title: Chronic Seizures Worsen Cognitive Function with Loss of Presenilin 2 Function in a Sex-Related Manner

abstract:
Patients with early-onset Alzheimers disease (EOAD) are at elevated risk for seizures, including patients with variants in presenilin 2 (PSEN2). Like patients with epilepsy, untreated seizures may also worsen cognitive function in AD. We therefore sought to define the impact of loss of normal PSEN2 function and chronic seizures on cognitive function in the aged brain. Male and female PSEN2 KO and age- and sex-matched wild-type (WT) mice were sham or corneal kindled beginning at 6-months-old. Kindled and sham-kindled mice were then challenged up to 6 weeks later in a battery of cognitive tests: non-habituated open field (OF), T-maze spontaneous alternation (TM), and Barnes maze (BM), followed by immunohistochemistry for markers of neuroinflammation and neuroplasticity. PSEN2 KO mice required significantly more stimulations to kindle (males: p<0.02; females: p<0.02). Chronic seizures more significantly worsened anxiety-like behaviors of female PSEN2 KO mice as measured by percentage of total time exploring the center of an OF. TM performance was significantly worsened in kindled female (p=0.024), but not male, PSEN2 KO mice. Male BM performance was generally worsened by seizures (p=0.038), but not by genotype. Conversely, kindled PSEN2 KO females made the most BM errors (p=0.007). Chronic seizures also significantly altered expression of hippocampal neuroinflammation and neuroplasticity markers in a sex-specific manner. This study demonstrates that hippocampus-dependent cognitive function may be only worsened by uncontrolled chronic seizures in female, but not male, PSEN2 KO mice. Our work aligns with clinical evidence of sex-specific worsening of AD burden and supports sex-specific anticonvulsant interventions in AD.

Significance StatementIt is critical to understand the interaction between Alzheimers disease (AD)-associated genetic risk factors (i.e., PSEN2) and chronic seizures to implement disease modifying strategies for AD. Patients with EOAD are at up to 87-fold elevated risk of having focal impaired awareness seizures, but whether the seizures are a cause, or a consequence of functional decline is less clear. This study demonstrates that investigator-initiated chronic seizures specifically worsen cognitive function in female mice challenged in a battery of tasks, as well as shines renewed focus on the disproportionate burden of AD in the female brain. This work provides clear evidence that chronic seizures worsen cognitive performance in mice with an AD-associated genotype and suggests that seizure-induced functional deficits may be sex-specific.


title: Neural signature of everyday function in older adults at-risk of cognitive impairment

abstract: Assessment of everyday activities are central to the diagnosis of pre-dementia and dementia. Yet, little is known about the brain substrates and processes that contribute to everyday functional impairment, particularly during early stages of cognitive decline. We investigated everyday function using a complex gait task in normal older adults stratified by risk of cognitive impairment. We applied a novel EEG approach, which combines electroencephalographic with 3D-body tracking technology to measure brain-gait dynamics with millisecond precision while participants are in motion. Twenty-six participants (mean age = 74.9 years) with cognitive and everyday functional profiles within the normal range for their age and sex were ranked for risk of cognitive impairment. We used the Montreal Cognitive Assessment battery, a global index of cognition with a range from 0 to 30, to classify individuals as being at higher (22-26) and lower risk (27+). Individuals walking on a treadmill were exposed to visual perturbation designed to destabilize gait. Assuming that brain changes precede behavioral decline, we predicted that older adults increase step width to gain stability, yet the underlying neural signatures would be different for lower versus higher risk individuals. When pooling across risk groups, we found that step width increased and fronto-parietal activation shifted from transient, during swing phases, to sustained across the gait cycle during visually perturbed input. As predicted, step width increased in both groups but underlying neural signatures were different. Fronto-medial theta (3-7Hz) power of gait-related brain oscillations were increased in higher risk individuals during both perturbed and unperturbed inputs. On the other hand, left central gyri beta (13-28Hz) power was decreased in lower risk individuals, specifically during visually perturbed input. Finally, relating MoCA scores to spectral power pooled across fronto-parietal regions, we found associations between increased theta power and worse MoCA scores and between decreased beta power and better MoCA scores.Able-bodied older adults at-risk of cognitive impairment are characterized by unique neural signatures of mobility. Stronger reliance on frontomedial theta activation in at-risk individuals may reflect higher-order compensatory responses for deterioration of basic sensorimotor processes. Region and spectral-specific signatures of mobility may provide brain targets for early intervention against everyday functional decline.

category: neuroscience


title: Accumulation of Treg cells is detrimental in late-onset (aged) mouse model of multiple sclerosis

abstract:
Although typically associated with onset in young adults, multiple sclerosis (MS) also attacks aged people, which is termed late-onset MS. The disease can be recapitulated and studied in the aged mouse model of experimental autoimmune encephalomyelitis (EAE). The onset of induced EAE is delayed in aged mice, but the disease severity is increased relative to standard EAE in young mice. Given that CD4+FoxP3+ regulatory T (Treg) cells play an ameliorative role in MS/EAE severity and the aged immune system accumulates Treg cells, failure of these cells to prevent or ameliorate EAE disease is enigmatic. When analyzing the distribution of Treg cells in EAE mice, the aged mice exhibited a higher proportion of polyclonal(pan) Treg cells and a lower proportion of antigen-specific-Treg cells in their periphery, but lower proportions of pan- and antigen-specific-Treg cells in the central nervous system (CNS). Furthermore, in the aged CNS, Treg cells exhibited a higher plasticity and T effector (Teff) cells exhibited a greater clonal expansion, which disrupted the Treg/Teff balance. Transiently inhibiting FoxP3 expression in peripheral Treg cells partially ameliorated the disease and corrected Treg distribution in the aged mice. These results provide evidence that accumulated aged Treg cells play a detrimental role in neuronal inflammation of aged MS.

HighlightsO_ST_ABSQuestionC_ST_ABSCD4+ regulatory T (Treg) cells typically play an ameliorative role in multiple sclerosis (MS) onset and severity. However, why aged immune system has accumulated peripheral Treg cells, but the elderly has more severe MS symptoms?

FindingsAged Treg cells cannot easily distribute to the CNS of aged EAE mice, and those aged Treg cells that did enter the CNS exhibited increased plastic features. However, transient inhibition of peripherally accumulated Treg cells corrected Treg distribution and partially ameliorated the disease in the aged mice.

Conclusion and mechanistic insightsAccumulated aged Treg cells within an "inflammaging" condition do not play an ameliorative role but are potentially detrimental for inflamed CNS repair processes in aged EAE mice due to impeding the trafficking of immune cells into the inflamed CNS.

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category: immunology


title: Superinfection Exclusion of Alphaherpesviruses Interferes with Virion Trafficking

abstract:
Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection interferes with secondary viral infections within that same cell. Although SIE has been observed across many viruses, it has remained relatively understudied. A recently characterized glycoprotein D (gD) -independent SIE of alphaherpesviruses presents a novel mechanism of co-infection restriction for Herpes Simplex Virus Type 1 (HSV-1) and Pseudorabies virus (PRV). In this study, we evaluated the role of multiplicity of infection (MOI), receptor expression, and trafficking of virions to gain greater insight into potential mechanisms of alphaherpesvirus SIE.

We observed that high MOI secondary viral infections were able to overcome SIE in a manner that was independent of receptor availability. Utilizing recombinant viruses expressing fluorescent protein fusions, we assessed virion localization during SIE through live fluorescent microscopy of dual-labeled virions and localization of capsid assemblies. Analysis of these assemblies confirmed changes in the distribution of capsids during SIE. These results indicate that SIE during PRV infection inhibits viral entry or fusion while HSV-1 SIE inhibits infection through a post-entry mechanism. Although the timing and phenotype of SIE is similar between alphaherpesviruses, the related viruses implement different mechanisms to restrict coinfection.

IMPORTANCEMost viruses utilize a form of superinfection exclusion to conserve resources and control population dynamics. gD-dependent superinfection exclusion in alphaherpesviruses is well-documented. However, the under-characterized gD-independent SIE provides new insight into how alphaherpesviruses limit sequential infection. The observations described here demonstrate that gD-independent SIE differs between PRV and HSV-1. Comparing these differences provide new insights into the underlying mechanisms of SIE implemented by two related viruses.

category: microbiology


title: Nuclear lamins promote protrusion dynamics and collective, confined migration in vivo

abstract: Cells migrate collectively through confined environments during development and cancer metastasis. While the nucleus, a large and stiff organelle, impedes cell migration between non-deformable pillars in vitro, its function in vivo may vary depending on the microenvironment. Further, it is unknown how nuclei contribute to collective migration in vivo and whether nuclei in different positions within cell collectives experience different forces. Here, we use border cell migration in the fly ovary as an in vivo model to investigate the effects of confined, collective migration on nuclei and the contribution of nuclear lamins to migration. We found severe yet transient nuclear deformations occur, particularly in the leading cell, as border cells squeeze through tiny crevices between germline cells, termed nurse cells. Leading cells extend protrusions between nurse cells, which may pry open space to allow the cluster to advance. Here we report that the leading cell nuclei deformed as they moved into leading protrusions. Then as protrusions widened, the nucleus recovered a more circular shape. These data suggest that lead cell nuclei may help protrusions expand and thereby enlarge the migration path. To test how nuclei might promote or impede border cell migration, we investigated nuclear lamins, proteins that assemble into intermediate filaments and structurally support the nuclear envelope. Depletion of the Drosophila B-type lamin, Lam, from the outer, motile border cells, but not the inner, nonmotile polar cells, impeded border cell migration, whereas perturbations of the A-type lamin, LamC, did not. While wild type border cell clusters typically have one large leading protrusion as they delaminate from the anterior follicular epithelium, clusters depleted of B-type lamin had multiple, short-lived protrusions, resulting in unproductive cluster movement and failure to progress along the migration path. Further, border cell nuclei depleted of B-type lamins were small, formed blebs, and ruptured. Together, these data indicate that B-type lamin is requied for nuclear integrity, which in turn stabilizes the leading protrusion and promotes overall cluster polarization and collective movement through confined spaces.

category: cell biology


title: Caveolae mediated endocytosis of VLDL particles in macrophages requires NPC1 and STARD3 for further lysosomal processing

abstract:
Macrophages accumulate triglycerides under certain pathological conditions such as atherosclerosis. Triglycerides are carried in the bloodstream as part of very low-density lipoproteins (VLDL) and chylomicrons. How macrophages take up and process VLDL-lipids is not very well known. Here, using VLDL-sized triglyceride-rich emulsion particles, we aimed to study the mechanism by which VLDL-triglycerides are taken up, processed, and stored in macrophages. Our results show that macrophage uptake of emulsion particles mimicking VLDL (VLDLm) is dependent on lipoproteins lipase (LPL) and requires the lipoprotein-binding C-terminal domain of LPL but not the catalytic N-terminal domain. Subsequent internalization of VLDLm-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by triglyceride hydrolysis catalyzed by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for subsequent storage as triglycerides is mediated by Stard3, whereas NPC1 was found to promote the extracellular efflux of fatty acids from lysosomes. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete part of the internalized triglycerides as fatty acids.

SummaryTriglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a pro-inflammatory effect on macrophage. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. We show that uptake by macrophages of VLDL-like particles is dependent on the enzyme lipoproteins lipase via its C-terminal domain. Subsequent internalization of VLDL-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by hydrolysis by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for lipid storage is mediated by Stard3, while NPC1 promotes the extracellular efflux of fatty acids. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete internalized triglycerides as fatty acids.

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title: The probability of joint monophyly of all species in an arbitrary species tree.

abstract: Monophyly is a feature of a set of genetic lineages in which every lineage in the set is more closely related to all other members of the set than it is to any lineage outside the set. Multiple sets of lineages that are separately monophyletic are said to be reciprocally monophyletic, or jointly monophyletic. The prevalence of reciprocal monophyly, or joint monophyly, has been used to evaluate phylogenetic and phylogeographic hypotheses, as well as to delimit species. These applications often make use of a probability of joint monophyly under models of gene lineage evolution. Studies in coalescent theory have computed this joint monophyly probability for small numbers of separate groups in arbitrary species trees, and for arbitrary numbers of separate groups in trivial species trees. Here, generalizing existing results on monophyly probabilities under the multispecies coalescent, we derive the probability of joint monophyly for arbitrary numbers of separate groups in arbitrary species trees. We illustrate how our result collapses to previously examined cases. We also study the effect of tree height, sample size, and number of species on the probability of joint monophyly. The result also enables computation of relatively simple lower and upper bounds on the joint monophyly probability. Our results expand the scope of joint monophyly calculations beyond small numbers of species, subsuming past formulas that have been used in simpler cases.

category: evolutionary biology


title: Genomic characteristics of recently recognized Vibrio cholerae El Tor lineages associated with cholera in Bangladesh, 1991-2017

abstract:
Comparative genomic analysis of Vibrio cholerae El Tor associated with endemic cholera in Asia revealed two distinct lineages, one dominant in Bangladesh and the other in India. An in depth whole genome study of V. cholerae El Tor clinical strains isolated during endemic cholera in Bangladesh (1991 - 2017) included reference genome sequence data obtained online. Core genome phylogeny established using single nucleotide polymorphisms (SNPs) showed V. cholerae El Tor strains comprised two lineages, BD-1 and BD-2, which, according to Bayesian phylodynamic analysis, originated from paraphyletic group BD-0 around 1981. BD-1 and BD-2 lineages overlapped temporally but were negatively associated as causative agents of cholera 2004-2017. Genome wide association study (GWAS) revealed 140 SNPs and 31 indels, resulting in gene alleles unique to BD-1 and BD-2. Regression analysis of root to tip distance and year of isolation indicated early BD-0 strains at the base, whereas BD-1 and BD-2 subsequently emerged and progressed by accumulating SNPs. Pangenome analysis provided evidence of gene acquisition by both BD-1 and BD-2, of which six crucial proteins of known function were predominant in BD-2. BD-1 and BD-2 diverged and have distinctively different genomic traits, namely heterogeneity in VSP-2, VPI-1, mobile elements, toxin encoding elements, and total gene abundance. In addition, the observed phage-inducible chromosomal island-like element (PLE1), and SXT ICE elements (ICETET) in BD-2 presumably provided a fitness advantage for the lineage to outcompete BD-1 as the etiological agent of the endemic cholera in Bangladesh, with implications for global cholera epidemiology.

ImportanceCholera is a global disease with specific reference to the Bay of Bengal Ganges Delta where Vibrio cholerae O1 El Tor, causative agent of the disease showed two circulating lineages, one dominant in Bangladesh and the other in India. Results of in-depth genomic study of V. cholerae associated with endemic cholera during the past 27 years (1991 - 2017) indicate emergence and succession of the two lineages, BD-1 and BD-2, arising from a common ancestral paraphylatic group, BD-0, comprising the early strains and short-term evolution of the bacterium in Bangladesh. Among the two V. cholerae lineages, BD-2 supersedes BD-1 and is predominant in the most recent endemic cholera in Bangladesh. The BD-2 lineage contained significantly more SNPs and indels, and showed richness in gene abundance, including antimicrobial resistance genes, gene cassettes, and PLE to fight against bacteriophage infection, acquired over time. These findings have important epidemic implications at a global scale.

category: genomics


title: Characterizing the variation in chromosome structure ensembles in the context of the nuclear microenvironment

abstract: Inside the nucleus, chromosomes are subjected to direct physical interaction between different components, active forces, and thermal noise, leading to the formation of an ensemble of three-dimensional structures. However, it is still not well understood to what extent and how the structural ensemble varies from one chromosome region or cell-type to another. We designed a statistical analysis technique and applied it to single-cell chromosome imaging data to reveal the heterogeneity of individual chromosome structures. By analyzing the resulting structural landscape, we find that the largest dynamic variation is the overall radius of gyration of the chromatin region, followed by domain reorganization within the region. By comparing different human cell-lines and experimental perturbation data using this statistical analysis technique and a network-based similarity quantification approach, we identify both cell-type and condition-specific features of the structural landscapes. We identify a relationship between epigenetic state and the properties of chromosome structure fluctuation and validate this relationship through polymer simulations. Overall, our study suggests that the types of variation in a chromosome structure ensemble are cell-type as well as region-specific and can be attributed to constraints placed on the structure by factors such as variation in epigenetic state.


title: Galvanotaxis of ciliates: Spatiotemporal dynamics of Coleps hirtus under electric fields

abstract: Galvanotaxis describes the functional response of organisms to electric fields. In ciliates, the electric field influences the electrophysiology and thus the cilia beat dynamics. This leads to a change of the swimming direction towards the cathode. The dynamical response to electric fields of Coleps hirtus has not been studied since the observations of Verworn in 1890 (1). While galvanotaxis has been studied in other cilitates, C. hirtus exhibit properties not found elsewhere, such as biomineralization-processes of alveolar plates with impact on the intracellular calcium regulation and a bimodal resting membrane potential, which leads unique electrophysiological driven bimodal swimming dynamics. Here, we statistically analyze the galvanotactic dynamics of C. hirtus by automated cell tracking routines. We found that the number of cells that show a galvanotactic response, increases with the increase of the applied electric field strength with a mean at about 2.1 V/cm. The spatiotemporal swimming dynamics change and lead to a statistical increase of linear elongated cell trajectories that point toward the cathode. Further, the increase of the electric fields decreases the mean velocity variance for electric fields larger than about 1.3 V/cm, while showing no significant change in the absolute velocity for any applied electric field. Fully functional galvanotactic responses were observed at a minimum extracellular calcium concentration of 20 {micro}M. The results add important insights to the current understanding of cellular dynamics of ciliates and suggest that the currently accepted model lags the inclusion of the swimming dynamics and the complex calcium regulatory system of the cell. The results of this study do not only extend the fundamental understanding of C. hirtus dynamics, but also open possibilities for technical applications, such as biosensors or microrobots in the future.

category: biophysics


title: Water soluble polysaccharides from Spirulina platensis exhibited cyto/DNA protection and suppress the growth of gastric cancer cells via modulation of galectin-3

abstract: Polysaccharides from natural sources play a significant role in the management of different cancer types including gastric cancer. In this study, we reported the effect of spirulina polysaccharide (Sp) on galectin-3 modulatory activity on gastric cancer cells. The polysaccharide was isolated from the spirulina biomass, characterized, and the in silico, in vitro studies are carried out to assess the bioactivities. The isolated Sp possessed average molecular weight of 1457 kDa, and galactose (42%) as major sugar along with Rhamnose, Arabinose, Xylose, and Mannose. Further, characterization of Sp by FT-IR and NMR spectrum indicated the presence of ({beta}1-4D) galactose sugar with galactoarabinorhamnoglycan backbone. Among the monosaccharides, galactose showed highest binding affinity with galectin-3 protein as evidenced by the in silico interaction study. The obtained Sp, inhibited the proliferation of AGS gastric cancer cells by 48 % without affecting normal NIH/3T3 cells as opposed to doxorubicin, a known anticancer drug. Also, Sp exhibited galectin-3 mediated haemagglutination inhibition with MIC of 9.37 g/mL compared to galactose 6.25 g/mL, sugar specific to galectin-3. The Sp treatment significantly (p<0.05) lowered the expression of galectin-3 by 32 % compared to untreated control cells. In addition, Sp exhibited the potent cytoprotection in RBCs, Buccal cells, and DNA exposed to oxidants. Thus, the findings suggest that the polysaccharide from spirulina offer a promising therapeutic strategy in the management of gastric cancer in addition to its currently known nutritional and pharmaceutical applications.

category: cancer biology


title: Assessment and Comparative Study of Biofilm Formation with frequency of Multi Drug Resistance in strains of Staphylococcus aureus

abstract:
BackgroundThe study was conducted to identify the role of biofilms in the antibiotic susceptibility in the strains of Staphylococcus aureus. A total of 19 non repeated pus/wound swab samples from different anatomic locations and 17 samples that were previously identified as S. aureus and preserved in the labs were included in the study. The Staphylococcus aureus was identified on the basis of colony morphology, Grams stain, biochemical tests (catalase and coagulase tests) and molecular identification through PCR amplification.

MethodologyA total of 26 samples were recovered out of the 31 samples. Kirby-Bauer disk diffusion susceptibility test was used to determine the sensitivity or resistance of S. aureus to methicillin. Out of the 26 strains, 4 were highly resistant, 10 were moderately resistant and 12 strains were sensitive. Three different protocols (Tube Method, Congo Red Agar Method and Tissue Culture plate method) were used for the detection of biofilm formation for both resistant and sensitive strains.

ResultComparative analysis of the antibiotic susceptibility and biofilm formation by different protocols showed that 70% strains that are resistant to antibiotic methicillin produced moderate-strong biofilms. 50% have produced the moderate-strong biofilms in all 3 protocols. In case of sensitive, 50% strains had produced none-weak biofilms in all 3 protocols.

DecisionsThe strains that had zone of inhibition of close to resistance produced weak-strong biofilms but they all produced weak biofilms in CRA method. It can be concluded that the strains of S. aureus that have the ability to produce biofilms become methicillin resistant.


title: Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling

abstract:
Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18S rRNA and 73 sites in 25S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.

HighlightsO_LImethod enabling single-molecule profiling of RNA modifications is developed and reveals heterogeneous classes of modified ribosomes.
C_LIO_LIrRNA 2O methylation and pseudouridylation modifications are independent of each other.
C_LIO_LIin functional centers of the ribosome are modified in a concerted fashion.
C_LIO_LIof function for RNA helicases Dbp3 and Prp43 produce discrete overlapping subpopulations of incompletely modified ribosomes.
C_LIO_LImodification profiles are resilient to rapidly changing nutrient conditions and perturbation of translation
C_LI

category: molecular biology


title: Sibling rivalry among the ZBTB transcription factor family: homo vs. heterodimers

abstract: The BTB domain is an oligomerization domain found in over 200 proteins encoded in the human genome. In the family of BTB domain and Zinc Finger-containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site while the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed in vitro binding assays with fluorescent protein-BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family.


title: Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single cell sequencing

abstract:
BackgroundImmune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited.

MethodsIn this study, we compared the single-cell transcriptomes of 77{square}957 immune cells from 12 species using single-cell RNA-sequencing (scRNA-seq). Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells.

ResultsResults revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular crosstalk and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells.

ConclusionsThis study is the first to provide a comprehensive analysis of the cross-species single-cell atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders.


title: Global plant-frugivore trait matching is shaped by climate and biogeographic history

abstract: Species interactions are influenced by the trait structure of local multi-trophic communities. However, it remains unclear whether mutualistic interactions in particular can drive trait patterns at the global scale, where climatic constraints and biogeographic processes gain importance. Here we evaluate global relationships between traits of frugivorous birds and palms (Arecaceae), and how these relationships are affected, directly or indirectly, by assemblage richness, climate and biogeographic history. We leverage a new and expanded gape size dataset for nearly all avian frugivores, and find a positive relationship between gape size and fruit size, i.e., trait matching, which is influenced indirectly by palm richness and climate. We also uncover a latitudinal gradient in trait matching strength, which increases towards the tropics and varies among zoogeographic realms. Taken together, our results suggest trophic interactions have consistent influences on trait structure, but that abiotic, biogeographic and richness effects also play important, though sometimes indirect, roles in shaping the functional biogeography of mutualisms.

category: ecology


title: Sugar-rich larval diet promotes lower adult pathogen load and higher survival after infection in a polyphagous fly

abstract:
Nutrition is a central factor influencing immunity and resistance to infection, but the extent to which nutrition during development affects adult responses to infections is poorly understood. Our study investigated how the nutritional composition of the larval diet affects the survival, pathogen load, and food intake of adult fruit flies, Bactrocera tryoni, after bacterial septic infection. We found a sex-specific effect of larval diet composition on survival post-infection: survival rate was higher and bacterial load was lower for infected females fed sugar-rich larval diet compared with females fed protein-rich larval diet, an effect that was absent in males. Both males and females were heavier when fed a balanced larval diet compared to protein- or sugar-rich diet, while body lipid reserves were higher in the sugar-rich larval diet compared with other diets. Body protein reserve was lower for sugar-rich larval diets compared to other diets in males, but not females. Both females and males shifted their nutrient intake to ingest a sugar-rich diet when infected compared with sham-infected flies without any effect of the larval diet, suggesting that sugar-rich diets can be beneficial to fight off bacterial infection. Overall, our findings show that nutrition during early life can shape individual fitness in adulthood.

Summary statementDevelopmental conditions influence adulthood. Here we showed that when the larval diet is rich in sugar, resistance to infection is increased in females at adulthood, in a polyphagous fly.


title: Evolutionary traits of Tick-borne encephalitis virus: Pervasive non-coding RNA structure conservation and molecular epidemiology

abstract: Tick-borne encephalitis virus (TBEV) is the etiological agent of tick-borne encephalitis, an infectious disease of the central nervous system that is often associated with severe sequelae in humans. While TBEV is typically classified into three subtypes, recent evidence suggests a more varied range of TBEV subtypes and lineages that differ substantially in the architecture of their 3 untranslated region (3UTR). Building on comparative genomics approaches and thermodynamic modelling, we characterize the TBEV UTR structureome diversity and propose a unified picture of pervasive non-coding RNA (ncRNA) structure conservation. Moreover, we provide an updated phylogeny of TBEV, building on more than 220 publicly available complete genomes, and investigate the molecular epidemiology and phylodynamics with Nextstrain, a web-based visualization framework for real-time pathogen evolution.


title: The role of pseudo-overdominance in maintaining inbreeding depression

abstract: Classical models ignoring linkage predict that deleterious recessive mutations purge or fix within inbred populations, yet these often retain moderate to high segregating load. True overdominance generates balancing selection that sustains inbreeding depression even in inbred populations but is rare. In contrast, arrays of mildly deleterious recessives linked in repulsion may occur commonly enough to generate pseudo-overdominance and sustain segregating load. We used simulations to explore how long pseudo-overdominant regions (PODs) persist following their creation via hybridization between populations fixed for alternative mutations at linked loci. Balancing haplotype loads, tight linkage, and moderate to strong cumulative selective effects serve to maintain PODs, suggesting that PODs may most often arise and persist in low recombination regions (e.g., inversions). Selection and drift unbalance the load, eventually eliminating PODs, but this process is very slow when pseudo-overdominance is strong. Background selection across the genome accelerates the loss of weak PODs but reinforces strong PODs in inbred populations by disfavoring homozygotes. Further modeling and studies of POD dynamics within populations could help us understand how PODs affect persistence of the load and how inbred mating systems evolve.


title: A genome-edited β-cell model of Prader-Willi syndrome reveals chronic deficits in endoplasmic reticulum chaperones and insulin secretion

abstract: Prader-Willi syndrome (PWS) is a multisystem disorder caused by loss of expression of a cluster of paternally-expressed, imprinted genes. Neonatal failure to thrive is followed by childhood-onset hyperphagia, obesity, neurobehavioral abnormalities, and hormonal deficits. Prior evidence from a mouse model with a deletion of the orthologous PWS-gene domain identified abnormal pancreatic islet development with deficient insulin secretion, hypoglucagonemia, and postnatal onset of progressive, lethal hypoglycemia. To investigate the role of PWS-genes in {beta}-cell secretory function, we used CRISPR/Cas9 genome-editing to generate isogenic, clonal INS-1 insulinoma lines with 3.16 Mb deletions of the silent, maternal (control) or active, paternal (PWS) alleles. PWS {beta}-cells showed a significant reduction in basal and glucose-stimulated insulin secretion, signifying a deficiency in cell-autonomous insulin secretion. Parallel proteome and transcriptome studies revealed reduced levels of secreted peptides and twelve endoplasmic reticulum (ER) chaperones, including HSPA5 and HSP90B1. In contrast to the dosage compensation previously seen for ER chaperones in Hspa5 or Hsp90b1 gene knockouts, compensation is precluded by the widespread deficiency of ER chaperones in PWS {beta}-cells. Consistent with the reduced ER chaperone levels, PWS INS-1 {beta}-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. These results suggest that a chronic deficit of ER chaperones in PWS {beta}-cells leads to a delay in ER transit and/or folding of insulin and other cargo along the secretory pathway. The findings illuminate the pathophysiological basis of hormone deficits in PWS and implicate PWS-imprinted genes in {beta}-cell secretory pathway function.

category: genetics


title: Variation of female pronucleus reveal oocyte or embryo abnormality: An expert experience deep learning of non-dark box analysis

abstract:
BackgroundPronuclear assessment appears to have the ability to distinguish good and bad embryos in the zygote stage, but paradoxical results were obtained in clinical studies. This situation might be caused by the robust qualitative detection of the development of dynamic pronuclei. Here, we aim to establish a quantitative pronuclear measurement method by applying expert experience deep learning from large annotated datasets.

MethodsConvinced handle-annotated 2PN images (13419) were used for deep learning then corresponded errors were recorded through handle check for subsequent parameters adjusting. We used 790 embryos with 52479 PN images from 155 patients for analysis the area of pronuclei and the pre-implantation genetic test results. Establishment of the exponential fitting equation and the key coefficient {beta} 1was extracted from the model for quantitative analysis for pronuclear(PN) annotation and automatic recognition.

FindingsBased on the female original PN coefficient {beta}1, the chromosome-normal rate in the blastocyst with biggest PN area is much higher than that of the blastocyst with smallest PN area (58.06% vs. 45.16%, OR=1.68 [1.07-2.64]; P=0.031). After adjusting coefficient {beta}1 by the first three frames which high variance of outlier PN areas was removed, coefficient {beta}1 at 12 hours and at 14 hours post-insemination, similar but stronger evidence was obtained. All these discrepancies resulted from the female propositus in the PGT-SR subgroup and smaller chromosomal errors.

Conclusion(s)The results suggest that detailed analysis of the images of embryos could improve our understanding of developmental biology.

FundingNone


title: Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

abstract: A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (pooled CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells and present a framework to power biological discovery with the resulting genotype-phenotype map. We use transcriptional phenotypes to predict the function of poorly-characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena - from RNA processing to differentiation. We leverage this ability to systematically identify the genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene function and cellular behavior.


title: The aphid BCR4 structure and activity uncover a new defensin peptide superfamily

abstract: Aphids (Hemiptera: Aphidoidea) are among the most injuring insects for agricultural plants and their management is a great challenge in agronomical research. A new class of proteins, called Bacteriocyte-specific Cysteine-Rich (BCR), provides an alternative to chemical insecticides for pest control. BCRs have been initially identified in the pea aphid Acyrthosiphon pisum. They are small disulfide bond-rich proteins expressed exclusively in aphid bacteriocytes, the insect derived cells that host intracellular symbiotic bacteria. Here, we show that one out of the A. pisum BCRs, BCR4, displays an outstanding insecticidal activity against the pea aphid, impairing insect survival and nymphal growth, providing evidence for its potential use as a new biopesticides. Our comparative genomics and phylogenetic analysis indicate that BCRs seem restricted to the aphid lineage. The 3D structure of the BCR4 reveals that this peptide belongs to a yet unknown structural class of peptides and defines a new superfamily of defensins.

category: biochemistry


title: Structure of EPCR in a non-canonical conformation

abstract: Structural motion and conformational flexibility are often linked to biological functions of proteins. Whether the endothelial protein C receptor (EPCR), like other molecules, is vulnerable to folding transitions or might adopt alternative conformations remains unknown. The current understanding points to a rigid molecular structure suitable for binding of its ligands, like the anticoagulant protein C, or the CIDR1 domains of Plasmodium falciparum. In this study, we have identified a novel conformation of EPCR, captured by X-ray diffraction analyses, whereby Tyr154 shows a dramatically altered structural arrangement, likely incompatible with protein C binding. Biolayer interferometry analysis confirms previous results supporting a critical role for this position in protein C binding. Importantly, the conformational change has no apparent effect in the bound lipid. We conclude these findings reveal a site of conformational vulnerability in EPCR and inform a highly malleable region that could modulate EPCR functions.


title: Amyloidogenesis of SARS-CoV-2 Spike Protein

abstract: SARS-CoV-2 infection is associated with a surprising number of morbidities. Uncanny similarities with amyloid-disease associated blood coagulation and fibrinolytic disturbances together with neurologic and cardiac problems led us to investigate the amyloidogenicity of the SARS-CoV-2 Spike protein (S-protein). Amyloid fibril assays of peptide library mixtures and theoretical predictions identified seven amyloidogenic sequences within the S-protein. All seven peptides in isolation formed aggregates during incubation at 37{degrees}C. Three 20-amino acid long synthetic Spike peptides (sequence 191-210, 599-618, 1165-1184) fulfilled three amyloid fibril criteria: nucleation dependent polymerization kinetics by ThT, Congo red positivity and ultrastructural fibrillar morphology. Full-length folded S-protein did not form amyloid fibrils, but amyloid-like fibrils with evident branching were formed during 24 hours of S-protein co-incubation with the protease neutrophil elastase (NE) in vitro. NE efficiently cleaved S-protein rendering exposure of amyloidogenic segments and accumulation of the peptide 193-202, part of the most amyloidogenic synthetic Spike peptide. NE is overexpressed at inflamed sites of viral infection and at vaccine injection sites. Our data propose a molecular mechanism for amyloidogenesis of SARS-CoV-2 S-protein in humans facilitated by endoproteolysis. The potential implications of S-protein amyloidogenesis in COVID-19 disease associated pathogenesis and consequences following S-protein based vaccines should be addressed in understanding the disease, long COVID-19, and vaccine side effects.


title: Fully Automatic Cell Segmentation with Fourier Descriptors

abstract: Cell segmentation is a fundamental problem in biology for which convolutional neural networks yield the best results nowadays. In this paper, we present FourierDist, a network, which is a modification of the popular StarDist and SplineDist architectures. While StarDist and SplineDist describe an object by the lengths of equiangular rays and control points respectively, our network utilizes Fourier descriptors, predicting a coefficient vector for every pixel on the image, which implicitly define the resulting segmentation. We evaluate our model on three different datasets, and show that Fourier descriptors can achieve a high level of accuracy with a small number of coefficients. FourierDist is also capable of accurately segmenting objects that are not star-shaped, a case where StarDist performs suboptimally according to our experiments.

category: bioinformatics


title: Enhancer RNA-based modeling of adverse events and objective responses of immunotherapy

abstract: Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 or CTLA-4 are emerging and effective immunotherapy strategies. However, ICI treated patients present heterogeneous responses and adverse events, thus demanding effective ways to assess benefit over risk before treatment. Here, by integrating pan-cancer clinical and molecular data, we tried to predict immune-related adverse events (irAEs, risk) and objective response rates (ORRs, benefit) based on enhancer RNAs (eRNAs) expression among patients receiving anti-PD-1/PD-L1 therapy. We built two effective regression models, explaining 71% variance (R=0.84) of irAEs with three eRNAs and 79% (R=0.89) of ORRs with five eRNAs. Interestingly, target genes of irAE-related enhancers, including upstream regulators of MYC, were involved in metabolism, inflammation, and immune activation, while ORR-related enhancers target PAK2 and DLG1 which directly participate in T cell activation. Our study provides references for the identification of immunotherapy-related biomarkers and potential therapeutic targets during immunotherapy.


title: Can Chemical Exchange Saturation Transfer (CEST MRI) be used as biomarker of disease progression in Prion Disease?

abstract: Human prion diseases are fatal neurodegenerative disorders that may have prolonged asymptomatic incubation periods. However, the underlying mechanism by which prions cause brain damage remains unclear. In turn, characterization of early pathological aspects would be of benefit for the diagnosis and potential treatment of these progressive neurodegenerative disorders. We investigated chemical exchange saturation transfer (CEST) MRI based on its exquisite sensitivity to cytosol protein content as a surrogate for prion disease pathology. Three groups of prion-infected mice at different stages of the disease underwent conventional magnetic resonance imaging and CEST MRI at 9.4T. For each mouse, chemical exchange contrasts were measured by applying five RF powers at various frequency offsets using magnetization transfer asymmetries. Relayed Nuclear Overhauser effects (NOE*) and amide proton transfer (APT*) were also assessed. For comparison, CEST MRI measurements were also made in healthy control mice brains. Here we show that alterations in CEST signal were detected before structural modifications or any clinical signs of prion disease. The detected CEST signal displayed different patterns at different stages of the disease indicating its potential for use as a longitudinal marker of disease progression. Highly significant correlations were found between CEST metrics and histopathological findings. A decline in NOE signal was positively correlated with abnormal prion protein deposition (R2 = 0.91) in the thalami of prion infected mice. Moreover, the NOE signal was negatively correlated with astrogliosis (R2 = 0.71) in the thalamus. No significant correlations were detected between NOE signals and spongiosis. MTR asymmetry at 3.5 ppm was also correlated with astrogliosis (R2 = 0.59), and prion protein deposition (R2 = 0.63) in thalamus. No significant changes were detected in APT* between prion-infected and control mice at all stages of the disease. Finally, MTR asymmetry between 2.8 and 3.2 ppm was correlated with prion protein deposition (R2 = 0.47) in the thalamus of prion -infected mice. To conclude, CEST MRI has potential utility as a biomarker of neurodegenerative processes in prion disease.


title: Effects of ozone on sickness and depressive-like behavioral and biochemical phenotypes and their relations to serum amyloid A and kynurenine in blood

abstract: Ozone (O3) is an air pollutant which primarily damages the lungs, but growing evidence supports that O3 exposure can also affect the brain. Serum amyloid A (SAA) and kynurenine have been identified as circulating factors that are upregulated by O3, and both can contribute to depressive-like behaviors in mice. However, little is known about the relations of O3 exposure to sickness and depressive-like behaviors in experimental settings. In this study, we evaluated O3 dose-, time- and sex-dependent changes in circulating SAA in context of pulmonary inflammation and damage, sickness and depressive-like behavioral changes, and systemic changes in kynurenine and indoleamine 2,3-dioxygenase (IDO), an enzyme that regulates kynurenine production and contributes to inflammation-induced depressive-like behaviors. Our results in Balb/c and CD-1 mice showed that 3ppm O3, but not 2 or 1ppm O3, caused elevations in serum SAA and pulmonary neutrophils, and these responses resolved by 48 hours. Sickness and depressive-like behaviors were observed at all O3 doses (1-3ppm), although the detection of certain behavioral changes varied by dose. We also found that Ido1 mRNA expression was increased in the brain and spleen 24 hours after 3ppm O3, and that kynurenine was increased in blood. Together, these findings indicate that acute O3 exposure induces transient symptoms of sickness and depressive-like behaviors which may occur in the presence or absence of overt pulmonary neutrophilia and systemic increases of SAA. We also present evidence that the IDO/kynurenine pathway is upregulated systemically following an acute exposure to O3 in mice.

category: physiology


title: Sex-specific Effects of SGLT2 Inhibition in a Kidney with Reduced Nephron Number: Modeling and Analysis

abstract: The kidney plays an essential role in regulating the homeostasis of electrolytes, acid-base species, and fluids. Kidney structure and function are significantly affected in diabetes. These pathophysiological changes include glomerular hyperfiltration and tubular hypertrophy, and ultimately leading to diabetic kidney disease. A class of medications that have shown promise in slowing the progression to diabetic kidney disease are the sodium-glucose cotransporter 2 (SGLT2) inhibitors. SGLT2 inhibitors target Na+ and glucose reabsorption along the proximal convoluted tubule, enhance urinary glucose, Na+ and fluid excretion, and lower hyperglycemia in diabetes. We postulate that both diabetes-induced and SGLT2 inhibition-induced changes in kidney may exhibit significant sex differences, because the distribution of renal transporters along the nephron may be markedly different between women and men, as recently shown in rodents. The goals of this study are to (i) analyze how kidney function is altered in male and female patients with diabetes, (ii) assess the renal effects, in women and men, of an anti-hyperglycemic therapy that inhibits the sodium-glucose cotransporter 2 (SGLT2) in the proximal convoluted tubules, and (iii) study how those renal effects are altered in uninephrectomy. To accomplish these goals, we have developed computational models of kidney function, separate for male and female patients with diabetes and/or uninephredctomy. The simulation results indicate that by inducing osmotic diuresis in the proximal tubules, SGLT2 inhibition reduces paracellular transport, eventually leading to diuresis and natriuresis.


title: Auxin-driven ecophysiological diversification of leaves in domesticated tomato

abstract:
Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalise the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato (Solanum lycopersicum) obscuravenosa (obv) mutant lacks BSEs. Here we identify the obv gene and the causative mutation, a non-synonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a rare polymorphism in the natural range of wild tomatoes, but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density and fruit shape, are controlled by OBV via changes in auxin signalling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR (ARF4) also results in defective BSE development, revealing an additional component of a novel genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selection.

One sentence summarydistribution of tomato heterobaric and homobaric leaves is controlled by a single-nucleotide polymorphism in an auxin-related transcription factor

category: plant biology


title: Towards a global perspective for Salvia L: Phylogeny, diversification, and floral evolution

abstract:
Premise of this studySalvia is the most species-rich genus in Lamiaceae, encompassing approximately 1000 species distributed all over the world. We sought a new evolutionary perspective for Salvia by employing macroevolutionary analyses to address the tempo and mode of diversification. To study the association of floral traits with speciation and extinction, we modeled and explored the evolution of corolla length and the lever-mechanism pollination system across our Salvia phylogeny.

MethodsWe reconstructed a multigene phylogeny for 366 species of Salvia in the broad sense including all major recognized lineages and numerous species from Iran, a region previously overlooked in studies of the genus. Our phylogenetic data in combination with divergence time estimates were used to examine the evolution of corolla length, woody vs. herbaceous habit, and presence vs. absence of a lever mechanism. We investigated the timing and dependence of Salvia diversification related to corolla length evolution through a disparity test and BAMM analysis. A HiSSE model was used to evaluate the dependency of diversification on the lever-mechanism pollination system in Salvia.

Key ResultsBased on recent investigations and classifications, Salvia is monophyletic and comprises [~]1000 species. Our inclusion, for the first time, of a comprehensive sampling for Iranian species of Salvia provides higher phylogenetic resolution for southwestern Asian species than obtained in previous studies. A medium corolla length (15-18mm) was reconstructed as the ancestral state for Salvia with multiple shifts to shorter and longer corollas. Macroevolutionary model analyses indicate that corolla length disparity is high throughout Salvia evolution, significantly different from expectations under a Brownian motion model during the last 28 million years of evolution. Our analyses show evidence of a higher diversification rate of corolla length for some Andean species of Salvia compared to other members of the genus. Based on our tests of diversification models, we reject the hypothesis of a direct effect of the lever mechanism on Salvia diversification.

ConclusionsUsing a broader species sampling than previous studies, we obtained a well- resolved phylogeny for southwest Asian species of Salvia. Corolla length is an adaptive trait throughout the Salvia phylogeny with a higher rate of diversification in the South American clade. Our results suggest caution in considering the lever-mechanism pollination system as one of the main drivers of speciation in Salvia.


title: OPEN leaf: an open-source cloud-based phenotyping system for tracking dynamic changes at leaf-specific resolution in Arabidopsis

abstract:
The first draft of the Arabidopsis genome was released more than 20 years ago and despite intensive molecular research, more than 30% of Arabidopsis genes remained uncharacterized or without an assigned function. This is in part due to gene redundancy within gene families or the essential nature of genes, where their deletion results in lethality (i.e., the dark genome).

High-throughput plant phenotyping (HTPP) offers an automated and unbiased approach to characterize subtle or transient phenotypes resulting from gene redundancy or inducible gene silencing; however, commercial HTPP platforms remain unaffordable. Here we describe the design and implementation of OPEN leaf, an open-source HTPP system with cloud connectivity and remote bilateral communication to facilitate data collection, sharing and processing.

OPEN leaf, coupled with the SMART imaging processing package was able to consistently document and quantify dynamic morphological changes over time at the whole rosette level and also at leaf-specific resolution when plants experienced changes in nutrient availability.

The modular design of OPEN leaf allows for additional sensor integration. Notably, our data demonstrate that VIS sensors remain underutilized and can be used in high-throughput screens to identify characterize previously unidentified phenotypes in a leaf-specific manner.

Significance StatementMany bottlenecks exist in high-throughput phenotyping involving computing power for processing and a lack of focus on abiotic stresses that has prevented an advancement in phenotyping on par with genotyping. Therefore, we create an automated HTP system that performs nutrient studies on Arabidopsis thaliana with cloud-based image processing that quantifies plant traits at a whole and leaf-level.


title: Discovering gene regulatory networks of multiple phenotypic groups using dynamic Bayesian networks

abstract: Dynamic Bayesian networks (DBNs) can be used for the discovery of gene regulatory networks from time series gene expression data. Here, we suggest a strategy for learning DBNs from gene expression data by employing a Bayesian approach that is scalable to large networks and is targeted at learning models with high predictive accuracy. Our framework can be used to learn DBNs for multiple groups of samples and highlight differences and similarities in their gene regulatory networks. We learn these DBN models based on different structural and parametric assumptions and select the optimal model based on the cross-validated predictive accuracy. We show in simulation studies that our approach is better equipped to prevent overfitting than techniques used in previous studies. We applied the proposed DBN-based classification approach to two time series transcriptomic datasets from the Gene Expression Omnibus database, each comprising data from distinct phenotypic groups of the same tissue type. In the first case, we used DBNs to characterize responders and non-responders to anti-cancer therapy. In the second case, we compared normal to tumor cells of colorectal tissue. The classification accuracy reached by the DBN-based classifier for both datasets was higher than reported previously. For the colorectal cancer dataset, our analysis suggested that GRNs for cancer and normal tissues have a lot of differences, which are most pronounced in the neighborhoods of oncogenes and known cancer tissue markers. The identified differences in gene networks of cancer and normal cells may be used for the discovery of targeted therapies.

category: systems biology


title: Diminished GAD67 Terminals and Ambient GABA Inhibition Associated with Reduced Motor Neuron Recruitment Threshold in the SOD1G93A ALS mouse

abstract: Pre-symptomatic studies in mouse models of the neurodegenerative motor neuron (MN) disease, Amyotrophic Lateral Sclerosis (ALS) highlight early alterations in intrinsic and synaptic excitability and have supported an excitotoxic theory of MN death. However, a role for synaptic inhibition in disease development is not sufficiently explored among other mechanisms. Since inhibition plays a role in both regulating motor output and in neuroprotection, we examined the age-dependent anatomical changes in inhibitory presynaptic terminals on MN cell bodies using fluorescent immunohistochemistry for GAD67 (GABA) and GlyT2 (glycine) presynaptic proteins comparing ALS-vulnerable trigeminal jaw closer (JC) motor pools with the ALS-resistant extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Our results indicate differential patterns of temporal changes of these terminals in vulnerable versus resilient MNs and relative differences between SOD1G93A and wild-type (WT) MNs. Notably, we found pre-symptomatic up-regulation in inhibitory terminals in the EO MNs while the vulnerable JC MNs mostly showed a decrease in inhibitory terminals. Specifically, there was a statistically significant decrease in the GAD67 somatic abuttal in the SOD1G93A JC MNs compared to WT around P12. Using in vitro patch-clamp electrophysiology, we found a parallel decrease in the ambient GABA-dependent tonic inhibition in the SOD1G93A JC MNs. While it is unclear if the two mechanisms are directly related, pharmacological blockade of specific subtype of GABAA-5 receptors suggests that tonic inhibition can control MN recruitment threshold. Furthermore, reduction in tonic GABA current as observed here in the mutant, identifies a putative molecular mechanism explaining our observations of hyperexcitable shifts in JC MN recruitment threshold in the SOD1G93A mouse. Lastly, we showcase non-parametric resampling-based bootstrap statistics for data analyses, and provide the Python code on GitHub for wider reuse.


title: Loss of Depalmitoylation Exaggerates Synaptic Upscaling and Leads to Neuroinflammation in a Lysosomal Storage Disease

abstract: Palmitoylation and depalmitoylation are the dichotomic processes of lipid modification regulating protein trafficking, recycling, and degradation, thereby controlling proteostasis. Despite our understanding of palmitoylation, depalmitoylation is far less studied. Here, we study a lysosomal depalmitoylating enzyme, palmitoyl-protein thioesterase 1 (PPT1), associated with the devastating neurodegenerative condition CLN1 disease and show that dark-rearing Ppt1-/- mice, which induces synaptic upscaling in vivo, worsen the symptoms. In Ppt1-/- cortical neurons, upscaling induction triggers exaggerated responses of synaptic calcium-permeable AMPA receptors composed of palmitoylated GluA1 subunits. Consequently, Ppt1-/- visual cortex exhibits hypersynchrony in vivo. Remarkably, we also find an overload of palmitoylated A-kinase anchor protein 5 (Akap5) in Ppt1-/- mouse brains, leading to microglial activation through NFAT. These findings indicate Ppt1 acts as a gatekeeper of homeostatic plasticity by regulating the proteostasis of palmitoylated synaptic proteins. Moreover, our results suggest that perturbed depalmitoylation results in neuroinflammation, which is common to neurodegenerative diseases.


title: Dynamic structural cell responses in the thymus to acute injury, regeneration, and age

abstract: The thymus, the primary site of T cell development, is extremely sensitive to insult but also harbors tremendous capacity for repair. Using single cell sequencing of thymic structural cells, as well as functional and structural analyses, we revealed distinct regenerative programs by endothelial and mesenchymal subsets after injury that stimulated epithelial repair; the compartment primarily supporting T cell development. Thymic function not only declined over lifespan, contributing to immune aging, but the capacity of the thymus to regenerate after damage also declined in old mice. This could be attributed to an inability of the old microenvironment to induce reparative programs; leading to reduced ability to restore tissue structure and function. These findings provide a detailed framework for the response of structural cells to aging and acute damage, which could have considerable implications for our understanding of aging immunity and recovery from treatments such as chemotherapy and bone marrow transplant.


title: Position-specific secondary acylation determines detection of lipid A by murine TLR4 and caspase-11

abstract: Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against Gram-negative bacterial infection, and a contributor to hyper-inflammatory response, leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor TLR4/MD2 or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited, and the precise features of lipid A that determine the differential activation of each receptor remain poorly defined. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we find that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4- or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4- or Casp11. Our findings indicate that TLR4 and Casp11 sense non-overlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.


title: Diverse and rapidly evolving viral strategies modulate arthropod vector gene expression

abstract: Vector-borne viral diseases threaten human and wildlife worldwide. Vectors are often viewed as a passive syringe injecting the virus, however to survive, replicate and spread, viruses must manipulate vector biology. While most vector-borne viral research focuses on vectors transmitting a single virus, in reality vectors often carry diverse viruses. Yet how viruses affect the vectors remains poorly understood. Here we focused on the varroa mite, an emergent parasite that vectors over 20 honey bee viruses, and has been responsible for colony collapses worldwide, as well as changes in global viral populations. Co-evolution of the varroa and the viral community makes it possible to investigate whether viruses affect vector gene expression, and whether these interactions affect viral epidemiology. Using a large set of available varroa transcriptomes we identified how abundances of individual viruses affect the vectors transcriptional network. Perhaps surprisingly, we found no evidence of competition between viruses, but rather that some virus abundances are positively correlated. Furthermore, we found a strong correlation between the extent to which a virus interacts with the vectors gene expression and co-occurrence with other viruses, suggesting that interactions with the vector affect epidemiology. We experimentally validated this observation by silencing candidate genes using RNAi and found that reduction in varroa gene expression was accompanied by a change in viral load. Combined, the meta-transcriptomic analysis and experimental results shed light on the mechanism by which viruses interact with each other and with their vector to shape the disease course.


title: Characterizing the genetic basis of trait evolution in the Mexican cavefish

abstract: Evolution in response to a change in ecology often coincides with various morphological, physiological, and behavioral traits. For most organisms little is known about the genetic and functional relationship between evolutionarily derived traits, representing a critical gap in our understanding of adaptation The Mexican tetra, Astyanax mexicanus, consists of largely independent populations of fish that inhabit at least 30 caves in Northeast Mexico, and a surface fish population, that inhabits the rivers of Mexico and Southern Texas. The recent application of molecular genetic approaches combined with behavioral phenotyping have established A. mexicanus as a model for studying the evolution of complex traits. Cave populations of A. mexicanus are interfertile with surface populations and have evolved numerous traits including eye degeneration, insomnia, albinism and enhanced mechanosensory function. The interfertility of different populations from the same species provides a unique opportunity to define the genetic relationship between evolved traits and assess the co-evolution of behavioral and morphological traits with one another. To define the relationships between morphological and behavioral traits, we developed a pipeline to test individual fish for multiple traits. This pipeline confirmed differences in locomotor activity, prey capture, and startle reflex between surface and cavefish populations. To measure the relationship between traits, individual F2 hybrid fish were characterized for locomotor behavior, prey-capture behavior, startle reflex and morphological attributes. Analysis revealed an association between body length and slower escape reflex, suggesting a trade-off between increased size and predator avoidance in cavefish. Overall, there were few associations between individual behavioral traits, or behavioral and morphological traits, suggesting independent genetic changes underlie the evolution of behavioral and morphological traits. Taken together, this approach provides a novel system to identify genes that underlie naturally occurring genetic variation in morphological and behavioral traits.


title: Variation in the transcriptome response and detoxification gene diversity drives pesticide tolerance in fishes

abstract: Pesticides are critical for invasive species management, but often have negative effects on non-target native biota. Tolerance to pesticides should have an evolutionary basis, but this is poorly understood. Invasive sea lamprey (Petromyzon marinus) populations in North America have been controlled with a pesticide lethal to them at lower concentrations than native fishes. We addressed how interspecific variation in gene expression and detoxification gene diversity confer differential pesticide sensitivity in two fish species. We exposed sea lamprey and bluegill (Lepomis macrochirus), a tolerant native species, to TFM, a pesticide commonly used in sea lamprey control, and used whole-transcriptome sequencing of gill and liver to characterize the cellular response. Comparatively, bluegill exhibited a larger number of detoxification genes expressed and a larger number of responsive transcripts overall, which likely contributes to greater tolerance to TFM. Understanding the genetic and physiological basis for pesticide tolerance is crucial for managing invasive species.


title: Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies

abstract:
The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an antiadherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 x103 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11x103 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their noncentrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7x103 and 11x103 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages.

We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research.

category: developmental biology


title: Comparison of different head and neck positions and behaviour in ridden elite dressage horses between warm-up and competition

abstract: Head and neck position (HNP) has been identified in literature as important influence on wellbeing. It was investigated in ridden elite dressage horses whether there is a relation between the HNP, ethological indicators and the grading in the warm-up area and in the test. 49 starters (83%) of a Grand-Prix Special (CDIO5 *) as part of the CHIO in Aachen 2018 and 2019 were examined. For each horse-rider pair, HNP (angle at vertical (AT), poll angle (PA), neck angel (NA)) used were analysed as well as conflict behavior for 3 minutes each in warm-up area and test. 6571 individual frames were used. The noseline was carried significantly less behind the vertical in test vs. warm-up (5.43 {degrees} {+/-} 4.19 vs. 11.01 {degrees} {+/-} 4.54 behind the vertical; T = 34.0; p < 0.05). The horses showed significantly less conflict behavior in the test vs. warm-up (123 {+/-} 54 vs. 160 {+/-} 75) (T = 76.00; p < 0.01). In the latter, a smaller PA and more defensive behaviour of the horses was observed compared to the test. A correlation between the grading of test and HNP was found (R = 0.38; p < 0.05). The further the noseline was behind the vertical, the higher was the chance of a good rating. The higher riders were ranked in the "FEI world ranking", the higher were their marks in the competition (2018: r = -0.69, p < 0.05; 2019: r = -0.76, p < 0.05). Horses of riders higher in world ranking tended to show more unusual oral behaviour (r = -0.30, p < 0.05), and a noseline stronger behind the vertical (r = - 0.37, p < 0.05) resulting in a smaller NA (r = 0.43, p < 0.05). This are from the point of view of animal welfare problematic results.

category: animal behavior and cognition


title: Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance

abstract: Epidemiological surveillance systems for pathogens in wild species have been proposed as a preventive measure for epidemic events. These systems can minimize the detrimental effects of an outbreak, but most importantly, passive surveillance systems are the best adapted to countries with limited resources. Therefore, the present study aims to evaluate the technical and infrastructural feasibility to establish this type of scheme in Costa Rica targeting thedetection of pathogens of zoonotic and conservation importance in wildlife. Between 2018 and 2020, 85 carcasses of free-ranging vertebrates were admitted for post mortem analysis and complementary laboratory analysis, representing a solid basis for the implementation of a passive surveillance system for wildlife diseases in the country. However, we encounter during this research significant constraints that affected the availability of carcasses for analysis, mainly related to the initial identification of cases, detection biases towards events in populated- or easily accessible-areas with nearby located wildlife management centers, further associated with financial disincentives, and limited local logistics capacity. Thus resulting in the exclusion of some geographic regions of the country. This epidemiological surveillance scheme allowed us to estimate the general state of health of the countrys wildlife, establishing the cause of death of the analyzed animals as follows: (i) 46 (54.1%) traumatic events, (ii) 23 (27.1%) infectious agents, (iii) two (2.4%) degenerative illness, (iv) three (3.5%) presumably poisoning, and (v) in 11 (12.9%)undetermined. It also allowed the detection of pathogens such as, canine distemper virus, Klebsiella pneumoniae, Toxoplasma gondii, Trypanosoma spp., Angiostrongylus spp., Dirofilaria spp., Baylisascaris spp., among others. As well as recognizing the circulation of these pathogens around national territory and also on those analyzed species. This strategy is crucial in geographical regions defined as critical for the appearance of diseases due to their great biodiversity and social conditions.

category: pathology


title: M-CSF-stimulated myeloid cells can convert into epithelial cells to participate in re-epithelialization and hair follicle regeneration during skin wound healing

abstract: Skin wound healing is a complex process which requires the interaction of many cell types and mediators in a highly sophisticated temporal sequence. Myeloid cells compose a significant proportion of the inflammatory cells recruited to a wound site and play important roles in clearance of damaged tissue and microorganisms. Myeloid cells have also been suggested to convert into fibro-blast-like cells and endothelial cells in participation of wound healing process. However, whether myeloid cells in wound skin can convert into epithelial cells and contribute to re-epithelialization and skin appendage regeneration is still unclear. In this study, we performed double immunofluorescent staining with antibodies for hematopoietic cells and keratinocytes as well as cell tracing technique to investigate hematopoietic cell conversion. The result show that during the healing process, some of the CD45-positive hematopoietic cells are overlapped with keratin 14, the markers of keratinocytes. Further, CD11b-positive myeloid cells seem the origin of converted epithelial cells. To confirm these results, we culture CD11b-posiitve myeloid cells from mouse splenocytes in a medium containing macrophage colony-stimulating factor (M-CSF) and dermal injection of these cells into the healthy skin when punch biopsy is created in mouse skin. Tracing injected labeled splenocyte-derived myeloid cells in skin, we confirm that myeloid cells able to convert into keratinocytes in repaired skin. Furthermore, our results from in vivo experiments provide new information on contribution of myeloid cells in hair follicle regeneration. In conclusion, this work highlights the myeloid cell contributions in wound repair and hair follicle regeneration in mice through conversion of M-CSF-stimulated CD11b+ myeloid cells into epithelial cells.


title: Kismet/CHD7/CHD8 affects gut biomechanics, the gut microbiome, and gut-brain axis in Drosophila melanogaster

abstract: The gut-brain axis may contribute to the pathophysiology of neurodevelopmental disorders, yet it is often unclear how risk genes associated with these disorders affect gut physiology in a manner that could impact microbial colonization. We addressed this question using Drosophila melanogaster with a null mutation in kismet, the ortholog of chromodomain helicase DNA-binding protein (CHD) family members CHD7 and CHD8. In humans, CHD7 and CHD8 are risk genes for neurodevelopmental disorders with co-occurring gastrointestinal symptoms. We found kismet mutant flies have a significant increase in gastrointestinal transit time, indicating functional homology of kismet with CHD7/CHD8 in vertebrates. To measure gut tissue mechanics, we used a high-precision force transducer and length controller, capable of measuring forces to micro-Newton precision, which revealed significant changes in the mechanics of kismet mutant guts, in terms of elasticity, strain stiffening, and tensile strength. Using 16S rRNA metagenomic sequencing, we also found kismet mutants have reduced diversity of gut microbiota at every taxonomic level and an increase in pathogenic taxa. To investigate the connection between the gut microbiome and behavior, we depleted gut microbiota in kismet mutant and control flies and measured courtship behavior. Depletion of gut microbiota rescued courtship defects of kismet mutant flies, indicating a connection between gut microbiota and behavior. In striking contrast, depletion of gut microbiome in the control strain reduced courtship activity. This result demonstrated that antibiotic treatment can have differential impacts on behavior that may depend on the status of microbial dysbiosis in the gut prior to depletion. We propose that Kismet influences multiple gastrointestinal phenotypes that contribute to the gut-brain axis to influence behavior. Based on our results, we also suggest that gut tissue mechanics should be considered as an element in the gut-brain communication loop, both influenced by and potentially influencing the gut microbiome and neuronal development.


title: The first Miocene fossils from coastal woodlands in the southern East African Rift

abstract:
The Miocene is a key time in the evolution of African mammals and their ecosystems witnessing the origin of the African apes and the isolation of eastern coastal forests through an expanding biogeographic arid corridor. Until recently, however, Miocene sites from the southeastern regions of the continent were unknown. Here we report discovery of the first Miocene fossil teeth from the shoulders of the Urema Rift in Gorongosa National Park, Mozambique, at the southern East African Rift System. We provide the first 1) radiometric age determinations of the fossiliferous Mazamba Formation, 2) reconstructions of past vegetation in the region based on pedogenic carbonates and fossil wood, and 3) description of fossil teeth from the southern rift. Gorongosa is unique in the East African Rift System in combining marine invertebrates, marine vertebrates, terrestrial mammals, and fossil woods in coastal paleoenvironments. The Gorongosa fossil sites offer the first evidence of persistent woodlands and forests on the coastal margins of southeastern Africa during the Miocene, and an exceptional assemblage of fossil vertebrates including new species. Further work will allow the testing of hypotheses positing the formation of a northeast-southwest arid corridor isolating species on the eastern coastal forests from those elsewhere in Africa.

BriefThe Miocene is a key time in the evolution of African mammals and their ecosystems encompassing hominine origins and the establishment of an arid corridor that isolated eastern Africas coastal forests. Until now, however, Miocene sites from southeastern Africa have been unknown. We report the discovery of the first Miocene fossil sites from Gorongosa National Park, Mozambique, and show that these sites formed in coastal settings. We provide radiometric ages for the fossiliferous sediments, reconstructions of past vegetation based on stable isotopes and fossil wood, and a description of the first fossil teeth from the region. Gorongosa is the only paleontological site in the East African Rift that combines fossil woods, marine invertebrates, marine vertebrates, and terrestrial mammals. Gorongosa offers the first evidence of persistent woodlands and forests on the coastal margins of southeastern Africa during the Miocene.

category: paleontology


title: The algal PETC-Pro171-Leu suppresses electron transfer in the cytochrome b6f under acidic lumenal condition

abstract:
Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane used to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH are variable, cyclic electron flow (CEF) between PSI and cytochrome b6f complex (b6f) produces extra ATP. The b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 mutant of the vascular plant Arabidopsis thaliana, carrying Pro194Leu in the b6f Rieske Iron-sulfur protein. In this work, we introduced pgr1 mutation in the green alga Chlamydomonas reinhardtii (PETC-P171L). Consistent with pgr1 phenotype, PETC-P171L displayed a limited photosynthesis along with slower photoautotrophic growth under high light conditions. Our data under low oxygen revealed that the {Delta}pH component in algae was already sufficient to trigger the effect in PETC-P171L in sub-saturating light conditions where the mutant b6f was more restricted to oxidize the PQ pool and revealed a diminished electron flow.

One sentence summaryChange of PETC to P171L via site directed mutagenesis alters the pH dependency of the photosynthetic control mechanism


title: Synaptosome microRNAs regulate synapse functions in Alzheimer's disease

abstract: MicroRNAs (miRNAs) are found in nerve terminals, synaptic vesicles, and synaptosomes, but it is unclear whether synaptic and cytosolic miRNA populations differ in Alzheimers disease (AD) or if synaptosomal miRNAs affect AD synapse activity. To address these questions, we generated synaptosomes and cytosolic fractions from postmortem brains of AD and unaffected control (UC) samples and analyzed them using a global Affymetrix miRNAs microarray platform. A group of miRNAs significantly differed (p<0.0001) with high fold changes variance (+/- >200-fold) in their expressions in different comparisons- 1) UC synaptosome vs UC cytosol, 2) AD synaptosomes vs AD cytosol, 3) AD cytosol vs UC cytosol, and 4) AD synaptosomes vs UC synaptosomes. MiRNAs data analysis revealed that some potential miRNAs were consistently different across sample groups. These differentially expressed miRNAs were further validated using AD postmortem brains, brains of APP transgenic (Tg2576), Tau transgenic (P301L), and wild type mice. The miR-501-3p, miR-502-3p and miR-877-5p were identified as potential synaptosomal miRNAs upregulated with disease progression based on AD Braak stages. Gene Ontology Enrichment and Ingenuity Pathway Analysis of synaptosomal miRNAs showed the involvement of miRNAs in nervous system development, cell junction organization, synapse assembly formation, and function of GABAergic synapse. This is the first description of synaptic versus cytosolic miRNAs in AD and their significance in synapse function.

category: neuroscience


title: A third vaccination with a single T cell epitope protects against SARS-CoV-2 infection in the absence of neutralizing antibodies

abstract:
Understanding the mechanisms and impact of booster vaccinations can facilitate decisions on vaccination programmes. This study shows that three doses of the same synthetic peptide vaccine eliciting an exclusive CD8+ T cell response against one SARS-CoV-2 Spike epitope protected all mice against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, while only a second vaccination with this T cell vaccine was insufficient to provide protection. The third vaccine dose of the single T cell epitope peptide resulted in superior generation of effector-memory T cells in the circulation and tissue-resident memory T (TRM) cells, and these tertiary vaccine-specific CD8+ T cells were characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping showed that a substantial fraction of the tertiary effector-memory CD8+ T cells developed from remigrated TRM cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

SummaryA third dose with a single T cell epitope-vaccine promotes a strong increase in tissue-resident memory CD8+ T cells and fully protects against SARS-CoV-2 infection, while single B cell epitope-eliciting vaccines are unable to provide protection.

category: immunology


title: Non-toxigenic Vibrio cholerae challenge strains for evaluating vaccine efficacy and inferring mechanisms of protection

abstract: Human challenge studies are instrumental for testing cholera vaccines, but these studies use outdated strains and require inpatient facilities. Here, we created next-generation isogenic Ogawa and Inaba V. cholerae challenge strains (ZChol strains) derived from a contemporary Zambian clinical isolate representative of current dominant pandemic V. cholerae. To minimize the risk of severe diarrhea these strains were rendered non-toxigenic, since antibody responses which limit V. cholerae colonization are the primary mechanism of immune protection. These strains did not cause diarrhea in infant mice and proved to accurately gauge reduction in intestinal colonization mediated by effective vaccination. They are also valuable as targets for measuring vibriocidal antibody responses. Using barcoded ZChol strains, we discovered that vaccination tightens the infection bottleneck without restricting pathogen expansion in vivo. ZChol strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera.

category: microbiology


title: Modelling the impact of urban and hospital wastewaters eco-exposomes on the antibiotic-resistance dynamics.

abstract:
Antibiotic-resistance emergence and selection have become major public health issues globally. The presence of antibiotic resistant bacteria (ARB) in natural and anthroposophical environments threatens to compromise the sustainability of care in human and animal populations. This study was undertaken to develop a simple model formalizing the selective impact of antibiotics and pollutants on the dynamics of bacterial resistance in water and use the model to analyze longitudinal spatiotemporal data collected in hospital and urban wastewaters. Longitudinal-sampling data were collected between 2012 and 2015 in four different locations in Haute-Savoie, France: hospital and urban wastewaters, before and after water-treatment plants. Concentration in three different types of compounds: 1) heavy metals 2) antibiotics and 3) surfactants; and abundance of 88 individual genes and mobile genetic elements, mostly conferring resistance to antibiotics, were simultaneously collected. A simple hypothesis-driven model describing the weekly ARB dynamics was proposed to fit available data by assuming normalized gene abundance to be proportional to ARB populations in water. Compounds impacts on the dynamics of 17 genes found in multiple sites were estimated. We found that while mercury and vancomycin had relevant effects on ARB dynamics, respectively positively affecting the dynamics of 10 and 12 identified genes, surfactants antagonistically affected genes dynamics (identified for three genes). This simple model enables analyzing the relationship between resistance-gene persistence in aquatic environments and specific compounds inherent to human activities. Applying our model to longitudinal data, we identified compounds that act as co-selectors for antibiotic resistance.

HighlightsO_LIWe analyzed longitudinal wastewater resistance genes and environmental data
C_LIO_LIWe developed a simple hypothesis-driven model to assess resistance selection
C_LIO_LIMercury and vancomycin were key drivers of antibiotic resistance in wastewater
C_LI


title: Visualizing translation dynamics at atomic detail inside a bacterial cell

abstract: Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here, we use cryo-electron tomography and sub-tomogram analysis to visualize the dynamics of translation inside the prokaryote Mycoplasma pneumoniae. We first obtain an in-cell atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves thirteen ribosome states that differ in conformation and composition and reflect intermediates during translation. Based on these states, we animate translation elongation and demonstrate how antibiotics reshape the translation landscape inside cells. During translation elongation, ribosomes often arrange in a defined manner to form polysomes. By mapping the intracellular three-dimensional organization of translating ribosomes, we show that their association into polysomes exerts a local coordination mechanism that is mediated by the ribosomal protein L9. Our work demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.


title: Mechanisms underlying sequence-dependent DNA hybridisation rates in the absence of secondary structure

abstract: The kinetics of DNA hybridisation are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridise at different rates are not well understood. Secondary structure is one predictable factor that influences hybridisation rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. Consequently, to achieve a good correlation with experimental data, current prediction algorithms require many parameters that provide little mechanistic insight into DNA hybridisation. In this context, we measured hybridisation rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify their hybridisation rates. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters, thus providing new insight into the physical factors underpinning DNA hybridisation rates.


title: Evidence for widespread cytoplasmic structuring into mesoscopic condensates

abstract: Eukaryotic cytoplasm organizes itself via both membrane-bound organelles and membrane-less biomolecular condensates (BMCs). Known BMCs exhibit liquid-like properties and are typically visualized on the scale of ~1 m. They have been studied mostly by microscopy, examining select individual proteins. Here, we investigate the global organization of native cytoplasm with quantitative proteomics, using differential pressure filtration, size exclusion, and dilution experiments. These assays reveal that BMCs form throughout the cytosplasm, predominantly at the mesoscale of ~100 nm. Our data indicate that at least 18% of the proteome is organized via such mesoscale BMCs, suggesting that cells widely employ dynamic liquid-like clustering to organize their cytoplasm, at surprisingly small length scales.