title: Structural basis of meiotic chromosome synaptic elongation through hierarchical fibrous assembly of SYCE2-TEX12

abstract: The synaptonemal complex (SC) is a supramolecular protein assembly that mediates synapsis between homologous chromosomes during meiosis. SC elongation along the chromosome length (up to 24 m) depends on its midline -fibrous component SYCE2-TEX12. Here, we report X-ray crystal structures of SYCE2-TEX12 as an individual building-block and upon assembly within a fibrous lattice. We combine these structures with mutagenesis, biophysics and electron microscopy to reveal the hierarchical mechanism of SYCE2-TEX12 fibre assembly. SYCE2-TEX12s building-blocks are 2:2 coiled-coils which dimerise into 4:4 hetero-oligomers and interact end-to-end and laterally to form 10-nm fibres, which intertwine within 40-nm bundled micrometre-long fibres that define the SCs midline structure. This assembly mechanism bears striking resemblance with intermediate filament proteins vimentin, lamin and keratin. Thus, SYCE2-TEX12 exhibits behaviour typical of cytoskeletal proteins to provide an -fibrous SC backbone that structurally underpins synaptic elongation along meiotic chromosomes.


title: Detectability of biotin tags by LC-MS/MS

abstract: The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin labeled peptide. Recent advances in labeling led to an increase in biotinylation efficiency and shifted the interest to detection of the site of biotinylation. This increased confidence in identification and provides additional structural information yet it requires efficient release of the biotinylated protein/peptide and sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics the effect of biotinylation on the chromatographic, ionization, and fragmentation behaviour and ultimate detection of peptides is not well understood. To address this we compare two commercially-available biotin labels EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter one containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase of hydrophobicity and charge reduction with increasing number of biotin labels attached. Based on our data we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.

category: biochemistry


title: Chromatin Interaction Neural Network (ChINN): A machine learning-based method for predicting chromatin interactions from DNA sequences

abstract: Chromatin interactions play important roles in regulating gene expression. However, the availability of genome-wide chromatin interaction data is limited. Various computational methods have been developed to predict chromatin interactions. Most of these methods rely on large collections of ChIP-Seq/RNA-Seq/DNase-Seq datasets and predict only enhancer-promoter interactions. Some of the  state-of-the-art methods have poor experimental designs, leading to over-exaggerated performances and misleading conclusions. Here we developed a computational method, Chromatin Interaction Neural Network (ChINN), to predict chromatin interactions between open chromatin regions by using only DNA sequences of the interacting open chromatin regions. ChINN is able to predict CTCF-, RNA polymerase II- and HiC-associated chromatin interactions between open chromatin regions. ChINN also shows good across-sample performances and captures various sequence features that are predictive of chromatin interactions. To apply our results to clinical patient data, we applied CHINN to predict chromatin interactions in 6 chronic lymphocytic leukemia (CLL) patient samples and a cohort of open chromatin data from 84 CLL samples that was previously published. Our results demonstrated extensive heterogeneity in chromatin interactions in patient samples, and one of the sources of this heterogeneity were the different subtypes of CLL.

category: bioinformatics


title: Sequencing of clinical samples reveals that adaptation keeps establishing during H7N9 virus infection in humans

abstract: The H7 subtype avian influenza viruses (AIV) have a much longer history and their adaptation through evolution pose continuous threat to humans 1. Since 2013 March, the novel reasserted H7N9 subtype have transmitted to humans through their repeated assertion in the poultry market. Through repeated transmission, H7N9 gradually became the second AIV subtype posing greater public health risk after H5N1 2,3. After infection, how the virus tunes its genome to adapt and evolve in humans remains unknown. Through direct amplification of H7N9 and high throughput (HT) sequencing of full genomes from the swabs and lower respiratory tract samples collected from infected patients in Shenzhen, China, we have analyzed the in vivo H7N9 mutations at the level of whole genomes and have compared with the genomes derived by in vitro cultures. These comparisons and frequency analysis against the H7N9 genomes in the public database, 40 amino acids were identified that play potential roles in virus adaptation during H7N9 infection in humans. Various synonymous mutations were also identified that might be crucial to H7N9 adaptation in humans. The mechanism of these mutations occurred in a single infection are discussed in this study.

category: microbiology


title: Evolution of lbx spinal cord expression and function

abstract:
Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many animals. Amniotes have two Lbx genes, but only Lbx1 is expressed in spinal cord. In contrast, teleosts have three lbx genes and we show here that zebrafish lbx1a, lbx1b and lbx2 are expressed by distinct spinal cell types, and that lbx1a is expressed in dI4, dI5 and dI6 interneurons, as in amniotes. Our data examining lbx expression in Scyliorhinus canicula and Xenopus tropicalis suggest that the spinal interneuron expression of zebrafish lbx1a is ancestral, whereas lbx1b has acquired a new expression pattern in spinal cord progenitor cells. lbx2 spinal expression was probably acquired in the ray-finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. We also show that the spinal function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction in the number of inhibitory spinal interneurons and an increase in the number of excitatory spinal interneurons, similar to mouse Lbx1 mutants. Interestingly, the number of inhibitory spinal interneurons is also reduced in lbx1b mutants, although in this case the number of excitatory interneurons is not increased. lbx1a;lbx1b double mutants have a similar spinal interneuron phenotype to lbx1a single mutants. Taken together these data suggest that lbx1b and lbx1a may be required in succession for correct specification of dI4 and dI6 spinal interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.

Research Highlightslbx1 spinal expression and function is conserved in vertebrates. In contrast, zebrafish lbx1b and lbx2 have novel spinal expression patterns that probably evolved in the ray-finned vertebrate lineage (lbx2) or teleosts (lbx1b).

category: developmental biology


title: MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

abstract:
Replication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of CoV-2. Modifications of full length S individually or in combination included two proline substitutions, mutations of the furin recognition site and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals.

SignificanceVaccines are required to control COVID-19 during the pandemic and possibly afterwards. Recombinant nucleic acids, proteins and virus vectors that stimulate immune responses to the CoV-2 S protein have provided protection in experimental animal or human clinical trials, though questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.


title: The high infectivity of SARS-CoV-2 B.1.1.7 is associated with increased interaction force between Spike-ACE2 caused by the viral N501Y mutation

abstract: The Spike glycoprotein receptor-binding domain (RBD) of SARS-CoV-2 mediates the viral particles binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of human cells. Therefore, Spike-ACE2 interaction is a crucial determining factor for viral infectivity. A new phylogenetic group of SARS-CoV-2 (lineage B.1.1.7) has been recently identified in the COVID-19 Genomics UK Consortium dataset, which features an amino acid substitution in the Spike RBD (N501Y mutation). Infections with the SARS-CoV-2 lineage B.1.1.7 have been overgrowing in recent weeks in the United Kingdom, indicating an even greater spread capacity than that seen with previous strains of the novel coronavirus. We hypothesized that this rapid spreading/infectivity of the B.1.1.7 lineage might be due to changes in the interaction force between the mutant Spike RBD and ACE2. This study employed in silico methods involving mutagenesis (N501Y mutation) and interface analysis focusing on the Spike RDB-ACE2 interaction. The results showed that the SARS-CoV-2 N501Y mutant (lineage B.1.1.7) establishes a more significant number of interactions relating to the mutant residue Y501 (Spike RDB) with residues Y41 and K353 (ACE2). This finding shows that the increased infectivity of SARS-CoV-2 lineage B.1.1.7 is associated with the interaction force between the Spike RBD Y501 mutant residue with the ACE2 receptor, which in this strain is increased.


title: MSM and sesame seed oil improve hyperlipidemia and PUFA metabolism in the db/db mouse

abstract:
BackgroundAn estimated 463 million people worldwide suffer from diabetes, and that number is projected to rise significantly to 700 million by 2045. One of the hallmarks of type 2 diabetes and metabolic syndrome is alterations in the lipid profile and polyunsaturated fatty acid metabolism.

ObjectiveThe objective of this study was to identify lipid alterations in leptin receptor deficient db/db mice, a type 2 diabetes model, to establish a baseline biological signature for the evaluation of natural products with purported lipid-altering activity.

MethodsSix-week old male db/db mice (n = 10/group) were randomized to the following groups: 1) diabetic control with no treatment, 2) methylsulfonylmethane (MSM) treatment (3.81 {+/-} 0.33 g/kg), 3) sesame seed oil (SSO) treatment (23.54 {+/-} 2.91 mg/kg), and 4) MSM and SSO combination treatment, receiving the same dosages of MSM and SSO as the single treatment groups. Eight-week old male C57BL6/J mice (n=10) were used as a non-diabetic control group.

ResultsSerum triglycerides and total cholesterol were significantly increased in the db/db model compared to nondiabetic control, mimicking diabetes in people. High-density lipoprotein (HDL) was significantly increased in all db/db treatment groups, with the most significant effect in the MSM and SSO combination group, with a corresponding decrease in non-HDL cholesterol. Serum total polyunsaturated fatty acid (PUFA) levels were significantly reduced in diabetic mice compared with control mice. In contrast, treatment with SSO alone reversed this effect such that fed mice exhibited serum PUFA levels comparable to control mice.

ConclusionsTreatment of db/db mice with MSM and SSO improved commonly measured clinical parameters in serum lipid panels. The combination of MSM and SSO treatments effects on HDL and non-HDL cholesterol and fatty acid metabolism could lead to improved clinical outcomes in diabetics, such as reduced incidence of atherosclerosis and hepatic steatosis.

category: pharmacology and toxicology


title: Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function.

abstract:
N-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin promotes pathological sequelae associated with GluN2B dysfunction.

HIGHLIGHTSO_LISpinophilin bidirectionally regulates GluN2B-containing NMDAR function.
C_LIO_LILoss of spinophilin in primary hippocampal neurons increases a pro-apoptotic marker.
C_LIO_LILoss of spinophilin in vivo decreases measures of spatial cognitive flexibility.
C_LI

Graphical AbstractSpinophilin increases the phosphorylation of Ser-1284 on GluN2B, thereby enhancing calcium influx through the GluN2B containing NMDARs. In contrast, spinophilin limits GluN2B-containing surface expression putatively due to modulation of GluN2B interactions with endocytotic proteins. Since the second effect of spinophilin occurs independent of the first, we observe an overall decrease in calcium influx through GluN2B containing NMDARs when spinophilin is present. This low, basal calcium influx is less likely to be promote calcium-dependent activation of caspase and downstream apoptotic pathways and permits flexible search strategies and behaviors. In the absence of spinophilin, the spinophilin-driven internalization of the receptors is decreased, more receptors are expressed on the surface and calcium influx into the cell is increased. This high levels of intracellular calcium triggers apoptotic pathways leading to cell death. This impact may be more dramatic in cells with high expression of GluN2B-containing NMDA receptors. This loss of spinophilin reduces cognitive flexibility in hippocampal dependent tasks.

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category: neuroscience


title: Cell type resolved co-expression networks of core clock genes in brain development.

abstract: The circadian clock regulates vital cellular processes by adjusting the physiology of the organism to daily changes in the environment. Rhythmic transcription of core Clock Genes (CGs) and their targets regulate these processes at the cellular level. Circadian clock disruption has been observed in people with neurodegenerative disorders like Alzheimers and Parkinsons. Also, ablation of CGs during development has been shown to affect neurogenesis in both in vivo and in vitro models. Previous studies on the function of CGs in the brain have used knock-out models of a few CGs. However, a complete catalog of CGs in different cell types of the developing brain is not available and it is also tedious to obtain. Recent advancements in single-cell RNA sequencing (scRNA-seq) has revealed novel cell types and elusive dynamic cell states of the developing brain. In this study by using publicly available single-cell transcriptome datasets we systematically explored CGs-coexpressing networks (CGs-CNs) during embryonic and adult neurogenesis. Our meta-analysis reveals CGs-CNs in human embryonic radial glia, neurons and also in lesser studied non-neuronal cell types of the developing brain.


title: Simultaneous inhibition of human CD4 and 4-1BB biogenesis suppresses cytotoxic T lymphocyte proliferation

abstract: The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive potential of CADA using in vitro activation models. CADA inhibited lymphocyte proliferation in a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads or phytohemagglutinin. The immunosuppressive effect of CADA involved both CD4+ and CD8+ T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited cell-mediated lympholysis. We discovered a direct down-modulatory effect of CADA on 4-1BB (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA blocked 4-1BB protein biosynthesis by inhibition of its co-translational translocation across the ER membrane in a signal peptide-dependent way. This study demonstrates that CADA, as potent down-modulator of human CD4 and 4-1BB, has promising in vitro immunomodulatory characteristics for future in vivo exploration as immunosuppressive drug.

category: immunology


title: Rapid progression is associated with lymphoid follicle dysfunction in SIV-infected infant rhesus macaques

abstract: HIV-infected infants are at an increased risk of progressing rapidly to AIDS in the first weeks of life. Here, we evaluated immunological and virological parameters in 25 SIV-infected infant rhesus macaques to understand the factors influencing a rapid disease outcome. Infant macaques were infected with SIVmac251 and monitored for 10 to 17 weeks post-infection. SIV-infected infants were divided into either typical (TypP) or rapid (RP) progressor groups based on levels of plasma anti-SIV antibody levels and SIV plasma viral load (with RP infants having low SIV-specific antibodies and high viral loads). Following SIV infection, 11 out of 25 infant macaques exhibited an RP phenotype, with 5 of these succumbing to AIDS-related infections. Interestingly, the TypP and RP infants were similar in their CD4 depletion and activation of CD8 T cells as measured by the levels of HLA-DR on the cell surface. However, differences between the two groups were identified in other immune cell populations, including a failure to expand activated memory (CD21-CD27+) B cells in peripheral blood in RP infant macaques, as well as reduced levels of germinal center (GC) B cells and T follicular helper (Tfh) cells in spleens (4- and 10-weeks post-SIV). Reduced B cell proliferation in splenic germinal GCs was associated with increased SIV+ cell density and follicular type 1 interferon (IFN)-induced immune activation. Further analyses determined that at 2-weeks post SIV infection TypP infants exhibited elevated levels of the GC-inducing chemokine CXCL13 in plasma, as well as significantly lower levels of viral envelope diversity compared to RP infants. Our findings provide evidence that early viral and immunologic events following SIV infection contributes to impairment of B cells, Tfh cells and germinal center formation, ultimately impeding the development of SIV-specific antibody responses in rapidly progressing infant macaques.


title: Bovine neutrophils release extracellular traps and cooperate with macrophages in Mycobacterium avium subsp. paratuberculosis clearance

abstract: Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guerin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1{beta} and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1{beta} and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.


title: Bovine neutrophils release extracellular traps and cooperate with macrophages in Mycobacterium avium subsp. paratuberculosis clearance in vitro


title: Cyclic AMP signalling and glucose metabolism mediate pH taxis by African trypanosomes

abstract: The collective movement of African trypanosomes on semi-solid surfaces, known as social motility, is presumed to be due to migration factors and repellents released by the parasites. Here we show that procyclic (insect midgut) forms acidify their environment as a consequence of glucose metabolism, generating pH gradients by diffusion. Early and late procyclic forms exhibit self-organising properties on agarose plates. While early procyclic forms are repelled by acid and migrate outwards, late procyclic forms remain at the inoculation site. Furthermore, trypanosomes respond to exogenously formed pH gradients, with both early and late procyclic forms being attracted to alkali. pH taxis is mediated by multiple cyclic AMP effectors: deletion of one copy of adenylate cyclase ACP5, or both copies of the cyclic AMP response protein CARP3, abrogates the response to acid, while deletion of phosphodiesterase PDEB1 completely abolishes pH taxis. The ability to sense pH is biologically relevant as trypanosomes experience large changes as they migrate through their tsetse host. Supporting this, a CARP3 null mutant is severely compromised in its ability to establish infections in flies. Based on these findings, we propose that the expanded family of adenylate cyclases in trypanosomes might govern other chemotactic responses in their two hosts.


title: Integrative analysis reveals unique features of the Smc5/6 complex

abstract: Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for Smc5/6s distinct functions is currently poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that while the complex shares a similar overall organization with other SMC complexes, it possesses several unique features. In contrast to the reported folded-arm structures of cohesin and condensin, our data suggest that Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5-Nse6 subcomplex. Further, we show that Nse5-Nse6 subcomplex adopts a novel structure with an extensive dimerization interface and multiple domains contacting other subunits of the Smc5/6 complex. We also provide evidence that the Nse5-Nse6 module uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative multi-scale study identifies distinct structural features of the Smc5/6 complex and functional cooperation amongst its co-evolved unique subunits.

category: molecular biology


title: The structural basis for Ulp2 recruitment to the kinetochore

abstract: The step-by-step process of chromosome segregation defines the stages of the cell division cycle. In eukaryotes, signaling pathways that control these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to the centromere of each chromosome. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for the SUMO protease, Ulp2. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.

category: cell biology


title: Sliding of kinetochore fibers along bridging fibers helps center the chromosomes on the spindle

abstract: Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores towards the center. We developed speckle microscopy in human spindles and confirmed the key prediction that kinetochore fiber flux is length-dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux markedly slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers promote chromosome alignment.


title: Microtubule poleward flux promotes chromosome centering on the spindle


title: Coordinated poleward flux of sister kinetochore fibers drives chromosome alignment


title: Fatty acid metabolic reprogramming promotes C. elegans development

abstract:
Acetylcholine signaling has been reported to play essential roles in animal metabolic regulation and disease affected by diets. However, the underlying mechanisms that how diets regulate animal physiology and health are not well understood. Here we found that the acetylcholine receptor gene eat-2 was expressed in most of the pharyngeal muscles, which is in accordance to our previous report that EAT-2 received synaptic signals not only from pharyngeal MC neurons. The expression of fatty acid synthesis genes was significantly increased in both eat-2 and tmc-1 fast-growth mutants on CeMM food environment, compared to the wild-type. Excitingly, dietary fatty acids such as 15-methyl-hexadecanoic acid (C17ISO), palmitic acid (PA, C16:0) and stearic acid (SA, C18:0) supplementation, significantly accelerated wild-type worm development on CeMM, indicating that the fatty acid synthesis reprogramming is an essential strategy for C. elegans to regulate its development and growth on CeMM diet. Furthermore, we found that fatty acid elongase gene elo-6 knock-out significantly attenuated eat-2 mutant fast growth, while overexpression of elo-6 could rescue the eat-2; elo-6 double mutant slow development, which suggested that elo-6 played a major role in the above metabolic remodeling. Taken together, our report indicates that diets regulate neuromuscular circuit and modulate C. elegans development via fatty acid metabolic reprogramming. As most of the key genes and metabolites found in this study are conserved in both invertebrate and vertebrate animals, we believed that our results might provide essential clues to the molecular mechanisms underlying interactions among animal nutrition sensation, metabolism reprogramming and developmental regulation.

Significance StatementDiets and nutritional composition affect animal development and human health, however the underlying mechanisms remain elusive. We demonstrate that the acetylcholine receptor gene eat-2 is expressed in most of pharyngeal muscles, and the expression of fatty acid synthesis genes is significantly increased in both eat-2 and tmc-1 fast-growth mutants on the synthetic chemical defined CeMM food environment. Dietary supplementation of several fatty acids significantly speed up animal development. Furthermore, we demonstrate that the fatty acid elongase gene elo-6 knock-out attenuates eat-2 mutant fast growth, and overexpression of wild-type elo-6 promotes the eat-2; elo-6 double mutant slow development. Our findings describe that acetylcholine signaling coordinate nutrition sensation and developmental regulation through fatty acid metabolic remodeling.

category: genetics


title: Drosophila functional screening of de novo variants in autism uncovers deleterious variants and facilitates discovery of rare neurodevelopmental diseases

abstract: Individuals with autism spectrum disorders (ASD) exhibit an increased burden of de novo variants in a broadening range of genes. We functionally tested the effects of ASD missense variants using Drosophila through  humanization rescue and overexpression-based strategies. We studied 79 ASD variants in 74 genes identified in the Simons Simplex Collection and found 38% of them caused functional alterations. Moreover, we identified GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in eight previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes point to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.


title: Pan-cancer analysis of mRNA stability for decoding tumour post-transcriptional programs

abstract: RNA stability is a crucial and often overlooked determinant of gene expression. Some of the regulators of mRNA stability are long known as key oncogenic or tumour suppressor factors. Nonetheless, the extent to which mRNA stability contributes to transcriptome remodeling in cancer is unknown, and the factors that modulate mRNA stability during cancer development and progression are largely uncharacterized. Here, by decoupling transcriptional and post-transcriptional effects in RNA-seq data of 7760 samples from 18 cancer types, we present a pan-cancer view of the mRNA stability changes that accompany tumour development and progression. We show that thousands of genes are dysregulated at the mRNA stability level, and identify the potential factors that drive these changes, including >80 RNA-binding proteins (RBPs) and microRNAs (miRNAs). Most RBPs and miRNAs have cancer type-specific activities, but a few show recurrent inactivation across multiple cancers, including the RBFOX family of RBPs and miR-29. Analysis of cell lines with phenotypic activation or inhibition of RBFOX1 and miR-29 confirms their role in modulation of genes that are dysregulated across multiple cancers, with functions in calcium signaling, extracellular matrix organization, and stemness. Overall, our study highlights the critical role of mRNA stability in shaping the tumour transcriptome, with recurrent post-transcriptional changes that are ~30% as frequent as transcriptional events. These results provide a resource for systematic interrogation of cancer-associated stability drivers and pathways.

category: genomics


title: Muscle coordination retraining inspired by musculoskeletal simulations: a study on reducing knee loading

abstract: Humans typically coordinate their muscles to meet movement objectives like minimizing energy expenditure. In the presence of pathology, new objectives gain importance, like reducing loading in an osteoarthritic joint, but people often do not change their muscle coordination patterns to meet these new objectives. Here we use musculoskeletal simulations to identify simple changes in coordination that can be taught by providing feedback of electromyographic activity to achieve a therapeutic goal--reducing joint loading. Our simulations predicted that changing the relative activation of the redundant ankle plantarflexors could reduce knee contact force during walking, but it was unclear whether humans could re-coordinate redundant muscles during a complex task like walking. With simple biofeedback of electromyographic activity, healthy individuals reduced the ratio of gastrocnemius to soleus muscle activation by 25{+/-}15% (p=0.004). The resulting "gastrocnemius avoidance" gait pattern reduced the late-stance peak of simulation-estimated knee contact force by 12{+/-}12% (p=0.029). Simulation-informed muscle coordination retraining could be a promising treatment for knee osteoarthritis and a powerful tool for optimizing coordination for a variety of rehabilitation and performance applications.

category: bioengineering


title: Impact of Cymbopogon flexuosus (Poaceae) essential oil and primary components on the eclosion and larval development of Aedes aegypti (Diptera: Culicidae)

abstract: The current study describes the effects of sub-lethal concentrations and constituent compounds (citral and geranyl acetate) of Cymbopogon flexuosus essential oil (EO) on the development of Aedes aegypti (L.) eggs and larvae. To demonstrate the ovicidal activity of EO, we treated embryonated eggs with 6, 18, and 30 mg.L-1 and larvae with 3 and 6 mg.L-1 EO concentrations. Citral and geranyl acetate were evaluated at 18, 30, and 42 mg.L-1 and compared with commercial growth inhibitors (diflubenzuron and methoprene) at 3 and 6 mg.L-1 concentrations. For each treatment, we measured larval head diameter, siphon length, and body length. To complement the morphological analysis, we examined concentrations of moult hormone (MH) and juvenile hormone III (JH III) using high-performance liquid chromatography (HPLC) coupled to mass spectrometry. The EO decreased egg hatching at all concentrations: 6 mg.L-1 in 45.3%; 18 mg.L-1 in 23.3%, and 30 mg.L-1 in 34.6%. EO also altered molting among larval instars and between larvae and pupae, with an increase in the length (3 mg.L-1: 6 {+/-} 0.0 mm; 6 mg.L-1: 6 {+/-} 0.7 mm) and head width (3 mg.L-1: 0.8 {+/-} 0 mm; 6 mg.L-1: 0.8 {+/-} 0.0 mm) compared with the control group (length: 5.3 {+/-} 0 mm; head width: 0.7 {+/-} 0.0 mm). We did not detect chromatographic signals of MH and JH III in larvae treated with C. flexuosus EO or their major compounds. The sub-lethal concentrations C. flexuosus EO caused a similar effect to diflubenzuron, decreasing hormone concentration, extending the larval period, and death.


title: The characteristics of intracellular Ca2+ signals in vivo necessitate a new model for salivary fluid secretion

abstract: Salivary fluid secretion involves an intricate choreography to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. Given this incompatibility to the previous model, a new mathematical model was constructed to explain how salivary secretion can be efficiently stimulated by apically localized Ca2+ signals.

category: physiology


title: Hippocampal representations of distance, space, and direction and their plasticity predict navigational performance

abstract: The hippocampus is implicated in episodic memory and allocentric spatial navigation. However, spatial selectivity is insufficient to navigate; one needs information about the distance and direction to the reward on a specific journey. The nature of these representations, whether they are expressed in an episodic-like sequence, and their relationship with navigational performance are unknown. We recorded single units from dorsal CA1 of the hippocampus while rats navigated to an unmarked reward zone defined solely by distal visual cues, similar to the classic water maze. The allocentric spatial selectivity was substantially weaker than in typical real world tasks, despite excellent navigational performance. Instead, the majority of cells encoded path distance from the start of trials. Cells also encoded the rats allocentric position and head angle. Often the same cells multiplexed and encoded path distance, head direction and allocentric position in a sequence, thus encoding a journey-specific episode. The strength of neural activity and tuning strongly correlated with performance, with a temporal relationship indicating neural responses influencing behavior and vice versa. Consistent with computational models of associative Hebbian learning, neural responses showed increasing clustering and became better predictors of behaviorally relevant variables, with neurometric curves exceeding and converging to psychometric curves. These findings demonstrate that hippocampal neurons multiplex and exhibit highly plastic, task- and experience-dependent tuning to path-centric and allocentric variables to form an episode, which could mediate navigation.


title: Mirror Neurons are Modulated by Reward Expectation and Grip Force in the Sensorimotor Cortices (S1, M1, PMd, PMv)

abstract: Mirror Neurons (MNs) respond similarly when primates make, or observe, grasping movements. Recent work indicates that reward expectation influences rostral M1 (rM1) during manual, observational, and Brain Machine Interface (BMI) reaching movements. Previous work showed MNs are modulated by subjective value. Here we expand on the above work utilizing two non-human primates (NHPs), one male Macaca Radiata (NHP S) and one female Macaca Mulatta (NHP P), that were trained to perform a cued reward level isometric grip-force task, where the NHPs had to apply visually cued grip-force to move and transport a virtual object. We found a population of (S1 area 1-2, rM1, PMd, PMv) units that significantly represented grip-force during manual and observational trials. We found the neural representation of visually cued force was similar during observational trials and manual trials for the same units, however, the representation was weaker during observational trials. Comparing changes in neural time lags between manual and observational tasks indicated that a subpopulation fit the standard MN definition of observational neural activity lagging the visual information. Neural activity in (S1 areas 1-2, rM1, PMd, PMv) significantly represented force and reward expectation. In summary, we present results indicating that sensorimotor cortices have MNs for visually cued force and value.


title: Mirror Neurons are Modulated by Grip Force and Reward Expectation in the Sensorimotor Cortices (S1, M1, PMd, PMv)


title: A low-cost mouse cage warming system provides improved intra-ischemic and post-ischemic body temperature control &ndash: application for reducing variability in experimental stroke studies

abstract:
Experimental guidelines have been proposed to improve the rigor and reproducibility of experimental stroke studies in rodents. As brain temperature is a strong determinant of ischemic injury, tight management of brain or body temperature (Tcore) during the experimental protocol is highly recommended. However, little guidance is provided regarding how or for how long temperature support should be provided. We compared a commonly used heat support method (cage on heating pad) with a low-cost custom built warm ambient air cage (WAAC) system. Both heat support systems were evaluated for the middle cerebral artery occlusion (MCAo) model in mice. The WAAC system provided improved temperature control (more normothermic Tcore and less Tcore variation) during the intra-ischemic period (60 min) and post-ischemic period (3 hrs). Neurologic deficit score showed significantly less variance at post-stroke day 1 (PSD1) in WAAC system mice. Mean infarct volume was not statistically different by heat support system, however, standard deviation was 54% lower in the WAAC system group. In summary, we provide a simple low-cost heat support system that provides superior Tcore management in mice during the intra-ischemic and post-ischemic periods, which results in reduced variability of experimental outcomes.

HighlightsO_LIWe describe the fabrication of a low-cost mouse cage warming system (warmed ambient air cage; WAAC system) that can be assembled and applied in any stroke laboratory.
C_LIO_LIThe WAAC system provides more precise control of post-stroke mouse body temperature compared with traditional heating pad warming system.
C_LIO_LIThe more precise control of post-stroke core temperature reduces variability in some experimental measures in more severely injured mice.
C_LI


title: Single neuron activity predicts behavioral performance of individual animals during memory retention

abstract: In 1972 Rescorla and Wagner formulated their model of classical Pavlovian conditioning postulating that the associative strength of a stimulus is expressed directly in the behavior it elicits1. Many biologists and psychologists were inspired by this model, and numerous experiments thereafter were interpreted assuming that the magnitude of the conditioned response (CR) reflects an associative effect at the physiological level. However, a correlation between neural activity and the expression of the CR in individual animals has not yet been reported. Here we show that, following differential odor conditioning, the change in activity of single mushroom body output neurons (MBON) of the honeybee predicts the behavioral performance of the individual during memory retention. The encoding of the stimulus-reward association at the mushroom body output occurs about 600 ms before the initiation of the CR. We conclude that the MB provides a stable representation of the stimulus-reward associative strength, and that this representation is required for behavioral decision-making during memory retention.


title: Mechanical Stretch Inhibition SensitizesProprioceptors to Compressive Stresses

abstract: A repetitive gait cycle is an archetypical component within the behavioural repertoire of many if not all animals including humans. It originates from mechanical feedback within proprioceptors to adjust the motorprogram during locomotion and thus leads to a periodic orbit in a low dimensional space. Here, we investigate the mechanics, molecules and neurons responsible for proprioception in Caenorhabditis (C.) elegans to gain insight into how mechanosensation shapes the orbital trajectory to a well-defined limit cycle. We used genome editing, force spectroscopy and multiscale modeling and found that alternating tension and compression with the spectrin network of a single proprioceptor encodes body posture and informs TRP-4/NOMPC and TWK-16/TREK2 homologs of mechanosensitive ion channels during locomotion. In contrast to a widely accepted model of proprioceptive  stretch reception, we found that proprioceptors activated under compressive stresses in vivo and in vitro, and speculate that this property is conserved across function and species.


title: Increased theta/alpha synchrony in the habenula-prefrontal network with negative emotional stimuli in human patients

abstract: Lateral habenula is believed to encode negative motivational stimuli and plays key roles in the pathophysiology of psychiatric disorders. However, how habenula activities are modulated during the perception and processing of emotional information is still poorly understood. We recorded local field potentials from bilateral habenula areas with simultaneous cortical magnetoencephalography in nine patients with psychiatric disorders during an emotional picture viewing task. Oscillatory activity in the theta/alpha band (5-10 Hz) within the habenula and prefrontal cortical regions, as well as the coupling between these structures, are increased during the perception and processing of negative emotional stimuli compared to positive emotional stimuli. The evoked increase in theta/alpha band synchronization in the frontal cortex-habenula network correlated with the emotional valence not the arousal score of the stimuli. These results provide direct evidence for increased theta/alpha synchrony within the habenula area and prefrontal cortex-habenula network in the perception of negative emotion in human participants.


title: Synaptopodin is required for stress fiber and contractomere assembly at the epithelial junction

abstract:
The apical junction of epithelial cells can generate force to control cell geometry and perform contractile processes while maintaining barrier function and adhesion. Yet, the structural basis for force generation at the apical junction is not fully understood. Here, we describe 2 synaptopodin-dependent actomyosin structures that are spatially, temporally, and structurally distinct. The first structure is formed by retrograde flow of synaptopodin initiated at the apical junction, creating sarcomeric stress fibers that lie parallel to the junction and insert into junctional complexes on the apical plane. Retrograde flow of synaptopodin is also seen at vinculin-decorated basal junctions on the basal plane. Contractions of apical stress fibers is associated with clustering of membrane complexes via side-on synaptopodin linkers whereas contractions of stress fibers inserted at the apical junction via head-on synaptopodin linkers results in junction shortening. Upon junction maturation, apical stress fibers are disassembled. In mature epithelial monolayer, a motorized "contractomere" complex capable of "walking the junction" is formed at junction vertices. Contractomere motility results in changes in junctional length, altering the overall shape of the cell and packing geometry within the monolayer. We propose a model of epithelial homeostasis that utilizes contractomere motility to preserve the permeability barrier during intercellular movement and junctional processes.

Summary StatementSynaptopodin retrograde flows initiate the assembly of apical and basal stress fibers from the apical and basal junctions. In mature apical junction, a motorized junctional complex, we termed the contractomere, allows the apical junction to change length and organize cell geometry within a confluent monolayer.


title: Fluctuations in population densities inform stability mechanisms in host-parasitoid interactions

abstract: Population dynamics of host-parasitoid interactions has been traditionally studied using a discrete-time formalism starting from the classical work of Nicholson and Bailey. It is well known that differences in parasitism risk among individual hosts can stabilize the otherwise unstable equilibrium of the Nicholson-Bailey model. Here, we consider a stochastic formulation of these discrete-time models, where the host reproduction is a random variable that varies from year to year and drives fluctuations in population densities. Interestingly, our analysis reveals that there exists an optimal level of heterogeneity in parasitism risk that minimizes the extent of fluctuations in the host population density. Intuitively, low variation in parasitism risk drives large fluctuations in the host population density as the system is on the edge of stability. In contrast, high variation in parasitism risk makes the host equilibrium sensitive to the host reproduction rate, also leading to large fluctuations in the population density. Further results show that the correlation between the adult host and parasitoid densities is high for the same year, and gradually decays to zero as one considers cross-species correlations across different years. We next consider an alternative mechanism of stabilizing host-parasitoid population dynamics based on a Type III functional response, where the parasitoid attack rate accelerates with increasing host density. Intriguingly, this nonlinear functional response makes qualitatively different correlation signatures than those seen with heterogeneity in parasitism risk. In particular, a Type III functional response leads to uncorrelated adult and parasitoid densities in the same year, but high cross-species correlation across successive years. In summary, these results argue that the cross-correlation function between population densities contains signatures for uncovering mechanisms that stabilize consumer-resource population dynamics.

category: ecology


title: RNA editing controls meiotic drive by a Neurospora Spore killer

abstract: Neurospora Sk-2 is a complex meiotic drive element that is transmitted to offspring through sexual reproduction in a biased manner. Sk-2s biased transmission mechanism involves spore killing, and recent evidence has demonstrated that spore killing is triggered by a gene called rfk-1. However, a second gene, rsk, is also critically important for meiotic drive by spore killing because it allows offspring with an Sk-2 genotype to survive the toxic effects of rfk-1. Here, we present evidence demonstrating that rfk-1 encodes two protein variants: a 102 amino acid RFK-1A and a 130 amino acid RFK-1B, but only RFK-1B is toxic. We also show that expression of RFK-1B requires an early stop codon in rfk-1 mRNA to undergo adenosine-to-inosine (A-to-I) mRNA editing. Finally, we demonstrate that RFK-1B is toxic when expressed within vegetative tissue of Spore killer sensitive (SkS) strains, and that this vegetative toxicity can be overcome by co-expressing Sk-2s version of RSK. Overall, our results demonstrate that Sk-2 uses RNA editing to control when its spore killer is produced, and that the primary killing and resistance functions of Sk-2 can be conferred upon an SkS strain by the transfer of only two genes.


title: Inference of emergent spatio-temporal processes from single-cell sequencing reveals feedback between de novo DNA methylation and chromatin condensates

abstract:
Recent breakthroughs in single-cell genomics allow probing molecular states of cells with unprecedented detail along the sequence of the DNA. Biological function relies, however, on emergent processes in the three-dimensional space of the nucleus, such as droplet formation through phase separation. Here, we use single-cell multi-omics sequencing to develop a theoretical framework to rigorously map epigenome profiling along the DNA sequence onto a description of the emergent spatial dynamics in the nucleus. Drawing on scNMT-seq multi-omics sequencing in vitro and in vivo we exemplify our approach in the context of exit from pluripotency and global de novo methylation of the genome. We show how DNA methylation patterns of the embryonic genome are established through the interplay between spatially correlated DNA methylation and topological changes to the DNA. This feedback leads to the predicted formation of 30-40nm sized condensates of methylated DNA and determines genome-scale DNA methylation rates. We verify these findings with orthogonal single cell multi-omics data that combine the methylome with HiC measurements. Notably, this scale of chromatin organization has recently been described by super-resolution microscopy. Using this framework, we identify local methylation correlations in gene bodies that precede transcriptional changes at the exit from pluripotency. Our work provides a general framework of how mechanistic insights into emergent processes underlying cell fate decisions can be gained by the combination of single-cell multi-omics and methods from theoretical physics that have not been applied in the context of genomics before.

HighlightsO_LIWe develop methodology to infer collective spatio-temporal processes in the physical space of the nucleus from single-cell methylome sequencing experiments.
C_LIO_LIWe show that DNA methylation relies on a feedback between de novo methylation and nanoscale changes in DNA topology, leading to the formation of methylation condensates.
C_LIO_LIChromatin condensates at this scale have recently been described by high-resolution microscopy but have remained without mechanistic explanation.
C_LIO_LIUsing this framework, we identify changes in the distribution of DNA methylation marks in gene bodies that precede gene silencing at the exit from pluripotency.
C_LI


title: Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

abstract: Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

category: biophysics


title: Apomixis and genetic background affect distinct traits in Hieracium pilosella L. grown under competition

abstract:
BackgroundApomixis, the asexual reproduction through seeds, occurs in over 40 plant families and avoids the hidden cost of sex. Apomictic plants are thought to have an advantage in sparse populations and when colonizing new areas but may have a disadvantage in changing environments because they propagate via fixed genotypes.

In this study, we separated the influences of different genetic backgrounds (potentially reflecting local adaptation) from those of the mode of reproduction, i.e., sexual vs. apomictic, on nine fitness-related traits in Hieracium pilosella L. We aimed to test whether apomixis per se may provide a fitness advantage in different competition environments in a common garden setting.

ResultsTo separate the effects of genetic background from those of reproductive mode, we generated five families of apomictic and sexual full siblings by crossing two paternal with four maternal parents. Under competition, apomictic plants showed reproductive assurance (probability of seeding, fertility), while offspring of sexual plants with the same genetic background had a higher germination rate. Sexual plants grew better (biomass) than apomictic plants in the presence of grass as a competitor but apomictic plants spread further vegetatively (maximum stolon length) when their competitors were sexual plants of the same species. Furthermore, genetic background as represented by the five full-sibling families influenced maximum stolon length, the number of seeds, and total fitness. Under competition with grass, genetic background influenced fecundity, the number of seeds, and germination rate.

ConclusionsOur results suggest that both the mode of reproduction as well as the genetic background affect the success of H. pilosella in competitive environments. Total fitness, the most relevant trait for adaptation, was only affected by the genetic background. However, we also show for the first time that apomixis per se has effects on fitness-related traits that are not confounded by -- and thus independent of -- the genetic background.

category: plant biology


title: Analysis of Scholarly Discourse in Biosecurity from 1986-2020

abstract: Scholarly discourse in Biosecurity is complex and interdisciplinary. The term  Biosecurity has different meanings depending on the discipline. This term most often strives to capture efforts to prepare for and respond to threats posed by infectious organisms1. Increasing complexity of discourse in this interdisciplinary field and lack of common definitions can pose communication challenges. We use text mining of scholarly articles to characterize the interdisciplinary field of Biosecurity and the uses of this term over the past 35 years; a context evolution model is built to describe major changes in this field, and it shows a progression toward health security in recent years. Understanding the shifts in discourse predominance helps to illuminate the major challenges facing biosecurity research and academic community, and contributes to enhancing communication across disciplines within the Biosecurity field.

category: scientific communication and education


title: All-or-none disconnection of pyramidal inputs onto parvalbumin-positive interneurons gates ocular dominance plasticity

abstract: Disinhibition is an obligatory initial step in the remodeling of cortical circuits by sensory experience. Our investigation on disinhibitory mechanisms in the classical model of ocular dominance plasticity uncovered an unexpected novel form of experience-dependent circuit plasticity. In layer 2/3 of mouse visual cortex monocular deprivation triggers a complete, "all-or-none", elimination of connections from pyramidal cells onto nearby parvalbumin-positive interneurons (PyrPV). This circuit plasticity is unique as it is transient, local and discrete. It lasts only one day, and it does not manifest as widespread changes in synaptic strength, rather, only about half of local connections are lost and the remaining ones are not affected in strength. Mechanistically, the deprivation-induced loss of PyrPV is contingent on a reduction of the protein neuropentraxin2 (NPTX2). Functionally, the loss of PyrPV is absolutely necessary for ODP. We surmise, therefore, that this "all-or-none" loss of local PyrPV circuitry gates experience-dependent cortical plasticity.


title: The gastrin-releasing peptide regulates stress-enhanced fear and dopamine signaling

abstract:
Fear extinction is an adaptive behavioral process critical for organisms survival, but deficiency in extinction may lead to PTSD. While the amygdala and its neural circuits are critical for fear extinction, the molecular identity and organizational logic of cell types that lie at the core of these circuits remain unclear. Here we report that mice deficient for amygdala-enriched gastrin-releasing peptide gene (Grp-/-) exhibit enhanced neuronal activity in the basolateral amygdala (BLA) and stronger fear conditioning, as well as deficient extinction in stress-enhanced fear learning (SEFL). rAAV2-retro-based tracing combined with visualization of the GFP knocked in the Grp gene showed that BLA receives GRPergic or conditioned stimulus projections from the indirect auditory thalamus-to-auditory cortex pathway, ventral hippocampus and ventral tegmental area. Transcription of dopamine-related genes was decreased in BLA of Grp-/- mice following SEFL extinction recall, suggesting that the GRP may mediate fear extinction regulation by dopamine.

Impact statementMice deficient for the amygdala-enriched gastrin-releasing peptide gene are susceptible to stress-enhanced fear, a behavioral protocol with relevance to PTSD, and show a decrease in dopamine-related gene transcription.


title: Olfactory coding in the bumble bee antennal lobe

abstract: Sociality is classified as one of the major transitions in evolution, with the largest number of eusocial species found in the insect order Hymenoptera, including the Apini (honey bees) and the Bombini (bumble bees). Bumble bees and honey bees not only differ in their social organization and foraging strategies, but comparative analyses of their genomes demonstrated that bumble bees have a slightly less diverse family of olfactory receptors than honeybees, suggesting that their olfactory abilities have adapted to different social and/or ecological conditions. However, unfortunately, no precise comparison of olfactory coding has been performed so far between honey bees and bumble bees, and little is known about the rules underlying olfactory coding in the bumble bee brain. In this study, we used in vivo calcium imaging to study olfactory coding of a panel of floral odorants in the antennal lobe (AL) of the bumble bee Bombus terrestris. Our results show that odorants evoke consistent neuronal activity in the bumble bee antennal lobe. Each odorant evokes a different glomerular activity pattern revealing this molecules chemical structure, i.e. its carbon chain length and functional group. Response intensity as well as odor-similarity relationships were highly correlated to those measured in honey bees. This study thus suggests that bumble bees, like honey bees, are equipped to respond to odorants according to their chemical features.


title: CO2 signalling mediates neurovascular coupling in the cerebral cortex

abstract: The mechanisms of neurovascular coupling remain incompletely understood. Here we show in experimental animal models that the neuronal activity-dependent increases in local cerebral blood flow in the somatosensory cortex are abolished by saturation of brain CO2-sensitive vasodilatory mechanism or disruption of brain HCO3-/CO2 transport, independently of baseline cerebral perfusion and brain tissue pH. These results suggest that increases in metabolic CO2 production and CO2 signalling play an important role in the development of neurovascular coupling response.


title: Widespread inhibitory projections from the interposed cerebellar nucleus

abstract: The cerebellum consists of parallel parasagittal modules that contribute to diverse behaviors, spanning motor to cognitive. Recent work employing cell-type specific tracing has identified circumscribed output channels of the cerebellar nuclei that could confer tight functional specificity. These studies have largely focused on excitatory projections of the cerebellar nuclei, however, leaving open the question of whether inhibitory neurons also constitute multiple output modules. We mapped output and input patterns to intersectionally restricted cell types of the interposed and adjacent interstitial nuclei. In contrast to the widespread assumption of primarily excitatory outputs and restricted inferior olive-targeting inhibitory output, we found that inhibitory neurons from this region ramified widely within the brainstem, targeting both motor- and sensory-related nuclei, distinct from excitatory output targets. Despite differences in output targeting, monosynaptic rabies tracing revealed largely shared afferents to both cell classes. We discuss the potential novel functional roles for inhibitory outputs in the context of cerebellar theory.


title: Widespread inhibitory projections from the cerebellar interposed nucleus


title: A Hot-Coal theory of Working Memory

abstract: Working memory allows us to selectively remember and flexibly manipulate a limited amount of information. Importantly, once we learn a certain operation, it generalizes to any memory object, not just the objects it has been trained on. Here we propose a conceptual model for how this might be achieved on the neural network level. It relies on spatial computing, in which sensory information flows spatially within the network over time. As a result, information about, for instance, object order can be retrieved agnostically to the detailed synaptic connectivity responsible for encoding specific memory items. This spatial flow is reflected in low-dimensional brain activity complementing high-dimensional activity that accounts for storing the sensory information itself. By comparing the dimensionality of local field potentials and spiking activity from prefrontal cortex of rhesus macaques performing multi-item working memory tasks we verify predictions from this model. We discuss how spatial computing may be a principle to aid generalization and zero-shot learning by utilizing spatial dimensions as an additional information encoding dimension. The new model also helps explain several aspects of neurophysiological activity related to working memory control, including dimensionality, context-dependent selectivity as well as persistent and non-persistent delay activity.


title: Zebrafish Polycomb repressive complex-2 critical roles are largely Ezh2- over Ezh1-driven and concentrate during early embryogenesis

abstract: Polycomb repressive complex-2 (PRC2) methylation of histone H3 lysine-27 (H3K27me) is associated with stable transcriptional repression. PRC2 famously silences Hox genes to maintain anterior-posterior segment identities but also enables early cell fate specification, restrains progenitor cell differentiation, and canalizes cell identities. Zebrafish PRC2 genetic studies have focused on ezh2, which, with its paralog ezh1, encodes the H3K27 methyltransferase component. ezh2 loss-of-function mutants reinforce essential vertebrate PRC2 functions during early embryogenesis albeit with limited contributions to body plan establishment. However, redundancy with ezh1 and the lethality of maternal-zygotic homozygous ezh2 nulls could obscure additional early developmental and organogenesis roles of PRC2. Here, we combine new and existing zebrafish ezh1 and ezh2 alleles to show collective maternal/zygotic ezh2 exclusively provides earliest embryonic PRC2 H3K27me3 activity. Zygotic ezh1, which becomes progressively expressed as ezh2 levels dissipate, has minor redundant and noncompensatory larval roles but itself is not required for viability or fertility. Zygotic Ezh2/PRC2 promotes correct craniofacial bone shape and size by maintaining proliferative pre-osteoblast pools. An ezh2 allelic series including disrupted maternal ezh2 uncovers axial skeleton homeotic transformations and pleiotropic organogenesis defects. Further, once past a critical early window, we show zebrafish can develop near normally with minimal bulk H3K27me3. Our results suggest Ezh2-containing PRC2 stabilizes rather than instructs early developmental decisions while broadly contributing to organ size and embellishment.


title: Simultaneous Identification of Brain Cell Type and Lineage via Single Cell RNA Sequencing

abstract: Acquired mutations are sufficiently frequent such that the genome of a single cell offers a record of its history of cell divisions. Among more common somatic genomic alterations are loss of heterozygosity (LOH). Large LOH events are potentially detectable in single cell RNA sequencing (scRNA-seq) datasets as tracts of monoallelic expression for constitutionally heterozygous single nucleotide variants (SNVs) located among contiguous genes. We identified runs of monoallelic expression, consistent with LOH, uniquely distributed throughout the genome in single cell brain cortex transcriptomes of F1 hybrids involving different inbred mouse strains. We then phylogenetically reconstructed single cell lineages and simultaneously identified cell types by corresponding gene expression patterns. Our results are consistent with progenitor cells giving rise to multiple cortical cell types through stereotyped expansion and distinct waves of neurogenesis. Compared to engineered recording systems, LOH events accumulate throughout the genome and across the lifetime of an organism, affording tremendous capacity for encoding lineage information and increasing resolution for later cell divisions. This approach can conceivably be computationally incorporated into scRNA-seq analysis and may be useful for organisms where genetic engineering is prohibitive, such as humans.


title: Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses

abstract:
Viruses have evolved means to manipulate the hosts ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.

Author summaryDue to the limited size of virus genomes, virus replication critically relies on host cell components. In addition to the host cells energy metabolism and its DNA replication and protein synthesis apparatus, the protein degradation machinery is an attractive target for viral re-appropriation. Certain viral factors divert the specificity of host ubiquitin ligases to antiviral host factors, in order to mark them for destruction by the proteasome, to lift intracellular barriers to virus replication. Here, we present molecular details of how the simian immunodeficiency virus accessory protein Vpr interacts with a substrate receptor of host Cullin4-RING ubiquitin ligases, and how this interaction redirects the specificity of Cullin4-RING to the antiviral host factor SAMHD1. The studies uncover the mechanism of Vpr-induced SAMHD1 recruitment and subsequent ubiquitylation. Moreover, by comparison to related accessory proteins from other immunodeficiency virus species, we illustrate the surprising variability in the molecular strategies of SAMHD1 counteraction, which these viruses adopted during evolutionary adaptation to their hosts. Lastly, our work also provides deeper insight into the inner workings of the hosts Cullin4-RING ubiquitylation machinery.

category: biochemistry


title: A Constitutively Closed Pannexin1 Channel in Lipid Bilayer Nanodiscs Assembles as a Large-Pore Heptamer

abstract:
Pannexin 1 (Panx1) channels are widely expressed and play important roles in apoptotic cell clearance, inflammation, blood pressure regulation, neurological disorders, opiate withdrawal, and cancer progression and metastasis. We performed (1) physicochemical analysis on a constitutively closed Panx1 channel (designated fPanx1{Delta}C) to examine the entire population of particles to detect multiple oligomeric states and (2) cryoEM in the membrane mimetics amphipol A8-35 and lipid bilayer nanodiscs. Our results reveal that the dominant if not exclusive oligomeric state of fPanx1{Delta}C is a heptamer, in solution and by cryoEM. The Panx1 heptamer provides further structural diversity within the family of large-pore channels, including hexameric LRRC8 (SWELL1) channels and connexin hemichannels, octameric CALHM1 channels and innexin hemichannels, and undecameric CALHM2 channels. Conserved structural themes are a large cytoplasmic vestibule with a diameter that corresponds roughly with the oligomeric state and a 4-helix bundle protomer, albeit with noncanonical helical packing for CALHM1 and CALHM2.

In BriefThe 4-helix bundle protomer of a constitutively closed pannexin1 channel assembles as a heptamer in solution and by cryoEM.


title: A persistent invasive phenotype in post-hypoxic tumor cells is revealed by novel fate-mapping and computational modeling

abstract: Hypoxia is a critical factor in solid tumors that has been associated with cancer progression and aggressiveness. We recently developed a hypoxia-fate mapping system that allowed the tracing of post-hypoxic cells within a tumor for the first time. This novel approach, based on an oxygen-dependent fluorescent switch, made the investigation of the post-hypoxic phenotype possible. The system allowed us to measure key biological features such as oxygen distribution, cell proliferation and migration. Using this data, we developed a computational model to investigate the motility and phenotypic persistence of hypoxic and post-hypoxic cells during tumor progression. The behavior of hypoxic and post-hypoxic cells was defined by phenotypic persistence time, cell movement bias and the fraction of cells that respond to an enhanced migratory stimulus. Our studies revealed that post-hypoxic cells have an enhanced persistent migratory phenotype that promotes the formation of invasive structures ("plumes") expanding towards the oxygenated tumor regions. This work combined advanced cell tracking and imaging techniques with mathematical modeling, and revealed for the first time that a persistent invasive migratory phenotype that develops under hypoxic conditions enhances their escape into non-hypoxic tumor regions to invade the surrounding tissue.

category: cancer biology


title: Neuron tracing and quantitative analyses of dendritic architecture reveal symmetrical three-way-junctions and phenotypes of git-1 in C. elegans

abstract:
Complex dendritic trees are a distinctive feature of neurons. Alterations to dendritic morphology are associated with developmental, behavioral and neurodegenerative changes. The highly-arborized PVD neuron of C. elegans serves as a model to study dendritic patterning; however, quantitative, objective and automated analyses of PVD morphology are missing. Here, we present a method for neuronal feature extraction, based on deep-learning and fitting algorithms. The extracted neuronal architecture is represented by a database of structural elements for abstracted analysis. We obtain excellent automatic tracing of PVD trees and uncover that dendritic junctions are unevenly distributed. Surprisingly, these junctions are three-way-symmetrical on average, while dendritic processes are arranged orthogonally. We quantify the effect of mutation in git-1, a regulator of dendritic spine formation, on PVD morphology and discover a localized reduction in junctions. Our findings shed new light on PVD architecture, demonstrating the effectiveness of our objective analyses of dendritic morphology and suggest molecular control mechanisms.

Author SummaryNerve cells (neurons) collect input signals via branched cellular projections called dendrites. A major aspect of the study of neurons, dating back over a century, involves the characterization of neuronal shapes and of their dendritic processes.

Here, we present an algorithmic approach for detection and classification of the tree-like dendrites of the PVD neuron in C. elegans worms. A key feature of our approach is to represent dendritic trees by a set of fundamental shapes, such as junctions and linear elements. By analyzing this dataset, we discovered several novel structural features. We have found that the junctions connecting branched dendrites have a three-way-symmetry, although the dendrites are arranged in a crosshatch pattern; and that the distribution of junctions varies across distinct sub-classes of the PVDs dendritic tree. We further quantified subtle morphological effects due to mutation in the git-1 gene, a known regulator of dendritic spines. Our findings suggest molecular mechanisms for dendritic shape regulation and may help direct new avenues of research.

category: systems biology


title: The growing topology of the C. elegans connectome

abstract:
Probing the developing neural circuitry in Caenorhabditis elegans has enhanced our understanding of nervous systems. The C. elegans connectome, like those of other species, is characterized by a rich club of densely connected neurons embedded within a small-world architecture. This organization of neuronal connections, captured by quantitative network statistics, provides insight into the systems capacity to perform integrative computations. Yet these network measures are limited in their ability to detect weakly connected motifs, such as topological cavities, that may support the systems capacity to perform segregated computations. We address this limitation by using persistent homology to track the evolution of topological cavities in the growing C. elegans connectome throughout neural development, and assess the degree to which the growing connec-tomes topology is resistant to biological noise. We show that the developing connectome topology is both relatively robust to changes in neuron birth times and not captured by similar growth models. Additionally, we quantify the consequence of a neurons specific birth time and ask if this metric tracks other biological properties of neurons. Our results suggest that the connectomes growing topology is a robust feature of the developing con-nectome that is distinct from other network properties, and that the growing topology is particularly sensitive to the exact birth times of a small set of predominantly motor neurons. By utilizing novel measurements that track biological features, we anticipate that our study will be helpful in the construction of more accurate models of neuronal development in C. elegans.

Author SummaryNetwork analyses have identified several local and global properties of the C. elegans connectome that are relevant to the organisms function and its capacity for information processing. Recent work has extended those investigations by focusing on the connectomes growth, in an effort to uncover potential drivers of connectome formation. Here we investigate connectome growth from the perspective of applied algebraic topology, by tracking both changing and persistent homology. In doing so, we are able to measure the resilience of the growth process to perturbations, and assess spatial variations in that resilience throughout the organisms body. Our findings provide new insights regarding the development of this simple natural connectome, as we have determined the existence of a robust and topologically simple network feature that is unexplained by the presence of other notable features of the connectome.


title: Bulkhead-like apical membrane structures between hepatocytes are required for anisotropic lumen expansion and liver tissue morphogenesis

abstract: Lumen morphogenesis is key to the function of organs and results from the integration of molecular pathways and mechanical forces1-3. The mechanisms governing anisotropic lumen expansion remain elusive4-6. In contrast to epithelial cells which have simple apico-basal polarity and form tubes, hepatocytes are multi-polar and form narrow lumina that grow anisotropically between adjacent cells, collectively generating a complex 3D network of bile canaliculi (BC)7,8. Here, we studied lumen elongation and BC morphogenesis in differentiating primary mouse hepatoblasts in vitro. Remarkably, we discovered a pattern of specific extensions of the apical membrane traversing the lumen between adjacent hepatocytes and sealed by tight junctions, reminiscent of the bulkheads of boats. These structures were also present in the developing liver. A targeted screen revealed that silencing of Rab35 caused loss of the bulkheads, conversion of hepatocyte into simple epithelial polarity and formation of spherical lumina in vitro. Strikingly, we could re-engineer hepatocyte polarity and tissue morphogenesis in vivo in the embryonic liver, converting BC into simple epithelial tubes. Our results suggest that the apical bulkheads of hepatocytes are cell-intrinsic anisotropic mechanical elements that ensure stability of the elongating lumen between two cells, thus determining the structure of BC during liver tissue morphogenesis.


title: Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads


title: The novel leishmanial Copper P-type ATPase plays a vital role in intracellular parasite survival

abstract: Copper is essential for all life forms; however in excess it is extremely toxic. Toxic properties of copper are utilized by hosts against various pathogenic invasions. Leishmania, in its both free-living and intracellular forms, was found to exhibit appreciable tolerance towards copper-stress. To determine the mechanism of copper-stress evasion employed by Leishmania we identified and characterized the hitherto unknown Copper-ATPase in Leishmania major and determined its role in parasites survival in host macrophage cells. L. major Cu-ATPase, LmATP7, exhibits high homology with its orthologues at multiple conserved motifs. In promastigotes, LmATP7 localized to the plasma membrane with a fraction in intracellular puncta. Upon copper treatment, LmATP7 expression increases few folds. LmATP7 is capable of complementing copper transport in Cu-ATPase-{Delta} yeast strain. Promastigotes overexpressing LmATP7 exhibits higher survival upon copper stress indicating efficacious copper export compared to wild type and heterozygous LmATP7 knock-out parasites. We explored macrophage-Leishmanial interaction with respect to copper stress subjected by the host upon parasite and the parasites reciprocating response thereon to evade the stress. The Copper-P-type-ATPases ATP7A/7B serve as major copper exporters in mammals that maintain cellular copper levels. We found that Leishmania infection triggers ATP7A upregulation in macrophages. Additionally, as part of host response, ATP7A traffics from trans-Golgi network and transports copper to the endosomal and endolysosomal compartments harbouring the Leishmania amastigotes. Finally, we show LmATP7 overexpression in this parasite increased amastigote survivability within infected macrophages whereas knocking it down reduces that drastically, establishing its role in combating host-induced copper stress.


title: PEDF and Derived Peptides Prevent Apoptosis and Promote Differentiation of Retinal Photoreceptors

abstract: Pigment epithelium-derived factor (PEDF) is a cytoprotective protein for the retina. We hypothesize that this protein acts on neuronal survival and differentiation of photoreceptor cells in culture. The purpose of the present study was to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors. Results show that PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an inhibitor of the PEDF receptor or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. Our findings support the neurotrophic PEDF peptides as neuronal guardians for the retina, highlighting their potential as promoters of retinal differentiation, and inhibitors of retina cell death and its blinding consequences.


title: Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate

abstract: Proper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter-molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose, creating a superstructure that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. This observation suggests that the +TIP network might under some conditions form a biomolecular condensate. Previous studies have shown that overexpression of the +TIP CLIP-170 induces large "patch" structures containing CLIP-170 and other +TIPs. To test the hypothesis that these patches might be biomolecular condensates, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.


title: Estimation of cell cycle kinetics in higher plant root meristem with cellular fate and positional resolution.

abstract:
Plant root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To shape the organs structure signaling between the different cells and cell files must be highly coordinated. Thereby, diverging kinetics of chromatin remodeling and cell growth in these files need to be integrated and balanced by differential cell growth and local differences in cell proliferation frequency. Understanding the local differences in cell cycle duration in the RAM and its correlation with chromatin organization is crucial to build a holistic view on the different regulatory processes and requires a quantitative estimation of the chromatin geometry and underlying mitotic cell cycle phases timing at every cell file and every position. Unfortunately, so far precise methods for such analysis are missing.

This study presents a robust and straightforward pipeline to determine in parallel the duration of cell cycles key stages in all cell layers of a plants root and their nuclei organization. The methods combine marker-free techniques based on the detection of the nucleus, deep analysis of the chromatin phase transition, incorporation of 5-ethynyl-2'-deoxyuridine (EdU), and mitosis with a deep-resolution plant phenotyping platform to analyze all key cell cycle events kinetics.

In the Arabidopsis thaliana L. RAM S-phase duration was found to be as short as 20-30 minutes in all cell files. The subsequent G2-phase duration however depends on the cell type/position and varies from 3.5 hours in the pericycle to more than 4.5 hours in the epidermis. Overall, S+G2+M duration in Arabidopsis under our condition is 4 hours in the pericycle and up to 5.5 hours in the epidermis.

Endocycle duration was determined as the time required to achieve 100% EdU index in the transition zone and estimated to be in the range of 3-4 hours.

Besides Arabidopsis, we show that the presented technique is applicable also to root tips of other dicot and monocot plants (tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.) and wheat (Triticum aestivum L.).


title: Estimation of differential cell cycle kinetics in higher plant root meristem with cellular fate and positional resolution.


title: Estimation of cell cycle kinetics in higher plant root meristem links organ position with cellular fate and chromatin structure


title: Manipulating the murine Lgr5 locus using a rapid, efficient and flexible CRISPR/Cas9 pipeline

abstract: The maintenance of adult epithelial tissues, such as the lining of the intestine, depends on their periodical renewal. This is achieved through small populations of adult stem cells. Lgr5 serves as a marker for these cells. Here we report a novel non-variegated Lgr5 mouse model, which was generated via the CRISPR/Cas9 system. We show that this Lgr5-2A-CreERT2-2A-mOrange2 mouse line can be used for lineage tracing, as well as for directing gene expression to Lgr5+ cells. The introduction of the transgene affects neither the expression, nor the function of endogenous Lgr5. Therefore, this new tool will serve to mark and manipulate intestinal stem cells to gain new biological insights.

category: developmental biology


title: Precise genomic deletions using paired prime editing

abstract: Technologies that precisely delete genomic sequences in a programmed fashion can be used to study function as well as potentially for gene therapy. The leading contemporary method for programmed deletion uses CRISPR/Cas9 and pairs of guide RNAs (gRNAs) to generate two nearby double-strand breaks, which is often followed by deletion of the intervening sequence during DNA repair. However, this approach can be inefficient and imprecise, with errors including small indels at the two target sites as well as unintended large deletions and more complex rearrangements. Here we describe a prime editing-based method that we term PRIME-Del, which induces a deletion using a pair of prime editing gRNAs (pegRNAs) that target opposite DNA strands, effectively programming not only the sites that are nicked but also the outcome of the repair. We demonstrate that PRIME-Del achieves markedly higher precision in programming deletions than CRISPR/Cas9 and gRNA pairs. We also show that PRIME-Del can be used to couple genomic deletions with short insertions, enabling deletions whose junctions do not fall at protospacer-adjacent motif (PAM) sites. Finally, we demonstrate that lengthening the time window of expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful in enabling precise, flexible programming of genomic deletions, including in-frame deletions, as well as for epitope tagging and potentially for programming rearrangements.


title: The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates

abstract: SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.