposed and appear to improve accuracy as well as precision. In this paper, we propose a mixture model solution specifically designed for single-point estimation, that provides various advantages over the existing methodology. We use a 314 sample database, constructed with public datasets, to motivate and fit models for the conditional distribution of the observed intensities given allele specific copy numbers. With the estimated models in place we can compute posterior probabilities that provide a useful prediction rule as well as a confidence measure for each call. Software to implement this procedure will be available in the Bioconductor oligo packagehttp://www.bioconductor.org)

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology. Microscopic imaging and technolog

The Spinel LiCoMnO4: 5V cathode and conversion anode

LiCoMnO4 was made at 550 °C in 2 h using a novel biotemplating synthetic methodology. High temperature heat treatment under flowing N2 was then used to prepare the cation-disordered rock salt, LiCoMnO3. We demonstrate for the first time that both phases can operate as conversion anodes in lithium-ion batteries, operating at ~ 0.7 V with specific capacities of ~ 400 mAh g-1. We also demonstrate that 1,3-propane sultone can be used as an electrolytic additive to provide a modest boost to specific capacity in cells cycled at high potentials with LiCoMnO4 as the cathode

Seizure epicenter depth and translaminar field potential synchrony underlie complex variations in tissue oxygenation during ictal initiation

Whether functional hyperemia during epileptic activity is adequate to meet the heightened metabolic demand of such events is controversial. Whereas some studies have demonstrated hyperoxia during ictal onsets, other work has reported transient hypoxic episodes that are spatially dependent on local surface microvasculature. Crucially, how laminar differences in ictal evolution can affect subsequent cerebrovascular responses has not been thus far investigated, and is likely significant in view of possible laminar-dependent neurovascular mechanisms and angioarchitecture. We addressed this open question using a novel multi-modal methodology enabling concurrent measurement of cortical tissue oxygenation, blood flow and hemoglobin concentration, alongside laminar recordings of neural activity, in a urethane anesthetized rat model of recurrent seizures induced by 4-aminopyridine. We reveal there to be a close relationship between seizure epicenter depth, translaminar LFP synchrony and tissue oxygenation during the early stages of recurrent seizures, whereby deep layer seizures are associated with decreased cross laminar synchrony and prolonged periods of hypoxia, and middle layer seizures are accompanied by increased cross-laminar synchrony and hyperoxia. Through comparison with functional activation by somatosensory stimulation and graded hypercapnia, we show that these seizure-related cerebrovascular responses occur in the presence of conserved neural-hemodynamic and blood flow-volume coupling. Our data provide new insights into the laminar dependency of seizure-related neurovascular responses, which may reconcile inconsistent observations of seizure-related hypoxia in the literature, and highlight a potential layer-dependent vulnerability that may contribute to the harmful effects of clinical recurrent seizures. The relevance of our findings to perfusion-related functional neuroimaging techniques in epilepsy are also discussed

Structures of C1-IgG1 provide insights into how danger pattern recognition activates complement

Microscopic imaging and technolog