title: Macroevolutionary dynamics of climatic niche space

abstract: How and why lineages evolve along niche space as they diversify and adapt to different environments is fundamental to evolution. Progress has been hampered by the difficulties of linking a comprehensive empirical characterization of species niches with flexible evolutionary models that describe their evolution. Consequently, the relative influence of external episodic and biotic factors remains poorly understood. Here we characterize species two-dimensional temperature and precipitation niche space occupied (i.e., species niche envelope) as complex geometries and assess their evolution across a large vertebrate radiation (all Aves) using a model that captures heterogeneous evolutionary rates on time-calibrated phylogenies. We find that extant birds coevolved from warm, mesic climatic niches into colder and drier environments and responded to the K-Pg boundary with a dramatic increase in disparity. Contrary to expectations of subsiding rates of niche evolution as lineages diversify, our results show that overall rates have increased steadily, with some lineages experiencing exceptionally high evolutionary rates, associated with colonization of novel niche spaces, and others showing niche stasis. Both competition- and environmental change-driven niche evolution transpire and result in highly heterogeneous rates near the present. Our findings share the limitations of all work based purely on extant taxa but highlight the growing ecological and conservation insights arising from the model-based integration of increasingly comprehensive and robust environmental and phylogenetic information.

category: evolutionary biology


title: Photosynthesis and antioxidant metabolism modulate the low-temperature resistance of seed germination in maize

abstract:
Spring maize is usually subjected to low-temperature stress during seed germination, which retards seedling growth even if under a suitable temperature. However, the mechanism underlying maize seed germination under low-temperature stress modulating seedling growth after being transferred to normal temperature is still ambiguous. In this study, we used two maize inbred lines with different low-temperature resistance (SM and RM) to investigate the mechanism. The results showed that the SM line had higher lipid peroxidation and lower total antioxidant capacity and germination percentage than the RM line under low-temperature stress, which indicated that the SM line was more vulnerable to low-temperature stress. Further transcriptome analysis revealed that seed germination under low-temperature stress caused down-regulation of photosynthesis related gene ontology (GO) terms in two lines. Moreover, the SM line displayed down-regulation of ribosome and superoxide dismutase (SOD) related genes, whereas genes involved in SOD and vitamin B6 were up-regulated in the RM line. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that photosynthesis and antioxidant metabolism related pathways played important roles in seed germination in response to low-temperature stress, and the photosynthetic system displayed a higher damage degree in the SM line. Both qRT-PCR and physiological characteristics experiments showed similar results with transcriptome data. Taken together, we propose a model for maize seed germination in response to low-temperature stress.

One sentence summaryDamage degree of photosynthesis and total antioxidant capacity (especially SOD activity) determine diverse low-temperature resistance among maize inbred lines at the germination stage.

category: genomics


title: The SPEECHLESS-induced stomatal increase is required for the salt tolerance of oil palm

abstract: Oil palm is the most productive oil producing plant. Salt stress leads to growth damage and decrease in yield of oil palm. However, the physiological responses of oil palm to salt stress and their underlying mechanisms are not clear. RNA-Seq for leaf samples from young palms challenged under three levels of salts (100, 250 and 500 mM NaCl) and control for 14 days was conducted. Diverse signalling pathways were involved in responses to different levels of salt stress. All the three levels of salt stress activated EgSPCH expression and induced stomatal density of oil palm, which was contrasting to that in Arabidopsis. Under strong salt stress group, oil palm removed excessive salt via stomata. Overexpression of EgSPCH in Arabidopsis increased the stomatal production but lowered the salt tolerance. These data suggest that in oil palm, salt activates EgSPCH to generate more stomata in response to salt stress. Our results shed a light on the cellular response to salt stress of oil palm and provide new insights into the mechanisms of different salt-induced stomatal development between halophytes and glycophytes.

category: plant biology


title: Novel piece of the puzzle: ALI1 is required for oxo-C14-HSL priming in Arabidopsis

abstract: Quorum sensing (QS) molecules mediate communication between bacterial cells. N-acyl homoserine lactones (AHL) are one of the best-studied groups of QS molecules. In addition to bacterial communication, AHL are involved in interactions with eukaryotes. Short side-chain AHL are readily taken up by plants. They induce root elongation and growth promotion. Hydrophobic long side-chain AHL are usually not transported over long distances although, they may prime plants for enhanced resistance. Unfortunately, studies elucidating the plant factors required for response to AHL are sparse. Here, we provide evidence of a plant protein, namely the AHL-priming protein 1 (ALI1), indispensable for enhanced resistance response induced by the N-3-oxotetradecanoyl-homoserine lactone (oxo-C14-HSL). Comparing Col-0 and the ali1 mutant, we revealed loss of AHL-priming in ali1. This phenomenon is reverted with the reintroduction of ALI1 into ali1. Additional transcriptome analysis revealed that ali1 is less sensitive to oxo-C14-HSL treatment compared to the wild-type. Our results suggest, therefore, that ALI1 is required for oxo-C14-HSL-dependent priming for enhanced resistance in Arabidopsis.


title: Pharmacologic analysis of non-synonymous coding 5-HT2A SNPs reveals alterations psychedelic drug potencies and efficacies

abstract:
Serotonin (5-Hydroxytryptamine; 5-HT) 2A receptor (5-HT2AR) signaling is essential for the actions of classical psychedelic drugs. In this study, we examined whether random sequence variations in the gene (single nucleotide polymorphisms, SNPs) encoding the 5-HT2AR affect the signaling of four commonly used psychedelic drugs. We examined the in vitro pharmacology of seven non-synonymous SNPs, which give rise to S12N, T25N, D48N, I197V, A230T, A447V, and H452Y variant 5-HT2A serotonin receptors. We found that these non-synonymous SNPs exert statistically significant, although modest, effects on the efficacy and potency of four therapeutically relevant hallucinogens. Significantly, the in vitro pharmacological effects of the SNPs drug actions at 5-HT2AR are drug specific.

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category: neuroscience


title: Antagonism of D1, but not D2, dopamine receptors inhibits cued sucrose seeking by decreasing arousal

abstract: Dopamine facilitates approach to reward via its actions on dopamine receptors in the nucleus accumbens. For example, blocking either D1 or D2 dopamine receptors in the accumbens reduces the proportion of reward- predictive cues to which rats respond with cued approach. Recent evidence indicates that accumbens dopamine also promotes wakefulness and arousal, but the relationship between dopamines roles in arousal and reward seeking remains unexplored. Here, we show that the ability of systemic or intra-accumbens injections of the D1 antagonist SCH23390 to reduce cued approach to reward depends on the animals state of arousal. Handling the animal, a manipulation known to increase arousal, was sufficient to reverse the behavioral effects of the antagonist. In addition, SCH23390 reduced spontaneous locomotion and increased time spent in sleep postures, both consistent with reduced arousal, but also increased time spent immobile in postures inconsistent with sleep. In contrast, the ability of the D2 antagonist haloperidol to reduce cued approach was not reversible by handling. Haloperidol reduced spontaneous locomotion but did not increase sleep postures, instead increasing immobility in non-sleep postures. We place these results in the context of the extensive literature on dopamines contributions to behavior, and propose the arousal-motor hypothesis, a novel synthesis that accounts both for our findings and many previous results that have drawn disparate and conflicting conclusions.


title: Deficiency in endocannabinoid synthase DAGLB contributes to Parkinson's disease and dopaminergic neuron dysfunction

abstract: 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid (eCB) in the brain, regulates diverse neural functions. However, whether 2-AG deficiency contributes to Parkinsons disease (PD) and nigral dopaminergic neurons (DANs) dysfunction is unclear. Diacylglycerol lipase  and {beta} (DAGLA and DAGLB) mediate the biosynthesis of 2-AG. Using homozygosity mapping and whole-exome sequencing, we linked multiple homozygous loss-of-function mutations in DAGLB to a form of early-onset autosomal recessive PD. We then used RNA sequencing and fiber photometry with genetically encoded eCB sensors to demonstrate that DAGLB is the main 2-AG synthase in nigral DANs. Genetic knockdown of Daglb by CRISPR/Cas9 in mouse nigral DANs substantially reduces 2-AG levels in the substantia nigra (SN). The SN 2-AG levels are markedly correlated with the vigor of movement during the acquisition of motor skills, while Daglb-deficiency impairs motor learning. Conversely, pharmacological enhancement of 2-AG levels increases nigral DAN activity and dopamine release and improves motor learning. Together, we demonstrate that DAGLB-deficiency contributes to the etiopathogenesis of PD, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neural activity and dopamine release, and provide preclinical evidence for the beneficial effects of 2-AG augmentation in PD treatment.


title: Novel and known transcriptional targets of ALS/FTD protein TDP-43: Meta-analysis and interactive graphical databases

abstract: TDP-43 proteinopathy is the major pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal dementia (FTD). Mounting evidence implicates a loss of normal TDP-43 function in neurodegeneration, either resultant from or independent of TDP-43 aggregation. TDP-43 knockdown is therefore a common paradigm for modelling ALS and FTD. However, because TDP-43 can interact directly with thousands of mRNA targets and regulate the function of other RNA binding proteins, the phenotype of TDP-43 depletion is likely to differ depending on the proteomic and transcriptomic profile of the model cell type. Here, we conducted a meta-analysis of publicly available RNA-sequencing datasets that utilized TDP-43 knockdown to model ALS or FTD, and validated these against RNA-sequencing data from TDP-43-immunonegative neuronal nuclei from ALS/FTD brain. We present these analyses as easy-to-use interactive graphical databases. Of 9 TDP-43-knockdown datasets identified, 4 showed significant depletion of TARDBP (human HeLa and SH-SY5Y cell lines, induced human motor neurons, and mouse striatal tissue). There was little overlap in differentially expressed genes between TDP-43-knockdown model cell types, but PFKP, RANBP1, KIAA1324, ELAVL3, and STMN2 were among the common TDP-43 targets. Similarly, there were few genes that showed common patterns of differential exon usage between cell types and which validated in TDP-43-immunonegative neurons, but these included well-known targets POLDIP3, RANBP1, STMN2, and UNC13A, and novel targets EXD3, CEP290, KPNA4, and MMAB. Enrichment analysis showed that TDP-43 knockdown in different cell types affected a unique range of biological pathways. Together, these data identify novel TDP-43 targets, validate known TDP-43 targets, and show that TDP-43 plays both conserved and cell-type-specific roles in the regulation of gene expression and splicing. Identification of cell-type-specific TDP-43 targets will enable sensitive mapping of cell-autonomous TDP-43 dysfunction beyond just neurons, while shared TDP-43 targets are likely to have therapeutic value across myriad cell types.


title: Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content related to inflammation and recovery processes

abstract: Extracellular vesicles (EVs) are lipid bilayer enclosed structures that not only represent a newly discovered means for cell-to-cell communication but may also serve as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 hours after experimental stroke in mice and compared them to controls (shams) using the nCounter(R) Nanostring panels, with or without prior RNA isolation from BDEVs. We found that both panels show similar results when comparing upregulated mRNA in stroke. Notably, the higher upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, indicating that regenerative mechanisms are already taking place at this time-point. The five top overexpressed mRNAs in stroke mice compared to shams were confirmed by RT-qPCR and, interestingly, were found to be present as full-length open-reading frame in BDEVs. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and probably predominantly present in small BDEVs ([≤] 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter(R) panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation and that the mRNA cargo of BDEVs indicates their participation in inflammatory but also recovery processes after stroke.


title: Increased context adjustment is associated with auditory sensitivities but not with autistic traits

abstract:
Bayesian models of autism suggest that disruptions in context-sensitive prediction error weighting may underpin sensory perceptual alterations, such as hypersensitivities. We used an auditory oddball paradigm with pure tones arising from high or low uncertainity contexts to determine whether autistic individuals display differences in context adjustment relative to neurotypicals. We did not find group differences in early prediction error responses indexed by mismatch negativity. However, the autism group had larger evoked responses to outliers, at 300ms latency suggesting a greater reorienting of attention to surprising sounds. A dimensional approach revealed a positive correlation between context-dependent prediction errors and auditory sensitivities, but not with autistic traits. These findings suggest that autism studies may benefit from accounting for sensory sensitivities in group comparisons.

Lay SummaryWe find no differences in brain responses (EEG amplitudes) between autistic and neurotypical adults when listening to two contexts of tone patterns. However, we find a larger difference in the EEG amplitude when shifting between low to high uncertainity streams of tones, as sensory sensitivities (but not autistic traits) increase. These findings show that perceptual alterations maybe a function of sensory sensitivities, but not necessarily autistic traits. We suggest that future EEG studies in autism may benefit from accounting for sensory sensitivities.


title: The double-edged role of nitric oxide drives predictable microbial community organization according to the microenvironment

abstract: Microbial assemblages are omnipresent in the biosphere, forming communities on the surfaces of roots, rocks, and within living tissues. These communities can exhibit strikingly beautiful compositional structures, with certain members reproducibly occupying particular spatiotemporal microniches. Yet often, we lack the ability to explain the spatial patterns we see within them. To test the hypothesis that certain spatial patterns in microbial communities may be explained by the exchange of redox-active metabolites whose biological function is sensitive to environmental gradients, here we developed a simple community consisting of synthetic Pseudomonas aeruginosa strains with a partitioned denitrification pathway: a strict consumer and strict producer of nitric oxide (NO), a key pathway intermediate. Because NO can be both toxic or beneficial depending on the amount of oxygen present, this system provided an opportunity to investigate whether dynamic oxygen gradients can tune metabolic cross-feeding in a predictable fashion. Using a combination of genetic analysis, different growth environments and imaging, we show that oxygen availability controls whether NO cross-feeding is commensal or mutually beneficial, and that this organizing principle maps to the microscale. More generally, this work underscores the importance of considering the double-edged roles redox-active metabolites can play in shaping microbial communities.

category: microbiology


title: Engineering efficient termination of bacteriophage T7 RNA polymerase transcription

abstract: The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

category: molecular biology


title: A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia

abstract: The molecular regulation of human hematopoietic stem cell (HSC) maintenance is therapeutically important, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. Here, we have studied a rare genetic disorder due to MECOM haploinsufficiency, characterized by an early-onset absence of HSCs in vivo. By generating a faithful model of this disorder in primary human HSCs and coupling functional studies with integrative single-cell genomic analyses, we uncover a key transcriptional network involving hundreds of genes that is required for HSC maintenance. Through our analyses, we nominate cooperating transcriptional regulators and identify how MECOM prevents the CTCF-dependent genome reorganization that occurs as HSCs differentiate. Strikingly, we show that this transcriptional network is co-opted in high-risk leukemias, thereby enabling these cancers to acquire stem cell properties. Collectively, we illuminate a regulatory network necessary for HSC self-renewal through the study of a rare experiment of nature.

category: cell biology


title: Divalent metal ions boost effect of nucleic acids delivered by cell-penetrating peptides

abstract: Cell-penetrating peptides (CPPs) are promising tools for transfection of various substances, including nucleic acids, into cells. The aim of current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed of CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed an effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed with addition of the ions, without increasing cytotoxicity. We propose a novel strategy of preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, in the aim to increase CPP-mediated transfection efficiency and likelihood of using CPPs in clinics.


title: Estimating abundance of a recovering transboundary brown bear population with capture-recapture models

abstract: Abundance of small populations of large mammals may be assessed using complete counts of the different individuals detected over a time period, so-called minimum detected size (MDS). However, as population is growing larger and its distribution is expanding wider, the risk of under-estimating population size using MDS is increasing sharply due to the rarely fulfilled assumption of perfect detection of all individuals of the population, and as a result, the need to report uncertainty in population size estimates becomes crucial. We addressed these issues within the framework of the monitoring of the critically endangered Pyrenean brown bear population that was on the edge of extinction in the mid-1990s with only five individuals remaining, but was reinforced by 11 bears originated from Slovenia since then. We used Pollocks closed robust design (PCRD) capture-recapture models applied to the cross-border non-invasive sampling data from France, Spain and Andorra to provide the first published annual abundance estimates of the Pyrenean brown bear population and its trends over time. Annual population size increased and displayed a fivefold rise between 2008 and 2020, reaching > 60 individuals in 2020. Detection heterogeneity among individuals may stem from intraspecific home range size disparities making it more likely to find signs of individuals who move more. We found a lower survival rate in cubs than in adults and subadults, since the formers suffer from more mortality risks (such as infanticides, predations, mother death or abandonments) than the latters. Our study provides evidence that the PCRD capture-recapture modelling approach can provide reliable estimates of the size of and trend in large mammal populations, while minimizing bias due to inter-individual heterogeneity in detection probabilities and allowing the quantification of sampling uncertainty surrounding these estimates. Such information is vital for informing management decision-making and assessing population conservation status.

category: ecology


title: Differential effects of soil-based agricultural diversification on arthropods and crop yield

abstract:
O_LIAgricultural diversification often promotes biodiversity and ecosystem services by increasing habitat diversity. However, responses to agricultural diversification are context dependent, differentially impacting functional groups of service-providing organisms and crop yields.
C_LIO_LIConservation and no tillage are promoted as agricultural diversification practices that increase soil heterogeneity and habitat diversity. Here we investigated whether soil tillage practices in canola crop fields altered arthropod biodiversity or yield, and how effects of field-scale diversification compared to landscape-scale habitat context. We focused on effects of high, medium, or no tillage on five functional groups with unique diets and reproductive strategies: (i) herbivores, (ii) kleptoparasites, (iii) parasitoids, (iv) pollinators, and (v) predators.
C_LIO_LIEffects of agricultural diversification on arthropod abundance and diversity varied across functional groups. Pollinators responded to on-farm soil diversification, benefiting from medium tillage. Predators and herbivores responded most strongly to landscape-scale habitat composition and were more abundant in landscapes with more semi-natural habitat. However, variation in arthropod communities had little effect on canola crop yield, which was lowest in fields with no tillage.
C_LIO_LIPolicy implications: Our results indicate that natural history differences among arthropod functional groups mediate how habitat availability affects biodiversity. Crop yields, however, showed no response to biodiversity of ecosystem service providers. Our research highlights the need to determine the contexts in which soil diversification practices meet a multi-faceted goal of simultaneously supporting natural biodiversity, ecosystem services, and crop yield.
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title: Co-evolution of Leptospira and Tenrecidae (Afrotheria) on Madagascar

abstract: Leptospirosis is a bacterial zoonosis caused by pathogenic Leptospira that are maintained in the kidney lumen of infected animals acting as reservoirs and contaminating the environment via infected urine. The investigation of leptospirosis through a One Health framework has been stimulated by notable genetic diversity of pathogenic Leptospira together with a high infection prevalence in certain animal reservoirs. Studies of Madagascars native mammal fauna have revealed a diversity of Leptospira with high levels of host-specificity. Native rodents, tenrecids, and bats shelter several distinct lineages and species of Leptospira, some of which have also been detected in acute human cases. Specifically, Leptospira mayottensis, first discovered in humans on Mayotte, an island neighboring Madagascar, was subsequently identified in a few species of Malagasy tenrecids, an endemic family of small mammals. Distinct L. mayottensis lineages were identified in shrew tenrecs (Microgale cowani and Nesogale dobsoni) on Madagascar, and later in spiny tenrecs (Tenrec ecaudatus) on Mayotte. These findings suggest that L. mayottensis (i) has co-radiated with tenrecids on Madagascar, and (ii) has recently emerged in human populations on Mayotte following the introduction of T. ecaudatus from Madagascar. Hitherto, L. mayottensis has not been detected in spiny tenrecs on Madagascar. In the present study, we broaden the investigation of Malagasy tenrecids and describe the presence of L. mayottensis in Malagasy T. ecaudatus and M. thomasi. These results confirm the hypothesis that L. mayottensis was introduced to Mayotte, presumably via T. ecaudatus, and provide additional data on the co-radiation of Leptospira and Tenrecidae.


title: Co-radiation of Leptospira and Tenrecidae (Afrotheria) on Madagascar


title: Spodoptera littoralis genome mining brings insights on the dynamic of expansion of gustatory receptors in polyphagous noctuidae

abstract:
The bitter taste, triggered via gustatory receptors, serves as an important natural defense against the ingestion of poisonous foods in animals, and the diversity of food diet is usually linked to an increase in the number of gustatory receptor genes. This has been especially observed in polyphagous insect species, such as noctuid species from the Spodoptera genus. However, the dynamic and physical mechanisms leading to these gene expansions and the evolutionary pressures behind them remain elusive. Among major drivers of genome dynamics are the transposable elements but, surprisingly, their potential role in insect gustatory receptors expansion has not been considered yet.

In this work, we hypothesized that transposable elements and possibly positive selection would be involved in the active dynamic of gustatory receptor evolution in Spodoptera spp. We first sequenced de novo the full 465Mb genome of S. littoralis, and manually annotated all chemosensory genes, including a large repertoire of 373 gustatory receptor genes (including 19 pseudogenes). We also improved the completeness of S. frugiperda and S. litura gustatory receptor repertoires. Then, we annotated transposable elements and revealed that a particular category of class I retrotransposons, the SINE transposons, was significantly enriched in the vicinity of gustatory receptor gene clusters, suggesting a transposon-mediated mechanism for the formation of these clusters. Selection pressure analyses indicated that positive selection within the gustatory receptor gene family is cryptic, only 7 receptors being identified as positively selected.

Altogether, our data provide a new good quality Spodoptera genome, pinpoint interesting gustatory receptor candidates for further functional studies and bring valuable genomic information on the mechanisms of gustatory receptor expansions in polyphagous insect species.


title: Colonic Oxygen Microbubbles Augment Systemic Oxygenation and CO2 Removal in a Porcine Smoke Inhalation Model of Severe Hypoxia

abstract:
Inhalation injury can lead to pulmonary complications resulting in the development of respiratory distress and severe hypoxia. Respiratory distress is one of the major causes of death in critically ill patients with a reported mortality rate of up to 45%. The present study focuses on the effect of oxygen microbubble (OMB) infusion via the colon in a porcine model of smoke inhalation-induced lung injury. Juvenile female Duroc pigs (n=6 colonic OMB, n=6 no treatment) ranging from 39-51 kg in weight were exposed to smoke under general anesthesia for 2 h. Animals developed severe hypoxia 48 h after smoke inhalation as reflected by reduction in SpO2 to 66.3 % {+/-} 13.1% and PaO2 to 45.3 {+/-} 7.6 mmHg, as well as bilateral diffuse infiltrates demonstrated on chest x-ray. Colonic OMB infusion (75 - 100 mL/kg dose) resulted in significant improvements in systemic oxygenation as demonstrated by an increase in PaO2 of 13.2 {+/-} 4.7 mmHg and SpO2 of 15.2% {+/-} 10.0% out to 2.5 h, compared to no-treatment control animals that experienced a decline in PaO2 of 8.2 {+/-} 7.9 mmHg and SpO2 of 12.9% {+/-} 18.7% over the same timeframe. Likewise, colonic OMB decreased PaCO2 and PmvCO2 by 19.7 {+/-} 7.6 mmHg and 7.6 {+/-} 6.7 mmHg, respectively, compared to controls that experienced increases in PaCO2 and PmvCO2 of 17.9 {+/-} 11.7 mmHg and 18.3 {+/-} 11.2 mmHg. We conclude that colonic OMB therapy has potential to treat patients experiencing severe hypoxemic respiratory failure.

One Sentence SummaryEnteral oxygen microbubbles increase systemic oxygen and decrease carbon dioxide levels in acutely hypoxic pigs after smoke inhalation-induced respiratory failure.

category: bioengineering


title: DISEASES 2.0: a weekly updated database of disease-gene associations from text mining and data integration

abstract: The scientific knowledge about which genes are involved in which diseases grows rapidly, which makes it difficult to keep up with new publications and genetics datasets. The DISEASES database aims to provide a comprehensive overview by systematically integrating and assigning confidence scores to evidence for disease-gene associations from curated databases, genome-wide association studies (GWAS), and automatic text mining of the biomedical literature. Here, we present a major update to this resource, which greatly increases the number of associations from all these sources. This is especially true for the text-mined associations, which have increased by at least 9-fold at all confidence cutoffs. We show that this dramatic increase is primarily due to adding full-text articles to the text corpus, secondarily due to improvements to both the disease and gene dictionaries used for named entity recognition, and only to a very small extent due to the growth in number of PubMed abstracts. DISEASES now also makes use of a new GWAS database, TIGA, which considerably increased the number of GWAS-derived disease-gene associations. DISEASES itself is also integrated into several other databases and resources, including GeneCards/MalaCards, Pharos/TCRD, and the Cytoscape stringApp. All data in DISEASES is updated on a weekly basis and is available via a web interface at https://diseases.jensenlab.org, from where it can also be downloaded under open licenses.

category: bioinformatics


title: Coalescent tree recording with selection for fast forward-in-time simulations

abstract: Forward simulations are increasingly important in evolutionary genetics to simulate selection with realistic demography, mating systems and ecology. To reach the performance needed for genome-wide simulations a number of new simulation techniques have been developed recently. Kelleher et al. (2018) introduced a technique consisting in recording the entire genetic history of the population and placing mutations on the coalescent tree. This method cannot model selection. I recently introduced a simulation technique that speed up fitness calculation by assuming that fitness effects among haplotypes are multiplicative (Matthey-Doret, 2021). More precisely, fitness measures are stored for subsets of the genome and, at time of reproduction, if no recombination happen within a given subset, then the fitness for this subset for the offspring haplotype is directly inferred from the parental haplotype. Here, I present and benchmark a hybrid of the above two techniques. The algorithm records the genetic history of a species, directly places the mutations on the tree and infers fitness of subsets of the genome from parental haplotypes. At recombinant sites, the algorithm explores the tree to reconstruct the genetic data at the recombining segment. I benchmarked this new technique implemented in SimBit and report an important improvement of performance compared to previous techniques to simulate selection. This improvement is particularly drastic at low recombination rate. Such developments of new simulation techniques are pushing the horizon of the realism with which we can simulate species molecular evolution.


title: Investigating the conformational dynamics of Zika virus NS4B protein

abstract:
Zika virus (ZIKV) NS4B protein is a membranotropic protein having multifunctional roles such as evasion of host-immune system, and induction of host membrane rearrangement for viral genome replication and processing of polyprotein. Despite its versatile functioning, its topology and dynamics are not entirely understood. Presently, there is no NMR or X-ray crystallography-based structure available for any flaviviral NS4B protein. Therefore, in this study, we have investigated the structural dynamics of Zika Virus NS4B protein through 3D structure models using molecular dynamics (MD) simulations approach and experiments. Subsequently, we employed a reductionist approach to understand the dynamics of ZIKV NS4B protein. For this, we studied its N-terminal (residues 1-38), C-terminal (residues 194-251), and cytosolic (residues 131-169) regions in isolation. Further, we have performed experiments to prove the maximum dynamics in its cytosolic region. Using a combination of computational tools and circular dichroism (CD) spectroscopy, we validate the cytosolic region as an intrinsically disordered protein region (IDR). The microsecond-long all atoms molecular dynamics and replica-exchange MD simulations complement the experimental observations. We have also analysed its behaviour under the influence of differently charged liposomes and macromolecular crowding agents which may have significance on its overall dynamics. Lastly, we have proposed a ZIKV NS4B protein model illustrating its putative topology consisting of various membrane-spanning and non-membranous regions.

HighlightsO_LIMicrosecond simulations of NS4B N-terminus and cytosolic regions exposed their dynamic nature.
C_LIO_LIC-terminal region remains intact in presence lipid bilayer during 1 s simulations.
C_LIO_LISpectroscopic results also reveal the cytosolic region as an intrinsically disordered region.
C_LIO_LICytosolic region exhibits gain-of-structure property under helix inducing solvents.
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category: biophysics


title: Identification and Validation of Novel Microtubule Suppressors with an Imidazopyridine Scaffold through Structure Based Virtual Screening and Docking.

abstract: Targeting the colchicine binding site of alpha/beta tubulin microtubules can lead to suppression of microtubule dynamics, cell cycle arrest and apoptosis. Therefore, development of microtubule (MT) inhibitors is considered a promising route to anticancer agents. Our approach to identify novel scaffolds as MT inhibitors depends on a 3D-structure based pharmacophore approach and docking using three programmes MOE, Autodock and BUDE (Bristol University Docking Engine) to screen a library of virtual compounds. From this work we identified the compound 7-(3-Hydroxy-4-methoxy-phenyl)-3-(3-trifluoromethyl-phenyl)-6,7-dihydro-3H-imidazo[4,5-b] pyridin-5-ol (6) as a novel inhibitor scaffold. This compound inhibited several types of cancer cell proliferation at low micromolar concentrations with low toxicity. Compound 6 caused cell cycle arrest in the G2/M phase and blocked tubulin polymerization at low micromolar concentration (IC50 = 6 micromolar, inducing apoptosis via activation of caspase 9, increasing the level of the pro-apoptotic protein Bax and decreasing the level of the anti-apoptotic protein Bcl2. In summary, our approach identified a lead compound with potential antimitotic and antiproliferative activity.

category: cancer biology


title: Chronic AMPK inactivation slows SHH medulloblastoma progression by inhibiting mTORC1 signaling and depleting tumor stem cell populations

abstract: We show that inactivating AMPK in vivo in a genetic model of medulloblastoma depletes tumor stem cell populations and slows tumor progression. Medulloblastoma, the most common malignant pediatric brain tumor, grows as heterogenous communities comprising diverse types of tumor and stromal cells. We have previously shown that different types of cells in medulloblastomas show different sensitivities to specific targeted therapies. To determine if specific populations depend on AMPK, we analyzed mice with AMPK-inactivated medulloblastomas. We engineered mice with brain-wide, conditional deletion of the AMPK catalytic subunits Prkaa1 and Prkaa2 and conditional expression SmoM2, an oncogenic Smo allele that hyperactivates Sonic Hedgehog (SHH) signaling. We compared the medulloblastomas that formed in these mice to tumors that form in AMPK-intact mice with conditional SmoM2 expression. AMPK-inactivated tumors progressed more slowly, allowing longer event-free survival. AMPK inactivation altered the cellular heterogeneity, determined by scRNA-seq, increasing differentiation, decreasing tumor stem cell populations and reducing glio-neuronal multipotency. Mechanistically, AMPK inactivation altered glycolytic gene expression and decreased mTORC1 pathway activation. Hk2-deletion reproduced key aspects of the AMPK-inactivation phenotype, implicating altered glycolysis in the tumor suppressive effect of AMPK inactivation. Our results show that AMPK inactivation impairs tumor growth through mechanisms that disproportionately affect tumor stem cell populations. As stem cells are intrinsically resistant to current cytotoxic therapy that drives recurrence, finding ways to target these populations may prevent treatment failure. Our data suggest that targeted AMPK inactivation may produce therapeutic effects in tumor stem cell populations refractory to other therapeutic approaches.


title: Ca2+ oscillations, waves, and networks in islets from human donors with and without type 2 diabetes

abstract: Pancreatic islets are highly interconnected structures that produce pulses of insulin and other hormones, maintaining normal homeostasis of glucose and other nutrients. Normal stimulus-secretion and intercellular coupling are essential to regulated secretory responses and these hallmarks are known to be altered in diabetes. In the present study, we used calcium imaging of isolated human islets to assess their collective cell behavior. The activity occurred in the form of calcium oscillations, was synchronized across different regions of islets through calcium waves, and was glucose-dependent: higher glucose enhanced the activity, elicited a greater proportion of global calcium waves, and led to denser and less fragmented functional networks. Hub regions were identified in stimulatory conditions, and they represented the most active islet regions. Moreover, calcium waves were found to be initiated in different subregions and the roles of initiators and hubs did not overlap. In type 2 diabetes, glucose-dependence was retained, but a reduced activity, locally restricted waves, and more segregated networks were detected compared with control islets. Interestingly, hub regions seemed to suffer the most by losing a disproportionately large fraction of connections. These changes affected islets from donors with diabetes in a heterogeneous manner.

category: physiology


title: Phosphate-induced resistance to pathogen infection in Arabidopsis

abstract:
In nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudimentary. Here we show that MIR399 overexpression, loss-of-function of PHO2 (PHOSPHATE2), or treatment with high Pi, is accompanied by an increase in phosphate (Pi) content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g. miR399 overexpressor, pho2 mutant, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibiotrophic fungal pathogens. In the absence of pathogen infection, the expression level of genes in the salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling pathways was higher in high Pi plants compared to wild type plants, which is consistent with increased levels of SA and JA in non-infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while the ERF1-PDF1 branch positively responds to fungal infection, the MYC2/VSP2 branch is negatively regulated during pathogen infection in high Pi plants. This study supports that Pi accumulation promotes resistance to infection by fungal pathogens in Arabidopsis, while providing a basis to better understand crosstalk between Pi signaling and hormonal signalling pathways for modulation of plant immune responses.

Significance statementThis study highlights the importance of phosphate (Pi) in regulating immune responses, hence, disease resistance in Arabidopsis thaliana. Increasing Pi content either by MIR399 overexpression (or loss-of-function of PHOSPHATE2), as well as by Pi treatment enhances resistance to infection by necrotrophic and hemibiotrophic fungal pathogens through modulation of SA- and JA-dependent signaling pathways. These results also support that miR399 functions as a regulator of Arabidopsis immunity.


title: Can pyrethroid-piperonyl butoxide (PBO) nets reduce the efficacy of indoor residual spraying with pirimiphos-methyl against pyrethroid-resistant malaria vectors?

abstract: As the uptake of pyrethroid-PBO ITNs increases, their combination with IRS insecticides could become an operational reality in many malaria-endemic communities. Pirimiphos-methyl is a pro-insecticide requiring activation by mosquito cytochrome P450 enzymes to induce toxicity while PBO blocks activation of these enzymes in pyrethroid-resistant vector mosquitoes. PBO may thus antagonise the toxicity of pirimiphos-methyl IRS when combined with pyrethroid-PBO ITNs. The impact of combining two major brands of pyrethroid-PBO ITNs (Olyset(R) Plus, PermaNet(R) 3.0) with pirimiphos-methyl IRS (Actellic(R) 300CS) was evaluated against pyrethroid-resistant Anopheles gambiae sl in two parallel experimental hut trials in southern Benin in comparison to bendiocarb IRS and each intervention alone. The wild vector population was resistant to pyrethroids but susceptible to pirimiphos-methyl and bendiocarb. PBO pre-exposure partially restored deltamethrin toxicity but not permethrin. Mosquito mortality in experimental huts was significantly improved in the combinations of bendiocarb IRS with Olyset(R) Plus (33%) and PermaNet(R) 3.0 (38%) compared to bendiocarb IRS alone (14-16%, p<0.001), demonstrating an additive effect. Conversely, mortality was significantly reduced in the combinations of pirimiphos-methyl IRS with Olyset(R) Plus (59%) and PermaNet(R) 3.0 (55%) compared to pirimiphos-methyl IRS alone (77-78%, p<0.001), demonstrating an antagonistic effect. Combining pirimiphos-methyl IRS with the pyrethroid-PBO ITNs provided significantly improved mosquito mortality (55-59%) compared to the pyrethroid-PBO ITNs alone (22-26%) and improved blood-feeding inhibition relative to the IRS alone. This study provided evidence of an antagonistic effect when pyrethroid-PBO ITNs were combined with pirimiphos-methyl IRS in the same household resulting in lower levels of vector mosquito mortality compared to the IRS alone. Pirimiphos-methyl IRS also showed potential to significantly enhance malaria control when deployed to complement pyrethroid-PBO ITNs in an area where PBO fails to fully restore susceptibility to pyrethroids.

category: zoology


title: Early neural activity changes associated with visual conscious perception

abstract: The neural mechanisms of visual conscious perception have been investigated for decades. However, the spatiotemporal dynamics associated with the earliest neural responses following consciously perceived stimuli are still poorly understood. Using a dataset of intracranial EEG recordings, the current study aims to investigate the neural activity changes associated with the earliest stages of visual conscious perception. Subjects (N=10, 1,693 grey matter electrode contacts) completed a continuous performance task in which individual letters were presented in series and subjects were asked to press a button when they saw a target letter. Broadband gamma power (40-115Hz) dynamics were analyzed in comparison to baseline prior to stimulus and contrasted for target trials with button presses and non-target trials without button presses. Regardless of event type, we observed early gamma power changes within 30-150 ms from stimulus onset in a network including increases in bilateral occipital, fusiform, frontal (including frontal eye fields), and medial temporal cortex, increases in left lateral parietal-temporal cortex, and decreases in the right anterior medial occipital cortex. No significant differences were observed between target and non-target stimuli until >150 ms post-stimulus, when we saw greater gamma power increases in left motor and premotor areas, suggesting a possible role of these later signals in perceptual decision making and/or motor responses with the right hand. The early gamma power findings suggest a broadly distributed cortical visual detection network that is engaged at early times tens of milliseconds after signal transduction from the retina.


title: Directional Tuning of Phase Precession Properties in the Hippocampus

abstract: Running direction in the hippocampus is encoded by rate modulations of place field activity but also by spike timing correlations known as theta sequences. Whether directional rate codes and the directionality of place field correlations are related, however, has so far not been explored and therefore the nature of how directional information is encoded in the cornu ammonis remains unresolved. Here, using a previously published dataset that contains the spike activity of rat hippocampal place cells in the CA1, CA2 and CA3 subregions during free foraging of male Long-Evans rats in a 2D environment, we found that rate and spike timing codes are related. Opposite to a place fields preferred firing rate direction spikes are more likely to undergo theta phase precession and, hence, more strongly impact paired correlations. Furthermore, we identified a subset of field pairs whose theta correlations are intrinsic in that they maintain the same firing order when the running direction is reversed. Both effects are associated with differences in theta phase distributions, and are more prominent in CA3 than CA1. We thus hypothesize that intrinsic spiking is most prominent when the directionally modulated sensory-motor drive of hippocampal firing rates is minimal, suggesting that extrinsic and intrinsic sequences contribute to phase precession as two distinct mechanisms.


title: Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

abstract: Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1,2, which involve human-specific mechanisms3-6 that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors7. Single-cell transcriptomics (scRNA-seq) and comparison to independent NSCs8, showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks. Neuronal and glial maturation in iCoMoNSC-derived cultures was similar to that of cortical organoids9. Overexpression of wild-type TDP-43 in a minority of iCoMoNSC-derived neurons led to progressive fragmentation and aggregation, resulting in loss of function and neurotoxicity. scRNA-seq revealed a novel set of misregulated RNA targets coinciding in both TDP-43 overexpressing neurons and patient brains exhibiting loss of nuclear TDP-43. The strongest misregulated target encoded for the synaptic protein NPTX2, which was consistently misaccumulated in ALS and FTLD patient neurons with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby highlighting a new pathway of neurotoxicity.


title: In-depth characterization of the serum antibody epitope repertoire in inflammatory bowel disease using phage-displayed immunoprecipitation sequencing

abstract: Inflammatory bowel diseases (IBD), e.g. Crohns disease (CD) and ulcerative colitis (UC), are chronic immune-mediated inflammatory diseases. A comprehensive overview of an IBD-specific antibody epitope repertoire is, however, lacking. We leveraged a high-throughput phage-displayed immunoprecipitation sequencing (PhIP-seq) workflow to identify antibodies against 344,000 antimicrobial, immune and food antigens in 497 IBD patients as compared to 1,326 controls. IBD was characterized by 373 differentially abundant antibodies (202 overrepresented and 171 underrepresented), with 17% shared by both IBDs, 55% unique to CD and 28% unique to UC. Antibodies against bacterial flagellins dominated in CD and were associated with ileal involvement, fibrostenotic disease and anti-Saccharomyces cerevisiae antibody positivity, but not with fecal microbiome composition. Antibody epitope repertoires accurately discriminated CD from controls (AUC=0.89), and similar discrimination was achieved when using only ten antibodies (AUC=0.87). IBD patients thus show a distinct antibody repertoire against selected peptides, allowing patient stratification and discovery of immunological targets.

category: immunology


title: Quantitative Structural Analysis of Influenza Virus by Cryo-electron Tomography and Convolutional Neural Networks

abstract:
Influenza viruses pose severe public health threats; they cause millions of infections and tens of thousands of deaths annually in the US. Influenza viruses are extensively pleomorphic, in both shape and size as well as organization of viral structural proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships as well as aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and extensive viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we developed a cryoET processing pipeline leveraging convolutional neural networks (CNNs) to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza viral morphology, glycoprotein density, and conduct subtomogram averaging of HA glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.

Graphical abstract

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title: Noncanonical structural requirements of neurofibromin SUMOylation reveal a folding-deficiency of several pathogenic mutants

abstract: Neurofibromin (Nf1) is a large multidomain protein encoded by the tumour-suppressor gene NF1. NF1 is mutated in a frequently occurring genetic disease, neurofibromatosis type I, and in various cancers. The best described function of Nf1 is its Ras-GTPase activity, carried out by its GAP-related domain (GRD). SecPH, another structurally well-characterized domain of Nf1, is immediately adjacent to the GRD and interacts with lipids and proteins, thus connecting Nf1 to diverse signalling pathways. Here, we demonstrate, for the first time, that Nf1 and SecPH are substrates of the SUMO pathway. We identified a well-defined SUMOylation profile of SecPH and a main SUMOylation event on Lys1731 that appears to play a role in Ras-GAP activity. Our data allowed us to characterize a new set of pathogenic Nf1 missense mutants that exhibits a disrupted SUMOylation profile that may correlate with their unfolding. Accordingly, Lys1731 SUMOylation is mediated by a noncanonical structural motif, therefore allowing a read-out of SecPH conformation and folding status.


title: Wanted not, wasted not: Searching for non-target taxa in environmental DNA metabarcoding by-catch

abstract: Metabarcoding of environmental DNA is based on primers that are specific to the target taxa (e.g. bacteria, zooplankton, fishes). However, due to the nature of the commonly used protocols, regardless of the chosen primers, several sequences of non-target species will inevitably be generated, but are usually discarded in commonly used bioinformatics pipelines. These non-target sequences might contain important biological information about the presence of other species in the studied habitats and its potential for ecological studies is still poorly understood. Here, we analyzed the presence of mammal and bird species in aquatic environmental samples that were originally amplified targeting teleost fish species. After all cleaning and checking steps, we kept 21 amplicon sequence variants (ASVs) belonging to mammals and ten to birds. Most ASVs were taxonomic assigned to farm/domestic animals, such as cats, cows, and ducks. Yet, we were able to identify a native semi-aquatic mammal, the capybara, in the samples. Four native bird species and a non-native potentially invasive bird (Corvus sp.) were also detected. Although the data derived from these samples for mammals and birds are of limited use for diversity analyses, our results demonstrate the potential of aquatic samples to characterize non-aquatic birds and highlight the presence of a potentially invasive species that had not been recorded before in the region.


title: Life history adaptations to fluctuating environments: Combined effects of demographic buffering and lability of vital rates

abstract: Demographic buffering and lability have been identified as adaptive strategies to optimise fitness in a fluctuating environment. These are not mutually exclusive, however we lack efficient methods to measure their relative importance for a given life history. Here, we decompose the stochastic growth rate (fitness) into components arising from nonlinear responses and variance-covariance of demographic parameters to an environmental driver, which allows studying joint effects of buffering and lability. We apply this decomposition for 154 animal matrix population models under different scenarios, to explore how these main fitness components vary across life histories. Faster-living species appear more responsive to environmental fluctuations, either positively or negatively. They have the highest potential for strong adaptive demographic lability, while demographic buffering is a main strategy in slow-living species. Our decomposition provides a comprehensive framework to study how organisms adapt to variability through buffering and lability, and to predict species responses to climate change.


title: Life history adaptations to fluctuating environments: Combined effects of demographic buffering and lability of demographic parameters


title: Micro- and macroevolutionary change of colony-level traits in bryozoans

abstract: The evolution of trait variation among populations of animals is difficult to study due to the many overlapping genetic and environmental influences that control phenotypic expression. But, in a group of animals called bryozoans, it is possible to isolate genetic contributions to phenotypic variation, due to the modular nature of bryozoan colonies. Each bryozoan colony represents a snapshot of the phenotypes that correspond to a single genotype, which can be summarized as a phenotypic distribution. We test whether these phenotypic distributions are evolvable across a generation of colonies in two sister species of the bryozoan Stylopoma, grown and bred in a common garden breeding experiment. We find that components of phenotypic distributions, specifically median trait values of colony members, are evolvable between generations of colonies. Furthermore, this evolvability has macroevolutionary importance because it correlates with the morphological distance between these two species. Because parts of the phenotypic distributions are evolvable, and this evolvability corresponds to evolutionary divergence between species, we infer that these distributions can shift and change shape over macroevolutionary timescales. Such changes to phenotypic distributions across many generations of colonies may underpin the emergence of colony-level traits, like division of labor in colonies.


title: Evolvability and macroevolution of colony-level traits in two species of bryozoans


title: Epistatic genetic interactions govern morphogenesis during sexual reproduction and infection in a global human fungal pathogen

abstract:
Cellular development is orchestrated by evolutionarily conserved signaling pathways, which are often pleiotropic and involve intra- and inter-pathway epistatic interactions that form intricate, complex regulatory networks. Cryptococcus species are a group of closely-related human fungal pathogens that grow as yeasts yet transition to hyphae during sexual reproduction. Additionally, during infection they can form large, polyploid titan cells that evade immunity and develop drug resistance. Multiple known signaling pathways regulate cellular development, yet how these are coordinated and interact with genetic variation is less well understood. Here, we conducted quantitative trait locus (QTL) analyses of a mapping population generated by sexual reproduction of two parents, only one of which is unisexually fertile. We observed transgressive segregation of the unisexual phenotype among progeny, as well as a novel large-cell phenotype under mating-inducing conditions. These large-cell progeny were found to produce titan cells both in vitro and in infected animals. Two major QTLs and corresponding quantitative trait genes (QTGs) were identified: RIC8 (encoding a guanine-exchange factor) and CNC06490 (encoding a putative Rho-GTPase activator), both involved in G-protein signaling. The two QTGs interact epistatically with each other and with the mating-type locus in phenotypic determination. These findings provide insights into the complex genetics of morphogenesis during unisexual reproduction and pathogenic titan cell formation and illustrate how QTL analysis can be applied to identify epistasis between genes. This study shows that phenotypic outcomes are influenced by the genetic background upon which mutations arise, implicating dynamic, complex genotype-to-phenotype landscapes in fungal pathogens and beyond.

Significance statementCellular development is regulated by a complex web of signaling pathways that respond to both intracellular and extracellular cues. Morphological transitions in pathogenic fungi, such as those observed during sexual reproduction or in response to the host environment, offer tractable models for understanding the principles that govern eukaryotic cell development and morphogenesis. Using the human fungal pathogen Cryptococcus deneoformans as a model and applying QTL analysis, we defined novel genes and gene-gene interactions involved in the yeast-hyphal transition and titanization, two morphological developments that are important for adaptation, pathogenesis, and evolution of this fungal pathogen. Our study provides new insights into the conservation and complexity of key signaling pathways in regulating cell development in fungi, as well as other eukaryotes.

category: genetics


title: Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance

abstract:
BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms.

MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem susceptible Enterobacterales, isolated from patients from four U.S. hospitals over nearly five years.

ResultsOur CRE collection comprised a diverse range of species, lineages and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events.

Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment.

ConclusionsCollectively, the framework we developed provides a clearer, high resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.


title: A correlation map of genome-wide DNA methylation patterns between paired human brain and buccal samples

abstract: Epigenome-wide association studies (EWAS) assessing the link between DNA methylation (DNAm) and phenotypes related to structural brain measures, cognitive function, and neurodegenerative diseases are becoming increasingly more popular. Due to the inaccessibility of brain tissue in humans, several studies use peripheral tissues such as blood, buccal swabs, and saliva as surrogates. To aid the functional interpretation of EWAS findings in such settings, there is a need to assess the correlation of DNAm variability across tissues in the same individuals. In this study, we performed a correlation analysis between DNAm data of a total of n=120 matched post-mortem buccal and prefrontal cortex samples. We identified nearly 25,000 (3% of approximately 730,000) cytosine-phosphate-guanine (CpG) sites showing significant (False Discovery Rate q < 0.05) correlations between buccal and PFC samples. Correlated CpG sites showed a preponderance to being located in promoter regions and showed a significant enrichment of being determined by genetic factors, i.e. methylation quantitative trait loci (mQTL), based on buccal and dorsolateral prefrontal cortex mQTL databases. Our novel buccal-brain DNAm correlation map will provide a valuable resource for future EWAS using buccal samples for studying DNAm effects on phenotypes relating to the brain. All correlation results are made freely available to the public online.


title: Immobilization of C. elegans on cultivation plates by thermoelectric cooling for high-throughput subcellular-resolution microscopy

abstract: Despite its profound impact on biology, most high-resolution in vivo microscopy approaches remain low throughput because current immobilization techniques require significant manual effort. We greatly accelerate imaging of the nematode Caenorhabditis elegans by implementing a simple cooling approach to easily immobilize entire populations directly on their cultivation plates. We optimize and characterize cooling immobilization. Counterintuitively, relatively warmer temperatures immobilize animals significantly more effectively than colder temperatures utilized in prior studies. This enhanced immobilization enables clear submicron-resolution fluorescence imaging, which is challenging to achieve with most current immobilization techniques. We demonstrate 64x magnification 3D imaging and timelapse recording of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. By obviating individual animal manipulation, our approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows.


title: Strong immobilization of C. elegans en masse on cultivation plates by thermoelectric cooling for high-throughput submicron-resolution microscopy


title: High-throughput submicron-resolution microscopyof entire C. elegans populations under strongimmobilization by cooling cultivation plates


title: Empowering Engineered Muscle in Biohybrid Pump by Extending Connexin 43 Duration with Reduced Graphene Oxides

abstract: Engineered skeletal muscle act as therapeutics invaluable to treat injured or diseased muscle and a "living" material essential to assemble biological machinery. For normal development, skeletal myoblasts should express connexin 43, one of the gap junction proteins that promote myoblast fusion and myogenesis, during the early differentiation stage. However, myoblasts cultured in vitro often down-regulate connexin 43 before differentiation, limiting myogenesis and muscle contraction. This study demonstrates that tethering myoblasts with reduced graphene oxide (rGO) slows connexin 43 regression during early differentiation and increases myogenic mRNA synthesis. The whole RNA sequencing also confirms that the rGO on cells increases regulator genes for myogenesis, including troponin, while decreasing negative regulator genes. The resulting myotubes generated a three-fold larger contraction force than the rGO-free myotubes. Accordingly, a valveless biohybrid pump assembled with the rGO-tethered muscle increased the fluid velocity and flow rate considerably. The results of this study would provide an important foundation for developing physiologically relevant muscle and powering up biomachines that will be used for various bioscience studies and unexplored applications.


title: BioImageIT: Open-source framework for integration of image data-management with analysis

abstract: Open science and FAIR principles have become major topics in the field of bioimaging. This is due to both new data acquisition technologies that generate large datasets, and new analysis approaches that automate data mining with high accuracy. Nevertheless, data are rarely shared and rigorously annotated because it requires a lot of manual and tedious management tasks and software packaging. We present BioImageIT, an open-source framework for integrating data management according to FAIR principles with data processing.


title: Canalized morphogenesis driven by inherited tissue asymmetries in Hydra regeneration

abstract: The emergence and stabilization of a body axis is a major step in animal morphogenesis, determining the symmetry of the body plan as well as its polarity. To advance our understanding of the emergence of body-axis polarity we study regenerating Hydra. Axis polarity is strongly memorized in Hydra regeneration even in small tissue segments. What type of processes confer this memory? To gain insight into the emerging polarity, we utilize frustrating initial conditions by studying regenerating tissue strips which fold into hollow spheroids by adhering their distal ends, of opposite original polarities. Despite the convoluted folding process and the tissue rearrangements during regeneration, these tissue strips develop a new organizer in a reproducible location preserving the original polarity and yielding an ordered body plan. These observations suggest that the integration of mechanical and biochemical processes supported by their mutual feedback attracts the tissue dynamics towards a well-defined developmental trajectory biased by weak inherited cues from the parent animal. Hydra thus provide an example of dynamic canalization in which the dynamic rules themselves are inherited, in contrast to the classical picture where a detailed developmental trajectory is pre-determined.


title: Kinetic characteristics of transcriptional bursting in a complex gene model with cyclic promoter structure

abstract: While transcription occurs often in a bursty manner, various possible regulations can lead to complex promoter patterns such as promoter cycles, giving rise to an important issue: How do promoter kinetics shape transcriptional bursting kinetics? Here we introduce and analyze a general model of the promoter cycle consisting of multi-OFF states and multi-ON states, focusing on the effects of multi-ON mechanisms on transcriptional bursting kinetics. The derived analytical results indicate that bust size follows a mixed geometric distribution rather than a single geometric distribution assumed in previous studies, and ON and OFF times obey their own mixed exponential distributions. In addition, we find that the multi-ON mechanism can lead to bimodal burst-size distribution, antagonistic timing of ON and OFF, and diverse burst frequencies, each further contributing to cell-to-cell variability in the mRNA expression level. These results not only reveal essential features of transcriptional bursting kinetics patterns shaped by multi-state mechanisms but also can be used to the inferences of transcriptional bursting kinetics and promoter structure based on experimental data.

category: systems biology


title: Task Demands Change Motion Information Coding in Area MT of the Macaque

abstract: Neurons in the primate Middle Temporal (MT) area signal information about visual motion and work together with the lateral prefrontal cortex (LPFC) to support memory-guided comparisons of visual motion direction. These areas are reciprocally connected and both contain neurons that signal visual motion direction in the strength of their responses. Previously, LPFC was shown to display marked changes in stimulus coding with altered task demands. Since MT and LPFC work together, we sought to determine if MT neurons display similar changes with heightened task demands. We hypothesized that heightened working-memory task demands would improve the task-relevant information and precipitate memory-related signals in MT. Here we show that engagement in a motion direction comparison task altered non-sensory activity and improved stimulus encoding by MT neurons. We found that this improvement in stimulus information transmission was largely due to preferential reduction in trial-to-trial variability within a sub-population of highly direction-selective neurons. We also found that a divisive normalization mechanism accounted for seemingly contradictory effects of task-demands on a heterogeneous population of neurons.


title: Distribution map of peristaltic waves in the chicken embryonic gut reveals importance of ENS and inter-region cross talks along the gut axis

abstract:
Gut peristaltic movements recognized as the wave-like propagation of a local contraction are crucial for effective transportation and digestion/absorption of ingested materials. Although the physiology of gut peristalsis has been well studied in adults, it remains largely unexplored how the cellular functions underlying these coordinated tissue movements are established along the rostral-caudal gut axis during development. The chicken embryonic gut serves as an excellent experimental model for elucidating the endogenous potential and regulation of these cells since peristalsis occurs even though no ingested material is present in the moving gut. By combining video-recordings and kymography, we provide a spatial map of peristaltic movements along the entire gut posterior to the duodenum: midgut (jejunum and ileum), hindgut, caecum, and cloaca. Since the majority of waves propagate bidirectionally at least until embryonic day 12 (E12), the sites of origin of peristaltic waves (OPWs) can unambiguously be detected in the kymograph. The spatial distribution map of OPWs has revealed that OPWs become progressively confined to specific regions/zones along the gut axis during development by E12, and that such specific zones are largely conserved between different individuals implying genetic regulation for OPW determination. We have also found that the enteric nervous system (ENS) is essential for the OPW patterning since an ablation of ENS or blocking neural activity by tetrodotoxin disrupts the confined pattern of OPWs, resulting in a failure of transportation of inter-luminally injected ink. Finally, we have discovered a functional coupling of the endpoint of hindgut with the cloaca. When surgically separated, the cloaca ceases its acute contractions that would normally occur concomitantly with the peristaltic rhythm of the hindgut. Our findings shed light on the intrinsic regulations of gut peristalsis, including unprecedented ENS contribution and inter-region cross talk along the gut axis.

Contribution to the field statementIt has been well accepted that the gut peristalsis is important in adults, where a luminal content (ingested material) mechanically influences peristalsis. However, the endogenously regulated cellular mechanisms that initiate and sustain peristalsis have poorly been explored, and this might be a reason why few therapeutic treatments have been available for gut diseases associated with peristaltic dysfunction. Recent studies have shown that peristaltic movements occur in the embryonic gut, suggestive of genetic involvement. However, how the peristaltic movement is coordinately patterned along the gut axis remains unknown, because most studies have used isolated fragments of the gut. In our study, we examined the entire gut posterior to the duodenum of chicken embryos to produce a spatial map of peristaltic movements during gut development. Using the map of origins of peristaltic waves (OPWs), we found previously unappreciated roles of enteric nervous system for the OPW patterning and for the physiological functions of embryonic gut tissues. Furthermore, we propose that peristaltic movements might mediate inter-region crosstalk along the gut axis. Our findings provide novel insights into the mechanisms by which the gut peristalsis is established during development at the cellular basis.

category: developmental biology


title: Harnessing gene-gene interactions via an RNA-Sequencing network analysis framework improves precision medicine prediction in rheumatoid arthritis

abstract:
The study of gene-gene interactions in RNA-Sequencing (RNA-Seq) data has traditionally been hard owing the large number of genes detectable by Next-Generation Sequencing (NGS). However, differential gene-gene pairs can inform our understanding of biological processes and yield improved prediction models. Here, we utilised four well curated pathway repositories obtaining 10,537 experimentally evaluated gene-gene interactions. We then extracted specific gene-gene interaction networks in synovial RNA-Seq to characterise histologically-defined pathotypes in early rheumatoid arthritis patients. Specific gene-gene networks were also leveraged to predict response to methotrexate-based disease-modifying anti-rheumatic drug (DMARD) therapy in the Pathobiology of Early Arthritis Cohort (PEAC). We statistically evaluated the differential interactions identified within each network using robust linear regression models, and the ability to predict response was evaluated by receiver operating characteristic (ROC) curve analysis.

The analysis comparing different histological pathotypes showed a coherent molecular signature matching the histological changes and highlighting novel pathotype-specific gene interactions and mechanisms. Analysis of responders vs non-responders revealed higher expression of apoptosis regulating gene-gene interactions in patients with good response to conventional synthetic DMARD. Detailed analysis of interactions between pairs of network-linked genes identified the SOCS2/STAT2 ratio as predictive of treatment success, improving ROC area under curve (AUC) from 0.62 to 0.78. In conclusions, we demonstrate a novel, powerful method which harnesses gene interaction networks for leveraging biologically relevant gene-gene interactions leading to improved models for predicting treatment response.

category: genomics


title: The Presence of the Temporal Horn Exacerbates the Vulnerability of Hippocampus during Head Impacts

abstract: Hippocampal injury is common in traumatic brain injury (TBI) patients, but the underlying pathogenesis remains elusive. In this study, we hypothesize that the presence of the adjacent fluid-containing temporal horn exacerbates the biomechanical vulnerability of the hippocampus. Two finite element models of the human head were used to investigate this hypothesis, one with and one without the temporal horn, and both including a detailed hippocampal subfield delineation. A fluid-structure interaction coupling approach was used to simulate the brain-ventricle interface, in which the intraventricular cerebrospinal fluid was represented by an arbitrary Lagrangian-Eulerian multi-material formation to account for its fluid behavior. By comparing the response of these two models under identical loadings, the model that included the temporal horn predicted increased magnitudes of strain and strain rate in the hippocampus with respect to its counterpart without the temporal horn. This specifically affected cornu ammonis (CA) 1 (CA1), CA2/3, hippocampal tail, subiculum, and the adjacent amygdala and ventral diencephalon. These computational results suggest that the presence of the temporal horn exacerbate the vulnerability of the hippocampus, highlighting the mechanobiological dependency of the hippocampus on the temporal horn.


title: A mosaic bulk-solvent model improves density maps and the fit between model and data

abstract:
Bulk solvent is a major component of bio-macromolecular crystals and therefore contributes significantly to diffraction intensities. Accurate modeling of the bulk-solvent region has been recognized as important for many crystallographic calculations, from computing of R-factors and density maps to model building and refinement. Owing to its simplicity and computational and modeling power, the flat (mask-based) bulk-solvent model introduced by Jiang & Brunger (1994) is used by most modern crystallographic software packages to account for disordered solvent. In this manuscript we describe further developments of the mask-based model that improves the fit between the model and the data and aids in map interpretation. The new algorithm, here referred to as mosaic bulk-solvent model, considers solvent variation across the unit cell. The mosaic model is implemented in the computational crystallography toolbox and can be used in Phenix in most contexts where accounting for bulk-solvent is required. It has been optimized and validated using a sufficiently large subset of the Protein Data Bank entries that have crystallographic data available.

SynopsisA mosaic bulk-solvent method models disordered solvent more accurately than current flat bulk solvent model. This improves the fit between the model and the data, improves map quality and allows for the solution of problems previously inaccessible.


title: Predicting the Functional Impact of KCNQ1 Variants with Artificial Neural Networks

abstract:
Recent advances in experimental and computational protein structure determination have provided access to high-quality structures for most human proteins and mutants thereof. However, linking changes in structure in protein mutants to functional impact remains an active area of method development. If successful, such methods can ultimately assist physicians in taking appropriate treatment decisions. This work presents three artificial neural network (ANN)-based predictive models that classify four key functional parameters of KCNQ1 variants as normal or dysfunctional using PSSM-based evolutionary and/or biophysical descriptors. Recent advances in predicting protein structure and variant properties with artificial intelligence (AI) rely heavily on the availability of evolutionary features and thus fail to directly assess the biophysical underpinnings of a change in structure and/or function. The central goal of this work was to develop an ANN model based on structure and physiochemical properties of KCNQ1 potassium channels that performs comparably or better than algorithms using only on PSSM-based evolutionary features. These biophysical features highlight the structure-function relationships that govern protein stability, function, and regulation. The input sensitivity algorithm incorporates the roles of hydrophobicity, polarizability, and functional densities on key functional parameters of the KCNQ1 channel. Inclusion of the biophysical features outperforms exclusive use of PSSM-based evolutionary features in predicting activation voltage dependence and deactivation time. As AI is increasing applied to problems in biology, biophysical understanding will be critical with respect to  explainable AI, i.e., understanding the relation of sequence, structure, and function of proteins. Our model is available at www.kcnq1predict.org.

Author summaryHeartbeat is maintained by electrical impulses generated by ion-conducting channel proteins in the heart such as the KCNQ1 potassium channel. Pathogenic variants in KCNQ1 can lead to channel loss-of-function and predisposition to fatal life-threatening irregularities of heart rhythm (arrhythmia). Machine learning methods that can predict the outcome of a mutation on KCNQ1 structure and function would be of great value in helping to assess the risk of a heart rhythm disorder. Recently, machine learning has made great progress in predicting the structures of proteins from their sequences. However, there are limited studies that link the effect of a mutation and change in protein structure with its function. This work presents the development of neural network models designed to predict mutation-induced changes in KCNQ1 functional parameters such as peak current density and voltage dependence of activation. We compare the predictive ability of features extracted from sequence, structure, and physicochemical properties of KCNQ1. Moreover, input sensitivity analysis connects biophysical features with specific functional parameters that provides insight into underlying molecular mechanisms for KCNQ1 channels. The best performing neural network model is publicly available as a webserver, called Q1VarPredBio, that delivers predictions about the functional phenotype of KCNQ1 variants.


title: Glucose dynamics during ozone exposure measured using radiotelemetry: Stress drivers and human concordance

abstract:
BackgroundStress-related neurobehavioral and metabolic disorders are associated with altered circulating adrenal-derived hormones and hyperglycemia. Temporal assessment of glucose and these hormones is critical for insights on an individuals health.

ObjectivesHere we used implantable-telemetry in rats to assess real-time changes in circulating glucose during and after exposure to the air pollutant ozone, and link responses to circulating neuroendocrine stress and metabolic hormones. We also compared rodent glucose and corticosterone (cortisol in humans) responses to humans exposed to ozone.

MethodsFirst, using a cross-over design, we monitored glucose levels during single or repeated ozone exposures (0.0, 0.2, 0.4 and 0.8-ppm) and non-exposure periods in male Wistar-Kyoto-rats implanted with glucose-telemeters. A second cohort of un-implanted rats was exposed to ozone (0.0, 0.4 or 0.8-ppm) for 30-min, 1-hour, 2-hour, or 4-hour with hormones measured immediately after exposure. Then we assessed glucose metabolism in sham and adrenalectomized rats with or without pharmacological interventions of adrenergic and glucocorticoid receptors. Finally, we assessed glucose and cortisol in serum samples from a clinical study involving exposure of human volunteers to air or 0.3 ppm ozone.

ResultsOzone (0.8-ppm) caused hyperglycemia and hypothermia beginning 90-min into exposure, with reversal of effects 4-6 hours post-exposure. Glucose monitoring during four daily 4-hour ozone exposures revealed duration of hyperglycemia, adaptation, and diurnal variations. Ozone-induced hyperglycemia was preceded by increased adrenocorticotropic hormone, corticosterone, and epinephrine, but depletion of thyroid-stimulating, prolactin, and luteinizing hormones. Hyperglycemia was inhibited in rats that were adrenalectomized and/or treated with glucocorticoid inhibitor. The depletion of cortisol was dampened in humans exposed to ozone during intermittent exercise.

DiscussionWe demonstrate for the first time the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced metabolic dysfunction as exposure occurs. Real-time glucose monitoring with stress hormones assessment may be useful in identifying interactions among pollutants and stress-related illnesses.

category: pharmacology and toxicology


title: Glucose dynamics during ozone exposure measured using radiotelemetry: Stress drivers


title: A fungal plant pathogen overcomes conserved broad-spectrum disease resistance by rapid gene loss

abstract: Hosts and pathogens typically engage in an evolutionary arms race. This also applies to phytopathogenic powdery mildew fungi, which can rapidly overcome plant resistance and perform host jumps. Using experimental evolution, we show that the powdery mildew pathogen Blumeria graminis f.sp. hordei is capable of breaking the agriculturally important broad-spectrum resistance conditioned by barley loss-of-function mlo mutants. Partial mlo virulence is associated with a distinctive pattern of adaptive mutations, including small-sized (8-40 kb) deletions, one of which likely affects spore morphology. The detected mutational spectrum comprises the same loci in at least two independent mlo-virulent isolates, indicating convergent multigenic evolution. This work highlights the dynamic genome evolution of an obligate biotrophic plant pathogen with a transposonenriched genome.

category: plant biology


title: Time-dependent memory and individual variation in Arctic brown bears (Ursus arctos)

abstract: 1Animal movement modelling provides unique insight about how animals perceive their landscape and how this perception may influence space use. When coupled with data describing an animals environment, ecologists can fit statistical models to location data to describe how spatial memory informs movement. We performed such an analysis on a population of brown bears (Ursus arctos) in the Canadian Arctic using a model incorporating time-dependent spatial memory patterns. Brown bear populations in the Arctic lie on the periphery of the species range, and as a result endure harsh environmental conditions. In this kind of environment, effective use of memory to inform movement strategies could spell the difference between survival and mortality. The model we fit tests four alternate hypotheses (some incorporating memory; some not) against each other, and we found a high degree of individual variation in how brown bears used memory. We found that 52% (11 of 21) of the bears used complex, time-dependent spatial memory to inform their movement decisions. These results, coupled with existing knowledge on individual variation in the population, highlight the diversity of foraging strategies for Arctic brown bears while also displaying the inference that can be drawn from this innovative movement model.


title: Minimal cross-over between mutations associated with Omicron variant of SARS-CoV-2 and CD8+ T cell epitopes identified in COVID-19 convalescent individuals

abstract:
There is a growing concern that ongoing evolution of SARS-CoV-2 could lead to variants of concern (VOC) that are capable of avoiding some or all of the multi-faceted immune response generated by both prior infection or vaccination, with the recently described B.1.1.529 (Omicron) VOC being of particular interest. Peripheral blood mononuclear cell samples from PCR-confirmed, recovered COVID-19 convalescent patients (n=30) infected with SARS-CoV-2 in the United States collected in April and May 2020 who possessed at least one or more of six different HLA haplotypes were selected for examination of their anti-SARS-CoV-2 CD8+ T-cell responses using a multiplexed peptide-MHC tetramer staining approach. This analysis examined if the previously identified viral epitopes targeted by CD8+ T-cells in these individuals (n=52 distinct epitopes) are mutated in the newly described Omicron VOC (n=50 mutations). Within this population, only one low-prevalence epitope from the Spike protein restricted to two HLA alleles and found in 2/30 (7%) individuals contained a single amino acid change associated with the Omicron VOC. These data suggest that virtually all individuals with existing anti-SARS-CoV-2 CD8+ T-cell responses should recognize the Omicron VOC, and that SARS-CoV-2 has not evolved extensive T-cell escape mutations at this time.

ImportanceThe newly identified Omicron variant of concern contains more mutations than any of the previous variants described to date. In addition, many of the mutations associated with the Omicron variant are found in areas that are likely bound by neutralizing antibodies, suggesting that the first line of immunological defense against COVID-19 may be compromised. However, both natural infection and vaccination develop T-cell based responses, in addition to antibodies. This study examined if the parts of the virus, or epitopes, targeted by the CD8+ T-cell response in thirty individuals who recovered from COVID-19 in 2020 were mutated in the Omicron variant. Only one of 52 epitopes identified in this population contained an amino acid that was mutated in Omicron. These data suggest that the T-cell immune response in previously infected, and most likely vaccinated individuals, should still be effective against Omicron.


title: Plant-based expression of SARS-CoV-2 antigens for use in an oral vaccine

abstract: Oral and intra-nasal vaccines represent a key means of inducing mucosal-based immunity against infection with SARS-CoV-2, yet such vaccines represent only a minority of candidates currently in development. In this brief communication, we assessed the expression of the SARS-CoV-2 Receptor Binding Domain (RBD) subunit of the surface-exposed Spike glycoprotein in the leaves of nine edible plant species (lettuce, spinach, collard greens, tomato, cucumber, radish, arugula, pepper, and Coho greens), with a goal of identifying a suitable candidate for the development of an oral vaccine against COVID-19. We report lettuce (Lactuca sativa L. cv. Hilde II Improved) to be a preferred host to support in planta expression of SARS-CoV-2 RBD, representing an important first step towards development of a plant-based oral vaccine.


title: The UV RESISTANCE LOCUS 8-Mediated UV-B Response Is Required Alongside CRYPTOCHROME1 For Plant Survival Under Sunlight In The Field

abstract: As sessile organisms, plants are subjected to fluctuating sunlight including potentially detrimental ultraviolet-B radiation (UV-B). In Arabidopsis thaliana, experiments under controlled conditions have shown that UV RESISTANCE LOCUS 8 (UVR8) controls photomorphogenic responses for acclimation and tolerance to UV-B; however, its long-term impacts on plant performance remain poorly understood in naturally fluctuating environments. Here we quantified the survival and reproduction of different Arabidopsis mutant genotypes in diverse field and laboratory conditions. We found that uvr8 mutants produced more fruits than wild type in growth chambers with artificial low UV-B conditions but not in natural field conditions. Importantly, independent double mutants of UVR8 and the blue-light photoreceptor gene CRYPTOCHROME 1 (CRY1) in two genetic backgrounds showed a drastic reduction in fitness in the field. UV-B attenuation experiments in field conditions and supplemental UV-B in growth chambers demonstrated that UV-B caused the conditional cry1 uvr8 lethality phenotype. RNA sequencing in different conditions revealed a large number of genes with statistical interaction of UVR8 and CRY1 mutations in the presence of UV-B in the field. Among them, Gene Ontology analysis identified enrichment of categories related to UV-B response, oxidative stress, photoprotection and DNA damage repair. Our study demonstrates the functional importance of the UVR8-mediated response across life stages in natura, which is partially redundant with CRY1, and provides an integral picture of gene expression associated with plant environmental responses under diverse environmental conditions.


title: Predictive profiling of SARS-CoV-2 variants by deep mutational learning

abstract: The continual evolution of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the emergence of variants that show resistance to vaccines and neutralizing antibodies (1-4) threaten to prolong the coronavirus disease 2019 (COVID-19) pandemic (5). Selection and emergence of SARS-CoV-2 variants are driven in part by mutations within the viral spike protein and in particular the ACE2 receptor-binding domain (RBD), a primary target site for neutralizing antibodies. Here, we develop deep mutational learning (DML), a machine learning-guided protein engineering technology, which is used to interrogate a massive sequence space of combinatorial mutations, representing billions of RBD variants, by accurately predicting their impact on ACE2 binding and antibody escape. A highly diverse landscape of possible SARS-CoV-2 variants is identified that could emerge from a multitude of evolutionary trajectories. DML may be used for predictive profiling on current and prospective variants, including highly mutated variants such as omicron (B.1.1.529), thus supporting decision making for public heath as well as guiding the development of therapeutic antibody treatments and vaccines for COVID-19.

category: synthetic biology


title: Simultaneous analysis of antigen-specific B and T cells after SARS-CoV-2 infection and vaccination

abstract: Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B And T cell Tandem Lymphocyte Evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an optimized Activation Induced Marker (AIM) T cell assay and dual-color B cell antigen probes. Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection.