title: Oncogene-induced matrix reorganization controls CD8+ T cell function in the tumor microenvironment

abstract: CD8+ T cell dysfunction is a critical barrier to anti-tumor immunity, but molecular mechanisms underlying the regulation of T cell dysfunction in solid tumors are diverse and complex. Extracellular matrix (ECM) composition facilitates solid tumor progression in part by inhibiting T cell migration/infiltration; however, the impact of individual ECM molecules on T cell function in the tumor microenvironment (TME) is virtually unknown. Moreover, upstream regulators of aberrant ECM deposition/organization in solid tumors are poorly defined. Therefore, we investigated the regulation and effects of ECM composition on CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS). This immunologically "hot" soft-tissue sarcoma exhibits durable responses to checkpoint therapy in some human patients, suggesting it may provide insights into strategies for optimizing T cell function and improving immunotherapy efficacy. Using an autochthonous model of UPS and data from multiple human patient cohorts, we discovered a multi-pronged mechanism wherein oncogene-induced remodeling of the TME promotes CD8+ T cell dysfunction, suppresses T cell-mediated cytolysis, and enhances immune evasion. Specifically, we observed that the transcriptional co-activator Yap1, which we previously linked to UPS progression, promotes the aberrant deposition of collagen VI in the UPS TME. In turn, collagen VI induces CD8+ T cell dysfunction by inhibiting T cell autophagic flux and remodeling fibrillar collagen architecture in the TME. Furthermore, collagen type I opposed ColVI in this setting, acting as tumor suppressor. Thus, our findings reveal that CD8+ T cell-mediated anti-tumor immunity in solid cancers is dependent upon oncogene-mediated ECM composition and remodeling in the TME.

category: cancer biology


title: Protein Kinase C Delta Regulates Mononuclear Phagocytes and Hinders Response to Immunotherapy in Cancer

abstract: Checkpoint immunotherapy unleashes T cell antitumor potential which has revolutionized cancer treatment showing unprecedented long-term responses. However, most patients do not respond to immunotherapy which often correlates with a dysfunctional or immunosuppressive myeloid compartment. The mononuclear phagocyte system (MPS) is a sub-class of myeloid cells comprising monocytes, macrophages and dendritic cells which plays a crucial role in tissue homeostasis. However, accumulating evidence suggests that mononuclear phagocytes contribute to all phases of tumorigenesis including orchestrating inflammatory events during de novo carcinogenesis, contribution to the progression of established tumors and promotion of resistance to checkpoint blockade. Thus, targeting the MPS could be an effective strategy to enhance checkpoint blockade efficacy and promote control of tumors. Here, we found that protein kinase C delta (PKC{delta}), a serine/threonine kinase, is abundantly expressed by mononuclear phagocytes in several human and mouse tumors. PKC{delta}-/- mice were more resistant to growth of various cancers compared to wild-type mice and were more responsive to anti-PD-1 immunotherapy. Furthermore, we found that tumors from PKC{delta}-/- mice harbor a Th-1-skewed immune landscape including increased antigen cross-presentation and T cell activation. Depletion of mononuclear phagocytes in vivo altered tumor growth in wild-type mice, but not in PKC{delta}-/- mice. In addition, coinjection of PKC{delta}-/--deficient M2-like macrophages with cancer cells into wild-type mice markedly delayed tumor growth and significantly increased intratumoral T cell activation compared to wild-type M2-like macrophages coinjected with cancer cells. Finally, intrinsic loss of PKC{delta}-/- functionally reprogrammed macrophages and dendritic cells by promoting their antigen presenting and cross-presenting capacity and triggered type I and type II interferon signaling. Thus, PKC{delta} might be targeted to reprogram mononuclear phagocytes and augment checkpoint blockade efficacy.

category: immunology


title: Sialophorin is an essential host element for vaccine immunity against pulmonary fungal infections

abstract:
The global burden of fungal infections is alarming, primarily due to the increasing immune-compromised population. The immuno-preventive/therapeutic measures, including vaccines, are necessary to prevent or control fungal diseases. Identifying a protective host element as a functional phenotypic marker is immensely valuable. We identified a host element, sialophorin, preferentially associated with antifungal memory T cells. We investigated its role in vaccine immunity using a mouse model of pulmonary fungal infection. We found that sialophorin was essential to bolster CD8+ T-cell responses to the vaccine by enhancing their differentiation and expanding cytokine-producing cells required for immunity. Using a gain-of-function approach, activating sialophorin using mAb augmented the CD8+ T cell responses, and sialophorin-sufficient CD8+ T cells were competitively superior in differentiation and expansion to the deficient cells. Sialophorin-mediated vaccine immunity was independent of the T cell trafficking effect. Finally, we show that sialophorin is a potential functional phenotypic marker of fungal vaccine-potency and immunity. Our study revealed that sialophorin is an essential host-target element to bolster vaccine responses and serves as a potential biomarker of fungal immunity.

Author SummaryFungal infections have been rising in recent years due to increased immunocompromised individuals. Vaccination of at-risk individuals helps counter the infections. Thus, suitable vaccine platforms are needed with apt adjuvants, and a phenotypic marker of vaccine immunity will bolster the efforts. We identified a phenotypic marker, sialophorin, associated with T cell vaccine immunity to fungal infection. Our findings show an essential role of sialophorin for fungal immunity, as a target of adjuvanticity, and as a potential biomarker of vaccine immunity against many fungal infections.


title: A ROS-dependent mechanism to drive progression through S phase

abstract: Long considered as cytotoxic reagents, reactive oxygen species (ROS) at the right concentration promote cell proliferation in cell culture, stem cells and model organisms. However, how ROS signaling is coordinated with cell cycle progression and integrated into the cell cycle control machinery on the molecular level remains unsolved. Here, we report oscillations of mitochondrial ROS during the cell cycle that target cyclin-dependent kinase 2 (CDK2). Chemical and metabolic interference with ROS production decrease T-loop phosphorylation on CDK2, impeding its full activation and thus efficient DNA replication. ROS regulate CDK2 activity through oxidation of a conserved cysteine residue in close proximity to the T-loop, which prevents binding of the T-loop phosphatase KAP. Together our data reveal how ROS couple mitochondrial metabolism to DNA replication and cell cycle progression, and provide a solution to the longstanding conundrum of how KAP activity towards CDKs can be cell cycle-regulated.

category: cell biology


title: Understanding the transcriptional response to ER stress in Chinese hamster ovary cells using multiplexed single cell RNA-seq

abstract:
Single cell RNA-seq (scRNA-seq) has recently been shown to provide a powerful method for the analysis of transcriptional heterogeneity in Chinese hamster ovary (CHO) cells. A potential drawback of current scRNA-seq platforms is that the cost can limit the complexity of experimental design and therefore the utility of the approach. In this manuscript, we report the use of oligonucleotide barcoding to perform multiplexed CHO cell scRNA-seq to study the impact of tunicamycin (TM), an inducer of the unfolded protein response (UPR). For this experiment, we treated a CHO-K1 GS cell line with 10g/ml tunicamycin and acquired samples at 1, 2, 4 and 8 hr post-treatment as well as a non-treated TM-control. We transfected cells with sample-specific polyadenylated ssDNA oligonucleotide barcodes enabling us to pool all cells for scRNA-seq. The sample from which each cell originated was subsequently determined by the oligonucleotide barcode sequence. Visualisation of the transcriptome data in a reduced dimensional space confirmed that cells were not only separable by sample but were also distributed according to time post-treatment. These data were subsequently utilised to perform weighted gene co-expression analysis (WGCNA) and uncovered groups of genes associated with TM treatment. For example, the expression of one group of coexpressed genes was found to increase over the time course and were enriched for biological processes associated with ER stress. The use of multiplexed single cell RNA-seq has the potential to reduce the cost associated with higher sample numbers and avoid batch effects for future studies of CHO cell biology.

HighlightsO_LIPolyadenylated ssDNA oligonucleotide labelling is a viable strategy for multiplexed CHO cell scRNA-seq analysis.
C_LIO_LITo demonstrate the effectiveness of the method we conducted an experiment to study the CHO cell response to tunicamycin treatment.
C_LIO_LIscRNA-seq was carried out on an untreated control and at 4 time points post tunicamycin treatment. Cells from each sample were transfected with a unique oligonucleotide barcode and pooled for single cell transcriptomics.
C_LIO_LIEach sample was demultiplexed post-sequencing and gene expression profiles of > 5,300 cells were obtained across the experiment. Following dimensionality reduction and visualisation, the cells were distributed according to sample identity.
C_LIO_LIAnalysis of the resulting data enabled improved understanding of the transcriptional response to tunicamycin treatment. Three gene coexpression modules were found to be correlated with the tunicamycin time course. Gene set enrichment analysis revealed the over representation of genes related to biological processes associated with ER stress, and protein misfolding in one of these groups of coexpressed genes.
C_LIO_LIFurther use of this approach will enable the CHO cell biology community to perform increasingly complex single cell experiments in a cost-effective manner.
C_LI


title: AMPK-p38 axis converts human pluripotent stem cells to naïve state

abstract: Pluripotent stem cells (PSCs) have been reported to exhibit two stages of pluripotency,  primed and  naive states. Typical human PSCs (hPSCs) are in the primed state. Though several methods for conversion from primed to naive state have been reported, the mechanism of the process is not fully understood. Here, we report that 5 adenosine monophosphate-activated protein kinase (AMPK) and its downstream p38 is a signaling axis that can induce the naive conversion of hPSCs with single pathway activation. The simple addition of an AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR), or overexpression of a constitutive active form of p38 (CA-p38) alone in primed hPSCs induced naive hPSCs that satisfied naive state criteria: differentiation ability to three germ layers and naive state-specific transcriptional expression, epigenomic resetting, and mitochondrial activity. RNA-seq analysis demonstrated that our AICAR- or CA-p38-induced naive hPSCs show closely similar gene expression patterns to naive state human embryonic stem cells (HNES1) derived from human inner cell mass (ICM). This novel and simple naive conversion method provides new avenues for understanding and elucidating the fundamental mechanism of naive conversion.


title: Early Endosome Capture Proteomics and its Application to Amyloid Precursor Protein Intramembrane Processing by β and γ-Secretases

abstract: Degradation and recycling of plasma membrane-localized proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. The endolysosomal system also participates in intracellular membrane protein trafficking, signaling, and protein degradation via autophagy. Our understanding of the endolysosomal system has been significantly advanced through the development of methods for selective capture of lysosomes (termed Lyso-IP), but methods for analogous isolation of early intermediates in the endolysosomal system are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of early endosomes in action. In addition to resident endosomal components, we identify numerous recycling, regulatory and membrane fusion complexes as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes that we distinguished from lysosomes via comparative proteomics. The approach allows capture of internalized transferrin within minutes of its addition to cells, indicating the utility of the approach for dynamic analysis of early/sorting endosomes. Among the endocytic cargo identified by Endo-IP was the amyloid precursor protein (APP) genetically linked with Alzheimers disease. Processing of APP to amyloidogenic A{beta} peptides by {beta}- and {gamma}-Secretases can occur within the endolysosomal system among other compartments, but methods for spatial quantification of A{beta} products in individual organelles are lacking. We combined Endo- and Lyso-IP with targeted proteomics to provide a spatial digital snapshot of A{beta} products. This analysis revealed that products of A{beta} processing by {beta}- and {gamma}-Secretases, and alterations in the specificity of cleavage by small molecule {gamma}-Secretase modulators, can be quantified in both early/sorting endosomes and lysosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of the many processes that are coordinated on early endosomes.


title: The molecular organization of flat and curved caveolae indicates bendable structural units at the plasma membrane.

abstract: Caveolae are small coated inner plasma membrane invaginations found in many cell types. Their diverse functions span from endocytosis to signaling, regulating key cellular processes including lipid uptake, pathogen entry, and membrane tension. Caveolae undergo shape changes from flat to curved. It is unclear which proteins regulate this process. To address this gap, we studied the shapes of caveolae with platinum replica electron microscopy in six common cell types. Next, we developed a correlative multi-color stimulated emission depletion (STED) fluorescence and platinum replica EM imaging (CLEM) method to image caveolae-associated proteins at caveolae of different shapes at the nanoscale. Caveolins and cavins were found at all caveolae, independent of their curvature. EHD2, a classic caveolar neck protein, was strongly detected at both curved and flat caveolae. Both pacsin2 and the regulator EHBP1 were found only at a subset of caveolae. Pacsin2 was localized primarily to areas surrounding flat caveolae, whereas EHBP1 was mostly detected at spheres. Contrary to classic models, dynamin was absent from caveolae and localized only to clathrin-coated structures. Cells lacking dynamin showed no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. Together, we provide a mechanistic map for the molecular control of caveolae shape by eight of the major caveolae-associated coat and regulatory proteins. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by more intermittent associations with pacsin2 and EHBP1. These complexes can flatten and curve, capturing membrane to enable lipid traffic and changes to the surface area of the cell.


title: MicroRNA-eQTLs in the developing human neocortex link miR-4707-3p expression to brain size

abstract: Expression quantitative trait loci (eQTL) data have proven important for linking non-coding loci to protein-coding genes. But eQTL studies rarely measure microRNAs (miRNAs), small noncoding RNA known to play a role in human brain development and neurogenesis. Here, we perform small-RNA sequencing across 212 mid-gestation human neocortical tissue samples, profiled 907 miRNAs, discovering 111 novel early brain-expressed miRNAs, and identified 85 local-miRNA-eQTLs. Colocalization of miRNA-eQTLs with GWAS summary statistics yielded one robust colocalization of miR-4707-3p expression with educational attainment and head size phenotypes, where the miRNA expression increasing allele was associated with decreased head size. Exogenous expression of miR-4707-3p in primary human neural progenitor cells led to increased proliferative and neurogenic gene markers, indicating miR-4707-3p modulates progenitor proliferation. Integrating miRNA-eQTLs with existing GWAS yielded discovery of a miRNA modulating developmental fate decisions that alter human brain size.

category: genetics


title: Prioritization of driver genes in cancer-associated copy number alterations identifies B4GALT5 as a glycooncogene

abstract: Cancer is a disease resulting from aberrant communication between cells of a multicellular organism. The glycan coat that surrounds the cells is an important player in cellular communication. While altered cell surface glycans are known biomarkers for cancer, glycan biosynthesis itself has not been considered a potential oncogenic pathway. So, to understand the oncogenic potential of the glycan biosynthetic pathways we have analyzed the copy number alterations (CNA) of genes encoding for glycosylation regulators (glycogenes) in cancer genome datasets and identify novel glyco-oncogenes and glyco-tumor suppressor genes (TSGs). CNA of oncogenes and TSGs is an important cancer-associated genetic alteration that associates with worst prognosis. Nevertheless, identity of the driver genes in the copy number altered segments of the genome remains obscure in most cases. We developed a prioritization pipeline based on bioinformatic and experimental criteria to identify putative driver genes. In addition to correctly identifying several well-established oncogenes/TSGs, this pipeline discerns several novel oncogenes and TSGs, some of which are glycogenes. Further, among glyco-oncogenes there is an enrichment for glycosphingolipid biosynthetic pathway and trans-Golgi associated lysosomal sorting machinery and among glyco-TSGs there is an enrichment for early N-glycan biosynthetic enzymes. As a proof-of-principle we show that one of the identified glycooncogene B4GALT5, encoding a key enzyme in the glycosphingolipid pathway exhibits oncogenic property of promoting increased growth of hepatocellular carcinoma cells. Thus, this study identifies glycosylation pathways with oncoregulatory properties and opens up a new group of enzymes as potential therapeutic targets for cancer.


title: Timing Is Everything In Autumn - Coordination Of Senescence Onset By A Transcriptional Program In Response To Environmental And Phytohormone Signals

abstract:
European aspen (Populus tremula L.) undergoes a coordinated senescence program during autumn; however, it is not known what exactly triggers it. To identify the cellular program leading to senescence, we utilized natural variation among Swedish aspen genotypes in a common garden to study senescence timing and the underlying changes in leaf phytohormone and transcriptome profiles. Apart from the patterns of major transcriptional cascade that was similar between the genotypes and closely associated with cytokinin and auxin metabolite levels and gradually decreasing air temperature during autumn, we detected patterns that consistently preceded or coincided with senescence onset in individual genotypes. Another cascade seemed to respond to short-term changes in weather conditions that re-wired the transcriptional network; the up-regulation of genes related to ethylene and abiotic stress, programmed cell death and translation occurred first in the early-senescing genotypes and later in the late one. Network analyses displayed a connection between the two cascades, metabolic stress and immunity responses mediated by salicylic acid (SA)-signalling pathway that was repressed along with SA levels at senescence onset. We propose that autumn senescence in aspen trees is affected by environmental variation that evokes stress and the timing is fine-tuned by their stress tolerance mechanism.

One sentence summarySalicylic acid signalling pathway fine-tunes the timing of senescence onset under challenging environmental conditions in autumn.

category: plant biology


title: Prostaglandin D2 pathway in a transgenic rat model of Alzheimer's disease: therapeutic potential of timapiprant a DP2 antagonist

abstract: The cyclooxygenase pathway, a key mediator of inflammation, is implicated in Alzheimers disease (AD). A deeper investigation is required into the contributions of this pathway to the neuropathology of AD. Cyclooxygenases produce prostaglandins, which have multiple receptors and functions including inflammation, nociception, sleep, cardiovascular maintenance and reproduction. In the brain, prostaglandin D2 (PGD2) is the most abundant prostaglandin, increases the most under pathological conditions, and plays roles in sleep, stroke and inflammation. PGD2 signals through its DP1 and DP2 receptors and their activation can be protective or detrimental. We address the relationship between the PGD2 pathway and AD neuropathology with F344-AD transgenic (Tg-AD) rats that exhibit age-dependent and progressive pathology similar to AD patients. We analyzed the PGD2 pathway in the hippocampus of wild type (WT) rats and their Tg-AD littermates, at the age of 11 months, when Tg-AD rats exhibit plaques and perform significantly worse in hippocampal-dependent cognitive tasks than WT rats. Using mass spectrometry, we determined that PGD2 levels were at least 14.5-fold higher than PGE2, independently of genotype. Immunohistochemistry established that microglial DP1 receptors were more abundant and neuronal DP2 receptors were fewer in Tg-AD than in WT rats. RNA sequencing profiling of 33 genes involved in the PGD2 and PGE2 pathways revealed that mRNA levels were the highest for L-PGDS, the major PGD2 synthase in the brain. To evaluate the pathophysiological significance of our findings on the PGD2 pathway, we treated a subset of rats (WT and Tg-AD males) with timapiprant, a potent and highly selective oral DP2 antagonist being developed as a once-daily oral treatment in patients with allergic inflammation. We conclusively show that timapiprant significantly mitigated some of the AD pathology exhibited by the Tg-AD male rats. More comprehensive studies are necessary to support the therapeutic potential of timapiprant and that of other PGD2-related compounds in the treatment of AD.

category: neuroscience


title: Spatial modeling reveals nuclear phosphorylation and subcellular shuttling of YAP upon drug-induced liver injury

abstract:
The Hippo signaling pathway controls cell proliferation and tissue regeneration via its transcriptional effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). In this context, the canonical pathway topology is characterized by sequential phosphorylation of kinases in the cytoplasm that define the subcellular localization of YAP and TAZ. However, the molecular mechanisms controlling the nuclear/cytoplasmic shuttling dynamics of both factors under physiological and tissue-damaging conditions are poorly understood. By implementing experimental data, partial differential equation (PDE) modeling, as well as automated image analysis, we demonstrate that nuclear phosphorylation contributes to differences between YAP and TAZ localization in the nucleus and cytoplasm. Treatment of hepatocyte-derived cells with hepatotoxic acetaminophen (APAP) overdose induces a biphasic protein phosphorylation eventually leading to nuclear protein enrichment of YAP but not TAZ. APAP-dependent regulation of nuclear/cytoplasmic YAP shuttling is not an unspecific cellular response but relies on the sequential induction of reactive oxygen species (ROS), RAC-alpha serine/threonine-protein kinase (AKT, synonym: protein kinase B), as well as elevated nuclear interaction between YAP and AKT. Mouse experiments confirm this consecutive sequence of events illustrated by the expression of ROS-, AKT-, and YAP-specific gene signatures upon APAP administration. In summary, our data illustrate the importance of nuclear processes in the regulation of Hippo pathway activity. YAP and TAZ exhibit different shuttling dynamics, which explains distinct cellular responses of both factors under physiological and tissue-damaging conditions.

SignificanceWe show that canonical view on the Hippo pathway must be extended by additional regulatory processes in cell nuclei. These processes significantly contribute to the activity of YAP and TAZ under unchallenged conditions (e.g., with cell density as physiological regulator of the Hippo kinase cassette) or under cell damaging conditions (e.g., after administration of APAP overdose). APAP-induced cellular damage activates YAP via distinct molecular processes as part of a cell-protective response.

category: molecular biology


title: Protons in gating the Kv1,2 channel: a calculated conformational change in response to addition of a proton, and a proposed path from voltage sensing domain to gate

abstract: We have in the past proposed that protons constitute the gating current in the potassium channel Kv1.2. Here we present a quantum calculation of a protonation change in a 311 atom section of intracellular S4-S5 linker, together with part of the T1 intracellular moiety of the channel. This proton shift leads to a hinge rotation in the linker, which in turn produces a separation of two amino acids, K312 and R326 (using the numbering of the 3Lut pdb structure). Two complete proton wires can then be proposed that would fully account for the gating mechanism with protons; the proton wires have as yet not been completely calculated. However, the path seems reasonably evident, based on the amino acids in the S4-S5 linker, which connects to the pore transmembrane S6 segment as well, and the T1 moiety of the channel, which is part of one proton path. This therefore also accounts for the T1 effect on gating. We had earlier shown how a proton could be generated from the VSD. Taken together the paths from the VSD to the gate show how the VSD can couple to the gating mechanism by having protons move between the VSD and the gate, closing the channel by both producing the hinge rotation and providing electrostatic repulsion to an incoming K+ ion. The protons move under the influence of membrane polarization/depolarization. Taken together, this makes our previous model much more detailed, specifying the role of particular amino acids.

category: biophysics


title: PIEZO1-dependent erythrocyte dehydration as the mechanism for selection of an allele protecting from severe malaria.

abstract:
PIEZO1 is a cation specific mechanoreceptor channel implicated in red blood cell (RBC) volume homeostasis. Several PIEZO1 gain of function (GoF) variants demonstrate delayed channel inactivation and can cause hereditary xerocytosis (HX), a disease characterized by hemolytic anemia, RBC dehydration, and shape distortion. The milder PIEZO1E756del GoF variant, prevalent in populations of African descent, protects carriers from severe malaria caused by Plasmodium falciparum and ameliorate disease in a rodent malaria model. To explore the mechanism of this malaria protection, P. falciparum infection of human PIEZO1E756del RBC was analyzed in shear-stressed and static cultures with and without Yoda1, a PIEZO1 agonist. RBC dehydration was a common pathophysiological factor affecting parasite replication in both culture conditions. PIEZO1 channel opening by either Yoda1 or shear stress produced dehydration-dependent cell hemolysis, inhibiting P. falciparum infection. Since the physiological activator of PIEZO1 in circulating RBC is shear stress, we propose that shear stress-induced dehydration, disproportionally affecting RBC of GoF PIEZO1 E756del carriers, makes erythrocytes less habitable for P. falciparum to the point of hemolysis, and thus ameliorates malaria in GoF PIEZO1E756del carriers. More generally, RBC dehydration processes may be a pathway for protection from the severe form of malaria common to several hematological disorders, including sickle cell trait.

Key pointsO_LIPIEZO1E756del activation in African American donor RBC provokes dehydration-dependent cell hemolysis, impairing P. falciparum replication.
C_LIO_LIRBC dehydration could be a malaria ameliorating factor in several known RBC hematological disorders, including sickle cell trait.
C_LI


title: mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM

abstract:
Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state.

TOC GRAPHICS O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: One-sided design of protein-protein interaction motifs using deep learning

abstract:
Protein-protein interactions are part of most processes in life and thereby the ability to generate new ones to either control, detect or inhibit them has universal applications. However, to develop a new binding protein to bind to a specific site at atomic detail without any additional input is a challenging problem. After DeepMind entered the protein folding field, we have seen rapid advances in protein structure predictions thanks to the implementation of machine learning algorithms. Neural networks are part of machine learning and they can learn the regularities from their input data. Here, we took advantage of their capabilities by training multiple neural networks on co-crystal structures of natural protein complexes. Inspired by image caption algorithms, we developed an extensive set of NN-based models, referred to as iNNterfaceDesign. It predicts the positioning and the secondary structure for the new binding motifs and then designs the backbone atoms followed by amino acid sequence design. Our methods are capable of recapitulating native interactions, including antibody-antigen interactions, while they also capable to produce more diverse solutions to binding at the same sites. As it was trained on natural complexes, it learned their features and can therefore also highlight preferential binding sites, as found in natural protein-protein interactions. Our method is generally applicable, and we believe that this is the first deep learning model for one-sided design of protein-protein interactions.

Abstract figure

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category: bioinformatics


title: Computational design of ultrasensitive flexible peptide:receptor signaling complexes for enhanced chemotaxis

abstract: Engineering biosensors that sensitively recognize specific biomolecules and trigger functional cellular responses is a holy grail of diagnostics and synthetic cell biology. Biosensor design approaches have mostly focused on binding structurally well-defined molecules. Coupling the sensing of flexible compounds to complex cellular responses would considerably expand biosensor applications, but remains challenging. We developed a computational strategy for designing highly sensitive receptor biosensors of flexible peptides. Using the method, we created ultrasensitive chemotactic GPCR:peptide pairs capable of eliciting potent chemotaxis in human primary T cells. Through mutual induced fit, our flexible structure design approach enhanced peptide contacts with binding and allosteric sites in the ligand pocket to achieve unprecedented signaling efficacy and potency. The approach paves the road for designing peptide-sensing receptors and expanding the biosensor toolbox for basic and therapeutic applications.


title: High temperature molecular motions within a model protomembrane architecture

abstract: Modern phospholipid membranes are known to be in a functional, physiological state, corresponding to the liquid crystalline phase, only under very precise external conditions. The phase is characterised by specific lipid motions, which seem mandatory to permit sufficient flexibility and stability for the membrane. It can be assumed that similar principles hold for proto-membranes at the origin of life although they were likely composed of simpler, single chain fatty acids and alcohols. In the present study we investigated molecular motions of four types of model membranes to shed light on the variations of dynamics and structure as a function of temperature as protocells might have existed close to hot vents. We find a clear hierarchy among the flexibilities of the samples, where some structural parameters seem to depend on the lipids used while others do not.


title: Hand-foot coordination is significantly influenced by motion direction in individuals with autism spectrum disorder

abstract:
Abstract and keywordsGenerally, when individuals attempt to move two limbs rhythmically in the opposite direction (e.g., flex the left hand and extend the left foot along the sagittal plane), the movements tend to be instead performed in the same direction. This phenomenon, known as directional constraint, can be harnessed to examine the difficulties in movement coordination exhibited by most individuals with autism spectrum disorder (ASD). While such difficulties have already been investigated through standardized clinical assessments, they have not been examined through kinematic methods. Thus, we employed a clinical assessment scale in an experimentally controlled environment to investigate whether stronger directional constraint during the rhythmic movement of two limbs is more pronounced and associated with decreased movement coordination in individuals with ASD. ASD and typically developing (TD) participants were asked to rhythmically move two limbs either in the same or opposite directions. In addition, the coordination skills of participants were assessed using the Bruininks-Oseretsky Test of Motor Proficiency Second Edition (BOT-2). Subjects with ASD showed significantly stronger directional constraint than TD participants during the contralateral and ipsilateral movement of the hand and foot. According to the pooled data from both groups, participants who showed stronger directional constraint during these two movement conditions also exhibited poorer coordinated movement skills in the BOT-2. Furthermore, stronger directional constraint was associated with severe autistic traits. These results suggest that people with ASD may have difficulties in inhibiting the neural signals that synchronize the direction of inter-limb movements, thus resulting in coordination disabilities.

Lay SummaryIndividuals with autism spectrum disorder (ASD) often exhibit difficulties in coordinated movements. We asked those with ASD and healthy participants to move two limbs (e.g., left hand and left foot) either in the same or the opposite direction. Results demonstrated that participants with ASD had more difficulties in counteracting the tendency of their hand and foot to synchronously move in the same direction. Our findings suggested that difficulties to suppress synchronized movements of the hand and foot result in coordination disabilities.


title: A neuromodulatory model for determining the effect of emotion-respiration-cognition coupling on the time-to-respond

abstract: Respiration and emotional stimuli modulate cognitive ability and the reaction time to generate bodily movement. To understand mechanisms for emotion-respiration-cognition coupling, first, we considered a schematic feed-forward neural network, in which neurons was biased by respiratory-relevant sensory input and the activation function of a neuron was modulated by a neuromodulator, such as norepinephrine (NE). Furthermore, we assumed that the neural model received a stimulus input and generated a response action upon the activity of the output neuron exceeding a certain threshold. Time-to-respond (TTR) was equivalently modulated by the intensity of the input bias and the neuromodulator strength for small action execution threshold; however, it was dominantly modulated by only the neuromodulator for high threshold. Second, we implemented a comprehensive model comprising a cardio-respiration relevant neuromechanical-gas system, a respiratory central pattern generator (CPG), NE dynamics to modulate neurocognitive dynamics, and a locus coeruleus (LC) circuit, which was the primary nucleus for controlling NE. The LC neurons received pCO2 or synaptic current from an inspiratory neurons, which resulted in shortened TTR by a stimulus input during inhalation. By contrast, upon receiving pulmonary stretch information, the TTR was shortened by a stimulus input during exhalation. In humans, TTR is shortened when a fear-related stimulus is presented during inhalation, and likewise, TTR is weakly-shortened when surprise-related stimulus is presented during exhalation. Hence, we conclude that emotional stimuli in humans may switch the gating strategies of information and the inflow to LC to change the attention or behavior strategy.


title: Electrophysiological signatures reveal spinal learning mechanisms for a lasting sensorimotor adaptation

abstract: Neurocircuits within the spinal cord are essential for movement automaticity. However, spinal mechanisms that underlie lasting sensorimotor adjustments remain unclear. Here, we establish a quantitative kinematic framework to characterize a conditioning behavior in which spinal circuits without brain input learn to adapt motor output upon multimodal sensory integration, undergo extinction, and reinforcement of learned behavior with repetitive training. In-vivo Neuropixel spinal cord recordings from awake behaving mice reveal learning phase-tuned single unit activities. In addition, optically identified unit recordings and a loss-of-function experiment demonstrate an essential function of a class of spinal inhibitory interneurons in this learning paradigm. Together, these data reveal neuronal underpinnings that shape lasting sensorimotor adaptation where stable sensory dissemination regulates learning and the existence of neuronal assembly that retains learned behavior.


title: Excitatory and inhibitory effects of HCN channel modulation on excitability of layer V pyramidal cells

abstract: Dendrites of cortical pyramidal cells are densely populated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, a.k.a. Ih channels. Ih channels are targeted by multiple neuromodulatory pathways, and thus are one of the key ion-channel populations regulating the pyramidal cell activity. Previous observations and theories attribute opposing effects of the Ih channels on neuronal excitability due to their mildly hyperpolarized reversal potential. These effects are difficult to measure experimentally due to the fine spatiotemporal landscape of the Ih activity in the dendrites, but computational models provide an efficient tool for studying this question in a reduced but generalizable setting. In this work, we build upon existing biophysically detailed models of thick-tufted layer V pyramidal cells and model the effects of over- and under-expression of Ih channels as well as their neuromodulation by dopamine (gain of Ih function) and acetylcholine (loss of Ih function). We show that Ih channels facilitate the action potentials of layer V pyramidal cells in response to proximal dendritic stimulus while they hinder the action potentials in response to distal dendritic stimulus at the apical dendrite. We also show that the inhibitory action of the Ih channels in layer V pyramidal cells is due to the interactions between Ih channels and a hot zone of low voltage-activated Ca2+ channels at the apical dendrite. Our simulations suggest that a combination of Ih-enhancing neuromodulation at the proximal apical dendrite and Ih-inhibiting modulation at the distal apical dendrite can increase the layer V pyramidal excitability more than any of the two neuromodulators alone. Our analyses uncover the effects of Ih-channel neuromodulation of layer V pyramidal cells at a single-cell level and shed light on how these neurons integrate information and enable higher-order functions of the brain.


title: A recurrent network architecture explains tectal activity dynamics and experience-dependent behaviour

abstract: The ongoing activity of neuronal populations represents an internal brain state that influences how sensory information is processed to control behaviour. Conversely, external sensory inputs perturb network dynamics, resulting in lasting effects that persist beyond the duration of the stimulus. However, the relationship between these dynamics and circuit architecture and their impact on sensory processing, cognition and behaviour are poorly understood. By combining cellular-resolution calcium imaging with mechanistic network modelling, we aimed to infer the spatial and temporal network interactions in the zebrafish optic tectum that shape its ongoing activity and state-dependent responses to visual input. We showed that a simple recurrent network architecture, wherein tectal dynamics are dominated by fast, short range, excitation countered by long-lasting, activity-dependent suppression, was sufficient to explain multiple facets of population activity including intermittent bursting, trial-to-trial sensory response variability and spatially-selective response adaptation. Moreover, these dynamics also predicted behavioural trends such as selective habituation of visually evoked prey-catching responses. Overall, we demonstrate that a mechanistic circuit model, built upon a uniform recurrent connectivity motif, can estimate the incidental state of a dynamic neural network and account for experience-dependent effects on sensory encoding and visually guided behaviour.


title: Computational analysis of memory consolidation following inhibitory avoidance (IA) training in adult and infant rats: critical roles of CaMKIIα and MeCP2

abstract:
Key features of long-term memory (LTM), such as its stability and persistence, are acquired during processes collectively referred to as consolidation. The dynamics of biological changes during consolidation are complex. In adult rodents, consolidation exhibits distinct periods during which the engram is more or less resistant to disruption. Moreover, the ability to consolidate memories differs during developmental periods. Although the molecular mechanisms underlying consolidation are poorly understood, the initial stages rely on interacting signaling pathways that regulate gene expression, including brain-derived neurotrophic factor (BDNF) and Ca2+/calmodulin-dependent protein kinase II  (CaMKII) dependent feedback loops. We investigated the ways in which these pathways may contribute to developmental and dynamical features of consolidation. A computational model of molecular processes underlying consolidation following inhibitory avoidance (IA) training in rats was developed. Differential equations described the actions of CaMKII, multiple feedback loops regulating BDNF expression, and several transcription factors including methyl-CpG binding protein 2 (MeCP2), histone deacetylase 2 (HDAC2), and SIN3 transcription regulator family member A (Sin3a). This model provides novel explanations for the (apparent) rapid forgetting of infantile memory and the temporal progression of memory consolidation in adults. Simulations predict that dual effects of MeCP2 on the expression of bdnf, and interaction between MeCP2 and CaMKII, play critical roles in the rapid forgetting of infantile memory and the progress of memory resistance to disruptions. These insights suggest new potential targets of therapy for memory impairment.

Author SummaryLong-term memories (LTMs) are enduring and resistant to disruption These features are acquired via processes collectively referred to as consolidation. In adults, the initial stages of consolidation follow complex dynamics that are believed to emerge from interacting biochemical signaling pathways [1], including BDNF and CaMKII dependent feedback loops. Similarly, the acquisition of ability to consolidate memory in infantile animals is believed to emerge from the functional maturation of these molecular pathways [2]. Here, the ways in which these pathways contribute to consolidation were investigated using a computational model. This model provides novel explanations for the apparent rapid forgetting of infantile memory and for development of resistance to disruption during memory consolidation.


title: High-contrast en-bloc staining of mouse whole-brain samples for EM-based connectomics

abstract: Connectomes of human cortical gray matter require high-contrast homogeneously stained samples sized at least 2-3 mm on a side, and a whole-mouse brain connectome requires samples sized at least 5-10 mm on a side. Here we report en-bloc staining and embedding protocols for these and other applications, removing a key obstacle for connectomic analyses at mammalian whole-brain level.


title: Unraveling axonal mechanisms of traumatic brain injury

abstract: Axonal swellings (AS) are the neuropathological hallmark of axonal injury in several disorders from trauma to neurodegeneration. Current evidence proposes a role of perturbed Ca2+ homeostasis in AS formation, involving impaired axonal transport and focal distension of the axons. Mechanisms of AS formation, in particular moments following injury, however, remain unknown. Here we show that AS form independently from intra-axonal Ca2+ changes, which are required primarily for the persistence of AS in time. We further show that the majority of axonal proteins undergoing de/phosphorylation immediately following injury belong to the cytoskeleton. This correlates with an increase in the distance of the actin/spectrin periodic rings and with microtubule tracks remodeling within AS. Observed cytoskeletal rearrangements support axonal transport without major interruptions. Our results demonstrate that the earliest axonal response to injury consists in physiological adaptations of axonal structure to preserve function rather than in immediate pathological events signaling axonal destruction.


title: Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics of human antibody secreting cells.

abstract:
Antibody-secreting cells (ASCs) secrete IgM, IgA, or IgG antibodies and are key components of humoral immunity; however, little is known about unique characteristics of the Ig-classes due to limited availability of material and challenges to quantify many intracellular molecular modalities at a single-cell resolution. We combined a method to in vitro differentiate peripheral B-cells into ASCs with integrated multi-omic single-cell sequencing technologies to quantify subclass-specific hallmark surface markers, transcriptional profiles and signaling transduction pathway components. Our approach detected differential expression of plasmablast and plasma cell markers, homing receptors and IL-2, IL-6, JAK/STAT and mTOR signaling activity across Ig-subclasses. Taken together, our integrated multi-omics approach allowed high-resolution phenotypic characterization of single cells in a complex sample of in vitro differentiated human ASCs. Our strategy is expected to further our understanding of human ASCs in healthy and diseased samples and provide a valuable tool to identify novel biomarkers and potential drug targets.

TeaserIntegrated single-cell analysis allows tri-modal phenotypic analysis of in-vitro generated human antibody-secreting cells.


title: Evaluating impact of experimental design on accuracy of parameter estimation and model selection efficiency: T cell clustering as a case study

abstract: Mathematical modeling provides a rigorous way to quantify immunological processes and to discriminate between alternative mechanisms driving specific biological phenomena. In our experience it is typical that mathematical models of immunological phenomena are developed to explain specific sets of experimental data after the data have been collected by experimental collaborators. Whether the available data are sufficient to accurately estimate model parameters or to discriminate between alternative models is not typically investigated; in fact, in our experience, previously collected data often do not allow to discriminate between alternative models. As a case study we develop a series of power analyses to determine optimal sample sizes that allow for accurate estimation of model parameters and for discrimination between alternative models describing clustering of T cells around Plasmodium liver stages. In our typical experiments, mice are infected intravenously with Plasmodium sporozoites that invade hepatocytes (liver cells), and then activated T cells are transferred into the infected mice. The number of T cells found in the vicinity of individual infected hepatocytes at different times after T cell transfer is counted using intravital microscopy. We previously developed a series of mathematical models aimed to explain highly variable number of T cells per parasite; one of such models, the density-dependent recruitment (DDR) model, fitted the data better than alternative models such as the density-independent exit (DIE) model. Here we show that the ability to discriminate between these alternative models depends on the number of parasites imaged in the analysis; analysis of about n = 50 parasites at 2, 4, and 8 hours after T cell transfer will allow for over 95% probability to select the correct model. The type of data collected also has an impact; following T cell clustering around individual parasites over time (called as longitudinal (LT) data) allows for a more precise and less biased estimates of parameters of the DDR model than a more traditional way of imaging individual parasites in different liver areas/mice (cross-sectional (CS) data). However, LT imaging comes at a cost of a need to keep the mice alive under the microscope for hours which may be ethically difficult. We finally show that the number of time points at which measurements are taken also impacts precision of estimation of DDR model parameters; in particular, measuring T cell clustering at one time point does not allow to accurately estimate all parameters of the DDR model. Taken together, we illustrate how stochastic simulations of an assumed underlying process and random sampling (power analysis) allow to determine the optimal size of the dataset needed to accurately estimate model parameters and to discriminate between alternative models.


title: The cardiopharyngeal mesoderm contributes to lymphatic vessel development

abstract: Lymphatic vessels are crucial for tissue homeostasis and immune responses in vertebrates. Recent studies have demonstrated that lymphatic endothelial cells (LECs) arise from both venous sprouting (lymphangiogenesis) and de novo production from non-venous origins (lymphvasculogenesis), which is similar to blood vessel formation through angiogenesis and vasculogenesis. However, the contribution of LECs from non-venous origins to lymphatic networks is considered to be relatively small. Here, we identify the Islet1 (Isl1)-expressing cardiopharyngeal mesoderm (CPM) as a non-venous origin of craniofacial and cardiac LECs. Genetic lineage tracing with Isl1-Cre and Isl1-MerCreMer mice suggested that a subset of CPM cells gives rise to LECs. These CPM-derived LECs are distinct from venous-derived LECs in terms of their developmental processes and anatomical locations. Later, they form the craniofacial and cardiac lymphatic vascular networks in collaboration with venous-derived LECs. Collectively, our results demonstrate that there are two major sources of LECs, the cardinal vein and the CPM. As the CPM is evolutionarily conserved, these findings may improve our understanding of the evolution of lymphatic vessel development across species. Most importantly, our findings may provide clues to the pathogenesis of lymphatic malformations, which most often develop in the craniofacial and mediastinal regions.

category: developmental biology


title: Lethal and sub-lethal concentrations of pesticides detected in milkweeds purchased from nurseries across the United States

abstract: The decline of monarch butterflies in both the eastern and western United States has garnered widespread public interest. Planting milkweed has been promoted as one action that individuals can take, but very little is known with respect to potential pesticide contamination of store-bought milkweeds during the process of production and transport to market. In this study, we collected 235 milkweed leaf samples from 33 retail nurseries across the US to screen for pesticides. Across all samples, we detected 61 different pesticides with an average of 12.2 compounds per leaf. While most of the compounds have unknown effects on caterpillars, 48 samples contained a pesticide at a concentration that exceeds a published lepidopteran LD50 and 89 contained a pesticide above a concentration shown to have a sub-lethal effect for a monarch. We detected only modest effects of store size and milkweed species; however, plants with labels advertising their value for wildlife were more likely to contain pesticides at concentrations known to have negative effects on monarchs. These results demonstrate the extensiveness of pesticide exposure within nursery milkweeds and the potential impacts on monarchs and other insects consuming store-bought plants.

category: ecology


title: The interplay between habitat fragmentation and life traits affects specialization in lichen symbioses

abstract:
O_LIInteractions between organisms are determined by species traits and differ in specialization, from generalist to highly specialized relationships. Although we expect that the effect of habitat loss and fragmentation on the abundance and survival of species depends on their degree of specialization, few studies have deepened into the interplay between interaction specialization and habitat loss.
C_LIO_LIHere, we investigate the combined effect of habitat fragmentation and life traits (growth type and reproductive mode) on the specialization of lichen-forming fungi (mycobionts) towards its photosynthetic partners (photobionts) in lichen symbioses.
C_LIO_LIWe studied mycobiont specialization in epiphytic lichen communities present in ten fragments of Quercus rotundifolia trees embedded in an agricultural matrix. Both mycobionts and photobionts were identified through DNA sequencing and mycobiont specialization was measured through interaction parameters calculating the relative number of interactions (normalized degree; ND) and the specialization of each species based on its discrimination from a random selection of partners (d). Phylogenetic generalized linear mixed models were used to analyze the effect of patch size as well as the life traits growth type (crustose, foliose, fruticose) and reproduction mode (sexual vs asexual) on mycobiont specialization.
C_LIO_LIBoth mycobiont and photobiont richness along the patch size gradient followed a hump-back pattern, which was more pronounced in photobionts. Mycobionts forming crustose biotypes established the largest number of interactions. Mycobiont specialization (d) was larger for fruticose and foliose biotypes and for species with vegetative reproduction. Along the gradient of fragment size, the relative number of interactions decreased and the specialization of mycobionts with vegetative reproduction increased.
C_LIO_LISynthesis: The analysis of mycobiont specialization towards their photobionts in communities of epiphytic lichens in a fragmented Mediterranean forest revealed that the interaction between species life traits and habitat loss on specialization cannot be neglected. The results also pointed to the ability of some species to modulate their interactions to certain extent, suggesting that species might have a greater resilience to abiotic changes than expected, presumably creating extinction debt or even avoiding extinction processes to some extent.
C_LI

ResumenO_LILas interacciones entre organismos estan determinadas por los rasgos funcionales de las especies y difieren en su grado de especializacion, desde interacciones generalistas hasta las altamente especializadas. Aunque se espera que el efecto de la perdida y fragmentacion del habitat sobre la abundancia y la supervivencia de las especies dependa de su grado de especializacion, pocos estudios han profundizado en la interaccion entre la especializacion de las interacciones y la perdida de habitat.
C_LIO_LIEn este articulo investigamos el efecto combinado de la fragmentacion del habitat y los rasgos funcionales de las especies (tipo de crecimiento y modo reproductivo) en la especializacion de los hongos liquenizados (micobiontes) hacia sus socios fotosinteticos (fotobiontes) en las simbiosis de liquenes.
C_LIO_LISe estudio la especializacion de micobiontes en comunidades de liquenes epifitos presentes en diez fragmentos de arboles de Quercus rotundifolia incrustados en una matriz agricola. Tanto los micobiontes como los fotobiontes fueron identificados mediante la secuenciacion del ADN y la especializacion de los micobiontes se midio a traves de parametros de interaccion calculando el numero relativo de interacciones (grado normalizado; ND) y la especializacion de cada especie en funcion de su discriminacion de una seleccion aleatoria de simbiontes (d). Se utilizaron modelos lineales mixtos generalizados filogeneticos para analizar el efecto del tamano del parche, asi como los rasgos de tipo de crecimiento (crustaceo, folioso, frutal) y el modo de reproduccion (sexual frente a asexual) sobre la especializacion de los micobiontes.
C_LIO_LITanto la riqueza de micobiontes como de fotobiontes a lo largo del gradiente de tamano de los parches siguio un patron de U inversa, que fue mas pronunciado en los fotobiontes. Los micobiontes que forman biotipos crustaceos establecieron el mayor numero de interacciones. La especializacion de los micobiontes (d) fue mayor para los biotipos fruticulosos y foliosos y para las especies con reproduccion vegetativa. A lo largo del gradiente del tamano del fragmento, el numero relativo de interacciones disminuyo y la especializacion de los micobiontes con reproduccion vegetativa aumento.
C_LIO_LISintesis: El analisis de la especializacion de los micobiontes hacia sus fotobiontes en comunidades de liquenes epifitos en un bosque mediterraneo fragmentado revelo que no se puede descartar la interaccion entre los rasgos de vida de las especies y la perdida de habitat sobre la especializacion. Los resultados tambien apuntaron a la capacidad de algunas especies para modular sus interacciones hasta cierto punto, sugiriendo que las especies podrian tener una resiliencia a los cambios abioticos mayor de lo esperado, presumiblemente creando una deuda de extincion o incluso evitando los procesos de extincion hasta cierto punto. Palabras clave: Bosque mediterraneo, epifitos, fotobiontes, hongos liquenizados, selectividad.
C_LI


title: Introduced crops supplement rather than replace indigenous crops in an African center of agrobiodiversity

abstract:
O_LICrop diversity plays a major role in underpinning food security. It is especially important to smallholder and subsistence farmers, who often rely on crop diversity for stable and resilient production. Despite this, global expansion of a small pool of major crops and the associated homogenisation of global agricultural systems may decrease on-farm crop diversity.
C_LIO_LIWe surveyed 1,369 subsistence farms stratified across climate gradients in the Ethiopian Highlands, to characterise the richness and cultivated area of the 83 edible crops they contained. We further categorise these crops by their period of introduction to Ethiopia. We apply non-metric multidimensional scaling and mixed effects modelling to characterise agrisystem composition and test the impact of crop introductions.
C_LIO_LIWe find a significant positive relationship between introduced and indigenous crop richness, suggesting that crop introductions have tended to supplement rather than replace or reduce indigenous crop diversity. Geographically matched farms with higher proportions of introduced crops, had significantly higher overall crop richness. Analysis of socio-economic drivers indicates that both poverty and low accessibility are associated with reduced cultivation of modern introductions.
C_LIO_LIWe conclude that global patterns of major crop expansion do not necessarily result in agrobiodiversity loss for subsistence farmers, in our Ethiopian case study. Importantly, socioeconomic factors may strongly influence the farmers propensity to adopt novel species, suggesting targets for agricultural extension policies. Given the rapid climatic, economic and demographic changes impacting global food systems and the threats to food security these entail, robust indigenous agricultural systems supplemented with diverse introduced crops may enhance resilience.
C_LI


title: Climate adaptation and vulnerability of foundation species in a global change hotspot

abstract: Climate change is altering species ranges, and abundances within ranges, as populations become differentially adapted and vulnerable to the climates they face. Hence, characterising current ranges, whether species harbour and exchange adaptive genetic variants, and how variants are distributed across landscapes undergoing rapid change, is crucial to predicting responses to future climates and informing conservation strategies. Such insights are nonetheless lacking for most species of conservation concern. We characterise genomic patterns of neutral variation, climate adaptation, and climate vulnerability (the amount of genomic change needed to track climate change by adaptation) in sister foundation species, the endemic marine tubeworms Galeolaria caespitosa and Galeolaria gemineoa, across a sentinel region for climate change impacts. First, species are shown to be partly sympatric despite previous support for non-overlapping ranges, and genetically isolated despite known capacity for hybrid crosses to yield viable early offspring. Second, species show signals of polygenic adaptation, but to differing components of temperature and involving mostly different loci. Last, species are predicted to be differentially vulnerable to climate change, with G. gemineoa -- the less genetically diverse species -- needing double the adaptation to track projected changes in temperature compared to its sister species. Together, our findings provide new insights into climate adaptation and its potential disruption by climate change for foundation species that enhance local biodiversity, with implications for evolutionarily-enlightened management of coastal ecosystems.

category: evolutionary biology


title: Evolutionary relationships and range evolution of greenhood orchids (subtribe Pterostylidinae): insights from plastid phylogenomics

abstract:
Australia harbours a rich and highly endemic orchid flora with over 90% of native species found nowhere else. However, little is known about the assembly and evolution of Australias orchid flora. Here, we used a phylogenomic approach to infer evolutionary relationships and range evolution in Pterostylidinae (Orchidoideae), the second largest subtribe in the Australian orchid flora, comprising the genera Pterostylis and Achlydosa. Phylogenetic analysis of 75 plastid genes provided well-resolved and supported phylogenies. Intrageneric relationships in Pterostylis were clarified and monophyly of eight of ten sections supported. Achlydosa was found to not form part of Pterostylidinae and instead merits recognition at subtribal level, as Achlydosinae.

Pterostylidinae were inferred to have originated in temperate eastern Australia in the early Oligocene, coinciding with the complete separation of Australia from Antarctica and the onset of the Antarctic Circumpolar Current, which led to profound changes in the worlds climate. Divergence of all major lineages occurred during the Miocene, accompanied by increased aridification and seasonality of the Australian continent, resulting in transformational vegetational changes from rainforest to more open sclerophyllous vegetation. The majority of extant species were inferred to have originated in the Quaternary, from the Pleistocene onwards. The rapid climatic oscillations during the Pleistocene are regarded as an important driver of speciation in Pterostylidinae. The subtribe underwent lineage diversification mainly within its ancestral range, in temperate eastern Australia. Long-distance dispersals to southwest Australia commenced from the late Miocene onwards, after the establishment of the Nullarbor Plain, which constitutes a strong edaphic barrier to mesic plants. Range expansions from the mesic into the arid zone of eastern Australia (Eremaean region) commenced from the early Pleistocene onwards. Extant distributions of Pterostylidinae in other Australasian regions, such as New Zealand and New Caledonia, are of more recent origin, resulting from long-distance dispersals from the Pliocene onwards. Temperate eastern Australia was identified as key source area for dispersals to other Australasian regions.


title: Convergent genomic signatures of adaptation to an extreme environment in cyprinoid fishes

abstract: Cyprinoids are a worldwide distributed and diverse group of freshwater fish with more than 3,000 species. Although primarily freshwater, some cyprinoid species had convergently evolved to thrive in extreme environments and underlying genetic mechanisms remain unclear. Here, we leveraged 32 cyprinoid fish genomes to identify common genomic changes associated with convergent adaptation to highly saline and alkaline water in two East Asian cyprinoid fish species, Gymnocypris przwalskii and Leuciscus waleckii, representing two independent extremophile fish lineages. We found that genome-wide rate of nonsynonymous substitution and signal of intensified selection is higher in extremophile relative to non-extremophile fish taxa. We further tested gene-wide molecular convergence and found hundreds of genes tended to experience convergent shifts in selections in extremophile fish taxa, including convergent acceleration and positive selection. These genes were associated with several key functions, such as nervous system development, reproduction, ion transport and immune response, and included genes that previously have been implicated for saline or alkaline tolerance in fish. Additionally, comparative transcriptomic analyses defined the convergent roles of differentially expressed genes under selection in extremophile fish taxa during convergent adaptation. Taken together, our work provides insights into the genomic basis of convergent adaptation to extreme environments in fish.


title: Fold Evolution by Creative Destruction

abstract:
Mechanisms by which new protein folds emerge and diverge pose central questions in biological sciences. Incremental mutation and step-wise adaptation explain relationships between topologically similar protein folds. However, the universe of folds is diverse and riotous, suggesting roles of more potent and creative forces. Sequence and structure similarity are observed between topologically distinct folds, indicating that proteins with distinct folds may share common ancestry.

We found evidence of common ancestry between three distinct {beta}-barrel folds: OB, SH3 and cradle loop barrel (CLB). The data suggest a mechanism of fold evolution that interconverts SH3, OB and CLB. This mechanism, which we call creative destruction, can be generalized to explain other examples of fold evolution including circular permutation. In creative destruction, an open reading frame duplicates or otherwise merges with another to produce a fused polypeptide. A merger forces two ancestral domains into a new sequence and spatial context. The fused polypeptide can explore folding landscapes that are inaccessible to either of the independent ancestral domains. However, the folding landscapes of the fused polypeptide are not fully independent of those of the ancestral domains. Creative destruction is thus partially conservative in that a daughter fold would inherit some motifs from the ancestral folds. After a merger and refolding, adaptive processes such as mutation and loss of extraneous segments optimize the new daughter fold.

SignificanceMechanisms of emergence and early diversification of structured proteins present deep and difficult problems in evolutionary biology. Here we excavate the deepest evolutionary history, found within the translation machinery, which is an ancient molecular fossil and the birthplace of all proteins. We demonstrate common origins of some of the simplest, oldest and most common protein folds. Furthermore, the data suggest a mechanism, that we call creative destruction, that explains at molecular level how simple folds spawn more complex folds. In this mechanism, new folds emerge from old folds via gene duplication, expression, exploration of new folding landscapes and adaptation. Creative destruction explains the facile emergence of complex from simple architectures in a funneled exploration.


title: The Evolution of Protein Folds by Creative Destruction


title: Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces

abstract: Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary leading to the diversification of this genus over the last 20 million years. Here, we report the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.


title: Insights into the domestication of avocado and potential genetic contributors to heterodichogamy

abstract:
O_LIThe domestication history of avocado (Persea americana) remains unclear, in part due to a lack of suitable genomic tools.
C_LIO_LIWe created a reference genome from the Gwen varietal, which is closely related to the economically dominant Hass varietal. We also compiled a database of 34 resequenced accessions that represented the three botanical races of P. americana.
C_LIO_LIOur genome assembly had an N50 of 3.37 megabases, a BUSCO score of 91% and was scaffolded with a genetic map, producing 12 pseudo-chromosomes with 49,450 genes. We used the Gwen genome as a reference to investigate the population genomics of avocado. Our analyses were consistent with three separate domestication events; we estimated that the Mexican race diverged from the Lowland (formerly known as  West Indian) and Guatemalan races >1 million years ago. We also identified putative targets of selective sweeps in domestication events; within the Guatemalan race, putative candidate genes were enriched for fruit development and ripening. We also investigated divergence between heterodichogamous flowering types.
C_LIO_LIWith the help of a new reference genome, we inferred the domestication history of avocado and identified genes that may contribute to heterodichogamy, including genes with functions in pollination and floral development.
C_LI

category: genomics


title: Felidae call type and species identification based on acoustic features

abstract: The cat family Felidae is one of the most successful carnivore lineages today. However, the study of the evolution of acoustic communication between felids remains a challenge due to the lack of fossils, the limited availability of audio recordings because of their largely solitary and secretive behavior, and the underdevelopment of computational models and methods needed to address acoustic evolutionary questions. This study is a first attempt at developing a machine learning-based approach to the classification of felid calls as well as the identification of acoustic features that distinguish felid call types and species from one another. A felid call dataset was developed by extracting audio clips from diverse sources. The audio clips were manually annotated for call type and species. Due to the limited availability of samples, this study focused on the Pantherinae subfamily. Time-frequency features were then extracted from the Pantherinae dataset. Finally, several classification algorithms were applied to the resulting data. We achieved 91% accuracy for this Pantherinae call type classification. For the species classification, we obtained 86% accuracy. We also obtained the most predictive features for each of the classifications performed. These features can inform future research into the evolutionary acoustic analysis of the felid group.

category: animal behavior and cognition


title: Improved performance of biohybrid muscle-based bio-bots doped with piezoelectric boron nitride nanotubes

abstract: Biohybrid robots, or bio-bots, integrate living and synthetic materials following a synergistic strategy to acquire some of the unique properties of biological organisms, like adaptability or bio-sensing, which are difficult to obtain exclusively using artificial materials. Skeletal muscle is one of the preferred candidates to power bio-bots, enabling a wide variety of movements from walking to swimming. Conductive nanocomposites, like gold nanoparticles or graphene, can provide benefits to muscle cells by improving the scaffolds mechanical and conductive properties. Here, we integrate boron nitride nanotubes (BNNTs), with piezoelectric properties, in muscle-based bio-bots and demonstrate an improvement in their force output and motion speed. We provide a full characterization of the BNNTs, and we confirm their piezoelectric behavior with piezometer and dynamometer measurements. We hypothesize that the improved performance is a result of an electric field generated by the nanocomposites due to stresses produced by the cells during differentiation, which in turns improves their maturation. We back this hypothesis with finite element simulations supporting that this stress can generate a non-zero electric field within the matrix. With this work, we show that the integration of nanocomposite into muscle-based bio-bots can improve their performance, paving the way towards stronger and faster bio-hybrid robots.

category: bioengineering


title: An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences

abstract: T cell receptor (TCR) repertoires can be profiled using next generation sequencing (NGS) to monitor dynamical changes in response to disease and other perturbations. Several strategies for profiling TCRs have been recently developed with different benefits and drawbacks. Genomic DNA-based bulk sequencing, however, remains the most cost-effective method to profile TCRs. The major disadvantage of this method is the need for multiplex target amplification with a large set of primer pairs with potentially very different amplification efficiencies. One approach addressing this problem is by iteratively adjusting the concentrations of the primers based on their efficiencies, and then computationally correcting any remaining bias. Yet there are no standard, publicly available protocols to process and analyze raw sequencing data generated by this method. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both approaches, we show that the concordance between bulk clonality metrics obtained from using the commercial kits and that developed herein is high. Therefore, we suggest the method presented here as an inexpensive and non-commercial alternative for measuring and monitoring adaptive dynamics in TCR clonotype repertoire.


title: The dynamical Matryoshka model: 3. Diffusive nature of the atomic motions contained in a new dynamical model for deciphering local lipid dynamics

abstract: In accompanying papers [Bicout et al., BBA - Biomembr. Submitted; Cisse et al., BBA - Biomembr. Submitted], a new model called Matryoshka model has been proposed to describe the geometry of atomic motions in phospholipid molecules in bilayers and multilamellar vesicles based on their quasielastic neutron scattering (QENS) spectra. Here, in order to characterize the relaxational aspects of this model, the energy widths of the QENS spectra of the samples were analyzed first in a model-free way. The spectra were decomposed into three Lorentzian functions, which are classified as slow, intermediate, and fast motions depending on their widths. The analysis provides the diffusion coefficients, residence times, and geometrical parameters for the three classes of motions. The results corroborate the parameter values such as the amplitudes and the mobile fractions of atomic motions obtained by the application of the Matryoshka model to the same samples. Since the current analysis was carried out independently of the development of the Matryoshka model, the present results enhance the validity of the model. The model will serve as a powerful tool to decipher the dynamics of lipid molecules not only in model systems, but also in more complex systems such as mixtures of different kinds of lipids or natural cell membranes.


title: Response to immune checkpoint blockade improved in pre-clinical model of breast cancer after bariatric surgery

abstract: Bariatric surgery is becoming more prevalent as a sustainable weight loss approach, with vertical sleeve gastrectomy (VSG) being the first line of surgical intervention. We and others have shown that obesity exacerbates tumor growth while diet-induced weight loss impairs obesity-driven progression. It remains unknown how bariatric surgery-induced weight loss impacts cancer progression or alters responses to therapy. Using a pre-clinical model of diet induced obesity followed by VSG or diet-induced weight loss, breast cancer progression and immune checkpoint blockade therapy was investigated. Weight loss by bariatric surgery or weight matched dietary intervention before tumor engraftment protected against obesity-exacerbated tumor progression. However, VSG was not as effective as dietary intervention in reducing tumor burden despite achieving a similar extent of weight and adiposity loss. Circulating leptin did not associate with changes in tumor burden. Uniquely, tumors in mice that received VSG displayed elevated inflammation and immune checkpoint ligand, PD-L1. Further, mice that received VSG had reduced tumor infiltrating T lymphocytes and cytolysis suggesting an ineffective anti-tumor microenvironment. VSG-associated elevation of PD-L1 prompted us to next investigate the efficacy of immune checkpoint blockade in lean, obese, and formerly obese mice that lost weight by VSG or weight matched controls. While obese mice were resistant to immune checkpoint blockade, anti-PD-L1 potently impaired tumor progression after VSG through improved anti-tumor immunity. Thus, in formerly obese mice, surgical weight loss followed by immunotherapy reduced breast cancer burden.


title: Multi-omic profiling of the leukemic microenvironment shows bone marrow interstitial fluid is distinct from peripheral blood plasma

abstract:
The bone marrow is the place of hematopoiesis with a microenvironment that supports lifelong maintenance of stem cells and high proliferation. It is not surprising that this environment is also favourable for malignant cells emerging in the bone marrow or metastasizing to it. While the cellular composition of the bone marrow microenvironment has been extensively studied, the extracellular matrix and interstitial fluid components have received little attention. Since the sinusoids connect the bone marrow interstitial fluid to the circulation, it is often considered to have the same composition as peripheral blood plasma. Stark differences in the cellular composition of the bone marrow and peripheral blood with different secretory capacities would however suggest profound differences.

In this study we set out to better define if and how the bone marrow interstitial fluid (BMIF) compares to the peripheral blood plasma (PBP) and how both are remodeled during chemotherapy. We applied a multi-omic strategy to quantify the metabolite, lipid and protein components as well as the proteolytic modification of proteins to gain a comprehensive understanding of the two compartments. We found that the bone marrow interstitial fluid is clearly distinct from peripheral blood plasma, both during active pediatric acute lymphoblastic leukemia and following induction chemotherapy. Either compartment was shaped differently by active leukemia, with the bone marrow interstitial fluid being rich in extracellular vesicle components and showing protease dysregulation while the peripheral blood plasma showed elevation of immune regulatory proteins. Following chemotherapy, the BMIF showed signs of cellular remodeling and impaired innate immune activation while the peripheral blood plasma was characterized by restored lipid homeostasis.


title: Evolutionary gain and loss of a plant pattern-recognition receptor for HAMP recognition

abstract: Pattern recognition receptors (PRR) recognize distinct pathogen and herbivore-associated molecular patterns (PAMPs and HAMPs) and mediate activation of immune responses, but the evolution of new PRR sensing functions is not well understood. We employed comparative genomics and functional analysis to define evolutionary events leading to the sensing of the peptide HAMP inceptin (In11) by the PRR Inceptin Receptor (INR). Existing and de novo genome assemblies revealed that the presence of a functional INR gene corresponded with In11 response across 55 million years (my) of legume evolution, and that In11 recognition is unique to the clade of Phaseoloid legumes. The INR loci of certain Phaseoloid and non-Phaseoloid species also contain diverse INR-like homologues, suggesting that the evolution of INR receptor function [~]28 mya occurred after an ancestral gene insertion [~]32 mya. Functional analysis of chimeric and ancestrally reconstructed receptors revealed that specific AA differences in the C1 leucine-rich repeat (LRR) domain and C2 intervening motif likely mediated gain of In11 recognition. In summary, we present a conceptual model for the evolution of a defined PRR function based on patterns of INR variation in legumes.


title: Dissecting Succulence: Crassulacean Acid Metabolism and Hydraulic Capacitance are Independent Adaptations in Clusia Leaves

abstract:
O_LISucculence is found across the world as an adaptation to water-limited niches. The fleshy organs of succulent plants develop via enlarged photosynthetic chlorenchyma and/or achlorophyllous water storage hydrenchyma cells. The precise mechanism by which anatomical traits contribute to drought tolerance is unclear, as the effect of succulence is multifaceted. Large cells are believed to provide space for nocturnal storage of malic acid fixed by crassulacean acid metabolism (CAM), whilst also buffering water potentials by elevating hydraulic capacitance (CFT). Furthermore, the effect of CAM and elevated CFT on growth and water conservation have not been compared, despite the assumption that these adaptations often occur together.
C_LIO_LIWe assessed the relationship between succulent anatomical adaptations, CAM and CFT, across the genus Clusia. In addition, we simulated the effects of CAM and CFT on growth and water conservation during drought using the Photo3 model.
C_LIO_LIWithin Clusia leaves, CAM and CFT are independent traits: CAM requires large palisade chlorenchyma cells, whereas hydrenchyma tissue governs interspecific differences in CFT. In addition, our model suggests that CAM supersedes CFT as a means to maximise CO2 assimilation and minimise transpiration during drought.
C_LIO_LIOur study challenges the assumption that CAM and CFT are mutually dependent traits within succulent leaves.
C_LI


title: Discussion-based DEI education to help create inclusive and open BME research lab environments

abstract: Diversity in teams has been shown to enhance creativity and innovation, particularly in teams where all members felt a sense of belonging. Creating an inclusive environment in a lab setting that provides a sense of belonging to all is challenging. This is particularly true in a field like Biomedical Engineering/Bioengineering where diversity is multifaceted and includes peoples diverse personal/cultural backgrounds and also diverse scientific backgrounds. In a research lab, there is additional diversity in training, as most labs contain trainees at different levels (undergrad, grad, postdoc, high school). To aid in creating a sense of belonging in a research lab, we have devised a novel initiative based upon open group discussions on diversity, equity, and inclusion topics. The initiative included a first presentation/discussion by the PI to set the stage for defining diversity, equity, and inclusion, and provided examples of existing DEI issues within our field, such as lack of racial diversity in awards like the NIH New Innovator Award and among conference speakers. After the initial presentation, trainee-led, bi-weekly-structured discussions were maintained. First, discussions focused broadly on any DEI related topic to enhance general knowledge on DEI issues and provide discussion comfort among the group. Second, there was a period to deepen knowledge of a specific topic (in our example, microaggressions), which was followed up with a period of discussions on potential solutions (such as how to react when observing a microaggression and what to do in response to realizing ones own microaggression). Students reported that our discussions were the only ones they have had in their training thus far, and felt that these discussions made them feel like they belonged in the lab, made the lab more inclusive, enhanced their awareness of how to create inclusive spaces, and taught them about a variety of DEI topics. Overall, our DEI discussions have fostered open conversations within our lab group about DEI-related issues and topics at the lab, department, university, and countrywide levels and has established a space where students feel safe to voice their opinions and ask questions. We hope to ultimately use these DEI discussions to create actionable steps for addressing topics, e.g., microaggressions, in different scenarios that can be applied by group members in their future careers.

category: scientific communication and education


title: Defining Predictors of Successful Early Career to Independent Funding Conversion Among Surgeon-Scientists

abstract:
IntroductionThe National Institutes of Health (NIH) provides research funding to scientists at different stages of their career through a range of grant awards. Early-stage researchers are eligible for mentored Career Development (K) awards, to aid in the transition to independent NIH funding. Factors such as education, subspecialty, and time to funding have been studied as predictors of obtaining independent awards in nonsurgical specialties. However, in surgery, the importance of these factors has yet to be clearly elucidated. We aim to identify predictors of K to independent award conversion among surgeon-scientists to understand how to better support early-stage researchers transitioning to independent careers.

Materials and MethodsIn July 2020, the NIH Research Portfolio Online Reporting Tools database was queried for individuals affiliated with surgery departments who received NIH Career Development Awards (between 2000 and 2020). The following factors were analyzed: publications, institution, degrees, year of completion of training, and gender.

ResultsBetween 2000 and 2020, 228 surgeons received K Awards, of which 44% transitioned to independent funding. On average, surgeons received a K award 4.0 years after completing fellowship training and an independent award 5.4 years after receiving a K grant. The time to receiving a K award was predictive of successfully achieving independent funding, and those with independent funding had a significantly greater number of publications per year of their K-award.

ConclusionSurgeons successful in transitioning to independent NIH awards do so approximately 9 years after finishing fellowship. Publication track record is the main factor associated with successful conversion from a K award. Surgery departments should emphasize manuscript productivity and develop strategies to minimize time to independent funding to help K-awardees begin independent research careers.


title: Pad&eacute SSA: A frequency domain method for estimating the dynamics of stochastic reaction networks

abstract: Dynamic analysis and control of living cells relies on mathematical representations of cellular processes that are themselves modelled as biomolecular reaction networks. Stochastic models for biomolecular reaction networks are commonly employed for analysing intracellular networks having constituent species with low-copy numbers. In such models, the main object of interest is the probability distribution of the state vector of molecular counts which evolves according to a set of ordinary differential equations (ODEs) called the Chemical Master Equation (CME). Typically this set is very large or even infinite, making the CME practically unsolvable in most cases. Hence the outputs based on the CME solution, like the statistical moments of various state components, are generally estimated with Monte Carlo (MC) procedures by simulating the underlying Markov chain with Gillespies Stochastic Simulation Algorithm (SSA). However to obtain statistical reliability of the MC estimators, often a large number of simulated trajectories are required, which imposes an exorbitant computational burden. The aim of this paper is to present a frequency domain method for mitigating this burden by exploiting a small number of simulated trajectories to robustly estimate certain intrinsic eigenvalues of the stochastic dynamics. This method enables reliable estimation of time-varying outputs of interest from a small number of sampled trajectories and this estimation can be carried out for several initial states without requiring additional simulations. We demonstrate our method with a couple of examples.

category: systems biology


title: A fluctuation-based approach to infer kinetics and topology of cell-state switching

abstract: In the noisy cellular environment, RNAs and proteins are subject to considerable stochastic fluctuations in copy numbers over time. As a consequence, single cells within the same isoclonal population can differ in their expression profile and reside in different phenotypic states. The dynamic nature of this intercellular variation, where individual cells can transition between different states over time makes it a particularly hard phenomenon to characterize. Here we propose a novel fluctuation-test approach to infer the kinetics of transitions between cell states. More specifically, single cells are randomly drawn from the population and grown into cell colonies. After growth for a fixed number of generations, the number of cells residing in different states is assayed for each colony. In a simple system with reversible switching between two cell states, our analysis shows that the extent of colony-to-colony fluctuations in the fraction of cells in a given state is monotonically related to the switching kinetics. Several closed-form formulas for inferring the switching rates from experimentally quantified fluctuations are presented. We further extend this approach to multiple cell states where harnessing fluctuation signatures can reveal both the topology and the rates of cell-state switching. In summary, our analysis provides a powerful approach for dissecting cell-state transitions based on a single time point measurement. This is especially important for scenarios where a measurement involves killing the cell (for example, performing single-cell RNA-seq or assaying whether a microbial/cancer cell is in a drug-sensitive or drug-tolerant state), and hence the state of the same cell cannot be measured at different time points.


title: The impact of sparsity in low-rank recurrent neural networks

abstract:
Neural population dynamics are often highly coordinated, allowing task-related computations to be understood as neural trajectories through low-dimensional subspaces. How the network connectivity and input structure give rise to such activity can be investigated with the aid of low-rank recurrent neural networks, a recently-developed class of computational models which offer a rich theoretical framework linking the underlying connectivity structure to emergent low-dimensional dynamics. This framework has so far relied on the assumption of all-to-all connectivity, yet cortical networks are known to be highly sparse. Here we investigate the dynamics of low-rank recurrent networks in which the connections are randomly sparsified, which makes the network connectivity formally full-rank. We first analyse the impact of sparsity on the eigenvalue spectrum of low-rank connectivity matrices, and use this to examine the implications for the dynamics. We find that in the presence of sparsity, the eigenspectra in the complex plane consist of a continuous bulk and isolated outliers, a form analogous to the eigenspectra of connectivity matrices composed of a low-rank and a full-rank random component. This analogy allows us to characterise distinct dynamical regimes of the sparsified low-rank network as a function of key network parameters. Altogether, we find that the low-dimensional dynamics induced by low-rank connectivity structure are preserved even at high levels of sparsity, and can therefore support rich and robust computations even in networks sparsified to a biologically-realistic extent.

Author summaryIn large networks of neurons, the activity displayed by the population depends on the strength of the connections between each neuron. In cortical regions engaged in cognitive tasks, this population activity is often seen to be highly coordinated and low-dimensional. A recent line of theoretical work explores how such coordinated activity can arise in a network of neurons in which the matrix defining the connections is constrained to be mathematically low-rank. Until now, this connectivity structure has only been explored in fully-connected networks, in which every neuron is connected to every other. However, in the brain, network connections are often highly sparse, in the sense that most neurons do not share direct connections. Here, we test the robustness of the theoretical framework of low-rank networks to the reality of sparsity present in biological networks. By mathematically analysing the impact of removing connections, we find that the low-dimensional dynamics previously found in dense low-rank networks can in fact persist even at very high levels of sparsity. This has promising implications for the proposal that complex cortical computations which appear to rely on low-dimensional dynamics may be underpinned by a network which has a fundamentally low-rank structure, albeit with only a small fraction of possible connections present.


title: The role of clearance in neurodegenerative diseases

abstract: Alzheimers disease, the most common form of dementia, is a systemic neurological disorder associated with the formation of toxic, pathological aggregates of proteins within the brain that lead to severe cognitive decline, and eventually, death. In normal physiological conditions, the brain rids itself of toxic proteins using various clearance mechanisms. The efficacy of brain clearance can be adversely affected by the presence of toxic proteins and is also known to decline with age. Motivated by recent findings, such as the connection between brain cerebrospinal fluid clearance and sleep, we propose a mathematical model coupling the progression of toxic proteins over the brains structural network and protein clearance. The model is used to study the interplay between clearance in the brain, toxic seeding, brain network connectivity, aging, and progression in neurodegenerative diseases such as Alzheimers disease. Our findings provide a theoretical framework for the growing body of clinical results showing that clearance plays an important role in the etiology, progression and treatment of Alzheimers disease.