Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review

Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments. Here, we review a portfolio of new methods for comparing labelling distributions between different compartments in one study group (method 1) and between the same compartments in two or more groups (method 2). Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures. The methods can be used to analyse gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments, but in method 1, membranes must be treated as organelles. With method 1, gold counts are combined with stereological estimators of compartment size to determine labelling density (LD). For volume-occupiers, LD can be expressed simply as golds per test point and, for surface-occupiers, as golds per test line intersection. Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions. For method 2, the raw gold particle counts distributed between compartments are simply compared across groups by contingency table and Chi-squared analysis. This identifies the main compartments responsible for the differences between group distributions. Finally, we discuss labelling efficiency (the number of gold particles per target molecule) and describe how it can be estimated for volume- or surface-occupiers by combining stereological data with biochemical determinations

At last, a Pennsylvanian stem-stonefly (Plecoptera) discovered

<p>Abstract</p> <p>Background</p> <p>Stem-relatives of many winged insect orders have been identified among Pennsylvanian fossils (Carboniferous Period). Owing to their presumed 'basal' position in insect phylogeny, stoneflies were expected to occur at this period. However, no relative has ever been designated convincingly.</p> <p>Results</p> <p>In this paper, we report specimens belonging to a new fossil insect species collected from the Tupo Formation (Pennsylvanian; China). The wing venation of <it>Gulou carpenteri </it><b>gen. et sp. nov</b>. exhibits character states diagnostic of the order Plecoptera, but lack character states shared by unequivocal representatives of the order. Derived from this identification, the delimitation of the fossil species is ascertained based on comparison of several extant stonefly species. This comparative analysis allowed a trait present in <it>G. carpenteri </it><b>gen. et sp. nov</b>., but rarely occurring in extant species, to be documented and highlighted as atavistic. Affinities of taxa formerly proposed as putative stem-stoneflies are reconsidered in the light of the new discovery.</p> <p>Conclusions</p> <p><it>Gulou carpenteri </it><b>gen. et sp. nov</b>. is considered the only genuine Plecoptera reported from the Pennsylvanian. Continuing efforts on the systematics of Pennsylvanian winged insects indicate a fauna more diverse than previously appreciated. It suggests that insects already had a long, yet undocumented, history by this time.</p

Is Fetal Growth Restriction Associated with a More Severe Maternal Phenotype in the Setting of Early Onset Pre-Eclampsia? A Retrospective Study

BACKGROUND: Both pre-eclampsia and fetal growth restriction are thought to result from abnormal placental implantation in early pregnancy. Consistent with this shared pathophysiology, it is not uncommon to see growth restriction further confound the course of pre-eclampsia and vice versa. It has been previously suggested that superimposed growth restriction is associated with a more severe pre-eclamptic phenotype, however this has not been a consistent finding. Therefore, we set out to determine whether the presence of fetal growth restriction among women with severe early-onset pre-eclampsia was associated with more severe maternal disease compared to those without a growth-restricted fetus. METHODS AND FINDINGS: We undertook a retrospective cohort study of women presenting to a tertiary hospital with severe early-onset pre-eclampsia (<34 weeks' gestation) between 2005-2009. We collected clinical data, including severity of pre-eclampsia, maternal and neonatal outcomes. Of 176 cases of severe pre-eclampsia, 39% (n = 68) were further complicated by fetal growth restriction. However, no significant difference was seen in relation to the severity of pre-eclampsia between those with or without a growth-restricted baby. The presence of concomitant growth restriction was however associated with a significantly increased risk of stillbirth (p = 0.003) and total perinatal mortality (p = 0.02). CONCLUSIONS: The presence of fetal growth restriction among women with severe early-onset pre-eclampsia is not associated with increased severity of maternal disease. However the incidence of stillbirth and perinatal death is significantly increased in this sub-population

Aquatic therapy for boys with Duchenne muscular dystrophy (DMD): an external pilot randomised controlled trial

Background: Standard treatment of Duchenne muscular dystrophy (DMD) includes regular physiotherapy. There are no data to show whether adding aquatic therapy (AT) to land-based exercises helps maintain motor function. We assessed the feasibility of recruiting and collecting data from boys with DMD in a parallel-group pilot randomised trial (primary objective), also assessing how intervention and trial procedures work. Methods: Ambulant boys with DMD aged 7–16 years established on steroids, with North Star Ambulatory Assessment (NSAA) score ≥8, who were able to complete a 10-m walk test without aids or assistance, were randomly allocated (1:1) to 6 months of either optimised land-based exercises 4 to 6 days/week, defined by local community physiotherapists, or the same 4 days/week plus AT 2 days/week. Those unable to commit to a programme, with >20% variation between NSAA scores 4 weeks apart, or contraindications to AT were excluded. The main outcome measures included feasibility of recruiting 40 participants in 6 months from six UK centres, clinical outcomes including NSAA, independent assessment of treatment optimisation, participant/therapist views on acceptability of intervention and research protocols, value of information (VoI) analysis and cost-impact analysis. Results: Over 6 months, 348 boys were screened: most lived too far from centres or were enrolled in other trials; 12 (30% of the targets) were randomised to AT (n = 8) or control (n = 4). The mean change in NSAA at 6 months was −5.5 (SD 7.8) in the control arm and −2.8 (SD 4.1) in the AT arm. Harms included fatigue in two boys, pain in one. Physiotherapists and parents valued AT but believed it should be delivered in community settings. Randomisation was unattractive to families, who had already decided that AT was useful and who often preferred to enrol in drug studies. The AT prescription was considered to be optimised for three boys, with other boys given programmes that were too extensive and insufficiently focused. Recruitment was insufficient for VoI analysis. Conclusions: Neither a UK-based RCT of AT nor a twice weekly AT therapy delivered at tertiary centres is feasible. Our study will help in the optimisation of AT service provision and the design of future research. Trial registration: ISRCTN4100295

Selective Reagent Ion Mass Spectrometric Investigations of the Nitroanilines

This paper presents an investigation of proton and charge transfer reactions to 2-, 3- and 4-nitroanilines (C6H6N2O2) involving the reagent ions H3O+·(H2O)n (n=0, 1 and 2) and O2+, respectively, as a function of reduced electric field (60-240 Td), using Selective Reagent Ion-Time-of-Flight-Mass Spectrometry (SRI-ToF-MS). To aid in the interpretation of the H3O+·(H2O)n experimental data, the proton affinities and gas-phase basicities for the three nitroaniline isomers have been determined using density functional theory. These calculations show that proton transfer from both the H3O+ and H3O+·H2O reagent ions to the nitroanilines will be exoergic and hence efficient, with the reactions proceeding at the collisional rate. For proton transfer from H3O+ to the NO2 sites, the exoergicities are 171 kJ mol-1 (1.8 eV), 147 kJ mol-1 (1.5 eV) and 194 kJ mol-1 (2.0 eV) for 2-, 3- and 4-nitroanilines, respectively. Electron transfer from all three of the nitroanilines is also significantly exothermic by approximately 4 eV. Although a substantial transfer of energy occurs during the ion/molecule reactions, the processes are found to predominantly proceed via non-dissociative pathways over a large reduced electric field range. Only at relatively high reduced electric fields (>180 Td) is dissociative proton and charge transfer observed. Differences in fragment product ions and their intensities provide a means to distinguish the isomers, with proton transfer distinguishing 2-nitroaniline (2-NA) from 3- and 4-NA, and charge transfer distinguishing 4-NA from 2- and 3-NA, thereby providing a means to enhance selectivity using SRI-ToF-MS.(VLID)4826158Version of recor

Early postoperative MRI overestimates residual tumour after resection of gliomas with no or minimal enhancement

Standards for residual tumour measurement after resection of gliomas with no or minimal enhancement have not yet been established. In this study residual volumes on early and late postoperative T2-/FLAIR-weighted MRI are compared. A retrospective cohort included 58 consecutive glioma patients with no or minimal preoperative gadolinium enhancement. Inclusion criteria were first-time resection between 2007 and 2009 with a T2-/FLAIR-based target volume and availability of preoperative, early (<48 h) and late (1-7 months) postoperative MRI. The volumes of non-enhancing T2/FLAIR tissue and diffusion restriction areas were measured. Residual tumour volumes were 22% smaller on late postoperative compared with early postoperative T2-weighted MRI and 49% smaller for FLAIR-weighted imaging. Postoperative restricted diffusion volume correlated with the difference between early and late postoperative FLAIR volumes and with the difference between T2 and FLAIR volumes on early postoperative MRI. We observed a systematic and substantial overestimation of residual non-enhancing volume on MRI within 48 h of resection compared with months postoperatively, in particular for FLAIR imaging. Resection-induced ischaemia contributes to this overestimation, as may other operative effects. This indicates that early postoperative MRI is less reliable to determine the extent of non-enhancing residual glioma and restricted diffusion volumes are imperativ

Intraspecies Transmission of BASE Induces Clinical Dullness and Amyotrophic Changes

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrPTSE), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrPTSE type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle

Vertical Binocular Disparity is Encoded Implicitly within a Model Neuronal Population Tuned to Horizontal Disparity and Orientation

Primary visual cortex is often viewed as a “cyclopean retina”, performing the initial encoding of binocular disparities between left and right images. Because the eyes are set apart horizontally in the head, binocular disparities are predominantly horizontal. Yet, especially in the visual periphery, a range of non-zero vertical disparities do occur and can influence perception. It has therefore been assumed that primary visual cortex must contain neurons tuned to a range of vertical disparities. Here, I show that this is not necessarily the case. Many disparity-selective neurons are most sensitive to changes in disparity orthogonal to their preferred orientation. That is, the disparity tuning surfaces, mapping their response to different two-dimensional (2D) disparities, are elongated along the cell's preferred orientation. Because of this, even if a neuron's optimal 2D disparity has zero vertical component, the neuron will still respond best to a non-zero vertical disparity when probed with a sub-optimal horizontal disparity. This property can be used to decode 2D disparity, even allowing for realistic levels of neuronal noise. Even if all V1 neurons at a particular retinotopic location are tuned to the expected vertical disparity there (for example, zero at the fovea), the brain could still decode the magnitude and sign of departures from that expected value. This provides an intriguing counter-example to the common wisdom that, in order for a neuronal population to encode a quantity, its members must be tuned to a range of values of that quantity. It demonstrates that populations of disparity-selective neurons encode much richer information than previously appreciated. It suggests a possible strategy for the brain to extract rarely-occurring stimulus values, while concentrating neuronal resources on the most commonly-occurring situations

Single-copy nuclear genes resolve the phylogeny of the holometabolous insects

Background: Evolutionary relationships among the 11 extant orders of insects that undergo complete metamorphosis, called Holometabola, remain either unresolved or contentious, but are extremely important as a context for accurate comparative biology of insect model organisms. The most phylogenetically enigmatic holometabolan insects are Strepsiptera or twisted wing parasites, whose evolutionary relationship to any other insect order is unconfirmed. They have been controversially proposed as the closest relatives of the flies, based on rDNA, and a possible homeotic transformation in the common ancestor of both groups that would make the reduced forewings of Strepsiptera homologous to the reduced hindwings of Diptera. Here we present evidence from nucleotide sequences of six single-copy nuclear protein coding genes used to reconstruct phylogenetic relationships and estimate evolutionary divergence times for all holometabolan orders. Results: Our results strongly support Hymenoptera as the earliest branching holometabolan lineage, the monophyly of the extant orders, including the fleas, and traditionally recognized groupings of Neuropteroidea and Mecopterida. Most significantly, we find strong support for a close relationship between Coleoptera (beetles) and Strepsiptera, a previously proposed, but analytically controversial relationship. Exploratory analyses reveal that this relationship cannot be explained by long-branch attraction or other systematic biases. Bayesian divergence times analysis, with reference to specific fossil constraints, places the origin of Holometabola in the Carboniferous (355 Ma), a date significantly older than previous paleontological and morphological phylogenetic reconstructions. The origin and diversification of most extant insect orders began in the Triassic, but flourished in the Jurassic, with multiple adaptive radiations producing the astounding diversity of insect species for which these groups are so well known. Conclusion: These findings provide the most complete evolutionary framework for future comparative studies on holometabolous model organisms and contribute strong evidence for the resolution of the 'Strepsiptera problem', a long-standing and hotly debated issue in insect phylogenetics