Biology subjects

Elmer, J.

Publications and source records attributed to Elmer, J..

9 recordsLinked to original sources

bioRxiv2022

Noninvasive, automated and reliable detection of spreading depolarizations in severe traumatic brain injury using scalp EEG

BackgroundNoninvasive detection of spreading depolarizations (SD), as a potentially treatable mechanism of worsening brain injuries after traumatic brain injuries (TBI), has remained elusive. Current methods to detect SDs are based on intracranial recording, an invasive method with limited spatial coverage. Less invasive methods to diagnose SD are needed to improve generalizability and application of this emerging science and to guide worsening brain injury treatments. Here, we demonstrate, for the first time, a signal processing paradigm that can enable automated detection of SDs using noninvasive electroencephalography (EEG). MethodsBuilding on our previously developed WAVEFRONT algorithm, we have designed a novel automated SD detection method. This algorithm, with learnable parameters and improved velocity estimation, extracts and tracks propagating power depressions, as well as near-DC shifts using low-density EEG. This modified WAVEFRONT is robust to the amplitude outliers and non-propagating depressions on the scalp. We show the feasibility of detecting SD events (700 total SDs) in continuous, low-density scalp EEG recording (95{+/-}42.2 hours with 19 electrodes) acquired from 12 severe TBI patients who underwent decompressive hemicraniectomy (DHC) and intracranial EEG that could be used as a ground truth for event detection. We quantify the performance of WAVEFRONT in terms of SD detection accuracy, including true positive rate (TPR) and false positive rate (FPR), as well as the accuracy of estimating the frequency of SDs. ResultsWAVEFRONT achieves the best average validation accuracy of 74% TPR (with 95% confidence interval of 70.8%-76.7%), with less than 1.5% FPR using Delta band EEG. Preliminary evidence suggests that WAVEFRONT can achieve a very good performance (regression with R2 [~=]0.71) in the estimation of SD frequencies. ConclusionsWe demonstrate feasibility and quantify the performance of noninvasive SD detection after severe TBI using an automated algorithm. WAVEFRONT can potentially be used for diagnosis and monitoring of worsening brain injuries to guide treatments by providing a measure of SD frequency. Extension of these results to patients with intact skulls requires further study.

neuroscience↗

bioRxiv2021

Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

PurposeTargeted regulation of transfected extra-chromosomal plasmid DNA typically requires the integration of 9 - 20 bp docking sites into the plasmid. Here, we report an elegant approach, The Dpn Adaptor Linked Effector (DAL-E) system, to target fusion proteins to 6-methyladenosine in GATC, which appears frequently in popular eukaryotic expression vectors and is absent from endogenous genomic DNA. Methods: The DNA-binding region from the DpnI endonuclease binds 6-methyladenosine within the GATC motif. We used a Dpn-transcriptional activator (DPN7-TA) fusion to induce gene expression from transiently transfected pDNAs. ResultsWe validated methylation-dependent activity of DPN7-TA with a panel of target pDNAs. We observed stronger transactivation when GATC targets were located upstream of the transcriptional start site in the target pDNA. Conclusion: DAL-E, consisting of a 108 aa, 12 kD DNA-binding adaptor and a 4 bp recognition site, offers a genetically-tractable, tunable system that can potentially be redesigned to recruit a variety of regulators (e.g. activators, silencers, epigenome editors) to transfected plasmid DNA. LAY SUMMARYTransfection of plasmid DNA (pDNA) is a commonly used method for introducing exogenous genetic material into mammalian cells. Once introduced into cells not all pDNAs express this genetic material at sufficient levels. Current techniques to improve transgene expression are limited and are not always feasible for all plasmids. This report presents a new method to improve gene expression from pDNA. The Dpn Adaptor Linked Effector (DAL-E) binds to methylated adenines in the pDNA resulting in increased expression. This technique has exciting implications for improved genetic engineering of mammalian cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=124 HEIGHT=200 SRC="FIGDIR/small/466616v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1116db0org.highwire.dtl.DTLVardef@1386f99org.highwire.dtl.DTLVardef@26c63corg.highwire.dtl.DTLVardef@1a09673_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Metformin Confers Cardiac and Renal Protection in Sudden Cardiac Arrest via AMPK Activation

Cardiac arrest (CA) causes high mortality due to multi-system organ damage attributable to ischemia-reperfusion injury. Recent work in our group found that among diabetic patients who experienced cardiac arrest, those taking metformin had less evidence of cardiac and renal damage after cardiac arrest when compared to those not taking metformin. To investigate the potential for metformin to impact cardiac arrest outcomes, the current study investigates metformin interventions on cardiac and renal outcomes in a non-diabetic CA mouse model. We found that two weeks of metformin pretreatment protects against reduced ejection fraction and reduces kidney ischemia-reperfusion injury at 24 hours post-arrest. This cardiac and renal protection depends on AMPK signaling, as demonstrated by outcomes in mice pretreated with the AMPK activator AICAR or metformin plus the AMPK inhibitor compound C. At this 24-hour time point, gene expression analysis showed that metformin pretreatment caused changes supporting autophagy, antioxidant response, and protein translation. Further investigation found associated improvements in mitochondrial structure and markers of autophagy. We also found that markers of protein nitrosylation between sham and arrest were not significantly different and thus unlikely to be a driver of gene expression differences. Notably, Western analysis indicated that protein synthesis was preserved in arrest hearts of animals pretreated with metformin. The AMPK activation-mediated protection of protein synthesis was also demonstrated in a hypoxia/reoxygenation cell culture model. Despite the positive impacts of pretreatment in vivo and in vitro, metformin did not preserve ejection fraction when deployed at resuscitation. Taken together, we propose that metformins in vivo cardiac preservation occurs through AMPK activation, requires adaptation before arrest, and is associated with preserved protein translation. New and NoteworthyWe leveraged a mouse model of cardiac arrest to study whether metformin pretreatment could protect cardiac function after arrest. We find that AMPK activity is crucial to this protection and characterize several pathways associated with this response. Signaling nodes with strong association may be precise targets to promote cardiac function in ischemia/reperfusion injury or in cardiac stunning.

physiology↗

bioRxiv2021

Transcriptomic Analysis of the Innate Immune Response to in vitro Transfection of Plasmid DNA

The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA-sequencing to reveal correlations between the transfection efficiencies of four cell types (PC-3, Jurkat, HEK-293T, and primary CD3+ T cells) and their innate immune responses to nonviral gene delivery. Overall, the highest transfection efficiency was observed in HEK-293T cells (87%), which upregulated only 142 genes with no known anti-viral functions. Lipofection upregulated a much larger number (n = 1,057) of cytokine-stimulated genes (CSGs) in PC-3 cells, which also exhibited a significantly lower transfection efficiency. However, the addition of serum during Lipofection and electroporation significantly increased transfection efficiencies and decreased the number of upregulated genes in PC-3 cells. Finally, while Lipofection of Jurkat and Primary T cells only upregulated a few genes, several anti-viral CSGs that were absent in HEK and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain their relatively low Lipofection efficiencies (8-21%). Indeed, overexpression of one such CSG (IFI16) significantly decreased transfection efficiency in HEK cells to 33%.

bioengineering↗

bioRxiv2020

Post release immune responses of Tasmanian devils vaccinated with an experimental devil facial tumour disease vaccine

Disease is increasingly becoming a driver of wildlife population declines and extinction risk. Vaccines have been one of the most successful health interventions in human history, but few have been tested for mitigating wildlife disease. The transmissible cancer, devil facial tumour disease (DFTD), triggered the Tasmanian devils (Sarcophilus harrisii) inclusion on the international endangered species list. Development of a protective DFTD vaccine would provide a valuable management approach for conservation of the species. In 2016, 33 devils from a DFTD-free insurance population were given an experimental DFTD vaccination prior to their release on the north coast of Tasmania. The release site was already home to an incumbent population of devils, including some individuals with DFTD. To determine the efficacy of the vaccination protocol and the longevity of the response it induced, six trapping trips took place at the site over the 2.5 years following release. Eight of the 33 vaccinated devils were re-trapped, and six of those developed DFTD within the monitoring period. Despite the apparent lack of protection provided by the vaccine for the re-trapped devils, we observed several signs of immune activation not usually found in unvaccinated devils. Firstly, sera collected from the eight devils showed that anti-DFTD antibodies persisted for up to two years post vaccination. Secondly, tumour infiltrating lymphocytes were found in three out of four biopsies collected from vaccinated devils which contrasts with the "immune deserts" typical of DFTs; only one out of twenty incumbent devils with DFTD trapped during the same period had a tumour biopsy exhibiting immune cell infiltrate. Thirdly, immunohistochemical analysis of tumour biopsies from the vaccinated devils identified the functional immune molecules associated with antigen presenting cells (MHC-II) and T cells (CD3), and the immune checkpoint molecule PD-1, all associated with anti-tumour immunity in other species. These results correlate with our previous study on captive devils in which a prophylactic vaccine primed the devil immune system and, following DFTD challenge and tumour growth, immunotherapy induced complete tumour regressions. The field trial results presented here provide further evidence that the devil immune system can be primed to recognise DFTD cells, but additional immune manipulation could be needed for complete protection or induction of tumour regressions.

immunology↗

bioRxiv2018

Use of MYB as a new synthetic activator to enhance transgene expression within repressed Polycomb chromatin

Epigenetic silencing of transgenes has been a persistent challenge for mammalian cell engineering. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cells genome. To identify elements that mitigate epigenetic silencing, we tested components from the c-myb and NF-kB transcriptional regulation systems in transiently transfected DNA and at chromosomally integrated transgenes in PC-3 and HEK293 cells. DNA binding sites for MYB (c-myb) placed upstream of a minimal promoter strongly enhanced expression from transiently transfected plasmid DNA. We targeted p65 and MYB fusion proteins to chromosomal transgenes that were silenced by ectopic Polycomb chromatin or by uncharacterized endogenous chromatin. Transient expression of Gal4-MYB induced sustained activation of the Polycomb-silenced UAS-Tk-luciferase transgene. We used custom guide RNAs and dCas9-MYB to target MYB to different sites. Transgene activation within ectopic Polycomb chromatin required proximity of dCas9-MYB to the transcriptional start site, while activation at the naturally repressed transgene was position-independent. Our report is the first to demonstrate the use of MYB in the context of the CRISPR-activation system. The results demonstrate that DNA elements and fusion proteins derived from c-myb can mitigate epigenetic silencing to improve transgene expression in engineered cell lines.

synthetic biology↗

bioRxiv2018

An algorithm for automated, noninvasive detection of cortical spreading depolarizations based on EEG simulations

We present a novel signal processing algorithm for automated, noninvasive detection of Cortical Spreading Depolarizations (CSDs) using electroencephalography (EEG) signals and validate the algorithm on simulated EEG signals. CSDs are waves of neurochemical changes that suppress neuronal activity as they propagate across the brains cortical surface. CSDs are believed to mediate secondary brain damage after brain trauma and cerebrovascular diseases like stroke. We address key challenges in detecting CSDs from EEG signals: (i) decay of high spatial frequencies as they travel from the cortical surface to the scalp surface; and (ii) presence of sulci and gyri, which makes it difficult to track the CSD waves as they travel across the cortex. Our algorithm detects and tracks \"wavefronts\" of the CSD wave, and stitches together data across space and time to decide on the presence of a CSD wave. To test our algorithm, we provide different models and complex patterns of CSD waves, including different widths of CSD suppressions, and use these models to simulate scalp EEG signals using head models of 4 subjects from the OASIS dataset. Our results suggest that the average width of suppression that a low-density EEG grid of 40 electrodes can detect is 1.1 cm, which includes a vast majority of CSD suppressions, but not all. A higher density EEG grid having 340 electrodes can detect complex CSD patterns as thin as 0.43 cm (less than minimum widths reported in prior works), among which single-gyrus propagation is the hardest to detect because of its small suppression area.

bioengineering↗

bioRxiv2018

Small Molecule Inhibition of the Innate Immune Response Increases Transgene Expression

Foreign molecules like plasmid DNA trigger a complex and potent innate immune response comprised of highly redundant signal transduction cascades that result in the activation of transcription factors and the production of inflammatory cytokines. Unfortunately, this defense mechanism can hinder gene therapy by inhibiting transgene expression. The goal of this study was to increase transgene expression by inhibiting key components of the innate immune response ({beta}-catenin, NF-{kappa}B/AP1, TBK1, TLR9, and p38 MAPK) with small molecule inhibitors (iCRT-14, curcumin, BX-795, E6446, and VX-702 respectively). The effects of each drug on transgene (luciferase) expression, inflammatory cytokine (IL-6) levels, and cell viability were quantified in prostate (PC3), breast (MCF-7), and murine bladder (MB49) cancer cell lines. The {beta}-catenin inhibitor iCRT-14 (1 M) provided the highest enhancement of 35.5 {+/-} 19-fold in MCF-7 cells, while the other inhibitors increased transgene expression at a more modest level (2-9 fold). The optimal concentrations of iCRT-14, curcumin, and VX-702 showed no significant effect on cell proliferation; however, optimal concentrations of BX-795 and E6446 did significantly reduce cell proliferation. Nonetheless, inhibition of the innate immune response by iCRT-14 and curcumin was confirmed by a concomitant decrease in IL-6 production in PC3 cells. These results demonstrate that these inhibitors can improve gene therapy by preventing an inflammatory innate immune response.

bioengineering↗

bioRxiv2018

Enhancement of Transgene Expression by NF-Y and CTCF

If a transgene is effectively delivered to a cell, its expression may still be limited by epigenetic mechanisms that silence the transgene. Indeed, once the transgene reaches the nucleus, it may be bound by histone proteins and condensed into heterochromatin or associated with repressor proteins that block transcription. In this study, we sought to enhance transgene expression by adding binding motifs for several different epigenetic enzymes either upstream or downstream of two promoters (CMV and EF1). Screening these plasmids revealed that luciferase expression was enhanced 10-fold by the addition of a CCAAT box just upstream of the EF1 promoter to recruit nuclear transcription factor Y (NF-Y), while inserting a CCCTC-binding factor (CTCF) motif downstream of the EF1 promoter enhanced expression 14-fold (14.03 {+/-} 6.54). ChIP assays confirmed that NF-Y and CTCF bound to the motifs that were added to each plasmid, but the presence of NF-Y and CTCF did not significantly affect the levels of histone acetylation (H3K9ac). Overall, these result show that transgene expression from the EF1 promoter can be significantly increased with motifs that recruit NF-Y or CTCF.

bioengineering↗