Orchestrated Excitatory and Inhibitory Learning Rules Lead to the Unsupervised Emergence of Self-sustained and Inhibition-stabilized Dynamics Self-sustaining neural activity maintained through local recurrent connections is of fundamental importance to cortical function. We show that Up-states--an example of self-sustained, inhibition-stabilized network dynamics--emerge in cortical circuits across three weeks of ex vivo development, establishing the presence of unsupervised learning rules capable of generating self-sustained dynamics. Previous computational models have established that four sets of weights (WE[&lt;-]E, WE[&lt;-]I, WI[&lt;-]E, WI[&lt;-]I) must interact in an orchestrated manner to produce Up-states, but have not addressed how a family of learning rules can operate in parallel at all four weight classes to generate self-sustained inhibition-stabilized dynamics. Using numerical and analytical methods we show that, in part due to the paradoxical effect, standard homeostatic rules are only stable in a narrow parameter regime. In contrast, we show that a family of biologically plausible learning rules based on "cross-homeostatic" plasticity robustly lead to the emergence of self-sustained, inhibition-stabilized dynamics.
Recruitment of control and representational components of the semantic system during successful and unsuccessful access to complex factual knowledge Our ability to effectively retrieve complex semantic knowledge meaningfully impacts our daily lives, yet the neural processes that underly successful access and transient failures in access remain only partially understood. In this fMRI study, we contrast activation during successful semantic access, unsuccessful semantic access due to transient access-failures (i.e.,  tip-of-the-tongue,  feeling-of-knowing), and trials where the semantic knowledge was not possessed. Twenty-four human participants (14 female) were presented 240 trivia-based questions relating to person, place, object or scholastic knowledge-domains. Analyses of the recall event indicated a relatively greater role of a dorsomedial section of the prefrontal cortex in unsuccessful semantic access and relatively greater recruitment of the pars orbitalis of the inferior frontal gyrus in successful access. Successful access was also associated with increased activation in knowledge-domain selective areas. Generally, knowledge-domain selective areas showed increased responses for both preferred and non-preferred stimulus classes. The exception was place-selective regions (PPA, TOS and RSC), which were recruited during unsuccessful access attempts for all stimulus domains. Collectively, these results suggest that prefrontal semantic control systems and classical spatial-knowledge selective regions work together to locate relevant information and that access to complex knowledge results in a broad activation of semantic representation extending to regions selective for other knowledge domains.
Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Ultrahigh-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultrahigh-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 M 1-hydroxymidazolam and 12 M dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with ~30s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115,200 samples from 300 microtiter plate reads consecutively over 5.54 hours with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high throughput compound screening.

For Table of Contents Only

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/465730v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@5f916corg.highwire.dtl.DTLVardef@4473b4org.highwire.dtl.DTLVardef@82e8forg.highwire.dtl.DTLVardef@128b57b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Redefining colorectal cancer classification and clinical stratification through a single-cell atlas Colorectal cancer (CRC), a disease of high incidence and mortality, has had few treatment advances owing to a large degree of inter- and intratumoral heterogeneity. Attempts to classify subtypes of colorectal cancer to develop treatment strategies has been attempted by Consensus Molecular Subtypes (CMS) classification. However, the cellular etiology of CMS classification is incompletely understood and controversial. Here, we generated and analyzed a single-cell transcriptome atlas of 49,859 CRC cells from 16 patients, validated with an additional 31,383 cells from an independent CRC patient cohort. We describe subclonal transcriptomic heterogeneity of CRC tumor epithelial cells, as well as discrete stromal populations of cancer-associated fibroblasts (CAFs). Within CRC CAFs, we identify the transcriptional signature of specific subtypes (CAF-S1 and CAF-S4) in more than 1,500 CRC patients using bulk transcriptomic data that significantly stratifies overall survival in multiple independent cohorts. We also uncovered two CAF-S1 subpopulations, ecm-myCAF and TGF{beta}-myCAF, known to be associated with primary resistance to immunotherapies. We demonstrate that scRNA analysis of malignant, stromal, and immune cells exhibit a more complex picture than portrayed by bulk transcriptomic-based Consensus Molecular Subtypes (CMS) classification. By demonstrating an abundant degree of heterogeneity amongst these cell types, our work shows that CRC is best represented in a transcriptomic continuum crossing traditional classification systems boundaries. Overall, this CRC cell map provides a framework to re-evaluate CRC tumor biology with implications for clinical trial design and therapeutic development. SARS-CoV-2 B.1.617.2 Delta variant replication, sensitivity to neutralising antibodies and vaccine breakthrough The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD), in particular to the clinically approved bamlavinimab and imdevimab monoclonal antibodies. B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems as compared to B.1.1.7, associated with B.1.617.2 spike being in a predominantly cleaved state compared to B.1.1.7. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed substantially reduced ChAdOx-1 vaccine efficacy against B.1.617.2 relative to non-B.1.617.2. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era. The perceptual relevance of the speech envelope-following response in normal hearing and age-related hearing impaired listeners The aim of this work was to investigate the perceptual relevance of the frequency following response to the syllable /da/ for speech intelligibility in noise based on age and hearing deficits. Recordings of the auditory evoked potential from young normal hearing (NH) and older individuals with both normal hearing and high-frequency (HF) hearing loss were analyzed. EFR metrics obtained in quiet and noise condition were calculated and correlated with speech reception. The envelope following responses were analyzed in terms of amplitude, latency and noise robustness. The response was first simulated to form predictions on the effect of cochlear synaptopathy and outer hair cell loss on the EFR. The experimental findings were in line with the computational predictions in the found observation that the EFR was reduced as a consequence of ageing and HF hearing loss. Both the audiogram and the speech EFR magnitude fell short in the individual prediction of SRT in stationary noise, but they accounted well for group performance. We also obtained within-group EFR latency with a cross covariance matrix. Validation of the method confirmed that speech EFR latency was predictive of click ABR Wave V peak latency. Moreover, statistical analysis not only showed that the robustness of the EFR obtained in the noise condition was dependent on the degree of high-frequency hearing loss in the older NH adults, but also dependent on the EFR magnitude in the NH younger adults. These findings provide evidence towards the important role of the EFR in speech-in-noise perception. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Mapping densely packed GPIIb/IIIa receptors in murine blood platelets with expansion microscopy Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed GPIIb/IIIa receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of GPIIb/IIIa clusters in resting platelets, which are not affected by platelet activation indicating that they contribute to the rapid platelet response during platelet clotting.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/431449v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@c4e38forg.highwire.dtl.DTLVardef@37c1adorg.highwire.dtl.DTLVardef@8c8597org.highwire.dtl.DTLVardef@196bd69_HPS_FORMAT_FIGEXP  M_FIG C_FIG Navigating the amino acid sequence space between functional proteins using a deep learning framework MotivationShedding light on the relationships between protein se-quences and functions is a challenging task with many implications in protein evolution, diseases understanding, and protein design. Protein sequence / function space is however hard to comprehend due to its com-plexity. Generative models help to decipher complex systems thanks to their abilities to learn and recreate data specificity. Applied to protein sequences, they can point out relationships between protein positions and functions capture the sequence patterns associated with functions or navigate through uncharted area of molecular evolution.

ResultsIn this study, an unsupervised generative approach based on adversarial auto-encoder (AAE) is proposed to generate and explore new sequences with respect to their functions thanks to the prior distribution allowing a continuous exploration of the latent space. AAEs are tested on three protein families known for their multiple functions. Clustering re-sults on the encoded sequences from the latent space computed by AAEs display high level of homogeneity regarding the protein sequence func-tions. The study also reports and analyzes for the first time two sampling strategies based on latent space interpolation and latent space arithmetic to generate intermediate protein sequences sharing sequential and functional properties of original sequences issued from different families and functions. Generated sequences by interpolation between latent space data points demonstrate the ability of the AAE to generalize and to pro-duce meaningful biological sequences from an evolutionary uncharted area of the biological sequence space. Finally, 3D structure models generated by comparative modelling between different combinations of structures of different sub-families and of generated sequences from latent space or sub-family sequences point out to the ability of the latent space arithmetic to successfully transfer functional properties between sub-families. All in all this study confirms the ability of deep learning frameworks to model biological complexity and bring new tools to explore amino acid sequence and functional spaces.

AvailabilityCode and data used for this study are freely available at https://github.com/T-B-F/aae4seq.

Contacttristan@bitardfeildel.fr

Supplementary informationSupplementary data are available at online. A human antibody reveals a conserved site on beta-coronavirus spike proteins and confers protection against SARS-CoV-2 infection Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses ({beta}-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 [A] resolution and found that the peptide adopted a mainly helical structure. Conserved residues in {beta}-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on {beta}-CoV spike proteins for protective antibodies that may facilitate the development of pan-{beta}-CoV vaccines.

SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-{beta}-CoV vaccine design strategies Temperature Increase Drives Critical Slowing Down of Fish Ecosystems Fish ecosystems perform ecological functions that are critically important for the sustainability of marine ecosystems, such as global food security and carbon stock. During the 21st century, significant global warming caused by climate change has created pressing challenges for fish ecosystems that threaten species existence and global ecosystem health. Here, we study a coastal fish community in Maizuru Bay, Japan, and investigate the relationships between fluctuations of ST, abundance-based species interactions and salient fish biodiversity. Observations show that a local 20% increase in temperature from 2002 to 2014 underpins a long-term reduction in fish diversity ([~]25%) played out by some native and invasive species (e.g. Chinese wrasse) becoming exceedingly abundant; this causes a large decay in commercially valuable species (e.g. Japanese anchovy) coupled to an increase in ecological productivity. The fish community is analyzed considering five temperature ranges to understand its atemporal seasonal sensitivity to ST changes, and long-term trends. An optimal information flow model is used to reconstruct species interaction networks that emerge as topologically different for distinct temperature ranges and species dynamics. Networks for low temperatures are more scale-free compared to ones for intermediate (15-20{degrees}C) temperatures in which the fish ecosystem experiences a first-order phase transition in interactions from locally stable to metastable and globally unstable for high temperatures states as suggested by abundance-spectrum transitions. The dynamic dominant eigenvalue of species interactions shows increasing instability for competitive species (spiking in summer due to intermediate-season critical transitions) leading to enhanced community variability and critical slowing down despite higher time-point resilience. Native competitive species whose abundance is distributed more exponentially have the highest total directed interactions and are keystone species (e.g. Wrasse and Horse mackerel) for the most salient links with cooperative decaying species. Competitive species, with higher eco-climatic memory and synchronization, are the most affected by temperature and play an important role in maintaining fish ecosystem stability via multitrophic cascades (via cooperative-competitive species imbalance), and as bioindicators of change. More climate-fitted species follow temperature increase causing larger divergence divergence between competitive and cooperative species. Decreasing dominant eigenvalues and lower relative network optimality for warmer oceans indicate fishery more attracted toward persistent oscillatory states, yet unpredictable, with lower cooperation, diversity and fish stock despite the increase in community abundance due to non-commercial and venomous species. We emphasize how changes in species interaction organization, primarily affected by temperature fluctuations, are the backbone of biodiversity dynamics and yet for functional diversity in contrast to taxonomic richness. Abundance and richness manifest gradual shifts while interactions show sudden shift. The work provides data-driven tools for analyzing and monitoring fish ecosystems under the pressure of global warming or other stressors. Abundance and interaction patterns derived by network-based analyses proved useful to assess ecosystem susceptibility and effective change, and formulate predictive dynamic information for science-based fishery policy aimed to maintain marine ecosystems stable and sustainable. Prediction of the sex-determination gene in tunas (Thunnus fishes) Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex-determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies, and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex-determination gene in Thunnus fishes, or at least functions at a crucial point in the sex-differentiation cascade. Precartilage condensation during limb skeletogenesis occurs by tissue phase separation controlled by a bistable cell-state switch with suppressed oscillatory dynamics The tetrapod limb skeleton is initiated in unpatterned limb bud mesenchyme by the formation of precartilage condensations. Here, based on time-lapse videographic analysis of a forming condensation in a high-density culture of chicken limb bud mesenchyme, we observe a phase transition to a more fluidized state for cells within spatial compacted foci (protocondensations that will progress to condensations), as reflected in their spatial confinement, cell-substratum interaction and speed of motion. Previous work showed that galectin-8 and galectin-1A, two proteins of the galactoside-binding galectin family, are the earliest determinants of this process in the chicken limb bud, and that their interactions in forming skeletogenic patterns of condensations can be interpreted mathematically through a reaction-diffusion-adhesion framework. Based on this framework, we use an ordinary differential equation-based approach to analyze the core switching modality of the galectin reaction network and characterize the states of the network independent of the diffusive and adhesive arms of the patterning mechanism. We identify two steady states where the concentrations of both galectins are respectively, negligible, and very high. An explicit Lyapunov function shows that there are no periodic solutions. For sigmoidal galectin production terms, the model exhibits a bistable switch that arises from a monostable state via saddle-node bifurcation. Our model therefore predicts that the galectin network exists in low and high expression states separated in space or time without any intermediate states. This provides a causal basis for the observed outside vs. inside transition observed in the in vitro video data. We performed a quantitative analysis of the distribution of galectin-1A in cultures of condensing chick limb mesenchymal cells and found that the interior of the protocondensations had concentrations of this protein (compared to the immediate exterior) over and above that expected from its higher cell density, consistent with the models predictions. The galectin-based patterning network is thus suggested, on theoretical grounds, to incorporate a core switch independent of any spatial or temporal dynamics, that drives the chondrogenic cell state transition in limb skeletogenesis. Natural selection promotes the evolution of recombination 2: during the process of natural selection The ubiquity of sex and recombination in nature has eluded unified explanation since the time of Darwin. Conditions that promote the evolution of recombination, broadly defined as any form of genetic mixing, are fairly well understood: it is favored when genomes tend to contain more selectively mismatched combinations of alleles than can be explained by chance alone. Yet, while a variety of theoretical approaches have been put forth to explain why such conditions would prevail in natural populations, each has turned out to be of limited scope and applicability. Here, we show, simply and surprisingly, that natural selection acting on standing heritable variation always creates conditions favoring the evolution of recombination, in expectation. Specifically, we find that, in expectation: 1) the mean selective advantage of recombinants is non-negative, 2) the mean selective advantage of a recombination-competent modifier is non-negative, and 3) the asymptotic frequency of a recombination-competent modifier is close to one and is independent of the strength of selection. Remarkably, these findings are independent of the distribution of genic fitnesses in the standing heritable variation upon which natural selection acts, implying that the source of this variation is immaterial. Taken together, our findings indicate that: 1) the evolution of recombination should be promoted in expectation wherever natural selection is operating, and 2) sex and recombination may have evolved more as a byproduct than as a catalyst of natural selection. Haplotype-resolved powdery mildew resistance loci reveal the impact of heterozygous structural variation on NLR genes in Muscadinia rotundifolia Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries two powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. In this study, we identified, phased, and reconstructed the two haplotypes of each resistance-associated locus. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat (NLR) genes. Structural variations also impacted the domain composition of some NLRs. These findings emphasize the need of generating haplotype-resolved sequences instead of using consensus sequences for identifying haplotype-specific candidate genes. Comparison of the NLRs in the Run1.2 and Run2.2 loci indicated that the two loci are composed of a different number and classes of NLR genes. We provide a list of candidate NLR genes from the Run1.2b and Run2.2 loci, whose expression suggests a role in powdery mildew resistance in Trayshed. These first complete and haplotype-resolved resistance-associated loci, and their candidate NLR genes, represent new resources to develop powdery mildew-resistant grape cultivars. CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7 V-ATPases are rotary proton pumps that serve as signaling hubs with numerous proposed binding partners in cells. We used cryoEM combined with exhaustive focused classification to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 shows that mEAK-7s TLDc domain interacts with V-ATPases stator while its C-terminal  helix binds V-ATPases rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike inhibition of yeast V-ATPase by the TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK-7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. Together, these results show that differences in V-ATPase binding by TLDc domain-containing proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzymes activity. Mouse lemur, a new primate model to investigate cardiac pacemaker activity. BackgroundGray mouse lemurs (Microcebus murinus) are one of the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since Microcebus murinus replicates similar senescence processes of humans, it constitutes a useful model also for studying cardiovascular dysfunctions. However, its cardiac physiology is unknown. Thus, we investigated sinoatrial node (SAN) pacemaker activity of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.

Methods and ResultsWe recorded the heart automaticity in freely moving mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of electrical impulses in the SAN tissue. Conversely, their ventricular depolarization is more similar to large mammals. Mouse lemur SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (ICa,L) and T-type (ICa,T) calcium currents. Moreover, mouse lemur SAN myocytes display spontaneous ryanodine receptor (RyR)-dependent Ca2+ transients. Using muscle-derived stem cells (MDSCs) from the Microcebus murinus skeletal muscle, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).

ConclusionsIn conclusion, our characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans, and for pharmacologic testing of molecules that could assist in modulating age-related dysfunctions of automaticity. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. EGFR inhibition leads to enhanced desmosome assembly and cohesion in cardiomyocytes The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase, which is dysregulated in several kinds of cancer and heart diseases. Arrhythmogenic cardiomyopathy (AC) is a familial heart disease in part caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity, may provide potential new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of EGFR inhibition on cardiomyocyte cohesion under physiological and pathophysiological conditions using the murine plakoglobin knockout AC model, in which EGFR was upregulated. EGFR inhibition led to enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and AFM revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. Thus, inhibiting EGFR, thereby stabilizing desmosome integrity, might provide new treatment options for AC.

SummaryShoykhet et al. show EGFR inhibition-enhanced cardiomyocyte cohesion. This positive adhesiotropy is achieved via an increase in desmosomal assembly and DP/DSG2 localization at cell borders and is the first step towards a novel therapeutic approach for arrhythmogenic cardiomyopathy. Determinants of Murine Skin Microbiota Composition in Homeostasis and Wound Healing Animal microbiota have complex interactions with hosts and environment that determines its composition. Yet the ability of hosts to determine their microbiota composition is less well studied. In this study, to investigate the role host genetics in determining skin microbiota, we used 30 different mouse strains from the recombinant inbred panel, the Collaborative Cross. Murine skin microbiota composition was strongly dependent on murine strain with > 50% of the variation explained by murine strain. In particular, a quantitative trait locus on chromosome 4 associates both with Staphylococcus abundance and principal-component multi-trait analyses. Additionally, excisional wound associated changes in microbiota composition were not uniform across mouse strains and were host-specific, the genetic background accounting for about 40% of the variation in microbiota. Genetic background also had the highest effect on the healing speed of wounds accounting for over 50% of the variation while mouse age and microbiota composition change accounted only for 20% and 5% of the healing speed despite reaching statistical significance. In conclusion, host genetics has a significant impact on the skin microbiota composition during both homeostasis and wound healing. These findings have long reaching implications in our understanding of associations between microbiota dysbiosis and disease. The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition Although light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition, the process of light-induced photodamage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, that thrives under extreme high light and is highly resistant to photoinhibition. Therefore, C. ohadii is an ideal model for studying the molecular mechanisms underlying protection against photoinhibition. Comparison of the thylakoids of C. ohadii cells that were grown under low light versus extreme high light intensities, found that the alga employs all three known photoinhibition protection mechanisms: i) massive reduction of the PSII antenna size; ii) accumulation of protective carotenoids; and iii) very rapid repair of photo-damaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms and shows how photoinhibition protection mechanisms evolved to marginal conditions, enabling photosynthesis-dependent life in severe habitats.

One Sentence HighlightAnalysis of the photosynthetic properties of a desert algae that thrives at extreme high light intensities revealed protection from photoinhibition driven by the remarkable enhancement of three protection mechanisms. The Hippocampus uses cryptographic principles rather than plasticity to enable animals to secretly cache and retrieve their food For animals, the ability to hide and retrieve valuable information, such as the location of food, can mean the difference between life and death. Here, we propose that to achieve this, their brain uses spatial cells similarly to how we utilize encryption for data security. Some animals are able to cache hundreds of thousands of food items annually by each individual and later retrieve most of what they themselves stashed. Rather than memorizing their cache locations as previously suggested, we propose that they use a single cryptographic-like mechanism during both caching and retrieval. The model we developed is based on hippocampal spatial cells, which respond to an animals positional attention, such as when the animal enters a specific region (place-cells) or gazes at a particular location (spatial-view-cells). We know that the region that activates each spatial cell remains consistent across subsequent visits to the same area but not between areas. This remapping, combined with the uniqueness of cognitive maps, produces a persistent crypto-hash function for both food caching and retrieval. We also show that the model stores temporal information that helps animals in food caching order preference as previously observed. This behavior, which we refer to as crypto-taxis, might also explain consistent differences in decision-making when animals are faced with a large number of alternatives such as in foraging.