Biology subjects

Nelson, A.

Publications and source records attributed to Nelson, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Platelet response to influenza vaccination reflects effects of aging

Platelets are uniquely positioned as mediators of not only hemostasis but also innate immunity, but how age and alterations in functional status such as frailty influence platelet function during an immune response remains unclear. We assessed the platelet transcriptome in younger (age 21-35) and older (age [≥] 65) adults (including frail and non-frail individuals) following influenza vaccination. Prior to vaccination, we identified an age- and frailty-associated increase in expression of platelet activation and mitochondrial RNAs and decrease in RNAs encoding proteins mediating translation. Using tensor decomposition analysis, we also elucidated dynamic post-vaccination platelet activation and translation signatures associated with age and frailty. At the protein level, enhanced platelet activation was found in non-frail older adults, compared to young individuals both prior to and post-vaccine; but frail adults showed decreased platelet activation compared to non-frail that could reflect the influence of decreased translation RNA expression. Our results reveal an age-dependent alteration in platelet function prior to and post-vaccination that may contribute to age-associated chronic inflammation.

immunology↗

bioRxiv2021

A trans-acting long non-coding RNA represses flowering in Arabidopsis

Eukaryotic genomes give rise to thousands of long non-coding RNAs (lncRNAs), yet the purpose of lncRNAs remains largely enigmatic. Functional characterization of lncRNAs is challenging due to multiple orthogonal hypothesis for molecular activities of lncRNA loci. Here, we identified a flowering associated intergenic lncRNA (FLAIL) that represses flowering in Arabidopsis. An allelic series of flail loss-of-function mutants generated by CRISPR/Cas9 and T-DNA mutagenesis showed an early flowering phenotype. Gene expression analyses in flail mutants revealed differentially expressed genes linked to the regulation of flowering. A genomic rescue fragment of FLAIL introduced in flail mutants complemented gene expression defects and early flowering, consistent with trans-acting effects of the FLAIL RNA. Knock-down of FLAIL RNA levels using the artificial microRNA approach revealed an early flowering phenotype shared with genomic mutations, indicating a trans-acting role of FLAIL RNA in the repression of flowering time. Genome-wide detection of FLAIL-DNA interactions by ChIRP-seq suggested that FLAIL may directly bind genomic regions. FLAIL bound to genes involved in regulation of flowering that were differentially expressed in flail, consistent with the interpretation of FLAIL as a trans-acting lncRNA directly shaping gene expression. Our findings highlight FLAIL as a trans-acting lncRNA that affects flowering in Arabidopsis, likely through mediating transcriptional regulation of genes directly bound by FLAIL.

genomics↗

bioRxiv2021

Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases

The targeted deletion, replacement, integration, or inversion of DNA sequences at specified locations in the genome could be used to study or treat many human genetic diseases. Here, we describe twin prime editing (twinPE), a method for the programmable replacement or excision of DNA sequence at endogenous human genomic sites without requiring double-strand DNA breaks. TwinPE uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences. We show that twinPE in human cells can perform precise deletions of at least 780 bp and precise replacements of genomic DNA sequence with new sequences of at least 108 bp. By combining single or multiplexed twinPE with site-specific serine recombinases either in separate steps or in a single step, we demonstrate targeted integration of gene-sized DNA plasmids (>5,000 bp) into safe-harbor loci including AAVS1, CCR5, and ALB in human cells. To our knowledge, these results represent the first RNA-programmable insertion of gene-sized DNA sequences into targeted genomic sites of unmodified human cells without requiring double-strand breaks or homology-directed repair. Twin PE combined with recombinases also mediated a 40,167-bp inversion at IDS that corrects a common Hunter syndrome allele. TwinPE expands the capabilities of precision gene editing without requiring double-strand DNA breaks and synergizes with other tools to enable the correction or complementation of large or complex pathogenic alleles in human cells.

genomics↗

bioRxiv2021

GLS1 is a Protective Factor Rather than a Molecular Target in ARID1A-Mutated Ovarian Clear Cell Carcinoma

Targeting glutamine metabolism has emerged as a novel therapeutic strategy for several human cancers, including ovarian cancer. The primary target of this approach is the kidney isoform of glutaminase, glutaminase 1 (GLS1), a key enzyme in glutamine metabolism that is overexpressed in several human cancers. A first-in-class inhibitor of GLS1, called CB839 (Telaglenastat), has been investigated in several clinical trials, with promising results. The first clinical trial of CB839 in platinum-resistant ovarian cancer patients is forthcoming. ARID1A-mutated ovarian clear cell carcinoma (OCCC) is a relatively indolent and chemoresistant ovarian cancer histotype. In OCCC-derived cells in vitro and mouse models, loss of ARID1A leads to upregulation of GLS1. Thus, targeting of GLS1 with CB839 has been suggested as a targeted approach for OCCC patients with tumors harboring ARID1A-mutations. Here, we investigated whether GLS1 is differentially expressed between OCCC patients whose tumors are ARID1A positive and patients whose tumors are ARID1A negative. In clinical specimens of OCCC, we found that GLS1 overexpression was not correlated with ARID1A loss. In addition, GLS1 overexpression was associated with better clinical outcomes. Our findings suggest that GLS1 expression in OCCC may be a protective factor and that caution should be taken when considering the use of CB839 to treat OCCC patients.

cancer biology↗

bioRxiv2021

Activating P2Y1 receptors improves function in arteries with repressed autophagy

ObjectiveThe importance of endothelial cell (EC) autophagy to vascular homeostasis is evolving. Earlier we reported that purinergic 2Y1 receptor (P2Y1-R) activation rejuvenates shearstress induced nitric oxide (NO) generation in bovine aortic endothelial cells that is otherwise compromised after pharmacological and genetic autophagy repression. Here we determined the translational and functional relevance of these findings. Approach and ResultsFirst we assessed translational relevance using older humans and mice that exhibit blunted EC autophagy at rest together with impaired arterial function vs. appropriate controls. Rhythmic handgrip exercise elevated radial artery shear rate similarly in adult and older males for 60-min. Compared to baseline, autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs from adult but not older subjects. Regarding mice, indexes of autophagy and p-eNOSS1177 activation were robust in ECs from adult but not older mice in response to 60-min treadmill-running. Next we questioned whether an inability to initiate EC autophagy precipitates arterial dysfunction. Age-associated reductions in intraluminal flow-mediated vasodilation observed in older vs. adult mice were recapitulated in arteries from adult mice by : (i) NO synthase inhibition; (ii) autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) P2Y1-R blockade; and (v) germline depletion of P2Y1-Rs. Importantly, P2Y1-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from : (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy. ConclusionsArterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y1-Rs.

physiology↗

bioRxiv2021

Frequent development of broadly neutralizing antibodies in early life in a large cohort of HIV-infected children

Recent studies conducted in small cohorts of children have indicated that broadly neutralizing antibodies (bnAbs) may develop earlier after HIV infection compared to adults. To define the frequency and kinetics of bnAb responses in a larger pediatric cohort, we evaluated plasma from 212 ART-naive, children living with HIV aged 1 to 3 years. Neutralization breadth and potency was assessed using a panel of 10 tier-2 viruses and compared to those of adults with chronic HIV. Further, the magnitude, epitope specificity and IgG subclass distribution of Env-specific antibodies were also assessed using a binding antibody multiplex assay. We found that 1-year-old children demonstrated neutralization breadth comparable to that of chronically-infected adults, and breadth continued to increase with age such that the pediatric cohort overall exhibited significantly greater neutralization breadth than adults (p= 0.014). Similarly, binding antibody responses increased with age, and the levels in 2 to 3 year-old children were comparable to those of adults. Overall, there was no significant difference in antibody specificities or IgG subclass distribution between the pediatric and adult cohorts. Interestingly, the neutralization activity was mapped to a single epitope (CD4 binding site, V2 or V3 glycans) in only 5 of 38 pediatric broadly neutralizing samples, suggesting a polyclonal neutralization response may develop in most children. These results contribute to a growing body of evidence suggesting that the early life immune system may present advantages for the development of an effective HIV vaccine. Author summaryUnderstanding the development of broadly neutralizing antibodies during natural HIV infection is critical to provide insights for the development of an effective vaccine. In this study, we used a large cohort (n=212) of HIV infected children aged 1-3 years old to define the frequency and kinetics of broad neutralization in comparison to adults with chronic HIV. We also evaluated the magnitude and epitope specificity of HIV Env-specific antibodies in children and adults. We observed that both the binding and neutralizing antibody responses increased with age. However, by one year of age, children had neutralization breadth comparable to that of chronically infected adults; and overall, 1-3 year-old children demonstrated higher neutralization breadth than adults. Only slight differences in epitope-specificity and IgG subclass distribution were observed between children and adults, suggesting that these factors are not major contributors to the observed age-related difference in neutralization breadth. The epitope specificity of the neutralizing antibodies could not be mapped in the majority of children, suggesting that their response may be polyclonal. Our study contributes to the body of work indicating a potential advantage of the early life immune system for the elicitation of broad neutralization.

immunology↗

bioRxiv2021

A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes

We performed a comprehensive analysis of the transcriptional changes within and across cell populations during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using the single cell RNA-seq combinatorial barcoding method SPLiT-seq, we sequenced >20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. As expected, differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in differentiation population composition and cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.

cell biology↗

bioRxiv2021

The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

BackgroundA hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early pro-inflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and ResultsThe role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction (TAC) surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after TAC surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2-KO mice displayed a pro-inflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, TAC surgery led to the death of all mice by day 6 that was associated with myocardial hyper-inflammation and vascular leakage. ConclusionsTogether, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

cell biology↗

bioRxiv2021

Metabolism Control in 3D Printed Living Materials

The three-dimensional printing of cells offers an attractive opportunity to design and develop innovative biotechnological applications, such as the fabrication of biosensors or modular bioreactors. Living materials (LMs) are cross-linked polymeric hydrogel matrices containing cells, and recently, one of the most deployed LMs consists of F127-bis-urethane methacrylate (F127-BUM). The material properties of F127-BUM allow reproducible 3D printing and stability of LMs in physiological environments. These materials are permissible for small molecules like glucose and ethanol. However, no information is available for oxygen, which is essential-- for example, towards the development of aerobic bioprocesses using microbial cell factories. To address this challenge, we investigated the role of oxygen as a terminal electron acceptor in the budding yeasts respiratory chain and determined its permissibility in LMs. We quantified the ability of cell-retaining LMs to utilize oxygen and compared it with cells in suspension culture. We found that the cells ability to consume oxygen was heavily impaired inside LMs, indicating that the metabolism mostly relied on fermentation instead of respiration. To demonstrate an application of these 3D printed LMs, we evaluated a comparative brewing process. The analysis showed a significantly higher (3.7%) ethanol production using 3D printed LMs than traditional brewing, indicating an efficient control of the metabolism. Towards molecular and systems biology studies using LMs, we developed a highly reliable method to isolate cells from LMs for flow cytometry and further purified macromolecules (proteins, RNA, and DNA). Our results show the application of F127-BUM-based LMs for microaerobic processes and envision the development of diverse bioprocesses using versatile LMs in the future.

bioengineering↗

bioRxiv2020

Long-term assessment of ecosystem services at ecological restoration sites using Landsat time series

Reversing ecological degradation through restoration activities is a key societal challenge of the upcoming decade. However, lack of evidence on the effectiveness of restoration interventions leads to inconsistent, delayed, or poorly informed statements of success, hindering the wise allocation of resources, representing a missed opportunity to learn from previous experiences. This study contributes to a better understanding of spatial and temporal dynamics of ecosystem services at ecological restoration sites. We developed a method using Landsat satellite images combined with a Before-After-Control-Impact (BACI) design, and applied this to an arid rural landscape, the Baviaanskloof in South Africa. Since 1990, various restoration projects have been implemented to halt and reverse degradation. We applied the BACI approach at pixel-level comparing the conditions of each intervened pixel (impact) with 20 similar control pixels. By evaluating the conditions before and after the intervention, we assessed the effectiveness of long-term restoration interventions distinguishing their impact from environmental temporal changes. The BACI approach was implemented with Landsat images that cover a 30-year period at a spatial resolution of 30 m. We evaluated the impact of three interventions (revegetation, livestock exclusion, and the combination of both) on three ecosystem services; forage provision, erosion prevention, and presence of iconic vegetation. We also evaluated whether terrain characteristics could partially explain the variation in impact of interventions. The resulting maps showed spatial patterns of positive and negative effects of interventions on ecosystem services. Intervention effectiveness differed between land cover vegetation clusters, terrain aspect, and soil parent material. Our method allows for spatially explicit quantification of the long-term restoration impact on ecosystem service supply, and for the detailed visualization of impact across an area. This pixel-level analysis is specifically suited for heterogeneous landscapes, where restoration impact not only varies between but also within restoration sites.

ecology↗

bioRxiv2020

Corticospinal neurons encode complex motor signals that are broadcast to dichotomous striatal circuits

Sensorimotor cortex controls movement in part through direct projections to the spinal cord. Here we show that these corticospinal neurons (CSNs) possess axon collaterals that innervate many supraspinal brain regions critical for motor control, most prominently the main input to the basal ganglia, the striatum. Corticospinal neurons that innervate the striatum form more synapses on D1-than D2-striatal projection neurons (SPNs). This biased innervation strategy corresponds to functionally distinct patterns of termination in spinal cord. CSNs are strongly driven during a striatum-dependent sequential forelimb behavior, and often represent high level movement features that are not linearly related to kinematic output. Copies of these activity patterns are relayed in a balanced fashion to both D1 and D2 projection pathways. These results reveal a circuit logic by which motor cortex corticospinal neurons relay both kinematic-related and unrelated signals to distinct striatal and spinal cord pathways, where postsynaptic connectivity ultimately dictates motor specificity. HighlightsO_LICorticospinal neurons send axon collaterals most abundantly to the striatum C_LIO_LIBiases in striatal innervation correspond to biases in spinal innervation C_LIO_LICSNs represent complex movement sequence information C_LIO_LICorollary motor sequence signals are relayed to both striatal projection pathways C_LI eTOC BlurbNelson, A. et al. detail the organization of corticospinal neurons and their coordinated cell type-specific targets in the dorsolateral striatum and spinal cord. Corticospinal neurons encode both kinematic-related and unrelated signals during motor sequences, and relay this information in a balanced fashion to dichotomous striatal pathways.

neuroscience↗

bioRxiv2020

Neural correlates of the shamanic state of consciousness

Despite the use of shamanism as a healing practice for several millennia, few empirical studies of the shamanic state of consciousness exist. We investigated the neural correlates of shamanic trance using high-density electroencephalography (EEG) in 24 shamanic practitioners and 24 healthy controls during rest, shamanic drumming, and classical music listening, followed by a validated assessment of altered states of consciousness. EEG data were used to assess changes in absolute power, connectivity, signal diversity, and criticality, which were correlated with assessment measures. We also compared assessment scores to those of individuals in a previous study under the influence of psychedelics. Shamanic practitioners were significantly different from controls in several domains of altered states of consciousness, with scores comparable to or exceeding that of healthy volunteers under the influence of psychedelics. Practitioners also displayed increased gamma power during drumming that positively correlated with elementary visual alterations. Furthermore, shamanic practitioners had decreased low alpha and increased low beta connectivity during drumming and classical music, and decreased neural signal diversity in the gamma band during drumming that inversely correlated with insightfulness. Finally, criticality in practitioners was increased during drumming in the low and high beta and gamma bands, with increases in the low beta band correlating with complex imagery and elementary visual alterations. These findings suggest that psychedelic drug-induced and non-pharmacologic alterations in consciousness have overlapping phenomenal traits but are distinct states of consciousness, as reflected by the unique brain-related changes during shamanic trance compared to previous literature investigating the psychedelic state.

neuroscience↗

bioRxiv2020

Chemogenetics reveal an anterior cingulate-thalamic pathway for attending to task-relevant information

In a changing environment, we need to decide when to select items that resemble previously rewarded stimuli and when it is best to switch to other stimulus types. Here, we used chemogenetic techniques to provide causal evidence that activity in the rodent anterior cingulate cortex and its efferents to the anterior thalamic nuclei modulate the ability to attend to reliable predictors of important outcomes. Rats were tested on an attentional set-shifting paradigm that first measures the ability to master serial discriminations involving a constant stimulus dimension that reliably predicts reinforcement (intradimensional-shift), followed by the ability to shift attention to a previously irrelevant class of stimuli when reinforcement contingencies change (extradimensional-shift). Chemogenetic silencing of the anterior cingulate cortex (Experiment 1) as well as selective inactivation of anterior cingulate efferents to the anterior thalamic nuclei (Experiment 2) impaired intradimensional learning but, facilitated two sets of extradimensional-shifts. This pattern of results signals the loss of a cortico-thalamic system for cognitive control that preferentially processes stimuli resembling those previously associated with reward. Previous studies highlight a separate prefrontal system that promotes switching to hitherto inconsistent predictors of reward when contingencies change. Competition between these two systems regulates cognitive flexibility and choice.

neuroscience↗

bioRxiv2020

Cell states beyond transcriptomics: integrating structural organization and gene expression in hiPSC-derived cardiomyocytes

We present a quantitative co-analysis of RNA abundance and sarcomere organization in single cells and an integrated framework to predict subcellular organization states from gene expression. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes expressing mEGFP-tagged alpha-actinin-2 to develop quantitative image analysis tools for systematic and automated classification of subcellular organization. This captured a wide range of sarcomeric organization states within cell populations that were previously difficult to quantify. We performed RNA FISH targeting genes identified by single cell RNA sequencing to simultaneously assess the relationship between transcript abundance and structural states in single cells. Co-analysis of gene expression and sarcomeric patterns in the same cells revealed biologically meaningful correlations that could be used to predict organizational states. This study establishes a framework for multi-dimensional analysis of single cells to study the relationships between gene expression and subcellular organization and to develop a more nuanced description of cell states. Graphical AbstractTranscriptional profiling and structural classification was performed on human induced pluripotent stem cell-derived cardiomyocytes to characterize the relationship between transcript abundance and subcellular organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/081083v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@8f89c4org.highwire.dtl.DTLVardef@19dc813org.highwire.dtl.DTLVardef@1ba7c20org.highwire.dtl.DTLVardef@2b3daa_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach and represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

genomics↗

bioRxiv2020

Physical confinement impacts cellular phenotype within living materials

Additive manufacturing allows three-dimensional printing of polymeric materials together with cells, creating living materials for applications in biomedical research and biotechnology. However, understanding the cellular phenotype within living materials is lacking and a key limitation for their wider application. Herein, we present an approach to characterize the cellular phenotype within living materials. We immobilized the budding yeast Saccharomyces cerevisiae in three different photocross-linkable triblock polymeric hydrogels containing F127-bis-urethane methacrylate, F127-dimethacrylate, or poly(alkyl glycidyl ether)-dimethacrylate. Using optical and scanning electron microscopy, we showed that hydrogels based on these polymers were stable under physiological conditions, but yeast colonies showed differences in the interaction within the living materials. We found that the physical confinement, imparted by compositional and structural properties of the hydrogels, impacted the cellular phenotype by reducing the size of cells in living materials compared with suspension cells. These properties also contributed to the differences in immobilization patterns, growth of colonies, and colony coatings. We observed that a composition-dependent degradation of polymers was likely possible by cells residing in the living materials. In conclusion, our investigation highlights the need for a holistic understanding of the cellular response within hydrogels to facilitate the synthesis of application-specific polymers and the design of advanced living materials in the future.

bioengineering↗

bioRxiv2019

Interrogating mechanisms of ssDNA binding to a viral HUH-endonuclease by alanine scanning of an electrostatic patch

HUH endonucleases (dubbed "HUH-tags") are small protein domains capable of forming covalent adducts with ssDNA in a sequence-specific manner. Because viral HUH-tags are relatively small, react quickly, and require no chemical modifications to their ssDNA substrate, they have great value as protein fusion tags in biotechnologies ranging from genetic engineering to single-molecule studies. One of the greatest assets of these tags is sequence-specificity to their unique, native Ori sequence in vivo, introducing the possibility of using multiple HUH-tags in multiplexed "one-pot" reactions. However, their mechanism of ssDNA sequence binding and specificity is poorly understood, and there is noted cross-reactivity between tags of closely related species. In order to understand the mechanism of ssDNA binding, we performed an alanine scan along a positively-charged patch of one such HUH-tag, replication-associated protein from Wheat Dwarf Virus (WDV Rep), and characterized the enzymatic activity in both the rate and extent of the reaction. In molecular beacon Stopped-Flow experiments, single point mutants of WDV showed a more than 60% decrease in reaction rate constant, and gel shift assays showed an almost complete lack of activity for some variants for single nucleotide ssDNA substitutions. In all, these findings help allow us to highlight key interactions in WDV-ssDNA binding, and we gain further insight into potential rational engineering of HUH-endonucleases to bind desired sequences of DNA.

biochemistry↗

bioRxiv2019

Stable encoding of visual cues in the mouse retrosplenial cortex

The rodent retrosplenial cortex functions as an integrative hub for sensory and motor signals, serving roles in both navigation and memory. While retrosplenial cortex (RSC) is reciprocally connected with the sensory cortex, the form in which sensory information is represented in the retrosplenial cortex and how it interacts with behavioural state is unclear. Here, we used 2-photon cellular imaging of neural activity of putative excitatory (CaMKII expressing) and inhibitory (parvalbumin expressing) neurons to measure visual and running evoked activity in RSC and compare it to primary visual cortex (V1). We found that stimulus position and orientation information was preserved between V1 and RSC, and additionally that positional information was organised topographically. Stimulus directional preference was biased towards nasal-temporal flow. Locomotion modulation of activity of single neurons, both in darkness and light, was also more pronounced in RSC than V1, and strongest in parvalbumin-positive neurons. Longitudinal measurements of single neurons showed that these response features were stably maintained over many weeks. These data provide evidence for stable representations of visual cues in retrosplenial cortex which are highly spatially selective. These may provide sensory data to contribute to the formation of memories of spatial information.

neuroscience↗