Proteomic assessment of a cell model of spinal muscular atrophy

Background Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES) cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease. Results When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8) in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament) and motor neuron markers (Hb9, Islet-1, and ChAT). Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells), the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived. Conclusion SMN-deficient ES cells provide a cell-culture model for SMA. SMN deficiency activates cellular stress pathways, causing a dysregulation of energy metabolism, protein degradation, and cytoskeleton stability

A review of biophysical and socio-economic effects of unconventional oil and gas extraction - implications for South Africa

The impacts associated with unconventional oil and gas (UOG) extraction will be cumulative in nature and will most likely occur on a regional scale, highlighting the importance of using strategic decision-making and management tools. Managing possible impacts responsibly is extremely important in a water scarce country such as South Africa, versus countries where more water may be available for UOG extraction activities. This review article explains the possible biophysical and socioeconomic impacts associated with UOG extraction within the South African context and how these complex impacts interlink. Relevant policy and governance frameworks to manage these impacts are also highlighted.The Water Research Commission, South Africa, is thanked for providing funding for this research.http://www.elsevier.com/locate/jenvman2017-12-31hb2017Geolog

Vulnerability mapping as a tool to manage the environmental impacts of oil and gas extraction

Various biophysical and socio-economic impacts may be associated with unconventional oil and gas (UOG) extraction. A vulnerability map may assist governments during environmental assessments, spatial planning and the regulation of UOG extraction, as well as decision-making around UOG extraction in fragile areas. A regional interactive vulnerability map was developed for UOG extraction in South Africa. This map covers groundwater, surface water, vegetation, socio-economics and seismicity as mapping themes, based on impacts that may emanate from UOG extraction. The mapping themes were developed using a normative approach, where expert input during the identification and classification of vulnerability indicators may increase the acceptability of the resultant map. This article describes the development of the interactive vulnerability map for South Africa, where UOG extraction is not yet allowed and where regulations are still being developed to manage this activity. The importance and policy implications of using vulnerability maps for managing UOG extraction impacts in countries where UOG extraction is planned are highlighted in this article.The Water Research Commission, South Africahttp://rsos.royalsocietypublishing.orgam2018Geolog

The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology

Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7−/− mice. Early postnatal Tdrd7−/− animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7−/− lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7−/− cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7−/− lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7−/− cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7−/− lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7−/− fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7–ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7’s novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts

Proteomic assessment of a cell model of spinal muscular atrophy

Abstract Background Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES) cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease. Results When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8) in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament) and motor neuron markers (Hb9, Islet-1, and ChAT). Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells), the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived. Conclusion SMN-deficient ES cells provide a cell-culture model for SMA. SMN deficiency activates cellular stress pathways, causing a dysregulation of energy metabolism, protein degradation, and cytoskeleton stability.</p

Monitoring of unconventional oil and gas extraction and its policy implications : a case study from South Africa

Biophysical and socio-economic monitoring during unconventional oil and gas (UOG) extraction is important to assess change and to have reference conditions against which to identify UOG extraction activity impacts. The large-scale cumulative impacts of UOG extraction makes standardised monitoring across geographic and socio-political regions important. This article emphasises the importance of a robust monitoring framework that must serve as a guideline for planning monitoring activities during UOG extraction. A case study from South Africa is presented to illustrate important aspects to address during the development of a UOG extraction monitoring framework. The South African case is critically assessed and resultant policy implications are discussed. Important policy considerations include performing baseline monitoring during UOG extraction, performing UOG extraction monitoring in an integrated, systematic, and standardised manner, ensuring that proper resources are available to perform the monitoring and implementing an adaptive management plan that is linked to UOG extraction monitoringThe Water Research Commission , South Africahttp://www.elsevier.com/locate/enpol2019-07-01hj2018Geolog

Vulnerability mapping as a tool to manage the environmental impacts of oil and gas extraction

Various biophysical and socio-economic impacts may be associated with unconventional oil and gas (UOG) extraction. A vulnerability map may assist governments during environmental assessments, spatial planning and the regulation of UOG extraction, as well as decision-making around UOG extraction in fragile areas. A regional interactive vulnerability map was developed for UOG extraction in South Africa. This map covers groundwater, surface water, vegetation, socio-economics and seismicity as mapping themes, based on impacts that may emanate from UOG extraction. The mapping themes were developed using a normative approach, where expert input during the identification and classification of vulnerability indicators may increase the acceptability of the resultant map. This article describes the development of the interactive vulnerability map for South Africa, where UOG extraction is not yet allowed and where regulations are still being developed to manage this activity. The importance and policy implications of using vulnerability maps for managing UOG extraction impacts in countries where UOG extraction is planned are highlighted in this article.The Water Research Commission, South Africahttp://rsos.royalsocietypublishing.orgam2018Geolog