title: Distinct progressions of neuronal activity changes underlie the formation and consolidation of a gustatory associative memory

abstract:
Acquiring new memories is a multi-stage process. Ample of studies have convincingly demonstrated that initially acquired memories are labile, and only stabilized by later consolidation processes. These multiple phases of memory formation are known to involve modification of both cellular excitability and synaptic connectivity, which in turn change neuronal activity at both the single neuron and ensemble levels. However, the specific mapping between the known phases of memory and the observed changes in neuronal activity remains unknown. Here we address this unknown in the context of conditioned taste aversion learning by continuously tracking gustatory cortex (GC) neuronal taste responses from alert rats in the 24 hours following a taste-malaise pairing. We found that the progression of neuronal activity changes in the GC depend on the neuronal organizational level. The population response changed continuously; these changes, however, were only reflected in the population mean amplitude during the acquisition and consolidation phases, and in the known quickening of the ensemble state dynamics after the time of consolidation. Together our results demonstrate how complex dynamics in different representational level of cortical activity underlie the formation and stabilization of memory within the cortex.

Significant StatementMemories are formed through a multi-phase process; an early initial acquired memory consolidates into a stable form over hours and days. While the underlying phase-specific molecular pathways are fairly known, the neuronal activity changes during these different phases remain elusive. Here we studied this unknown by tracking cortical neuronal activity over 24h as the taste becomes aversive following association with malaise. We found that that the progression of activity changes is organization-level dependent: The population response changed continuously; the population mean amplitude was time-locked to the acquisition and consolidation phases, and the quickening of the known ensemble state dynamics appear only after consolidation. Our results reveal the complex organizational-level neuronal interactions that underlie the progression of memory formation.


title: The degree of context un/familiarity impacts the emotional feeling and preaware cardiac-brain activity: a study with emotionally salient naturalistic paradigm using DENS Dataset

abstract: Emotion experiments with naturalistic paradigms are emerging and giving new insights into dynamic brain activity. Context familiarity is considered as an important dimensions of emotion processing by appraisal theorists. However, how the context un/familiarity of the naturalistic stimuli influences the central and autonomic activity is not probed yet [check it]. Hence, we tried to address this issue in this work by breaking it down into three questions. 1) What is the relation between context un/familiarity with the neural correlates of self-assessment affective dimensions viz. valence and arousal; 2) the influence of context un/familiarity in cardiac-brain mutual interaction during emotion processing; 3.) brain network reorganization to accommodate the degree of context familiarity. We found that the less-context familiarity is primarily attributed to negative emotion feeling mediated by lack of predictability of sensory experience. Whereas, with high-context familiarity, both positive and negative emotions are felt. For less-context familiarity, the arousal activity is negatively correlated with EEG power. In addition, the cardiac activity for both high and less context familiarity is modulated before the reported self-awareness of emotional feeling. The correlation of cortical regions with cardiac activity and connectivity patterns reveals that ECG is modulated by salient feature during pre-awareness and correlates with AIC and conceptual hub in high-familiarity. Whereas, for the low familiarity, the cardiac activity is correlated with the exteroceptive sensory regions. In addition, we found that OFC and dmPFC have high connectivity with less-context familiarity, whereas AIC has high connectivity with high-context familiarity. To the best of our knowledge, the context familiarity and its influence on cardiac and brain activity have never been reported with a naturalistic paradigm. Hence, this study significantly contributes to understanding automatic processing of emotions by analyzing the effect of context un/familiarity on affective feelings, the dynamics of cardiac-brain mutual interaction, and the brains effective connectivity during pre-awareness.

category: neuroscience


title: How Filopodia Respond to Calcium in the Absence of a Calcium-binding Structural Protein: They Use Rapid Transit

abstract: During directional locomotion, cells must reorient themselves in response to attractive or repulsive cues. Filopodia are narrow actin-based protrusions whose prevalence at the leading edge of a migrating cell is related to the persistence of locomotion. Although there is a marked absence of calcium-binding components in their structure, they responded to store-operated calcium entry (SOCE). Here, we used a two-phase protocol to determine how they responded. In the first phase, extracellular calcium was removed and ER calcium lowered by blocking reuptake through the calcium pump. This was known to activate stromal interacting molecule (STIM) and cause its microtubule-mediated translocation to the cell surface. In the second phase, extracellular calcium and calcium influx into the ER were restored. ER depletion caused filopodia to increase, followed by a spontaneous decrease that was blocked by inhibiting endocytosis. The intracellular calcium concentration increased during depletion, while the size of the exchangeable compartment of vesicles, measured by fluid-phase marker uptake, shrank. When SOCE mediators and the aquaporin, AQP4, were localized, STIM and transient receptor potential canonical (TPRC) channels occupied vesicular profiles side-by-side in linear arrays. STIM1 was translocated, as expected. TRPC1 was initially in a rapidly recycling pool (RRP) where it partially colocalized with Vamp2. Calcium restoration caused TRPC1 exocytosis, while STIM1 reverted toward its original pattern associated with the ER. The exchangeable compartment was restored and this enabled filopodia extension, which was blocked by inhibitors of TRPC1/4/5 and endocytosis. That vesicle recycling was essential for extension during calcium readdition was indicated by reversal of the effect of endocytosis inhibitors in the depletion and readdition phases. The results suggest that SOCE regulates the size of the RRP in epithelial cells, and vesicle recycling is the immediate mechanism affecting filopodia extension. The conclusions are discussed in light of factors regulating protrusion formation, namely surface tension and vesicle trafficking.

category: cell biology


title: Overexpression of SHORT-ROOT2 transcription factor enhanced the outgrowth of mature axillary buds in poplar trees

abstract:
O_LIPlant branching is usually prevented by an actively proliferating apex. In poplars, one GRAS family member, SHORT-ROOT2 (PtSHR2), was preferentially expressed in axillary buds (AXBs) and was inducible during bud maturation and activation. Overexpression of PtSHR2 (PtSHR2OE) in hybrid poplar impaired the apical dominance and simultaneously promoted the outgrowth of axillary branches below the maturation point (BMP), accompanied by regulated expression of genes critical for axillary meristem initiation and bud formation.
C_LIO_LIFollowing a detained phenotypic characterization, we compared the IAA and trans-zeatin levels in apical shoots and AXBs of wild-type and PtSHR2OE trees, together with gene expression analyses and defoliation, decapitation, and hormone reapplication assays.
C_LIO_LIPtSHR2OE AXBs contained a significantly lower ratio of auxin to cytokinin than wild-type AXBs, particularly in those below the BMP. Decapitation induced a faster bud burst in PtSHR2OE trees than in wild-type plants, and it could be strongly inhibited by exogenously applied auxin and cytokinin biosynthesis inhibitor, but only partially inhibited by N-1-naphthylphthalamic acid (NPA).
C_LIO_LIAn impaired basipetal auxin transport, rather than an insufficient auxin biosynthesis or auxin insensitivity, disturbed the local hormonal homeostasis in PtSHR2OE AXBs, which in turn enhanced the axillary bud initiation and promoted the bud release.
C_LI

HighlightOverexpression of PtSHR2 in poplar impaired the apical dominance and promoted axillary bud outgrowth below the maturation point through disturbing the basipetal auxin transport and auxin and cytokinin homeostasis.

category: genetics


title: Unravelling Form and Function: Improved function of engineered cardiac tissue through extra-cellular anisotropy

abstract: Cardiac tissue engineering is a promising therapeutic option for myocardial repair after injury, however, so far engineered heart patches have shown limited translational utility due to poor electrical integration and tissue contractility. Emerging research suggests that scaffolds that recapitulate the three-dimensional structure of the native myocardium improve physiological function. Complex scaffold fabrication remains a technical challenge and the isolated impact of scaffold architecture on tissue function and cellular physiology is poorly understood. Here, we provided a direct comparison between isotropic and aligned collagen scaffold morphologies where all confounding physio-mechanical features, such as strut wall thickness and surface roughness are conserved. This enabled the independent and systematic assessment of the effects of pore macro-architecture on global tissue function and cellular maturation. We seeded our scaffolds with embryonic stem cell derived cardiomyocytes (hESC-CM) and measured tissue function through calcium signal transduction and dynamic contractile strain. The aligned tissue constructs facilitated improved signalling synchronicity and directional contractility. We further examined the influence of scaffold macrostructure on intercellular organization and intracellular development. Cells on aligned constructs conformed to the orientation of the scaffold macro-structure and were found to have phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macro-structure on engineered tissue function at multiple length scales. These findings inform the design of optimized cardiac tissue and expand the potential for engineered tissue in regenerative and model medical systems by reducing the gaps in tissue functionality that limit their utility.

category: bioengineering


title: Machine Learning Optimization of Photosynthetic Microbe Cultivation and Recombinant Protein Production

abstract:
BackgroundArthrospira platensis (commonly known as spirulina) is a promising new platform for low-cost manufacturing of biopharmaceuticals. However, full realization of the platforms potential will depend on achieving both high growth rates of spirulina and high expression of therapeutic proteins.

ObjectiveWe aimed to optimize culture conditions for the spirulina-based production of therapeutic proteins.

MethodsWe used a machine learning approach called Bayesian black-box optimization to iteratively guide experiments in 96 photobioreactors that explored the relationship between production outcomes and 17 environmental variables such as pH, temperature, and light intensity.

ResultsOver 16 rounds of experiments, we identified key variable adjustments that approximately doubled spirulina-based production of heterologous proteins, improving volumetric productivity between 70% to 100% in multiple bioreactor setting configurations.

ConclusionAn adaptive, machine learning-based approach to optimize heterologous protein production can improve outcomes based on complex, multivariate experiments, identifying beneficial variable combinations and adjustments that might not otherwise be discoverable within high-dimensional data.


title: Older adults generalize their movements across walking contexts more than young during gradual and abrupt split-belt walking

abstract: Generalization of movements from experienced to novel situations is a critical aspect of motor learning. It has been demonstrated that the training period when a movement is learned influences the movements generalization to untrained situations. However, little is known about how healthy aging affects these processes. For example, young adults exhibit greater generalization of movements learned on a device (e.g. split-belt treadmill or robotic arm) to movements without it (e.g, overground walking or unconstrained reaching) when participants experience small vs. large perturbations on the training device. Here, we investigated whether a similar effect would be observed in older adults. To this end, we compared the generalization of split-belt adaptation to overground walking in older (75.9{+/-}4.8 years old) and young adults (24.7{+/-}5.9 years old) when adapted gradually (i.e., small perturbations) vs. abruptly (i.e., large perturbations). We found that both age groups adapted more to the abrupt condition compared to the gradual condition, which resulted in greater adaptation effects (i.e., aftereffects) on the treadmill in the abrupt than the gradual groups. We also found that older adults generalize more than young adults, regardless of the perturbation schedule (i.e., gradual or abrupt). Our results suggest that abrupt perturbations during adaptation do not limit the generalization of movement in older adults-perhaps because they are more likely to attribute them to their own faulty movements. These results suggest that large perturbations are better than small when training older clinical populations since abrupt disturbances would lead to more adaptation and generalization of corrected movements in older people.


title: Autologous human immunocompetent white adipose tissue-on-chip

abstract: Obesity and associated diseases, such as diabetes, have reached epidemic proportions globally. In the era of  diabesity and due to its central role for metabolic and endocrine processes, adipose tissue (specifically white adipose tissue; WAT) has become a target of high interest for therapeutic strategies. To gain insights in cellular and molecular mechanisms of adipose (patho-)physiology, researchers traditionally relied on animal models since in vitro studies on human WAT are challenging due to the large size, buoyancy, and fragility of mature white adipocytes. Leveraging the Organ-on-Chip technology, we introduce a next-generation microphysiological in vitro model of human WAT based on a tailored microfluidic platform featuring vasculature-like perfusion. The platform integrates a 3D tissue comprising all major WAT-associated cellular components in an autologous manner, including not only mature adipocytes but also organotypic endothelial barriers and stromovascular cells featuring tissue-resident innate immune cells, specifically adipose tissue macrophages. This microphysiological tissue model recapitulates pivotal WAT functions, such as energy storage and mobilization as well as endocrine and immunomodulatory activities. The combination of all individual cell types with extra cellular matrix-like hydrogels in a precisely controllable bottom-up approach enables the generation of a multitude of replicates from the same donors circumventing issues of inter-donor variability and paving the way for personalized medicine. Moreover, it allows to adjust the models degree of complexity to fit a specific purpose via a flexible mix- and-match approach with different cell component modules. This novel WAT-on-chip system constitutes a human-based, autologous and immunocompetent in vitro model of adipose tissue that recapitulates almost full tissue heterogeneity. In the future, the new WAT-on-chip model can become a powerful tool for human-relevant research in the field of metabolism and its associated diseases as well as for compound testing and personalized- and precision medicine applications.


title: Printed Texts Tracking and Following for a Finger-Wearable Electro-Braille System Through Opto-electrotactile Feedback

abstract: This paper presents our recent development on a portable and refreshable text reading and sensory substitution system for the blind or visually impaired (BVI), called Finger-eye. The system mainly consists of an opto-text processing unit and a compact electro-tactile based display that can deliver text-related electrical signals to the fingertip skin through a wearable and Braille-dot patterned electrode array and thus delivers the electro-stimulation based Braille touch sensations to the fingertip. To achieve the goal of aiding BVI to read any text not written in Braille through this portable system, in this work, a Rapid Optical Character Recognition (R-OCR) method is firstly developed for real-time processing text information based on a Fisheye imaging device mounted at the finger-wearable electrotactile display. This allows real-time translation of printed text to electro-Braille along with natural movement of users fingertip as if reading any Braille display or book. More importantly, an electro-tactile neuro-stimulation feedback mechanism is proposed and incorporated with the R-OCR method, which facilitates a new opto-electrotactile feedback based text line tracking control approach that enables text line following by user fingertip during reading. Multiple experiments were designed and conducted to test the ability of blindfolded participants to read through and follow the text line based on the opto-electrotactile-feedback method. The experiments show that as the result of the opto-electrotactile-feedback, the users were able to maintain their fingertip within a 2mm distance of the text while scanning a text line. This research is a significant step to aid the BVI users with a portable means to translate and follow to read any printed text to Braille, whether in the digital realm or physically, on any surface.

category: bioinformatics


title: Strong regulatory effects of vgll3 genotype on reproductive axis gene expression in immature male Atlantic salmon

abstract: Age at maturity is a major contributor to the diversity of life history strategies in organisms. The process of maturation is influenced by both genetics and the environment, and includes changes in levels of sex hormones and behavior, but the specific factors leading to variation in maturation timing are not well understood. gnrh1 regulates the transcription of gonadotropin genes at the onset of puberty in many species, but this gene is lacking in certain teleost species including Atlantic salmon (Salmo salar), which raises the possibility of the involvement of other important regulatory factors during this process. Earlier research has reported a strong association of alternative alleles of the vgll3 gene with maturation timing in Atlantic salmon, suggesting it as a potential candidate regulating reproductive axis genes. Here, we investigated the expression of reproductive axis genes in immature Atlantic salmon males with different vgll3 genotypes during the spawning period. We detected strong vgll3 genotype-dependent differential expression of reproductive axis genes (such as fshb, lhb, amh and igf3) tested in the pituitary, and testis of one-year-old immature male Atlantic salmon. In addition, we observed differential expression of jun (ap1) and nr5a1b (sf1), potential upstream regulators of gonadotropins in the pituitary, as well as axin2, id3, insl3, itch, ptgs2a and ptger4b, the downstream targets of amh and igf3 in the testis. Hereby, we provide evidence of strong vgll3 genotype-dependent transcriptional regulation of reproductive axis genes prior to sexual maturation together with models for distinct actions of vgll3 genotypes on the molecular processes controlling spermatogenesis in Atlantic salmon.

category: physiology


title: Modeling Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism dementia Complex with Microglia Containing Cerebral Organoids Derived from Induced Pluripotent Stem Cells

abstract:
Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-dementia Complex (ALS-PDC) is a neurodegenerative disease linked to the traditional consumption of cycad seeds by the Chamorro people of Guam. Little is known about the etiological role of cycad toxin in ALS-PDC. Patient-derived induced pluripotent stem cells were derived from age- and sex-matched affected and unaffected patient lymphoid cells then differentiated into cerebral organoids. After three months, the ALS-PDC affected organoids were smaller, their neurons had less extensive neurite outgrowth, and the organoids had more reactive astrocytes and M1 microglia, fewer resting and M2 microglia, and more open extracellular space. Most of these phenomena could be recapitulated by exposing unaffected organoids to {beta}-methylamino L-alanine (BMAA), a toxic amino acid produced by cyanobacteria living with cycad plants. Furthermore, ALS-PDC affected organoids exhibited an exacerbated neuroinflammatory response to BMAA exposure via activation of caspase1/NLRP3 inflammasome. A genome-wide transcriptome analysis of the organoids showed that the most down-regulated pathways were taurine, alanine, aspartate, and glutamate metabolism; protein digestion; and absorption. The most down-regulated biological processes were type I interferon signaling, regulation of neuron differentiation and extracellular matrix organization. Our results suggested that the etiology of ALS-PDC is due to metabolic disorders that shifted microglia to a more pro-inflammatory M1 state instead of a non-inflammatory, repairing M2 state, which exacerbated inflammation and reduced extracellular matrix strength. Supplementation of transforming growth factor beta (TGF-{beta}) to ALS-PDC affected organoids increased the expression of interferon-induced transmembrane proteins (IFITMs) and restored M2 microglia populations and extracellular matrix organization. Organoids containing networks of neurons, astrocytes, microglia derived from iPSC with our protocol provides an excellent cellular model for neurodegenerative disease modeling.

Significance StatementO_LIWestern Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-dementia Complex (ALS-PDC) cerebral organoids containing networks of neurons, astrocytes, and microglia were generated from patient specific lymphoid derived induced pluripotent stem cells.
C_LIO_LIALS-PDC affected organoids were smaller, with neurons had less extensive neurite outgrowth, more reactive astrocytes and M1 microglia, fewer resting and M2 microglia, and more open extracellular matrix spaces when compared to ALS-PDC unaffected organoids.
C_LIO_LIGenome-wide transcriptome analysis indicated that ALS/PDC affected organoids had significantly lower expression of genes related to vitamin B6, amino acid and protein glycation metabolisms, down-regulated type I interferon signaling, the regulation of neuron differentiation and extracellular matrix production.
C_LIO_LIOur results implicated that the etiology of ALS-PDC is due to metabolic disorders that led the shift of microglia to more pro-inflammatory M1 state and less non-inflammatory resting, repairing M2 state of microglia subpopulation, which primed the exacerbated inflammation and reduced extracellular matrix strength. TGF-{beta} promoted interferon-induced transmembrane protein (IFITMs) expression and restored the repairing M2 state of microglia population and extracellular matrix organization.
C_LI


title: The emergence of highly fit SARS-CoV-2 variants accelerated by recombination

abstract: The SARS-CoV-2 pandemic recently entered an alarming new phase with the emergence of the variants of concern (VOC) and understanding their biology is paramount to predicting future ones. Current efforts mainly focus on mutations in the spike glycoprotein (S), but changes in other regions of the viral proteome are likely key. We analyzed more than 900,000 SARS-CoV-2 genomes with a computational systems biology approach including a haplotype network and protein structural analyses to reveal lineage-defining mutations and their critical functional attributes. Our results indicate that increased transmission is promoted by epistasis, i.e., combinations of mutations in S and other viral proteins. Mutations in the non-S proteins involve immune-antagonism and replication performance, suggesting convergent evolution. Furthermore, adaptive mutations appear in geographically disparate locations, suggesting that either independent, repeat mutation events or recombination among different strains are generating VOC. We demonstrate that recombination is a stronger hypothesis, and may be accelerating the emergence of VOC by bringing together cooperative mutations. This emphasizes the importance of a global response to stop the COVID-19 pandemic.

category: evolutionary biology


title: The emergence of highly fit SARS-CoV-2 variants accelerated by epistasis and caused by recombination


title: Rapid expansion of SARS-CoV-2 variants of concern is a result of adaptive epistasis


title: Broad-spectrum in vitro antiviral activity of ODBG-P-RVn: an orally-available, lipid-modified monophosphate prodrug of remdesivir parent nucleoside (GS-441524)

abstract: The intravenous administration of remdesivir for COVID-19 confines its utility to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524) against viruses that cause diseases of human public health concern, including SARS-CoV-2. ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had near-equivalent activity to remdesivir in primary-like human small airway epithelial cells. Our results warrant investigation of ODBG-P-RVn efficacy in vivo.

category: pharmacology and toxicology


title: High genetic barrier to escape from human polyclonal SARS-CoV-2 neutralizing antibodies

abstract: The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in SARS-CoV-2 convalescent and vaccinated individuals are key determinants of neutralization breadth and, consequently, the genetic barrier to viral escape. Using chimeric viruses and antibody-selected viral mutants, we show that multiple neutralizing epitopes, within and outside the viral receptor binding domain (RBD), are variably targeted by polyclonal plasma antibodies and coincide with sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic  polymutant spike proteins that resisted polyclonal antibody neutralization to a similar degree as currently circulating variants of concern (VOC). Importantly, by aggregating VOC-associated and plasma-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in SARS-CoV-2 spike are sufficient to confer near-complete resistance to the polyclonal neutralizing antibodies generated by convalescents and mRNA vaccine recipients. Strikingly however, plasma from individuals who had been infected and subsequently received mRNA vaccination, neutralized this highly resistant SARS-CoV-2 polymutant, and also neutralized diverse sarbecoviruses. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against future sarbecovirus pandemics.

category: microbiology


title: VPS29 exerts opposing effects on endocytic viral entry

abstract: Emerging zoonotic viral pathogens threaten global health and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic and pandemic threat coronaviruses as well as ebolavirus. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. VPS29 deficiency caused changes endosome morphology, and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence viral susceptibility and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases.


title: A novel method for robust estimation of ants' walking speed and curvature on convoluted trajectories derived from their gait pattern

abstract: Accurate measurements of travel distance and speed are crucial for the analysis of animal movements. Measuring the movements of ants entails measuring the change in locations registered at time intervals. This process involves dilemma of setting the proper time window: a short time window is vulnerable to spatial errors in observation, while a long time window leads to underestimation of the travel distance. To overcome these difficulties, we propose a novel algorithm that successively interpolates two consecutive points of ants trajectory for a given time window by embracing the alternating tripod gait of ants. We demonstrate that this algorithm is more reliable compared to the conventional method of travel distance estimation based on the sum of the consecutive straight-line displacements (SLD). After obtaining speed estimates for a range of sampling time windows, we applied a fitting method that can estimate the actual speed without prior knowledge of spatial error distribution. We compared results from several methods of speed and curvature extracted from the empirical data of ant trajectories. We encourage empirical scientists to utilize the proposed methods rather than the conventional SLD method of speed estimation as this process is a more reliable and subjective selection of the sampling time window can be avoided.

category: animal behavior and cognition


title: Spatial representability of neuronal activity

abstract: A common approach to interpreting spiking activity is based on identifying the firing fields--regions in physical or configuration spaces that elicit responses of neurons. Common examples include hippocampal place cells that fire at preferred locations in the navigated environment, head direction cells that fire at preferred orientations of the animals head, view cells that respond to preferred spots in the visual field, etc. In all these cases, firing fields were discovered empirically, by trial and error. We argue that the existence and a number of properties of the firing fields can be established theoretically, through topological analyses of the neuronal spiking activity.


title: Learning Orientations: a Discrete Geometry Model

abstract: In the mammalian brain, many neuronal ensembles are involved in representing spatial structure of the environment. In particular, there exist cells that encode the animals location and cells that encode head direction. A number of studies have addressed properties of the spatial maps produced by these two populations of neurons, mainly by establishing correlations between their spiking parameters and geometric characteristics of the animals environments. The question remains however, how the brain may intrinsically combine the direction and the location information into a unified spatial framework that enables animals orientation. Below we propose a model of such a framework, using ideas and constructs from algebraic topology and synthetic affine geometry.


title: Transmit Field Bias Correction of T1w/T2w Myelin Maps

abstract: T1-weighted divided by T2-weighted (T1w/T2w) myelin maps were initially developed for neuroanatomical analyses such as identifying cortical areas, but they are increasingly used in statistical comparisons across individuals and groups with other variables of interest. Existing T1w/T2w myelin maps contain radiofrequency transmit field (B1+) biases, which may be correlated with these variables of interest, leading to potentially spurious results. Here we propose two empirical methods for correcting these transmit field biases using either explicit measures of the transmit field or alternatively a 'pseudo-transmit' approach that is highly correlated with the transmit field at 3T. We find that the resulting corrected T1w/T2w myelin maps are both better neuroanatomical measures (e.g., for use in cross-species comparisons), and more appropriate for statistical comparisons of relative T1w/T2w differences across individuals and groups (e.g., sex, age, or body-mass-index) within a consistently acquired study at 3T. We recommend that investigators who use the T1w/T2w approach for mapping cortical myelin use these B1+ transmit field corrected myelin maps going forward.


title: Empirical Transmit Field Bias Correction of T1w/T2w Myelin Maps


title: Cell-Type Specific Responses to Associative Learning in the Primary Motor Cortex

abstract: The primary motor cortex (M1) is known to be a critical site for movement initiation and motor learning. Surprisingly, it has also been shown to possess reward-related activity, presumably to facilitate reward-based learning of new movements. However, whether reward-related signals are represented among different cell types in M1, and whether their response properties change after cue-reward conditioning remains unclear. Here, we performed longitudinal in vivo two-photon Ca2+ imaging to monitor the activity of different neuronal cell types in M1 while mice engaged in a classical conditioning task. Our results demonstrate that most of the major neuronal cell types in M1 showed robust but differential responses to both cue and reward stimuli, and their response properties undergo cell-type specific modifications after associative learning. PV-INs responses became more reliable to the cue stimulus, while VIP-INs responses became more reliable to the reward stimulus. PNs only showed robust response to the novel reward stimulus, and they habituated to it after associative learning. Lastly, SOM-IN responses emerged and became more reliable to both conditioned cue and reward stimuli after conditioning. These observations suggest that cue- and reward-related signals are represented among different neuronal cell types in M1, and the distinct modifications they undergo during associative learning could be essential in triggering different aspects of local circuit reorganization in M1 during reward-based motor skill learning.


title: Activation of T cell checkpoint pathways during beta-cell antigen presentation by engineered dendritic cells promotes protection of non-obese diabetic mice from type 1 diabetes

abstract: Defective immune regulation has been recognized in type 1 diabetes (T1D). Immune regulatory T cell check-point receptors, which are generally upregulated on activated T cells, have been the molecules of attention as therapeutic targets for enhancing immune response in tumor therapy. We reported that dendritic cells (DCs) that are engineered to express selective ligands for checkpoint receptors can induce effective T cell tolerance in antigen-immunization models. Here, we show that pancreatic {beta} -cell antigen (BcAg) presentation by engineered tolerogenic-DCs (tDCs) that express CTLA4 selective ligand (B7.1wa) or a combination of CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1, and HVEM-CRD1 respectively; multiligand-DCs) causes an increase in regulatory cytokine and T cell (Treg) responses and suppression of the effector T cell function as compared to engineered control-DCs. Non-obese diabetic (NOD) mice treated with BcAg-pulsed CTLA4-ligand-DCs and multiligand-DCs at pre-diabetic and early-hyperglycmic stages showed significantly lower degree of insulitis, higher frequencies of insulin-positive islets, profound delay in, and reversal of, hyperglycemia for a significant duration. Immune cells from the tDC treated mice not only produced lower amounts of IFN{gamma} and higher amounts of IL10 and TGF{beta}1 upon BcAg challenge, but also failed to induce hyperglycemia upon adoptive transfer. While both CTLA4-ligand-DCs and multiligand-DCs were effective in inducing tolerance, multiligand-DC treatment produced an overall higher suppressive effect on effector T cell function and disease outcome. These studies show that enhanced engagement of T cell checkpoint receptors during BcAg presentation can modulate T cell function and suppress autoimmunity and progression of the disease in T1D.

category: immunology


title: Induction of cross-reactive antibody responses against the RBD domain of the spike protein of SARS-CoV-2 by commensal microbiota

abstract: The commensal microflora is a source for multiple antigens that may induce cross-reactive antibodies against host proteins and pathogens. However, whether commensal bacteria can induce cross-reactive antibodies against SARS-CoV-2 remains unknown. Here we report that several commensal bacteria contribute to the generation of cross-reactive IgA antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. We identified SARS-CoV-2 unexposed individuals with RBD-binding IgA antibodies at their mucosal surfaces. Conversely, neutralising monoclonal anti-RBD antibodies recognised distinct commensal bacterial species. Some of these bacteria, such as Streptococcus salivarius, induced a cross-reactive anti-RBD antibodies upon supplementation in mice. Conversely, severely ill COVID-19 patients showed reduction of Streptococcus and Veillonella in their oropharynx and feces and a reduction of anti-RBD IgA at mucosal surfaces. Altogether, distinct microbial species of the human microbiota can induce secretory IgA antibodies cross-reactive for the RBD of SARS-CoV-2.


title: The Cell Adhesion Molecule TMIGD1 Binds to Moesin and Regulates Tubulin Acetylation and Cell Migration

abstract:
BackgroundThe cell adhesion molecule transmembrane and immunoglobulin (Ig) domain containing1 (TMIGD1) is a novel tumor suppressor that plays important roles in regulating cell-cell adhesion, cell proliferation and cell cycle. However, the mechanisms of TMIGD1 signaling are not yet fully elucidated.

ResultsTMIGD1 binds to the ERM family proteins moesin and ezrin, and an evolutionarily conserved RRKK motif on the carboxyl terminus of TMIGD1 mediates the interaction of TMIGD1 with the N-terminal ERM domains of moesin and ezrin. TMIGD1 governs the apical localization of moesin and ezrin, as the loss of TMIGD1 in mice altered apical localization of moesin and ezrin in epithelial cells. In cell culture, TMIGD1 inhibited moesin-induced filopodia-like protrusions and cell migration. More importantly, TMIGD1 stimulated the Lysine (K40) acetylation of -tubulin and promoted mitotic spindle organization and CRISPR/Cas9-mediated knockout of moesin impaired the TMIGD1-mediated acetylation of -tubulin and filamentous (F)-actin organization.

ConclusionsTMIGD1 binds to moesin and ezrin, and regulates their cellular localization. Moesin plays critical roles in TMIGD1-dependent acetylation of -tubulin, mitotic spindle organization and cell migration. Our findings offer a molecular framework for understanding the complex functional interplay between TMIGD1 and the ERM family proteins in the regulation of cell adhesion and mitotic spindle assembly, and have wide-ranging implications in physiological and pathological processes such as cancer progression.


title: Tankyrase interacts with the allosteric site of glucokinase and inhibits its glucose-sensing function in the beta cell

abstract: Insulin secretion in the pancreatic beta cell is rate-limited by glucokinase (GCK), the glucose sensor that catalyzes the first step of glucose metabolism. GCK consists of two lobes connected by a flexible hinge that allows the kinase to exhibit a spectrum of conformations ranging from the active, closed form to several inactive, less-compact forms. Activating GCK mutations can cause hyperinsulinemia and hypoglycemia in infants. A similar phenotype exhibited by tankyrase (TNKS)-deficient mice prompted us to investigate whether TNKS might modulate the glucose-sensing function of GCK. We found that TNKS colocalizes and directly interacts with GCK. Their interaction is mediated by two ankyrin-repeat clusters (ARC-2 and -5) in TNKS and a tankyrase-binding motif (TBM, aa 63-68) in the GCK hinge. This interaction is conformation sensitive, human GCK variants that cause hyperglycemia (V62M) or hypoglycemia (S64Y) enhance or diminish the interaction respectively, even though they have no impact on TNKS interaction in the context of a GCK peptide (V62M) or a peptide library (S64Y). Moreover, the TNKS-GCK interaction is inhibited by high glucose concentrations, which are known to stabilize GCK in the active (closed, glucose-avid) conformation. Conversely, glucose phosphorylation by GCK in vitro is inhibited by TNKS. To study this in vitro inhibitory effect in the MIN6 beta cells, we showed that glucose-stimulated insulin secretion is suppressed upon stabilization of the TNKS protein and is conversely enhanced upon TNKS knockdown. Based on these findings as well as by contrasting with hexokinase-2, we propose that TNKS is a physiological GCK inhibitor in pancreatic beta cells that acts by trapping the kinase in the open (inactive) conformation.


title: Mechanisms of ecological divergence with gene flow in a reef-building coral on an isolated atoll in Western Australia

abstract: Understanding the mechanisms driving phenotypic variation in traits facing intensified selection from climate change is a crucial step in developing effective conservation and restoration initiatives. This is particularly true for reef-building corals, which are among the most vulnerable to climate change and are in dramatic decline globally. At the Rowley Shoals in Western Australia, the prominent reef flat becomes exposed on low tide and the stagnant water in the shallow atoll lagoons heats up, creating a natural laboratory for characterising the mechanisms that control phenotypic responses to different environments. We combined whole genome re-sequencing, common garden heat stress experiments, transcriptome-wide gene expression analyses, and symbiont metabarcoding to explore the mechanisms that facilitate survival in contrasting habitat conditions. Our data show that, despite high gene flow between habitats, spatially varying selection drives subtle shifts in allele frequencies at hundreds of loci. These changes were concentrated into several islands of divergence spanning hundreds of SNPs that showed strong linkage disequilibrium and were associated with a coordinated increase in minor allele frequencies in corals taken from the lagoon habitat, where the range of environmental conditions is greatest. Common garden heat stress assays showed individuals from the lagoon exhibited higher bleaching resistance than colonies from the reef slope, and RNAseq identified pronounced physiological differences between the corals from the two habitats, primarily associated with molecular pathways including cell signalling, ion transport and metabolism. Despite the pronounced physioloigical and environmental differences between habitats, metabarcoding of the Symbiodiniaceae ITS2 region revealed all colonies to be associated exclusively with the genus Cladocopium, with no detectable differences between habitats. This study contributes to the growing number of studies documenting the complex mechanisms that facilitate coral survival in extreme environments, and showcases the utility of combining multiple sequencing techniques to unravel complex climate-related traits.

category: genomics


title: Cell type determination for cardiac differentiation occurs soon after seeding of human induced pluripotent stem cells

abstract: Cardiac directed differentiation of human induced pluripotent stem cells consistently produces a mixed population of cardiomyocytes and non-cardiac cell types even when using very well-characterized protocols. We wondered whether differentiated cell types might result from intrinsic differences in hiPS cells prior to the onset of differentiation. By associating individual differentiated cells that share a common hiPS cell precursor, we were able to test whether expression variability in differentiated cells was pre-determined from the hiPS cell state. Although within a single experiment, differentiated cells that share an hiPS cell progenitor were more transcriptionally similar to each other than to other cells in the differentiated population, when the same hiPS cells were differentiated in parallel, we did not observe high transcriptional similarity across differentiations. Additionally, we found that substantial cell death occurred during differentiation in a manner that suggested that all cells were equally likely to survive or die, suggesting that there was no intrinsic selection bias for cells descended from particular hiPS cell progenitors. These results led us to wonder about how cells grow out spatially during the directed differentiation process. Labeling cells by their expression of a few canonical cell type marker genes, we showed that cells expressing the same marker tended to occur in patches observable by visual inspection, suggesting that cell type determination across multiple cell types, once initiated, is maintained in a cell-autonomous manner for multiple divisions. Altogether, our results show that while there is substantial heterogeneity in the initial hiPS cell population, that heterogeneity is not responsible for heterogeneous outcomes, and that the window during which cell type specification occurs is likely to begin shortly after the seeding of hiPS cells for differentiation.


title: Modeling the C. elegans Germline Stem Cell Genetic Network using Automated Reasoning

abstract: Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible model system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation. We model the "stem cell fate" versus entry into the "meiotic development" pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply a formal reasoning framework to derive predictive networks for control of differentiation. Using this approach we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision-making. This work lays a foundation for developing realistic whole tissue models of the C. elegans germ line where each cell in the model will execute a synthesized genetic network.

category: systems biology


title: Segregation of neural crest specific lineage trajectories from a heterogeneous neural plate border territory only emerges at neurulation

abstract: The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harbouring neural crest and cranial placode progenitors. Here we profile avian epiblast cells as a function of time using single-cell RNA-seq to define transcriptional changes in the emerging  neural plate border. The results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridisation. Developmental trajectory analysis shows that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined lineages.

category: developmental biology


title: Analyzing the Perceptual Attributes of Electro-tactile Stimuli as Function of Various Signal Properties

abstract: This study is designed to determine the proper electrical signals that induce various perceptual qualities. These perceptions are chosen from a wide range of feelings, comfort and even surface qualities such as smoothness or flatness. The goal is to derive the relationship between the electrical signal properties (i.e. voltage, frequency and duty cycle) and the perceived qualities which are understood by human observers and reveal any pattens in their reports. A total of 144 measurements are collected from 8 participants. The results are analyzed using numerous statistical methods. In the end, the governing factors are identified and a signal with specific properties is is determined for each of the perception qualities. A great number of plots are included in this study to facilitate the process of choosing the appropriate signal for each factor.


title: KnowMore: An Automated Knowledge Discovery Tool for the FAIR SPARC Datasets

abstract: This manuscript provides the methods and outcomes of KnowMore, the Grand Prize winning automated knowledge discovery tool developed by our team during the 2021 NIH SPARC FAIR Data Codeathon. The National Institutes of Health Stimulating Peripheral Activity to Relieve Conditions (NIH SPARC) program generates rich datasets from neuromodulation researches, curated according to the Findable, Accessible, Interoperable, and Reusable (FAIR) SPARC data standards. These datasets are publicly available through the SPARC Data Portal at sparc.science. Currently, the process of simultaneously comparing and analyzing multiple SPARC datasets is tedious because it requires investigating each dataset of interest individually and downloading all of them to conduct cross-analyses. It is crucial to enhance this process to enable rapid discoveries across SPARC datasets. To fill this need, we created KnowMore, a tool integrated into the SPARC Portal that only requires the user to select their datasets of interest to launch an automated discovery process. KnowMore uses several SPARC resources (Pennsieve, o2S2PARC, SciCrunch, protocols.io, Biolucida), data science methods, and machine learning algorithms in the back end to generate various visualizations in the front end intended to help the user identify potential similarities, differences, and relations across the datasets. These visualizations can lead to a new discovery, new hypothesis, or simply guide the user to the next logical step in their discovery process. The outcome of this project is a SPARC portal-ready code architecture that helps researchers to use SPARC datasets more efficiently and fully leverages their FAIR characteristics. The tool has been built and documented such that more data analysis methods and visualization items could be easily added. The potential for automated discoveries from SPARC datasets is huge given the unique SPARC data ecosystem promoting FAIR data practices, and KnowMore has only demonstrated a small highlight of what could be achieved to speed up discoveries from SPARC datasets.


title: SIRT5 is the desuccinylase of LDHA as novel cancer metastatic stimulator in aggressive prostate cancer

abstract: Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men worldwide. Among patients who developed advanced PCa, 80% suffered from bone metastasis, with a sharp drop in the survival rate. Despite great efforts, the details of the mechanisms underlying castration-resistant PCa (CRPC) remains unclear. SIRT5, an NAD+-dependent desuccinylase, is hypothesized to be a key regulator of various cancers. However, compared to other SIRTs, the role of SIRT5 in cancer has not been extensively studied. Here, we showed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages. The correlation between the decrease in the SIRT5 level and the patients survival rate was also confirmed. Using quantitative global succinylome analysis, we characterized a significant increase of lysine 118 succinylation (K118su) of lactate dehydrogenase A (LDHA), which plays a role in increasing LDH activity. As a substrate of SIRT5, LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients. This study investigated the reduction of SIRT5 and LDHA-K118su as a novel mechanism involved in PCa progression. It can also be proposed as a new target that can prevent castration-resistant PCa progression, which is key to PCa treatment.

category: cancer biology


title: Extent and complexity of RNA processing in the development of honey bee queen and worker castes revealed by Nanopore direct RNA sequencing

abstract: The distinct honey bee (Apis mellifera) worker and queen castes have become a model for the study of genomic mechanisms of phenotypic plasticity. Prior studies have explored differences in gene expression and methylation during development of the two castes, but thus far no study has performed a genome-wide analysis of differences in RNA processing. To address this here we performed a Nanopore-based direct RNA sequencing with exceptionally long reads to compare the mRNA transcripts between honey bee queen and workers at three points during their larval development. We found thousands of significantly differentially expressed isoforms (DEIs) between queen and worker larvae. Most DEIs contained alternative splicing, and many of them contained at least two types of alternative splicing patterns, indicating complex RNA processing in honey bee caste differentiation. We found a negative correlation between poly(A) length and DEI expression, suggesting that poly(A) tails participate in the regulation of isoform expression. Hundreds of isoforms uniquely expressed in either queens or workers during their larval development, and isoforms were expressed at different points in queen and worker larval development demonstrating a dynamic relationship between isoform expression and developmental mechanisms. These findings show the full complexity of RNA processing and transcript expression in honey bee phenotypic plasticity.


title: Powerful eQTL mapping through low coverage RNA sequencing

abstract: Mapping genetic variants that regulate gene expression (eQTL mapping) in large-scale RNA sequencing (RNA-seq) studies is often employed to understand functional consequences of regulatory variants. However, the high cost of RNA-Seq limits sample size, sequencing depth, and therefore, discovery power. In this work, we demonstrate that, given a fixed budget, eQTL discovery power can be increased by lowering the sequencing depth per sample and increasing the number of individuals sequenced in the assay. We perform RNA-Seq of whole blood tissue across 1490 individuals at low-coverage (5.9 million reads/sample) and show that the effective power is higher than that of an RNA-Seq study of 570 individuals at high-coverage (13.9 million reads/sample). Next, we leverage synthetic datasets derived from real RNA-Seq data to explore the interplay of coverage and number individuals in eQTL studies, and show that a 10-fold reduction in coverage leads to only a 2.5-fold reduction in statistical power. Our study suggests that lowering coverage while increasing the number of individuals is an effective approach to increase discovery power in RNA-Seq studies.


title: Genetic Diversity in the IZUMO1-JUNO Protein-Receptor Pair Involved in Human Reproduction

abstract:
Fertilization in mammals begins with the union of egg and sperm, an event that starts a cascade of cellular processes. The molecular-level understanding of these processes can guide the development of new strategies for controlling and/or promoting fertilization, and inform researchers and medical professional on the best choice of interventions. The proteins encoded by the IZUMO1 and JUNO genes form a ligand-receptor protein pair involved in the recognition of sperm and egg. Due to their role in the fertilization process, these proteins are potential targets for the development of novel anti-contraceptive, as well as infertility treatments. Here we present a comprehensive analysis of these gene sequences, with the objective of identifying evolutionary patterns that may support their relevance as targets for preventing or improving fertility among humans. JUNO and IZUMO1 gene sequences were identified within the genomes of over 2,000 humans sequenced in the 1000 Genomes Project. The human sequences were subjected to analyses of nucleotide diversity, selection neutrality, population-based differentiation (FST), haplotype inference, and whole chromosome scanning for signals of positive or of balancing selection. Derived alleles were determined by comparison to archaic hominin and other primate genomes. The potential effect of common non-synonymous variants on protein-protein interaction was also assessed. IZUMO1 displays higher variability among human individuals than JUNO. Genetic differentiation between continental population pairs was within whole-genome estimates for all but the JUNO gene in the African population group with respect to the other 4 population groups (American, East Asian, South Asian, and European). Tajimas D values demonstrated deviation from neutrality for both genes in comparison to a group of genes identified in the literature as under balancing or positive selection. Tajimas D for IZUMO1 aligns with values calculated for genes presumed to be under balancing selection, whereas JUNOs value aligned with genes presumed to be under positive selection. These inferences on selection are both supported by SNP density, nucleotide diversity and haplotype analysis. A JUNO haplotype carrying 3 derived alleles out of 5, one of which is a missense mutation implicated in polyspermy, was found to be significant in a population of African ancestry. Polyspermy has a disadvantageous impact on fertility and its presence in approximately 30% of the population of African ancestry may be associated to a potentially beneficial role of this haplotype. This role has not been established and may be related to a non-reproductive role of JUNO. The high degree of conservation of the JUNO sequence combined with a dominant haplotype across multiple population groups supports JUNO as a potential target for the development of contraceptive treatments. In addition to providing a detailed account of human genetic diversity across these 2 important and related genes, this study also provides a framework for large population-based studies investigating protein-protein interactions at the genome level.

AUTHOR SUMMARYFertilization in mammals depends on egg and sperm connecting to each other. Two proteins that are essential for this process are eggs JUNO and sperms IZUMO1. These proteins are encoded by genes known as JUNO and IZUMO1, which are present in the genomes of all humans. We analyzed the publicly available genomes of over 2,000 individuals from different continental populations (African (AFR), American (AMR), East Asian (EAS), European (EUR) and South Asian (SAS)) to investigate how well-conserved these genes are among humans, and to look for any signs that genetic variation on these genes could impact the binding of their encoded proteins to one another which, in turn, could impact the fertilization process. We found that the JUNO gene in the African population sampled diverged the most compared to the other population groups, and this divergence was above what is generally observed across the genome. Around 30% of the people of African ancestry investigated here carry a mutation that is associated to the abnormal fertilization of an egg by more than one sperm (known as polyspermy). Why this mutation is so common in the African population should be further investigated. We speculate that this mutation confers a genetic advantage related to the role of this receptor-ligand pair in cells not involved in reproduction.


title: αS oligomers generated from polyunsaturated fatty acid and dopamine metabolite differentially interact with Aβ to enhance neurotoxicity

abstract: It is increasingly becoming clear that neurodegenerative diseases are not as discrete as originally thought to be but rather display significant overlap in histopathological and clinical presentations. For example, nearly half of the patients with Alzheimer disease (AD) and synucleinopathies such as Parkinson disease (PD) show symptoms and pathological features of one another. Yet, the molecular events and features that underlie such comorbidities in neurodegenerative diseases remain poorly understood. Here, inspired to uncover the molecular underpinnings of the overlap between AD and PD, we investigated the interactions between amyloid-{beta} (A{beta}) and -synuclein (S), aggregates of which form the major components of amyloid plaques and Lewy bodies, respectively. Specifically, we focused on S oligomers generated from the dopamine metabolite called dihydroxyphenylacetaldehyde (DOPAL), and a polyunsaturated fatty acid docosahexaenoic acid (DHA). Both S oligomers showed structural and conformational differences confirmed by their disparity in size, secondary structure, susceptibility to proteinase K digestion and cytotoxicity. More importantly, the two oligomers differentially modulated A{beta} aggregation. While both oligomers inhibited A{beta} aggregation to varying extents, they induced structurally different A{beta} assemblies. Furthermore, A{beta} seeded with DHA-derived S oligomers showed greater toxicity than DOPAL-derived S oligomers in SH-SY5Y neuroblastoma cells. These results provide insights into the interactions between two amyloid proteins with empirically distinctive biophysical and cellular manifestations, enunciating a basis for potentially ubiquitous cross-amyloid interactions across many neurodegenerative diseases.

category: biophysics


title: BCL6-dependent TCF-1+ progenitor cells maintain effector and helper CD4 T cell responses to persistent antigen

abstract: Shortly after priming, the fate of activated CD4 T cells is segregated into BCL6+ follicular helper T (Tfh) and BCL6- effector (Teff) cells. However, it remains unknown how these subsets are sustained in the presence of chronic antigen stimulation. Using a combination of single cell- and population-based approaches, we show that in chronic viral infection, activated CD4 T cells differentiate into BCL6-dependent TCF-1+ progenitor cells with superior capacity to expand and give rise to both Teff and Tfh. They share properties with progenitor-exhausted CD8 T cells and are required for the continued generation of Teff cells as antigen persists. In response to tumors, an analogous CD4 T cell population develops in draining lymph nodes. Our study reveals the heterogeneity and plasticity of CD4 T cells upon encountering persistent antigen and highlights their population dynamics through a stable bipotent intermediate state.


title: In vitro activity of bedaquiline and imipenem against actively growing, nutrient-starved, and intracellular Mycobacterium abscessus

abstract: Mycobacterium abscessus lung disease is difficult to treat due to intrinsic drug resistance and the persistence of drug-tolerant bacteria. Currently, the standard of care is a multi-drug regimen with at least 3 active drugs, preferably including a {beta}-lactam (imipenem or cefoxitin). These regimens are lengthy, toxic, and have limited efficacy. The search for more efficacious regimens led us to evaluate bedaquiline, a diarylquinoline licensed for treatment of multidrug-resistant tuberculosis. We performed in vitro time-kill experiments to evaluate the activity of bedaquiline alone and in combination with the first-line drug imipenem against M. abscessus under various conditions. Against actively growing bacteria, bedaquiline was largely bacteriostatic and antagonized the bactericidal activity of imipenem. Contrarily, against nutrient-starved persisters, bedaquiline was bactericidal, while imipenem was not, and bedaquiline drove the activity of the combination. In an intracellular infection model, bedaquiline and imipenem had additive bactericidal effects. Correlations between ATP levels and the bactericidal activity of imipenem and its antagonism by bedaquiline were observed. Interestingly, the presence of Tween 80 in the media affected the activity of both drugs, enhancing the activity of imipenem and reducing that of bedaquiline. Overall, these results show that bedaquiline and imipenem interact differently depending on culture conditions. Previously reported antagonistic effects of bedaquiline on imipenem were limited to conditions with actively multiplying bacteria and/or the presence of Tween 80, whereas the combination was additive or indifferent against nutrient-starved and intracellular M. abscessus, where promising bactericidal activity of the combination suggests it may have a role in future treatment regimens.


title: The Global Virome in One Network (VIRION): an atlas of vertebrate-virus associations

abstract: Data cataloguing viral diversity on Earth have been fragmented across sources, disciplines, formats, and various degrees of open collation, posing challenges for research on macroecology, evolution, and public health. Here, we solve this problem by establishing a dynamically-maintained database of vertebrate-virus associations, called The Global Virome in One Network (VIRION). The VIRION database has been assembled through both reconciliation of static datasets and integration of dynamically-updated databases. These data sources are all harmonized against one taxonomic backbone, including metadata on host and virus taxonomic validity and higher classification; additional metadata on sampling methodology and evidence strength are also available in a harmonized format. In total, the VIRION database is the largest open-source, open-access database of its kind, with roughly half a million unique records that include 9,521 resolved virus "species" (of which 1,661 are ICTV ratified), 3,692 resolved vertebrate host species, and 23,147 unique interactions between taxonomically-valid organisms. Together, these data cover roughly a quarter of mammal diversity, a tenth of bird diversity, and ~6% of the estimated total diversity of vertebrates, and a much larger proportion of their virome than any previous database. We show how these data can be used to test hypotheses about microbiology, ecology, and evolution, and make suggestions for best practices that address the unique mix of evidence that coexists in these data.


title: Early-life telomere length covaries with life-history traits and scales with chromosome length in birds

abstract: Telomeres, the short DNA sequences that protect chromosome ends, are an ancient molecular structure, which is highly conserved across most eukaryotes. Species differ in their telomere lengths, but the causes of this variation are not well understood. Here, we demonstrate that mean early-life telomere length is an evolutionary labile trait across 58 bird species (representing 35 families in 12 orders) with the greatest trait diversity found among passerines. Among these species, telomere length is significantly negatively associated with the fast-slow axis of life-history variation, suggesting that telomere length may have evolved to mediate trade-offs between physiological requirements underlying the diversity of pace-of-life strategies in birds. Curiously, within some species, larger individual chromosome size predicts longer telomere lengths on that chromosome, leading to the suggestion that telomere length also covaries with chromosome length across species. We show that longer mean chromosome length or genome size tends to be associated with longer mean early-life telomere length (measured across all chromosomes) within a phylogenetic framework constituting up to 32 bird species. Combined, our analyses generalize patterns previously found within a few species and provide potential adaptive explanations for the 10-fold variation in telomere lengths observed among birds.

category: molecular biology


title: Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography

abstract:
Neurons navigate long distances and extend axons to form the complex circuitry of the mature brain. This depends on the coordinated response and continuous remodelling of the microtubule and F-actin networks in the axonal growth cone. Growth cone architecture remains poorly understood at nanoscales. We therefore investigated mouse hippocampal neuron growth cones using cryo-electron tomography to directly visualise their three-dimensional subcellular architecture with molecular detail. Our data show the hexagonal arrays of actin bundles that form filopodia penetrate the growth cone interior and terminate in the transition zone. We directly observe the modulation of these and other growth cone actin bundles by alteration of individual F-actin helical structures. Blunt-ended microtubules predominate in the growth cone, frequently contain lumenal particles and carry lattice defects. Investigation of the effect of absence of doublecortin, a neurodevelopmental cytoskeleton regulator, on growth cone cytoskeleton shows no major anomalies in overall growth cone organisation or in F-actin subpopulations. However, our data suggest that microtubules sustain more structural defects, highlighting the importance of microtubule integrity during growth cone migration.

Summary statementCryo-electron tomographic reconstruction of neuronal growth cone subdomains reveals distinctive F-actin and microtubule cytoskeleton architectures and modulation at molecular detail.


title: Cross-seasonal weather effects interact with breeding conditions to impact reproductive success in an alpine songbird

abstract:
O_LIIn alpine habitats, fluctuating early-season weather conditions and short breeding seasons limit reproductive opportunities, such that arriving and breeding earlier or later than the optimum may be particularly costly for migratory species. Given early-season energy limitations, the influence of environmental conditions across the annual cycle on breeding phenology may have pronounced fitness consequences, yet our understanding of cross-seasonal dynamics in alpine breeding organisms is severely limited.
C_LIO_LIFor an alpine-breeding, migratory population of horned lark (Eremophila alpestris) in northern British Columbia, Canada (54.8{degrees}N latitude) we assessed how spatially explicit weather conditions from across the annual cycle influenced clutch initiation date and offspring development. We also addressed how cross-seasonal effects on breeding parameters interact to influence reproductive fitness.
C_LIO_LIWith 12 years of intensive breeding data and 3 years of migration data from archival light-level geolocators, we used a sliding window approach to identify critical points during the annual cycle where weather events most influenced breeding phenology and offspring development. Consequences for reproductive success were assessed using nest survival simulations.
C_LIO_LIAverage clutch initiation varied up to 11 days among years but did not advance from 2003 to 2019. Colder temperatures with greater precipitation at wintering habitats, as well as colder temperatures upon arrival at the breeding site delayed clutch initiation, independent of arrival time. Extreme cold (sub-zero temperatures) within a staging area just prior to arrival at the breeding site carried over to prolong offspring development rate, potentially by influencing parental investment. Nest survival decreased with both later clutch initiation and prolonged offspring development, such that females that nested earlier and fledged offspring at a younger age were up to 45% more likely to reproduce successfully.
C_LIO_LIWe demonstrate pronounced carry-over effects acting through mechanisms that influence breeding phenology and offspring development independently. We also highlight the potential importance of staging areas for alpine songbirds, particularly given that environmental conditions are becoming increasingly decoupled across seasons. Understanding the cross-seasonal mechanisms shaping breeding decisions in stochastic environments like the alpine enables more accurate predictions of future individual- and population-level responses to climate change.
C_LI

category: ecology


title: A kinetic error filtering mechanism for enzyme-free copying of nucleic acid sequences

abstract: Accurate copying of nucleic acid sequences is essential for self-replicating systems. Modern cells achieve error ratios as low as 10-9 with sophisticated enzymes capable of kinetic proofreading. In contrast, experiments probing enzyme-free copying of RNA and DNA as potential prebiotic replication processes find error ratios on the order of 10%. Given this low intrinsic copying fidelity, plausible scenarios for the spontaneous emergence of molecular evolution require an accuracy-enhancing mechanism. Here, we study a  kinetic error filtering scenario that dramatically boosts the likelihood of producing exact copies of nucleic acid sequences. The mechanism exploits the observation that initial errors in template-directed polymerization of both DNA and RNA are likely to trigger a cascade of consecutive errors and significantly stall downstream extension. We incorporate these characteristics into a mathematical model with experimentally estimated parameters, and leverage this model to probe to what extent accurate and faulty polymerization products can be kinetically discriminated. While limiting the time window for polymerization prevents completion of erroneous strands, resulting in a pool in which full-length products show an enhanced accuracy, this comes at the price of a concomitant reduction in yield. We show that this fidelity-yield trade-off can be circumvented via repeated copying attempts in cyclically varying environments such as the temperature cycles occurring naturally in the vicinity of hydrothermal systems. This setting could produce exact copies of sequences as long as 50mers within their lifetime, facilitating the emergence and maintenance of catalytically active oligonucleotides.


title: A flexible summary-based colocalization method with application to the mucin Cystic Fibrosis lung disease modifier locus

abstract: Mucus obstruction is a central feature in the Cystic Fibrosis (CF) airways. A genome-wide association study (GWAS) of lung disease by the CF Gene Modifier Consortium (CFGMC) identified a significant locus containing two mucin genes, MUC20 and MUC4. Expression quantitative trait locus (eQTL) analysis using human nasal epithelial (HNE) from 94 CF Canadians in the CFGMC demonstrated MUC4 eQTLs that mirrored the lung association pattern in the region, suggesting that MUC4 expression may mediate CF lung disease. Complications arose, however, with colocalization testing using existing methods: the locus is complex and the associated SNPs span a 0.2Mb region with high linkage disequilibrium and evidence of eQTLs for multiple genes and tissues (heterogeneity). We previously developed the Simple Sum (SS), a powerful colocalization test in regions with heterogeneity, but SS assumed eQTLs to be present to achieve type I error control. Here we propose a two-stage SS (SS2) colocalization test that avoids a prior eQTL assumptions, accounts for multiple hypothesis testing and the composite null hypothesis and enables meta-analysis. We compare SS2 to published approaches through simulation and demonstrate type I error control for all settings with the greatest power in the presence of high LD and heterogeneity. Applying SS2 to the MUC20/MUC4 CF lung disease locus with eQTLs from CF HNE revealed significant colocalization with MUC4 (p = 1.71x10-5) rather than MUC20. The SS2 is a powerful method to inform the responsible gene(s) at a locus and guide future functional studies. SS2 has been implemented in the application LocusFocus (locusfocus.research.sickkids.ca).


title: Impact of sickle cell trait hemoglobin on the intraerythrocytic transcriptional program of Plasmodium falciparum

abstract:
Sickle-trait hemoglobin (HbAS) confers near-complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses of Plasmodium falciparum in normal (HbAA) and HbAS erythrocytes during both in vitro cultivation of reference parasite strains and naturally-occurring P. falciparum infections in Malian children with HbAA or HbAS. During in vitro cultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasites maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurers clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses of P. falciparum transcriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially-expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screen in vitro and in vivo indicates that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severe P. falciparum, modulation of these responses may offer avenues by which to neutralize P. falciparum parasites.

ImportanceSickle-trait hemoglobin (HbAS) confers near-complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we use RNA-seq to measure the impact of HbAS on the blood stage transcriptome of Plasmodium falciparum in in vitro time series experiments and in vivo samples from natural infections. Our in vitro time series data reveal that, during its blood stage, P. falciparums gene expression in HbAS is impacted primarily through alterations in the abundance of gene products as opposed to variations in the timing of gene expression. Collectively, our in vitro and in vivo data indicate that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Due to the persistent association of HbAS and protection from severe disease, these processes that are modified in HbAS may offer strategies to neutralize P. falciparum.


title: Minimalistic 3D Chromatin Models: Sparse Interactions in Single Cells Drive the Chromatin Fold and Form Many-Body Units

abstract: Computational modeling of 3D chromatin plays an important role in understanding the principles of genome organization. We discuss methods for modeling 3D chromatin structures, with focus on a minimalistic polymer model which inverts population Hi-C into high-resolution, high-coverage single-cell chromatin conformations. Utilizing only basic physical properties such as nuclear volume and no adjustable parameters, this model uncovers a few specific Hi-C interactions (15-35 for enhancerrich loci in human cells) that can fold chromatin into individual conformations consistent with single-cell imaging, Dip-C, and FISH-measured genomic distance distributions. Aggregating an ensemble of conformations also reproduces population Hi-C interaction frequencies. Furthermore, this single-cell modeling approach allows quantification of structural heterogeneity and discovery of specific many-body units of chromatin interactions. This minimalistic 3D chromatin polymer model has revealed a number of insights: 1) chromatin scaling rules are a result of volume-confined polymers; 2) TADs form as a byproduct of 3D chromatin folding driven by specific interactions; 3) chromatin folding at many loci is driven by a small number of specific interactions; 4) cell subpopulations equipped with different chromatin structural scaffolds are developmental stage-dependent; and 5) characterization of the functional landscape and epigenetic marks of many-body units which are simultaneously spatially co-interacting within enhancer-rich, euchromatic regions. The implications of these findings in understanding the genome structure-function relationship are also discussed.


title: Preferential interactions of a crowder protein with the specific binding site of a native protein complex

abstract:
The crowded cellular environments provide ample opportunities for proteins to interact with bystander macromolecules, yet direct evidence, let alone residue-specific information, for such nonspecific binding is rare. Here, by combining NMR spectroscopy and atomistic modeling, we investigated how crowders influence the association equilibrium and kinetics of two protein partners, EIN and HPr. Ficoll-70 increases the EIN-HPr binding affinity whereas bovine serum albumin (BSA) decreases the affinity. The opposite effects of the two crowders are quantitatively explained by atomistic modeling, which shows that the stabilizing effect of Ficoll-70 arises from volume exclusion favoring the bound state. In contrast, the destabilizing effect of BSA arises from preferential soft interactions with the free state; notably, BSA has favorable electrostatic interactions with positively charged HPr residues within the EIN-binding site. Some of the residues from this site indeed experience significant chemical shift perturbation when titrated with BSA, while the relaxation rates of HPr backbone amides exhibit overall elevation. Furthermore, relaxation dispersion data indicate that Ficoll-70 and BSA both slow down the EIN-HPr association rate, but change the dissociate rate in opposite directions. The observations on kinetics are accounted for by two effects of the crowders: increasing the solution microviscosity and reshaping the EIN-HPr interaction energy surface. The kind of preferential interactions between BSA and HPr that leads to competition with EIN should be prevalent in cellular environments. Our NMR results and atomistic modeling provide benchmarks, at both qualitative and quantitative levels, for the effects of crowded cellular environments on protein-protein specific interactions.

Significance StatementAlthough nonspecific binding of crowder macromolecules with functional proteins is likely prevalent in vivo, direct evidence is rare. Here we present NMR characterizations showing that bovine serum albumin preferentially interacts with a specific binding site on HPr, leading to competition with the latters partner EIN. The preferential interactions result in destabilization of the EIN-HPr native complex and speedup of its dissociation, contrary to expectations from excluded-volume and viscosity effects. Atomistic modeling of macromolecular crowding rationalizes the experimental observations, and provides qualitative and quantitative insight into the influences of the crowded cellular environment on protein-protein specific interactions. Our work also has implications for evolution, regarding how nonspecific binding can be either minimized or exploited for gaining new functions.


title: Viscoelasticity and Noise Properties Reveal the Formation of Biomemory in Cells

abstract: Living cells are neither perfectly elastic nor liquid and return a viscoelastic response to external stimuli. Nanoindentation provides force distance curves allowing the investigation of cell mechanical properties, and yet, these curves can differ from point to point on cell surface revealing its inhomogeneous character. In the present work, we propose a mathematical method to estimate both viscoelastic and noise properties of cells, as these are depicted on the values of the scaling exponents of relaxation function and power spectral density respectively. The method uses as input the time derivative of the response force in a nanoindentation experiment. Generalized moments method and/or rescaled range analysis are used to study the resulting time series depending on their non-stationary or stationary nature. We conducted experiments in living Ulocladium Chartarum spores. We found that spores, in the approaching phase present a viscoelastic behavior with the corresponding scaling exponent in the range 0.25-0.52, and in the retracting phase present a liquid-like behavior with exponents in the range 0.67-0.85. This substantial difference of the scaling exponents in the two phases suggests the formation of biomemory as response of the spores to the indenting AFM mechanical stimulus. The retracting phase may be described as a process driven by bluish noises, while the approaching one is driven by persistent noise.


title: Integrating multi-omics data reveals function and therapeutic potential of deubiquitinating enzymes

abstract: Deubiquitinating enzymes (DUBs) are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. Inhibition of DUBs promises to make classically undruggable targets such as the tumor suppressor TP53 and oncogene c-Myc amenable to regulation by small molecules. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper, which are browsable online via the DUB Portal, promise to improve understanding of DUBs as a family as well as the activities of specific DUBs such as USP14, UCHL5 and USP7, which have been targeted with investigational cancer therapeutics.


title: Immunohistochemical assays for bladder cancer molecular subtyping: Optimizing parsimony and performance using Lund taxonomy

abstract: Transcriptomic and proteomic profiling reliably classifies bladder cancers into luminal and basal molecular subtypes. Based on their prognostic and predictive associations, these subtypes may improve clinical management of bladder cancers. However, the complexity of published subtyping algorithms has limited their translation into practice. Here we optimize and validate compact subtyping algorithms based on the Lund taxonomy. We reanalyzed immunohistochemistry (IHC) expression data of muscle-invasive bladder cancer samples from Lund 2017 (n=193) and 2012 (n=76) cohorts. We characterized and quantified IHC expression patterns, and determined the simplest, most accurate decision tree models to identify subtypes. We tested the utility of a previously published algorithm using routine antibody assays commonly available in surgical pathology laboratories (GATA3, KRT5 and p16) to identify basal/luminal subtypes and to distinguish between luminal subtypes, Urothelial-Like (Uro) and Genomically Unstable (GU). We determined the dominant decision tree classifiers using four-fold cross-validation with separate uniformly distributed train (75%) and validation (25%) sets. Using the three-antibody algorithm resulted in 86-95% accuracy across training and validation sets for identifying basal/luminal subtypes, and 67-86% accuracy for basal/Uro/GU subtypes. Although antibody assays for KRT14 and RB1 are not routinely used in pathology practice, these features achieved the simplest and most accurate models to identify basal/luminal and Uro/GU/basal subtypes, achieving 93-96% and 85-86% accuracies, respectively. When translated to a more complex model using eight antibody assays, accuracy was comparable to simplified models, with 86% (train) and 82% (validation). We conclude that a simple immunohistochemical classifier can accurately identify luminal (Uro, GU) and basal subtypes and pave the way for clinical implementation.

category: pathology


title: Titin switches from an extensible spring to a mechanical rectifier upon muscle activation

abstract: In contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm-1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.


title: Ciliary IFT88 safeguards coordinated epiphyseal vascularisation, resorption and ossification from disruptive physiological mechanical forces

abstract: In the musculoskeletal system, appropriate cell and tissue responses to mechanical force delineate morphogenesis and ensure lifelong health. Despite this, how mechanical cues are integrated into biological programmes remains unclear. Primary cilia are microtubule-based organelles that tune a range of cell activities, including signalling cascades activated or modulated, by extracellular biophysical cues. Here, we demonstrate that the inducible, cartilage-specific deletion of Intraflagellar transport protein 88 (IFT88), which reduces ciliation in the adolescent mouse growth plate (GP), uncouples chondrocyte differentiation from cartilage resorption and mineralisation in a mechano-dependent manner. Targeting IFT88, inhibits hypertrophic chondrocyte VEGF expression, vascular recruitment, osteoclastic activity and the replacement of cartilage with bone. These effects are largely restricted to peripheral tibial regions beneath the load-bearing compartments of the knee. Increases in physiological loading, in control mice, also impairs ossification in the peripheral GP, mimicking the effects of IFT88 deletion. Strikingly, limb immobilisation rescues disrupted VEGF and restores epiphyseal dynamics in Ift88cKO mice. These data indicate, that during this pivotal phase in adolescent skeletal maturation that defines the cessation of growth, ciliary IFT88 protects the coordinated ossification of the growth plate from an otherwise disruptive heterogeneity of physiological mechanical forces.


title: Good and bad lettuce leaf microbes? Unravelling the genetic architecture of the microbiome to inform plant breeding for enhanced food safety and reduced food waste

abstract: Lettuce is a high value food crop, consumed raw around the world. Engineering of the leaf microbiome could provide significant benefits for enhanced crop yield and stress resistance and help to reduce food waste caused by microbial spoilage. Lettuce leaves also act as a vector for human pathogens, implicated in several high-profile food-borne disease outbreaks. Since host genotype helps determine microbiome composition, we hypothesize that leaf surface traits can be defined that associate with  good bacterial microbiomes providing benefits to the crop and that  bad microbiomes, where spoilage organisms and human pathogens are abundant, can also be associated to underlying leaf genetics, providing key targets for future crop breeding. Using a Recombinant Inbred Line (RIL) population, we show that cultivated and wild parental genotypes differ with reduced bacterial diversity, larger leaves and fewer, larger stomata, smaller epidermal cells and more hydrophilic leaf surfaces found in the cultivated compared to wild lettuce. Functional analysis of the associated microbiome revealed increased abundance of genes associated with disease virulence for the cultivated lettuce genotype, suggesting domestication has had broad impacts on leaf and associated bacterial microbiome traits. We defined the core lettuce bacterial microbiome from 171 RILs, comprised of 45 taxa in the phyla Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi and Deinococcus-Thermus. Leaf surface characteristics important in influencing bacterial diversity and abundance were identified as stomatal size (length and width), epidermal cell area and number and leaf surface hydrophobicity of the abaxial leaf surface. Quantitative trait loci (QTL) for leaf surface traits, frequently mapped alongside those for the extended phenotype of bacterial taxa abundance, including for human pathogens Campylobacter spp., Escherichia-Shigella spp., Clostridium spp. (LG 4, 5 and 6) and spoilage bacteria, Pseudomonas spp. (LG 1, 3, 4, 6 and 9). Candidate genes underlying these QTL were enriched in GO terms for cell wall assembly and modification, defence response, hormone-mediated signalling and biosynthesis and anatomical structure development. This work provides the first insight into the genetic architecture of host surface traits in a leafy crop alongside the mapped genetic architecture of bacterial communities and has identified areas of the lettuce genome as important targets for future microbiome engineering.

category: plant biology


title: Endogenous auxin directs development of embryonic stem cells into somatic proembryos in Arabidopsis

abstract:
Somatic embryogenesis (SE) is the process by which embryos develop from in vitro cultured vegetative tissue explants. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is widely used for SE induction, but SE can also be induced by overexpression of specific transcription factors, such as AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). 2,4-D and AHL15 both trigger the biosynthesis of the natural auxin indole-3-acetic acid (IAA). However, the role of this endogenously produced auxin in SE is yet not well understood. In this study we show that the induction of embryonic stem cells from explants does not require IAA biosynthesis, whereas an increase in IAA levels is essential to maintain embryo identity and for embryo formation from these stem cells. Further analysis showed that YUCCA (YUC) genes involved in the IPyA auxin biosynthesis pathway are up-regulated in embryo-forming tissues. Chemical inhibition of the IPyA pathway significantly reduced or completely inhibited the formation of somatic embryos in both 2,4-D-and AHL15-dependent systems. In the latter system, SE could be restored by exogenous IAA application, confirming that the biosynthesis-mediated increase in IAA levels is important. Our analyses also showed that PIN1 and AUX1 are the major auxin carriers that determine respectively auxin efflux and influx during SE. This auxin transport machinery is required for the proper transition of embryonic cells to proembryos and, later, for correct cell fate specification and differentiation. Taken together, our results indicate that auxin biosynthesis in conjunction with its polar transport are required during SE for multicellular somatic proembryo development and differentiation.

One sentence summarySomatic embryogenesis in Arabidopsis requires auxin biosynthesis and polar auxin transport only after the acquisition of embryonic competence for somatic proembryo development and differentiation.


title: Apoplastic effector candidates of a foliar forest pathogen trigger cell death in host and non-host plants

abstract: Forests are under threat from pests, pathogens, and changing climate. One of the major forest pathogens worldwide is Dothistroma septosporum, which causes dothistroma needle blight (DNB) of pines. D. septosporum is a hemibiotrophic fungus related to well-studied Dothideomycete pathogens, such as Cladosporium fulvum. These pathogens use small secreted proteins, termed effectors, to facilitate the infection of their hosts. The same effectors, however, can be recognised by plants carrying corresponding immune receptors, resulting in resistance responses. Hence, effectors are increasingly being exploited to identify and select disease resistance in crop species. In gymnosperms, however, such research is scarce. We predicted and investigated apoplastic D. septosporum candidate effectors (DsCEs) using bioinformatics and plant-based experiments. We discovered secreted proteins that trigger cell death in the angiosperm Nicotiana spp., suggesting their recognition by immune receptors in non-host plants. In a first for foliar forest pathogens, we also developed a novel protein infiltration method to show that tissue-cultured pine shoots can respond with a cell death response to one of our DsCEs, as well as to a reference cell death-inducing protein. These results contribute to our understanding of forest pathogens and may ultimately provide clues to disease immunity in both commercial and natural forests.