title: Characterizing the effect of small-scale topographic variability on co-existing native and invasive species in a heterogeneous grassland using airborne hyperspectral remote sensing

abstract:
O_LICharacterizing the distribution, mechanism, and behaviour of invasive species is crucial to implementing an effective plan for the protection and management of native grassland ecosystems. Hyperspectral remote sensing has been used for mapping and monitoring invasive species at various spatial and temporal scales. However, most studies focus either on invasive tree species mapping or on the landscape-level using low-spatial resolution remote sensing imagery. These low-resolution images are not fine enough to distinguish individual invasive grasses, especially in a heterogeneous environment where invasive species are small, fragmented, and co-existing with native plants with similar color and texture.
C_LIO_LITo capture the small yet highly dynamic invasive plants at different stages of the growing season and under various topography and hydrological conditions, we use airborne high-resolution narrow-band hyperspectral imagery (HrHSI) to map invasive species in a heterogeneous grassland ecosystem in southern Ontario, Canada.
C_LIO_LIThe results show that there is high spectral and textural separability between invasive species and between invasive and native plants, leading to an overall species classification accuracy of up to 89.6%. The combination of resultant species-level maps and the digital elevation model (DEM) showed that seasonality is the dominant factor that drives the distribution of invasive species at the landscape level, while small-scale topographic variations partially explain local patches of invasive species.
C_LIO_LIThis study provides insights into the feasibility of using HrHSI in mapping invasive species in a heterogeneous ecosystem and offers the means to understand the mechanism and behaviour of invasive species for a more effective grassland management strategy.
C_LI


title: Emergent RNA-RNA interactions can promote stability in a nascent phototrophic endosymbiosis

abstract:
Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms which act to stabilise nascent endosymbioses - between two fundamentally selfish biological organisms - are unclear. Theory suggests that enforcement mechanisms, which punish misbehaviour, may act to stabilise such interactions by resolving conflict. However, how such mechanisms can emerge in a nascent endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model nascent endosymbiosis, Paramecium bursaria - Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived mRNA released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock-down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knock-down in response to endosymbiont-derived RNA processing by host RNAi factors, which we term  RNAi-collisions, represents a mechanism which can promote stability in a nascent eukaryote-eukaryote endosymbiosis. By imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions and symbiotic status.

SIGNIFICANCE STATEMENTStable endosymbiosis between eukaryotic microbes has driven the evolution of further cellular complexity. Yet the mechanisms which can act to stabilise a nascent eukaryote-eukaryote endosymbiosis are unclear. Using the model nascent endosymbiotic system, Paramecium bursaria-Chlorella, we demonstrate that endosymbiont-host RNA-RNA interactions can drive a cost to host growth upon endosymbiont digestion, punishing the host for misbehaviour. These RNA-RNA interactions are facilitated by the host RNA-interference system. For endosymbiont mRNA sharing a high-level of sequence identity with host transcripts, this process can result in host gene knock-down. We propose that these endosymbiont-host RNA-RNA interactions- RNAi collisions-represent a viable enforcement mechanism to sanction the host for breakdown of the endosymbiosis, promoting the stability of a nascent endosymbiotic interaction.

category: evolutionary biology


title: Recurring adaptive introgression of a supergene that determines social organization

abstract: Introgression has been proposed as an essential source of adaptive genetic variation. However, a key barrier to adaptive introgression is that recombination can break down combinations of alleles that underpin many traits. This barrier might be overcome in supergene regions, where suppressed recombination leads to joint inheritance across many loci. Here, we study the evolution of a large supergene region that determines a major social and ecological trait in Solenopsis fire ants: whether colonies have one queen or multiple queens. Using coalescent-based phylogenies built from the genomes of 365 haploid fire ant males, we show that the supergene variant responsible for multiple-queen colonies evolved in one species and repeatedly spread to other species through introgressive hybridization. This finding highlights how supergene architecture can enable a complex adaptive phenotype to recurrently permeate species boundaries.


title: Recurring adaptive introgression of a supergene variant that determines social organization


title: The Population Genetics of Gene Regulatory Networks

abstract: The evolution of diverse phenotypes both involves and is constrained by molecular interaction networks. When these networks influence patterns of expression, we refer to them as gene regulatory networks (GRNs). Here, we develop a quasi-species model of GRN evolution. With this model, we prove that-across a broad spectrum of viability and mutation functions-the dynamics converge to a stationary distribution over GRNs. Next, we show from first principles how the frequency of GRNs at equilibrium will be proportional to each GRNs eigenvector centrality in the genotype network. Finally, we determine the structural characteristics of GRNs that are favored in response to a range of selective environments and mutational constraints. Our work connects GRN evolution to quasi-species models, and thus can provide a mechanistic explanation for the topology of GRNs experiencing various evolutionary forces.


title: The Ensemble of Gene Regulatory Networks at Mutation-Selection Balance


title: Adhesion dynamics regulate cell intercalation behaviour in an active tissue

abstract:
Cell intercalation is a key cell behaviour of morphogenesis and wound healing, where local cell neighbour exchanges can cause dramatic tissue deformations such as body axis extension. Here, we develop a mechanical model to understand active cell intercalation behaviours in the context of an epithelial tissue. Extending existing descriptions, such as vertex models, the junctional actomyosin cortex of every cell is modelled as a continuum morphoelastic rod, explicitly representing cortices facing each other at bicellular junctions. Cells are described directly in terms of the key subcellular constituents that drive dynamics, with localised stresses from the contractile actomyosin cortex and adhesion molecules coupling apposed cortices. This multi-scale apposed-cortex formulation reveals key behaviours that drive tissue dynamics, such as cell-cell shearing and flow of junctional material past cell vertices. We show that cell neighbour exchanges can be driven by purely junctional mechanisms. Active contractility and viscous turnover in a single bicellular junction are sufficient to shrink and remove a junction. Next, the 4-way vertex is resolved and a new, orthogonal junction extends passively. The adhesion timescale defines a frictional viscosity that is an important regulator of these dynamics, modulating tension transmission in the tissue as well as the speeds of junction shrinkage and growth. The model additionally predicts that rosettes, which form when a vertex becomes common to many cells, are likely to occur in active tissues with high adhesive friction.

SIGNIFICANCECell intercalation, or neighbour exchange, is a crucial behaviour that can drive tissue deformations, dissipate stress and facilitate wound healing. Substantial experimental work has identified the key molecular players facilitating intercalation, but there remains a lack of consensus and understanding of their physical roles. Existing biophysical models that represent cell-cell contacts with single edges cannot study the continuous dynamics of intercalation, involving shear between coupled cell cortices. Deriving a continuum description of the cell cortex, explicitly coupling neighbouring cortices with adhesions, we define the biophysical conditions required for successful neighbour exchanges. Furthermore, we show how the turnover of adhesion molecules specifies a viscous friction that regulates active tissue dynamics.

category: biophysics


title: Hsp40s display class-specific binding profiles, serving complementary roles in the prevention of tau amyloid formation

abstract: The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles, forming insoluble, amyloid-type aggregates associated with neurodegenerative conditions, such as Alzheimers disease. Tau aggregation, however, can be prevented in the cell by a class of proteins known as molecular chaperones, which play important roles in maintaining protein homeostasis. While numerous chaperones are known to interact with tau, though, little is known about the detailed mechanisms by which these prevent tau aggregation. Here, we describe the effects of the ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid fiber formation and compare these to the well-studied small heat shock protein HSPB1. We find that each chaperone prevents tau aggregation differently, by interacting with distinct sets of tau species along the aggregation pathway and thereby affecting their incorporation into fibers. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone tau conformers and even mature fibers, thus efficiently preventing formation of tau amyloids. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, different site in CTDI. These results provide important insight into the molecular mechanism by which the different members of the Hsp40 chaperone family counteract the formation, propagation, and toxicity of tau aggregates. Furthermore, our findings highlight the fact that chaperones from different families and different classes play distinct, but complementary roles in preventing pathological protein aggregation.


title: Analysis of Conformational Exchange Processes using Methyl-TROSY-Based Hahn Echo Measurements of Quadruple-Quantum Relaxation

abstract: Transverse nuclear spin relaxation can be a sensitive probe of chemical exchange on timescales on the order of microseconds to milliseconds. Here we present an experiment for the simultaneous measurement of the relaxation rates of two four-spin transitions in selectively protonated methyl groups within perdeuterated biomacromolecules, alongside control experiments for measurement of 1H and 13C chemical shift anisotropies. We show that analysis of the static magnetic field dependence of zero-, double- and quadruple-quantum Hahn echo relaxation rates provides a robust indication of chemical exchange and determines the signed relative magnitudes of proton and carbon chemical shift differences between ground and excited states. The analysis can be combined with CPMG relaxation dispersion measurements to provide improved precision, particularly in the determination of 1H chemical shift differences.


title: An evidence-based 3D reconstruction of Asteroxylon mackiei the most complex plant preserved from the Rhynie chert

abstract: The 407-million-year-old Rhynie chert preserves the earliest terrestrial ecosystem and informs our understanding of early life on land. However, our knowledge of the 3D structure, and development of these plants is still rudimentary. Here we used digital 3D reconstruction techniques to produce the first complete reconstruction of the lycopsid Asteroxylon mackiei, the most complex plant in the Rhynie chert. The reconstruction reveals the organisation of the three distinct axes types - leafy shoot axes, root-bearing axes and rooting axes - in the body plan. Combining this reconstruction with developmental data from fossilised meristems, we demonstrate that the A. mackiei rooting axis - a transitional lycophyte organ between the rootless ancestral state and true roots - developed from root-bearing axes by anisotomous dichotomy. Our discovery demonstrates how this unique organ developed, and highlights the value of evidence-based reconstructions for understanding the development and evolution of the first complex plants on Earth.

category: paleontology


title: Resolving leaf level metabolism of C4 Setaria viridis acclimated to low light through a biochemical model

abstract:
When C4 plants are exposed to low light, CO2 concentration in the bundle sheath (BS) decreases, causing an increase in photorespiration, leakiness (the ratio of CO2 leak rate out of the BS over the rate of supply via C4 acid decarboxylation), and a consequent reduction in biochemical efficiency. This can to some extent be mitigated by complex acclimation syndromes, which are of primary importance for crop productivity, but not well studied. We unveil a strategy of leaf-level low light acclimation involving regulation of electron transport processes. Firstly, we characterise anatomy, gas-exchange and electron transport of Setaria viridis grown under low light. Through a newly developed biochemical model we resolve the photon fluxes, and reaction rates to explain how these concerted acclimation strategies sustain photosynthetic efficiency. Smaller BS in low light-grown plants limited leakiness but sacrificed light harvesting and ATP production. To counter ATP shortage and maintain high assimilation rates, plants facilitated light penetration through mesophyll and upregulated cyclic electron flow in the BS. This novel shade tolerance mechanism based on optimisation of light reactions is more efficient than the known mechanisms involving the rearrangement of dark reactions and can potentially lead to innovative strategies for crop improvement.

SignificanceWe mechanistically link the optical cross section with the rate of electron transport, the engagement of cyclic electron flow, the relative rate of ATP and NADPH generation, and ultimately the apportioning of dark reactions between mesophyll and bundle sheath cells. The striking capacity of S. viridis to acclimate light reactions presents a novel and perhaps the most efficient shade tolerance strategy, potentially leading to novel possibilities for crop improvement.

category: plant biology


title: The neural correlates of semantic and grammatical encoding during sentence production in a second language: evidence from an fMRI study using syntactic priming

abstract: Japanese English learners have difficulty speaking Double Object (DO; give B A) than Prepositional Object (PO; give A to B) structures which neural underpinning is unknown. In speaking, syntactic and phonological processing follow semantic encoding, conversion of non-verbal mental representation into a structure suitable for expression. To test whether DO difficulty lies in linguistic or prelinguistic process, we conducted functional magnetic resonance imaging. Thirty participants described cartoons using DO or PO, or simply named them. Greater reaction times and error rates indicated DO difficulty. DO compared with PO showed parieto-frontal activation including left inferior frontal gyrus, reflecting linguistic process. Psychological priming in PO produced immediately after DO and vice versa compared to after control, indicated shared process between PO and DO. Cross-structural neural repetition suppression was observed in occipito-parietal regions, overlapping the linguistic system in pre-SMA. Thus DO and PO share prelinguistic process, whereas linguistic process imposes overload in DO.

category: neuroscience


title: Rapid appetitive transitions are sculpted by amygdala to accumbens pathways

abstract:
Foraging, pursuit, and predation rapidly transition into behavioral quiescence during reward capture and consumption. While appetitive-consummatory dissociations are embedded at both psychological and neural levels, the mechanisms controlling switches or transitions between appetitive seeking and consummatory behaviors remain poorly understood. Here we identify the BLA[->]AcbSh pathway as critical to these transitions by showing that this pathway inhibits the appetitive seeking response in the presence of consummatory demands. Using an appetitive cue-discrimination task in male rats, we show that reward delivery is a significant driver of seeking inhibition and that a BLA[->]AcbSh pathway mediates this inhibition. This role in suppressing seeking responses during periods of consumption was not due to a general suppression of behavior because responding to other cues during the same test was unaffected. Moreover, it was specific to the BLA[->]AcbSh pathway, because the contribution of the BLA[->]AcbC pathway to appetitive switching was distinct and modest. State-dependent silencing of BLA[->]AcbSh revealed that the modulation of seeking before and after reward delivery are co-dependent. Finally, we found that BLA terminals in AcbSh have functional connectivity to LH-projecting AcbSh neurons, thereby identifying a BLA[->]AcbSh[->]LH pathway as a putative route for the rapid regulation of appetitive behaviors. Taken together, these findings suggest that the BLA[->]AcbSh pathway is a core component of an appetitive switching system, recruited under conditions requiring rapid or dynamic shifts in appetitive behavior, and that this pathway enables these shifts by actively inhibiting seeking.

Significance statementForaging, pursuit, and predation quickly transition into behavioral quiescence during reward capture and consumption. These transitions are critical for flexible and responsive sequences of behavior. Here we show that behavioral transitions are actively controlled at the limbic-striatal interface. We identify reward receipt as a proximal trigger for transition between reward seeking and taking, we identify active inhibition as the functional operation of this transition, and we identify the basolateral amygdala[->]accumbens shell pathway as critical to this functional operation.


title: Complete neuroanatomy and sensor maps of Odonata wings for fly-by-feel flight control

abstract: Can mechanosensors in animal wings allow reconstruction of the wing aeroelastic states? Little is known about how flying animals utilize wing mechanosensation to monitor the dynamic state of their highly deformable wings. Odonata, dragonflies and damselflies, are a basal lineage of flying insects with excellent flight performance, and their wing mechanics have been studied extensively. Here, we present a comprehensive map of the wing sensory system for two Odonata species, including both the external sensor morphologies and internal neuroanatomy. We identified eight morphological classes of sensors; most were mechanosensors innervated by a single neuron. Their innervation patterns and morphologies minimize axon length and allow morphological latency compensation. We further mapped the major veins of another 13 Odonata species across 10 families and identified consistent sensor distribution patterns, with sensor count scaling with wing length. Finally, we constructed a high-fidelity finite element model of a dragonfly wing for structural analysis. Our dynamic loading simulations revealed features of the strain fields that wing sensor arrays could detect to encode different wing deformation states. Taken together, this work marks the first step toward an integrated understanding of fly-by-feel control in animal flight.


title: Complete neuroanatomy and sensor maps of dragonfly wings for fly-by-feel monitoring of aeroelasticity


title: Complete neuroanatomy of dragonfly wings suggests direct sensing of aeroelastic deformations


title: A complex feature-based representation of vocalizations emerges in the superficial layers of primary auditory cortex

abstract: Early in auditory processing, neural responses faithfully reflect acoustic input. At higher stages of auditory processing, however, neurons become selective for particular call types, eventually leading to specialized regions of cortex that preferentially process calls at the highest auditory processing stages. We previously proposed that an intermediate step in how non-selective responses are transformed into call-selective responses is the detection of informative call features. But how neural selectivity for informative call features emerges from non-selective inputs, whether feature selectivity gradually emerges over the processing hierarchy, and how stimulus information is represented in non-selective and feature-selective populations remain open questions. In this study, using unanesthetized guinea pigs, a highly vocal and social rodent, as an animal model, we characterized the neural representation of calls in three auditory processing stages - the thalamus (vMGB), and thalamorecipient (L4) and superficial layers (L2/3) of primary auditory cortex (A1). We found that neurons in vMGB and A1 L4 did not exhibit call-selective responses and responded throughout the call durations. However, A1 L2/3 neurons showed high call-selectivity with about a third of neurons responding to only one or two call types. These A1 L2/3 neurons only responded to restricted portions of calls suggesting that they were highly selective for call features. Receptive fields of these A1 L2/3 neurons showed complex spectrotemporal structures that could underlie their high call feature selectivity. Information theoretic analysis revealed that in A1 L4 stimulus information was distributed over the population and was spread out over the call durations. In contrast, in A1 L2/3, individual neurons showed brief bursts of high stimulus-specific information, and conveyed high levels of information per spike. These data demonstrate that a transformation in the neural representation of calls occurs between A1 L4 and A1 L2/3, leading to the emergence of a feature-based representation of calls in A1 L2/3. Our data thus suggest that observed cortical specializations for call processing emerge in A1, and set the stage for further mechanistic studies.


title: Lower promoter activity of the ST8SIA2 gene has been favored in evolving human collective brains

abstract: ST8SIA2 is the main factor regulating expression of the phenotype involved in schizophrenia. Lowered promoter activity of the ST8SIA2 gene is considered to be protective against schizophrenia by conferring tolerance to psychosocial stress. Here, we examined the promoter type composition of anatomically modern humans (AMHs) and archaic humans (AHs; Neanderthals and Denisovans), and compared the promoter activity at the population level (population promoter activity; PPA) between them. In AMHs, the TCT-type, showing the second lowest promoter activity, was most prevalent in the ancestral population of non-Africans. However, the detection of only the CGT-type from AH samples and recombination tracts in AH sequences showed that the CGT- and TGT-types, exhibiting the two highest promoter activities, were common in AH populations. Furthermore, interspecies gene flow occurred into AMHs from AHs and into Denisovans from Neanderthals, influencing promoter-type compositions independently in both AMHs and AHs. The difference of promoter-type composition makes PPA unique in each population. East and Southeast Asian populations show the lowest PPA. This results from the selective increase of the CGC-type, showing the lowest promoter activity, in these populations. Every non-African population shows significantly lower PPA than African populations, resulting from the TCT-type having the highest prevalence in the ancestral population of non-Africans. In addition, PPA reduction is also found among subpopulations within Africa via a slight increase of the TCT-type. These findings indicate a trend toward lower PPA in the spread of AMHs, interpreted as a continuous adaptation to psychosocial stress arising in migration. This trend is considered as genetic tuning for the evolution of collective brains. The inferred promoter-type composition of AHs differed markedly from that of AMHs, resulting in higher PPA in AHs than in AMHs. This suggests that the trend toward lower PPA is a unique feature in AMH spread.


title: Ahead of Maturation: Enhanced Speech Envelope Training Boosts Rise Time Discrimination in Pre-Readers at Cognitive Risk for Dyslexia

abstract:
Dyslexia has frequently been related to atypical auditory temporal processing and speech perception. Results of studies emphasizing speech onset cues and reinforcing the temporal structure of the speech envelope, i.e. envelope enhancement, demonstrated reduced speech perception deficits in individuals with dyslexia. The use of this strategy as an auditory intervention might thus reduce some of the deficits related to dyslexia. Importantly, interventions are most effective when they are provided during kindergarten and first grade. Hence, we provided a tablet-based 12-week preventive auditory and phonics-based intervention to pre-readers at cognitive risk for dyslexia and investigated the effect on auditory temporal processing with a rise time discrimination task. Ninety-one pre-readers at cognitive risk for dyslexia (aged 5-6) were assigned to two groups receiving a phonics-based intervention and playing a story listening game either with (n = 31) or without (n = 31) envelope enhancement or a third group playing control games and listening to non-enhanced stories (n = 29). Rise time discrimination was measured directly before, directly after and one year after the intervention. While the groups listening to non-enhanced stories mainly improved after the intervention during first grade, the group listening to enhanced stories improved during the intervention in kindergarten and subsequently remained stable during first grade. Hence, an envelope enhancement intervention improves auditory processing skills important for the development of phonological skills. This occurred before the onset of reading instruction, preceding the maturational improvement of these skills, hence giving at risk children a head start when learning to read.

Research highlightsO_LIThe first investigation of speech envelope enhancement as a potential preventive intervention strategy in pre-readers at cognitive risk for dyslexia
C_LIO_LISpeech envelope enhancement increases the rise time sensitivity of children at cognitive risk for dyslexia
C_LIO_LIRise time discrimination can be enhanced before formal reading instruction, a crucial period in development
C_LI


title: Maternal diet-induced obesity during pregnancy alters lipid supply to fetuses and changes the cardiac tissue lipidome in a sex- dependent manner

abstract: Maternal obesity during pregnancy has immediate and long-term detrimental effects on the offspring heart. In this study, we characterized the cardiac and circulatory lipid profiles in fetuses of diet-induced obese pregnant mice and established the changes in lipid abundance and fetal cardiac transcriptomics. We used untargeted and targeted lipidomics and transcriptomics to define changes in the serum and cardiac lipid composition and fatty acid metabolism in male and female fetuses. From these analyses we observed: (1) maternal obesity affects the maternal and fetal serum lipidome distinctly; (2) female heart lipidomes are more sensitive to maternal obesity than male fetuses; (3) changes in lipid supply might contribute to early expression of lipolytic genes in mouse hearts exposed to maternal obesity. These results highlight the existence of sexually dimorphic responses of the fetal heart to the same in utero obesogenic environment and identify lipids species that might mediate programming of cardiovascular health.

category: developmental biology


title: A muscle-related contractile tissue specified by MRTF-activity in Porifera

abstract: Muscle-based movement is a hallmark of animal biology, but the evolutionary origins of myocytes - the cells that comprise muscle tissues - are unknown. Sponges (Porifera) provide an opportunity to reconstruct the earliest periods of myocyte evolution. Although sponges are believed to lack muscle, they are capable of coordinated whole-body contractions that purge debris from internal water canals. This behavior has been observed for decades, but their contractile tissues remain uncharacterized; it is an open question whether they have affinity to muscle or non-muscle contractile tissues in other animals. Here, we characterize the endothelial-like lining of water canals (the endopinacoderm) as a primary contractile tissue in the sponge Ephydatia muelleri. We find tissue-wide organization of contractile actin-bundles that contain striated-muscle myosin II and transgelin, and that contractions are regulated by the release of internal Ca2+ stores upstream of the myosin-light-chain-kinase (MLCK) pathway. Further, we show that the endopinacoderm is developmentally specified by myocardin-related transcription factor (MRTF) as part of an environmentally-inducible transcriptional complex that otherwise is known to function in muscle development, plasticity, and regeneration in other animals. We conclude that both muscle tissues and the endopinacoderm evolved from a common, multifunctional contractile tissue in the animal stem-lineage. Furthermore, as an actin-regulated force-sensor, MRTF-activity offers a mechanism for how water canals dynamically remodel in response to flow and can re-form normally from stem-cells in the absence of the intrinsic positional cues characteristic of embryogenesis in other animals.


title: A muscle-related contractile tissue specified by myocardin-related transcription factor activity in Porifera


title: CRISPR Knock-in Designer: automatic oligonucleotide design software to introduce point mutations using CRISPR/Cas9

abstract: Knock-in of precise point mutations into protein-coding genes has been one of the earliest and most important applications of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9. The ability to perform such precise gene editing is crucial to interrogate the function of specific protein residues and to create models of human diseases caused by protein amino acid changes. The homologous protein residues can be mutated in model animal species, and the consequences of these mutations can be studied, leading to a better understanding of the disease in question. Design of point mutation knock-in strategies has been a combination of manual steps assisted by several computational tools resulting in a time-consuming process and preventing a single rapid and integrated solution. We have therefore designed CRISPR Knock-in Designer, which can perform rapid and automatic design of point mutation knock-in DNA oligonucleotides upon provision of the mutation, a guide RNA, and essential identifier or sequence information. The tool supports most experimentally established CRISPR types and has multiple options for the resulting oligonucleotides to satisfy the needs of most users. We also provide allele-specific PCR-based and restriction enzyme-based genotyping strategies as part of the program output. CRISPR Knock-in Designer adjusts to the genomic context of any target codon and tries to design knock-in strategies when a codon straddles two exons, a situation we explored in whole genomes of several model species. CRISPR Knock-in Designer output can also be adapted for use with some of the newer Prime Editing design tools to facilitate the introduction of a specific mutation sequence using this advanced technology.

category: genetics


title: Experiment level curation identifies high confidence transcriptional regulatory interactions in neurodevelopment

abstract:
To facilitate the development of large-scale transcriptional regulatory networks (TRNs) that may enable in-silico analyses of disease mechanisms, a reliable catalogue of experimentally verified direct transcriptional regulatory interactions (DTRIs) is needed for training and validation. There has been a long history of using low-throughput experiments to validate single DTRIs. Therefore, we hypothesize that a reliable set of DTRIs could be produced by curating the published literature for such evidence. In our survey of previous curation efforts, we identified the lack of details about the quantity and the types of experimental evidence to be a major gap, despite the importance of such details for the identification of bona fide DTRIs. We developed a curation protocol to inspect the published literature for support of DTRIs at the experiment level, focusing on genes important to the development of the mammalian nervous system. We sought to record three types of low-throughput experiments: Transcription factor (TF) perturbation, TF-DNA binding, and TF-reporter assays. Using this protocol, we examined a total of 1,310 papers to assemble a collection of 1,499 unique DTRIs, involving 251 TFs and 825 target genes, many of which were not reported in any other DTRI resource. The majority of DTRIs (965, 64%) were supported by two or more types of experimental evidence and 27% were supported by all three. Of the DTRIs with all three types of evidence, 170 had been tested using primary tissues or cells and 44 had been tested directly in the central nervous system. We used our resource to document research biases among reports towards a small number of well-studied TFs. To demonstrate a use case for this resource, we compared our curation to a previously published high-throughput perturbation screen and found significant enrichment of the curated targets among genes differentially expressed in the developing brain in response to Pax6 deletion. This study demonstrates a proof-of-concept for the assembly of a high confidence DTRI resource in order to support the development of large-scale TRNs.

Author SummaryThe capacity to computationally reconstruct gene regulatory networks using large-scale biological data is currently limited by the absence of a high confidence set of one-to-one regulatory interactions. Given the lengthy history of using small scale experimental assays to investigate individual interactions, we hypothesize that a reliable collection of gene regulatory interactions could be compiled by systematically inspecting the published literature. To this end, we developed a curation protocol to examine and record evidence of regulatory interactions at the individual experiment level. Focusing on the area of brain development, we applied our pipeline to 1,310 publications. We identified 3,601 individual experiments, providing detailed information about 1,499 regulatory interactions. Many of these interactions have verified activity specifically in the embryonic brain. By capturing reports of regulatory interactions at this level of granularity, we present a resource that is more interpretable than other similar resources.

category: genomics


title: Energy expenditure does not explain step length-width choices during walking

abstract: Healthy young adults have a most preferred walking speed, step length, and step width that are close to energetically optimal. However, people can choose to walk with a multitude of different step lengths and widths, which can vary in both energy expenditure and preference. Here we further investigate step length-width preferences and their relationship to energy expenditure. In line with a growing body of research, we hypothesized that peoples preferred stepping patterns would not be fully explained by metabolic energy expenditure. To test this hypothesis we used a two-alternative forced-choice paradigm. Fifteen participants walked on an oversized treadmill. Each trial participants experienced two stepping patterns and then chose the pattern they preferred. Over time, we adapted the choices such that there was 50% chance of choosing one pattern over another (equally preferred). If peoples preferences are based solely on metabolic energy expenditure, then these equally preferred stepping patterns should have equal energy expenditure. We found that energy expenditure differed across equally preferred step length-width patterns (p < 0.001). On average, longer steps with higher energy expenditures were preferred over shorter and wider steps with lower energy expenditures (p < 0.001). We also asked participants to rank a set of shorter, wider, and longer steps from most preferred to least preferred, and from most energy expended to least energy expended. Only 7/15 participants had the same rankings for their preferences and perceived energy expenditure. Our results suggest that energy expenditure is not the only factor influencing a persons conscious gait choices.


title: Human Fronto-Striatal Connectivity is Organized into Discrete Functional Subnetworks

abstract: The striatum is interconnected with the cerebral cortex via multiple recurrent loops that play a major role in many neuropsychiatric conditions. Primate cortico-striatal connections can be precisely mapped using invasive tract-tracing. However, noninvasive human research has not mapped these connections with anatomical precision, limited by the practice of averaging neuroimaging data across individuals. Here we utilized highly-sampled resting-state functional connectivity MRI for individually-specific precision functional mapping of cortico-striatal connections. We identified ten discrete, individual-specific subnetworks linking cortex--predominately frontal cortex--to striatum. These subnetworks included previously unknown striatal connections to the human language network. The discrete subnetworks formed a stepped rostral-caudal gradient progressing from nucleus accumbens to posterior putamen; this organization was strongest for projections from medial frontal cortex. The stepped gradient organization fit patterns of fronto-striatal connections better than a smooth, continuous gradient. Thus, precision subnetworks identify detailed, individual-specific stepped gradients of cortico-striatal connectivity that include human-specific language networks.


title: Direct neural evidence for the contrastive roles of the complementary learning systems in adult acquisition of native vocabulary.

abstract: The Complementary Learning Systems (CLS) theory provides a powerful framework for considering the acquisition, consolidation and generalisation of new knowledge. We tested this proposed neural division of labour in adults through an investigation of the consolidation and long-term retention of newly-learned native vocabulary with post-learning functional neuroimaging. Newly-learned items were compared to two conditions: (i) previously known items to highlight the similarities and differences with established vocabulary; and (ii) unknown/untrained items to provide a control for non-specific perceptual and motor-speech output. Consistent with the CLS, retrieval of newly-learned items was supported by a combination of regions associated with episodic memory (including left hippocampus) and the language-semantic areas that support established vocabulary (left inferior frontal gyrus and left anterior temporal lobe). Furthermore, there was a shifting division of labour across these two networks in line with the items consolidation status; faster naming was associated with more activation of language-semantic areas and lesser activation of episodic memory regions. Hippocampal activity during naming predicted more than half the variation in naming retention six months later.


title: Direct neural evidence for the contrastive roles of the complementary learning systems in adult acquisition of native vocabulary


title: Direct and Indirect Pathway Neurons in Ventrolateral Striatum Differentially Regulate Licking Movement and Nigral Responses

abstract: Drinking behavior in rodents is characterized by stereotyped, rhythmic licking movement, which is regulated by the basal ganglia. It is unclear how direct and indirect pathways control the lick bout and individual spout contact. We find that inactivating D1 and D2 receptors-expressing medium spiny neurons (MSNs) in the ventrolateral striatum (VLS) oppositely alters the number of licks in a bout. D1- and D2-MSNs exhibit different patterns of lick sequence-related activity and different phases of oscillation time-locked to the lick cycle. On timescale of a lick cycle, transient inactivation of D1-MSNs during tongue protrusion reduces spout contact probability, whereas transiently inactivating D2-MSNs has no effect. On timescale of a lick bout, inactivation of D1-MSNs (D2-MSNs) causes rate increase (decrease) in a subset of basal ganglia output neurons that decrease firing during licking. Our results reveal the distinct roles of D1- and D2-MSNs in regulating licking at both coarse and fine timescales.


title: New insights of glycosylation role on variable domain of antibody structures

abstract:
N-glycosylation at antibody variable domain (FvN-glyco) has emerged as an important modification for antibody function such as stability and antigen recognition, but it is also associated with autoimmune disease and IgE-mediated hypersensitivity reaction. However, the information related to its role and regulation is still scarce. Therefore, we investigated new insights in this regarding using solved antibodies structures presenting in the Protein Data Bank (PDB). From 130 FvN-glyco structures, we observed significant findings as a higher prevalence of N-glycosylation in human and mouse antibodies containing IGHV1-8 and IGHV2-2 germline genes, respectively. We also speculate the influence of activation-induced cytidine deaminase (AID) in introducing N-glycosylation sites during somatic hypermutation, specifically on threonine to asparagine substitution. Moreover, we highlight the enrichment of anti-HIV antibodies containing N-glycosylation at antibody variable domain and where we showed a possible important role of N-glycosylation, besides to antigen-antibody interactions, in antibody chain pair and antibody-antibody interactions. These could be a positive secondary effect of glycosylation to enhance antigen binding and further neutralization, including an additional mechanism to form Fab-dimers. Overall, our findings extend the knowledge on the characteristics and diverse role of N-glycosylation at antibody variable domain.

Key PointsO_LIPrevalence of FvN-glyco in human IGHV1-8 and mouse IGHV2-2 germline genes.
C_LIO_LIEnrichment of antibody FvN-glyco against virus, especially anti-HIV-1.
C_LIO_LIFvN-glyco in the interface with antigen, antibody pair chain, and another antibody.
C_LI

category: immunology


title: A noncatalytic activity of the H4K20 demethylase DPY-21 regulates condensin DC binding

abstract: Condensin is a multi-subunit SMC complex that binds to and compacts chromosomes. Here we addressed the regulation of condensin binding dynamics using C. elegans condensin DC, which represses X chromosomes in hermaphrodites for dosage compensation. We established fluorescence recovery after photobleaching (FRAP) using the SMC4 homolog DPY-27 and showed that a well-characterized ATPase mutation abolishes its binding. Next, we performed FRAP in the background of several chromatin modifier mutants that cause varying degrees of X-chromosome derepression. The greatest effect was in a null mutant of the H4K20me2 demethylase DPY-21, where the mobile fraction of condensin DC reduced from [~]30% to 10%. In contrast, a catalytic mutant of dpy-21 did not regulate condensin DC mobility. Hi-C data in the dpy-21 null mutant showed little change compared to wild type, uncoupling Hi-C measured long-range DNA contacts from transcriptional repression of the X chromosomes. Together, our results indicate that DPY-21 has a non-catalytic role in regulating the dynamics of condensin DC binding, which is important for transcription repression.

category: cell biology


title: The H4K20 demethylase DPY-21 regulates thedynamics of condensin DC binding


title: Using random effects in generalized linear mixed-effects models: assessing a common "rule of thumb"

abstract: As generalized linear mixed-effects models (GLMMs) have become a widespread tool in ecology, the need to guide the use of such tools is increasingly important. One common guideline is that one needs at least five levels of a random effect. Having such few levels makes the estimation of the variance of random effects terms (such as ecological sites, individuals, or populations) difficult, but it need not muddy ones ability to estimate fixed effects terms - which are often of primary interest in ecology. Here, I simulate ecological datasets and fit simple models and show that having too few random effects terms does not influence the parameter estimates or uncertainty around those estimates for fixed effects terms. Thus, it should be acceptable to use fewer levels of random effects if one is not interested in making inference about the random effects terms (i.e. they are  nuisance parameters used to group non-independent data). I also use simulations to assess the potential for pseudoreplication in (generalized) linear models (LMs), when random effects are explicitly ignored and find that LMs do not show increased type-I errors compared to their mixed-effects model counterparts. Instead, LM uncertainty (and p values) appears to be more conservative in an analysis with a real ecological dataset presented here. These results challenge the view that it is never appropriate to model random effects terms with fewer than five levels - specifically when inference is not being made for the random effects, but suggest that in simple cases LMs might be robust to ignored random effects terms. Given the widespread accessibility of GLMMs in ecology and evolution, future simulation studies and further assessments of these statistical methods are necessary to understand the consequences of both violating and blindly following simple guidelines.

category: ecology


title: Including random effects in statistical models in ecology: fewer than five levels?


title: Direct reconstitution and study of SUN protein interactions in vitro using mammalian cell-free expression

abstract:
Membrane proteins perform numerous important functions in cells and tissues. Approximately 20% of the human genome encodes for membrane proteins, which represent the majority of targets for clinically relevant small molecules. Consequently, understanding their structure and structure-function relationships is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional membrane proteins in supported lipid bilayers. Despite having shown the directional reconstitution of CFE-synthesized FL inner nuclear membrane SUN proteins (i.e. SUN1 and SUN2), which directly interact with outer nuclear membrane KASH proteins within the nuclear envelope lumen to form linker of nucleoskeleton and cytoskeleton (LINC) complexes that mechanically couple the cytoskeleton and nucleus, the mechanism underlying regulated LINC complex assembly remains unclear. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment (e.g. calcium ions and redox state) on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and hetero-typic interactions that can occur between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies of membrane protein biochemistry and function, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.

ABSTRACT GRAPHIC O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: biochemistry


title: In vitro synthesis and reconstitution using mammalian cell-free lysates enables the systematic study of the regulation of LINC complex assembly


title: Broadly-reactive IgG responses to heterologous H5 prime-boost influenza vaccination are shaped by antigenic relatedness to priming strains

abstract:
Prime-boost vaccinations of humans with different H5 strains have generated broadly protective antibody levels. However, the effect of an individuals H5 exposure history on antibody responses to subsequent H5 vaccination is poorly understood. To investigate this, we analyzed the IgG response to H5 A/Indonesia/5/2005 (Ind05) vaccination in three cohorts: (1) a double primed group that received two H5 vaccinations: A/Vietnam/203/2004 (Vie04) 5 years ago and A/Hong Kong/156/1997 (HK97) 11 years ago, (2) a single primed group that received Vie04 5 years ago, and (3) an H5-naive group that received two doses of the Ind05 vaccine 28 days apart. Hemagglutinin (HA)-reactive IgG levels were estimated by multiplex assay against an HA panel that included 21 H5 strains and 9 other strains representing H1, H3, H7, and H9 subtypes. Relative HA antibody landscapes were generated to quantitatively analyze the magnitude and breadth of antibody binding after vaccination. We found that short-interval prime-boosting with the Ind05 in the naive group generated a low anti-H5 response. Both primed groups generated robust antibody responses reactive to a broad range of H5 strains after boosting with Ind05; IgG antibody levels persisted longer in subjects who had been double primed years ago. Notably, the IgG responses were strongest against the first priming H5 strain, that reflecting influenza virus immune imprinting. Finally, the broad anti-H5 IgG response was stronger against strains having a small antigenic distance to the initial priming strain.

IMPORTANCEThe antigenic shift and draft of hemagglutinin (HA) in influenza viruses is accepted as one of the major reasons for immune evasion. The analysis of B cell immune responses to influenza infection and vaccination is complicated by the impact of exposure history and antibody cross-reaction between antigenically similar influenza strains. To assist in such analyses, the influenza "antibody landscape" method has been used to analyze and visualize the relationship of antibody mediated immunity to the antigenic distance between influenza strains. In this study, we describe a "relative antibody landscape" method, calculating the antigenic distance between the vaccine influenza strain and other H5 strains, and using this relative antigenic distance to plot with the anti-H5 IgG levels post-vaccination. This new method quantitatively estimates and visualizes the correlation between the humoral response to a particular influenza strain, and the antigenic distance to other strains. Our findings demonstrate the effect of H5 exposure history on H5 vaccine responses quantified by the relative antibody landscape method.


title: Why most Principal Component Analyses (PCA) in population genetic studies are wrong

abstract: Principal Component Analysis (PCA) is a multivariate analysis that allows reduction of the complexity of datasets while preserving data covariance and visualizing the information on colorful scatterplots, ideally with only a minimal loss of information. PCA applications are extensively used as the foremost analyses in population genetics and related fields (e.g., animal and plant or medical genetics), implemented in well-cited packages like EIGENSOFT and PLINK. PCA outcomes are used to shape study design, identify, and characterize individuals and populations, and draw historical and ethnobiological conclusions on origins, evolution, dispersion, and relatedness. The replicability crisis in science has prompted us to evaluate whether PCA results are reliable, robust, and replicable. We employed an intuitive color-based model alongside human population data for eleven common test cases. We demonstrate that PCA results are artifacts of the data and that they can be easily manipulated to generate desired outcomes. PCA results may not be reliable, robust, or replicable as the field assumes. Our findings raise concerns about the validity of results reported in the literature of population genetics and related fields that place a disproportionate reliance upon PCA outcomes and the insights derived from them. We conclude that PCA may have a biasing role in genetic investigations. An alternative mixed-admixture population genetic model is discussed.


title: DAnIEL: A User-Friendly Web Server for Fungal ITS Amplicon Sequencing Data

abstract: Trillions of microbes representing all kingdoms of life are resident in, and on, humans holding essential roles for host development and physiology. The last decade over a dozen online tools and servers, accessible via public domain, have been developed for the analysis of bacterial sequences, however, the analysis of fungi is still in its infancy. Here we present a web server dedicated to the comprehensive analysis of the human mycobiome for (i) translating raw sequencing reads to data tables and high-standard figures; (ii) integrating statistical analysis and machine learning with a manually curated relational database; (iii) comparing the users uploaded datasets with publicly available from the Sequence Read Archive. Using 2,048 publicly available ITS samples, we demonstrated the utility of DAnIEL web server on large scale datasets and show the differences in fungal communities between human gut, skin, nasopharynx, and oral body sites.

category: bioinformatics


title: Utilizing scp for the analysis and replication of single-cell proteomics data

abstract:
IntroductionMass spectrometry-based proteomics is actively embracing quantitative, single-cell level analyses. Indeed, recent advances in sample preparation and mass spectrometry (MS) have enabled the emergence of quantitative MS-based single-cell proteomics (SCP). While exciting and promising, SCP still has many rough edges. The current analysis workflows are custom and built from scratch. The field is therefore craving for standardized software that promotes principled and reproducible SCP data analyses.

Areas coveredThis special report is the first step toward the formalization and standardization of SCP data analysis. scp, the software that accompanies this work, successfully replicates one of the landmark SCP studies and is applicable to other experiments and designs. We created a repository containing the replicated workflow with comprehensive documentation in order to favor further dissemination and improvements of SCP data analyses.

Expert opinionReplicating SCP data analyses uncovers important challenges in SCP data analysis. We describe two such challenges in detail: batch correction and data missingness. We provide the current state-of-the-art and illustrate the associated limitations. We also highlight the intimate dependence that exists between batch effects and data missingness and offer avenues for dealing with these exciting challenges.

1 Article highlightsO_LISingle-cell proteomics (SCP) is emerging thanks to several recent technological advances, but further progress is still lagging due to the lack of principled and systematic data analysis.
C_LIO_LIThis work offers a standardized solution for the processing of SCP data demonstrated by the replication of a landmark SCP work.
C_LIO_LITwo important challenges remain: batch effects and data missingness. Furthermore, these challenges are not independent and therefore need to be modeled simultaneously.
C_LI


title: Replication of single-cell proteomics data reveals important computational challenges


title: Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain

abstract: Cytoplasmic dynein is activated by dynactin and cargo adapters in vitro, and the activation also needs LIS1 (Lissencephaly 1) in vivo. How this process is regulated remains unclear. Here we found in Aspergillus nidulans that a dynein AAA4 arginine-finger mutation bypasses the requirement of LIS1 for dynein activation driven by the early endosomal adapter HookA. As the AAA4 arginine-finger is implicated in AAA3 ATP hydrolysis, we examined AAA3 mutants defective in ATP binding and hydrolysis respectively. Astonishingly, blocking AAA3 ATP hydrolysis allows dynein activation by dynactin in the absence of LIS1 or HookA. As a consequence, dynein accumulates at microtubule minus ends while early endosomes stay near the plus ends. On the other hand, blocking AAA3 ATP binding abnormally prevents LIS1 from being dissociated from dynein upon motor activation. Thus, the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.


title: VHL inhibitor binding increases intracellular level of VHL

abstract: The von-Hippel Lindau (VHL) protein is a tumour suppressor protein frequently mutated in the VHL disease, which functions as substrate recognition subunit of a Cul2 E3 ubiquitin ligase (CRL2VHL). CRL2VHL plays an important role in oxygen sensing, by binding and targeting Hypoxia Inducible Factor-alpha subunits (HIF-alpha) for ubiquitination and degradation. VHL is also commonly hijacked by heterobifunctional degrader molecules known as proteolysis-targeting chimeras (PROTACs). In previous work we reported the structure-based design and functional characterisation of VHL inhibitors (VH032 and VH298) that induce the HIF response in cells. Here, we use unbiased quantitative mass spectrometry to identify the proteomic changes elicited by the VHL inhibitor and compare this to hypoxia or broad-spectrum prolyl-hydroxylase domain (PHD) enzyme inhibitor IOX2. Our results demonstrate the VHL inhibitor selectively activates the HIF response that is also present in the hypoxia- and IOX2-induced proteomic changes. Interestingly, VHL inhibitors were found to selectively upregulate a single protein, which is VHL itself. Our analysis revealed that this occurs via protein stabilisation of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn to reduced levels of HIF-1 protein. Our results demonstrate the high specificity of VHL inhibitors and suggest that use of these inhibitors would not produce overtly side effects due to prolonged HIF stabilisation. They also exemplify the concept that small-molecule binding induced protein stabilisation can increase protein levels inside cells.


title: Acyl Carrier Protein is essential for MukBEF action in Escherichia coli chromosome organization-segregation

abstract: Structural Maintenance of Chromosomes (SMC) complexes contribute ubiquitously to chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE accessory proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, is essential for in vivo function and is regulated by interactions with its dimeric kleisin, MukF, and KITE, MukE. Here we demonstrate that, in addition, MukB interacts with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction site at the joint of the MukB coiled-coil and show that the interaction is essential for MukB ATPase and for MukBEF function in vivo. Therefore, AcpP is an essential co-factor for MukBEF action in chromosome organization-segregation.


title: An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens

abstract: The trafficking of specific protein cohorts to the correct subcellular location at the correct time is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or FACS. To broadly empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identify genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in the mislocalization and destabilization of mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

category: bioengineering


title: Intracellular transport of electrotransferred DNA cargo is governed by coexisting ergodic and non ergodic anomalous diffusion

abstract: The ability of exogenous DNA cargo to overcome the active and viscoelastic eukaryotic cytoplasm is a principal determinant for the gene delivery efficacy. During DNA electrotransfer, DNA forms complexes with the membrane (DNA cargo) which is transported through the cytoplasm through a combination of passive diffusion and active transport. However, this process is poorly understood limiting rational optimization of DNA cargo to be delivered to different cell types. We have investigated the intracellular transport of DNA cargo (of sizes 100 bp, 250 bp and 500 bp) delivered by electrotransfer to non-cancerous and cancerous mammalian cells. We demonstrate that intracellular DNA cargo transport is governed by coexisting ergodic and non ergodic anomalous diffusion for all the tested DNA sizes and cell types. The apparent diffusion coefficient of the electrotransferred DNA cargo in the cytoplasm decreases when the DNA size is increased from 100 bp to 500 bp. Interestingly, the electrotransferred DNA cargo (500 bp) transport is strongly dependent on the cells cancer state. Intracellular electrotransferred DNA cargo transport has a higher probability of superdiffusive transport and lower probability of caging in metastatic cells compared to malignant cells followed by benign cells.


title: Cell Surface Multimeric Assemblies Regulate Canonical and Noncanonical EphA2 Receptor Tyrosine Kinase Signaling

abstract: The EphA2 receptor tyrosine kinase mediates ligand-induced canonical signaling associated with tumor suppression and ligand-independent noncanonical signaling implicated in tumor progression. Using time-resolved fluorescence spectroscopy in live cells, we find that unliganded EphA2 receptors pre-assemble into multimers, which is mediated by two symmetric and one asymmetric interfaces in the ectodomain. Upon ligand binding, EphA2 receptors are further assemble into large clusters that also requires the three interfaces. Functionally, disrupting either the symmetric or asymmetric contacts individually blocks the autorecycling of the EphA2 apo receptor. However, only symmetric contact disruption promotes noncanonical signaling and inhibits ligand-induced catalytic activation and endocytosis, which are associated with increased cell migration in vitro and reduced survival in a syngeneic murine glioblastoma model. Our results reveal the pivotal role of EphA2 assembly in dictating canonical vs. noncanonical signaling, and identify the precise molecular interfaces that mediate the formation of the EphA2 signaling clusters.


title: Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

abstract: The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches. In these two structures, Ubr1 adopts different conformations to facilitate the transfer of Ub from Ubc2 to either an N-degron peptide or a monoubiquitinated degron. These structures not only reveal the architecture of the Ubr1 complex but also provide mechanistic insights into the initiation and elongation steps of ubiquitination catalysed by Ubr1.


title: Cypripedium wardii (Orchidaceae) employs pseudopollen with both reward and deception to attract both flis and bees as pollinators

abstract:
O_LIFlowering plants always attract animals providing rewards or deceptive signals to gain reproductive success. However, there is no well-documented reporting about a pollination mechanism with both rewards and deceptive signals by a same object. We found Cypripedium wardii flowers seem to attract visitors by the white pseudopollenlike trichomes on labella in our preliminary field observation.
C_LIO_LITo explore the pollination mechanism of Cypripedium wardii, especially, the ecological function of the pseudopollen-like trichomes, we conducted field observations, analyses of the traits of visitors and flowers, and breeding system experiments.
C_LIO_LIThe white trichomes composed by multicellular moniliform hairs on the floral labella played a crucial role to attract pollinators, causing a high natural fruit set ratio in C. wardii. We established the direct connection of the white trichomes and real pollen.
C_LIO_LIWe propose that flowers of C. wardii provide pseudopollen to attract suitable bees and hoverflies as pollinators. And our evidence indicate that the pseudopollen owns both deceptive and rewarding ecological functions. Our study provide a clear pollination mechanism with both rewards and deceptive signals by a same object in angiosperm for the first time. However, an inbreeding depression seem to be caused by this strategy. And we speculated that the pollen mimicry strategy with both rewarding and deceptive functions in C. wardii may be an adaptation to the habitat fragmentation of this species to gain a reproductive assurance.
C_LI


title: The TIR-NBS-LRR protein CSA1 is required for autoimmune cell death in Arabidopsis pattern recognition co-receptor bak1 and bir3 mutants

abstract: The BRI1-associated kinase BAK1/SERK3 is a positive regulator of multiple leucine rich receptor kinase-mediated signaling pathways including pattern triggered immunity (PTI). Absence or overexpression of BAK1 leads to spontaneous cell death formation. BAK1-interacting receptors (BIR) constitutively interact with BAK1, and plants lacking or overexpressing BIR proteins phenocopy the cell death symptoms observed in bak1 knock outs or overexpressors. In the interactome of BIR3, the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) was identified by mass spectrometry. CSA1 physically interacts with BIR proteins and can be detected in complexes with BAK1. Direct interaction was shown only for CSA1 with BIR proteins but not BAK1. Double mutant bak1 bir3 genotypes develop strong dwarfism and cell death symptoms that are dependent on EDS1 and salicylic acid. Loss of CSA1 blocks bak1 and bak1 bir3-mediated cell death formation thus demonstrating that CSA1 is causal for this type of cell death. We propose that CSA1 guards BIR proteins and initiates autoimmune cell death that is observed when BAK1 BIR complexes are impaired. Our findings reveal how cell death in the absence of BAK1 and BIR3 is executed and links BAK1, a common co-receptor of many pattern recognition receptors, to NLR proteins typically implicated in effector-triggered immunity.


title: Detection of sleep apnea from single-channel electroencephalogram (EEG) using an explainable convolutional neural network

abstract: Sleep apnea (SA) is a common disorder involving the cessation of breathing during sleep. It can cause daytime hypersomnia, accidents, and, if allowed to progress, serious, chronic conditions. Continuous positive airway pressure is an effective SA treatment. However, long waitlists impede timely diagnosis; overnight sleep studies involve trained technicians scoring a polysomnograph, which comprises multiple physiological signals including multi-channel electroencephalography (EEG). Therefore, it is important to develop simplified and automated approaches to detect SA. We have developed an explainable convolutional neural network (CNN) to detect SA from single-channel EEG recordings which generalizes across subjects. The network architecture consisted of three convolutional layers. We tuned hyperparameters using the Hyperband algorithm, optimized parameters using Adam, and quantified network performance with subjectwise 10-fold cross-validation. Our CNN performed with an accuracy of 76.7% and a Matthews correlation coefficient (MCC) of 0.54. This performance was reliably above the conservative baselines of 50% (accuracy) and 0.0 (MCC). To explain the mechanisms of our trained network, we used critical-band masking (CBM): after training, we added bandlimited noise to test recordings; we parametrically varied the noise band center frequency and noise intensity, quantifying the deleterious effect on performance. We reconciled the effects of CBM with lesioning, wherein we zeroed the trained networks 1st-layer filter kernels in turn, quantifying the deleterious effect on performance. These analyses indicated that the network learned frequency-band information consistent with known SA biomarkers, specifically, delta and beta band activity. Our results indicate single-channel EEG may have clinical potential for SA diagnosis.


title: Isolation of sensory hair cell specific exosomes in human perilymph

abstract: Evaluation of hearing loss patients using clinical audiometry has been unable to give a definitive cellular or molecular diagnosis, hampering the development of treatments of sensorineural hearing loss. However, biopsy of inner ear tissue without losing residual hearing function for pathologic diagnosis is extremely challenging. In a clinical setting, perilymph can be accessed, so alternative methods for molecular characterization of the inner ear may be developed. Recent approaches to improving inner ear diagnostics have been focusing on the evaluation of the proteomic or miRNA profiles of perilymph. Inspired by recent characterization and classification of many neurodegenerative diseases using exosomes which not only are produced in locally in diseased tissue but are transported beyond the blood brain barrier, we demonstrate the isolation of human inner ear specific exosomes using a novel ultrasensitive immunomagnetic nano pom-poms capture-release approach. Using perilymph samples harvested from surgical procedures, we were able to isolate exosomes from sensorineural hearing loss patients in only 2-5 L of perilymph. By isolating sensory hair cell derived exosomes through their expression level of myosin VII, we for the first time sample material from hair cells in the living human inner ear. This work sets up the first demonstration of immunomagnetic capture-release nano pom-pom isolated exosomes for liquid biopsy diagnosis of sensorineural hearing loss. With the ability to isolate exosomes derived from different cell types for molecular characterization, this method also can be developed for analyzing exosomal biomarkers from more accessible patient tissue fluids such as plasma.


title: Single-cell atlas of progressive supranuclear palsy reveals a distinct hybrid glial cell population

abstract: Progressive supranuclear palsy (PSP) is a neurodegenerative disorder whose molecular complexity at a single cell level has not been evaluated. Here we analyzed 45,559 high quality nuclei from the subthalamic nucleus and associated basal ganglia regions from post-mortem human PSP brains with varying degrees of tau pathology compared to controls (n=3 per group). We identified novel astrocyte-oligodendrocyte hybrid cell populations that overexpress neurotropic factors in conjunction with suppression of the unfolded protein response pathway. Notably, trajectory analysis identified subpopulations of hybrid cells with distinct astrocytic, oligodendrocytic and hybrid molecular states that change from a neuroprotective hybrid cell to an astrocytic cell with impaired homeostatic function in PSP. Our single nucleus transcriptomic data provides insights into the cell-type-specific contributions to the disease for investigating the molecular and cellular basis of PSP.


title: Development of highly potent neutralising nanobodies against multiple SARS-CoV-2 variants including the variant of concern B.1.351

abstract: The pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. During the years of 2020-2021, millions of humans have died due to SARS-CoV-2 infection and severe economic damage to the global economy has occurred. Unprecedented rapid investments in vaccine development have been made to counter the spread of SARS-CoV-2 among humans. While vaccines are a key pillar of modern medicine, SARS-CoV-2 has mutated as it spread among humans. Vaccines previously developed and approved by regulators are becoming less effective against new variants. One variant of SARS-CoV-2 known as B.1.351 that was first reported to be present in South Africa significantly reduces the efficacy of vaccines developed to date. Therapeutic options that work against the B.1.351 variant are therefore urgently needed to counteract reduced vaccine efficacy. We present here the discovery of recombinant alpaca antibodies that neutralise live virus of B.1.351 and other SARS-CoV-2 variants potently. The antibodies described here may be a useful tool for clinicians who are treating patients infected with B.1.351 and other SARS-CoV-2 for which there is currently no known highly effective treatment.


title: Plaque Size Tool: an automated plaque analysis tool for simplifying and standardising bacteriophage plaque morphology measurements

abstract: Bacteriophage plaque size measurement is essential for phage characterisation, but their manual size estimation requires a considerable amount of time and effort. In order to ease the work of phage researchers, we have developed an automated command-line application called Plaque Size Tool (PST) that can detect plaques of different morphology on the images of Petri dishes and measure plaque area and diameter. Plaque size measurements using PST showed no difference to those obtained with manual plaque size measurement in Fiji, indicating future results using PST are backwards compatible with prior measurements in the literature. PST can be applied to a range of lytic bacteriophages producing oval-shaped plaques, including bulls-eye morphology. The application can also be used for titer calculation if most of the plaques are stand-alone. As laboratory automation becomes more commonplace, standardised and flexible open-source analytical tools like PST will be important parts of biofoundry and cloud lab bacteriophage workflows.

category: microbiology


title: Combinatorial approach with mass spectrometry and lectin microarray dissected glycoproteomic features of virion-derived spike protein of SARS-CoV-2

abstract: The COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2, has a global impact on public health. Since glycosylation of the viral envelope glycoproteins is known to be deeply associated with their immunogenicity, intensive studies on the glycans of its major glycoprotein, S protein, have been conducted. Nevertheless, the detailed site-specific glycan compositions of virion-associated S protein have not yet been clarified. Here, we conducted intensive glycoproteomic analyses of SARS-CoV-2 S protein using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarray. Using our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, we identified 1,759 site-specific glycan compositions. The most frequent was HexNAc:Hex:Fuc:NeuAc:NeuGc = 6:6:1:0:0, suggesting a tri-antennary N-glycan terminating with LacNAc and having bisecting GlcNAc and a core fucose, which was found in 20 of 22 glycosylated sites. The subsequent lectin microarray analysis emphasized intensive outer arm fucosylation of glycans, which efficiently complemented the glycoproteomic features. The present results illustrate the high-resolution glycoproteomic features of SARS-CoV-2 S protein and significantly contribute to vaccine design, as well as the understanding of viral protein synthesis.

category: molecular biology


title: CST interacts with the cohesin complex and promotes chromosome cohesion

abstract: Sister chromatid cohesion (SCC) is established during DNA replication by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. How SCC is established and maintained until mitosis remains incompletely understood and emerging evidence suggests that replication stress can lead to premature SCC loss. Here, we report that the single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. Following depletion of CST subunits, we observed an increase in the complete loss of SCC. Additionally, we determined that CST interacts with the cohesin complex. Unexpectedly, we did not find evidence of defective cohesion establishment or mitotic progression in the absence of CST. However, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Since replication stress was recently shown to induce SCC loss, we supposed that CST may be required to maintain SCC following fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells following exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.


title: Visualization of loop extrusion by DNA nanoscale tracing in single cells

abstract: The spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensation and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random chromatin structures such as topologically associating domains2, 3. However, whether the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we developed LoopTrace, a fluorescence imaging workflow for high-resolution reconstruction of 3D genome architecture that conserves chromatin structure at the nanoscale and can resolve the 3D-fold of chromosomal DNA with better than 5-kb precision in single human cells. Our results show that the chromatin fibre behaves as a random coil up to the megabase scale and is further structured by contacts between sites that anchor loops. Our single cell folds reveal that such looping interactions are sparse and lead to a large heterogeneity of folds with one or two dynamically positioned loop bases as the main reproducible feature of megabase scale chromosomal regions. Clustering folds by their 3D conformations revealed a series of structures consistent with progressive loop extrusion between major anchor sites. Consistently, the looping interactions and their non-random positioning depend on the presence of the loop extrusion enzyme cohesin and its anchor protein CTCF, respectively. Our approach is scalable and will be instrumental to image the functional 3D architecture of the genome directly at the nanoscale.


title: Visualization of loop extrusion by DNA nanoscale tracing in single human cells


title: Vacuole phase-partitioning boosts mitochondria activity and cell lifespan through an inter-organelle lipid pipeline

abstract: Functional linkage between mitochondria and lysosomes is crucial for survival under starvation and lifespan extension. Despite such co-dependency, the supportive pathways connecting mitochondria and lysosomes remain unclear. Here, we identify an inter-organelle lipid trafficking pathway linking yeast vacuole and mitochondria that results in increased mitochondria growth and respiratory activity under glucose starvation. The pathway depends on vacuolar phase-separated, lipid domains, which provide zones for: activation of the vacuolar proton pump; lipid droplet (LD) docking and internalization; and, lipid transfer from vacuole-to-ER-to-mitochondria. Partitioned vacuolar domains form through a specialized type of macro-autophagy, triggered only under acute glucose starvation, that delivers sterol-rich, endosomal-derived lipids to the vacuole. To balance this lipid influx, the vacuole reroutes lipids back to the ER to support both LD biogenesis and mitochondria growth and activity. Energy produced by enhanced mitochondrial activity then feeds back to support the inter-organelle lipid trafficking pathways to ensure survival under nutrient stress.


title: Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells

abstract: The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is mediated at least in part by the junctional component DLG-1 discs large, which appears to restrict protrusive activity to the apical domain. Finally, we demonstrate that the Rho-family GTPases CED-10 Rac and CDC-42 can regulate seam cell outgrowth and may also function downstream of LET-413. Our data uncover multiple essential functions for LET-413 in larval development and shed new light on the regulation of polarized outgrowth of the seam cells.


title: Predictors of genomic differentiation within a hybrid taxon

abstract: Hybridization is increasingly recognized as an important evolutionary force. Novel genetic methods now enable us to address how the genomes of parental species are combined to build hybrid genomes. However, we still do not know the relative importance of contingencies, genome architecture and local selection in shaping hybrid genomes. Here, we take advantage of the genetically divergent island populations of Italian sparrow on Crete, Corsica and Sicily to investigate the predictors of genomic variation within a hybrid taxon. We test if differentiation is affected by recombination rate, selection, or variation in ancestry proportion from each parent species. We find that the relationship between recombination rate and differentiation is less pronounced within hybrid lineages than between the parent species, as expected if purging of minor parent ancestry in low recombination regions reduces the variation available for differentiation. In addition, we find that differentiation between islands is correlated with differences in selection in two out of three comparisons. Patterns of within-island selection are correlated across all islands, suggesting that shared selection may mould genomic differentiation. The best predictor of strong differentiation within islands is the degree of differentiation from house sparrow, and hence loci with Spanish sparrow ancestry may vary more freely. Jointly, this suggests that constraints and selection interact in shaping the genomic landscape of differentiation in this hybrid species.


title: Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation in severe disease

abstract:
Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth-stunting, and diminished oral vaccine responses. While EE has been shown to be the by-product of recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE severity by performing high-throughput single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE from Lusaka, Zambia (8 HIV-negative, 3 HIV-positive), 6 adults without EE in Boston, USA, and 2 adults from Durban, South Africa, which we complemented with published data from 3 additional South African adults from the same clinical site. By using these data to reanalyze previously-defined bulk-transcriptomic signatures of reduced villus height and decreased plasma LPS levels in EE, we found that these signatures may be driven by an increased abundance of surface mucosal cells - a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we identified several cell subsets whose fractional abundances associate with histologically determined EE severity, small intestinal region, and HIV infection. Furthermore, by comparing distal duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells but with increased pro-inflammatory cytokine expression in the EE cohort. Altogether, our work illuminates epithelial and immune correlates of EE and provides new molecular targets for intervention.

One Sentence SummaryUsing single-cell RNA-sequencing, we characterize the pathophysiology of environmental enteropathy (EE) - a highly prevalent condition of the small intestine that is thought to be a primary cause of global growth-stunting cases and a key contributing factor to childhood malnutrition and diminished oral vaccine responses - to derive insights into the epithelial and immune correlates of disease severity, suggesting new therapeutic targets for future investigation.


title: Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLRthat is shed by Furin

abstract: The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote-carriers of ASGR1-deletions exhibited [~]34% lower risk of coronary artery disease and [~]10-14% non-HDL-cholesterol reduction. Since PCSK9 is a major degrader of LDLR, the regulation of LDLR and/or PCSK9 by ASGR1 was studied. Thus, we investigated the role of endogenous/overexpressed ASGR1 on LDLR degradation and functionality by Western-blot and immunofluorescence in HepG2 naive and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR and both interact with/enhance the degradation of the receptor independently. Such lack of cooperativity between PCSK9 and ASGR1 on LDLR expression was confirmed in livers of wild-type (WT) versus Pcsk9-/- mice. ASGR1-knockdown in HepG2 naive cells significantly increased total ([~]1.2-fold) and cell-surface ([~]4-fold) LDLR protein. In HepG2-PCSK9-knockout cells ASGR1-silencing led to [~]2-fold higher levels of LDLR protein and DiI-LDL uptake, associated with [~]9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 reduced primarily the immature non-O-glycosylated LDLR ([~]110 kDa), whereas the triple Gln240/Trp244/Glu253 Ala-mutant (loss of carbohydrate-binding) reduced the mature form of the LDLR ([~]150 kDa), suggesting that ASGR1 binds the LDLR in sugar-dependent and -independent fashion. Furin sheds ASGR1 at RKMK103{downarrow} into a secreted form, likely resulting in a loss-of-function on LDLR. LDLR is the first example of a liver-receptor ligand of ASGR1. Additionally, we demonstrate that lack of ASGR1 enhances LDLR levels and DiI-LDL incorporation, independently of PCSK9. Overall, silencing of ASGR1 and PCSK9 may lead to higher LDL-uptake by hepatocytes, thereby providing a novel approach to further reduce LDL-cholesterol.


title: Revealing the threat of emerging SARS-CoV-2 mutations to antibody therapies

abstract: The ongoing massive vaccination and the development of effective intervention offer the long-awaited hope to end the global rage of the COVID-19 pandemic. However, the rapidly growing SARS-CoV-2 variants might compromise existing vaccines and monoclonal antibody (mAb) therapies. Although there are valuable experimental studies about the potential threats from emerging variants, the results are limited to a handful of mutations and Eli Lilly and Regeneron mAbs. The potential threats from frequently occurring mutations on the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) to many mAbs in clinical trials are largely unknown. We fill the gap by developing a topology-based deep learning strategy that is validated with tens of thousands of experimental data points. We analyze 261,348 genome isolates from patients to identify 514 non-degenerate RBD mutations and investigate their impacts on 16 mAbs in clinical trials. Our findings, which are highly consistent with existing experimental results about variants from the UK, South Africa, Brazil, US-California, and Mexico shed light on potential threats of 95 high-frequency mutations to mAbs not only from Eli Lilly and Regeneron but also from Celltrion and Rockefeller University that are in clinical trials. We unveil, for the first time, that high-frequency mutations R346K/S, N439K, G446V, L455F, V483F/A, E484Q/V/A/G/D, F486L, F490L/V/S, Q493L, and S494P/L might compromise some of mAbs in clinical trials. Our study gives rise to a general perspective about how mutations will affect current vaccines.


title: Luminal Flow Actuation Generates Coupled Shear and Strain in a Microvessel-on-Chip

abstract:
In the microvasculature, blood flow-derived forces are key regulators of vascular structure and function. Consequently, the development of hydrogel-based microvessel-on-chip systems that strive to mimic the in vivo cellular organization and mechanical environment has received great attention in recent years. However, despite intensive efforts, current microvessel- on-chip systems suffer from several limitations, most notably failure to produce physiologically relevant wall strain levels. In this study, a novel microvessel-on-chip based on the templating technique and using luminal flow actuation to generate physiologically relevant levels of wall shear stress and circumferential stretch is presented. Normal forces induced by the luminal pressure compress the surrounding soft collagen hydrogel, dilate the channel, and create large circumferential strain. The fluid pressure gradient in the system drives flow forward and generates realistic pulsatile wall shear stresses. Rigorous characterization of the system reveals the crucial role played by the poroelastic behavior of the hydrogel in determining the magnitudes of the wall shear stress and strain. The experimental measurements are combined with an analytical model of flow in both the lumen and the porous hydrogel to provide an exceptionally versatile user manual for an application-based choice of parameters in microvessels-on-chip. This unique strategy of flow actuation adds a dimension to the capabilities of microvessel-on-chip systems and provides a more general framework for improving hydrogel-based in vitro engineered platforms.

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title: Prediction and evolution of the molecular fitness of SARS-CoV-2 variants: Introducing SpikePro

abstract: The understanding of the molecular mechanisms driving the fitness of the SARS-CoV-2 virus and its mutational evolution is still a critical issue. We built a simplified computational model, called SpikePro, to predict the SARS-CoV-2 fitness from the amino acid sequence and structure of the spike protein. It contains three contributions: the viral transmissibility predicted from the stability of the spike protein, the infectivity computed in terms of the affinity of the spike protein for the ACE2 receptor, and the ability of the virus to escape from the human immune response based on the binding affinity of the spike protein for a set of neutralizing antibodies. Our model reproduces well the available experimental, epidemiological and clinical data on the impact of variants on the biophysical characteristics of the virus. For example, it is able to identify circulating viral strains that, by increasing their fitness, recently became dominant at the population level. SpikePro is a useful instrument for the genomic surveillance of the SARS-CoV-2 virus, since it predicts in a fast and accurate way the emergence of new viral strains and their dangerousness. It is freely available in the GitHub repository github.com/3BioCompBio/SpikeProSARS-CoV-2.


title: Early Patellofemoral Cartilage and Bone Degeneration in a Rat Model of Noninvasive Anterior Cruciate Ligament Rupture

abstract:
BackgroundAnterior cruciate ligament rupture (ACLR) is a well-known risk factor for the development of post-traumatic osteoarthritis (PTOA). While clinical and pre-clinical studies have characterized the onset and progression of PTOA in the tibiofemoral joint compartment, very little is known about degenerative changes in the patellofemoral compartment after ACL injury.

Hypothesis/PurposeTo evaluate the extent to which ACL rupture induces acute patellofemoral joint degeneration by quantifying articular cartilage morphology and remodeling of subchondral and trabecular bone microarchitecture in the patellofemoral compartment.

Study DesignDescriptive laboratory study.

MethodsAdult female Lewis rats were randomized to undergo either a non-surgical ACL rupture or a Sham procedure (n = 6 per group). Ex vivo contrast-enhanced micro-computed tomography ({micro}CT) and histological evaluation of the patellofemoral compartment were performed at 2-weeks post-injury, representing a timepoint of documented early PTOA in the tibiofemoral compartment in this model.

ResultsACL rupture causes osteophyte formation in the patella and mild degeneration in the superficial zone of articular cartilage (AC), including surface fibrillation, fissures, increased cellularity, and abnormal chondrocyte clustering at two weeks post-injury. Contrast-enhanced {micro}CT analysis demonstrates significant increases in AC thickness of patellar and trochlear cartilage. Loss of subchondral bone thickness, bone volume fraction, and tissue mineral density, as well as changes to trabecular microarchitecture in both the patella and trochlea, were indicative of catabolic bone remodeling.

ConclusionThese results demonstrate that the patellofemoral joint develops mild but evident degenerative changes in the acute time period following ACL rupture, extending the utility of this rat model to the study of degenerative patellofemoral changes following joint trauma.

Clinical RelevanceACL rupture causes mild degeneration and swelling of articular cartilage, coupled with catabolic bone remodeling in the patellofemoral compartment. Characterizing the pathophysiology of patellofemoral PTOA in its early stages may provide a better understanding of disease progression and provide opportunities for preventative therapeutic intervention.

category: physiology