Abstract Adipose-derived stem cells were isolated from rats and differentiated to a Schwann cell-like phenotype in vitro . The differentiated cells (dADSCs) underwent self-alignment in a tethered ...type-1 collagen gel, followed by stabilisation to generate engineered neural tissue (EngNT-dADSC). The pro-regenerative phenotype of dADSCs was enhanced by this process, and the columns of aligned dADSCs in the aligned collagen matrix supported and guided neurite extension in vitro . EngNT-dADSC sheets were rolled to form peripheral nerve repair constructs that were implanted within NeuraWrap conduits to bridge a 15 mm gap in rat sciatic nerve. After 8 weeks regeneration was assessed using immunofluorescence imaging and transmission electron microscopy and compared to empty conduit and nerve graft controls. The proportion of axons detected in the distal stump was 3.5 fold greater in constructs containing EngNT-dADSC than empty tube controls. Our novel combination of technologies that can organise autologous therapeutic cells within an artificial tissue construct provides a promising new cellular biomaterial for peripheral nerve repair.
Abstract A new combination of tissue engineering techniques provides a simple and effective method for building aligned cellular biomaterials. Self-alignment of Schwann cells within a tethered type-1 ...collagen matrix, followed by removal of interstitial fluid produces a stable tissue-like biomaterial that recreates the aligned cellular and extracellular matrix architecture associated with nerve grafts. Sheets of this engineered neural tissue supported and directed neuronal growth in a co-culture model, and initial in vivo tests showed that a device containing rods of rolled-up sheets could support neuronal growth during rat sciatic nerve repair (5 mm gap). Further testing of this device for repair of a critical-sized 15 mm gap showed that, at 8 weeks, engineered neural tissue had supported robust neuronal regeneration across the gap. This is, therefore, a useful new approach for generating anisotropic engineered tissues, and it can be used with Schwann cells to fabricate artificial neural tissue for peripheral nerve repair.
An 11-residue peptide with the sequence DS̱LEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl ...transferasecatalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an α-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.
Adipogenesis is a complex process that involves the differentiation of preadipocytes into mature adipocytes. We have developed two-dimensional (2D) and three-dimensional (3D) cell culture systems for ...the purpose of culturing and differentiating primary preadipocytes
in vitro. Differentiating preadipocytes show multiple lipid droplet accumulation and comparable protein expression patterns to mature adipocytes
in vivo. We report that in both
in vitro systems terminally differentiated adipocytes show characteristics similar to those of mature adipocytes
in vivo, assessed by the expression of the S100α/β protein, insulin receptor and caveolin-1, and receptors for inflammatory mediators, namely tumor necrosis factor-α receptors I and II (TNFRI and TNFRII) and chemokine receptor 5 (CCR5). Our results demonstrate that the S100 protein, caveolin-1, and insulin receptor are expressed and up-regulated in differentiating and terminally differentiated cells. In addition, the receptors for TNFα are not present in preadipocytes but are expressed in differentiating preadipocytes and in differentiated adipocytes. Similarly, CCR5 was exclusively expressed in differentiating preadipocytes and terminally differentiated adipocytes, but not in preadipocytes. Both 2D and 3D culture models are highly robust and reproducible and offer the potential to study adipogenesis and cellular interactions closely resembling and comparable to those
in vivo. Our 3D collagen system offers a distinct advantage over the 2D system in that the adipocytes remain confined within the matrix and remain intact during biochemical analysis. Moreover, the collagen matrix allows adipocytes to closely simulate morphological characteristics and behavior as
in vivo whilst permitting manipulation of the microenvironment
in vitro to study adipogenesis.
Efficient histological quantification of tumour-infiltrating T and B lymphocyte (TIL) subsets in archival tissues would greatly facilitate investigations of the role of TIL in human cancer biology. ...We sought to develop such a method.
Ten ×40 digital images of 4 μ sections of 16 ductal invasive breast carcinomas immunostained for CD3, CD4, CD8, and CD20 were acquired (a total of 640 images). The number of pixels in each image matching a partition of Lab colour space corresponding to immunostained cells were counted using the ‘Color range’ and ‘Histogram’ tools in Adobe Photoshop 7. These pixel counts were converted to cell counts per mm
2 using a calibration factor derived from one, two, three or all 10 images of each case/antibody combination.
Variations in the number of labelled pixels per immunostained cell made individual calibration for each case/antibody combination necessary. Calibration based on two fields containing the most labelled pixels gave a cell count minimally higher (+
5.3%) than the count based on 10-field calibration, with 95% confidence limits −
14.7 to +
25.3%. As TIL density could vary up to 100-fold between cases, this accuracy and precision are acceptable.
The methodology described offers sufficient accuracy, precision and efficiency to quantify the density of TIL sub-populations in breast cancer using commonly available software, and could be adapted to batch processing of image files.
De novo mutations in protein-coding genes are a well-established cause of developmental disorders
. However, genes known to be associated with developmental disorders account for only a minority of ...the observed excess of such de novo mutations
. Here, to identify previously undescribed genes associated with developmental disorders, we integrate healthcare and research exome-sequence data from 31,058 parent-offspring trios of individuals with developmental disorders, and develop a simulation-based statistical test to identify gene-specific enrichment of de novo mutations. We identified 285 genes that were significantly associated with developmental disorders, including 28 that had not previously been robustly associated with developmental disorders. Although we detected more genes associated with developmental disorders, much of the excess of de novo mutations in protein-coding genes remains unaccounted for. Modelling suggests that more than 1,000 genes associated with developmental disorders have not yet been described, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of genes associated with developmental disorders.
Mapping the transport and deposition of tephra is important for the assessment of an eruption’s impact on health, transport, vegetation and infrastructure, but it is challenging at large distances ...from a volcano (> 1000 km), where it may not be visible to the naked eye. Here we describe a range of methods used to quantify tephra deposition and impact on air quality during the 21–28 May 2011 explosive basaltic eruption of Grímsvötn volcano, Iceland. Tephra was detected in the UK with tape-on-paper samples, rainwater samples, rainwater chemistry analysis, pollen slides and air quality measurements. Combined results show that deposition was mainly in Scotland, on 23–25 May. Deposition was patchy, with adjacent locations recording different results. Tape-on-paper samples, collected by volunteer citizen scientists, and giving excellent coverage across the UK, showed deposition at latitudes >55°N, mainly on 24 May. Rainwater samples contained ash grains mostly 20–30
μ
m
long (maximum recorded grainsize 80
μ
m
) with loadings of up to 116
g
r
a
i
n
s
c
m
-2
. Analysis of rainwater chemistry showed high concentrations of dissolved Fe and Al in samples from N Scotland on 24–27 May. Pollen slides recorded small glass shards (3–4
μ
m
long) deposited during rainfall on 24–25 May and again on 27 May. Air quality monitoring detected increased particulate matter concentrations in many parts of the country. An hourly concentration of particles < 10
μ
m
in diameter (PM
10
) of ∼413
μ
g
m
-3
, was measured in Aberdeen at 02:00hrs on 24 May 2011. Significant peaks of non-anthropogenic PM, which is most likely to have a volcanic origin, could be tracked as far south as the English Midlands (> 53°N) on 24 May but no negative effects on health were reported. Although the eruption column reached altitudes of 20 km above sea level, air mass trajectories suggest that only tephra from the lowest 4 km above sea level of the eruption plume was transported to the UK. This demonstrates that even low plumes could deliver tephra to the UK and suggests that the relative lack of basaltic tephra in the tephrochronological record is not due to transport processes.
•We found a high asymptomatic rate in vulnerable people with epilepsy.•Enhanced surveillance allows to quickly contain outbreaks.•We report a low rate of COVID-19 morbidity and mortality in a ...long-term care facility.•Preventative measures allow reducing resident-to-resident and -to-caregiver transmission.•Children and young adults appear to have lower infection rates.
In this cohort study, we aim to compare outcomes from coronavirus disease 2019 (COVID-19) in people with severe epilepsy and other co-morbidities living in long-term care facilities which all implemented early preventative measures, but different levels of surveillance.
During 25-week observation period (16 March–6 September 2020), we included 404 residents (118 children), and 1643 caregivers. We compare strategies for infection prevention, control, and containment, and related outcomes, across four UK long-term care facilities. Strategies included early on-site enhancement of preventative and infection control measures, early identification and isolation of symptomatic cases, contact tracing, mass surveillance of asymptomatic cases and contacts. We measured infection rate among vulnerable people living in the facilities and their caregivers, with asymptomatic and symptomatic cases, including fatality rate.
We report 38 individuals (17 residents) who tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive, with outbreaks amongst residents in two facilities. At Chalfont Centre for Epilepsy (CCE), 10/98 residents tested positive: two symptomatic (one died), eight asymptomatic on weekly enhanced surveillance; 2/275 caregivers tested positive: one symptomatic, one asymptomatic. At St Elizabeth’s (STE), 7/146 residents tested positive: four symptomatic (one died), one positive during hospital admission for symptoms unrelated to COVID-19, two asymptomatic on one-off testing of all 146 residents; 106/601 symptomatic caregivers were tested, 13 positive. In addition, during two cycles of systematically testing all asymptomatic carers, four tested positive. At The Meath (TM), 8/80 residents were symptomatic but none tested; 26/250 caregivers were tested, two positive. At Young Epilepsy (YE), 8/80 children were tested, all negative; 22/517 caregivers were tested, one positive.
Infection outbreaks in long-term care facilities for vulnerable people with epilepsy can be quickly contained, but only if asymptomatic individuals are identified through enhanced surveillance at resident and caregiver level. We observed a low rate of morbidity and mortality, which confirmed that preventative measures with isolation of suspected and confirmed COVID-19 residents can reduce resident-to-resident and resident-to-caregiver transmission. Children and young adults appear to have lower infection rates. Even in people with epilepsy and multiple co-morbidities, we observed a high percentage of asymptomatic people suggesting that epilepsy-related factors (anti-seizure medications and seizures) do not necessarily lead to poor outcomes.
The construct validity of self‐reported counterproductive work behaviors (CPBs) and organizational citizenship behaviors (OCBs) was explored by separating item content from item wording, through the ...confirmatory factor analysis of three scales: a CPB scale containing only negatively worded items, an OCB scale with only positively worded items, and the supervision subscale of the Job Descriptive Index (JDI) (Smith, Kendall and Hulin 1969) comprised of both negatively and positively worded items. Results (N = 475) suggest that self‐report measures of CPBs and OCBs are affected by method variance, but that the presence of such an influence does not compromise the substantive interpretation of these scales. Consequently, these scales do appear to be unique constructs.
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