Direct observation of the gene organization of the complement C4 and 21-hydroxylase loci by pulsed field gel electrophoresis.

Pulsed field gel electrophoresis and enzymes that cut genomic DNA infrequently have been used to define large RFLPs at the human C4 loci. With the enzymes BssH II or Sac II, and C4 or 21-hydroxylase DNA probes, it has been possible to observe directly the number of C4 genes present on a haplotype, and also whether the C4 genes are long (6-7-kb intron present) or short (6-7-kb intron absent). Haplotypes that have either two long C4 genes or one long and one short C4 gene generate BssH II fragments of approximately 115 or approximately 105 kb, respectively. Haplotypes that have either a single long or a single short C4 gene generate BssH II fragments of approximately 80 or approximately 70 kb, respectively. This technique has been used to analyze the DNA isolated from PBMC and allows the complete definition of the C4 gene organization of an individual without the need for family studies

Development of three-dimensional collagen scaffolds with controlled architecture for cell migration studies using breast cancer cell lines

Cancer is characterized by cell heterogeneity and the development of 3D $\textit{in vitro }$ assays that can distinguish more invasive or migratory phenotypes could enhance diagnosis or drug discovery. 3D collagen scaffolds have been used to develop analogues of complex tissues $\textit{in vitro }$ and are suited to routine biochemical and immunological assays. We sought to increase 3D model tractability and modulate the migration rate of seeded cells using an ice-templating technique to create either directional/anisotropic or non-directional/isotropic porous architectures within cross-linked collagen scaffolds. Anisotropic scaffolds supported the enhanced migration of an invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. In addition, MDA-MB-468 showed increased migration upon epithelial-to-mesenchymal transition (EMT) in anisotropic scaffolds. The provision of controlled architecture in this system may act both to increase assay robustness and as a tuneable parameter to capture detection of a migrated population within a set time, with consequences for primary tumour migration analysis. The separation of invasive clones from a cancer biomass with in vitro platforms could enhance drug development and diagnosis testing by contributing assay metrics including migration rate, as well as modelling cell-cell and cell-matrix interaction in a system compatible with routine histopathological testing.The authors gratefully acknowledge the financial support of the ERC Advanced Grant 320598 3D-E and the Newton Trust. A.H. held a Daphne Jackson Fellowship funded by the University of Cambridge for part of the work. R.D.H. is funded through a NC3Rs studentship

Cross Section Ratios between different CM energies at the LHC: opportunities for precision measurements and BSM sensitivity

The staged increase of the LHC beam energy provides a new class of interesting observables, namely ratios and double ratios of cross sections of various hard processes. The large degree of correlation of theoretical systematics in the cross section calculations at different energies leads to highly precise predictions for such ratios. We present in this letter few examples of such ratios, and discuss their possible implications, both in terms of opportunities for precision measurements and in terms of sensitivity to Beyond the Standard Model dynamics.Comment: 19 pages, 9 figure

Recent history, current status, conservation and management of native mammalian carnivore species in Great Britain

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record After historical declines in population sizes and ranges, we compare and contrast the recent history and contemporary variation in the status of Great Britain's eight native mammalian carnivore species from the 1960s to 2017. Wildcat Felis silvestris conservation status is unfavourable and is masked by hybridisation with domestic cats Felis catus. Red foxes Vulpes vulpes remain widespread but are currently declining. European otter Lutra lutra, European pine marten Martes martes and European polecat Mustela putorius populations are characterised by rapid recovery. Otters have almost completely recolonised Great Britain, polecats have expanded their range throughout southern Britain from refugia in Wales and pine martens have expanded their range from the Scottish Highlands. European badgers Meles meles have generally increased in population density. Status assessments of stoats Mustela erminea and weasels Mustela nivalis are data-deficient but available evidence suggests that stoats may have increased while weasels may have declined. Anthropogenic processes influencing carnivore status include legal protections, habitat quality, reintroductions, predator control, pollutants, hybridisation and diseases and their associated control practices. Population effects of contaminants, such as anticoagulant rodenticides, remain poorly characterised. The widespread interface with domestic and feral cats makes the wildcat's situation precarious. Recent declines in rabbit Oryctolagus cuniculus populations are a concern, given that several carnivore species depend on them as food. We conclude that, with the exception of the wildcat, the status of Great Britain's mammalian carnivores has markedly improved since the 1960s. Better understanding of the social aspects of interactions between humans and expanding predator populations is needed if conflict is to be avoided and long-term co-existence with people is to be possible.Vincent Wildlife TrustUniversity of Exete

b-Initiated processes at the LHC: a reappraisal

Several key processes at the LHC in the standard model and beyond that involve $b$ quarks, such as single-top, Higgs, and weak vector boson associated production, can be described in QCD either in a 4-flavor or 5-flavor scheme. In the former, $b$ quarks appear only in the final state and are typically considered massive. In 5-flavor schemes, calculations include $b$ quarks in the initial state, are simpler and allow the resummation of possibly large initial state logarithms of the type $\log \frac{{\cal Q}^2}{m_b^2}$ into the $b$ parton distribution function (PDF), ${\cal Q}$ being the typical scale of the hard process. In this work we critically reconsider the rationale for using 5-flavor improved schemes at the LHC. Our motivation stems from the observation that the effects of initial state logs are rarely very large in hadron collisions: 4-flavor computations are pertubatively well behaved and a substantial agreement between predictions in the two schemes is found. We identify two distinct reasons that explain this behaviour, i.e., the resummation of the initial state logarithms into the $b$-PDF is relevant only at large Bjorken $x$ and the possibly large ratios ${\cal Q}^2/m_b^2$'s are always accompanied by universal phase space suppression factors. Our study paves the way to using both schemes for the same process so to exploit their complementary advantages for different observables, such as employing a 5-flavor scheme to accurately predict the total cross section at NNLO and the corresponding 4-flavor computation at NLO for fully exclusive studies.Comment: Fixed typo in Eq. (A.10) and few typos in Eq. (C.2) and (C.3

Delayed onset of changes in soma action potential genesis in nociceptive A-beta DRG neurons in vivo in a rat model of osteoarthritis

<p>Abstract</p> <p>Background</p> <p>Clinical data on osteoarthritis (OA) suggest widespread changes in sensory function that vary during the progression of OA. In previous studies on a surgically-induced animal model of OA we have observed that changes in structure and gene expression follow a variable trajectory over the initial days and weeks. To investigate mechanisms underlying changes in sensory function in this model, the present electrophysiological study compared properties of primary sensory nociceptive neurons at one and two months after model induction with properties in naïve control animals. Pilot data indicated no difference in C- or Aδ-fiber associated neurons and therefore the focus is on Aβ-fiber nociceptive neurons.</p> <p>Results</p> <p>At one month after unilateral derangement of the knee by cutting the anterior cruciate ligament and removing the medial meniscus, the only changes observed in Aβ-fiber dorsal root ganglion (DRG) neurons were in nociceptor-like unresponsive neurons bearing a hump on the repolarization phase; these changes consisted of longer half width, reflecting slowed dynamics of AP genesis, a depolarized Vm and an increased AP amplitude. At two months, changes observed were in Aβ-fiber high threshold mechanoreceptors, which exhibited shorter AP duration at base and half width, shorter rise time and fall time, and faster maximum rising rate/maximum falling rate, reflecting accelerated dynamics of AP genesis.</p> <p>Conclusion</p> <p>These data indicate that Aβ nociceptive neurons undergo significant changes that vary in time and occur later than changes in structure and in nociceptive scores in this surgically induced OA model. Thus, if changes in Aβ-fiber nociceptive neurons in this model reflect a role in OA pain, they may relate to mechanisms underlying pain associated with advanced OA.</p

Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

STUDY QUESTION: Can label-free, non-invasive optical imaging by hyperspectral autofluorescence microscopy discern between euploid and aneuploid cells within the inner cell mass (ICM) of the mouse preimplantation embryo? SUMMARY ANSWER: Hyperspectral autofluorescence microscopy enables discrimination between euploid and aneuploid ICM in mouse embryos. WHAT IS KNOWN ALREADY: Euploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilized to diagnose aneuploidy and mosaicism in clinical in vitro fertilization. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the foetal lineage ICM of some blastocyst embryos. STUDY DESIGN, SIZE, DURATION: The impact of aneuploidy on cellular autofluorescence and metabolism of primary human fibroblast cells and mouse embryos was assessed using a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (4-6 independent replicates, euploid n = 467; aneuploid n = 969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n = 39; reversine n = 44) and chimeric blastocysts (eight independent replicates: control n = 34; reversine n = 34; 1:1 (control:reversine) n = 30 and 1:3 (control:reversine) n = 37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n = 13 euploid and n = 9 aneuploid). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two models were employed: (i) primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the four- to eight-cell division. Individual blastomeres were dissociated from control and reversine-treated eight-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Individual blastomeres and embryos were interrogated by hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic co-factors (inferred from their autofluorescence signature): NAD(P)H and flavins with the subsequent calculation of the optical redox ratio (ORR: flavins/[NAD(P)H + flavins]). Autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features from each cell/blastomere/ICM. This was used to discriminate between different cell populations. MAIN RESULTS AND THE ROLE OF CHANCE: An increase in the relative abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for aneuploid cells in primary human fibroblasts and reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of endogenous cell autofluorescence achieved separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres cells, (iii) control and reversine-treated chimeric blastocysts, (iv) 1:1 and 1:3 chimeric blastocysts and (v) confirmed euploid and aneuploid ICM from mouse blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.97, 0.99, 0.87, 0.88 and 0.93, respectively. We believe that the role of chance is low as mathematical features separated euploid from aneuploid in both human fibroblasts and ICM of mouse blastocysts.N/A. LIMITATIONS, REASONS FOR CAUTION: Although we were able to discriminate between euploid and aneuploid ICM in mouse blastocysts, confirmation of this approach in human embryos is required. While we show this approach is safe in mouse, further validation is required in large animal species prior to implementation in a clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: We have developed an original, accurate and non-invasive optical approach to assess aneuploidy within the ICM of mouse embryos in the absence of fluorescent tags. Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid human fibroblast and mouse blastocysts (ICM). This approach may potentially lead to a new diagnostic for embryo analysis. STUDY FUNDING/COMPETING INTEREST(S): K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003) and the National Health and Medical Research Council (APP2003786). The authors declare that there is no conflict of interest