Biology subjects

Wagner, M.

Publications and source records attributed to Wagner, M..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

A new twist in ABC transporter mediated multidrug resistance - Pdr5 is a drug/proton co-transporter

The two major efflux pump systems are involved in multidrug resistance (MDR): (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ABC transporters that are involved in MDR and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of transport and specificity remain elusive. Here, we provide electrophysiological data on reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

biochemistry↗

bioRxiv2021

A simple coloring method to distinguish colonies of the yeasts Lachancea thermotolerans and Saccharomyces cerevisiae on agar media

The yeast Lachancea thermotolerans converts consumed sugar partly to lactic acid instead of ethanol and is therefore used together with Saccharomyces cerevisiae to produce wines with a lower alcohol content. Being able to distinguish these yeasts is important for quality control and quantitative assessment of the contributions of both yeasts to wine fermentations. Commonly used methods to routinely distinguish these organisms are indirect or rely on commercial products of undisclosed composition. Here we describe that adding bromocresol purple to agar media induces Lachancea colonies to develop a brown color, whereas Saccharomyces colonies remain white.

microbiology↗

bioRxiv2021

Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co- occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest substrate affinity of any characterized AOA, which are similar to previously determined affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values strongly supports the hypothesis that - like for AOB - ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

microbiology↗

bioRxiv2021

Gene flux and acid-imposed selection are the main drivers of antimicrobial resistance in broiler chicks infected with Salmonella enterica serovar Heidelberg

Antimicrobial resistance (AR) spread is a worldwide health challenge, stemming in large part, from the ability of microbes to share their genetic material through horizontal gene transfer (HGT). Overuse and misuse of antibiotics in clinical settings and in food production have been linked to this increased prevalence and spread of AR. Consequently, public health and consumer concerns have resulted in a remarkable recent reduction in antibiotics used for food animal production. This is driven by the assumption that removing this selective pressure will favor the recovery of antibiotic susceptible taxa and will limit AR sharing through HGT, allowing the currently available antibiotic arsenal to be effective for a longer period. In this study we used broiler chicks raised antibiotic-free and Salmonella enterica serovar Heidelberg (SH), as a model food pathogen, to test this hypothesis. Our results show that neonatal broiler chicks challenged with an antibiotic susceptible SH strain and raised without antibiotics carried susceptible and multidrug resistance SH strains 14 days after challenge. SH infection perturbed the microbiota of broiler chicks and gavaged chicks acquired antibiotic resistant SH at a higher rate. We determined that the acquisition of a plasmid from commensal Escherichia coli population conferred multidrug resistance phenotype to SH recipients and carriage of this plasmid increased the fitness of SH under acidic selection pressure. These results suggest that HGT of AR shaped the evolution of SH and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella. ImportanceThe reported increase in antibiotic resistant bacteria in humans have resulted in a major shift away from antibiotics use in food animal production. This has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, and this in turn will allow the currently available antibiotic arsenal to be more effective. This shift in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic susceptible Salmonella Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggests that simply removing antibiotics from food-animal production might not be sufficient to limit the spread of antimicrobial resistance.

microbiology↗

bioRxiv2021

Extracellular riboflavin induces anaerobic biofilm formation in Shewanella oneidensis

Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalysed by the terminal reductases and as endogenous electron shuttles. In the model organism Shewanella oneidensis, riboflavin production and excretion triggers a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescin decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations which by far exceed the necessary threshold value. ImportanceFerric iron is the fourth most abundant element of the earth crust. It occurs at neutral pH in the form insoluble iron minerals. The dissimilatory reduction of these minerals is an import part of global geological cycles and is catalyzed by microorganisms through extended respiratory chains to the cell surface. Shewanella oneidensis is one of the best understood model organisms for this kind of extracellular respiration. Flavins are important for the reduction of extracellular electron acceptors by S. oneidensis. since they have a function as (I) cofactors of the terminal reductases and (II) electron shuttles. In this study we reveal that flavin molecules are further employed as quorum sensing molecules. They are excreted by the organisms in a growth dependent manner and lead to anaerobic biofilm formation as a specific response at a certain threshold concentration. Although we know multiple examples of quorum sensing mechanisms, the use of riboflavin was so far not described and at least in S. oneidensis proceeds via a new regulatory routine that proceeds on the trancriptomic and posttranscriptomic level.

microbiology↗

bioRxiv2021

The Relationship between Birth Timing, Circuit Wiring, and Physiological ResponseProperties of Cerebellar Granule Cells

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular properties that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birthtime and the depth of the molecular layer to which their axons (parallel fibers) project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control, early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multi-depth 2-photon Ca2+ imaging of parallel fibers of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with differences in the proportions of parallel fibers encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber[->]GrC circuitry, but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also suggest that GrCs can represent general aversiveness, in addition to recently described reward representations. Significance StatementCerebellar granule cells (GrCs) comprise the majority of all neurons in the mammalian brain and are usually regarded as a uniform cell type. However, the birth timing of an individual GrC dictates where its axon projects. Using viral-genetic techniques, we find that early- and late-born GrCs receive different proportions of inputs from the same set of input regions. Using in vivo multi-depth 2-photon Ca2+ imaging of axons of early- and late-born GrCs, we found that both populations represent diverse task variables and stimuli, with differences in the proportions of axons in encoding of a subset of movement and reward parameters. These results indicate that birth timing contributes to the input selection and physiological response properties of GrCs.

neuroscience↗

bioRxiv2021

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across interdomain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

microbiology↗

bioRxiv2021

Molecular profiling of retinal pigment epithelial cell differentiation for therapeutic use

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, detailed single cell mapping of the transient cellular and molecular dynamics from the pluripotent state to mature RPE has not been performed. Here we conduct single-cell transcriptomic analyses of 25,718 cells during differentiation as well as in embryonic and adult retina references, revealing differentiation progression through an un-expected initial cell diversification recapitulating early embryonic development before converging towards an RPE lineage. We also identified NCAM1 to track and capture an intermediate retinal progenitor with the potential to give rise to multiple neuroepithelial lineages. Finally, we profiled hESC-RPE cells after subretinal transplantation into the rabbit eye, uncovering robust in vivo maturation towards an adult state. Our detailed evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

developmental biology↗

bioRxiv2020

De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability

High-impact pathogenic variants in more than 1,000 protein-coding genes cause Mendelian forms of neurodevelopmental disorders (NDD), including the newly reported AGO2 gene. This study describes the molecular and clinical characterization of 28 probands with NDD harboring heterozygous AGO1 coding variants. De novo status was always confirmed when parents were available (26/28). A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Some variants were recurrently identified in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linkers domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behavior and additional behavioral manifestations. Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to AGO2 phenotype.

genetics↗

bioRxiv2020

Potassium Carbonate (K2CO3) - A cheap, non-toxic and high-density floating solution for microplastic isolation from beach sediments

Beaches are good indicators for microplastic distribution and local microplastic pollution. Multiple methods have been developed for extracting microplastics from sediment, mainly through density separation. However, the chemicals applied are often expensive and harmful for the user or to the environment. We briefly review the problems associated with the use of these chemicals and present a new floatation solution, potassium carbonate (K2CO3) that has many advantages over available media. It is non-toxic and cheap, and with a density of 1.54 g/cm3 the K2CO3 solution yielded a mean recovery rate of around 92% for PVC, one of the densest polymers, that cannot be easily extracted with alternative floatation agents. We propose that the use of K2CO3 is particularly promising for long term and large-scale monitoring studies, not least because it allows an increasing involvement of citizen scientists, hopefully leading to an increased public awareness of the plastic problem in the seas.

ecology↗

bioRxiv2020

Biomolecular models of EPI-X4 binding to CXCR4 allow the rational optimization of peptides with therapeutic potential

The Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) is a body-own fragment of albumin and specific antagonist of the CXC-motif-chemokine receptor 4 (CXCR4). CXCR4 signaling is induced by its sole chemokine ligand CXCL12 and is involved in a plethora of functions including cell homing, differentiation, survival and angiogenesis. Consequently, dysregulation of CXCR4 is involved in a variety of disorders, such as cancer or inflammatory diseases, making CXCR4 an attractive drug target. EPI-X4 and derivatives with increased CXCR4 binding affinities represent promising leads as CXCR4 antagonists and have shown therapeutic activity in mouse models of inflammatory diseases. However, it is currently unclear how EPI-X4 and its derivatives interact with CXCR4. Here, by combining biomolecular simulations with experimental mutagenesis and activity studies we investigated the binding behavior of EPI-X4 to CXCR4 at the molecular level. Our work allowed us to show that the EPI-X4 peptide interacts primarily in the minor pocket of CXCR4 through its N-terminal residues. The biomolecular interactions highlighted by the computational studies are in good agreement with the experimental mutagenesis data. Moreover, we found that the N-terminal seven amino-acids of EPI-X4 (a 16-mer) and its improved derivatives (12-mers) are sufficient for CXCR4 binding, which led to the development of shorter leads with optimized CXCR4 antagonizing properties. Collectively, we here established how EPI-X4 binds to its receptor and used this knowledge for rational drug design. The new peptide variants developed by us are more potent in terms of inhibiting CXCR4-downstream signaling and cancer cell migration, without toxic effects.

molecular biology↗

bioRxiv2020

Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes

BackgroundThe ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. ObjectiveTo expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles. MethodsExome sequencing was used to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3A Gal4 trap null allele was generated using CRISPR-Cas9 genome editing technology to aid interpretation of variants. ResultsWe report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. Four of the identified missense variants, p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val), were inherited in trans to loss-of-function alleles. A fifth missense variant, p.(Arg327Pro), was homozygous. Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, and Arg327Pro are severe loss-of-function alleles leading to early developmental lethality and neurogenesis defects, whereas Leu77Val and Arg170Trp are partial loss of function alleles that cause progressive locomotion defects. Moreover, Leu77Val and Arg170Trp expression leads to an increase in autophagy and mitophagy in adult muscles. ConclusionOur findings expand the allelic spectrum of ATAD3A variants, and exemplify the use of a functional assay in Drosophila to aid variant interpretation.

genetics↗

bioRxiv2020

Functional dynamics of de-afferented early visual cortex in glaucoma

fMRI studies in macular degeneration (MD) and retinitis pigmentosa (RP) demonstrated that responses in the lesion projection zones (LPZ) of V1 are task related, indicating significant limits of bottom-up visual system plasticity in MD and RP. In advanced glaucoma (GL), a prevalent eye disease and leading cause of blindness, the scope of visual system plasticity is currently unknown. We performed 3T fMRI in patients with extensive visual field defects due to GL (n=5), RP (n=2) and healthy controls (n=7; with simulated defects). Participants viewed contrast patterns drifting in 8 directions alternating with uniform gray and performed 3 tasks: (1) passive viewing (PV), (2) one-back task (OBT) and (3) fixation-dot task (FDT). During PV, they passively viewed the stimulus with central fixation, during OBT they reported the succession of the same two motion directions, and during FDT a change in the fixation color. In GL, LPZ responses of the early visual cortex (V1, V2 and V3) shifted from negative during PV to positive for OBT [p (corrected): V1(0.006); V2(0.04); V3(0.008)], while they were negative in the controls simulated LPZ for all stimulation conditions. For RP a similar pattern as for GL was observed. Consequently, activity in the de-afferented visual cortex in glaucoma is, similar to MD and RP, task-related. In conclusion, the lack of bottom-up plasticity appears to be a general feature of the human visual system. These insights are of importance for the development of treatment and rehabilitation schemes in glaucoma. HighlightsO_LIFunctional dynamics of early visual cortex LPZ depend on task demands in glaucoma C_LIO_LIBrain activity in deprived visual cortex suggests absence of large-scale remapping C_LIO_LILimited scope of bottom-up plasticity is a general feature of human visual system C_LIO_LIVisual system stability and plasticity is of relevance for therapeutic advances C_LI

neuroscience↗

bioRxiv2020

An optimized derivative of an endogenous CXCR4 antagonist prevents atopic dermatitis and airway inflammation

BackgroundAberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes including chronic inflammatory diseases. Thus, the chemokine receptor CXCR4 is a promising target for the therapy of inflammatory disorders, such as atopic dermatitis or allergic asthma. A natural fragment of serum albumin, named EPI-X4, has previous been identified as endogenous peptide antagonist and inverse agonist of CXCR4. The endogenous CXCR4 antagonist provides a promising basis for the development of improved analogues for the therapy of inflammatory diseases. ObjectiveTo increase the anti-CXCR4 activity of EPI-X4 and to evaluate the therapeutic potential of optimized analogs in mouse models of atopic dermatitis and asthma. MethodsMolecular docking analysis of the interaction of EPI-X4 with CXCR4 was performed to define critical interaction motifs and to rationally design analogs with increased activity. EPI-X4 derivatives were synthesized and CXCR4 binding and antagonizing activity determined in assays for antibody competition, inhibition of CXCR4-mediated HIV-1 infection, CXCL12-dependent Ca2+ mobilization, ERK and AKT phosphorylation and cell migration. Toxicity of peptides was evaluated in zebrafish embryos. The therapeutic efficacy of the lead peptide EPI-X4 JM#21 was determined in mouse models of atopic dermatitis and asthma. ResultsDocking analysis identified key interaction motifs of EPI-X4/CXCR4. Rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4 and resulted in the generation of the lead analog JM#21, which bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. JM#21 did not exert toxic effects in zebrafish embryos and efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Moreover, EPI-X4 and its improved derivative suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. ConclusionThe rationally designed EPI-X4 derivative JM#21 is a potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2020

A novel oxidase from Alcaligenes sp. HO-1 oxidizes hydroxylamine to N2

Hydroxylamine is a key intermediate of microbial ammonia oxidation and plays an important role in the biogeochemical cycling of N-compounds. Hydroxylamine is oxidized to NO or N2O by hydroxylamine oxidases or cytochrome P460 from heterotrophic or autotrophic bacteria, but its enzymatic oxidation to N2 has not yet been observed. Here, we report on the discovery of a novel oxidase that converts hydroxylamine to N2 from the newly isolated heterotrophic nitrifier Alcaligenes strain HO-1. Strain HO-1 accumulated hydroxylamine and produced N2 from ammonia oxidation. Using transcriptome analysis and heterologous expression via fosmid library screening, we identified three genes (dnfABC) of strain HO-1 that enabled E. coli cells not only to produce hydroxylamine from 15N-labelled ammonium but also to further convert it to 15N2. The three genes were individually cloned and expressed, and their translational products DnfA, DnfB, and DnfC were purified. In vitro DnfA bound to hydroxylamine and catalyzed the conversion of hydroxylamine to N2 in the presence of FAD, NADH and O2. Thus, DnfA was identified as a novel hydroxylamine oxidase and catalyzed a previously unknown N-N bond forming reaction with a yet-to-be discovered mechanism. DnfA homologs were detected in different bacterial groups, suggesting that hydroxylamine oxidation to nitrogen might occur in additional microbial taxa.

microbiology↗

bioRxiv2020

An endogenous scaling mechanism in zebrafish appendages that controls two-pore potassium-leak channel activity to regulate morphogen and growth factor transcription.

The increase in activity of the two-pore potassium-leak channel Kcnk5b maintains allometric juvenile growth of adult zebrafish appendages. However, it remains unknown how this channel maintains allometric growth and how its bioelectric activity is regulated to scale these anatomical structures. We show the activation of Kcnk5b is sufficient to activate several genes that are part of important development programs. We provide in vivo transplantation evidence that the activation of gene transcription is cell autonomous. We also show that Kcnk5b will induce the expression of different subsets of the tested developmental genes in different cultured mammalian cell lines, which may explain how one electrophysiological stimulus can coordinately regulate the allometric growth of diverse populations of cells in the fin that use different developmental signals. We also provide evidence that the post-translational modification of serine 345 in Kcnk5b by calcineurin regulates channel activity to scale the fin. Thus, we show how an endogenous bioelectric mechanism can be regulated to promote coordinated developmental signaling to generate and scale a vertebrate appendage.

developmental biology↗

bioRxiv2020

GluD2- and Cbln1-mediated Competitive Synaptogenesis Shapes the Dendritic Arbors of Cerebellar Purkinje Cells

The synaptotrophic hypothesis posits that synapse formation stabilizes dendritic branches, yet this hypothesis has not been causally tested in vivo in the mammalian brain. Presynaptic ligand cerebellin-1 (Cbln1) and postsynaptic receptor GluD2 mediate synaptogenesis between granule cells and Purkinje cells in the molecular layer of the cerebellar cortex. Here we show that sparse but not global knockout of GluD2 causes under-elaboration of Purkinje cell dendrites in the deep molecular layer and overelaboration in the superficial molecular layer. Developmental, overexpression, structure-function, and genetic epistasis analyses indicate that dendrite morphogenesis defects result from competitive synaptogenesis in a Cbln1/GluD2-dependent manner. A generative model of dendritic growth based on competitive synaptogenesis largely recapitulates GluD2 sparse and global knockout phenotypes. Our results support the synaptotrophic hypothesis at initial stages of dendrite development, suggest a second mode in which cumulative synapse formation inhibits further dendrite growth, and highlight the importance of competition in dendrite morphogenesis.

neuroscience↗

bioRxiv2020

Genomic and kinetic analysis of novel Nitrospinae enriched by cell sorting

Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) are key players in global nitrogen and carbon cycling. Members of the phylum Nitrospinae are the most abundant, known NOB in the oceans. To date, only two closely affiliated Nitrospinae species have been isolated, which are only distantly related to the environmentally abundant uncultured Nitrospinae clades. Here, we applied live cell sorting, activity screening, and subcultivation on marine nitrite-oxidizing enrichments to obtain novel marine Nitrospinae. Two binary cultures were obtained, each containing one Nitrospinae strain and one alphaproteobacterial heterotroph. The Nitrospinae strains represent two new genera, and one strain is more closely related to environmentally abundant Nitrospinae than previously cultured NOB. With an apparent half-saturation constant of 8.7{+/-}2.5 {micro}M, this strain has the highest affinity for nitrite among characterized marine NOB, while the other strain (16.2{+/-}1.6 {micro}M) and Nitrospina gracilis (20.1{+/-}2.1 {micro}M) displayed slightly lower nitrite affinities. The new strains and N. gracilis share core metabolic pathways for nitrite oxidation and CO2 fixation but differ remarkably in their genomic repertoires of terminal oxidases, use of organic N sources, alternative energy metabolisms, osmotic stress and phage defense. The new strains, tentatively named "Candidatus Nitrohelix vancouverensis" and "Candidatus Nitronauta litoralis", shed light on the niche differentiation and potential ecological roles of Nitrospinae.

microbiology↗