title: NT-CRISPR: Combining natural transformation and CRISPR/Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens

abstract: The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for a wide range of projects. To fully harness the potential of this fascinating bacterium, convenient and highly efficient genome editing methods are indispensable to create novel strains, tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This process, called natural transformation, was tamed for genome editing. It displays a high efficiency and is able to mediate uptake of multiple DNA fragments, thereby allowing multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR/Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR/Cas9, targeting the wild type sequence, leading to death of non-edited cells. Through highly efficient cell killing with efficiencies of up to 99.999 %, integration of antibiotic resistance markers became dispensable and thus enabled scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100 % and further demonstrated its applicability for the simultaneous deletion of multiple chromosomal regions. Lastly, we demonstrated that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby massively broadens the target spectrum.


title: Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity

abstract: The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. Here we systematically investigated the sequence determinants governing CRISPR/Cas9 specificity by measuring the off-on ratios of 1,902 gRNAs on 13,314 target sequences using an improved synthetic system with dual-target design. Our study revealed a comprehensive set of rules including 3 factors in CRISPR/Cas9 off-targeting: 1) the nucleotide context and position of a single mismatch; 2) an "epistasis-like" combinatorial effect of multiple mismatches; and 3) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context. Notably, the combinatorial effect and GMT are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. Based on these rules, we developed a model-based off-target prediction tool (MOFF), which showed superior performance compared to the existing methods.

category: systems biology


title: Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

abstract:
There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3, 5-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required for cholesterol metabolism. Finally, in this work the pharmacokinetic properties of Rv1625c agonists are optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a novel role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

Author SummaryThe recalcitrance of Mycobacterium tuberculosis (Mtb) to conventional antibiotics has created a need to identify novel pharmacological mechanisms to inhibit Mtb pathogenesis. There is a growing understanding of the metabolic adaptations Mtb adopts during infection to support its survival and pathogenesis. This has generated interest in identifying small molecule compounds that effectively inhibit these in vivo metabolic adaptations, while overcoming challenges like poor pharmacokinetic properties or redundancy in target pathways. The Mtb cholesterol utilization pathway has repeatedly been speculated to be a desirable antibiotic target, but compounds that successfully inhibit this complex pathway and are suitable for use in vivo are lacking. Here, we establish that stimulating cAMP synthesis in Mtb is a mechanism that is sufficient to block cholesterol utilization by the bacterium, preventing the release of key metabolic intermediates that are derived from breakdown of the cholesterol molecule. For the first time, we identify small molecule agonists of the Mtb adenylyl cyclase Rv1625c that have promising pharmacological properties and are suitable for use during in vivo studies. These Rv1625c agonists increase cAMP synthesis, inhibit cholesterol utilization by Mtb, and disrupt Mtb pathogenesis in mouse models of chronic infection.

category: microbiology


title: Selection for non-specific adhesion is a driver of FimH evolution increasing Escherichia coli biofilm capacity

abstract: Bacterial interactions with surfaces rely on the coordinated expression and interplay of surface exposed adhesion factors. However, how bacteria dynamically modulate their vast repertoire of adhesins to achieve surface colonization is not yet well-understood. We used experimental evolution and positive selection for improved adhesion to investigate how an initially poorly adherent Escherichia coli strain increased its adhesion capacities to abiotic surfaces. We showed that all identified evolved clones acquired mutations located almost exclusively in the lectin domain of fimH, the gene coding for the -D-mannose-specific tip adhesin of type 1 fimbriae. While most of these fimH mutants showed reduced mannose- binding ability, they all displayed enhanced binding to abiotic surfaces, indicating a trade-off between FimH-mediated specific and non-specific adhesion properties. Several of the identified mutations were already reported in FimH lectin domain of pathogenic and environmental E. coli, suggesting that, beyond patho-adaptation, FimH microevolution favoring non-specific surface adhesion could constitute a selective advantage for natural E. coli isolates. Consistently, although E. coli deleted for the fim operon still evolves an increased adhesion capacity, mutants selected in the {Delta}fim background are outcompeted by fimH mutants revealing clonal interference for adhesion. Our study therefore provides insights into the plasticity of E. coli adhesion potential and shows that evolution of type 1 fimbriae is a major driver of the adaptation of natural E. coli to colonization.


title: Genetic screens identify connections between ribosome recycling and nonsense mediated decay

abstract:
The decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in S. cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well as NMD4 and EBS1, we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations in S. cerevisiae expand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1, 2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3UTR correlate with a loss of NMD.

Author SummaryIn this work, we aim to understand the mechanism of targeting mRNAs for decay via the long-studied nonsense mediated decay (NMD) pathway. We demonstrate that efficient large and small subunit ribosome recycling are necessary components of NMD. We go on to provide evidence that either scanning or actively translating ribosomes in the 3UTR disrupt the decay of NMD targets. Our work highlights the importance of the composition of the 3UTR in NMD signaling and emphasizes the need for this region to indeed be untranslated for NMD to occur. Exon junction complexes (EJCs) in the 3UTR are known to induce NMD, however, in the budding yeast system used here, the NMD targets are EJC-free. Therefore, our data support a model in which factors other than EJCs may accumulate in the 3UTR and provide a signal for NMD.

category: molecular biology


title: Effects of Guangzhou seasonal climate change on the development of Aedes albopictus and its susceptibility to DENV-2

abstract:
The susceptibility of Asian tiger mosquitoes to DENV-2 in different seasons was observed in simulated field environments as a reference to design dengue fever control strategies in Guangzhou. The life table experiments of mosquitoes in four seasons were carried out in the field. The susceptibility of Ae. albopictus to dengue virus was observed in both environments in Guangzhou in summer and winter. Ae. albopictus was infected with dengue virus by oral feeding. On day 7 and 14 after infection, the viral load in the head, ovary, and midgut of the mosquito was detected using real-time fluorescent quantitative PCR. Immune-associated gene expression in infected mosquitoes was performed using quantitative real-time reverse transcriptase PCR. The hatching rate and pupation rate of Ae. albopictus larvae in different seasons differed significantly. The winter hatching rate of larvae was lower than that in summer, and the incubation time was longer than in summer. In the winter field environment, Ae. albopictus still underwent basic growth and development processes. Mosquitoes in the simulated field environment were more susceptible to DENV-2 than those in the simulated laboratory environment. In the midgut, viral RNA levels on day 7 in summer were higher than those on day 7 in winter (F = 14.459, P = 0.01); ovarian viral RNA levels on day 7 in summer were higher than those on day 7 in winter (F = 8.656, P < 0.001), but there was no significant difference in the viral load at other time points (P > 0.05). Dicer-2 mRNA expression on day 7 in winter was 4.071 times than that on day 7 in summer: the viral load and Dicer-2 expression correlated moderately. Ae. albopictus could still develop and transmit dengue virus in winter in Guangzhou. Mosquitoes under simulated field conditions were more susceptible to DENV-2 than those under simulated laboratory conditions.

Author summaryAe. albopictus is the only vector of dengue fever in Guangzhou. Observational data of Ae. albopictus development under natural conditions is important for monitoring vector population expansion, dengue virus transmission, and disease outbreak prevention. We found that Ae. albopictus could still develop in winter in Guangzhou. In addition, the viral load of mosquitoes in the simulated winter environment was lower than that in summer on the 7th day after infection; however, the difference in the viral load of mosquitoes in winter and summer on the 14th day after infection was not statistically significant. Mosquitoes in simulated field conditions were more susceptible to DENV-2 than those in simulated laboratory conditions. The results suggested that Ae. albopictus retains the ability to transmit dengue virus in winter in Guangzhou, and that improved dengue epidemic prevention and control measures are required. The findings of this study represent an important reference for the design of dengue fever control strategies in Guangzhou in response to global climate change.

category: ecology


title: High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

abstract:
Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple GFP insertions, 60 - 100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.

Author SummaryGermlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.

category: genetics


title: Abundances of transfer RNA modifications and transcriptional levels for tRNA-modifying enzymes are sex-specific in mosquitoes

abstract: As carriers of multiple human diseases, understanding the mechanisms behind mosquito reproduction may have implications for remediation strategies. Transfer RNA (tRNA) acts as the adapter molecule of amino acids and are key components in protein synthesis and a critical factor in the function of tRNAs is chemical modifications. Here, we provide an assessment of tRNA modifications between sexes for three mosquito species and examine correlated transcript levels underlying key proteins involved in tRNA modification. Thirty-three tRNA modifications were detected among mosquito species and most of these modifications are higher in females compared to males. Analysis of previous male and female RNAseq datasets indicated a similar increase in tRNA modifying enzymes in females, supporting our observed female enrichment of tRNA modifications. Tissues-specific expressional studies revealed high transcript levels for tRNA modifying enzymes in the ovaries for Aedes aegypti, but not male reproductive tissues. These studies suggest that tRNA modifications may be critical to reproduction in mosquitoes, representing a potential novel target for control.

category: biochemistry


title: A cortical circuit for abrupt visual learning in the primate brain

abstract: Visual plasticity declines sharply after the critical period, yet we easily learn to recognize new faces and places even as adults. Such learning is often characterized by a "moment of insight", an abrupt and dramatic improvement in recognition. The mechanisms that support abrupt learning are unknown, but one hypothesis is that they involve changes in synchronization between brain regions. To test this hypothesis, we used a behavioral task in which non-human primates rapidly learned to recognize novel images and to associate them with specific responses. Simultaneous recordings from inferotemporal and prefrontal cortices revealed a transient synchronization of neural activity between these areas that peaked around the moment of insight. Synchronization was strongest between inferotemporal sites that encoded images and reward-sensitive prefrontal sites. Moreover, its magnitude intensified gradually over image exposures, suggesting that abrupt learning culminates from the search for informative signals within a circuit linking sensory information to task demands.

category: neuroscience


title: Long-range cortical synchronization supports abrupt visual learning


title: Hippocampus and striatum encode distinct task regularities that guide human timing behavior

abstract: The brain encodes the statistical regularities of the environment in a task-specific yet flexible and generalizable format. Here, we seek to understand this process by converging two parallel lines of research, one centered on sensorimotor timing, and the other on cognitive mapping in the hippocampal system. By combining functional magnetic resonance imaging (fMRI) with a fastpaced time-to-contact (TTC) estimation task, we found that the hippocampus signaled behavioral feedback received in each trial as well as performance improvements across trials along with reward-processing regions. Critically, it signaled performance improvements independent from the tested intervals, and its activity accounted for the trial-wise regression-to-the-mean biases in TTC estimation. This suggests that the hippocampus supports the rapid encoding of temporal context even on short time scales in a behavior-dependent manner. Our results emphasize the central role of the hippocampus in statistical learning and position it at the core of a brain-wide network updating sensorimotor representations in real time for flexible behavior.


title: Rapid encoding of task regularities in the human hippocampus guides sensorimotor timing


title: Single Cell Atlas of Human Dura Reveals Cellular Meningeal Landscape and Insights into Meningioma Immune Response

abstract: Recent investigation of the meninges, specifically the dura layer, has highlighted its importance in CNS immune surveillance beyond a purely structural role. However, most of our understanding of the meninges stems from the use of pre-clinical models rather than human samples. In this study, we use single cell RNA-sequencing to perform the first characterization of both non-tumor-associated human dura and meningioma samples. First, we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition, through T cell receptor sequencing, we show significant TCR overlap between matched dura and meningioma samples. We also identify a functionally heterogeneous population of non-immune cell types and report copy-number variant heterogeneity within our meningioma samples. Our comprehensive investigation of both the immune and non-immune cell landscapes of human dura and meningioma at a single cell resolution provide new insight into previously uncharacterized roles of human dura.

category: immunology


title: Functional non-coding SNPs in human endothelial cells fine-map vascular trait associations

abstract: Functional consequences of genetic variation in the non-coding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of functional non-coding SNPs with effects in human endothelial cells (EC), we utilized molecular Quantitative Trait Locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional non-coding SNPs. Together with information from genome-wide association studies for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5,592 variants validated, whose enriched attributes included: 1) mutations to TF binding motifs for ETS or AP1 that are regulators of EC state, 2) location in accessible and H3K27ac-marked EC chromatin, and 3) molQTLs associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of non-coding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlight evidence suggesting mechanisms by which non-coding SNPs at two loci affect risk for Pulse Pressure/Large Artery Stroke, and Abdominal Aortic Aneurysm through respective effects on transcriptional regulation of POU4F1 and LDAH. Together, we highlight the attributes and context dependence of functional non-coding SNPs, and provide new mechanisms underlying vascular disease risk.


title: Modulating D1 rather than D2 receptor-expressing spiny-projection neurons corresponds to optimal antipsychotic effect

abstract: Overactive dopamine transmission in psychosis is predicted to unbalance striatal output via D1- and D2-dopamine receptor-expressing spiny-projection neurons (SPNs). Antipsychotic drugs are thought to re-balance this output by blocking D2-receptor signaling. Here we imaged D1- and D2-SPN Ca2+ dynamics in mice to determine the neural signatures of antipsychotic effect. Initially we compared effective (clozapine and haloperidol) antipsychotics to a candidate drug that failed in clinical trials (MP-10). Clozapine and haloperidol normalized hyperdopaminergic D1-SPN dynamics, while MP-10 only normalized D2-SPN activity. Clozapine, haloperidol or chemogenetic manipulations of D1-SPNs also normalized sensorimotor gating. Given the surprising correlation between clinical efficacy and D1-SPN modulation, we evaluated compounds that selectively target D1-SPNs. D1R partial agonism, antagonism, or positive M4 cholinergic receptor modulation all normalized the levels of D1-SPN activity, locomotion, and sensorimotor gating. Our results suggest that D1-SPN activity is a more relevant therapeutic target than D2-SPN activity for the development of effective antipsychotics.


title: A vibrissa pathway that activates the limbic system

abstract: Vibrissa sensory inputs play a central role in driving rodent behavior. These inputs transit through the sensory trigeminal nuclei, which give rise to the ascending lemniscal and paralemniscal pathways. While lemniscal projections are somatotopically mapped from brain stem to cortex, those of the paralemniscal pathway are more widely distributed. Yet the extent and topography of paralemniscal projections are unknown, along with the potential role of these projections in controlling behavior. Here we used viral tracers to map paralemniscal projections. We find that this pathway broadcasts vibrissa-based sensory signals to brain stem regions that are involved in the regulation of autonomic functions and to forebrain regions that are involved in the expression of emotional reactions. We further provide evidence that GABAergic cells of the Kolliker-Fuse nucleus gate trigeminal sensory input in the paralemniscal pathway via a mechanism of presynaptic or extrasynaptic inhibition.


title: Sound improves neuronal encoding of visual stimuli in mouse primary visual cortex

abstract:
In everyday life, we integrate visual and auditory information in routine tasks such as navigation and communication. While it is known that concurrent sound can improve visual perception, the neuronal correlates of this audiovisual integration are not fully understood. Specifically, it remains unknown whether improvement due to sound of detection and discriminability of visual stimuli is reflected in the neuronal firing patterns in the primary visual cortex (V1). Furthermore, presentation of the sound can induce movement in the subject, but little is understood about whether and how sound-induced movement contributes to V1 neuronal activity. Here, we investigated how sound and movement interact to modulate V1 visual responses in awake, head-fixed mice and whether this interaction improves neuronal encoding of the visual stimulus. We presented visual drifting gratings with and without simultaneous auditory white noise to awake mice while recording mouse movement and V1 neuronal activity. Sound modulated the light-evoked activity of 80% of light-responsive neurons, with 95% of neurons exhibiting increased activity when the auditory stimulus was present. Sound consistently induced movement. However, a generalized linear model revealed that sound and movement had distinct and complementary effects of the neuronal visual responses. Furthermore, decoding of the visual stimulus from the neuronal activity was improved with sound, an effect that persisted even when controlling for movement. These results demonstrate that sound and movement modulate visual responses in complementary ways, resulting in improved neuronal representation of the visual stimulus. This study clarifies the role of movement as a potential confound in neuronal audiovisual responses and expands our knowledge of how multimodal processing is mediated at a neuronal level in the awake brain.

Significance statementSound and movement are both known to modulate visual responses in the primary visual cortex, however sound-induced movement has remained unaccounted for as a potential confound in audiovisual studies in awake animals. Here, authors found that sound and movement both modulate visual responses in an important visual brain area, the primary visual cortex, in distinct, yet complementary ways. Furthermore, sound improved encoding of the visual stimulus even when accounting for movement. This study reconciles contrasting theories on the mechanism underlying audiovisual integration and asserts the primary visual cortex as a key brain region participating in tripartite sensory interactions.


title: Reactivation of transposable elements following hybridization in fission yeast

abstract: Hybridization is thought to reactivate transposable elements (TEs) that were efficiently suppressed in the genomes of the parental hosts. Here, we provide evidence for this  genomic shock hypothesis in the fission yeast Schizosaccharomyces pombe. In this species, two divergent lineages (Sp and Sk) have experienced recent, likely human induced, hybridization. We used long-read sequencing data to assemble genomes of 37 samples derived from 31 S. pombe strains spanning a wide range of ancestral admixture proportions. A comprehensive TE inventory revealed exclusive presence of long terminal repeat (LTR) retrotransposons. Sequence analysis of active full-length elements, as well as solo-LTRs, revealed a complex history of homologous recombination. Population genetic analyses of syntenic sequences placed insertion of many solo-LTRs prior to the split of the Sp and Sk lineages. Most full-length elements were inserted more recently after hybridization. With the exception of a single full-length element with signs of positive selection, both solo-LTRs, and in particular, full-length elements carried signatures of purifying selection indicating effective removal by the host. Consistent with reactivation upon hybridization, the number of full-length LTR retrotransposons, varying extensively from zero to 87 among strains, significantly increased with the degree of genomic admixture. This study gives a detailed account of global TE diversity in S. pombe, documents complex recombination histories within TE elements and provides evidence for the  genomic shock hypothesis.

category: evolutionary biology


title: Cytokinin-CLAVATA crosstalk is an ancient mechanism regulating shoot meristem homeostasis in land plants

abstract: Plant shoots grow from stem cells within Shoot Apical Meristems (SAMs), which produce lateral organs while maintaining the stem cell pool. In the model flowering plant Arabidopsis, the CLAVATA (CLV) pathway functions antagonistically with cytokinin signaling to control the size of the multicellular SAM via negative regulation of the stem cell organizer WUSCHEL (WUS). Although comprising just a single cell, the SAM of the model moss Physcomitrium patens (formerly Physcomitrella) performs equivalent functions during stem cell maintenance and organogenesis, despite the absence of WUS-mediated stem cell organization. Our previous work showed that the stem cell-delimiting function of the CLV pathway receptors CLAVATA1 (CLV1) and RECEPTOR-LIKE PROTEIN KINASE2 (RPK2) is conserved in the moss P. patens. Here, we use P. patens to assess whether CLV-cytokinin crosstalk is also an evolutionarily conserved feature of stem cell regulation. Genetic analyses reveal that CLV1 and RPK2 regulate SAM proliferation via separate pathways in moss. Surprisingly, cytokinin receptor mutants also form ectopic stem cells in the absence of cytokinin signaling. Through modeling, we identified regulatory network archtectures that recapitulated the stem cell phenotypes of clv1 and rpk2 mutants, cytokinin application, cytokinin receptor mutations, and higher-order combinations of these perturbations. These models predict that CLV1 and RPK2 act through separate pathways wherein CLV1 represses cytokinin-mediated stem cell initiation and RPK2 inhibits this process via a separate, cytokinin-independent pathway. Our analysis suggests that crosstalk between CLV1 and cytokinin signaling is an evolutionarily conserved feature of SAM homeostasis that preceded the role of WUS in stem cell organization.

category: plant biology


title: Strigo-D2 - a bio-sensor for monitoring the spatio-temporal pattern of strigolactone signaling in intact plants

abstract: Strigolactones (SLs) are a class of plant hormones modulating developmental programs in response to endogenous and exogenous stimuli and mediating biotic interactions. However, a comprehensive view on the spatio-temporal pattern of SL signaling has not been established and tools for a systematic in planta analysis do not exist. Here, we present Strigo-D2, a genetically encoded ratiometric SL signaling sensor, allowing the examination of SL signaling distribution with cellular resolution and its rapid response to altered SL levels in intact plants. By monitoring the abundance of a truncated and fluorescently labeled SUPPRESSOR OF MAX2 1-LIKE 6 (SMXL6) protein, a proteolytic target of the SL signaling machinery, we show that all cell types investigated have the capacity to respond to changes in SL levels but with very different dynamics. In particular, SL signaling is pronounced in vascular cells but low in guard cells and the meristematic region of the root. We also show that other hormones leave Strigo-D2 activity unchanged indicating that initial SL signaling steps work in isolation from other hormonal signaling pathways. Specificity and spatio-temporal resolution of Strigo-D2 underline the value of the sensor for monitoring SL signaling in a broad range of biological contexts and with highly instructive analytical depth.


title: Uptake of Fe-fraxetin complexes, an IRT1 independent strategy for iron acquisition in Arabidopsis thaliana.

abstract:
O_LIIron (Fe) is a micronutrient essential for plant growth and development. Iron uptake in alkaline soil is a challenge for most plants. In this study, we investigated the role of the catechol coumarins fraxetin and esculetin in plant Fe acquisition and their Fe chelating properties.
C_LIO_LIMass spectrometry and capillary electrophoresis were used to characterize Fe-coumarin complexes. To understand the role of these complexes, genetic, molecular and biochemical approaches were deployed.
C_LIO_LIWe demonstrated that catechol coumarins are taken up by Arabidopsis thaliana root via an ATP dependent mechanism and that plants defective in IRT1 activity (the main high affinity Fe importer) or bHLH121 (a key regulator of Fe deficiency responses) can be complemented by exogenous supply of fraxetin and to a lesser extent of esculetin. We also showed that Fe and fraxetin can form stable complexes at neutral to alkaline pH that can be taken up by the plant.
C_LIO_LIOverall, these results indicate that at high pH, fraxetin can improve Fe nutrition by directly transporting Fe(III) into the root, circumventing the FRO2/IRT1 system, in a similar way as phytosiderophores do in grasses. This strategy may explain how non-grass species can thrive in alkaline soils.
C_LI


title: Kinetic modelling of β-cell metabolism reveals control points in the insulin-regulating pyruvate cycling pathways

abstract: Insulin, a key hormone in the regulation of glucose homeostasis, is secreted by pancreatic {beta}-cells in response to elevated glucose levels. Insulin is released in a biphasic manner in response to glucose metabolism in {beta}-cells. The first phase of insulin secretion is triggered by an increase in the ATP:ADP ratio; the second phase occurs in response to both a rise in ATP:ADP as well as other key metabolic signals, including a rise in the NADPH:NADP+ ratio. Experimental evidence indicates that pyruvate-cycling pathways play an important role in the elevation of the NADPH:NADP+ ratio in response to glucose. In this work we developed a kinetic model for the tricarboxylic acid cycle and pyruvate cycling pathways. We successfully validated our model against recent experimental observations and performed local and global sensitivity analysis to identify key regulatory interactions in the system. The model predicts that the dicarboxylate carrier (DIC) and pyruvate transporter (PYC) are the most important regulators of pyruvate cycling and NADPH production. In contrast, our analysis showed that variation in the pyruvate carboxylase (PC) flux was compensated by a response in the activity of mitochondrial isocitrate dehydrogenase (ICDm) resulting in minimal effect on overall pyruvate cycling flux. The model predictions suggest starting points for further experimental investigation, as well as potential drug targets for treatment of type 2 diabetes.


title: Reduced expression of the psychiatric risk gene DLG2 (PSD93) impairs hippocampal synaptic integration and plasticity

abstract: Background: Genetic variations indicating loss of function in the DLG2 gene have been associated with markedly increased risk for schizophrenia, autism spectrum disorder, and intellectual disability. DLG2 encodes the postsynaptic scaffolding protein DLG2 (PSD93) that interacts with NMDA receptors, potassium channels, and cytoskeletal regulators but the net impact of these interactions on synaptic plasticity, likely underpinning cognitive impairments associated with these conditions, remains unclear. Methods: Hippocampal CA1 neuronal excitability and synaptic function were investigated in a novel clinically relevant heterozygous Dlg2+/- rat model using ex vivo patch-clamp electrophysiology, pharmacology, and computational modelling. Results: Dlg2+/- rats had increased NMDA receptor-mediated synaptic currents and, conversely, impaired associative long-term potentiation. This impairment resulted from an increase in potassium channel function leading to a decrease in input resistance and reduced supra-linear dendritic integration during induction of associative long-term potentiation. Enhancement of dendritic excitability by blockade of potassium channels or activation of muscarinic M1 receptors with selective allosteric agonist 77-LH-28-1 reduced the threshold for dendritic integration and 77-LH-28-1 rescued the associative long-term potentiation impairment in the Dlg2+/- rats. Conclusions: Despite increasing synaptic NMDA receptor currents, the combined impact of reduced DLG2 impairs synaptic integration in dendrites resulting in disrupted associative synaptic plasticity. This biological phenotype can be reversed by compound classes used clinically such as muscarinic M1 receptor agonists and is therefore a potential target for therapeutic intervention.


title: Low amplitude burst detection of catecholamines

abstract: Machine learning advances in electrochemical detection have recently produced sub-second and concurrent detection of dopamine and serotonin during perception and action tasks in conscious humans. Here, we present a new machine learning approach to sub-second, concurrent separation of dopamine, norepinephrine, and serotonin. The method exploits a low amplitude burst protocol for the controlled voltage waveform and we demonstrate its efficacy by showing how it separates dopamine-induced signals from norepinephrine induced signals. Previous efforts to deploy electrochemical detection of dopamine in vivo have not separated the dopamine-dependent signal from a norepinephrine-dependent signal. Consequently, this new method can provide new insights into concurrent signaling by these two important neuromodulators.


title: Neurotensin neurons in the central extended amygdala control energy balance

abstract: Overeating and a sedentary life style are major causes of obesity and related metabolic disorders. Identification of the neurobiological processes that regulate energy balance will facilitate development of interventions for these disorders. Here we show that the Neurotensin-expressing neurons in the mouse IPAC (IPACNts), a nucleus of the central extended amygdala, bidirectionally coordinate hedonic feeding and physical activity, thereby regulating energy balance, metabolic processes and bodyweight. IPACNts are preferentially activated by consumption of highly palatable food or exposure to its taste and smell. Activating IPACNts promotes food intake in a palatability-dependent manner and decreases locomotion. Conversely, inhibiting IPACNts selectively reduces palatable food intake and dramatically enhances physical activity and energy expenditure, and in parallel stimulates physiological responses that oppose diet-induced obesity and metabolic dysfunctions. Thus, a single neuronal population, Neurotensin-expressing neurons in the IPAC, acts to control obesogenic and leptogenic processes by synergistically coordinating energy intake and expenditure with metabolism.


title: Widely naturalized species are not more promiscuous in the use of different nitrogen forms, but benefit more from inorganic nitrogen

abstract:
Nitrogen (N) is an essential mineral nutrient necessary for plant growth and has been considered a crucial factor influencing invasion success. Former studies mainly focused on responses of alien plants to different levels of N availability. However, in natural soils, N comes in different forms. Few experimental studies have explored responses of naturalized alien species to different N forms, and whether such responses are related to their naturalization success globally.

We selected 22 common herbaceous species native to Germany that have all become naturalized and thus been introduced elsewhere in the world. We grew these species under six different N conditions that differed in the amount or form of N, and assessed their growth performance in a greenhouse experiment.

We found that plants produced more biomass when grown under high N levels than under low N levels, and when N was provided in inorganic form than when provided in organic form. Neither biomass production nor promiscuity to different N forms was related to naturalization success of the species. However, the biomass response to inorganic N, relative to organic N, was stronger for the widely naturalized species than for the less widely naturalized ones.

Our study shows that although the widely naturalized alien species were not more promiscuous than the less widely naturalized species, they took more advantage of the inorganic-N forms. This indicates that naturalization success might be partly driven by a species ability to take advantage of increased inorganic N levels.


title: Long-distance dispersal of pigeons and doves generated new ecological opportunities for host-switching and adaptive radiation by their parasites

abstract: Adaptive radiation is an important mechanism of organismal diversification, and can be triggered by new ecological opportunities. Although poorly studied in this regard, parasites present an ideal system to study adaptive radiations because of their close associations with host species. Both experimental and comparative studies suggest that the ectoparasitic wing lice of pigeons and doves have undergone an adaptive radiation, resulting in differences in both body size and overall coloration. Here we show that long-distance dispersal by dove hosts was important for parasite diversification, providing new ecological opportunities for parasites to speciate by host-switching. We further show that these new opportunities for host-switching decreased over time, and cospeciation became the more dominant mode of parasite speciation. Taken together, our results suggest that host dispersal, followed by host-switching, provided novel ecological opportunities that facilitated adaptive radiation by parasites.


title: Non-Viral Engineering of CAR-NK and CAR-T cells using the Tc Buster Transposon System™

abstract:
Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome.

Here, we used the newly developed hyperactive Tc Buster (Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function.

Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

category: bioengineering


title: Rapid Prototyping of Cell Culture Microdevices Using Parylene-Coated 3D Prints

abstract: Fabrication of microfluidic devices by photolithography generally requires specialized training and access to a cleanroom. As an alternative, 3D printing enables cost-effective fabrication of microdevices with complex features that would be suitable for many biomedical applications. However, commonly used resins are cytotoxic and unsuitable for devices involving cells. Furthermore, 3D prints are generally refractory to elastomer polymerization such that they cannot be used as master molds for fabricating devices from polymers (e.g. polydimethylsiloxane, or PDMS). Different post-print treatment strategies, such as heat curing, ultraviolet light exposure, and coating with silanes, have been explored to overcome these obstacles, but none have proven universally effective. Here, we show that deposition of a thin layer of parylene, a polymer commonly used for medical device applications, renders 3D prints biocompatible and allows them to be used as master molds for elastomeric device fabrication. When placed in culture dishes containing human neurons, regardless of resin type, uncoated 3D prints leached toxic material to yield complete cell death within 48 hours, whereas cells exhibited uniform viability and healthy morphology out to 21 days if the prints were coated with parylene. Diverse PDMS devices of different shapes and sizes were easily casted from parylene-coated 3D printed molds without any visible defects. As a proof-of-concept, we rapid prototyped and tested different types of PDMS devices, including triple chamber perfusion chips, droplet generators, and microwells. Overall, we suggest that the simplicity and reproducibility of this technique will make it attractive for fabricating traditional microdevices and rapid prototyping new designs. In particular, by minimizing user intervention on the fabrication and post-print treatment steps, our strategy could help make microfluidics more accessible to the biomedical research community.


title: Reproducibility and reusability limitations in Regulatory Circuits: analysis and solutions

abstract:
The Regulatory Circuits project is among the most recent and the most complete attempts to identify cell-type specific regulatory networks in Human. It is one of the largest efforts of public genomics data integration, based on data from the major consortia FANTOM5, ENCODE and Roadmap Epigenomics. This project is a main provider of biological data, cited more than 224 times (Google Scholar) and its resulting networks were used in at least 42 other articles.

For such a general resource, reproducibility of both the outputs (regulation networks) and methods (data integration pipeline) is a major issue, since biological data are updated regularly. In addition, users may want to introduce new data into the Regulatory Circuits framework to provide networks about previously uncharacterized cell types or to add information about specific regulators, which require to re-execute the whole pipeline on the new data.

In this article, we analyze the various factors limiting reproducibility of the Regulatory Circuits data and methods. Starting from a factual description of our understanding of the methods used in Regulatory Circuits, our contribution is two-fold: we propose (1) a characterization of the different levels of reusability, reproducibility and conceptual issues in the original workflow and (2) a new implementation of the workflow ensuring its consistency with the published description and allowing for an easier reuse and reproduction of the published outputs. Both are applicable beyond the case of Regulatory Circuits.

category: bioinformatics


title: High-resolution reconstruction and deconvolution of array detector images

abstract: Array detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high resolution image by linearly combining sub-detector images. To overcome the limitations of these techniques, we propose a new reconstruction method that takes the full stack of spatially reassigned detector signals as input. We show on both calibration slides and real data that our deconvolution method allows to achieve a better reconstruction performance in terms of resolution, image contrast, and spatial intensity homogeneity. The tested algorithms are available in an open source software.


title: A comparison of high resolution image reconstruction methods from confocal array detector with a new deconvolution algorithm


title: ANDOR and beyond: dynamically switchable logic gates as modules for flexible information processing

abstract: Understanding how complex (bio-)chemical pathways and regulatory networks may be capable of processing information in efficient, flexible, and robust ways is a key question with implications touching fields across biology, systems biology, biochemistry, synthetic biology, dynamical systems theory, and network science. Considerable effort has been focused on the identification and characterization of structural motifs in these signaling networks, and companion efforts have instead sought to cast their operation as controlled by dynamical modules that appear out of dynamical correlations during information processing. While both these approaches have been successful in many examples of biological information processing, cases in which the signaling or regulatory network exhibits multi-functionality or context dependence remain problematic. We here propose a small set of higher-order effective modules that simultaneously incorporate both network structure and the attendant dynamical landscape. In so doing, we render effective computational units that can perform different logical operations based purely on the basin of attraction in which the network dynamics resides or is steered to. These dynamically switchable biochemical logic gates require fewer chemical components or gene products overall than their traditional analogs where static, separate gates are used for each desired function. We demonstrate the applicability and limits of these flexible gates by determining a robust range of parameters over which they correctly operate and further characterize the resilience of their function against intrinsic noise of the constituent reactions using the theory of large deviations. We also show the capability of this framework for general computations by designing a binary adder/subtractor circuit composed of only six components.

category: biophysics


title: Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling

abstract: Microglia are brain-resident immune cells with a repertoire of functions in the developing, mature and pathological brain. Their wide-ranging roles in physiology include the clearance of cellular debris, elimination of excess synapses, regulation of neuronal activity and contributions to blood vessel development. Despite these known roles for microglia, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, using in vivo acute and longitudinal two-photon imaging in transgenic mice combined with electron microscopy, fixed tissue immunohistochemistry, pharmacological treatments and laser speckle imaging, we document the steady-state interactions between ramified CX3CR1+ myeloid cell somata and capillaries in the brain. We first confirm that these myeloid cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing and contrasting them with parenchymal microglia (PCMs) in their static, dynamic and chronic morphological activities including during microglial depletion and repopulation. Molecularly, we identify microglial-specific purinergic P2RY12 receptors as a receptor regulating CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, to elucidate roles for microglia in vascular structure and function, we eliminated microglia and showed that this triggered capillary dilation, blood flow increase, and impaired vasodilative responses. We find that P2RY12-/- and PANX1-/- mice recapitulate these vascular impairments suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function.


title: Linking Individual Differences in Personalized Functional Network Topography to Psychopathology in Youth

abstract: The spatial layout of large-scale functional brain networks differs between individuals and is particularly variable in association cortex that has been implicated in a broad range of psychiatric disorders. However, it remains unknown whether this variation in functional topography is related to major dimensions of psychopathology in youth. Capitalizing on a large sample with 27-minutes of high-quality functional MRI data (n=790, ages 8-23 years) and advances in machine learning, we examined associations between functional topography and four correlated dimensions of psychopathology (fear, psychosis, externalizing, anxious-misery) as well as an overall psychopathology factor. We found that functional topography significantly predicted individual differences in dimensions of psychopathology, driven mainly by robust associations between topography and overall psychopathology. Reduced cortical representations of association networks were among the most important features of the model. Our results emphasize the value of considering systematic differences in functional neuroanatomy for personalized diagnostics and therapeutics in psychiatry.


title: Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

abstract: Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.


title: Auditory cortex activation is modulated nonlinearly by stimulation duration: A functional near-infrared spectroscopy (fNIRS) study

abstract: Functional near-infrared spectroscopy (NIRS) is an increasingly popular method in hearing research. However, few studies have considered efficient stimulation parameters for fNIRS auditory experimental design. The objectives of our study are (1) to characterize the auditory hemodynamic responses to trains of white noise with increasing stimulation durations (8s, 10s, 15s, 20s) in terms of amplitude and response linearity; (2) to identify the most-efficient stimulation duration using fNIRS; and (3) to generalize results to more ecological environmental stimuli. We found that cortical activity is augmented following the increments in stimulation durations and reaches a plateau after about 15s of stimulation. The linearity analysis showed that this augmentation due to stimulation duration is not linear in the auditory cortex, the non-linearity being more pronounced for longer durations (15s and 20s). The 15s block duration that we propose as optimal precludes signal saturation, is associated with a high response amplitude and a relatively short total experimental duration. Moreover, the 15s duration remains optimal independently of the nature of presented sounds. The sum of these findings suggests that 15s stimulation duration used in the appropriate experimental setup allows researchers to acquire optimal fNIRS signal quality.


title: Neural representations of stereotype content predict social decisions

abstract: To guide social interaction, people often rely on expectations about the traits of other people based on markers of social group membership, i.e., stereotypes. Although the influence of stereotypes on social behavior is widespread, key questions remain about how traits inferred from social group membership are instantiated in the brain and incorporated into neural computations that guide social behavior. Here, we show that the human lateral orbitofrontal cortex (OFC) represents the content of stereotypes about members of different social groups in the service of social decision-making. During fMRI scanning, participants decided how to distribute resources across themselves and members of a variety of social groups in a modified Dictator Game. Behaviorally, we replicated our recent finding that perceptions of others traits, captured by a two-dimensional framework of stereotype content (warmth and competence), biased participants monetary allocation choices in a context-dependent manner: recipients warmth increased advantageous inequity aversion and their competence increased disadvantageous inequity aversion. Neurally, representational similarity analysis (RSA) revealed that perceptions of others traits in the two-dimensional space were represented in the temporoparietal junction and superior temporal sulcus, two regions associated with mentalizing, and in the lateral OFC, known to represent latent environmental features during goal-directed outcome inference outside the social domain. Critically, only the latter predicted individual choices, suggesting that the effect of stereotypes on behavior is mediated by inference-based, domain-general decision-making processes in the OFC.


title: Neural representations of others' traits predict social decisions


title: Dynamic Cognitive States Predict Individual Variability in Behavior and Modulate with EEG Functional Connectivity during Working Memory

abstract:
Fluctuations in strategy, attention, or motivation can cause large variability in performance across task trials. Typically, this variability is treated as noise, and assumed to cancel out, leaving supposedly stable relationships among behavior, neural activity, and experimental task conditions. Those relationships, however, could change with a participants internal cognitive states, and variability in performance may carry important information regarding those states, which cannot be directly measured. Therefore, we used a mathematical, state-space modeling framework to estimate internal states from measured behavioral data, quantifying each participants sensitivity to factors such as past errors or distractions, to predict their reaction time fluctuations. We show how modeling these states greatly improves trial-by-trial prediction of behavior. Further, we identify EEG functional connectivity features that modulate with each state. These results illustrate the potential of this approach and how it could enable quantification of intra- and inter-individual differences and provide insight into their neural bases.

Statement of RelevanceCognitive behavioral performance and its neural bases vary both across individuals and within individuals over time. Understanding this variability may be key to the success of clinical or educational interventions. Internal cognitive states reflecting differences in strategy, attention, and motivation may drive much of these inter- and intra-individual differences, but often cannot be reliably controlled or measured in cognitive neuroscience research. The mathematical modeling framework developed here uses measured data to estimate a participants dynamic, internal cognitive states, with each state derived from specific factors hypothesized to affect attention, motivation or strategy. The results highlight potential sources of behavioral variability and reveal EEG features that modulate with each state. Our method quantifies and characterizes individual behavioral differences and highlights their underlying neural mechanisms, which could be used for future targeted training or neuromodulation therapies to improve cognitive performance.


title: Influence of anatomical features of different brain regions on the spatial localization of fiber photometry signals

abstract: Fiber photometry is widely used in neuroscience labs for in vivo detection of functional fluorescence from optical indicators of neuronal activity with a simple optical fiber. The fiber is commonly placed next to the region of interest to both excite and collect the fluorescence signal. However, the path of both excitation and fluorescence photons is altered by the uneven optical properties of the brain, due to local variation of the refractive index, different cellular types, densities and shapes. Nonetheless, the effect of the local anatomy on the actual shape and extent of the volume of tissue that interfaces with the fiber has received little attention so far. To fill this gap, we measured the size and shape of fiber photometry efficiency field in the primary motor and somatosensory cortex, in the hippocampus and in the striatum of the mouse brain, highlighting how their substructures determine the detected signal and the depth at which photons can be mined. Importantly, we show that the information on the spatial expression of the fluorescent probes alone is not sufficient to account for the contribution of local subregions to the overall collected signal, and it must be combined with the optical properties of the tissue adjacent to the fiber tip.


title: Involvement of ASIC1a channels in the spinal processing of pain information by deep projection neurons

abstract: Dorsal horn of the spinal cord is an important crossroad of pain neuraxis, especially for the neuronal plasticity mechanisms that can lead to chronic pain states. Windup is a well-known spinal pain facilitation process initially described several decades ago, but which exact mechanism is still not fully understood. Here, we combine both ex vivo and in vivo electrophysiological recordings of spinal neurons with computational modelling to demonstrate a role for ASIC1a-containing channels in the windup process. Spinal application of the ASIC1a inhibitory venom peptides mambalgin-1 and psalmotoxin-1 (PcTx1) significantly reduces the ability of deep wide dynamic range (WDR) neurons to develop windup in vivo. All deep WDR-like neurons recorded from spinal slices exhibit an ASIC current with biophysical and pharmacological characteristics consistent with functional expression of ASIC1a/ASIC2 heteromeric channels. A computational model of WDR neuron supplemented with heteromeric ASIC1a/ASIC2 channel parameters accurately reproduces the experimental data, further supporting a positive contribution of these channels to windup. It also predicts a calcium-dependent windup decrease for elevated ASIC conductances, a phenomenon that was experimentally validated using either a combination of calcium-activated potassium channel inhibitory peptides (apamin and iberiotoxin), or the Texas coral snake ASIC-activating toxin (MitTx). This study demonstrates a possible dual contribution to windup of calcium permeable ASIC1a/ASIC2 channels in deep laminae projecting neurons, promoting it upon moderate channel activity, but ultimately leading to calcium-dependent windup inhibition associated to potassium channels when activity increases.


title: Learning shapes cortical dynamics to enhance integration of relevant sensory input

abstract:
Adaptive sensory behavior is thought to depend on processing in recurrent cortical circuits, but how dynamics in these circuits shapes the integration and transmission of sensory information is not well understood. Here, we study neural coding in recurrently connected networks of neurons driven by sensory input. We show analytically how information available in the network output varies with the alignment between feedforward input and the integrating modes of the circuit dynamics. In light of this theory, we analyzed neural population activity in the visual cortex of mice that learned to discriminate visual features. We found that over learning, slow patterns of network dynamics realigned to better integrate input relevant to the discrimination task. This realignment of network dynamics could be explained by changes in excitatory-inhibitory connectivity amongst neurons tuned to relevant features. These results suggest that learning tunes the temporal dynamics of cortical circuits to optimally integrate relevant sensory input.

HighlightsO_LIA new theoretical principle links recurrent circuit dynamics to optimal sensory coding
C_LIO_LIPredicts that high-SNR input dimensions activate slowly decaying modes of dynamics
C_LIO_LIPopulation dynamics in primary visual cortex realign during learning as predicted
C_LIO_LIStimulus-specific changes in E-I connectivity in recurrent circuits explain realignment
C_LI


title: Structural (dys)connectivity associates with cholinergic cell density of the nucleus basalis of Meynert in Alzheimer's disease

abstract: Cognitive deficits in Alzheimers disease, specifically amnestic (memory dominant) deficits, are associated with cholinergic degeneration in the basal forebrain. The cholinergic nucleus within the basal forebrain, the nucleus basalis of Meynert, exhibits local atrophy and reduced cortical tract integrity on MRI, and reveals amyloid-{beta} and phosphorylated-tau pathology at autopsy. To better understand the pathophysiology of nucleus basalis of Meynert atrophy and its neocortical projections in Alzheimers disease, we utilized a combined post-mortem in-situ MRI and histopathology approach. A total of 19 Alzheimers disease (10 amnestic and 9 non-amnestic) and 9 non-neurological control donors underwent 3T T1-weighted MRI for anatomical delineation and volume assessment of the nucleus basalis of Meynert, and diffusion-weighted imaging for microstructural assessment of the nucleus and its projections. At subsequent brain autopsy, tissue dissection and immunohistochemistry were performed for amyloid-{beta}, phosphorylated-tau and choline acetyltransferase. Compared to controls, we observed an MRI-derived volume reduction and altered microstructural integrity of the nucleus basalis of Meynert in Alzheimers disease donors. Furthermore, decreased cholinergic cell density was associated with reduced integrity of the nucleus and its tracts to the temporal lobe, specifically to the temporal pole of the superior temporal gyrus, and the parahippocampal gyrus. The association between cholinergic cell density and alteration to cortical tracts was specific for amnestic, compared to non-amnestic Alzheimers disease donors. Our study illustrates that the nucleus basalis of Meynert is severely affected in amnestic Alzheimers disease, both in terms of pathology within the nucleus, but also in terms of damage to its cortical projections, specifically to the temporal lobe, which may contribute to the observed cognitive deterioration.


title: Modeling minimum viable population size with multiple genetic problems of small populations

abstract: An important goal for conservation is to define minimum viable population (MVP) sizes for long-term persistence. Although many MVP size estimates focus on ecological processes, with increasing evidence for the role of genetic problems in population extinction, conservation practitioners have also increasingly started to incorporate inbreeding depression (ID). However, small populations also face other genetic problems such as mutation accumulation (MA) and loss of genetic diversity through genetic drift that are usually factored into population viability assessments only via verbal arguments. Comprehensive quantitative theory on interacting genetic problems is missing. Here we develop eco-evolutionary quantitative models that track both population size and levels of genetic diversity. Our models assume a biallelic multilocus genome whose loci can be under either a single or interacting genetic forces. In addition to mutation-selection-drift balance (for loci facing ID and MA), we include three forms of balancing selection (for loci where variation is lost through genetic drift). We define MVP size as the lowest population size that avoids an eco-evolutionary extinction vortex after a time sufficient for an equilibrium allele frequency distribution to establish. Our results show that MVP size decreases rapidly with increasing mutation rates for populations whose genomes are only under balancing selection, while for genomes under mutation-selection-drift balance, the MVP size increases rapidly. MVP sizes also increase rapidly with increasing number of loci under the same or different selection mechanisms until a point is reached at which even arbitrarily large populations cannot survive anymore. In the case of fixed number of loci under selection, interaction of genetic problems did not necessarily increase MVP sizes. To further enhance our understanding about interaction of genetic problems, there is need for more empirical studies to reveal how different genetic processes interact in the genome.


title: Genome sequencing and assembly of Tinospora cordifolia (Giloy) plant

abstract: During the ongoing COVID-19 pandemic Tinospora cordifolia also known as Giloy gained immense popularity and use due to its immunity-boosting function and anti-viral properties. T. cordifolia is among the most important medicinal plants that has numerous therapeutic applications in health due to the production of a diverse array of secondary metabolites. Therefore, to gain genomic insights into the medicinal properties of T. cordifolia, the first genome sequencing was carried out using 10x Genomics linked read technology and the draft genome assembly comprised of 1.01 Gbp. This is also the first genome sequenced from the plant family Menispermaceae. We also performed the first genome size estimation for T. cordifolia and was found to be 1.13 Gbp. The deep sequencing of transcriptome from the leaf tissue was also performed followed by transcriptomic analysis to gain insights into the gene expression and functions. The genome and transcriptome assemblies were used to construct the gene set in T. cordifolia that resulted in 19,474 coding gene sequences. Further, the phylogenetic position of T. cordifolia was also determined through the construction of a genome-wide phylogenetic tree using 35 other dicot species and one monocot species as an outgroup species.

category: genomics


title: Uncertainty quantification in subject-specific estimation of local vessel mechanical properties

abstract: Quantitative estimation of local mechanical properties remains critically important in the ongoing effort to elucidate how blood vessels establish, maintain, or lose mechanical homeostasis. Recent advances based on panoramic digital image correlation (pDIC) have made high-fidelity 3D reconstructions of small-animal (e.g., murine) vessels possible when imaged in a variety of quasi-statically loaded configurations. While we have previously developed and validated inverse modeling approaches to translate pDIC-measured surface deformations into biomechanical metrics of interest, our workflow did not heretofore include a methodology to quantify uncertainties associated with local point estimates of mechanical properties. This limitation has compromised our ability to infer biomechanical properties on a subject-specific basis, such as whether stiffness differs significantly between multiple material locations on the same vessel or whether stiffness differs significantly between multiple vessels at a corresponding material location. In the present study, we have integrated a novel uncertainty quantification and propagation pipeline within our inverse modeling approach, relying on empirical and analytic Bayesian techniques. To demonstrate the approach, we present illustrative results for the ascending thoracic aorta from three mouse models, quantifying uncertainties in constitutive model parameters as well as circumferential and axial tangent stiffness. Our extended workflow not only allows parameter uncertainties to be systematically reported, but also facilitates both subject-specific and group-level statistical analyses of the mechanics of the vessel wall.


title: IntroSpect: motif-guided immunopeptidome database building tool to improve the sensitivity of HLA binding peptide identification

abstract: Although database search tools originally developed for shotgun proteome have been widely used in immunopeptidomic mass spectrometry identifications, they have been reported to achieve undesirably low sensitivities and/or high false positive rates as a result of the hugely inflated search space caused by the lack of specific enzymic digestions in immunopeptidome. To overcome such a problem, we have developed a motif-guided immunopeptidome database building tool named IntroSpect, which is designed to first learn the peptide motifs from high confidence hits in the initial search and then build a targeted database for refined search. Evaluated on three representative HLA class I datasets, IntroSpect can improve the sensitivity by an average of 80% comparing to conventional searches with unspecific digestions while maintaining a very high accuracy ([~]96%) as confirmed by synthetic validation experiments. A distinct advantage of IntroSpect is that it does not depend on any external HLA data so that it performs equally well on both well-studied and poorly-studied HLA types, unlike a previously developed method SpectMHC. We have also designed IntroSpect to keep a global FDR that can be conveniently controlled, similar to conventional database search engines. Finally, we demonstrate the practical value of IntroSpect by discovering neoantigens from MS data directly. IntroSpect is freely available at https://github.com/BGI2016/IntroSpect.


title: Targeted in silico characterization of fusion transcripts in tumor and normal tissues via FusionInspector

abstract:
BackgroundGene fusions play a key role as driving oncogenes in tumors, and their reliable discovery and detection is important for cancer research, diagnostics, prognostics and guiding personalized therapy. While discovering gene fusions from genome sequencing can be laborious and costly, the resulting "fusion transcripts" can be recovered from RNA-seq data of tumor and normal samples. However, alleged and putative fusion transcript can arise from multiple sources in addition to the chromosomal rearrangements yielding fusion genes, including cis- or trans-splicing events, experimental artifacts during RNA-seq or computational errors of transcriptome reconstruction methods. Understanding how to discern, interpret, categorize, and verify predicted fusion transcripts is essential for consideration in clinical settings and prioritization for further research. Here, we present FusionInspector for in silico characterization and interpretation of candidate fusion transcripts from RNA-seq, enabling exploration of sequence and expression characteristics of fusions and their partner genes.

ResultsWe applied FusionInspector to thousands of tumor and normal transcriptomes, and identified statistical and experimental features enriched among biologically impactful fusions. Through clustering and machine learning, we identified large collections of fusions potentially relevant to tumor and normal biological processes. We show that biologically relevant fusions are enriched for relatively high expression of the fusion transcript, imbalanced fusion allelic ratios, and canonical splicing patterns, and are deficient in sequence microhomologies detected between partner genes.

ConclusionWe demonstrate FusionInspector to accurately in silico validate fusion transcripts, and to help identify numerous understudied fusions in tumor and normal tissues samples. FusionInspector is freely available as open source for screening, characterization, and visualization of candidate fusions via RNA-seq. We believe that this work will continue driving the discipline of transparent explanation and interpretation of machine learning predictions and tracing the predictions to their experimental sources.


title: Effects of Cholesterol on the mechanism of fengycin, a biofungicide

abstract: Fengycins are a class of antifungal lipopeptides synthesized by the bacteria Bacillus subtilis, commercially available as the primary component of the agricultural fungicide Serenade(R). They are toxic to fungi, but far less to mammalian cells. One key difference between mammalian and fungal cell membranes is the presence of cholesterol only in the former; recent experimental work showed that the presence of cholesterol reduces fengycin-induced membrane leakage. (1) Since our previous all-atom and coarse-grained simulations (2, 3) suggested that aggregation of membrane-bound fengycin is central to its ability to disrupt membranes, we hypothesized that cholesterol might reduce fengycin aggregation. Here, we test this hypothesis using coarse-grained molecular dynamics simulations, with sampling enhanced via the weighted ensemble method. The results indicate that cholesterol subtly alters the size distribution for fengycin aggregates, limits the lateral range of their membrane disordering, and reduces the ability of aggregates to bend the membrane. Taken together, these phenomena may account for cholesterols affects on fengycin activity.


title: The sodium leak channel NALCN encodes the major background sodium ion conductance in murine anterior pituitary cells

abstract: The pituitary gland, the so-called "master gland" produces and secretes a variety of hormones essential for regulating growth and development, metabolic homeostasis, reproduction and the stress response. The interplay between the brain and peripheral feedback signals controls hormone secretion from pituitary cells by regulating the properties of ion channels, and in turn, cell excitability. Endocrine anterior pituitary cells fire spontaneous action potentials to regulate their intracellular calcium level and eventually hormone secretion. However, the molecular identity of the non-selective cationic leak channel involved in maintaining the resting membrane potential at the firing threshold remained unknown. Here, we show that the sodium leak channel NALCN, known to modulate neuronal excitability, also regulates excitability in murine anterior pituitary cells. Using viral transduction combined with electrophysiology and calcium imaging we show that NALCN encodes the major Na+ leak conductance which tunes the resting membrane potential close to firing threshold to sustain the intrinsically-regulated firing in endocrine pituitary cells. Genetic interruption of NALCN channel activity, hyperpolarised the membrane potential drastically and stopped the firing activity and consequently abolished the cytosolic calcium oscillations. Moreover, we found that NALCN conductance forms a very small fraction of the total cell conductance yet has a profound impact on modulating pituitary cell excitability. Taken together, our results demonstrate that, NALCN is a crucial regulator of pituitary cell excitability and supports spontaneous firing activity to consequently regulate hormonal secretion. Our results suggest that receptor-mediated and potentially circadian changes in NALCN conductance can powerfully affect pituitary activity and hormone secretion.

category: physiology


title: Myofiber injury induces capillary disruption and regeneration of disorganized microvascular networks

abstract: Myofibers regenerate following injury, however the microvasculature must also recover to restore skeletal muscle function. We aimed to define the nature of microvascular damage and repair during skeletal muscle injury and regeneration induced by BaCl2. To test the hypothesis that microvascular disruption occurred secondary to myofiber injury in mice, isolated microvessels were exposed to BaCl2 or the myotoxin was injected into the gluteus maximus (GM) muscle. In isolated microvessels, BaCl2 depolarized smooth muscle cells and endothelial cells while increasing [Ca2+]i, but did not elicit cell death. At 1 day post injury (dpi) of the GM, capillary fragmentation coincided with myofiber degeneration while arteriolar and venular networks remained intact; neutrophil depletion before injury did not prevent capillary damage. Perfused capillary networks reformed by 5 dpi in association with more terminal arterioles and were dilated through 10 dpi; with no change in microvascular area or branch point number in regenerating networks, fewer capillaries aligned with myofibers and capillary networks were no longer organized into microvascular units. By 21 dpi, capillary orientation and organization had nearly recovered to that in uninjured GM. We conclude that following their disruption secondary to myofiber damage, capillaries regenerate as disorganized networks that remodel while regenerated myofibers mature.


title: Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

abstract: Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. The mechanism of secretion remains largely unknown, because hydrophobic metabolites, which may form oil droplets or crystals in cytosol, inducing cell death, cannot be directly secreted by transporters. Moreover, some secondary metabolic lipids react with cytosolic components leading to their decomposition. Lipophilic metabolites should thus be solubilized by matrix lipids and compartmentalized by membrane lipids. The mechanism of lipophilic metabolite secretion was assessed using shikonin, a red naphthoquinone lipid, in Lithospermum erythrorhizon. Cell secretion of shikonin also involved the secretion of about 30% of triacylglycerol (TAG), composed predominantly of saturated fatty acids. Shikonin production was associated with the induction of large amounts of the membrane lipid phosphatidylcholine. Together with in vitro reconstitution, these findings suggest a novel role for TAG as a matrix lipid for the secretion of lipophilic metabolites.


title: Integrative Gene Expression and Metabolic Analysis tool IgemRNA

abstract: Genome scale metabolic modelling is widely used technique to research metabolism impacts on organisms properties. Additional omics data integration enables a more precise genotype-phenotype analysis for biotechnology, medicine and life sciences. Transcriptome data amounts rapidly increase each year. Many transcriptome analysis tools with integrated genome scale metabolic modelling are proposed. But these tools have own restrictions, compatibility issues and the necessity of previous experience and advanced user skills. We have analysed and classified published tools, summarized possible transcriptome pre-processing, and analysis methods and implemented them in the new transcriptome analysis tool IgemRNA. Tool novelty is the possibility of transcriptomics data pre-processing approach, analysis of transcriptome with or without genome scale metabolic models and different thresholding and gene mapping approach availability. In comparison with usual Gene set enrichment analysis methods, IgemRNA options provide additional transcriptome data validation, where minimal metabolic network connectivity and flux requirements are met. IgemRNA allows to process transcriptome datasets, compare data between different phenotypes, execute multiple analysis and data filtering functions. All this is done via graphical user interface. IgemRNA is compatible with Cobra Toolbox 3.0 and uses some of its functions for genome scale metabolic model optimization tasks. IgemRNA is open access software available at https://github.com/BigDataInSilicoBiologyGroup/IgemRNA.


title: Event-Related Phase Synchronization Propagates Rapidly across Human VentralVisual Cortex

abstract: Visual inputs to early visual cortex integrate with semantic, linguistic and memory inputs in higher visual cortex, in a manner that is rapid and accurate, and enables complex computations such as face recognition and word reading. This implies the existence of fundamental organizational principles that enable such efficiency. To elaborate on this, we performed intracranial recordings in 82 individuals while they performed tasks of varying visual and cognitive complexity. We discovered that visual inputs induce highly organized posterior-to-anterior propagating patterns of phase modulation across the ventral occipitotemporal cortex. At individual electrodes there was a stereotyped temporal pattern of phase progression following both stimulus onset and offset, consistent across trials and tasks. The phase of low frequency activity in anterior regions was predicted by the prior phase in posterior cortical regions. This spatiotemporal propagation of phase likely serves as a feed-forward organizational influence enabling the integration of information across the ventral visual stream. This phase modulation manifests as the early components of the event related potential; one of the most commonly used measures in human electrophysiology. These findings illuminate fundamental organizational principles of the higher order visual system that enable the rapid recognition and characterization of a variety of inputs.


title: Rescue of behavioral and electrophysiological phenotypes in a Pitt-Hopkins syndrome mouse model by genetic restoration of Tcf4 expression

abstract: Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by monoallelic mutation or deletion in the transcription factor 4 (TCF4) gene. Individuals with PTHS typically present in the first year of life with developmental delay and exhibit intellectual disability, lack of speech, and motor incoordination. There are no effective treatments available for PTHS, but the root cause of the disorder, TCF4 haploinsufficiency, suggests that it could be treated by normalizing TCF4 gene expression. Here we performed proof-of-concept viral gene therapy experiments using a conditional Tcf4 mouse model of PTHS and found that postnatally reinstating Tcf4 expression in neurons improved anxiety-like behavior, activity levels, innate behaviors, and memory. Postnatal reinstatement also partially corrected EEG abnormalities, which we characterized here for the first time, and the expression of key TCF4-regulated genes. Our results support a genetic normalization approach as a treatment strategy for PTHS, and possibly other TCF4-linked disorders.


title: Shisa7 phosphorylation regulates GABAergic transmission and neurodevelopmental behaviors

abstract: GABA-A receptors (GABAARs) are crucial for development and regulation of the central nervous system. Altered GABAergic signaling is hypothesized to be involved in the pathophysiology of neurodevelopmental disorders. Nevertheless, how aberrant cellular and molecular mechanisms affect GABAARs in these diseases remain elusive. Recently, we identified Shisa7 as a GABAAR auxiliary subunit that modulates GABAAR trafficking, kinetics, and pharmacology, and discovered a phosphorylation site in Shisa7 (S405) critical for extrasynaptic 5-GABAAR trafficking and tonic inhibition. However, the role of S405 phosphorylation in the regulation of synaptic inhibition, plasticity, and behavior remains unknown. Here, we found that expression of a phospho-null mutant (Shisa7 S405A) in heterologous cells and neurons diminishes 2-GABAAR trafficking. Subsequently, we generate a Shisa7 S405A knock-in (KI) mouse line that displays reduced surface expression of GABAARs in hippocampal neurons. Importantly, both synaptic and tonic inhibition are decreased in KI mice. Moreover, chemically induced inhibitory long-term potentiation is impaired, highlighting a critical role of Shisa7 S405 in GABAergic plasticity. Lastly, KI mice exhibit enhanced locomotor activity and grooming associated with neurodevelopmental disorders. Collectively, our study reveals a phosphorylation site critical for Shisa7-dependent trafficking of synaptic and extrasynaptic GABAARs which contributes to behavioral endophenotypes displayed in neurodevelopmental disorders.


title: Burning for grassland pollination: recently burned patches promote plant flowering and insect pollinators

abstract: Grasslands are historically and evolutionarily associated with disturbances, such as fire, that drive biodiversity assembly patterns and biotic interactions. Disturbance suppression in fire- prone ecosystems usually leads to a decline in forb diversity and flowering due to biomass accumulation, which could jeopardize pollinator diversity. In this study, we investigated patterns and drivers of plant flowering and flower insect visitor communities in a chronosequence of patches from different time-since-fire categories in Southern Brazilian grasslands. Old-burnt patches (more than 2 years since fire) had taller vegetation, more biomass and grass cover than intermediate (about 1 year after fire) and freshly-burnt patches (less than 6 months since fire), which had increased bare soil cover. Forb flower abundance was much higher in freshly-burnt patches, directly predicted by the degree of habitat openness. Pollinator insects were then benefited by floral resource aggregation in freshly-burnt patches, increasing in abundance (bees and butterflies) and species richness (bees). Beetle communities were positively influenced by vegetation height. Furthermore, plant species flowering and bee species composition varied between freshly and old-burnt grasslands, with indicator species found for all recovery stages but mainly freshly-burnt patches. Altogether, these results indicate the importance of maintaining freshly-burnt patches in the grassland landscape: it helps to sustain flower diversity, pollination services, and flowering plant reproduction. Our findings support the idea that a mosaic of grasslands from different times-since- fire should be considered for grassland conservation.


title: Prediction performance of linear models and gradient boosting machine on complex phenotypes in outbred mice

abstract: Recent literature suggests machine learning methods can capture interactions between loci and therefore could outperform linear models when predicting traits with relevant epistatic effects. However, investigating this empirically requires data with high mapping resolution and phenotypes for traits with known non-additive gene action. The objective of the present study was to compare the performance of linear (GBLUP, BayesB and elastic net [ENET]) methods to a non-parametric tree-based ensemble (gradient boosting machine - GBM) method for genomic prediction of complex traits in mice. The dataset used contained phenotypic and genotypic information for 835 animals from 6 non-overlapping generations. Traits analyzed were bone mineral density (BMD), body weight at 10, 15 and 20 weeks (BW10, BW15 and BW20), fat percentage (FAT%), circulating cholesterol (CHOL), glucose (GLUC), insulin (INS) and triglycerides (TGL), and urine creatinine (UCRT). After quality control, the genotype dataset contained 50,112 SNP markers. Animals from older generations were considered as a reference subset, while animals in the latest generation as candidates for the validation subset. We also evaluated the impact of different levels of connectedness between reference and validation sets. Model performance was measured as the Pearsons correlation coefficient and mean squared error (MSE) between adjusted phenotypes and the models prediction for animals in the validation subset. Outcomes were also compared across models by checking the overlapping top markers and animals. Linear models outperformed GBM for seven out of ten traits. For these models, accuracy was proportional to the traits heritability. For traits BMD, CHOL and GLU, the GBM model showed better prediction accuracy and lower MSE. Interestingly, for these three traits there is evidence in literature of a relevant portion of phenotypic variance being explained by epistatic effects. We noticed that for lower connectedness, i.e., imposing a gap of one to two generations between reference and validation populations, the superior performance of GBM was only maintained for GLU. Using a subset of top markers selected from a GBM model helped for some of the traits to improve accuracy of prediction when these were fitted into linear and GBM models. The GBM model showed consistently fewer markers and animals in common among the top ranked than linear models. Our results indicate that GBM is more strongly affected by data size and decreased connectedness between reference and validation sets than the linear models. Nevertheless, our results indicate that GBM is a competitive method to predict complex traits in an outbred mice population, especially for traits with assumed epistatic effects.


title: SNP and Haplotype Regional Heritability Mapping (SNHap-RHM): joint mapping of common and rare variation affecting complex traits

abstract:
We describe a genome-wide analytical approach, SNP and Haplotype Regional Heritability Mapping (SNHap-RHM), that provides regional estimates of the heritability across locally defined regions in the genome. This approach utilises relationship matrices that are based on sharing of SNP and haplotype alleles at local haplotype blocks delimited by recombination boundaries in the genome. We implemented the approach on simulated data and show that the haplotype-based regional GRMs capture variation that is complementary to that captured by SNP-based regional GRMs, and thus justifying the fitting of the two GRMs jointly in a single analysis (SNHap-RHM). SNHap-RHM captures regions in the genome contributing to the phenotypic variation that existing genome-wide analysis methods may fail to capture. We further demonstrate that there are real benefits to be gained from this approach by applying it to real data from about 20,000 individuals from the Generation Scotland: Scottish Family Health Study. We analysed height and major depressive disorder (MDD). We identified seven genomic regions that are genome-wide significant for height, and three regions significant at a suggestive threshold (p-value < 1 x 10-5) for MDD. These significant regions have genes mapped to within 400kb of them. The genes mapped for height have been reported to be associated with height in humans. Similarly, those mapped for MDD have been reported to be associated with major depressive disorder and other psychiatry phenotypes. The results show that SNHap-RHM presents an exciting new opportunity to analyse complex traits by allowing the joint mapping of novel genomic regions tagged by either SNPs or haplotypes, potentially leading to the recovery of some of the "missing" heritability.

Author SummaryIn untangling the genetic contribution to observed phenotype differences, situations can arise where causative variants might be tagged by haplotypes and not in linkage disequilibrium with individual SNPs. This scenario is likely for relatively newly arisen and rarer variants. Here, we propose a regional heritability method, SNHap-RHM, that jointly fits haplotype-based and SNP-based genomic relationship matrices (GRMs) to capture genomic regions harbouring rare variants that the SNP-based GRMs might miss. By analysing ~20,000 Scottish individuals, we show by simulation that the two GRMs are very specific to the type of variant effects they can capture; - the haplotype-based GRMs specifically target haplotype effects which are mostly missed by SNP-based GRMs and vice versa. Applying the method to height and major depressive disorder led to the uncovering of regions in the genome that harbour genes associated with those traits. These results are uniquely important because first they confirm that effects tagged by haplotypes may be missed by conventional SNP-based methods. Secondly, our method, SNHap-RHM, presents an exciting new opportunity to analyse complex traits by allowing the joint mapping of genomic regions tagged by either SNPs or haplotypes, potentially leading to the recovery of some of the "missing" heritability.


title: Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f.sp. hordei

abstract:
In barley (Hordeum vulgare), the function of ROPs appears central to polar cell development and the interaction outcome with parasitic fungi but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP with GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant specific ROP nucleotide exchanger domain (PRONE). We performed phylogenetic analyses and annotated barley PRONE GEFs. HvGEF14 is expressed in leaf epidermal tissue and downregulated after inoculation with Blumeria graminis f.sp. hordei. Protein-protein interaction assays indicate that HvGEF14 and the type I barley ROP protein HvRACB can interact in yeast and in planta. Overexpression of HvGEF14 further recruited the ROP-GTP downstream interactor HvRIC171 to the cell periphery and let to interaction of HvRACB with an extended CRIB (Cdc42/Rac Interactive Binding motif) peptide of HvRIC171 in a similar manner as constitutively activated HvRACB. Finally, the over expression of HvGEF14 led to enhanced susceptibility to fungal entry while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells, which is crucial for fungal penetration success.

HighlightThe activated GTPase HvRACB is a susceptibility factor of barley in response to Blumeria graminis f.sp. hordei. The newly discovered guanine nucleotide exchange factor HvGEF14 is a HvRACB activator.