Biology subjects

Jung, C.

Publications and source records attributed to Jung, C..

50 records · Page 3Linked to original sources

bioRxiv2021

Adeno-associated virus (AAV) reduces cortical dendritic complexity in a TLR9-dependent manner

Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery in the nervous system. AAVs are widely used in research and are the basis of multiple FDA-approved gene therapies. Here, we find that the immune response to AAVs genome reduces dendritic complexity in mammalian cortex. Dendritic loss associated with AAV-mediated gene delivery occurs at experimentally-relevant titers, cannot be explained by responses to transgene expression or surgery, and is not restricted to a particular capsid serotype, encoded transgene, promoter, or production facility. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) and upregulation of immune molecules that can limit dendritic complexity and synaptic transmission. Blocking detection of unmethylated CpG-rich DNA via Toll-like receptor 9 (TLR9) protects dendritic complexity, suggesting that immunodetection of a core feature of the AAV genome triggers dendritic loss. These results reveal previously unsuspected impacts of AAV on neuronal structure and function and identify TLR9 inhibitors as important tools to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.

neuroscience↗

bioRxiv2021

Spike mutation T403R allows bat coronavirus RaTG13 to use human ACE2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats1. A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor2. Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.

microbiology↗

bioRxiv2021

Expansion of the prime editing modality with Cas9 from Francisella novicida

Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase (RT) after nick formation by CRISPR nickase. In this study, we developed a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas 9 [FnCas9(H969A)] nickase module, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing was dramatically extended, and accurate prime editing was induced specifically for the target genes.

bioengineering↗

bioRxiv2021

The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation

The blood-brain barrier (BBB) of Drosophila is comprised of a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity, by precisely regulating the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKAs molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments and not their total length is crucial for generating a proper paracellular seal. Based on these findings, we propose a model that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical for maintain tissue size and brain homeostasis during organogenesis.

developmental biology↗

bioRxiv2020

Genome-wide association study in the pseudocereal quinoa reveals selection pattern typical for crops with a short breeding history

Quinoa germplasm preserves useful and substantial genetic variation, yet it remains untapped due to a lack of implementation of modern breeding tools. We have integrated field and sequence data to characterize a large diversity panel of quinoa. Whole-genome sequencing of 310 accessions revealed 2.9 million polymorphic high confidence SNP loci. Highland and Lowland quinoa were clustered into two main groups, with FST divergence of 0.36 and fast LD decay of 6.5 and 49.8 Kb, respectively. A genome-wide association study uncovered 600 SNPs stably associated with 17 agronomic traits. Two candidate genes are associated with thousand seed weight, and a resistance gene analog is associated with downy mildew resistance. We also identified pleiotropically acting loci for four agronomic traits that are highly responding to photoperiod hence important for the adaptation to different environments. This work demonstrates the use of re-sequencing data of an orphan crop, which is partially domesticated to rapidly identify marker-trait association and provides the underpinning elements for genomics-enabled quinoa breeding.

genomics↗

bioRxiv2020

Natural Cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling

GPR15 is a G protein-coupled receptor proposed to play a role in mucosal immunity that also serves as entry cofactor for HIV and SIV. To discover novel endogenous GPR15 ligands, we screened a hemofiltrate-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of Cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2 and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that Cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this G protein-coupled receptor.

microbiology↗

bioRxiv2020

Context dependent activation/repression by Hunchback binding sites in Drosophila embryo

Hunchback (Hb) is considered a context-dependent transcription factor, able to activate or repress different enhancers during Drosophila embryo segmentation. The mechanism driving the contextdependent activity of Hb is however not well understood. Here we measure the activity of a large set of 20 synthetic enhancers that we design to elucidate the effect of Hb binding sites in Drosophila segmentation. We obtain quantitative data on the spatiotemporal dynamics of activity of all synthetic enhancers in-vivo, by using a quantitative and sensitive reporter system we recently developed. Our data reveal the dual role of Hb binding sites in segmentation enhancers: on the one hand, Hb act as a typical short range repressor by binding to its cognate sequences; on the other hand, we report a novel effect of a sequence containing multiple Hb binding sites, which is able to increase enhancer activity independently from Hb binding. This sequence, which contains multiple Poly-dA stretches, increases the activity of enhancers driven by different activators, possibly by disfavoring nucleosome occupancy. AUTHOR SUMMARYThe control of gene expression is a fundamental process that allows cells to respond to external stimuli and take on various identities in complex organisms. Enhancers are DNA sequences that play a key role in this process. In the simplest model of an enhancer, small parts of its sequence can be specifically bound by proteins called transcription factors and the occupancy pattern of these proteins on the enhancer determines the expression level of a specific gene. In this research work we have studied enhancers in the context of the development of a fruit fly embryo. We have built synthetic enhancer sequences containing binding sites for a few specific factors and measured their activity in living embryos using fluorescence microscopy. Our results revealed that binding sites for a particular protein, Hunchback, are able to influence the activity of the enhancer even independently from Hunchback binding to them. This discovery might help to explain the complex effects that have been observed when studying Hunchback binding sites in natural enhancers.

developmental biology↗

bioRxiv2020

Large-scale analysis of Drosophila core promoter function using synthetic promoters

The core promoter, the region immediately surrounding the transcription start site, plays a central role in setting metazoan gene expression levels, but how exactly it computes expression remains poorly understood. To dissect core promoter function, we carried out a comprehensive structure-function analysis to measure synthetic promoters activities, with and without an external stimulus (hormonal activation). By using robotics and a dual-luciferase reporter assay, we tested [~]3000 mutational variants representing 19 different Drosophila melanogaster promoter architectures. We explored the impact of different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, positioning, and flanking sequences. We observe strong effects of the mutations on activity, and a linear combination of the individual motif features can largely account for the combinatorial effects on core promoter activity. Our findings shed new light on the quantitative assessment of gene expression, a fundamental process in all metazoans.

systems biology↗

bioRxiv2020

Dynamic brain-to-brain concordance and behavioral mirroring as a mechanism of the patient-clinician interaction

The patient-clinician interaction can powerfully shape treatment out-comes such as pain, but is often considered an intangible 'art-of-medicine', and has largely eluded scientific inquiry. Although brain correlates of social processes such as empathy and theory-of-mind have been studied using single-subject designs, the specific behavioral and neural mechanisms underpinning the patient-clinician interaction are unknown. Using a two-person interactive design, we simultaneously recorded functional MRI (i.e. hyperscanning) in patient-clinician dyads, who interacted via live video while clinicians treated evoked pain in chronic pain patients. Our results show that patient analgesia is mediated by patient-clinician nonverbal behavioral mirroring and brain-to-brain concordance in circuitry implicated in theory-of-mind and social mirroring. Dyad-based analyses showed extensive dynamic coupling of these brain nodes with the partners' brain activity, yet only in dyads where clinical rapport had been established prior to the interaction. These findings point to a putatively key brain-behavioral mechanism for therapeutic alliance and psychosocial analgesia.

neuroscience↗

bioRxiv2020

Validation of suitable genes for normalization of diurnal gene expression studies in Chenopodium quinoa

Quinoa depicts high nutritional quality and abiotic stress resistance attracting strong interest in the last years. To unravel the function of candidate genes for agronomically relevant traits, studying their transcriptional activities by RT-qPCR is an important experimental approach. The accuracy of such experiments strongly depends on precise data normalization. To date, validation of potential candidate genes for normalization of diurnal expression studies has not been performed in C. quinoa. We selected eight candidate genes based on transcriptome data and literature survey, including conventionally used reference genes. We used three statistical algorithms (BestKeeper, geNorm and NormFinder) to test their stability and added further validation by a simulation-based strategy. We demonstrated that using different reference genes, including those top ranked by stability, causes significant differences among the resulting diurnal expression patterns, and that our novel approach overcomes failures related to stability-based selection of reference genes. Our results show that isocitrate dehydrogenase enzyme (IDH-A) and polypyrimidine tract-binding protein (PTB) are suitable genes to normalize diurnal expression data of two different quinoa accessions. The validated reference genes obtained in this study will improve the accuracy of RT-qPCR data normalization and facilitate gene expression studies in quinoa.

plant biology↗

bioRxiv2019

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeq-- nuclease digestion and deep sequencing--a massively parallel platform that measures cleavage kinetics and captures the time-resolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a--reported as a more specific nuclease in cells--has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

molecular biology↗

bioRxiv2019

Nod2 protects remote small intestinal sites in case of colonic inflammation.

NOD2 mutations are key risk factors for Crohns disease (CD). NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the roles of NOD2 during gut inflammation is not known. We initially observed that NOD2 expression was increased in epithelial cells remote from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation and gut permeability were examined in the small bowel of wild-type (WT), Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). In WT mice, Nod2 upregulation remote to rectal injury was associated with pro-inflammatory cytokine expression, recirculating CD4+ T-cells, increased paracellular permeability and myosin like chain kinase activity. Nod2 knockout or mutation led to duodenitis and ileitis demonstrating the remote protective role of Nod2. Bone morrow stem cell (BMSC) transplantations indicated that the small intestinal inflammation was due to NOD2 loss in both hematopoietic and non-hematopoietic compartments. As a whole, WT but not mutant NOD2 prevents disease extension at sites remote from the initial intestinal injury.

immunology↗

bioRxiv2018

Supercharging enables organized assembly of synthetic biomolecules

There are few methods for the assembly of defined protein oligomers and higher order structures that could serve as novel biomaterials. Using fluorescent proteins as a model system, we have engineered novel oligomerization states by combining oppositely supercharged variants. A well-defined, highly symmetrical 16-mer (two stacked, circular octamers) can be formed from alternating charged proteins; higher order structures then form in a hierarchical fashion from this discrete protomer. During SUpercharged PRotein Assembly (SuPrA), electrostatic attraction between oppositely charged variants drives interaction, while shape and patchy physicochemical interactions lead to spatial organization along specific interfaces, ultimately resulting in protein assemblies never before seen in nature.

bioengineering↗

bioRxiv2017

The Anatomical Tracings of Lesions After Stroke (ATLAS) Dataset - Release 1.1

Stroke is the leading cause of adult disability worldwide, with up to two-thirds of individuals experiencing long-term disabilities. Large-scale neuroimaging studies have shown promise in identifying robust biomarkers (e.g., measures of brain structure) of long-term stroke recovery following rehabilitation. However, analyzing large rehabilitation-related datasets is problematic due to barriers in accurate stroke lesion segmentation. Manually-traced lesions are currently the gold standard for lesion segmentation on T1-weighted MRIs, but are labor intensive and require anatomical expertise. While algorithms have been developed to automate this process, the results often lack accuracy. Newer algorithms that employ machine-learning techniques are promising, yet these require large training datasets to optimize performance. Here we present ATLAS (Anatomical Tracings of Lesions After Stroke), an open-source dataset of 304 T1-weighted MRIs with manually segmented lesions and metadata. This large, diverse dataset can be used to train and test lesion segmentation algorithms and provides a standardized dataset for comparing the performance of different segmentation methods. We hope ATLAS release 1.1 will be a useful resource to assess and improve the accuracy of current lesion segmentation methods.

neuroscience↗