title: Early evoked brain activity underlies auditory and audiovisual speech recognition deficits in schizophrenia

abstract:
ObjectivesPeople with Schizophrenia (SZ) show deficits in auditory and audiovisual speech recognition. It is possible that these deficits are related to aberrant early sensory processing, combined with an impaired ability to utilize visual cues to improve speech recognition. In this electroencephalography study we tested this by having SZ and healthy controls (HC) identify different unisensory auditory and bisensory audiovisual syllables at different auditory noise levels.

MethodsSZ (N = 24) and HC (N = 21) identified one of three different syllables (/da/, /ga/, /ta/) at three different noise levels (no, low, high). Half the trials were unisensory auditory and the other half provided additional visual input of moving lips. Task-evoked mediofrontal N1 and P2 brain potentials triggered to the onset of the auditory syllables were derived and related to behavioral performance.

ResultsIn comparison to HC, SZ showed speech recognition deficits for unisensory and bisensory stimuli. These deficits were primarily found in the no noise condition. Paralleling these observations, reduced N1 amplitudes to unisensory and bisensory stimuli in SZ were found in the no noise condition. In HC the N1 amplitudes were positively related to the speech recognition performance, whereas no such relationships were found in SZ. Moreover, no group differences in multisensory speech recognition benefits and N1 suppression effects for bisensory stimuli were observed.

ConclusionOur study shows that reduced N1 amplitudes relate to auditory and audiovisual speech processing deficits in SZ. The findings that the amplitude effects were confined to salient speech stimuli and the attenuated relationship with behavioral performance, compared to HC, indicates a diminished decoding of the auditory speech signals in SZs. Our study also revealed intact multisensory benefits in SZs, which indicates that the observed auditory and audiovisual speech recognition deficits were primarily related to aberrant auditory speech processing.

HighlightsSpeech processing deficits in schizophrenia related to reduced N1 amplitudes Audiovisual suppression effect in N1 preserved in schizophrenia Schizophrenia showed weakened P2 components in specifically audiovisual processing


title: Widespread distribution of collagens and collagen-associated domains in eukaryotes

abstract: The origin of collagen, the dominant structural component of metazoan extracellular matrix, has long been cited as a critical step in the evolution of metazoan multicellularity. While collagens were once thought to be found only in metazoans, scattered reports of collagen domains in Fungi, and more recently in close relatives of metazoans, have called into question whether collagens are truly unique to metazoans. Here, we take advantage of recently sequenced genomes and transcriptomes of diverse holozoans (the clade encompassing metazoans and their close relatives), as well as publicly available proteomes from diverse non-holozoan eukaryotes, to conduct a systematic search for collagen domains across eukaryotic diversity. We find that collagen domains are ubiquitous in choanoflagellates, the sister group of metazoans, and widespread across many other major eukaryotic taxa. Many predicted collagens in non-metazoans are comparable to metazoan collagens in length and proline content. Moreover, most are present in species that also encode putative prolyl 4-hydroxylase domains, suggesting that, like metazoan collagens, they may be stabilized through the hydroxylation of prolines. Fibrillar collagen and collagen IV appear to be unique to metazoans, and we posit that their ability to assemble into superstructures may have contributed to the origin of metazoan multicellularity.

category: evolutionary biology


title: Astrocytes encode complex behaviorally relevant information

abstract: Astrocytes, glial cells of the central nervous system, help to regulate neural circuit operation and adaptation. They exhibit complex forms of chemical excitation, most prominently calcium transients, evoked by neuromodulator and -transmitter receptor activation1-4. However, whether and how astrocytes contribute to cortical processing of complex behavior remains unknown1. One of the puzzling features of astrocyte calcium transients is the high degree of variability in their spatial and temporal patterns under behaving conditions. Here, we provide mechanistic links between astrocytes activity patterns, molecular signaling, and behavioral cognitive and motor activity variables by employing a visual detection task that allows for in vivo calcium imaging, robust statistical analyses, and machine learning approaches. We show that trial type and performance levels deterministically shape astrocytes spatial and temporal response properties. Astrocytes encode the animals decision, reward, and sensory properties. Our error analysis confirms that astrocytes carry behaviorally relevant information depending on and complementing neuronal coding. We also report that cell-intrinsic mechanisms curb astrocyte calcium activity. Additionally, we show that motor activity-related parameters strongly impact astrocyte responses and must be considered in sensorimotor study designs. Our data inform and constrain current models of astrocytes contribution to complex behavior and brain computation beyond their established homeostatic and metabolic roles.

category: neuroscience


title: Loss-of-function ROX1 mutations suppress the fluconazole susceptibility of upc2AΔ mutation in Candida glabrata, implicating additional positive regulators of ergosterol biosynthesis

abstract:
Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress induced transcription factors such as Upc2 and Upc2A as well as homologs of Sterol Response Element Binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2A{Delta} deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2A{Delta} mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14 methyl-ergosta-8,24(28)-dien-3{beta}, 6-diol, relative to upc2A{Delta}. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata.

ImportanceCandida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators of C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.s

category: microbiology


title: A resistome roadmap: from the human body to pristine environments

abstract:
A comprehensive characterization of the human body resistome (sets of antibiotic resistance genes (ARGs)) is yet to be done and paramount for addressing the antibiotic microbial resistance threat. Here, we study the resistome of 771 samples from five major body parts (skin, nares, vagina, gut and oral cavity) of healthy subjects from the Human Microbiome Project and addressed the potential dispersion of ARGs in pristine environments. A total of 28,731 ARGs belonging to 344 different ARG types were found in the HMP proteome dataset (n=9.1x107 proteins analyzed). Our study reveals a distinct resistome profile (ARG type and abundance) between body sites and high inter-individual variability. Nares had the highest ARG load ({approx}5.4 genes/genome) followed by the oral cavity, while the gut showed one of the highest ARG richness (shared with nares) but the lowest abundance ({approx}1.3 genes/genome). Fluroquinolone resistance genes were the most abundant in the human body, followed by macrolide-lincosamide-streptogramin (MLS) or tetracycline. Most of the ARGs belonged to common bacterial commensals and multidrug resistance trait was predominant in the nares and vagina. Our data also provide hope, since the spread of common ARG from the human body to pristine environments (n=271 samples; 77 Gb of sequencing data and 2.1x108 proteins analyzed) thus far remains very unlikely (only one case found in an autochthonous bacterium from a pristine environment). These findings broaden our understanding of ARG in the context of the human microbiome and the One-Health Initiative of WHO uniting human host-microbes and environments as a whole.

ImportanceThe current antibiotic resistance crisis affects our health and wealth at a global scale and by 2050 predictions estimate 10 million deaths attributed to antibiotic resistance worldwide. Remarkably, a comprehensive analysis of ARG diversity and prevalence in different human body sites is yet to be done. Undoubtedly, our body and human built-environment have antibiotic resistant bacteria than can also be transported to other environments. Hence, the analysis of Human Microbiome Project dataset provides us not only the opportunity to explore in detail the ARGs diversity and prevalence in different parts of our body but also to provide some insights into the dispersion of ARGs from human to natural populations inhabiting pristine environments. Thus, our data would help to stablish a baseline in ARG surveillance protocols to asses further changes in antibiotic resistances in our society.


title: A nesprin-4/kinesin-1 cargo model for nucleus positioning in cochlear outer hair cells

abstract: Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in-vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

category: cell biology


title: A nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells


title: Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

abstract: Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we designed a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid induction of organelle defunctionalization via remodeling of the membrane phospholipid composition. In particular, we identified a minimal, fully catalytic PLAAT with no unfavorable side effects. Chemically-induced translocation of the engineered PLAAT to the mitochondria surface resulted in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapted the molecular tool in peroxisomes, and observed leakage of matrix-resident functional proteins. The technique was compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should present a novel utility in organelle biology of diverse contexts.


title: Dual modulation of phase-transitioning licenses the Bicc1 network of ciliopathy proteins to bind specific target mRNAs

abstract: Perturbations in biomolecular condensates that form by phase-transitioning are linked to a growing number of degenerative diseases. For example, mutations in a multivalent interaction network of the Ankyrin (ANK) and sterile alpha motif (SAM) domain-containing ANKS3 and ANKS6 proteins with the RNA-binding protein Bicaudal-C1 (Bicc1) associate with laterality defects and chronic kidney diseases known as ciliopathies. However, insights into the mechanisms that control RNA condensation in ribonucleoprotein particles (RNPs) are scarce. Here, we asked whether heterooligomerization modulates Bicc1 binding to RNA. Reconstitution assays in vitro and live imaging in vivo show that a K homology (KH) repeat of Bicc1 self-interacts and synergizes with SAM domain self-polymerization independently of RNA to concentrate bound mRNAs in gel-like granules that can split or fuse with each other. Importantly, emulsification of Bicc1 by ANKS3 inhibited binding to target mRNAs, whereas condensation by ANKS6 co-recruitment increased it by liberating the KH domains from ANKS3. Our findings suggest that the perturbation of Bicc1-Anks3-Anks6 RNP dynamics is a likely cause of associated ciliopathies.

category: developmental biology


title: Inbreeding depression drives evolution of dispersal and polyandry

abstract: Inbreeding depression, defined as the reduction in fitness components of offspring of related individuals compared to offspring of unrelated individuals, is a widespread phenomenon and has profound demographic and evolutionary consequences. It can reduce the mean fitness of a population and increase extinction risk, and it can affect traits evolution. Inbreeding depression is widely hypothesized to be a key driver of the evolution of, among other traits, dispersal (individual movements potentially leading to spatial gene flow) and polyandry (female mating with multiple males within a single reproductive bout), as mechanisms to avoid inbreeding. In turn, both dispersal and polyandry can change the relatedness structure within and among populations, thus affecting opportunity for inbreeding and consequent evolution of inbreeding depression. However, despite this potential major shared driver, and despite the large amount of both theoretical and empirical work, evolution of dispersal and polyandry given inbreeding have been so far studied separately, and thus we still do not know whether and how dispersal and polyandry affect each others evolution, and how they may feed-back onto evolution of inbreeding depression itself. Here, using a genetically-explicit individual-based model, which models realistic distributions of selection and dominance coefficients of deleterious recessive mutations underpinning inbreeding depression, I show that: 1) inbreeding depression indeed drives evolution of dispersal and polyandry; 2) there is a negative feedback between dispersal evolution and polyandry evolution, which therefore evolve as alternative inbreeding avoidance strategies; 3) inbreeding depression is mainly shaped by the level of dispersal, while polyandry has a much more limited effect.


title: Virulence constrains transmission even in the absence of a genetic trade-off

abstract: The virulence-transmission trade-off predicts that parasite fitness peaks at intermediate virulence. However, whether this relationship is driven by the environment or genetically determined and if it depends on transmission opportunities remains unclear. We tackled these issues using inbred lines of the macro-parasitic spider-mite Tetranychus urticae. When transmission was not possible during the infection period, we observed a hump-shaped relationship between virulence and parasite fitness, as predicted by theory. This was environmentally driven, as no genetic correlation between traits was detected. However, when transmission to uninfected hosts occurred during the infection period, virulence was positively, environmentally and genetically correlated with parasite fitness. Therefore, the virulence-transmission trade-off depends on within-host dynamics and on the timing of transmission, rather than on a genetic correlation. This fundamental correlation may thus be easier to manipulate than previously thought.


title: The genetic history of Mayotte and Madagascar cattle breeds mirrors the complex pattern of human exchanges in Western Indian Ocean

abstract: Despite their central economic and cultural role, the origin of cattle populations living in Indian Ocean islands still remains poorly documented. Here, we unravel the demographic and adaptive histories of the extant Zebus from the Mayotte and Madagascar islands using high-density SNP genotyping data. We found that these populations are very closely related and both display a predominant indicine ancestry. They diverged in the 16th century at the arrival of European people who transformed the trade network in the area. Their common ancestral cattle population originates from an admixture between an admixed African zebu population and an Indian zebu that occurred around the 12th century at the time of the earliest contacts between human African populations of the Swahili corridor and Austronesian people from Southeast Asia in Comoros and Madagascar. A steep increase of the estimated population sizes from the beginning of the 16th to the 17th century coincides with the expansion of the cattle trade. By carrying out genome scans for recent selection in the two cattle populations from Mayotte and Madagascar, we identified sets of candidate genes involved in biological functions (cancer, skin structure and UV-protection, nervous system and behavior, organ development, metabolism and immune response) broadly representative of the physiological adaptation to tropical conditions. Overall, the origin of the cattle populations from Western Indian Ocean islands mirrors the complex history of human migrations and trade in this area.


title: Live Analysis of Position Effect Variegation Reveals Different Modes of Action for HP1 and Su(var)3-9

abstract:
AO_SCPLOWBSTRACTC_SCPLOWPosition Effect Variegation (PEV) results from the juxtaposition of euchromatic and heterochromatic components of eukaryotic genomes, silencing genes near the new euchromatin/heterochromatin junctions. Silencing is itself heritable through S phase, giving rise to distinctive random patterns of cell clones expressing the genes intermixed with clones in which the genes are silenced. Much of what we know about epigenetic inheritance in the soma stems from work on PEV aimed at identifying the components of the silencing machinery and its mechanism of inheritance. The roles of two central gene activities - the Su(var)3-9-encoded histone H3-Lysine-9 methyltransferase and the Su(var)205-encoded methyl-H3-Lysine-9 binding protein HP1a - have been inferred from terminal phenotypes, leaving considerable gaps in understanding how PEV behaves through development. Here, we investigate the PEV phenotypes of Su(var)3-9 and Su(var)205 mutations in live developing tissues. We discovered that mutation in Su(var)205 compromises the initial establishment of PEV in early embryogenesis. Later gains of heterochromatin-induced gene silencing are possible, but are unstable and lost rapidly. In contrast, a strain with mutation in Su(var)3-9 exhibits robust silencing early in development, but fails to maintain it through subsequent cell divisions. Our analyses show that while the terminal phenotypes of these mutations may appear identical, they have arrived at them through different developmental trajectories. We discuss how our findings expand and clarify existing models for epigenetic inheritance of heterochromatin-induced gene silencing.

SO_SCPLOWIGNIFICANCEC_SCPLOWCurrent concepts of epigenetic inheritance are exemplified by Position Effect Variegation, a phenomenon whereby heterochromatin can repress genes in clonal cell lineages. Heterochromatin is required for genome protection as it silences toxic transposable elements and prevents instability of repeat sequences. Histone H3 modified by methylation of Lysine-9 and HP1a are critical components of heterochromatin. Using live cell analysis of PEV in mutants in strains with mutations in Su(var)3-9, which encodes the histone methyltransferase, and Su(var)205, which encodes HP1a, we describe an unexpected dynamism in PEV, challenging current models of epigenetics, and revealing unexpectedly different modes of action of these two fundamental components of heterochromatin.

category: genetics


title: Live Analysis of Position Effect Variegation (PEV) in Drosophila Reveals Different Modes of Action for HP1a and Su(var)3-9


title: Computational and experimental studies of plasmodium falciparum protein PfAMA1 domain-II loop dynamics: implications in PfAMA1-PfRON2 binding event

abstract:
Protein-protein interactions are important targets for various drug discovery campaigns. One such promising and therapeutically pertinent protein-protein complex is PfAMA1-PfRON2 involved in malarial parasite invasion into human red blood cells. A thorough understanding of the interactions between these macromolecular binding partners is crucial for designing better therapeutics against this age-old disease. Although crystal structures of several PfAMA1-PfRON2 complexes are available, the mechanism of how domain II loop associates with PfRON2 is not clear. The current work investigates how the domain II loop of PfAMA1 exerts its effect on the alpha helix of the PfRON2, thus influencing the overall kinetics of this intricate recognition phenomenon. To this end, we have computationally simulated the dynamics and free energetics of domain II loop closing processes and identified a set of key amino acid residues of PfRON2 helix which are essential for binding. The subsequent evaluation of the binding affinity of Ala-substituted PfRON2 peptide ligands by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) validates the relative importance of the residues in context. Together, the combination of computational and experimental investigation reveals that the domain II loop of PfAMA1 is in fact responsible for arresting the PfRON2 molecule from egress, K2027 and D2028 of PfRON2 being the determinant residues for the capturing event. Our study provides a comprehensive understanding of the molecular recognition event between PfAMA1 and PfRON2, specifically in the post binding stage, which could potentially open up new avenues to drug discovery against malaria.

TOC GRAPHIC

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/463826v2_ufig1.gif" ALT="Figure 1">
View larger version (35K):
org.highwire.dtl.DTLVardef@1c26022org.highwire.dtl.DTLVardef@356357org.highwire.dtl.DTLVardef@10ceb04org.highwire.dtl.DTLVardef@1195633_HPS_FORMAT_FIGEXP  M_FIG C_FIG

category: biochemistry


title: Computational and experimental studies of the breathing motion of a protein loop: implications in PfAMA1-PfRON2 late-stage binding event


title: Membrane phosphoinositides stabilize GPCR-arrestin complexes and offer temporal control of complex assembly and dynamics

abstract: Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized some GPCRs may complex with arrestin, while others interact transiently; this difference affects receptor signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in arrestin recruitment and GPCR-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one requiring PIP-binding for arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes by promoting a receptor core-engaged state of the complex. As allosteric modulators of GPCR-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. The dependance on membrane PIPs provides a mechanism for arrestin release from transiently associated GPCRs, allowing their rapid recycling, while explaining how stably associated GPCRs are able to engage G proteins at endosomes.


title: Single Cell App: An App for Single Cell RNA-sequencing Data Visualization, Comparison and Discovery

abstract: The Single Cell App is a cloud-based application that allows visualisation and comparison of scRNA-seq data and is scalable according to use. Users upload their own or publicly available scRNA-seq datasets after pre-processing to be visualised using a web browser. The data can be viewed in two colour modes, Cluster - representing cell identity, and Values - level of expression, and data queried using keyword or gene identification number(s). Using the app to compare four different studies we determined that some genes frequently used as cell-type markers are in fact study specific. Phosphate transporter and hormone response genes were exemplary investigated with the app. This showed that the apparent cell specific expression of PHO1;H3 differed between GFP-tagging and scRNA-seq studies. Some phosphate transporter genes were induced by protoplasting, they retained cell specificity, indicating that cell specific stress responses (i.e. protoplasting). Examination of the cell specificity of hormone response genes revealed that 132 hormone responsive genes display restricted expression and that the jasmonate response gene TIFY8 is expressed in endodermal cells which differs from previous reports. It also appears that JAZ repressors have cell-type specific functions. These differences, identified using the Single Cell App, highlight the need for resources to enable researchers to find common and different patterns of cell specific gene expression. Thus, the Single Cell App enables researchers to form new hypothesis, perform comparative studies, allows for easy re-use of data for this emerging technology to provide novel avenues to crop improvement.

category: plant biology


title: The Activity of the Stress Modulated Arabidopsis Ubiquitin Ligases PUB46 and PUB48 is Partially Redundant

abstract: The Arabidopsis ubiquitin ligases PUB46, PUB47 and PUB48 are encoded by paralogus genes; pub46 and pub48 mutants display increased drought sensitivity compared to wild type (WT). Although the phenotype displayed in the single gene mutants, suggest that each has specific biological activity, PUB46 and PUB48 activity may be also redundant. To test functional redundancy between two gene products requires a double mutant. Unfortunately, the close proximity of the PUB46 and PUB48 gene loci precludes obtaining a double mutant by crossing the available single mutants. We thus applied microRNA technology to reduce the activity of all three gene products of the PUB46-48 subfamily by constructing an artificial microRNA (aMIR) targeted to this subfamily. Expressing aMIR46-48 in WT plants resulted in increased drought-sensitivity, a phenotype resembling that of the single pub46 and pub48 mutants, and enhanced sensitivity to methyl viologen, similar to that observed for the pub46 mutant. Furthermore, the WT plants expressing aMIR46-48 also revealed reduced inhibition by ABA at seed germination, a phenotype not evident in the single mutants. Expressing aMIR46-48 in pub46 and pub48 mutants further enhanced the drought sensitivity of each parental single mutant and of WT expressing aMIR46-48. Thus, whereas the gene-specific activity of the PUB46 and PUB48 E3s is partially redundant in that absence of either E3 leads to drought sensitivity, our ability to eliminate the activity of both PUB46 and PUB48 in the same plant reveals additional gene specific facets of their activity in the reaction to abiotic stress.


title: Meeting Measurement Precision Requirements for Effective Engineering of Genetic Regulatory Networks

abstract: Reliable, predictable engineering of cellular behavior is one of the key goals of synthetic biology. As the field matures, biological engineers will become increasingly reliant on computer models that allow for the rapid exploration of design space prior to the more costly construction and characterization of candidate designs. The efficacy of such models, however, depends on the accuracy of their predictions, the precision of the measurements used to parameterize the models, and the tolerance of biological devices for imperfections in modeling and measurement. To better understand this relationship, we have derived an Engineering Error Inequality that provides a quantitative mathematical bound on the relationship between predictability of results, model accuracy, measurement precision, and device characteristics. We apply this relation to estimate measurement precision requirements for engineering genetic regulatory networks given current model and device characteristics, recommending a target standard deviation of 1.5-fold. We then compare these requirements with the results of an interlaboratory study to validate that these requirements can be met via flow cytometry with matched instrument channels and an independent calibrant. Based on these results, we recommend a set of best practices for quality control of flow cytometry data and discuss how these might be extended to other measurement modalities and applied to support further development of genetic regulatory network engineering.

category: synthetic biology


title: Cell-type specific induction of cyclo-oxygenase-2 in layer II/III prefrontal cortical neurons mediates stress-induced anxiety phenotypes in mice.

abstract: The ability of the medial prefrontal cortex (mPFC) to exert top-down control of behavior is affected by stress. The molecular response of mPFC to stress is incompletely understood, however, in part because of the regions cellular heterogeneity. Here we used single nucleus RNA sequencing (snRNAseq) to map specific molecular cell types within the mPFC and to detect cell-type specific transcriptional changes to foot-shock stress. We identified Ptgs2, encoding cyclo-oxygenase 2, as an important candidate that is upregulated in layer II/III excitatory neurons after stress. Specifically, Ptgs2 was transiently upregulated with shock-induced fear learning and fear expression, along with Bdnf, Nptx2, and Lingo1, in a layer II/III neuronal population marked by the neuronal excitatory gene Slc17a7 and cell-type specific neuropeptide Penk. These dynamic cell-type specific expression patterns identified with snRNAseq were validated with quantitative fluorescent in situ hybridization. Using a pharmacological approach, we found that systemic lumiracoxib, a selective Ptgs2-inhibitor, led to a significant reduction in fear expression. Furthermore, genetic ablation of Ptgs2 in excitatory Camk2a-expressing neurons led to reduced stress-induced anxiety-like behaviors. Together these findings suggest that Ptgs2 is expressed in a dynamic, cell-type specific way in Layer II/III Penk+ neurons in mPFC, and that its role in prostaglandin and /or endocannabinoid regulation within these neurons may be an important mediator of stress-related behavior.


title: Investigating Emotion Dynamics and Its Association with Multimedia Content During Video Watching Through EEG Microstate Analysis

abstract: EEG microstates have been widely adopted to understand the complex and dynamic-changing process in dynamic brain systems, but how microstates are temporally modulated by emotion dynamics is still unclear. An investigation of EEG microstates under video-evoking emotion dynamics modulation would provide a novel insight into the understanding of temporal dynamics of functional brain networks. In the present study, we postulate that emotional states dynamically modulate the microstate patterns, and perform an in-depth investigation between EEG microstates and emotion dynamics under a video-watching task. By mapping from subjective-experienced emotion states and objective-presented stimulation content to EEG microstates, we gauge the comprehensive associations among microstates, emotions, and multimedia stimulation. The results show that emotion dynamics could be well revealed by four EEG microstates (MS1, MS2, MS3, and MS4), where MS3 and MS4 are found to be highly correlated to different emotion states (emotion task effect and level effect) and the affective information involved in the multimedia content (visual and audio). In this work, we reveal the microstate patterns related to emotion dynamics from sensory and stimulation dimensions, which deepens the understanding of the neural representation under emotion dynamics modulation and will be beneficial for the future study of brain dynamic systems.


title: EEG Microstate Correlates of Emotion Dynamics and Stimulation Content during Video Watching


title: Early mitochondrial stress and metabolic imbalance lead to photoreceptor cell death in retinal degeneration

abstract: Neurodegenerative diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. To deduce the mechanism leading to retinal degeneration, we adopted a temporal multi-omics approach and examined molecular and cellular events before the onset of photoreceptor cell death in the widely-used Pde6brd1/rd1 (rd1) mouse model. Transcriptome profiling of neonatal and developing rods revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial over-activation and related metabolic alterations as early determinants of pathology and implicate dysregulation of calcium signaling as the initiator of higher mitochondrial stress, which then transitions to mitochondrial damage and photoreceptor cell death in retinal degeneration. Our studies support the "one hit model" arguing against the cumulative damage hypothesis but suggest that cell death in neurodegenerative disease is initiated by specific rather than a random event.


title: Tear miRNAs identified in a murine model of Sjögren's Syndrome as potential diagnostic biomarkers and indicators of disease mechanism

abstract:
ObjectiveThe tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjogrens syndrome (SS), was analyzed to identify possible tear biomarkers of the disease.

MethodsMale NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as negative controls while age- and sex-matched male BALB/c mice served as healthy controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated using RT-qPCR in a separate mouse cohort, and the pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis.

ResultsIn comparison to tears from both healthy (male BALB/c) and negative control (female NOD) mice, initial analysis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were subsequently validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated in the male NOD mouse tears, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in the male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS.

ConclusionsA panel of differentially expressed miRNAs were identified in tears of SS model male NOD mice, including some never previously linked to SS. These may have potential utility as diagnostic biomarkers for ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis.

category: immunology


title: Extremely thermophilic endospores germinate and metabolise organic carbon in sediments heated to above 80°C

abstract: Endospores of thermophilic bacteria are widespread in cold seabed environments where they remain dormant during initial burial in accumulating sediments. The temperature increase during sedimentation can be simulated in experimental heating of sediments, resulting in the temperature-dependent activation of different endospore populations from the microbial seed bank. Here we investigated the response of endospore populations to heating at extreme high temperature (80- 99{degrees}C). Metabolites for germination and organic matter degradation (dipicolinic acid and organic acids) revealed both endospore germination and subsequent metabolism at [≥]80{degrees}C. Endospore-forming Firmicutes with the genomic potential for organic carbon and nitrogen transformation were recovered by genome-resolved metagenomics. Genomes from Symbiobacteriales, Thermosediminibacteriales, Moorellales and Calditerricolales encode multiple mechanisms for high temperature degradation of sedimentary organic carbon and features of necromass that accumulate during sediment burial including saccharides, amino and nucleic acids. The results provide insight into the metabolism of novel carbon cycling microorganisms activated at high temperature, and suggest that extremely thermophilic Firmicutes dispersed in the ocean are poised to germinate in response to sediment heating during burial and transform a wide range of organic substrates.


title: Unidirectional recruitment relations between proteins can be determined by a CRISPR/dCas9 recruitment assay

abstract:
Directional recruitment of protein complexes is critical for proper function of many nuclear processes. Here we present CRISPR-PITA (Protein Interaction and Telomere Recruitment Assay), an assay that determines the ability of a given protein to recruit any other nuclear factor. The protein of interest is directed via CRISPR/dCas9, a dead Cas9 that does not cut DNA, to a repeat sequence, such as telomeres, to obtain dots that are easily detectable by microscopy. The recruitment of endogenous nuclear proteins to these dots can then be visualized using specific antibodies. We determined recruitment abilities in CRISPR-PITA to methyl-CpG binding protein MeCP2, histone deacetylase 1 (HDAC1), heterochromatin protein 1 (HP1), and the latency-associated nuclear antigen (LANA) encoded by Kaposis sarcoma associated herpesvirus (KSHV, HHV-8). LANA was able to recruit its known interactors ORC2 and SIN3A to LANA-telomere dots. In contrast, LANA was unable to recruit MeCP2 whereas MeCP2 was able to recruit LANA. Similarly, HDAC1 that interacts with MeCP2 through the transcriptional-repression domain (TRD) same as LANA, was unable to recruit MeCP2, but MeCP2 recruited HDAC1. One important function of LANA is to tether the viral episomal genomes to the cellular chromosomes during cell division. The unidirectional recruitment of LANA by MeCP2, makes MeCP2 a candidate anchor for KSHV genome tethering by LANA. We found that cells derived from Rett syndrome and express a mutant MeCP2 (T158M), impaired in DNA binding, cannot support KSHV genome maintenance. In summary, we describe a broadly applicable protein recruitment assay based on CRISPR/dCas9.

Significance StatementCRISPR/Cas9 is a revolutionary system that has profoundly impacted biology research. Here we present another application for CRISPR/Cas9, in evaluating recruitment relations between nuclear proteins. A protein of interest is directed to a repeat sequence via the catalytically inactive Cas9 (dCas9) to generate easily detectable dots. Then, the recruitment of other nuclear proteins to these dots can be evaluated. Using this assay, we show that some interacting proteins have a unidirectional recruitment property, where only one of the proteins can recruit its partner. We propose that available interacting domains can force this unidirectional recruitment. Using this recruitment assay, we found unidirectional recruitment of the KSHV encoded LANA and HDAC1 by MeCP2. Furthermore, this unidirectional recruitment is critical for viral latency, since LANA fails to maintain the viral genomes in MeCP2 mutant cells.


title: High Resolution Slide-seqV2 Spatial Transcriptomics Enables Discovery of Disease-Specific Cell Neighborhoods and Pathways

abstract:
High resolution spatial transcriptomics is a transformative technology that enables mapping of RNA expression directly from intact tissue sections; however, its utility for the elucidation of disease processes and therapeutically actionable pathways remain largely unexplored. Here we applied Slide-seqV2 to mouse and human kidneys, in healthy and in distinct disease paradigms. First, we established the feasibility of Slide-seqV2 in human kidney by analyzing tissue from 9 distinct donors, which revealed a cell neighborhood centered around a population of LYVE1+ macrophages. Second, in a mouse model of diabetic kidney disease, we detected changes in the cellular organization of the spatially-restricted kidney filter and blood flow regulating apparatus. Third, in a mouse model of a toxic proteinopathy, we identified previously unknown, disease-specific cell neighborhoods centered around macrophages. In a spatially-restricted subpopulation of epithelial cells, we also found perturbations in 77 genes associated with the unfolded protein response (UPR). Our studies illustrate and experimentally validate the utility of Slide-seqV2 for the discovery of disease-specific cell neighborhoods.

One-Sentence SummaryHigh resolution Slide-seqV2 spatial transcriptomics in human and mouse kidneys.

category: molecular biology


title: An ancestral interaction module promotes oligomerisation in divergent mitochondrial ATP synthases

abstract: Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.


title: Inbreeding is associated with shorter early-life telomere length in a wild passerine

abstract: Inbreeding can have negative effects on survival and reproduction, which may be of conservation concern in small and isolated populations. However, the physiological mechanisms underlying inbreeding depression are not well-known. The length of telomeres, the DNA sequences protecting chromosome ends, has been associated with health or fitness in several species. We investigated effects of inbreeding on early-life telomere length in two small island populations of wild house sparrows (Passer domesticus) known to be affected by inbreeding depression. Using genomic and pedigree-based measures of inbreeding we found that inbred nestling house sparrows have shorter telomeres. This negative effect of inbreeding on telomere length may have been complemented by a heterosis effect resulting in longer telomeres in individuals that were less inbred than the population average. Furthermore, we found some evidence of stronger effects of inbreeding on telomere length in males than females. Thus, telomere length may reveal subtle costs of inbreeding in the wild and demonstrate a route by which inbreeding negatively impacts the physiological state of an organism already at early life-history stages.


title: Recoverability of Ancestral Recombination Graph Topologies

abstract: AO_SCPLOWBSTRACTC_SCPLOWRecombination is a powerful evolutionary process that shapes the genetic diversity observed in the populations of many species. Reconstructing genealogies in the presence of recombination from sequencing data is a very challenging problem, as this relies on mutations having occurred on the correct lineages in order to detect the recombination and resolve the placement of edges in the local trees. We investigate the probability of recovering the true topology of ancestral recombination graphs (ARGs) under the coalescent with recombination and gene conversion. We explore how sample size and mutation rate affect the inherent uncertainty in reconstructed ARGs; this sheds light on the theoretical limitations of ARG reconstruction methods. We illustrate our results using estimates of evolutionary rates for several biological organisms; in particular, we find that for parameter values that are realistic for SARS-CoV-2, the probability of reconstructing genealogies that are close to the truth is low.


title: The sperm-associated microbiota of crickets and their local variation and rapid turnover

abstract: While studying aspects of the sperm biology and immunity of two species of crickets, we encountered bacteria that were released from the male sperm container, the spermatophore, alongside sperm. We describe a presumably rich microbe flora in the sperm population ( sperm-associated microbiota). These sperm-associated microbiota differed between the two species of cricket and between different populations and showed functional diversity. Further, sperm-associated microbiota killed sperm, highlighting their potential role in fitness, especially since they are most likely transferred to females during mating.


title: Complex fitness landscape shapes variation in a hyperpolymorphic species

abstract: It is natural to assume that patterns of genetic variation in hyperpolymorphic species can reveal large-scale properties of the fitness landscape that are hard to detect by studying species with ordinary levels of genetic variation1,2. Here, we study such patterns in a fungus Schizophyllum commune, the most polymorphic species known3. Throughout the genome, short-range linkage disequilibrium caused by attraction of rare alleles is higher between pairs of nonsynonymous than of synonymous sites. This effect is especially pronounced for pairs of sites that are located within the same gene, especially if a large fraction of the gene is covered by haploblocks, genome segments where the gene pool consists of two highly divergent haplotypes, which is a signature of balancing selection. Haploblocks are usually shorter than 1000 nucleotides, and collectively cover about 10% of the S. commune genome. LD tends to be substantially higher for pairs of nonsynonymous sites encoding amino acids that interact within the protein. There is a substantial correlation between LDs at the same pairs of nonsynonymous sites in the USA and the Russian populations. These patterns indicate that selection in S. commune involves positive epistasis due to compensatory interactions between nonsynonymous alleles. When less polymorphic species are studied, analogous patterns can be detected only through interspecific comparisons.


title: Walking strides direct rapid and flexible recruitment of visual circuits for course control in <Drosophila>

abstract: Flexible mapping between activity in sensory systems and movement parameters is a hallmark of successful motor control. This flexibility depends on continuous comparison of short-term postural dynamics and the longer-term goals of an animal, thereby necessitating neural mechanisms that can operate across multiple timescales. To understand how such body-brain interactions emerge to control movement across timescales, we performed whole-cell patch recordings from visual neurons involved in course control in Drosophila. We demonstrate that the activity of leg mechanosensory cells, propagating via specific ascending neurons, is critical to provide a clock signal to the visual circuit for stride-by-stride steering adjustments and, at longer timescales, information on speed-associated motor context to flexibly recruit visual circuits for course control. Thus, our data reveal a stride-based mechanism for the control of high-performance walking operating at multiple timescales. We propose that this mechanism functions as a general basis for adaptive control of locomotion.


title: Human pluripotent stem cell-derived photoreceptors switch from cell autonomous axon extension to non-cell autonomous process pulling during synaptic marker redistribution.

abstract:
Photoreceptors (PRs) are the primary visual sensory cells, and their loss leads to blindness that is currently incurable. Cell replacement therapy holds promise as a therapeutic approach to restore vision to those who have lost PRs through damage or disease. While PR transplant research is ongoing in animal models, success is hindered by our limited understanding of PR axon growth during development and regeneration. Using a human pluripotent stem cell (hPSC) reporter line that labels PRs (WA09 CRX+/tdTomato), we generated retinal organoids in order to study mechanisms of PR process extension. We found that the earliest born PRs exhibit autonomous axon extension from dynamic terminals that appear similar to projection neuron growth cones. However, as hPSC-derived PRs age from 40 to 80 days of differentiation, they lose dynamic terminals in 2D plated cultures and within 3D retinal organoids, which does not correlate with cell birth date. Using a rod-specific hPSC reporter line (WA09 NRL+/eGFP), we further determined that rod PRs never form motile growth cones. Interestingly, PRs without motile terminals are still capable of extending axons, but neurites are generated from process stretching via their attachment to motile non-PR cells, which underlies the observed differences in PR neurite lengths on different substrata. While immobile PR terminals express actin, it is less polymerized and less organized than actin present in motile terminals. However, immobile PRs do localize synaptic proteins to their terminals, suggesting a normal developmental progression. These findings help inform the development of PR transplant therapies to treat blinding diseases and provide a platform to test treatments that restore autonomous PR axon extension.

Significance StatementLoss of photoreceptors (PRs) in the retina through damage or disease causes irreversible vision loss and blindness. One treatment approach is to replace lost cells with transplanted human stem cell-derived PRs, but this requires PR axons to integrate into the host retina to restore the required neural connections. For this strategy to succeed, we need to understand how PRs extend processes to their targets during development in situ, and whether dissociated human stem cell (hPSC)-derived PRs behave in a similar fashion. In this paper, we show that hPSC-PRs have only a short window during which they are capable of autonomous axon extension, which has implications for PR transplant efforts and for our basic understanding of human retinal development.


title: Human photoreceptors switch from autonomous axon extension to cell-mediated process pulling during synaptic marker redistribution.


title: Cell Adhesion-Dependent Biphasic Axon Outgrowth Elucidated by Femtosecond Laser Impulse

abstract:
Axon outgrowth is promoted by the mechanical coupling between the dynamic actin cytoskeleton and adhesive substrates via clutch and adhesion molecules in the axonal growth cone. In this study, we utilized a femtosecond laser-induced impulse to break the coupling between an axonal growth cone and an adhesive substrate, enabling us to evaluate the strength of the binding between proteins in the growth cone and a laminin substrate, and also determine the contribution of adhesion strength to axon outgrowth. We found that the adhesion strength of axonal L1 cell adhesion molecule (L1CAM)-laminin binding increased with the density of the laminin substrate. In addition, fluorescent speckle microscopy revealed that the retrograde flow of actin filaments in the axonal growth cone was dependent on the laminin density such that the flow speed reduced with increasing L1CAM-laminin binding. However, axon outgrowth did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, in which the outgrowth was suppressed by excessive L1CAM-laminin binding. Our quantitative evaluations of the adhesion strength suggest that the biphasic outgrowth is regulated by the balance between traction force and adhesion strength as a result of changes in the number of L1CAM-laminin interactions. These results imply that adhesion modulation is key to the regulation of axon guidance.

Significance StatementThere is a lack of a method to evaluate an adhesion strength of axonal growth cone. We evaluated the adhesion strength of axonal growth cones to a substrate by utilizing the force applied from a femtosecond laser impulse. This study shows that the adhesion strength between the growth cone and substrate is strengthened by L1CAM-laminin binding. Axon outgrowth did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, indicating that existence of suitable adhesion strength for axonal growth. Our findings suggest that the balance between growth cone adhesion strength and the traction force transmitted via cytoskeletal flow is a key factor in axon guidance.


title: Waning grasslands: a quantitative temporal evaluation of the grassland habitats across human-dominated upper Gangetic Plains, north India

abstract: Grassland habitats currently face severe anthropogenic exploitations leading to cascading effects on the survival of grassland-dependent biodiversity globally, particularly in non-protected areas. Significant amount of such biodiversity-rich grasslands in India are found outside protected areas but lack quantitative information on their status. We evaluated the current and historical (30 years) status of the grasslands using a combination of intensive field surveys and GIS tools across one of the most fertile, human-dominated region: the upper Gangetic Plains of north India. On-ground mapping and visual classifications revealed 57% decline in grassland habitats between 1985 (418 km2) and 2015 (178km2), mostly driven by conversion to croplands (74% contribution). Radio-telemetry data from the largest endemic cervid swamp deer (n=2) showed grassland-dominated average home range (50% BBMM) size of 1.02 km2. The animals highly preferred these patches (average Ivlevs index- 0.85) and showed highest temporal continuity (88%) compared to other LULC classes. Camera trapping within the core habitats suggests critical use of these patches as fawning/breeding grounds. Habitat suitability analysis indicates only [~]18% of the entire area along Ganges is suitable for swamp deer. Accurate mapping (86% accuracy) and characterization of four major grass species revealed a total 144.04 km{superscript 2} vegetation area, dominted by Saccharum sp. (35%). We recommend protection and recovery of these critical grassland patches to maintain  dynamic corridors and other appropriate management strategies involving multiple stakeholders to ensure survival of this critical ecosystem. Such evaluations, if spatially expanded, would be critical to restore this rapidly vanishing ecosystem worldwide.

category: ecology


title: The Japanese wolf is most closely related to modern dogs and its ancestral genome has been widely inherited by dogs throughout East Eurasia

abstract: The Japanese wolf (Canis lupus hodophilax Temminck, 1839) was a subspecies of the gray wolf that inhabited the Japanese Archipelago and became extinct 100-120 years ago. In this study, we determined the whole genomes of nine Japanese wolves from the 19th- early 20th centuries and 11 Japanese dogs and analyzed them along with both modern and ancient wolves and dogs. Genomic analyses indicate that the Japanese wolf was a unique subspecies of the gray wolf that was genetically distinct from both modern and ancient gray wolves, lacking gene flow with other gray wolves. A Phylogenetic tree that minimizes the effects of introgression shows that Japanese wolves are closest to the dog monophyletic group among the gray wolves. Moreover, Japanese wolves show significant genetic affinities with East Eurasian dogs. We estimated the level of introgression from the ancestor of the Japanese wolves to the ancestor of East Eurasian dogs that had occurred in the transitional period from the Pleistocene to the Holocene, at an early stage after divergence from West Eurasian dog lineages. Because of this introgression, Japanese wolf ancestry has been inherited by many dogs through admixture between East Eurasian dog lineages. As a result of this heredity, up to 5.5% of modern dog genomes throughout East Eurasia are derived from Japanese wolf ancestry.


title: Immunity to SARS-CoV-2 up to 15 months after infection

abstract:
BackgroundInformation concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) patients up to 15 months after symptoms onset.

MethodsThe levels of anti-spike and anti-receptor binding domain antibodies and neutralizing activities were tested in a total of 188 samples from 136 convalescent patients who experience mild to critical COVID-19. Specific memory B and T cell responses were measured in 76 peripheral blood mononuclear cell samples collected from 54 patients. Twenty-three vaccinated individuals were included for comparison.

FindingsFollowing a peak at day 15-28 post-infection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Plasma neutralizing activity against G614 was still detected in 87% of the patients at 6-15 months. Compared to G614, the median neutralizing titers against Beta, Gamma and Delta variants in plasma collected at early (15-103 days) and late (9-15 month) convalescence were 16- and 8-fold lower, respectively. SARS-CoV-2-specific memory B and T cells reached a peak at 3-6 months and persisted in the majority of patients up to 15 months although a significant decrease in specific T cells was observed between 6 and 15 months.

ConclusionThe data suggest that antiviral specific immunity especially memory B cells in COVID-19 convalescent patients is long-lasting, but some variants of concern, including the fast-spreading Delta variant, may at least partially escape the neutralizing activity of plasma antibodies.

FundingEU-ATAC consortium, the Italian Ministry of Health, the Swedish Research Council, SciLifeLab, and KAW.


title: Extracellular Fluid Flow Induces Shallow Quiescence through Physical and Biochemical Cues

abstract: The balance between cell quiescence and proliferation is fundamental to tissue physiology and homeostasis. Recent studies have shown that quiescence is not a passive and homogeneous state but actively maintained and heterogeneous. These cellular characteristics associated with quiescence were observed primarily in cultured cells under a static medium. However, cells in vivo face different microenvironmental conditions, particularly, under interstitial fluid flows distributed through extracellular matrices. Interstitial fluid flow exerts shear stress on cells and matrix strain, and results in continuous replacement of extracellular factors. In this study, by analyzing individual cells under varying fluid flow rates in microfluidic devices, we found that extracellular fluid flow alters cellular quiescence depth through flow-induced physical and biochemical cues. Specifically, increasing the flow rate drives cells to shallower quiescence and become more likely to reenter the cell cycle upon growth stimulation. Furthermore, we found that increasing shear stress or extracellular factor replacement individually, without altering other parameters, also results in shallow quiescence. We integrated the experimental results into a mathematical model to gain insight and predict the effects of varying extracellular fluid flow conditions on cellular quiescence depth. Our findings uncover a previously unappreciated mechanism that likely underlies the heterogeneous responses of quiescent cells for tissue repair and regeneration in physiological tissue microenvironments.


title: Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

abstract: Hsp70-family chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how Hsp70s differentiate between monomeric and oligomeric states of a target protein. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. We discover that electrostatic attraction enables the negatively charged BiP to bind positively charged E-peptide oligomers with low nanomolar affinity. We identify the specific BiP binding sites on proIGF2, and although some are positively charged, as monomers they bind BiP with characteristically low affinity in the micromolar range. We conclude that electrostatics enable BiP to preferentially recognize oligomeric states of proIGF2. Electrostatic targeting of Hsp70 to aggregates may be broadly applicable, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that are expected to be positively charged.


title: RSV immunizes bystander cells to IAV infection using interferons β and {lambda}

abstract:
We observed the interference between two prevalent respiratory viruses, respiratory syncytial virus (RSV) and influenza A virus (IAV, H1N1), and characterized its molecular underpinnings in alveolar epithelial cells (A549). We found that RSV induces higher interferon (IFN) {beta} production than IAV and that IFN{beta} priming confers higher protection against infection with IAV than with RSV. Consequently, we focused on the sequential infection scheme: RSV-then-IAV. Using the A549 WT, IFNAR1 KO, IFNLR1 KO, and IFNAR1-IFNLR1 double KO cell lines we found that both IFN{beta} and IFN{lambda} are necessary for maximum protection against subsequent infection. Immunostaining revealed that preinfection with RSV partitions the cell population into a subpopulation susceptible to subsequent infection with IAV and an IAV-proof subpopulation. Strikingly, the susceptible cells turned out to be those already compromised and efficiently expressing RSV, whereas the bystander, interferon-primed cells are resistant to IAV infection. Thus, the virus-virus exclusion at the cell population level is not realized through a direct competition for a shared ecological niche (single cell) but rather achieved with the involvement of specific cytokines induced within the host innate immune response.

Author summaryAfter being challenged with a pathogen, infected cells warn bystander cells about the potential threat by synthesizing and secreting cytokines, especially interferons. In this way, the not yet infected but warned cells are given an opportunity to reinforce their defense mechanisms before they encounter the pathogen replicating in the infected cells whose defense mechanisms have been overcome. Consequently, cell population is partitioned into the cells that may be more prone to infection with another pathogen and the cells that may be expected to be resistant to another pathogen. By studying at the single-cell level the sequential infection with two prevalent respiratory viruses, respiratory syncytial virus (RSV) and influenza A virus (IAV), we showed that the interferon-mediated resistance induced by infection with RSV protects non-infected cells from infection with IAV. The cells compromised by RSV turned out however to be susceptible to invasion of IAV. This demonstrates that, even in the absence of within-cell viral competition, the innate immune response can induct viral interference between primary infection with RSV and secondary infection with IAV.


title: Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of cytoplasmic vesicles and mitochondria during HSV-1 infection

abstract:
Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.

ImportanceUltrastructural changes to the morphology and organization of cellular compartments during herpes simplex virus-1 (HSV-1) infection have not previously been studied under near-physiological conditions. In this study, soft X-ray tomography was used to image the ultrastructure of vitrified cells during HSV-1 infection, identifying striking changes to the abundance and organization of cytoplasmic vesicles and mitochondria. The concentration of vesicles in the juxtanuclear region increased with time post infection, which could represent an increasing supply of vesicles to support capsid envelopment, and there is a transient increase in the size of lipid droplets in infected cells. Furthermore, we show that mitochondria elongate and form highly-branched networks as infection progresses. These findings offer insight into stages of virion morphogenesis and the cellular response to infection, highlighting the utility of cryo-soft-X-ray tomography for monitoring the near-native state ultrastructure of infected cells.


title: Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection


title: Nipah virus Bangladesh infection elicits organ-specific innate and inflammatory responses in the marmoset model

abstract: The common marmoset (Callithrix jacchus) has, in recent years, received more recognition as an ideal non-human primate (NHP) model not only for studies related to neuroscience, but also at high-biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Four marmosets were infected via the intranasal and intratracheal route and monitored for disease development. We obtained histopathological data, quantified genome copies on >25 tissue-types, and performed RNA-seq on six different organs from all infected and control marmosets. All four subjects developed fatal disease between days 8 and 11 post infection, with three animals developing pulmonary edema and hemorrhage as well as multi-focal hemorrhagic lymphadenopathy. One subject recapitulated neurologic clinical symptoms and cardiomyopathy on gross pathology. In all animals, syncytia were evident in endothelial cells in pulmonary vessels, cells in alveolar septum, and in splenic follicles or red pulp. To define the organ-specific innate and inflammatory responses, we performed RNA-seq on six different organs from all infected and compared to naive non-infected marmoset tissues. RNA-seq gave 17.4 million reads per sample on average, with the most highly infected tissue sample, the lung of one marmoset, containing >180,000 virus-specific reads. NiV V and W transcripts comprised [~]40% of all phosphoprotein-derived transcripts with V and W being very close to each other proportionally. Principal component analysis showed that in brain stem, the marmoset exhibiting neurological symptoms displayed a unique RNA transcriptome relative even to other infected marmosets. Upregulated genes in the various tissues belonged to distinct GO pathways. Additionally, one male and female animal had detectable viral reads in their ovaries (27,120) and testes (858). Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.


title: The spatial organization of replication is determined by cell size independently of chromosome copy number

abstract: We have studied how Escherichia coli coordinates replication and division cycles by tracking replisomes in individual cells through thousands of division cycles. By analysing the replication and division of the cells at various growth rates and with different genetic perturbations, we have reached four major conclusions: (1) Initiation is not based on titration of DnaA to the chromosomal binding sites because initiation is still coordinated with the division cycle several generations after DnaA synthesis is turned off. (2) We can rule out that initiation is triggered by a simple DnaA-ADP/ATP switch mechanism because initiation is still triggered at a well-defined cell size in a DARS1/DARS2/datA triple knockout if DnaA is overexpressed. (3) From the spatial organization of replisomes at different growth rates, it appears as if initiation is triggered by termination, but this hypothesis is ruled out by an NGS-based marker frequency analysis. (4) Overlapping replication cycles do not affect the spatial organization of the replication machinery and it appears as if each replication center contains a fixed number of DnaN molecules independently of how many replication forks are being processed.


title: Coping with oxidative stress in extreme environments: the distinctive roles played by Acinetobacter sp. Ver3 superoxide dismutases

abstract: Acinetobacter sp. Ver3 is a polyextremophilic strain characterized by a high tolerance to radiation and pro-oxidants. The Ver3 genome comprises the sodB and sodC genes encoding an iron (AV3SodB) and a copper/zinc superoxide dismutase (AV3SodC), respectively; however, the specific role(s) of these genes has remained elusive. We show that the expression of sodB remained unaltered in different oxidative stress conditions whereas sodC was up-regulated in the presence of blue light. Besides, we studied the changes in the in vitro activity of each SOD enzyme in response to diverse agents and solved the crystal structure of AV3SodB at 1.34 [A], one of the highest resolutions achieved for a SOD. Cell fractionation studies interestingly revealed that AV3SodB is located in the cytosol whereas AV3SodC is also found in the periplasm. Consistently, a bioinformatic analysis of the genomes of 53 Acinetobacter species pointed out the presence of at least one SOD type in each compartment, suggesting that these enzymes are separately required to cope with oxidative stress. Surprisingly, AV3SodC was found in an active state also in outer membrane vesicles, probably exerting a protective role. Overall, our multidisciplinary approach highlights the relevance of SOD enzymes when Acinetobacter spp. are confronted with oxidizing agents.


title: Candida albicans commensalism in the oral mucosa is favoured by limited virulence and metabolic adaptation .

abstract:
As part of the human microbiota, the fungus Candida albicans colonizes the oral cavity and other mucosal surfaces of the human body. Commensalism is tightly controlled by complex interactions of the fungus and the host to preclude fungal elimination but also fungal overgrowth and invasion, which can result in disease. As such, defects in antifungal T cell immunity render individuals susceptible to oral thrush due to interrupted immunosurveillance of the oral mucosa. The factors that promote commensalism and ensure persistence of C. albicans in a fully immunocompetent host remain less clear. Using an experimental model of C. albicans oral colonization in mice we explored fungal determinants of commensalism in the oral cavity. Transcript profiling of the oral isolate 101 in the murine tongue tissue revealed a characteristic metabolic profile tailored to the nutrient poor conditions in the stratum corneum of the epithelium where the fungus resides. Metabolic adaptation of isolate 101 was also reflected in enhanced nutrient acquisition when grown on oral mucosa substrates. Persistent colonization of the oral mucosa by C. albicans also correlated inversely with the capacity of the fungus to induce epithelial cell damage and to elicit an inflammatory response. Here we show that these immune evasive properties of isolate 101 are explained by a strong attenuation of a number of virulence genes, including those linked to filamentation. De-repression of the hyphal program by deletion or conditional repression of NRG1 abolished the commensal behaviour of isolate 101, thereby establishing a central role of this factor in the commensal lifestyle of C. albicans in the oral niche of the host.

AUTHOR SUMMARYThe oral microbiota represents an important part of the human microbiota and includes several hundreds to several thousands of bacterial and fungal species. One of the most prominent fungus colonizing the oral cavity is the yeast Candida albicans. While the presence of C. albicans usually remains unnoticed, the fungus can under certain circumstances cause lesions on the lining of the mouth referred to as oral thrush or contribute to other common oral diseases such as caries. Maintaining C. albicans commensalism in the oral mucosa is therefore of utmost importance for oral health and overall wellbeing. While overt fungal growth and disease is limited by immunosurveillance mechanisms during homeostasis, C. albicans strives to survive and evades elimination from the host. Here, we show that while commensalism in the oral cavity is characterized by a restricted fungal virulence and hyphal program, enforcing filamentation in a commensal isolate is sufficient for driving pathogenicity and fungus-induced inflammation in the oral mucosa thwarting persistent colonization. Our results further support a critical role for specialized nutrient acquisition allowing the fungus to thrive in the nutrient poor environment of the squamous epithelium. Together, this work revealed key determinants of C. albicans commensalism in the oral niche.


title: Short-Term Dairy Elimination and Reintroduction Minimally Perturbs the Gut Microbiota in Self-Reported Lactose Intolerant Adults

abstract: One of the outstanding questions regarding the human gut microbiota is how interventions designed to manipulate the microbiota may influence host phenotypic traits. Here, we employed a dietary intervention to probe this question in the context of lactose intolerance. To assess the effects of dairy elimination and reintroduction on the microbiota and host phenotype, we paired fecal 16S rRNA amplicon sequencing with a clinical assay for lactose intolerance, the hydrogen breath test. We studied 12 self-reported mildly lactose intolerant adults, each with tri-weekly collection of fecal samples over a 12-week study period (2 weeks baseline, 4 weeks of dairy elimination, 6 weeks of gradual milk reintroduction) and a hydrogen breath test before and after each phase. We found that although none of the subjects experienced a change in clinically defined status of lactose intolerant or tolerant, most subjects were qualitatively better able to tolerate dairy products with minimal symptoms at the end of the study compared to their baseline. Like clinical status, gut microbiota also resisted modification. Although the mean fraction of the genus Bifidobacterium, a group known to metabolize lactose, increased slightly with dairy reintroduction (from 0.0125 to 0.0206; Wilcoxon P = 0.068), the overall structure of each subjects gut microbiota remained highly individualized and largely stable. Our study is small, but it suggests the possibility of qualitatively improved tolerance in the absence of change in clinically defined tolerance nor major change in the gut microbiota.


title: Serine/arginine-rich splicing factor 7 plays oncogenic roles through specific regulation of m6A RNA modification

abstract: Serine/Arginine-Rich Splicing Factor 7 (SRSF7), which is previously recognized as a splicing factor, has been revealed to play oncogenic roles in multiple cancers. However, the mechanisms underlying its oncogenic roles have not been well addressed. Here, based on N6-methyladenosine (m6A) co-methylation network analysis across diverse cell lines, we found SRSF7 positively correlated with glioblastoma cell-specific m6A methylation. We then proved SRSF7 is a novel m6A regulator that specifically facilitates the m6A methylation near its binding sites on the mRNAs involved in cell proliferation and migration through recruiting methyltransferase complex. Moreover, SRSF7 promotes the proliferation and migration of glioblastoma cells largely dependent on the m6A methyltransferase. The two single-nucleotide m6A sites on PBK are regulated by SRSF7 and partially mediate the effects of SRSF7 on glioblastoma cells through recognition by IGF2BP2. Together, our discovery revealed a novel role of SRSF7 in regulating m6A and timely confirmed the existence and functional importance of RNA binding protein (RBP) mediated specific regulation of m6A.


title: HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

abstract: DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1-AS1 was retrieved as a top hit. Endogenous HIF1-AS1 reduced the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1-AS1 was down-regulated in pulmonary hypertension and loss-of-function approaches not only resulted in gene de-repression but also enhanced angiogenic capacity. As exemplified here with HIF1-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.


title: Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter

abstract: AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.


title: Genome-edited zebrafish model of ABCC8 loss-of-function disease

abstract:
KATP channel gain- (GOF) and loss-of-function (LOF) mutations in SUR1 or Kir6.2 underlie human neonatal diabetes mellitus (NDM) and congenital hyperinsulinism (CHI), respectively. Genetically modified mice with transgenic overexpression of GOF mutations recapitulate many features of human NDM and transgenic mice expressing incomplete KATP LOF do reiterate mild hyperinsulinism, although complete KATP knockout mice do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis in zebrafish are regulated by identical KATP channels to those in mammals, and to explore the possibility of using zebrafish for modeling CHI, we have examined SUR1 truncation mutation (K499X) introduced into the abcc8 gene. Patch-clamp analysis confirmed complete absence of channel activity in {beta}-cells from K499X (SUR1-/-) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish, nor in homozygous SUR1-/- fish, mimicking findings in SUR1 knockout mice. Mutant fish also demonstrated impaired glucose tolerance, similar to LOF mouse models. In paralleling features of mammalian hyperinsulinism resulting from loss-of-function mutations, this gene-edited animal provides a valid zebrafish model of KATP LOF driven-dependent pancreatic disease.

Key PointsO_LILoss-of function in the Kir6.2 (KCNJ11) and SUR1 (ABCC8)-encoded pancreatic islet {beta}-cell KATP channels underlie congenital hyperinsulinism. Mouse models reiterate key features, but zebrafish models could provide a powerful model for further analysis and therapy testing.
C_LIO_LIAn early nonsense mutation in exon 10 of SUR1 was generated by ENU mutagenesis.
C_LIO_LIPatch-clamp analysis revealed an absence of {beta}-cells of SUR1 truncation mutants.
C_LIO_LICa imaging demonstrated elevated basal [Ca]i in {beta}-cells with SUR1 truncation.
C_LIO_LIHomozygous SUR1 truncation mutants had normal fasting glucose but impaired glucose tolerance as adults, mimicking findings in mouse SUR1 knockouts.
C_LIO_LIIn paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent loss-of-function mutations, this gene-edited animal provides a valid zebrafish model of KATP -LOF dependent pancreatic diseases.
C_LI


title: JIP3 regulates bi-directional organelle transport in neurons through its interaction with dynein and kinesin-1

abstract: The conserved MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. Whether and how JIP3s interaction with dynein and kinesin-1 contributes to this critical organelle clearance function is unclear. Using purified recombinant human proteins, we show that dynein light intermediate chain (DLIC) binds to the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in a hydrophobic pocket of the RH1 domain, previously shown to abrogate RILPL2 binding to myosin Va, abrogates the binding of JIP3/4 and RILP to DLIC without perturbing the interaction between the JIP3 RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3s leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bi-directional organelle transport.


title: Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells

abstract: Cellular circadian rhythms confer daily temporal organisation upon behaviour and physiology that is fundamental to human health and disease. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body. Being naturally anucleate, RBC circadian rhythms share key elements of post-translational, but not transcriptional, regulation with other cell types. The physiological function and developmental regulation of RBC circadian rhythms is poorly understood, however, partly due to the small number of appropriate techniques available. Here, we extend the RBC circadian toolkit with a novel biochemical assay for haemoglobin oxidation status, termed "Bloody Blotting". Our approach relies on a redox-sensitive covalent haem-haemoglobin linkage that forms during cell lysis. Formation of this linkage exhibits daily rhythms in vitro, which are unaffected by mutations that affect the timing of circadian rhythms in nucleated cells. In vivo, haemoglobin oxidation rhythms demonstrate daily variation in the oxygen-carrying and nitrite reductase capacity of the blood, and are seen in human subjects under controlled laboratory conditions as well as in freely-behaving humans. These results extend our molecular understanding of RBC circadian rhythms and suggest they serve an important physiological role in gas transport.


title: Dynamin-2 Phosphorylation as A Critical Regulatory Target of Bin1 and GSK3α for Endocytosis in Muscle

abstract: Tight regulation of endocytosis ensures accurate control of cellular signaling and membrane dynamics, which are crucial for tissue morphogenesis and functions. Mutations of Bin1 and dynamin-2 (Dyn2), proteins that generate membrane curvature and sever endocytic invaginations, respectively, cause progressive hereditary myopathy. Here, we show that Bin1 inhibits Dyn2 via direct interaction of its SRC Homology 3 (SH3) domain with the proline-rich domain (PRD) of Dyn2. Phosphorylation of S848 of Dyn2 by GSK3, a kinase downstream of insulin signaling, relieves Dyn2 from the inhibition of Bin1 and promotes endocytosis in muscle. Mutations of Bin1 associated with centronuclear myopathy disrupt its inhibition of Dyn2, thereby exaggerating Dyn2 fission activity and causing excessive fragmentation of T-tubules in the muscle cells. Our work reveals how Bin1-Dyn2 interaction fine-tunes membrane remodeling at the molecular level, and lay the foundation for future exploration of endocytic regulation and hereditary muscle diseases.


title: Methylome inheritance and enhancer dememorization reset an epigenetic gate safeguarding embryonic programs

abstract: Drastic epigenetic reprogramming is essential to convert terminally-differentiated gametes to totipotent embryos. However, it remains puzzling why post-fertilization global DNA reprogramming occurs only in mammals but not in non-mammalian vertebrates. In zebrafish, global methylome inheritance is however accompanied by sweeping enhancer "dememorization" as they become fully methylated. By depleting maternal dnmt1 using oocyte microinjection in situ, we eliminated DNA methylation in zebrafish early embryos, which died around gastrulation with severe differentiation defects. Strikingly, methylation deficiency leads to extensive derepression of adult tissue-specific genes and CG-rich enhancers, which acquire ectopic TF binding and, unexpectedly, H3K4me3. By contrast, embryonic enhancers are generally CG-poor and evade DNA methylation repression. Hence, global DNA hypermethylation inheritance coupled with enhancer dememorization installs an epigenetic gate that safeguards embryonic programs and ensures temporally ordered gene expression. We propose that "enhancer dememorization" underlies and unifies distinct epigenetic reprogramming modes in early development between mammals and non-mammals.


title: Effects of background food on alternative grain uptake and zinc phosphide efficacy in wild house mice

abstract:
BACKGROUNDHouse mice (Mus musculus) cause significant, ongoing losses to grain crops in Australia, particularly during mouse plagues. Zinc phosphide (ZnP) coated grain is used for control, but with variable success. In a laboratory setting, we tested if mice would (1) switch from consumption of one grain type to another when presented with an alternative, and (2) consume ZnP-treated grains when presented as a choice with a different grain.

RESULTSMice readily switched from their background grain to an alternative grain, preferring cereals (wheat or barley) over lentils. Mice readily consumed ZnP-coated barley grains. Their mortality rate was significantly higher (86%, n=30) in the presence of a less-favoured grain (lentils) compared to their mortality rate (47%, n=29; and 53%, n=30) in the presence of a more-favoured grain (wheat and barley, respectively). Mice died between 4-112 h (median = 18 h) after consuming one or more toxic grains. Independent analysis of ZnP-coated grains showed variable toxin loading indicating that consumption of a single grain would not guarantee intake of a lethal dose. There was also a strong and rapid behavioural aversion if mice did not consume a lethal dose on the first night.

CONCLUSIONSThe registered dose rate of 25 g ZnP/kg wheat; [~] 1 mg ZnP/grain in Australia needs to be re-evaluated to determine what factors may be contributing to variation in efficacy. Further field research is also required to understand the complex association between ZnP dose, and quantity and quality of background food on efficacy of ZnP baits.


title: Natural history of a mouse model of X-linked myotubular myopathy

abstract: X-linked myotubular myopathy is a severe monogenetic disorder of the skeletal muscle caused by loss of expression/function mutations in the MTM1 (myotubularin) gene. There is a growing understanding of the pathologic and molecular abnormalities associated with loss of MTM1, and emerging therapeutic strategies that are in the process of translation to patients. Much of these data have been uncovered through experimentation in pre-clinical animal models of the disease. The most widely used model is an Mtm1 gene knockout mouse line; this line faithfully recapitulates the salient genetic and pathologic features of the disease. Despite the advances in aspects of XLMTM, there remain many unknowns related to disease pathomechanisms and to understanding of MTM1s function in normal muscle development, and a continued need for therapy identification and development. To address these barriers, and to lay the groundwork for future study, we performed a natural history study of the Mtm1 knockout mouse model of XLMTM. We show that certain molecular and pathologic changes precede overt phenotypic changes, while others, including abnormalities in triad structure, occur more coincident with muscle weakness in the mouse. In total, we provide a comprehensive longitudinal assessment of molecular and structural features of the murine XLMTM disease process.


title: Draft genomes of a male and female Australian jacky dragon (Amphibolurus muricatus)

abstract: Australia is remarkable for its lizard diversity, with very high endemicity because of continental-scale diversification and adaptive radiation. We employed 10X Genomics Chromium linked-reads technology to generate male and female draft genomes of the jacky dragon (Amphibolurus muricatus), an Australian dragon lizard (family Agamidae). The assemblies are 1.8 Gb in size and have a repeat content (38%) and GC content (42%) similar to other dragon lizards. The contig N50 values for the assemblies were 37.2 kb (female) and 28.8 kb (male), with corresponding scaffold N50 values of 720.5 kb and 369 kb. The longest scaffold was 6.5 Mb in each assembly. The BUSCO completeness percentages were 92.2% and 90.8% respectively. These statistics are comparable to other lizard genomes assembled using similar technology. Phylogenetic comparisons show that Australian dragon lizard species split from a common ancestor around 33.4 million years ago. The draft A. muricatus assemblies will be a valuable resource for understanding lizard sex determination and the evolution and conservation of Australian dragon lizards.

category: genomics


title: Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity

abstract: Infection from the opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments and lysosomal degradation. ExoU is a potent phospholipase which causes rapid destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitro than other antimicrobial classes. However, their overuse over the past decade has caused alarming rates of antibiotic resistance to emerge. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered tobramycin disrupted T3SS expression and inhibited both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-T cells even at concentrations below the minimal inhibitory concentrations (MIC). Fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and in particular did not subvert the cytotoxic effects of ExoS and ExoU.