Exploring the transcriptomic profile of human monkeypox virus via CAGE and native RNA sequencing approaches.

In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

Spatiotemporal transcriptomic mapping of regenerative inflammation in skeletal muscle reveals a dynamic multilayered tissue architecture.

Tissue regeneration is orchestrated by macrophages that clear damaged cells and promote regenerative inflammation. How macrophages spatially adapt and diversify their functions to support the architectural requirements of actively regenerating tissue remains unknown. In this study, we reconstructed the dynamic trajectories of myeloid cells isolated from acutely injured and early stage dystrophic muscles. We identified divergent subsets of monocytes/macrophages and DCs and validated markers (e.g., glycoprotein NMB [GPNMB]) and transcriptional regulators associated with defined functional states. In dystrophic muscle, specialized repair-associated subsets exhibited distinct macrophage diversity and reduced DC heterogeneity. Integrating spatial transcriptomics analyses with immunofluorescence uncovered the ordered distribution of subpopulations and multilayered regenerative inflammation zones (RIZs) where distinct macrophage subsets are organized in functional zones around damaged myofibers supporting all phases of regeneration. Importantly, intermittent glucocorticoid treatment disrupted the RIZs. Our findings suggest that macrophage subtypes mediated the development of the highly ordered architecture of regenerative tissues, unveiling the principles of the structured yet dynamic nature of regenerative inflammation supporting effective tissue repair.

High-resolution computed tomographic (HRCT) image series from 413 canid and 18 felid skulls.

Computed tomography (CT) is a non-invasive, three-dimensional imaging tool used in medical imaging, forensic science, industry and engineering, anthropology, and archaeology. The current study used high-resolution medical CT scanning of 431 animal skulls, including 399 dog skulls from 152 breeds, 14 cat skulls from 9 breeds, 14 skulls from 8 wild canid species (gray wolf, golden jackal, coyote, maned wolf, bush dog, red fox, Fennec fox, bat-eared fox), and 4 skulls from 4 wild felid species (wildcat, leopard, serval, caracal). This comprehensive and unique collection of CT image series of skulls can provide a solid foundation not only for comparative anatomical and evolutionary studies but also for the advancement of veterinary education, virtual surgery planning, and the facilitation of training in sophisticated machine learning methodologies.

Nonstereotypical Distribution and Effect of Ergosterol in Lipid Membranes.

Ergosterol, found in fungi and some protist membranes, is understudied compared with cholesterol from animal membranes. Generally, ergosterol is assumed to modulate membranes in the same manner as cholesterol, based on their similar chemical structures. Here we reveal some fundamental structural and dynamical differences between them. Neutron diffraction shows that ergosterol is embedded in the lipid bilayer much shallower than cholesterol. Ergosterol does not change the membrane thickness as much as cholesterol does, indicating little condensation effect. Neutron spin echo shows that ergosterol can rigidify and soften membranes at different concentrations. The lateral lipid diffusion measured by quasielastic neutron scattering indicates that ergosterol promotes a jump diffusion of the lipid, whereas cholesterol keeps the same continuous lateral diffusion as the pure lipid membrane. Our results point to quite distinct interactions of ergosterol with membranes compared with cholesterol. These insights provide a basic understanding of membranes containing ergosterol with implications for phenomena such as lipid rafts and drug interactions.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

The effect of environmental variation on the relationship between survival and risk-taking behaviour in a migratory songbird.

Temporal changes in environmental conditions may play a major role in the year-to-year variation in fitness consequences of behaviours. Identifying environmental drivers of such variation is crucial to understand the evolutionary trajectories of behaviours in natural contexts. However, our understanding of how environmental variation influences behaviours in the wild remains limited. Using data collected over 14 breeding seasons from a collared flycatcher (Ficedula albicollis) population, we examined the effect of environmental variation on the relationship between survival and risk-taking behaviour, a highly variable behavioural trait with great evolutionary and ecological significance. Specifically, using annual recapture probability as a proxy of survival, we evaluated the specific effect of predation pressure, food availability, and mean temperature on the relationship between annual recapture probability and risk-taking behaviour (measured as flight initiation distance [FID]). We found a negative trend, as the relationship between annual recapture probability and FID decreased over the study years and changed from positive to negative. Specifically, in the early years of the study, risk-avoiding individuals exhibited a higher annual recapture probability, whereas in the later years, risk-avoiders had a lower annual recapture probability. However, we did not find evidence that any of the considered environmental factors mediated the variation in the relationship between survival and risk-taking behaviour.

Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

Structure and function of Semaphorin-5A glycosaminoglycan interactions.

Integration of extracellular signals by neurons is pivotal for brain development, plasticity, and repair. Axon guidance relies on receptor-ligand interactions crosstalking with extracellular matrix components. Semaphorin-5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects on neuronal growth through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for GAG binding, however the underlying molecular basis and functions in vivo remain enigmatic. Here we dissect the structural basis for Sema5A:GAG specificity and demonstrate the functional significance of this interaction in vivo. Using x-ray crystallography, we reveal a dimeric fold variation for TSR4 that accommodates GAG interactions. TSR4 co-crystal structures identify binding residues validated by site-directed mutagenesis. In vitro and cell-based assays uncover specific GAG epitopes necessary for TSR association. We demonstrate that HS-GAG binding is preferred over CS-GAG and mediates Sema5A oligomerization. In vivo, Sema5A:GAG interactions are necessary for Sema5A function and regulate Plexin-A2 dependent dentate progenitor cell migration. Our study rationalizes Sema5A associated developmental and neurological disorders and provides mechanistic insights into how multifaceted guidance functions of a single transmembrane cue are regulated by proteoglycans.

Responses in the breeding parameters of the collared flycatcher to the changing climate.

Global climate change involves various aspects of climate, including precipitation changes and declining surface wind speeds, but studies investigating biological responses have often focused on the impacts of rising temperatures. Additionally, related long-term studies on bird reproduction tend to concentrate on breeding onset, even though other aspects of breeding could also be sensitive to the diverse weather aspects. This study aimed to explore how multiple aspects of breeding (breeding onset, hatching delay, breeding season length, clutch size, fledgling number) were associated with different weather components. We used an almost four-decade-long dataset to investigate the various aspects of breeding parameters of a collared flycatcher (Ficedula albicollis) population in the Carpathian Basin. Analyses revealed some considerable associations, for example, breeding seasons lengthened with the amount of daily precipitation, and clutch size increased with the number of cool days. Parallel and opposing changes in the correlated pairs of breeding and weather parameters were also observed. The phenological mismatch between prey availability and breeding time slightly increased, and fledgling number strongly decreased with increasing mistiming. Our results highlighted the intricate interplay between climate change and the reproductive patterns of migratory birds, emphasizing the need for a holistic approach. The results also underscored the potential threats posed by climate change to bird populations and the importance of adaptive responses to changing environmental conditions.

The estimation of additive genetic variance of body size in a wild passerine is sensitive to the method used to estimate relatedness among the individuals.

Assessing additive genetic variance is a crucial step in predicting the evolutionary response of a target trait. However, the estimated genetic variance may be sensitive to the methodology used, e.g., the way relatedness is assessed among the individuals, especially in wild populations where social pedigrees can be inaccurate. To investigate this possibility, we investigated the additive genetic variance in tarsus length, a major proxy of skeletal body size in birds. The model species was the collared flycatcher (Ficedula albicollis), a socially monogamous but genetically polygamous migratory passerine. We used two relatedness matrices to estimate the genetic variance: (1) based solely on social links and (2) a genetic similarity matrix based on a large array of single-nucleotide polymorphisms (SNPs). Depending on the relatedness matrix considered, we found moderate to high additive genetic variance and heritability estimates for tarsus length. In particular, the heritability estimates were higher when obtained with the genetic similarity matrix instead of the social pedigree. Our results confirm the potential for this crucial trait to respond to selection and highlight methodological concerns when calculating additive genetic variance and heritability in phenotypic traits. We conclude that using a social pedigree instead of a genetic similarity matrix to estimate relatedness among individuals in a genetically polygamous wild population may significantly deflate the estimates of additive genetic variation.

Lineage-determining transcription factor-driven promoters regulate cell type-specific macrophage gene expression.

Mammalian promoters consist of multifarious elements, which make them unique and support the selection of the proper transcript variants required under diverse conditions in distinct cell types. However, their direct DNA-transcription factor (TF) interactions are mostly unidentified. Murine bone marrow-derived macrophages (BMDMs) are a widely used model for studying gene expression regulation. Thus, this model serves as a rich source of various next-generation sequencing data sets, including a large number of TF cistromes. By processing and integrating the available cistromic, epigenomic and transcriptomic data from BMDMs, we characterized the macrophage-specific direct DNA-TF interactions, with a particular emphasis on those specific for promoters. Whilst active promoters are enriched for certain types of typically methylatable elements, more than half of them contain non-methylatable and prototypically promoter-distal elements. In addition, circa 14% of promoters-including that of Csf1r-are composed exclusively of 'distal' elements that provide cell type-specific gene regulation by specialized TFs. Similar to CG-rich promoters, these also contain methylatable CG sites that are demethylated in a significant portion and show high polymerase activity. We conclude that this unusual class of promoters regulates cell type-specific gene expression in macrophages, and such a mechanism might exist in other cell types too.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Microaneurysm detection using high-speed megahertz optical coherence tomography angiography in advanced diabetic retinopathy.

PURPOSE: To compare detection rates of microaneurysms (MAs) on high-speed megahertz optical coherence tomography angiography (MHz-OCTA), fluorescein angiography (FA) and colour fundus photography (CF) in patients with diabetic retinopathy (DR). METHODS: For this exploratory cross-sectional study, MHz-OCTA data were acquired with a swept-source OCT prototype (A-scan rate: 1.7 MHz), and FA and CF imaging was performed using Optos® California. MA count was manually evaluated on en face MHz-OCTA/FA/CF images within an extended ETDRS grid. Detectability of MAs visible on FA images was evaluated on corresponding MHz-OCTA and CF images. MA distribution and leakage were correlated with detectability on OCTA and CF imaging. RESULTS: 47 eyes with severe DR (n = 12) and proliferative DR (n = 35) were included. MHz-OCTA and CF imaging detected on average 56% and 36% of MAs, respectively. MHz-OCTA detection rate was significantly higher than CF (p < 0.01). The combination of MHz-OCTA and CF leads to an increased detection rate of 70%. There was no statistically significant association between leakage and MA detectability on OCTA (p = 0.13). For CF, the odds of detecting leaking MAs were significantly lower than non-leaking MAs (p = 0.012). Using MHz-OCTA, detection of MAs outside the ETDRS grid was less likely than MAs located within the ETDRS grid (outer ring, p < 0.01; inner ring, p = 0.028). No statistically significant difference between rings was observed for CF measurements. CONCLUSIONS: More MAs were detected on MHz-OCTA than on CF imaging. Detection rate was lower for MAs located outside the macular region with MHz-OCTA and for leaking MAs with CF imaging. Combining both non-invasive modalities can improve MA detection.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these are in turn determined by the multiscale structural features within condensates. However, structural characterizations of condensates are challenging, and hence rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and bespoke coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that mimic nucleolar granular components (GCs). We show that facsimiles of GCs are network fluids featuring spatial inhomogeneities across hierarchies of length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights, extracted from a combination of approaches, suggest that condensates formed by multivalent proteins share features with network fluids formed by associative systems such as patchy or hairy colloids.

Identification of herpesvirus transcripts from genomic regions around the replication origins.

Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a 'super regulatory center' in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries.

Domain crossover in the reductase subunit of NADPH-dependent assimilatory sulfite reductase.

NADPH-dependent assimilatory sulfite reductase (SiR) from Escherichia coli performs a six-electron reduction of sulfite to the bioavailable sulfide. SiR is composed of a flavoprotein (SiRFP) reductase subunit and a hemoprotein (SiRHP) oxidase subunit. There is no known high-resolution structure of SiR or SiRFP, thus we do not yet fully understand how the subunits interact to perform their chemistry. Here, we used small-angle neutron scattering to understand the impact of conformationally restricting the highly mobile SiRFP octamer into an electron accepting (closed) or electron donating (open) conformation, showing that SiR remains active, flexible, and asymmetric even with these conformational restrictions. From these scattering data, we model the first solution structure of SiRFP. Further, computational modeling of the N-terminal 52 amino acids that are responsible for SiRFP oligomerization suggests an eight-helical bundle tethers together the SiRFP subunits to form the SiR core. Finally, mass spectrometry analysis of the closed SiRFP variant show that SiRFP is capable of inter-molecular domain crossover, in which the electron donating domain from one polypeptide is able to interact directly with the electron accepting domain of another polypeptide. This structural characterization suggests that SiR performs its high-volume electron transfer through both inter- and intramolecular pathways between SiRFP domains and, thus, cis or trans transfer from reductase to oxidase subunits. Such highly redundant potential for electron transfer makes this system a potential target for designing synthetic enzymes.

Investigating the Effect of Reflectance Tuning on Photocatalytic Dye Degradation with Biotemplated ZnO Photonic Nanoarchitectures Based on Morpho Butterfly Wings.

Photonic nanoarchitectures of butterfly wings can serve as biotemplates to prepare semiconductor thin films of ZnO by atomic layer deposition. The resulting biotemplated ZnO nanoarchitecture preserves the structural and optical properties of the natural system, while it will also have the features of the functional material. The ZnO-coated wings can be used directly in heterogeneous photocatalysis to decompose pollutants dissolved in water upon visible light illumination. We used the photonic nanoarchitectures of different Morpho butterflies with different structural colors as biotemplates and examined the dependence of decomposition rates of methyl orange and rhodamine B dyes on the structural color of the biotemplates and the thickness of the ZnO coating. Using methyl orange, we measured a ten-fold increase in photodegradation rate when the 20 nm ZnO-coated wings were compared to similarly coated glass substrates. Using rhodamine B, a saturating relationship was found between the degradation rate and the thickness of the deposited ZnO on butterfly wings. We concluded that the enhancement of the catalytic efficiency can be attributed to the slow light effect due to a spectral overlap between the ZnO-coated Morpho butterfly wings reflectance with the absorption band of dyes, thus the photocatalytic performance could be changed by the tuning of the structural color of the butterfly biotemplates. The photodegradation mechanism of the dyes was investigated by liquid chromatography-mass spectroscopy.

The transcriptional control of the VEGFA-VEGFR1 (FLT1) axis in alternatively polarized murine and human macrophages.

Introduction: Macrophages significantly contribute to the regulation of vessel formation under physiological and pathological conditions. Although the angiogenesis-regulating role of alternatively polarized macrophages is quite controversial, a growing number of evidence shows that they can participate in the later phases of angiogenesis, including vessel sprouting and remodeling or regression. However, the epigenetic and transcriptional regulatory mechanisms controlling this angiogenesis-modulating program are not fully understood. Results: Here we show that IL-4 can coordinately regulate the VEGFA-VEGFR1 (FLT1) axis via simultaneously inhibiting the proangiogenic Vegfa and inducing the antiangiogenic Flt1 expression in murine bone marrow-derived macrophages, which leads to the attenuated proangiogenic activity of alternatively polarized macrophages. The IL-4-activated STAT6 and IL-4-STAT6 signaling pathway-induced EGR2 transcription factors play a direct role in the transcriptional regulation of the Vegfa-Flt1 axis. We demonstrated that this phenomenon is not restricted to the murine bone marrow-derived macrophages, but can also be observed in different murine tissue-resident macrophages ex vivo and parasites-elicited macrophages in vivo with minor cell type-specific differences. Furthermore, IL-4 exposure can modulate the hypoxic response of genes in both murine and human macrophages leading to a blunted Vegfa/VEGFA and synergistically induced Flt1/FLT1 expression. Discussion: Our findings establish that the IL-4-activated epigenetic and transcriptional program can determine angiogenesis-regulating properties in alternatively polarized macrophages under normoxic and hypoxic conditions.

Evolution of relative brain size in dogs-no effects of selection for breed function, litter size, or longevity.

Domestication is a well-known example of the relaxation of environmentally based cognitive selection that leads to reductions in brain size. However, little is known about how brain size evolves after domestication and whether subsequent directional/artificial selection can compensate for domestication effects. The first animal to be domesticated was the dog, and recent directional breeding generated the extensive phenotypic variation among breeds we observe today. Here we use a novel endocranial dataset based on high-resolution CT scans to estimate brain size in 159 dog breeds and analyze how relative brain size varies across breeds in relation to functional selection, longevity, and litter size. In our analyses, we controlled for potential confounding factors such as common descent, gene flow, body size, and skull shape. We found that dogs have consistently smaller relative brain size than wolves supporting the domestication effect, but breeds that are more distantly related to wolves have relatively larger brains than breeds that are more closely related to wolves. Neither functional category, skull shape, longevity, nor litter size was associated with relative brain size, which implies that selection for performing specific tasks, morphology, and life history does not necessarily influence brain size evolution in domesticated species.