Expression microdissection isolation of enriched cell populations from archival brain tissue

BACKGROUND: Laser capture microdissection (LCM) is an established technique for the procurement of enriched cell populations that can undergo further downstream analysis, although it does have limitations. Expression microdissection (xMD) is a new technique that begins to address these pitfalls, such as operator dependence and contamination. NEW METHOD: xMD utilises immunohistochemistry in conjunction with a chromogen to isolate specific cell types by extending the fundamental principles of LCM to create an operator-independent method for the procurement of specific CNS cell types. RESULTS: We report how xMD enables the isolation of specific cell populations, namely neurones and astrocytes, from rat formalin fixed-paraffin embedded (FFPE) tissue. Subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirms the enrichment of these specific populations. RIN values after xMD indicate samples are sufficient to carry out further analysis. COMPARISON WITH EXISTING METHOD: xMD offers a rapid method of isolating specific CNS cell types without the need for identification by an operator, reducing the amount of unintentional contamination caused by operator error, whilst also significantly reducing the time required by the current basic LCM technique. CONCLUSIONS: xMD is a superior method for the procurement of enriched cell populations from post-mortem tissue, which can be utilised to create transcriptome profiles, aiding our understanding of the contribution of these cells to a range of neurological diseases. xMD also addresses the issues associated with LCM, such as reliance on an operator to identify target cells, which can cause contamination, as well as addressing the time consuming nature of LCM

Transcriptomic analysis of human astrocytes in vitro reveals hypoxia-induced mitochondrial dysfunction, modulation of metabolism, and dysregulation of the immune response

Hypoxia is a feature of neurodegenerative diseases, and can both directly and indirectly impact on neuronal function through modulation of glial function. Astrocytes play a key role in regulating homeostasis within the central nervous system, and mediate hypoxia-induced changes in response to reduced oxygen availability. The current study performed a detailed characterization of hypoxia-induced changes in the transcriptomic profile of astrocytes in vitro. Human astrocytes were cultured under normoxic (5% CO2, 95% air) or hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 h, and the gene expression profile assessed by microarray analysis. In response to hypoxia 4904 genes were significantly differentially expressed (1306 upregulated and 3598 downregulated, FC ≥ 2 and p ≤ 0.05). Analysis of the significant differentially expressed transcripts identified an increase in immune response pathways, and dysregulation of signalling pathways, including HIF-1 (p = 0.002), and metabolism, including glycolysis (p = 0.006). To assess whether the hypoxia-induced metabolic gene changes observed affected metabolism at a functional level, both the glycolytic and mitochondrial flux were measured using an XF bioanalyser. In support of the transcriptomic data, under physiological conditions hypoxia significantly reduced mitochondrial respiratory flux (p = 0.0001) but increased basal glycolytic flux (p = 0.0313). However, when metabolically stressed, hypoxia reduced mitochondrial spare respiratory capacity (p = 0.0485) and both glycolytic capacity (p = 0.0001) and glycolytic reserve (p < 0.0001). In summary, the current findings detail hypoxia-induced changes in the astrocyte transcriptome in vitro, identifying potential targets for modifying the astrocyte response to reduced oxygen availability in pathological conditions associated with ischaemia/hypoxia, including manipulation of mitochondrial function, metabolism, and the immune response

Persistent DNA damage alters the neuronal transcriptome suggesting cell cycle dysregulation and altered mitochondrial function

Oxidative DNA damage induces changes in the neuronal cell cycle and activates a DNA damage response to promote repair, but these processes may be altered under a chronic oxidative environment, leading to the accumulation of unrepaired DNA damage and continued activation of a DNA damage response. Failure to repair DNA damage can lead to apoptosis or senescence, which is characterized by a permanent cell-cycle arrest. Increased oxidative stress and accumulation of oxidative DNA damage are features of brain ageing and neurodegeneration but the effects of persistent DNA damage in neurons are not well-characterized. We developed a model of persistent oxidative DNA damage in immortalized post-mitotic neurons in vitro by exposing them to a sub-lethal concentration of hydrogen peroxide following a “double stress” protocol, and performed a detailed characterization of the neuronal transcriptome using microarray analysis. Persistent DNA damage significantly altered the expression of genes involved in cell cycle regulation, DNA damage response and repair mechanisms, and mitochondrial function, suggesting an active DDR response to replication stress and alterations in mitochondrial electron transport chain. qPCR and functional validation experiments confirmed hyperactivation of mitochondrial Complex I in response to persistent DNA damage. These changes in response to persistent oxidative DNA damage may lead to further oxidative stress, contributing to neuronal dysfunction and ultimately neurodegeneration

Diving deep into digital literacy:emerging methods for research

Literacy studies approaches have tended to adopt a position which enables ethnographic explorations of a wide range of ‘literacies’. An important issue arising is the new challenge required for researchers to capture, manage, and analyse data that highlight the unique character of practices around texts in digital environments. Such inquiries, we argue, require multiple elements of data to be captured and analysed as part of effective literacy ethnographies. These include such things as the unfolding of digital texts, the activities around them, and features of the surrounding social and material environment. This paper addresses these methodological issues drawing from three educationally focused studies, and reporting their experiences and insights within uniquely different contexts. We deal with the issue of adopting new digital methods for literacy research through the notion of a ‘deep dive’ to explore educational tasks in classrooms. Through a discussion of how we approached the capture and analysis of our data, we present methods to better understand digital literacies in education. We then outline challenges posed by our methods, how they can be used more broadly for researching interaction in digital environments, and how they augment transdisciplinary debates and trends in research methods

Alpha-synuclein mRNA expression in oligodendrocytes in MSA

Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α‐Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser‐capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P < 0.0001). At the cellular level, MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls. GLIA 2014;62:964–97

Literacy in Lockdown: Learning and Teaching During COVID‐19 School Closures

Across the globe, students have been away from schools and their teachers, but literacy learning has continued. In many countries, students’ literacy proficiency is often measured via high‐stakes assessment tests. However, such tests do not make visible students’ literacy lives away from formal learning settings, so students are positioned as task responders, rather than as agentive readers and writers. The authors explore the fluidity and diversity of literacy events and practices for students and their teachers observed during the recent period of COVID‐19 lockdown restrictions

RNA-Seq profiling of neutrophil-derived microvesicles in Alzheimer’s disease patients identifies a miRNA signature that may impact blood–brain barrier integrity

(1) Background: Systemic infection is associated with increased neuroinflammation and accelerated cognitive decline in AD patients. Activated neutrophils produce neutrophil-derived microvesicles (NMV), which are internalised by human brain microvascular endothelial cells and increase their permeability in vitro, suggesting that NMV play a role in blood–brain barrier (BBB) integrity during infection. The current study investigated whether microRNA content of NMV from AD patients is significantly different compared to healthy controls and could impact cerebrovascular integrity. (2) Methods: Neutrophils isolated from peripheral blood samples of five AD and five healthy control donors without systemic infection were stimulated to produce NMV. MicroRNAs isolated from NMV were analysed by RNA-Seq, and online bioinformatic tools were used to identify significantly differentially expressed microRNAs in the NMV. Target and pathway analyses were performed to predict the impact of the candidate microRNAs on vascular integrity. (3) Results: There was no significant difference in either the number of neutrophils (p = 0.309) or the number of NMV (p = 0.3434) isolated from AD donors compared to control. However, 158 microRNAs were significantly dysregulated in AD NMV compared to controls, some of which were associated with BBB dysfunction, including miR-210, miR-20b-5p and miR-126-5p. Pathway analysis revealed numerous significantly affected pathways involved in regulating vascular integrity, including the TGFβ and PDGFB pathways, as well as Hippo, IL-2 and DNA damage signalling. (4) Conclusions: NMV from AD patients contain miRNAs that may alter the integrity of the BBB and represent a novel neutrophil-mediated mechanism for BBB dysfunction in AD and the accelerated cognitive decline seen as a result of a systemic infection

Observation of Scaling Violations in Scaled Momentum Distributions at HERA

Charged particle production has been measured in deep inelastic scattering (DIS) events over a large range of $x$ and $Q^2$ using the ZEUS detector. The evolution of the scaled momentum, $x_p$, with $Q^2,$ in the range 10 to 1280 $GeV^2$, has been investigated in the current fragmentation region of the Breit frame. The results show clear evidence, in a single experiment, for scaling violations in scaled momenta as a function of $Q^2$.Comment: 21 pages including 4 figures, to be published in Physics Letters B. Two references adde

D* Production in Deep Inelastic Scattering at HERA

This paper presents measurements of D^{*\pm} production in deep inelastic scattering from collisions between 27.5 GeV positrons and 820 GeV protons. The data have been taken with the ZEUS detector at HERA. The decay channel $D^{*+}\to (D^0 \to K^- \pi^+) \pi^+$ (+ c.c.) has been used in the study. The $e^+p$ cross section for inclusive D^{*\pm} production with $5<Q^2<100 GeV^2$ and $y<0.7$ is 5.3 \pms 1.0 \pms 0.8 nb in the kinematic region {$1.3<p_T(D^{*\pm})<9.0$ GeV and $| \eta(D^{*\pm}) |<1.5$}. Differential cross sections as functions of p_T(D^{*\pm}), $\eta(D^{*\pm}), W$ and $Q^2$ are compared with next-to-leading order QCD calculations based on the photon-gluon fusion production mechanism. After an extrapolation of the cross section to the full kinematic region in p_T(D^{*\pm}) and $\eta$(D^{*\pm}), the charm contribution $F_2^{c\bar{c}}(x,Q^2)$ to the proton structure function is determined for Bjorken $x$ between 2 $\cdot$ 10$^{-4}$ and 5 $\cdot$ 10$^{-3}$.Comment: 17 pages including 4 figure