De-noising Spatial Transcriptomics Data Based on Position and Image Information Spatial transcriptomics (ST) technologies provide gene expression close to or even superior to single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, the expression data captured by ST technologies suffer from high noise levels, as a result of the shallow coverage in each sequencing unit. The extra experimental steps for preserving the spatial locations of sequencing could result in even more severe noises, compared to regular single-cell RNA-sequencing (scRNA-seq). Fortunately, such noises could be largely removed by leveraging information from the physical locations of sequencing, and the tissue and cellular organization reflected by corresponding pathology images. In this work, we demonstrated the extensive levels of noise in ST data. We developed a mathematical model, named Sprod, to impute accurate ST gene expression based on latent space and graph learning of matched location and imaging data. We comprehensively validated Sprod and demonstrated its advantages over prior methods for removing drop-outs in scRNA-seq data. We further showed that, after adequate imputation by Sprod, differential expression analyses, pseudotime analyses, and cell-to-cell interaction inferences yield significantly more informative results. Overall, we envision denoising by Sprod to become a key first step to empower ST technologies for biomedical discoveries and innovations.
Fungal Ice2p is in the same superfamily as SERINCs, restriction factors for HIV and other viruses Ice2p is an integral endoplasmic reticulum (ER) membrane protein in budding yeast S. cerevisiae named ICE because it is required for Inheritance of Cortical ER. Ice2p has also been reported to be involved in an ER metabolic branch-point that regulates the flux of lipid either to be stored in lipid droplets or to be used as membrane components. Alternately, Ice2p has been proposed to act as a tether that physically bridges the ER at contact sites with both lipid droplets and the plasma membrane via a long loop on the proteins cytoplasmic face that contains multiple predicted amphipathic helices. Here we carried out a bioinformatic analysis to increase understanding of Ice2p. Firstly, regarding topology, we found that diverse members of the fungal Ice2 family have ten transmembrane helices, which places the long loop on the exofacial face of Ice2p, where it cannot form inter-organelle bridges. Secondly, we identified Ice2 as a full-length homologue of SERINC (serine incorporator), a family of proteins with ten transmembrane helices found universally in eukaryotes. Since SERINCs are potent restriction factors for HIV and other viruses, study of Ice2p may reveal functions or mechanisms that shed light on viral restriction by SERINCs.
Neprosin belongs to a new family of glutamic peptidase family based on in silico evidence Neprosin was first discovered in the insectivorous tropical pitcher plants of Nepenthes species as a novel protease with prolyl endopeptidase (PEP) activity. Neprosin has two uncharacterized domains of neprosin activation peptide and neprosin. A previous study has shown neprosin activity in hydrolyzing proline-rich gliadin, a gluten component that triggers celiac disease. In this study, we performed in silico structure-function analysis to investigate the catalytic mechanism of neprosin. Neprosin sequences lack the catalytic triad and motifs of PEP family S9. Protein structures of neprosins from Nepenthes x ventrata (NvNpr) and N. rafflesiana (NrNpr1) were generated by ab initio methods and comparatively assessed to obtain high-quality models. Structural alignment of models to experimental structures in the Protein Data Bank (PDB) found a high structural similarity to glutamic peptidases. Further investigations reveal other resemblances to the glutamic peptidases with low optimum pH that activates the enzyme via autoproteolysis for maturation. Two highly conserved glutamic acid residues, which are stable according to the molecular dynamics simulation, can be found at the active site of the substrate cleft. Protein docking demonstrated that mature neprosins bind well with potent antigen I-gliadin at the putative active site. Taken together, neprosins represent a new glutamic peptidase family, with a putative catalytic dyad of two glutamic acids. This study illustrates a hypothetical enzymatic mechanism of the neprosin family and demonstrates the useful application of an accurate ab initio protein structure prediction in the structure-function study of a novel protein family. If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context. In vitro evolution of uracil glycosylase towards DnaKJ and GroEL binding evolves different misfolded states Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK+DnaJ+ATP (DnaKJ) or E.coli cell extract (CE). Using binding to the eUNG inhibitor Ugi as probe for native foldedess, the CE selected population was further divided to Ugi binders (+U) (native) or non-binders (-U). We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection, as well as CE+U and natural evolution of eUNG has its own trajectory. While GroEL and CE-U selected for mutants highly sensitive to protease cleavage, DnaKJ selected for partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. CE+U selected for more neutral mutations than natural evolution. In a more general context, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds mutants misfolding-prone species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL and DnaKJ selected populations, while harboring also unique properties, explained by the existence of additional chaperones in CE, such as Tig, HtpG and ClpB. Darwin's small and medium ground finches might have taste preferences, but not for human foods Urbanization is rapidly changing ecological niches. On the inhabited Galapagos Islands, Darwins finches consume human-introduced foods preferentially; however, it remains unclear why. Here we presented pastry with flavour profiles typical of human foods (oily, salty, sweet) to small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis) to test if latent taste preferences might drive selection of human foods. If human-food flavours were consumed more than a neutral or bitter control only at sites with human foods, then we predicted tastes were acquired after urbanization; however, if no site-differences were found then this would indicate latent taste preferences. Contrary to both predictions, we found little evidence that human-food flavours were preferred compared to control flavours at any site. Instead, finches showed a weak aversion to oily foods, but only at remote (no human foods present) sites. This was further supported by behavioural responses, with beak wiping occurring more often at remote sites after finches tasted flavours associated with human foods. Our results suggest, therefore, that while Darwins finches may have acquired a tolerance to the flavours of human food, latent taste preferences are unlikely to have played a major role in their dietary response to increased urbanization. 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. Genetic interactions drive heterogeneity in causal variant effect sizes for gene expression and complex traits Despite the growing number of genome-wide association studies (GWAS), it remains unclear to what extent gene-by-gene and gene-by-environment interactions influence complex traits in humans. The magnitude of genetic interactions in complex traits has been difficult to quantify because GWAS are generally underpowered to detect individual interactions of small effect. Here, we develop a method to test for genetic interactions that aggregates information across all trait-associated loci. Specifically, we test whether SNPs in regions of European ancestry shared between European American and admixed African American individuals have the same causal effect sizes. We hypothesize that in African Americans, the presence of genetic interactions will drive the causal effect sizes of SNPs in regions of European ancestry to be more similar to those of SNPs in regions of African ancestry. We apply our method to two traits: gene expression in 296 African Americans and 482 European Americans in the Multi-Ethnic Study of Atherosclerosis (MESA) and low-density lipoprotein cholesterol (LDL-C) in 74K African Americans and 296K European Americans in the Million Veteran Program (MVP). We find significant evidence for genetic interactions in our analysis of gene expression; for LDL-C, we observe a similar point estimate although this is not significant, likely due to lower statistical power. These results suggest that gene-by-gene or gene-by-environment interactions modify the effect sizes of causal variants in human complex traits. Cellulomonas zeae sp. nov., Lelliottia zeae sp. nov., Paraburkholderia zeae sp. nov. and Sphingomonas zeigerminis sp. nov., four new bacterial species isolated from Zea mays and other hosts Four novel bacterial species collected from healthy tissues of corn, rice and soybean plants in the United States are described. These include Cellulomonas zeae sp. nov. isolated from corn in Indiana; Lelliottia zeae sp. nov. from corn in Indiana and rice in Arkansas; Paraburkholderia zeae sp. nov. isolated from corn in Iowa; and Sphingomonas zeigerminis sp. nov. from corn in Mississippi and soy in Arkansas. No pathogenic strains are known for any of the novel species based on genome comparisons to assemblies in GenBank. Environmental and genetic disease modifiers of haploinsufficiency of A20 Monogenic diseases can often manifest diverse clinical phenotypes and cause diagnostic dilemmas. While monoallelic loss-of-function variants in TNFAIP3 (Haploinsufficiency of A20; HA20) cause a highly penetrant autoinflammatory disease, the variable expressivity suggest a role for additional genetic and environmental disease modifiers. Here, we identify critically ill children who inherited a family-specific TNFAIP3 deletion from one of their otherwise healthy parents. Each of the probands also inherited in trans a subtle loss-of-function I207L TNFAIP3 variant that is common in Oceania, originally introgressed from Denisovans. Modelling this compound heterozgous state in mice under specific pathogen free conditions demonstrated a reduced threshold to break immune tolerance. Exaggerated immune responses were precipitated by inheriting the two genetic hits on the TNFAIP3 checkpoint coupled with increasing the microbial challenge to immune tolerance, either by co-housing with pet store mice carrying a wild microbial burden or by transient dietary exposure to a chemical that diminishes the intestinal mucin barrier separating gut microbes from immune sensing systems. These data illuminate second-hit genetic and environmental modifiers contributing to complex inflammatory and autoimmune disease. Increased mechanistic understanding of the presence and contribution of disease modifiers will aid diagnostic and prognostic patient stratification and potentially reveal novel therapeutic opportunities. Classification, Using Field Potential Images, of Pacemaker Micro-coordination in the Mouse Intestine The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals. Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia Cystic Fibrosis (CF) is caused by genetic defects that impair the cystic fibrosis transmembrane conductance regulator (CFTR) channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described application of 3D airway organoids in CFTR function assays have not been fully optimal. In this report we therefore describe an alternative method of culturing nasal brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1{beta} and interleukine-1{beta}, which enabled consistent detection of CFTR modulator responses in nasal airway organoids cultures from subjects with CF. 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. Satellitome comparison of two oedipodine grasshoppers highlights the contingent nature of satellite DNA evolution BackgroundThe full catalogue of satellite DNA (satDNA) within a same genome constitutes the satellitome. The Library Hypothesis predicts that satDNA in relative species reflects that in their common ancestor, but the evolutionary mechanisms and pathways of satDNA evolution have never been analyzed for full satellitomes. We compare here the satellitomes of two Oedipodine grasshoppers (Locusta migratoria and Oedaleus decorus) which shared their most recent common ancestor about 22.8 Ma ago.

ResultsWe found that about one-third of their satDNA families (near 60 in every species) showed sequence homology and were grouped into 12 orthologous superfamilies. The turnover rate of consensus sequences was extremely variable among the 20 orthologous family pairs analyzed in both species. The satDNAs shared by both species showed poor association with sequence signatures and motives frequently argued as functional, except for short inverted repeats allowing short dyad symmetries and non-B DNA conformations. Orthologous satDNAs frequently showed different FISH patterns at both intra- and interspecific levels. We defined indices of homogenization and degeneration and quantified the level of incomplete library sorting between species.

ConclusionsOur analyses revealed that satDNA degenerates through point mutation and homogenizes through partial turnovers caused by massive tandem duplications (the so-called satDNA amplification). Remarkably, satDNA amplification increases homogenization, at intragenomic level, and diversification between species, thus constituting the basis for concerted evolution. We suggest a model of satDNA evolution by means of recursive cycles of amplification and degeneration, leading to mostly contingent evolutionary pathways where concerted evolution emerges promptly after lineages split. BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance BackgroundDeclining proteostasis with aging contributes to increased susceptibility to neurodegenerative diseases, including Alzheimers disease (AD). Emerging studies implicate impairment of the endosome-lysosome pathway as a significant factor in the pathogenesis of these diseases. Our lab was the first to demonstrate that BAG3 regulates phosphorylated tau clearance. However, we did not fully define how BAG3 regulates endogenous tau proteostasis, especially in the early stages of disease progression.

MethodsMass spectrometric analyses were performed to identify neuronal BAG3 interactors. Multiple biochemical assays were used to investigate the BAG3-HSP70-TBC1D10B-Rab35-Hrs regulatory networks. Live-cell imaging was used to study the dynamic of endosomal pathway. Immunohistochemistry and immunoblotting were performed in human AD brains and BAG3 overexpressed P301S tau transgenic mice.

ResultsThe primary group of neuronal BAG3 interactors identified are involved in the endocytic pathway. Among them were key regulators of small GTPases, such as the Rab35 GTPase activating protein, TBC1D10B. We demonstrated that a BAG3-HSP70-TBC1D10B complex attenuates the ability of TBC1D10B to inactivate Rab35. Thus, BAG3 interacts with TBC1D10B to support the activation of Rab35 and recruitment of Hrs, initiating ESCRT-mediated endosomal tau clearance. Further, TBC1D10B shows significantly less co-localization with BAG3 in AD brains than in age-matched controls. Overexpression of BAG3 in P301S tau transgenic mice increased the co-localization of phospho-tau with the ESCRT III protein CHMP2B and reduced the levels of the mutant human tau.

ConclusionWe identified a novel BAG3-TBC1D10B-Rab35 regulatory axis that modulates ESCRT-dependent protein degradation machinery and tau clearance. Dysregulation of BAG3 could contribute to the pathogenesis of AD. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. Glucose dynamics during ozone exposure measured using radiotelemetry: Stress drivers BackgroundStress-related neurobehavioral and metabolic disorders are associated with altered circulating adrenal-derived hormones and hyperglycemia. Temporal assessment of glucose and these hormones is critical for insights on an individuals health.

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

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

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

DiscussionWe demonstrate for the first time the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced metabolic dysfunction as exposure occurs. Real-time glucose monitoring with stress hormones assessment may be useful in identifying interactions among pollutants and stress-related illnesses. Intragenomic rearrangements of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses Variation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses. Specifically, we report numerous genomic insertions of 5-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain 5-UTR-derived inserted sequences. In limited instances, there is mounting evidence that these insertions alter the fundamental biological properties of mutant viruses. Intragenomic rearrangements add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise.

SignificanceUnderstanding mechanisms of variation in coronaviruses is vital to control of their associated diseases. Beyond point mutations, insertions, deletions and recombination, we here describe for the first time intragenomic rearrangements and their relevance to changes in transmissibility, immune escape and/or virulence documented during the SARS-CoV-2 pandemic. Designing single-cell experiments to harvest fluctuation information while rejecting measurement noise Measurement error is a complicating factor that reduces or distorts the information contained in any experiment. This problem becomes even more serious in the context of experiments that are specifically designed to quantify single-cell gene expression heterogeneity, where important RNA and protein copy numbers are themselves subject to the inherently random fluctuations of biochemical reactions. It is not clear how measurement noise should be managed, in addition to other experiment design variables such as sampling size and frequency, to ensure that collected data will provide useful insights on gene expression mechanisms of interest. To address these fundamental single-cell experiment design challenges, we propose a computational framework that makes explicit use of measurement error modeling and Fisher Information Matrix-based criteria to decide between experimental methods. This unified approach not only quantifies how different noise characteristics affect uncertainty in parameter estimation, but also enables a systematic approach to design hybrid experiments that combine different measurement modalities. 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. HaloTag9: an engineered protein tag to improve fluorophore performance HaloTag9 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag9 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag9 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging. Frequency modulated timer regulates mammalian hibernation Mammalian hibernators decrease basal metabolism and body temperature (Tb) to minimize energy expenditure in harsh seasons. During hibernation, Tb drops to low temperature (<10 {degrees}C) and remains constant for days, known as deep torpor in small mammalian hibernators. Spontaneous interbout arousals interrupt torpor bouts, when Tb recovers to euthermic state ~37 {degrees}C. Torpor-interbout arousal event repeats during several months of hibernation. However, little is known about mechanisms governing Tb fluctuation across torpor-interbout arousal cycles during hibernation. Recent improvement in data-logging techniques enables us to monitor Tb for more than hundred days with high precision, opening up new avenues for quantitative analysis to address the principle governing Tb fluctuation. Here, we analyzed Tb fluctuation across torpor-interbout arousal cycle of Syrian hamster, which can hibernate in response to chronic cold and short photoperiod under a laboratory condition, using generalized harmonic analysis and discovered a model with frequency modulation quantitatively reproducing Tb fluctuation. This analysis also identified an unexpectedly longer period of 120-430 days as the period that modulates another period of several days, generating Tb fluctuation for Syrian hamster. We propose that concerted action of two endogenous periods governs torpor-interbout arousal cycles during hibernation. Dose-dependent thresholds of dexamethasone destabilize CAR T-cell treatment efficacy Chimeric antigen receptor (CAR) T-cell therapy is potentially an effective targeted immunotherapy for glioblastoma, yet there is presently little known about the efficacy of CAR T-cell treatment when combined with the widely used anti-inflammatory and immunosuppressant glucocorticoid, dexamethasone. Here we present a mathematical model-based analysis of three patient-derived glioblastoma cell lines treated in vitro with CAR T-cells and dexamethasone. Advanced in vitro experimental cell killing assay technologies allow for highly resolved temporal dynamics of tumor cells treated with CAR T-cells and dexamethasone, making this a valuable model system for studying the rich dynamics of nonlinear biological processes with translational applications. We model the system as a nonautonomous, two-species predator-prey interaction of tumor cells and CAR T-cells, with explicit time-dependence in the clearance rate of dexamethasone. Using time as a bifurcation parameter, we show that (1) dexamethasone destabilizes coexistence equilibria between CAR T-cells and tumor cells in a dose-dependent manner and (2) as dexamethasone is cleared from the system, a stable coexistence equilibrium returns in the form of a Hopf bifurcation. With the model fit to experimental data, we demonstrate that high concentrations of dexamethasone antagonizes CAR T-cell efficacy by exhausting, or reducing the activity of CAR T-cells, and by promoting tumor cell growth. Finally, we identify a critical threshold in the ratio of CAR T-cell death to CAR T-cell proliferation rates that predicts eventual treatment success or failure that may be used to guide the dose and timing of CAR T-cell therapy in the presence of dexamethasone in patients.

Author summaryBioengineering and gene-editing technologies have paved the way for advance immunotherapies that can target patient-specific tumor cells. One of these therapies, chimeric antigen receptor (CAR) T-cell therapy has recently shown promise in treating glioblastoma, an aggressive brain cancer often with poor patient prognosis. Dexamethasone is a commonly prescribed anti-inflammatory medication due to the health complications of tumor associated swelling in the brain. However, the immunosuppressant effects of dexamethasone on the immunotherapeutic CAR T-cells are not well understood. To address this issue, we use mathematical modeling to study in vitro dynamics of dexamethasone and CAR T-cells in three patient-derived glioblastoma cell lines. We find that in each cell line studied there is a threshold of tolerable dexamethasone concentration. Below this threshold, CAR T-cells are successful at eliminating the cancer cells, while above this threshold, dexamethasone critically inhibits CAR T-cell efficacy. Our modeling suggests that in the presence of high dexamethasone reduced CAR T-cell efficacy, or increased exhaustion, can occur and result in CAR T-cell treatment failure. Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts Three-dimensional scaffolds decellularized from native organs are a promising technique to establish engineered liver grafts and overcome the current shortage of donor organs. However, limited sources of bile duct cells and inappropriate cell distribution in bioengineered liver grafts have hindered their practical application. Organoid technology is anticipated to be an excellent tool for the advancement of regenerative medicine. In the present study, we reconstructed intrahepatic bile ducts in a rat decellularized liver graft by recellularization with liver ductal organoids. Using an ex vivo perfusion culture system, we demonstrated the biliary characteristics of repopulated mouse liver organoids, which maintained bile duct markers and reconstructed biliary tree-like networks with luminal structures. We also established a method for the co-recellularization with engineered bile ducts and primary hepatocytes, revealing the appropriate cell distribution to mimic the native liver. We then utilized this model in human organoids to demonstrate the reconstructed bile ducts. Our results show that liver ductal organoids are a potential cell source for bile ducts from bioengineered liver grafts using three-dimensional scaffolds. Intergenerational Effects of Early Life Starvation on Life-History, Consumption, and Transcriptome of a Holometabolous Insect Intergenerational effects, also known as parental effects in which the offspring phenotype is influenced by the parental phenotype, can occur in response to factors that occur not only in early but also in late parental life. However, little is known about how these parental life stage-specific environments interact with each other and with the offspring environment to influence offspring phenotype, particularly in organisms that realize distinct niches across ontogeny. We examined the effects of parental larval starvation and adult reproductive environment on offspring traits under matching or mismatching offspring larval starvation conditions using the holometabolous, haplo-diploid insect Athalia rosae (turnip sawfly). We show that the parental larval starvation treatment had trait-dependent intergenerational effects on both life-history and consumption traits of offspring larvae, partly in interaction with offspring conditions and sex, while there was no significant effect of parental adult reproductive environment. In addition, while offspring larval starvation led to numerous gene- and pathway-level expression differences, parental larval starvation impacted fewer genes and only the ribosomal pathway. Our findings reveal that parental starvation evokes complex intergenerational effects on offspring life-history traits, consumption patterns as well as gene expression, although the effects are less pronounced than those of offspring starvation. Hypertrophic Chondrocytes Serve as a Reservoir for Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.