Identifying the Molecular Cause of Extreme Endoplasmic Reticulum Dilation in Pediatric Osteosarcoma and Its Relationship to the Disease

Pediatric osteosarcoma tumors are characterized by an unusual abundance of grossly dilated endoplasmic reticulum and an immense genomic instability that has complicated identifying new effective molecular therapeutic targets. Here we report a novel molecular signature that encompasses the majority of 108 patient tumor samples, PDXs and osteosarcoma cell lines. These tumors exhibit reduced expression of four critical COPII vesicle proteins that has resulted in the accumulation of procollagen-I protein within ‘hallmark’ dilated ER. Using CRISPR activation technology, increased expression of only SAR1A and SEC24D to physiologically normal levels was sufficient to restore both collagen-I secretion and resolve dilated ER morphology to normal

Quit the Botching, Ohio: Exploring the Flaws in the Ohio Execution Protocol and the Need for Change

This note argues that the Ohio Department of Rehabilitation and Corrections (ODRC) should not obtain lethal injection drugs from unregulated compounding pharmacies. Ohio should only purchase drugs from an FDA-registered outsourcing facility. Part II explores the death sentence statute in Ohio and the use of compounding pharmacies. Part III compares Oklahoma\u27s statute in conjunction with Ohio and illustrates the adverse effects by utilizing compounding pharmacies. Part IV proposes recommendations to Ohio\u27s execution protocol. Part V provides a conclusion

Towards an allogeneic therapy for neural regeneration

Injuries to the central nervous system (CNS) can be devastating. CNS injuries include those to the spinal cord, where there can be a complete loss of function below the point of injury. Spinal cord injuries impact up to 500,000 people worldwide every year and where function is lost, quality of life can be severely limited. Olfactory ensheathing cells (OECs) are a promising cell therapy candidate for treatment of neurologic injury as they have been shown to promote neuronal survival and facilitate regeneration of severed axons. Despite their unique properties, OECs are very challenging cells to work with since they are difficult to isolate, difficult to sustain in culture for prolonged periods and there is still controversy around how to characterize their identity and potency. Due to the inherent variability of OEC yield in biopsies and difficulties in growing these cells, producing an autologous therapy is not currently a viable option. Therefore, we sought to develop allogeniec OEC lines using a conditional immortalization tool. We undertook characterization work to ensure they expressed key putative OEC markers (such as p75NTR and S100β) and were able to support neuron extension in in vitro models. Subsequently, extensive bioprocess development was undertaken to investigate parameters such as: Cell culture conditions, e.g. effect of different culture media, initial seeding densities and microcarrier-based expansion Characterization process parameters, e.g. effect of culture time and pre-selection on identity marker expression, impact of detergents on identity marker measurement, and finally their impact in functional assays (including neuron co-culture to identify the impact of processing on neuronal support activity). A number of important insights have been gained from this work including: identity markers are transient making characterization challenging; and analytics methodologies employed themselves affect what is measurable. However, these insights are informing our future approach to creating candidate cell lines for treatment of neurologic injury

Impact of dual cell co-culture and cell-conditioned media on yield and function of a human olfactory cell line for regenerative medicine

Olfactory ensheathing cells (OECs) are a promising candidate therapy for neuronal tissue repair. However, appropriate priming conditions to drive a regenerative phenotype are yet to be determined. We first assessed the effect of using a human fibroblast feeder layer and fibroblast conditioned media on primary rat olfactory mucosal cells (OMCs). We found that OMCs cultured on fibroblast feeders had greater expression of the key OEC marker p75NTR (25.1 ± 10.7 cells/mm2) compared with OMCs cultured on laminin (4.0 ± 0.8 cells/mm2, p = 0.001). However, the addition of fibroblast-conditioned media (CM) resulted in a significant increase in Thy1.1 (45.9 ± 9.0 cells/mm2 versus 12.5 ± 2.5 cells/mm2 on laminin, p = 0.006), an undesirable cell marker as it is regarded to be a marker of contaminating fibroblasts. A direct comparison between human feeders and GMP cell line Ms3T3 was then undertaken. Ms3T3 cells supported similar p75NTR levels (10.7 ± 5.3 cells/mm2) with significantly reduced Thy1.1 expression (4.8 ± 2.1 cells/mm2). Ms3T3 cells were used as feeder layers for human OECs to determine whether observations made in the rat model were conserved. Examination of the OEC phenotype (S100β expression and neurite outgrowth from NG108-15 cells) revealed that co-culture with fibroblast feeders had a negative effect on human OECs, contrary to observations of rat OECs. CM negatively affected rat and human OECs equally. When the best and worst conditions in terms of supporting S100β expression were used in NG108-15 neuron co-cultures, those with the highest S100β expression resulted in longer and more numerous neurites (22.8 ± 2.4 μm neurite length/neuron for laminin) compared with the lowest S100β expression (17.9 ± 1.1 μm for Ms3T3 feeders with CM). In conclusion, this work revealed that neither dual co-culture nor fibroblast-conditioned media support the regenerative OEC phenotype. In our case, a preliminary rat model was not predictive of human cell responses

Investigating the requirement for dual cell co-culture platforms in creating regenerative cell therapies for CNS injury

Injuries to the central nervous system (CNS) can be devastating. CNS injuries include those to the spinal cord, where there can be a complete loss of function below the point of injury. Spinal cord injury impacts up to 500,000 people worldwide every year and where function is lost, quality of life can be severely limited. Olfactory ensheathing cells (OECs) are a candidate cell therapy for spinal cord injury as they can promote neuronal survival and facilitate regeneration of severed axons. Despite their unique properties, OECs are very challenging cells to work with because they are difficult to isolate, difficult to sustain in culture for prolonged periods and there is still controversy around how to characterize their identity and potency. The overall aim of this project is to identify methods to enhance the survival, growth and function of OECs. It has been reported that for OECs to be truly functional they require interaction with fibroblasts. Therefore, we sought to investigate whether it is necessary to use fibroblasts as a feeder layer to support OECs via physical cell-cell contact, or whether paracrine soluble factors in the conditioned media from fibroblasts would provide the trophic support necessary to enhance OEC survival and growth. A human mucosal fibroblast cell line was used as a feeder layer. Primary rat OECs were cultured for 14 days on the feeder layer or control substrate (laminin-coated dishes). After 14 days, the morphology of cells was assessed and an algorithm generated using ImageJ was used to ascribe a mathematical value to OEC morphology to determine whether a correlation of morphology to expression of markers could be made. Cells cultured on feeders adopted a more spindle-like appearance compared with cells cultured on laminin, which adopted an enlarged morphology. The algorithm was used to analyse the circularity of cells that labelled positive for candidate identity marker S100b. It was found that cells cultured on feeders had a lower circularity, and therefore more elongated shape compared to those cultured on laminin (p=0.037). Additionally, a significant increase in p75NTR expression (a second candidate OEC marker) was observed (p=0.01) on feeders. To further investigate the relationship between the OECs and the feeders, cells were cultured in the presence of conditioned media from the fibroblasts. When cells were cultured in conditioned media there was a significant (p=0.002) upregulation of Thy1, an undesirable marker, and the significance of this will be investigated further with work underway to compare different feeder types and their impact on marker expression

Physiological Effects of Five Different Marine Natural Organic Matters (NOMs) and Three Different Metals (Cu, Pb, Zn) on Early Life Stages of the Blue Mussel (Mytilus galloprovincialis)

Metals are present in aquatic environments as a result of natural and anthropogenic inputs, and may induce toxicity to organisms. One of the main factors that influence this toxicity in fresh water is natural organic matter (NOM) but all NOMs are not the same in this regard. In sea water, possible protection by marine NOMs is not well understood. Thus, our study isolated marine NOMs by solid-phase extraction from five different sites and characterized them by excitation-emission fluorescence analysis—one inshore (terrigenous origin), two offshore (autochthonous origin), and two intermediate in composition (indicative of a mixed origin). The physiological effects of these five NOMS alone (at 8 mg/L), of three metals alone (copper, lead and zinc at 6 µg Cu/L, 20 µg Pb/L, and 25 µg Zn/L respectively), and of each metal in combination with each NOM, were evaluated in 48-h exposures of mussel larvae. Endpoints were whole body Ca2++Mg2+-ATPase activity, carbonic anhydrase activity and lipid peroxidation. By themselves, NOMs increased lipid peroxidation, Ca2++Mg2+-ATPase, and/or carbonic anhydrase activities (significant in seven of 15 NOM-endpoint combinations), whereas metals by themselves did not affect the first two endpoints, but Cu and Pb increased carbonic anhydrase activities. In combination, the effects of NOMs predominated, with the metal exerting no additional effect in 33 out of 45 combinations. While NOM effects varied amongst different isolates, there was no clear pattern with respect to optical or chemical properties. When NOMs were treated as a single source by data averaging, NOM had no effect on Ca2++Mg2+-ATPase activity but markedly stimulated carbonic anhydrase activity and lipid peroxidation, and there were no additional effects of any metal. Our results indicate that marine NOMs may have direct effects on this model marine organism, as well as protective effects against metal toxicity, and the quality of marine NOMs may be an important factor in these actions

Changing child health surveillance in Scotland : an exploration of the impact on preventive health care of pre-school children

The health service provides a Child Health Programme (CHP) to all children to help them attain their health and development potential. Core elements include screening, immunisations, growth and development surveillance, health promotion advice, and parenting support. The surveillance/advice/support components (known as Child Health Surveillance CHS) are delivered through a series of universally offered child health reviews mainly provided by Health Visitors (HVs) supplemented by additional support as required. Scottish policy issued in 2005 led to considerable changes to the CHP. The number of CHS reviews was substantially reduced to enable more intensive support of children who required it. A three category indicator of need was introduced at the same time to facilitate the identification of children requiring enhanced support. This thesis aims to explore the shift to more targeted provision of CHS that occurred from 2005 onwards, and to examine the impact of this on the preventive health care provided to pre-school children. The specific objectives are: · To describe the development of professional guidance on the CHP and how this has been adopted into Scottish policy. · To compare the CHP provided in Scotland to that offered in other high income countries. · To examine the impact of the changes to CHS on the coverage of universally offered child health reviews. · To explore, following the changes to CHS, which factors are associated with children being identified as in need of enhanced CHP support. · To assess the impact of the changes to CHS on the totality of preventive care provided to pre-school children by HVs and General Practitioners (GPs). The key methods used are literature review, policy analysis, and analysis of routine health data. Selected findings include the following: · All the high income countries studied provide the same basic elements as the Scottish CHP but the detail of the different programmes varies considerably. Some of the variation may reflect the different needs of different populations, but much seems to reflect different approaches to evidence interpretation and policy making in different settings. · Not all children offered ‘universal’ child health reviews actually receive them. Children from deprived areas are less likely to receive their reviews. Inequalities in review coverage have remained unchanged after the changes to CHS. · Many factors, including those reflecting infant and maternal health and family social risk, are associated with being identified by HVs as needing enhanced CHP support. The threshold at which children are identified as needing enhanced support varies between areas across Scotland. · GP provision of child health reviews has reduced after the changes to CHS as would be expected. Recorded GP provision of other preventive care consultations is uncommon and has not changed. Currently available routine data do not allow trends in the totality of HV provided care to be examined. In summary, the Child Health Programme makes an important contribution to supporting young children and their families but it is a complex service and considerable uncertainty about aspects of its content and delivery remain