Biology subjects

Jung, C.

Publications and source records attributed to Jung, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Novel fluorescence-based methods to determine infarct and scar size in murine models of reperfused myocardial infarction

BackgroundDetermination of infarct area and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in mice and other animal models. MethodsFor both, early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male C57BL/6J mice and hearts were extracted 24 hours, 7-, 21-, or 28-days following MI and stained with 2,3,5-Triphenyltetrazolium chloride (TTC) and Evans Blue, Hoechst, phalloidin, Sirius Red, Massons or Gomoris Trichrome or Wheat Germ Agglutinin (WGA). ResultsFluorescent staining combining Hoechst and phalloidin constitutes an alternative for TTC and Evans Blue, enabling a clear visualization of infarct area, area at risk, as well as remote area unaffected by MI. Infarct size early after reperfusion determined with TTC staining correlates strongly with that demarcated by phalloidin while combination of Hoechst and phalloidin staining can emulate classical TTC/Evans Blue staining 24 h post-MI. Moreover, WGA is equally accurate as the classical Sirius Red, Massons and Gomoris Trichrome stainings in identifying scar size in later phases (>7d) post-MI. Finally, we demonstrate feasibility of combining conventional fluorescence staining by localizing CD45+ leukocytes to specific regions of the infarcted myocardium. ConclusionWe established staining procedure is not inferior to classical TTC staining while providing substantial benefits including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/604384v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17ccc83org.highwire.dtl.DTLVardef@56d433org.highwire.dtl.DTLVardef@857f37org.highwire.dtl.DTLVardef@63297d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

Signatures in domesticated beet genomes pointing at genes under selection in a sucrose-storing root crop

The genus Beta encompasses important crops such as sugar, table, fodder, and leaf beets. All cultivated beets are believed to have originated from the wild sea beet, B. vulgaris subsp. maritima. Sugar beet, a recent crop dating back nearly 200 years, was selectively bred for enhanced root yield in combination with high sucrose content. We assembled a Beta diversity panel comprising wild and cultivated beet accessions. Whole-genome sequencing identified 10.3 million SNP markers. Four distinct genetic clusters were identified: table beet, sugar beet, Mediterranean sea beet, and Atlantic sea beet. A phylogenetic analysis revealed that cultivated beet accessions were genetically closer to Mediterranean than to Atlantic sea beet and that cultivated beets producing storage roots share a common ancestor. Cultivated beets exhibited genome regions with reduced nucleotide diversity compared to Mediterranean sea beets, indicating selection signatures. These regions contained putative candidate genes with potential roles in root development, suppression of lateral root formation, flowering time, and sucrose metabolism. A yet unknown sucrose transporter on chromosome 6 showed reduced nucleotide diversity exclusively in sugar beet accessions compared to other Beta types with low sucrose content, suggesting its role in sucrose storage. Within a region of high nucleotide diversity between accessions with contrasting root phenotypes, we found two genes encoding auxin response factors, which play a crucial role in root development. We reason these genes to be significant root thickening regulators in root crops.

genomics↗

bioRxiv2024

ZNF574 is a Quality Control Factor For Defective Ribosome Biogenesis Intermediates

Eukaryotic ribosome assembly is an intricate process that involves four ribosomal RNAs, 80 ribosomal proteins, and over 200 biogenesis factors that take part in numerous interdependent steps. This complexity creates a large genetic space in which pathogenic mutations can occur. "Dead-end" ribosome intermediates that result from biogenesis errors are rapidly degraded, affirming the existence of quality control pathway(s) that monitor ribosome assembly. However, the factors that differentiate between on-path and dead-end intermediates are unknown. We engineered a system to perturb ribosome assembly in human cells and discovered that faulty ribosomes are degraded via the ubiquitin proteasome system. We identified ZNF574 as a key component of a novel quality control pathway, which we term the Ribosome Assembly Surveillance Pathway (RASP). Loss of ZNF574 results in the accumulation of faulty biogenesis intermediates that interfere with global ribosome production, further emphasizing the role of RASP in protein homeostasis and cellular health.

cell biology↗

bioRxiv2024

SARS-CoV-2 Omicron Envelope T9I adaptation confers resistance to autophagy

To date, five variants of concern (VOCs) of SARS-CoV-2 have emerged that show increased fitness and/or immune evasion. While the continuously evolving escape from humoral immune responses has been analyzed in detail, adaptation of SARS-CoV-2 to human innate immune defenses such as autophagy is less understood. Here, we demonstrate that mutation T9I in the structural envelope (E) protein confers autophagy resistance of Omicron VOCs (BA.1, BA.5 and XBB.1.5) compared to 2020 SARS-CoV-2 or the Delta VOC. Mechanistic analyses revealed that Omicron-associated E T9I shows increased inhibition of autophagic flux and colocalization/interaction with autophagosomes, thus shielding incoming SARS-CoV-2 S pseudotyped virions from autophagy. Rare Omicron isolates carrying ancestral E T9 remain sensitive towards autophagy whereas recombinant early 2020 SARS-CoV-2 expressing E T9I shows increases resistance against autophagy. Our data indicate that the E T9I mutation drives autophagy resistance of the Omicron variants and thus may have contributed to their effective spread.

microbiology↗

bioRxiv2024

Oriented Cell Dataset: efficient imagery analyses using angular representation

In this work, we propose a new public dataset for cell detection in bright-field microscopy images annotated with Oriented Bounding Boxes (OBBs), named Oriented Cell Dataset (OCD). We show that OBBs provide a more accurate shape representation compared to standard Horizontal Bounding Boxes (HBBs), with slight overhead of one extra click in the annotation process. Our dataset also contains a subset of images with five independent expert annotations, which allows inter-annotation analysis to determine if the results produced by algorithms are within the expected variability of human experts. We investigated how to automate cell biology microscopy images by training seven popular OBB detectors in the proposed dataset, and focused our analyses on two main problems in cancer biology: cell confluence and polarity determination, the latter not possible through HBB representation. All models achieved statistically similar results to the biological applications compared to human annotation, enabling the automation of cell biology and cancer cell biology microscopy image analysis. Our code and dataset are available at https://github.com/LucasKirsten/Deep-Cell-Tracking-EBB.

bioinformatics↗

bioRxiv2023

Leaf and shoot apical meristem transcriptomes of quinoa (Chenopodium quinoa Willd.) in response to photoperiod and plant development

Our study aimed to identify candidate genes for flowering time regulation and photoperiod response in quinoa. We investigated the timing of photoperiod-driven floral transition and analyzed the transcriptomes of leaf and shoot apical meristems in photoperiod-sensitive and -insensitive quinoa accessions. Histology analysis of the apical meristem showed that floral transition in quinoa initiates two to three weeks after sowing. We found four groups of differentially expressed genes responding to plant development and floral transition, which were annotated in the QQ74-V2 reference genome, including (i) 222 genes differentially responding to photoperiod in leaves, (ii) 1,812 genes differentially expressed between accessions under long-day conditions in leaves, (iii) 57 genes responding to developmental changes between weeks under short-day conditions in leaves, and (iv) 911 genes responding to floral transition within the shoot apical meristem. Interestingly, out of the thousands of candidates, two putative FT orthologues and several others have been reported as key regulators of flowering time in other species (e.g., SOC1, COL, AP1). Additionally, we used co-expression networks to associate novel transcripts to a putative biological process based on the annotated genes within the same co-expression cluster. The candidate genes in this study would benefit quinoa breeding by identifying and integrating their beneficial haplotypes in crossing programs to develop adapted cultivars to diverse environmental conditions.

plant biology↗

bioRxiv2023

Inhibition of proline-rich-tyrosine kinase 2 restores cardioprotection by remote ischemic preconditioning in type 2 diabetes mellitus

BackgroundEndothelial function and cardioprotection through remote ischemic preconditioning (rIPC) are severely impaired in type 2 diabetes mellitus (T2DM). Proline-rich tyrosine kinase 2 (Pyk2), a downstream target of the insulin receptor, reduces endothelial nitric oxide synthase (eNOS) activity. Therapeutic options to rescue cardioprotection in T2DM and improve outcomes after acute myocardial infarction (AMI) are lacking. We hypothesized that vascular endothelium contributes to rIPC, and that inhibition of Pyk2 restores cardioprotection in T2DM through modulation of eNOS, thus limiting infarct size. MethodsNew Zealand Obese (NZO) mice were used as a polygenic model of T2DM. Effects of Pyk2-inhibition on endothelial function, remote ischemic preconditioning (rIPC), and infarct size (IS) after ischemia/reperfusion (I/R) were compared in NZO, eNOS KO, and C57Bl/6 (Bl6) mice. Plasma derived from mice and individuals with or without T2DM at baseline and after rIPC was transferred to isolated hearts and aortic rings to assess the effects of Pyk2-inhibition on remote tissue protection. ResultsTransfer experiments with plasma drawn from non-diabetic humans and mice exposed to rIPC demonstrate that endothelium-dependent signals for remote tissue protection are conveyed by plasma. Key features reflecting the glucometabolic spectrum in T2DM were detected in NZO mice, including hyperinsulinemia, insulin resistance, obesity, and impaired glucose tolerance. Similar to T2DM patients, these mice also revealed endothelial dysfunction with decreased flow-mediated dilation (FMD), reduced circulating nitrite levels, elevated arterial blood pressure, and larger infarct size after I/R. Pyk2 increased the phosphorylation of eNOS on its inhibitory site (Tyr656). Cardioprotective effects by rIPC were lost in NZO mice. Inhibition of Pyk2 restored endothelial function and rescued endothelium-dependent cardioprotection after rIPC displayed by lower IS and improved LV function post I/R. ConclusionEndothelial function contributing to remote tissue protection is severely impaired in diabetes mellitus. Proline-rich tyrosine kinase 2 is a novel target to rescue cardioprotection through endothelium-dependent remote ischemic preconditioning, advocating its role in limiting infarct size in diabetes mellitus. Clinical perspective What is new?O_LIVascular endothelium contributes to remote tissue protection in ischemic preconditioning, which is severely impaired in diabetes C_LIO_LIProline-rich tyrosine kinase 2 reduces eNOS-activity, causes endothelial dysfunction, and impairs cardioprotection through ischemic preconditioning C_LIO_LIInhibition of proline-rich tyrosine kinase 2 restores eNOS activity, endothelial function, and cardioprotective effects of remote ischemic preconditioning limiting infarct size in an experimental model of diabetes. C_LI What are the clinical implications?O_LIProper endothelial function is cirtical to maintain cardiovascular health. Endothelial dysfunction contributes to impaired remote tissue protection in diabetes. C_LIO_LIThese data demonstrate for the first time that endothelium-dependent cardioprotection in myocardial ischemia/reperfusion through remote ischemic preconditioning can be restored in diabetes. C_LIO_LIProline-rich tyrosine kinase 2 is a novel target to restore endothelium-dependent remote cardioprotection to improve the outcome of diabetic patients with acute myocardial infarction. C_LI

physiology↗

bioRxiv2023

Determinants of species-specific utilization of ACE2 by human and animal coronaviruses

Utilization of human ACE2 allowed several bat coronaviruses (CoVs), including the causative agent of COVID-19, to infect humans either directly or via intermediate hosts. Here, we analyzed the ability of Spike proteins from 24 human or animal CoVs to use ACE2 receptors across nine reservoir, potential intermediate and human hosts. We show that overall SARS-CoV-2 Omicron variants evolved more efficient ACE2 usage but mutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats. Spikes from most CoVs showed species-specific differences in ACE2 usage, partly due to variations in ACE2 residues 31, 41 or 354. Mutation of T403R allowed the RaTG13 bat CoV Spike to use all ACE2 orthologs analysed for viral entry. Sera from COVID-19 vaccinated individuals neutralized the Spike proteins of a range of bat Sarbecoviruses. Our results define determinants of ACE2 receptor usage of diverse CoVs and suggest that COVID-19 vaccination may protect against future zoonoses of SARS-CoV-related bat viruses. HighlightsO_LIMutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats C_LIO_LIVariations in ACE2 residues 31, 41 or 354 affect utilization by coronavirus Spike proteins C_LIO_LIResidue R403 in the Spike protein of bat coronavirus allow broad and effective ACE2 usage C_LIO_LISera from COVID-19 vaccinated individuals neutralize Spike proteins of bat Sarbecoviruses C_LI

molecular biology↗

bioRxiv2023

SARS-CoV-2 NSP5 Antagonizes the MHC II Antigen Presentation Pathway by Hijacking Histone Deacetylase 2

SARS-CoV-2 interferes with antigen presentation by downregulating MHC II on antigen presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. This downregulation of CIITA is independent of NSP5s proteolytic activity, but rather, NSP5 delivers HDAC2 to IRF3 at an IRF binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response. ImportanceSARS-CoV-2 alters the expression of many immunoregulatory proteins to limit and delay the host antiviral response, thereby producing a more severe and longer-lasting infection. Preventing and limiting the activation of helper T cells by reducing MHC II expression on antigen presenting cells is one of these strategies, but while this mechanism was identified early in the pandemic, the mechanism allowing SARS-CoV-2 to limit MHC II expression has remained unclear. Herein, we demonstrate that this occurs via a tripartite interaction between viral NSP5 and host HDAC2 and IRF3, where a complex of NSP5 and HDAC2 is recruited to IRF3 bound to the promoter of CIITA--the master regulator of MHC II expression--with the delivery of HDAC2 then mediating the deacetylation of the CIITA promoter and the suppression of MHC II expression.

immunology↗

bioRxiv2022

Direct access to millions of mutations by Whole Genome Sequencing of an oilseed rape mutant population

Induced mutations are an essential source of genetic variation in plant breeding. EMS mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In this study, a rapeseed M2 population was derived from M1 parent cultivar "Express" treated with EMS. Whole genomes were sequenced from fourfold (4x) pools of 1,988 M2 plants representing 497 M2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284,442 single nucleotide polymorphisms per M2 DNA pool were found resulting from EMS mutagenesis. 55% were C[->]T and G[->]A transitions, characteristic for EMS induced ( canonical) mutations, whereas the remaining SNPs were non-canonical transitions (15%) or transversions (30%). Additionally, we detected 88,725 high confidence insertions and deletions (InDels) per pool. On average, each M2 plant carried 39,120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Roughly 82% of such mutations were located either 5 kb upstream or downstream (~56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole-genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool "EMS Brassica" (http://www.emsbrassica.plantbreeding.uni-kiel.de/), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

genomics↗

bioRxiv2022

Reduced glucosinolate content in oilseed rape (Brassica napus L.) by random mutagenesis of BnMYB28 and BnCYP79F1 genes

The presence of anti-nutritive compounds like glucosinolates (GSLs) in the rapeseed meal severely restricts its utilization as animal feed. Therefore, reducing the GSL content to <18 {micro}mol/g dry weight in the seeds is a major breeding target. While candidate genes involved in the biosynthesis of GSLs have been described in rapeseed, comprehensive functional analyses are missing. By knocking out the aliphatic GSL biosynthesis genes BnMYB28 and BnCYP79F1 encoding an R2R3 MYB transcription factor and a cytochrome P450 enzyme, respectively, we aimed to reduce the seed GSL content in rapeseed. After expression analyses on single paralogs, we used an ethyl methanesulfonate (EMS) treated population of the inbred winter rapeseed Express617 to detect functional mutations in the two gene families. Our results provide the first functional analysis by knock-out for the two GSL biosynthesis genes in winter rapeseed. We demonstrate that independent knock-out mutants of the two genes possessed significantly reduced seed aliphatic GSLs, primarily progoitrin. Compared to the wildtype Express617 control plants (36.3 {micro}mol/g DW), progoitrin levels were decreased by 55.3% and 32.4% in functional mutants of BnMYB28 (16.20 {micro}mol/g DW) and BnCYP79F1 (24.5 {micro}mol/g DW), respectively. Our study provides a strong basis for breeding rapeseed with improved meal quality in the future.

plant biology↗

bioRxiv2022

Tumor necrosis factor is a necroptosis-associated alarmin

Necroptosis is a form of regulated cell death that can occur downstream of several immune pathways. While previous studies have shown that dysregulated necroptosis leads to strong inflammatory responses, little is known about the identity of the endogenous molecules that trigger necroptosis-mediated inflammatory responses. Using a reductionist in vitro model, we found that soluble TNF is strongly released in the context of necroptosis. On the one hand, necroptosis promotes TNF translation by inhibiting negative regulatory mechanisms acting at the post-transcriptional level. On the other hand, necroptosis markedly enhances TNF release by activating ADAM proteases. In studying TNF release at single-cell resolution, we found that TNF release triggered by necroptosis is activated in a switch-like manner and exceeds steady-state TNF processing in magnitude and speed. Although this shedding response precedes massive membrane damage, it is closely associated with lytic cell death. In fact, we found that lytic cell death induction using a pore-forming toxin also triggers TNF shedding, indicating that the activation of ADAM proteases is not strictly related to the necroptotic pathway but associated with biophysical changes of the cell membrane upon lytic cell death. These results demonstrate that lytic cell death, particularly necroptosis, is a critical trigger for TNF release and thus qualify TNF as a necroptosis-associated alarmin.

immunology↗

bioRxiv2022

An interaction between betaprime-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling

The essential COPI vesicular coat mediates retrieval of key transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAP proteins remain elusive. Biochemical and biophysical data reveal how {beta}-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity (KD ~1 {micro}M). Calorimetry data demonstrate both {beta}-COP propeller domains are required to bind Glo3 using electrostatic interactions. An acidic patch on {beta}-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (Binding of Coatomer, Cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or {beta}-COP abrogate the interaction in vitro, and loss of the {beta}-COP/Glo3 interaction drives Ste2 mis-sorting to the vacuole and aberrant Golgi morphology in budding yeast. Together, these data suggest cells require the {beta}-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where {beta}-COP may serve as a molecular platform to coordinate binding to multiple protein partners, including Glo3, Arf1, and the COPI F-subcomplex.

cell biology↗

bioRxiv2022

Determinants of Spike Infectivity, Processing and Neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2

The SARS-CoV-2 Omicron variant rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission, immune evasion and pathogenicity is thought to be driven by numerous mutations in the Omicron Spike protein. Here, we examined the impact of amino acid changes that are characteristic for the BA.1 and/or BA.2 Omicron lineages on Spike function, processing and susceptibility to neutralization. Individual mutations of S371F/L, S375F and T376A in the ACE2 receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes of G339D, D614G, N764K and L981F moderately enhanced it. Most mutations in the N-terminal region and the receptor binding domain reduced sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine or therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that allowed this SARS-CoV-2 variant to overtake the current pandemic. HIGHLIGHTSO_LIS371F/L, S373P and S375F impair Spike function and revert in some BA. 1 isolates C_LIO_LIChanges of Q954H and N969K in HR1 reduce while L981F enhances S-mediated infection C_LIO_LIOmicron-specific mutations in the NTD and RBD of Spike reduce neutralization C_LIO_LIN440K, G446S, E484A and Q493K confer resistance to bamlanivimab or imdevimab C_LI

microbiology↗

bioRxiv2022

High-density mapping of QTL controlling agronomically important traits in quinoa (Chenopodium quinoa Willd.)

Quinoa is a pseudocereal originating from the Andean regions. In spite of quinoas long cultivation history, genetic analysis of this crop is still in its infancy. We aimed to localize QTL contributing to the phenotypic variation of agronomically important traits. We crossed the Chilean accession PI-614889 and the Peruvian accession CHEN-109, which depicted significant differences in days to flowering, days to maturity, plant height, panicle length, thousand kernel weight (TKW), saponin content, and mildew susceptibility. We observed sizeable phenotypic variation across F2 plants and F3 families grown in the greenhouse and in the field, respectively. We used Skim-seq to genotype the F2 population and constructed a high-density genetic map with 133,923 SNPs. Fifteen QTL were found for ten traits. Two significant QTL, common in F2 and F3 generations, depicted pleiotropy for days to flowering, plant height, and TKW. The pleiotropic QTL harbored several putative candidate genes involved in photoperiod response and flowering time regulation. This study presents the first high-density genetic map of quinoa that incorporates QTL for several important agronomical traits. The pleiotropic loci can facilitate marker assisted selection in quinoa breeding programs. Key messageSkim-sequencing enabled the construction a high-density genetic map (133,923 SNPs) and fifteen QTL were detected for ten agronomically important traits.

genetics↗

bioRxiv2021

Cerebellar contributions to a brainwide network for flexible behavior

The cerebellum regulates nonmotor behavior, but the routes by which it exerts its influence are not well characterized. Here we report a necessary role for the posterior cerebellum in guiding a reversal learning task, acting through a network of diencephalic and neocortical structures. After chemogenetic inhibition of Purkinje cells in lobule VI or crus I, mice could learn a water Y-maze task but were impaired in their ability to reverse their initial choice. To map targets of perturbation, we imaged c-Fos activation in cleared whole brains using light-sheet microscopy. Reversal learning activated diencephalic regions and associative neocortical regions. Distinctive subsets of structures were altered by perturbation of lobule VI (including thalamus and habenula) and crus I (including hypothalamus and prelimbic/orbital cortex), and both perturbations influenced anterior cingulate and infralimbic cortex. To identify functional networks, we used correlated variation in c-Fos activation within each test group. Lobule VI inactivation weakened within-thalamus correlations, while crus I inactivation divided neocortical activity into well-separated sensorimotor and associative subnetworks. In both groups, high-throughput automated analysis of complex whole-body movement revealed deficiencies in across-day adaptation to an open field environment. Neither perturbation affected gait, within-day open-field adaptation, or location preference. Taken together, these experiments reveal brainwide systems for cerebellar influence that can affect multiple flexible responses. Significance statementThe cerebellum, a part of all vertebrate brains, provides feedback to the rest of the brain on a split-second basis in response to unexpected events. The consequent changes can range from minute adjustments in movement to broad changes in behavior. In mice, we investigated cerebellar regions that help guide flexible behavior. Inactivation of these regions prevented mice from responding flexibly to a changing Y-shaped maze. We used light-sheet microscopy of c-fos protein expression to map brainwide effects of different stages of learning and cerebellar inactivation. Inactivation caused changes in thalamocortical activity that were similar in pattern, but opposite in sign, from normal learning. Lobule VI inactivation weakened thalamic functional networks, while crus I inactivation divided sensorimotor from associative neocortical networks. The same inactivation also impaired multiday adaptation to an open arena, as tracked using machine vision. Our work shows how the cerebellums influence over flexible response can be mediated by a widespread brain network.

neuroscience↗

bioRxiv2021

Massively Parallel Selection of NanoCluster Beacons

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed the polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here we report a high-throughput selection method that takes advantage of repurposed next-generation-sequencing (NGS) chips to screen the activation fluorescence of [~]40,000 activator sequences. We find the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow and red POTs, respectively. Based on these findings, we propose a "zipper bag model" that explains how these hotspots lead to the creation of distinct silver cluster chromophores and contribute to the difference in chromophore chemical yields. Combining high-throughput screening with machine learning algorithms, we establish a pipeline to rationally design bright and multicolor NCBs.

bioengineering↗

bioRxiv2021

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidAs chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidAs chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N- chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCls chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

biochemistry↗