Robust and efficient online auditory psychophysics with the right auditory hygiene Most human auditory psychophysics research has historically been conducted in carefully controlled environments with calibrated audio-equipment, and over potentially hours of repetitive testing with expert listeners. Here, we operationally define such conditions as having high  auditory hygiene. Hereof, conducting auditory psychophysical paradigms online presents a serious challenge, in that results may hinge on absolute sound presentation level, reliably estimated perceptual thresholds, and sustained motivation and attention. We introduce a set of procedures that address these challenges and facilitate auditory hygiene for online auditory psychophysics. First, we establish a simple means of setting sound presentation levels. Across a set of four level-setting conditions conducted in person, we demonstrate the stability and robustness of this amplitude setting procedure in open air and controlled settings. Second, we test participants tone-in-noise thresholds using widely adopted online experiment platforms and demonstrate that reliable threshold estimates can be derived online in approximately one minute of testing. Third, using these amplitude and threshold setting procedures to establish participant-specific stimulus conditions, we show that an online implementation of the classic probe-signal paradigm can be used to demonstrate frequency-selective attention on an individual participant basis, using a third of the trials used in recent in-lab experiments. Finally, we show how threshold and attentional measures relate to well-validated assays of online participants in-task motivation, fatigue, and confidence. This demonstrates the promise of online auditory psychophysics for asking new auditory neuroscience questions quickly, efficiently, and with more diverse samples. Code for the tests is publicly available through Pavlovia and Gorilla.
The Impact of Neuron Morphology on Cortical Network Architecture It has become increasingly clear that the neurons in the cerebral cortex are not randomly interconnected. This wiring specificity can result from synapse formation mechanisms that interconnect neurons depending on their activity or genetically defined identity. Here we report that in addition to these synapse formation mechanisms, the structural composition of the neuropil provides a third prominent source by which wiring specificity emerges in cortical networks. This structurally determined wiring specificity reflects the packing density, morphological diversity and similarity of the dendritic and axonal processes. The higher these three factors are, the more recurrent the topology of the networks. Conversely, low density, diversity and similarity yield feedforward networks. These principles predict connectivity patterns from subcellular to network scales that are remarkably consistent with empirical observations from a rich body of literature. Thus, cortical network architectures reflect the specific morphological properties of their constituents to much larger degrees than previously thought.
Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Behavioral and postural analyses establish sleep-like states for mosquitoes that can impact host landing and blood feeding Sleep is an evolutionarily conserved process that has been described in different animal systems. For insects, sleep characterization has been primarily achieved using behavioral and electrophysiological correlates in a few systems. Sleep in mosquitoes, which are important vectors of disease-causing pathogens, has not been directly examined. This is surprising as circadian rhythms, which have been well studied in mosquitoes, influence sleep in other systems. In this study, we characterized sleep in mosquitoes using body posture analysis and behavioral correlates, and quantified the effect of sleep deprivation on sleep rebound, host landing and blood-feeding propensity. Body and appendage position metrics revealed a clear distinction between the posture of mosquitoes in their putative sleep and awake states for multiple species, which correlate with a reduction in responsiveness to host cues. Sleep assessment informed by these posture analyses indicated significantly more sleep during periods of low activity. Nighttime and daytime sleep deprivation resulting from the delivery of vibration stimuli induced sleep rebound in the subsequent phase in day and night active mosquitoes, respectively. Lastly, sleep deprivation suppressed host landing in both laboratory and field settings and also impaired blood feeding of a human host when mosquitoes would normally be active. These results suggest that quantifiable sleep states occur in mosquitoes, and highlight the potential epidemiological importance of mosquito sleep. Genomic inference of a human super bottleneck in Mid-Pleistocene transition The demographic history is a foundation of human evolutionary studies. However, the ancient demographic history during the Mid-Pleistocene is poorly investigated while it is essential for understanding the early origin of humankind. Here we present the fast infinitesimal time coalescent (FitCoal) process, which allows the analytical calculation of the composite likelihood of a site frequency spectrum and provides the precise inference of demographic history. We apply it to analyze 3,154 present-day human genomic sequences. We find that African populations have passed through a population super bottleneck, a small effective size of approximately 1,280 breeding individuals between 930 and 813 thousand years ago. Further analyses confirm the existence of the super bottleneck on non-African populations although it cannot be directly inferred. This observation, together with simulation results, indicates that confounding factors, such as population structure and selection, are unlikely to affect the inference of the super bottleneck. The time interval of the super bottleneck coincides with a gap in the human fossil record in Africa and possibly marks the origin of Homo heidelbergensis. Our results provide new insights into human evolution during the Mid-Pleistocene. Live Analysis of Position Effect Variegation (PEV) in Drosophila Reveals Different Modes of Action for HP1a and Su(var)3-9 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. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. IRI-CCE: A Platform for Evaluating CRISPR-based Base Editing Tools and Its Components Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs. The interplay of chromatin phase separation and lamina interactions in nuclear organization The genetic material of eukaryotes is segregated into transcriptionally active euchromatin and silent heterochromatin compartments. The spatial arrangement of chromatin compartments evolves over the course of cellular life in a process that remains poorly understood. The latest nuclear imaging experiments reveal a number of dynamical signatures of chromatin which are reminiscent of active multi-phase liquids. This includes the observations of viscoelastic response, coherent motions, Ostwald ripening and coalescence of chromatin compartments. There is also growing evidence that liquid-liquid phase separation of protein and nucleic acid components is the underlying mechanism for the dynamical behavior of chromatin. In order to dissect the organizational and dynamical implications of chromatins liquid behavior, we have devised a phenomenological field-theoretic model of nucleus as a multi-phase condensate of liquid chromatin types. Employing the liquid chromatin model of Drosophila nucleus, we have carried out an extensive set of simulations with an objective to shed light on the dynamics and chromatin patterning observed in the latest nuclear imaging experiments. Our simulations reveal the emergence of experimentally detected mesoscale chromatin channels and spheroidal droplets which arise from the dynamic interplay of chromatin type to type interactions and intermingling of chromosomal territories. We also quantitatively reproduce coherent motions of chromatin domains observed in displacement correlation spectroscopy measurements which are explained within the framework of our model by phase separation of chromatin types operating within constrained intra-chromosomal and inter-chromosomal boundaries. Finally, we illuminate the role of heterochromatin-lamina interactions in the nuclear organization by showing that these interactions enhance the mobility of euchromatin and indirectly introduce correlated motions of heterochromatin droplets.

SIGNIFICANCE STATEMENTThe latest super-resolution imaging experiments have revealed a surprisingly dynamic and stochastic nature of chromatin in eukaryotic nuclei which is reminiscent of multi-phase fluid behavior. As a result, the concepts from the theory of complex fluids such as phase separation, viscoelasticity, and droplet nucleation have found widespread utility in understanding salient features of nuclear organization. In order to understand and disentangle the complex interplay of forces that contribute to the emergent patterns of organization and dynamics, we have devised a phenomenological field-theoretic model of nucleus as a multi-phase condensate of liquid chromatin types. Armed with a mesoscopic model of nuclear chromatin, we have shed light on the distinct dynamical and structural contributions of chromatin type interactions intermingling of chromosomal territories and lamina binding. We also shed light on the dynamical heterogeneity and coherent motions of chromatin domains which are fully captured by an interplay of micro-phase separation of chromatin types and lamina binding. Effect of tag attachment on the flight performance of five raptor species Bio-logging devices play a fundamental and indispensable role in movement ecology studies, particularly in the wild. However, researchers are becoming increasingly aware of the influence that attaching devices can have on animals, particularly on their behaviour, energy expenditure and survival. The way a device is attached to an animals body has also potential consequences for the collected data, and quantifying the type and magnitude of such potential effects is fundamental to enable researchers to combine and compare data from different studies, as much as it is to improve animal welfare.

For over two decades, large terrestrial birds have been in the focus of long-term movement ecology research, employing bio-logging devices attached with different types of harnesses. However, comparative studies investigating the effects of different harness types used on these species are scarce.

In this study, we tested for potential differences in data collected by two commonly used harness types, backpack and leg-loop, on the flight performance of 10 individuals from five raptor species, equipped with high resolution bio-logging devices, in the same area and time. We explored the effect of harness type on vertical speed, horizontal speed, glide ratio, height above sea level, distance travelled, proportion of soaring and flapping behaviour, and VeDBA (a proxy for energy expenditure) between and within individuals, all used as fine-scale measures of flight performance. Birds equipped with leg-loops climbed up to 0.65 ms-1 faster, reached 19% greater heights while soaring, and spent less time in active flight compared to birds equipped with backpacks, suggesting that backpack harnesses, compared to leg-loops, might cause additional drag affecting the birds flight performance. A lower rate of sinking while gliding, a slightly higher glide ratio, higher horizontal speed while soaring, and lower VeDBA, were also indicative of less drag using leg-loops. Our results add to the existing literature highlighting the design-related advantages of leg-loops, and support the use of leg-loops as a better alternative to backpack harnesses for large soaring birds, when possible. Our study also highlights how apparently small changes in device attachment can lead to notable improvements in tagging practice, with implications for animal welfare, data interpretation and comparability. Regulatory perturbations of ribosome allocation optimize the growth proteome with a trade-off in adaptation capacity Bacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655 in glucose minimal medium. Mutants with only 3 of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Higher growth rates due to increased ribosome content affected resource allocation. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) reshape the proteome for growth with a concomitant fitness cost

HighlightsMutants with only 3 ribosomal operons grow faster than wild-type in minimal medium

 {Delta}4 rrn and rpoB mutants share phenotypic traits

Faster growth of mutants is achieved by increased ribosome content

Fast-growing mutants display reduced hedging expression and adaptation trade-offs

Despite similar ribosomal content in rich medium the mutants present growth defects Cluster-specific gene markers enhance Shigella and Enteroinvasive Escherichia coli in silico serotyping Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. The ability to differentiate Shigella and EIEC from each other is important for clinical diagnostic and epidemiologic investigations. The existing genetic signatures may not discriminate between Shigella and EIEC. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli. In this study, we identified 10 Shigella clusters, 7 EIEC clusters and 53 sporadic types of EIEC by examining over 17,000 publicly available Shigella/EIEC genomes. We compared Shigella and EIEC accessory genomes to identify the cluster-specific gene markers or marker sets for the 17 clusters and 53 sporadic types. The gene markers showed 99.63% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella/EIEC serotype specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70% and 99.81% cluster assignment accuracy for the assembled genomes and mapped reads respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity with 99.40% for assembled genomes and 99.38% for mapped reads for serotyping. The cluster markers and our new serotyping tool, ShigEiFinder (https://github.com/LanLab/ShigEiFinder), will be useful for epidemiologic and diagnostic investigations.

Impact statementThe differentiation of Shigella strains from enteroinvasive E. coli (EIEC) is important for clinical diagnosis and public health epidemiologic investigations. The similarities between Shigella and EIEC strains make this differentiation very difficult as both share common ancestries within E. coli. However, Shigella and EIEC are phylogenetically separated into multiple clusters, making high resolution separation using cluster specific genomic markers possible. In this study, we identified 17 Shigella or EIEC clusters including five that were newly identified through examination of over 17,000 publicly available Shigella and EIEC genomes. We further identified an individual or a set of cluster-specific gene markers for each cluster using comparative genomic analysis. These markers can then be used to classify isolates into clusters and were used to develop an in silico pipeline, ShigEiFinder (https://github.com/LanLab/ShigEiFinder) for accurate differentiation, cluster typing and serotyping of Shigella and EIEC from Illumina sequencing reads or assembled genomes. This study will have broad application from understanding the evolution of Shigella/EIEC to diagnosis and epidemiology.

Data summarySequencing data have been deposited at the National Center for Biotechnology Information under BioProject number PRJNA692536.

RepositoriesRaw sequence data are available from NCBI under the BioProject number PRJNA692536. Strength of interactions in the Notch gene regulatory network determines patterning and fate in the notochord Development of multicellular organisms requires the generation of gene expression patterns that determines cell fate and organ shape. Groups of genetic interactions known as Gene Regulatory Networks (GRNs) play a key role in the generation of such patterns. However, how the topology and parameters of GRNs determine patterning in vivo remains unclear due to the complexity of most experimental systems. To address this, we use the zebrafish notochord, an organ where coin-shaped precursor cells are initially arranged in a simple unidimensional geometry. These cells then differentiate into vacuolated and sheath cells. Using newly developed transgenic tools together with in vivo imaging, we identify jag1a and her6/her9 as the main components of a Notch GRN that generates a lateral inhibition pattern and determines cell fate. Making use of this experimental system and mathematical modeling we show that lateral inhibition patterning requires that ligand-receptor interactions are stronger within the same cell than in neighboring cells. Altogether, we establish the zebrafish notochord as an experimental system to study pattern generation, and identify and characterize how the properties of GRNs determine self-organization of gene patterning and cell fate. Adapting to extremes: reconstructing evolution in response to changing climate over time and space in the diverse Australian plant genus Acacia AimMacroevolutionary analysis is increasingly being used to study biodiversity responses to climate change, for example by using phylogenetic node ages to infer periods of diversification, or phylogenetic reconstruction of traits to infer adaptation to particular stresses. Here we apply a recently developed macroevolutionary method to investigate the responses of a diverse plant genus, Acacia, to increasing aridity and salinity in Australia from the Miocene to the present. We ask whether increase in tolerance of aridity and salinity coincided with periods of aridification, and if it allowed the radiation of Acacia into a wide range of niches.

TaxonAcacia

LocationAustralia

MethodsWe applied the Niche Evolution Model (NEMo), which combines Environmental (or Ecological) Niche Modelling (ENM) with phylogenetic comparative methods (PCM) in a single statistical framework, to a large database of Acacia presence-only records and presence-absence survey sites in order to infer current environmental tolerances of Australia Acacia species and reconstruct the evolution of environmental tolerance to increasing aridity and salinity.

ResultsWe find that patterns in evolution of Acacia, over time and across different habitat types, are consistent with the aridification history of Australia and suggests substantial ability to adapt to high aridity and salinity.

Main conclusionsOur results suggest that many Acacia lineages have been able to exploit new environments created during the aridification of Australia through evolution of environmental tolerance, resulting in their current dominance of many habitats across the continent. This study demonstrates that phylogenetic studies of the evolution of responses to changing environment can move beyond application of simple trait-based models, allowing the underlying processes of speciation, adaptation and dispersal to be explicitly modelled in a macroecological and macroevolutionary context.

Statement of significanceAcacia species are found throughout Australia, from rainforests to deserts, and are striking in their environmental adaptability, so they are a perfect case study for understanding evolution of tolerance to environmental extremes in a changing climate. We use the largest database of spatial distribution records yet assembled, using both surveys and atlas data, and a new analytical method that combines the strengths of environmental niche modelling with phylogenetic comparative methods, to demonstrate rapid evolution in aridity and salinity tolerance in response to aridification of the Australian continent during the Neogene and Quaternary. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI Erythropoietin supplementation ameliorates the immunological and hematological deficiencies of lysinuric protein intolerance in mice SLC7A7 encodes for y+LAT1, a cationic amino acid transporter expressed on the basolateral membrane of epithelial cells and macrophages. Mutations in human SLC7A7 cause lysinuric protein intolerance (LPI), a rare autosomal disorder with a variety of clinical symptoms. Citrulline and a low protein diet are widely used to treat the main symptoms of LPI; however, the hematological and immunological abnormalities in LPI remain poorly understood and difficult to manage by established treatments. Here, we show that erythropoietin levels are significantly diminished in mice lacking Slc7a7, which reduces the number of erythroblast precursors, resulting in mature red blood cells with a smaller mean corpuscular volume. Ex vivo analysis demonstrated that erythrocytes from Slc7a7-deficient mice were phagocytosed more by bone marrow-derived macrophages. Gene expression analysis of sorted red pulp macrophages (RPMs) from Slc7a7-deficient mice indicated that this cell population had higher erythrophagocytic activity, but preserved heme catabolic activity. The increase in erythrophagocytosis resulted in iron accumulation in macrophages and tissues, and ultimately led to RPM apoptosis. Surprisingly, ablation of Slc7a7 in myeloid cells failed to trigger tissue iron accumulation or changes in the RPM population, indicating that the expression of this gene in macrophages is not key for the development of iron accumulation in tissue. Finally, restoration of serum erythropoietin levels in Slc7a7-deficient mice re-established normal erythropoiesis and erythrophagocytosis and prevented the accumulation of iron in tissues. Thus, erythropoietin has a fundamental role in the immune-hematological alterations of LPI in mice, revealing a new crucial role for this hormone in this disease. Spatially explicit ecological modeling improves empirical characterization of dispersal Dispersal is a key ecological process, but remains difficult to measure. By recording numbers of dispersed individuals at different distances from the source one can acquire a dispersal gradient. Although dispersal gradients contain information on dispersal, they are influenced by the spatial extent of the source. How can we separate the two contributions to extract knowledge on dispersal?

One could use a small, point-like source for which a dispersal gradient represents a dispersal kernel, which quantifies the probability of an individual dispersal event from a source to a destination point. However, the validity of this approximation cannot be established before conducting measurements.

We formulated a theory that incorporates the spatial extent of sources to estimate dispersal kernels from dispersal gradients. We re-analyzed published dispersal gradients for three major plant pathogens. We also demonstrated using simulations that this approach provides more accurate estimates of dispersal kernels across biologically plausible scenarios. We concluded that the three plant pathogens disperse over substantially shorter distances compared to conventional estimates. Using this method, a significant proportion of published dispersal gradients can be re-analyzed to improve our knowledge about spatial scales of dispersal. Thus, our results can boost progress in characterization of dispersal across taxa. Main protease mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir (PF-07321332) The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics showed that these Mpro variants are similarly catalytically competent as the wildtype. We show that nirmatrelvir has similar potency against the variants as against the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants.

Graphical abstract

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org.highwire.dtl.DTLVardef@182cc6org.highwire.dtl.DTLVardef@1239d10org.highwire.dtl.DTLVardef@11ca6d9org.highwire.dtl.DTLVardef@df53b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Feline tooth resorption findings from a questionnaire survey on 8115 Finnish cats and a case-control study for a more defined population drawn from the survey Tooth resorption (TR) is one of the most common dental diseases in cats. It is a painful disease characterised by progressive dental destruction, which eventually results in loss of teeth. The aetiology of the disease remains unclear, but associations with old age, breed, other oral and dental diseases, and certain environmental factors have been suspected. We collected health and environmental information of 8115 Finnish cats from 41 breeds through an online survey for cat owners to study TR in Finland with an observational study. The frequency of TR that the owner reported as veterinarian-diagnosed was 3.9% in the entire data (316/8115) and 15% in cats that the owner reported as having veterinarian-diagnosed oral or dental disease (316/2070). We determined the risk factors and breed variation of feline TR by logistic regression with the entire data and a case-control study in a more defined population drawn from the entire data. In the entire data, we defined those cats that the owners had reported as being diagnosed with TR by a veterinarian as TR-cats and compared them to all other cats. In the case-control study, we included only cats with owner-reported veterinarian-diagnosed oral and dental diseases. We defined cats with TR-diagnosis made under sedation as TR-cases. Other veterinarian-diagnosed TR-cats were excluded. Cats with other oral and dental diseases were used as controls. The model with the entire population included 308 TR-cats compared with 7508 cats, and the case-control study included 198 TR-cats and 1683 controls. Although the diagnoses were reportedly made by a veterinarian, the owner answered the survey and might have understood, remembered, or interpreted the diagnoses in a way that could affect the results. The results indicated an increasing risk of TR with age. Dental calculus, gingivitis, and periodontitis were potential risk factors for TR. These findings and the interaction between dental calculus and gingivitis suggest that inflammatory changes caused by dental calculus increase the risk of TR. Keeping food available constantly was a potential protective factor. We found that Cornish Rex, European, and Ragdoll breeds were at higher risk for TR. The observed differences between breeds highlight a genetic contribution. Simpati: patient classifier identifies signature pathways based on similarity networks for the disease prediction BACKGROUNDPathway-based patient classification is a supervised learning task which implies a model learning pathways as features to predict the classes of patients. The counterpart of enrichment tools for the pathway analysis are fundamental methods for clinicians and biomedical scientists. They allow to find signature cellular functions which help to define and annotate a disease phenotype. They provide results which lead human experts to manually classify patients. It is a paradox that pathwaybased classifiers which natively resolve this objective are not strongly developed. They could simulate the human way of thinking, decipher hidden multivariate relationships between the deregulated pathways and the disease phenotype, and provide more information than a probability value. Instead, there are currently only two classifiers of such kind, they require a nontrivial hyperparameter tuning, are difficult to interpret and lack in providing new insights. There is the need of new classifiers which can provide novel perspectives about pathways, be easy to apply with different biological omics and produce new data enabling a further analysis of the patients.

RESULTSWe propose Simpati, an innovative and interpretable patient classifier based on pathway-specific patient similarity networks. The first classifier to adopt ad-hoc novel algorithms for such graph type. It standardizes the biological high-throughput dataset of patients profiles with a propagation algorithm that considers the interconnected nature of the cells molecules for inferring a new activity score. This allows Simpati to classify with dense, sparse, and non-homogenous omic data. Simpati organizes patients molecules in pathways represented by patient similarity networks for being interpretable, handling missing data and preserving the patient privacy. A network represents patients as nodes and a novel similarity measure determines how much every pair act co-ordinately in a pathway. Simpati detects signature biological processes based on how much the topological properties of the related networks separate the patient classes. In this step, it includes a new cohesive subgroup detection algorithm to handle patients not showing the same pathway activity as the other class members. An unknown patient is then classified by a unique recommender system which considers how much is similar to known patients and distant from being an outlier. Simpati outperforms previously published classifiers on five cancer datasets described with two biological omics, classifies well with sparse data, identifies more relevant pathways associated to the patients disease than the competitors and has the lowest computational requirements.

CONCLUSIONSimpati can serve as generic-purpose pathway-based classifier of patient classes. It provides signature pathways to unveil the altered biological mechanisms of a disease phenotype and to classify patients according to the learnt pathway-specific similarities. The signature condition and patient prediction can be deciphered considering the patient similarity networks which must reveal the members of a patient class more cohesive and similar than the non-members. Simpati divides the pathways in up and downinvolved. Upinvolved when the signaling cascades generated by the altered molecules of the disease patients impact stronger the pathway than the ones of the control class. We provide an R implementation, a graphical user interface and a visualization function for the patient similarity networks. The software is available at: https://github.com/LucaGiudice/Simpati Nutrient copper signaling promotes protein turnover by allosteric activation of ubiquitin E2D conjugases Nutrient copper supply is critical for cell growth and differentiation, and its disturbance is associated with major pathologies including cancer and neurodegeneration. Although increasing copper bioavailability in late Precambrian facilitated emergence of novel cuproproteins, their intricate regulation by this essential trace element remains largely cryptic. We found that subtle rises in cellular copper strikingly increase polyubiquitination and accelerate protein degradation within 30 minutes in numerous mammalian cell lines. We track this surprising observation to allostery induced in the UBE2D ubiquitin conjugase clade through a conserved CXXXC sub-femtomolar-affinity Cu+ binding motif. Thus, physiologic fluctuation in cytoplasmic Cu+ is coupled to the prompt degradation of UBE2D protein targets, including p53. In Drosophila harboring a larval-lethal knockdown of the nearly identical fly orthologue UbcD1, complementation with human UBE2D2 restored near-normal development, but mutation of its CXXXC Cu+ binding motif profoundly disrupted organogenesis. Nutrient Cu+ emerges as a trophic allosteric modulator of UBE2D activity through a structural motif whose evolution coincides with animal multicellularity.

One Sentence SummaryModulation of nutrient copper impacts protein turnover and animal morphogenesis through conserved allostery of ubiquitin E2D conjugases.

HilightsO_LINutrient copper supply is critical for cell growth and differentiation
C_LIO_LIThe E2D clade of ubiquitin conjugases contains a sub-femtomolar-affinity Cu+ binding motif
C_LIO_LIAllosteric activation by Cu+ markedly accelerates protein polyubiquitination
C_LIO_LIThis sensor couples physiologic fluctuations in cytoplasmic Cu+ with the degradation rate of E2D targets, including p53
C_LIO_LIThis metazoan signaling mechanism is critical for drosophila morphogenesis
C_LI

In BriefConserved allostery of ubiquitin E2D conjugases links nutrient copper signaling to protein degradation and animal morphogenesis.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/431211v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@17009fforg.highwire.dtl.DTLVardef@a07f8eorg.highwire.dtl.DTLVardef@11664c7org.highwire.dtl.DTLVardef@1327ab7_HPS_FORMAT_FIGEXP  M_FIG Graphical Abstract

C_FIG STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The cGAS-STING cytoplasmic double stranded DNA (dsDNA) sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties.

Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils and NK populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants the further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, CAR T cells, NK therapies or immune checkpoint blockade.

Significance statementModulation of the immune microenvironment is critical for immunosuppressive and therapy refractory tumors like glioblastoma. Activation of the STING pathway deeply remodels the brain tumor environment and attracts innate immune cells and natural killer cell populations, producing a robust antitumor effect with long-term immune memory. We further show that human glioblastoma tissue can respond to the therapy and lay the foundations for combined intracranial immunotherapies by using crosslinked biodegradable brain implants. Naming of human diseases on the wrong side of history BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Severe COVID-19 shares a common neutrophil activation signature with other acute inflammatory states Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaky disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflamatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention. Compendium of secondary metabolite biosynthetic diversity encoded in bacterial genomes Bacterial specialized metabolites are a proven source of antibiotics and cancer therapies, but whether we have sampled all the secondary metabolite chemical diversity of cultivated bacteria is not known. We analysed ~ 170,000 bacterial genomes and ~ 47,000 metagenome assembled genomes (MAGs) using a modified BiG-SLiCE and the new clust-o-matic algorithm. We found that only 3% of the natural products potentially encoded in bacterial genomes have been experimentally characterized. We show that the variation of secondary metabolite biosynthetic diversity drops significantly at the genus level, identifying it as an appropriate taxonomic rank for comparison. Equal comparison of genera based on Relative Evolutionary Distance revealed that Streptomyces bacteria encode the largest biosynthetic diversity by far, with Amycolatopsis, Kutzneria and Micromonospora also encoding substantial diversity. Finally we find that several less-well-studied taxa, such as Weeksellaceae (Bacteroidota), Myxococcaceae (Myxococcota), Pleurocapsa and Nostocaceae (Cyanobacteria), have potential to produce highly diverse sets of secondary metabolites that warrant further investigation.