Rescue of stalled clathrin-mediated endocytosis by asymmetric Arp2/3-mediated actin assembly Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.
Obesity affects mitochondrial metabolism and proliferative potential of equine endometrial progenitor cells. The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (Non-OB) and obese (OB) mares were characterized by cellular phenotype and multipotency. Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterized by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K and AKT, simultaneously with decreased signaling stabilizing cellular homeostasis (including mitofusin, SIRT1, FOXP3). Obesity alters functional features and the self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predict lower regenerative capacity if used as autologous transplants.
A generic hidden Markov model for multi-parent populations A common step in the analysis of multi-parent populations is genotype reconstruction: identifying the founder origin of haplotypes from dense marker data. This process often makes use of a probability model for the pattern of founder alleles along chromosomes, including the relative frequency of founder alleles and the probability of exchanges among them, which depend on a model for meiotic recombination and on the mating design for the population. While the precise experimental design used to generate the population may be used to derive a precise characterization of the model for exchanges among founder alleles, this can be tedious, particularly given the great variety of experimental designs that have been proposed. We describe an approximate model that can be applied for a variety of multi-parent populations. We have implemented the approach in the R/qtl2 software, and we illustrate its use in applications to publicly-available data on Diversity Outbred and Collaborative Cross mice BlenderPhotonics - an integrated open-source software environment for 3-D meshing and photon simulations in complex tissues SignificanceRapid advances in biophotonics techniques require quantitative, model-based computational approaches to obtain functional and structural information from increasingly complex and multi-scaled anatomies. The lack of efficient tools to accurately model tissue structures and subsequently perform quantitative multi-physics modeling greatly impedes the clinical translation of these modalities.

AimWhile the mesh-based Monte Carlo (MMC) method expands our capabilities in simulating complex tissues by using tetrahedral meshes, the generation of such domains often requires specialized meshing tools such as Iso2Mesh. Creating a simplified and intuitive interface for tissue anatomical modeling and optical simulations is essential towards making these advanced modeling techniques broadly accessible to the user community.

ApproachWe responded to the above challenge by combining the powerful, open-source 3-D modeling software, Blender, with state-of-the-art 3-D mesh generation and MC simulation tools, utilizing the interactive graphical user interface (GUI) in Blender as the front-end to allow users to create complex tissue mesh models, and subsequently launch MMC light simulations.

ResultsHere we present a tutorial to our newly developed Python-based Blender add-on - BlenderPhotonics - to interface with Iso2Mesh and MMC, allowing users to create, configure and refine complex simulation domains and run hardware-accelerated 3-D light simulations with only a few clicks. We provide a comprehensive introduction to this new tool and walk readers through 5 examples, ranging from simple shapes to sophisticated realistic tissue models.

ConclusionBlenderPhotonics is user-friendly and open-source, leveraging the vastly rich ecosystem of Blender. It wraps advanced modeling capabilities within an easy-to-use and interactive interface. The latest software can be downloaded at http://mcx.space/bp. Critical re-evaluation of the slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

Experimental ApproachFunctional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA, leading to wrong estimates of operational efficacy and affinity of an agonist. In contrast, functional analysis of OMA with Hill coefficient as a slope factor has not this flaw.

Key ResultsFitting of the OMA with Black et al. slope factor to concentration-response curves of several models (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) gives wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to concentration-response curves of these several models gives correct estimates of operational efficacy and affinity.

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias. Classification of daily crop phenology in PhenoCams using deep learning and hidden markov models Near surface cameras, such as those in the PhenoCam network, are a common source of ground truth data in modelling and remote sensing studies. Despite having locations across numerous agricultural sites, few studies have used near surface cameras to track the unique phenology of croplands. Due to management activities, crops do not have a natural vegetation cycle which many phenological extraction methods are based on. For example, a field may experience abrupt changes due to harvesting and tillage throughout the year. A single camera can also record several different plants due to crop rotations, fallow fields, and cover crops. Current methods to estimate phenology metrics from image time series compress all image information into a relative greenness metric, which discards a large amount of contextual information. This can include the type of crop present, whether snow or water is present on the field, the crop phenology, or whether a field lacking green plants consists of bare soil, fully senesced plants, or plant residue. Here we developed a modelling workflow to create a daily time series of crop type and phenology, while also accounting for other factors such as obstructed images and snow covered fields. We used a mainstream deep learning image classification model, VGG16. Deep learning classification models do not have a temporal component, so to account for temporal correlation among images our workflow incorporates a hidden markov model in the post-processing. The initial image classification model had out of sample F1 scores of 0.83-0.85, which improved to 0.86-0.91 after all post-processing steps. The resulting time series show the progression of crops from emergence to harvest, and can serve as a daily, local scale dataset of field states and phenological stages for agricultural research. Loci for prediction of penicillin and tetracycline susceptibility in Neisseria gonorrhoeae: a genome wide association study BackgroundWhile Neisseria gonorrhoeae poses an urgent public health threat because of increasing antimicrobial resistance, much of the circulating population remains susceptible to historical treatment regimens. Point-of-care diagnostics that report susceptibility could allow for reintroduction of these regimens, but development of such diagnostics has been limited to ciprofloxacin, for which susceptibility can be predicted from a single locus.

MethodsWe assembled a dataset of 12,045 N. gonorrhoeae genomes with phenotypic resistance data for tetracycline (n = 3,611) and penicillin (n = 6,935). Using conditional genome wide association studies (GWAS), we sought to define genetic variants associated with susceptibility to penicillin and tetracycline. We evaluated the sensitivity and specificity of these variants for predicting susceptibility and non-resistance in our collection of gonococcal genomes.

FindingsIn our conditional penicillin GWAS, the presence of a genetic variant defined by a non-mosaic penA allele without an insertion at codon 345 was significantly associated with penicillin susceptibility and had the highest negative effect size of significant variants (p = 5.0 x 10-14, {beta} = -2.5). In combination with the absence of blaTEM, this variant predicted penicillin susceptibility with high specificity (99.8%) and modest sensitivity (36.7%). For tetracycline, the wild type allele at rpsJ codon 57, encoding valine, was significantly associated with tetracycline susceptibility (p = 5.6 x 10-16, {beta} = -1.6) after conditioning on the presence of tetM. The combination of rpsJ codon 57 allele and tetM absence predicted tetracycline susceptibility with high specificity (97.2%) and sensitivity (88.7%).

InterpretationAs few as two genetic loci can predict susceptibility to penicillin and tetracycline in N. gonorrhoeae with high specificity. Molecular point-of-care diagnostics targeting these loci have the potential to increase available treatments for gonorrhea.

FundingNational Institute of Allergy and Infectious Diseases, the National Science Foundation, and the Smith Family Foundation

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with the terms "Neisseria gonorrhoeae" and "diagnostic" or "assay" plus "penicillin" or "tetracycline" for reports in any language published up to July 1, 2021. We additionally searched for "Neisseria gonorrhoeae" and "genome wide association study". We found that previously proposed molecular diagnostics for penicillin and tetracycline susceptibility either exclusively focused on plasmid-mediated resistance (i.e., targeting blaTEM or tetM) or did not include variants in genes encoding antibiotic targets (e.g., did not include penA or rpsJ). Targets for molecular surveillance have focused on resistance-associated alleles rather than susceptibility-associated alleles. We did not find any previous penicillin or tetracycline conditional genome wide association studies (GWAS) in N. gonorrhoeae.

Added value of this studyTo identify targets for molecular diagnostics that predict penicillin and tetracycline susceptibility, we conducted GWAS conditioning on the presence of plasmid-mediated resistance determinants to detect chromosomal loci with the highest association with susceptibility. We discovered a sequence (penA_01) that differentiates susceptible isolates from those with a resistance-associated insertion at codon 345 and from those with mosaic penA alleles, which is associated with penicillin susceptibility. We also found that rpsJ codon 57 was the chromosomal locus contributing the most to tetracycline susceptibility. The combination of these chromosomal loci and the absence of plasmid encoded determinants predicts penicillin and tetracycline susceptibility with high specificity in both a large global collection of N. gonorrhoeae and a validation dataset consisting of recently published genomes from CDCs Gonococcal Isolate Surveillance Program (GISP) surveillance collected in 2018.

Implications of all the available evidenceThe chromosomal loci penA_01 and rpsJ codon 57 in combination with plasmid loci blaTEM and tetM are candidates for the development of point-of-care molecular diagnostics for penicillin and tetracycline susceptibility. The loci may be combined with the currently available ciprofloxacin susceptibility diagnostics to predict susceptibility to multiple antibiotics. Additionally, our study suggests that conditional GWAS focused on variants associated with susceptibility may be a promising approach to identify minimal sets of loci for molecular diagnostics and surveillance. Learning and motor inhibitory control in crows and domestic chickens Cognitive abilities allow animals to navigate through complex, fluctuating environments. In the present study, we tested the performance of a captive group of eight crows, Corvus corone, and ten domestic chickens, Gallus gallus domesticus, in the cylinder task, as a test of motor inhibitory control, and reversal learning as a measure of learning ability and behavioural flexibility. Four crows and nine chickens completed the cylinder task, eight crows and six chickens completed the reversal learning experiment. Crows performed better in the cylinder task compared to chickens. In the reversal learning experiment, species did not significantly differ in the number of trials until learning criterion was reached. Performance in the reversal learning experiment did not correlate with performance in the cylinder task in chickens. Our results suggest crows to possess better motor inhibitory control compared to chickens. In contrast, learning performance in a reversal learning task did not differ between the species, indicating similar levels of behavioural flexibility. Interestingly, we describe notable individual differences in performance. We stress the importance not only to compare cognitive performance between species but also between individuals of the same species when investigating the evolution of cognitive skills. 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. DNA Strand Breaks and Gaps Direct Retroviral Integration Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and reverse transcribed (RT) copy DNA (cDNA). Previous studies demonstrated DNA site recognition limited intasome integration. Using single molecule Forster resonance energy transfer (smFRET), we show Prototype Foamy Virus (PFV) intasomes pause at DNA strand breaks and gaps. The break/gap discontinuities are similar to base excision repair (BER) lesion-processing intermediates, which affect retrovirus integration in vivo. Pausing targeted site-directed integration at the break/gap without inducing intasome conformational alterations. An 8-oxo-guanine lesion normally processes by BER and a G/T mismatch or a +T nucleotide insertion that induce flexibility or a bend in the DNA backbone did not promote intasome pausing or targeted integration. These results suggest that repair intermediates can modulate dynamic intasome-DNA interactions which target retroviral integration. Transfer learning framework for cell segmentation with incorporation of geometric features With recent advances in multiplexed imaging and spatial transcriptomic and proteomic technologies, cell segmentation is becoming a crucial step in biomedical image analysis. In recent years, Fully Convolutional Networks (FCN) have achieved great success in nuclei segmentation in in vitro imaging. Nevertheless, it remains challenging to perform similar tasks on in situ tissue images with more cluttered cells of diverse shapes. To address this issue, we propose a novel transfer learning, cell segmentation framework incorporating shape-aware features in a deep learning model, with multi-level watershed and morphological post-processing steps. Our results show that incorporation of geometric features improves generalizability to segmenting cells in in situ tissue images, using solely in vitro images as training data. Evolvability and macroevolution of colony-level traits in two species of bryozoans The evolution of trait variation among populations of animals is difficult to study due to the many overlapping genetic and environmental influences that control phenotypic expression. But, in a group of animals called bryozoans, it is possible to isolate genetic contributions to phenotypic variation, due to the modular nature of bryozoan colonies. Each bryozoan colony represents a snapshot of the phenotypes that correspond to a single genotype, which can be summarized as a phenotypic distribution. We test whether these phenotypic distributions are evolvable across a generation of colonies in two sister species of the bryozoan Stylopoma, grown and bred in a common garden breeding experiment. We find that components of phenotypic distributions, specifically median trait values of colony members, are evolvable between generations of colonies. Furthermore, this evolvability has macroevolutionary importance because it correlates with the morphological distance between these two species. Because parts of the phenotypic distributions are evolvable, and this evolvability corresponds to evolutionary divergence between species, we infer that these distributions can shift and change shape over macroevolutionary timescales. Such changes to phenotypic distributions across many generations of colonies may underpin the emergence of colony-level traits, like division of labor in colonies. TeamTree analysis: a new approach to evaluate scientific production Advances in science and technology depend on the work of research teams and the publication of results through peer-reviewed articles representing a growing socio-economic resource. Current methods to mine the scientific literature regarding a field of interest focus on content, but the workforce credited by authorship remains largely unexplored, and appropriate measures of scientific production are debated. Here, a new bibliometric approach named TeamTree analysis is introduced that visualizes the development and composition of the workforce driving a field. A new citation-independent measure that scales with the H index estimates impact based on publication record, genealogical ties and collaborative connections. This author-centered approach complements existing tools to mine the scientific literature and to evaluate research across disciplines. First detection of herpesvirus and frequency of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. Snrpb, the CCMS gene, is required in neural crest cells for proper splicing of genes essential for craniofacial morphogenesis Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). We show that Snrpb heterozygous embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5 splice site strength. We found increased exon-skipping in negative regulators of the P53-pathway, increased levels of nuclear P53, P53-target genes. However, removing TrP53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations. Force profile of the two-handed hardstyle kettlebell swing in novice older adults: an exploratory profile BackgroundUnderstanding the force profile of an exercise increases clinical confidence when assessing the benefits and potential risks of a prescribed exercise. This exploratory study presents the force profile of the hardstyle kettlebell swing in novice older adults and compares peak force with kettlebell deadlifts. These data will help inform healthcare providers and coaches who are considering prescribing kettlebell exercises for older adults.

MethodsThirty-five community-dwelling males and females (59-79 years) were recruited, from applicants to participate in the BELL trial. Two-handed hardstyle swings were performed with 8-16 kg. Deadlifts were performed with 8-24 kg and 8-32 kg for females and males, respectively. Ground reaction force was obtained from a floor-mounted force platform. Pairwise comparisons of peak force, forward force, rate of force development, swing cadence, sex, and kettlebell mass, were investigated for the kettlebell swing, with representative force-time curves described. Pairwise comparisons of peak force, sex and kettlebell mass were investigated for the deadlift, with comparisons of peak force, kettlebell mass, and sex, between swings and deadlifts.

ResultsFor kettlebells up to 16 kg, paired samples T-tests show a large exercise effect ({delta} > 1.4) with peak force higher for swings than deadlifts. Data shows: (i) higher peak force during swings than deadlifts ({delta} = 1.77), reaching 4.5 (1.0) N.kg-1, (ii) peak force during an 8 kg swing was greater than a 32kg deadlift, (iii) negligible difference in normalised peak force between males and females performing kettlebell swings, but a moderately large effect size during deadlifts (males > females, {delta} = 0.69), (iv) mean rate of force development of 19.9 (4.7) N.s-1.kg-1 with a very weak, positive correlation with kettlebell mass (y = 14.4 + 0.32x), and trivial effect of sex, (v) mean forward force equal to 5.5% of vertical force during swings, increasing from 3.8 (1.6) % with 8 kg to 7.1 (2.6) % with 16 kg.

ConclusionDuring kettlebell swings, there is negligible difference in normalised net peak force between novice males and females using the same absolute loads. Where ground reaction force is a therapeutic target, kettlebell swings with an 8 kg kettlebell could have similar effects to much heavier deadlifts (>24 kg). Kettlebell swings performed with lighter loads, could provider similar therapeutic value to much heavier deadlifts, and may be a more appealing, affordable, and convenient option for older adults. Late-acting self-incompatible system, preferential allogamy and delayed selfing in the heteromorphic invasive populations of Ludwigia grandiflora subsp. hexapetala AO_SCPLOWBSTRACTC_SCPLOWBreeding system influences local population genetic structure, effective size, offspring fitness and functional variation. Determining the respective importance of self- and cross-fertilization in hermaphroditic flowering plants is thus important to understand their ecology and evolution. The worldwide invasive species, Ludwigia grandiflora subsp. hexapetala (Lgh) presents two floral morphs: one self-compatible short-styled morph (S-morph) and one self-incompatible long-styled morph (L-morph). In this study, we identified the breeding systems of western European experimental and natural populations of Lgh by comparing structural characteristics of pollen and style, by studying self- and cross-pollen tube elongations and the viability of the resulting seeds and seedlings in both floral morphs. Our results showed no differences in pollen shape and stigma surfaces no matter the floral morph. In the self-incompatible L-morph flowers, self-pollen tubes were stopped tardily, in the ovarian area, and were unable to fertilize the ovules. This first formal identification of a late-acting, prezygotic self-incompatible system (LSI) in Ludwigia genus contributes a case of LSI in an additional family within the Myrtales order. In the self-compatible S-morph flowers, self-pollen always succeeded to self-fertilize the ovules that nearly all developed into viable seedlings. However, cross-pollen tubes always elongated faster than self-pollen tubes in both morphs. S-morph individuals may thus advantage preferential allogamy over selfing when cross-pollen is available despite its self-compatibility. As expected in late-acting self-incompatible systems, L-morph flowers authorised 0.2{per thousand} of selfed seeds during the uppermost flowering season, that increased to 1{per thousand} at the end of the flowering season. Such delayed selfing resulted in a significant quantity of viable floating seeds. They may contribute to the local regeneration, seed bank and propagation of the L-morph, which may contribute to explain its invasion success worldwide. Management plans of Lgh would gain to consider the breeding systems we identified. Nrg1/ErbB Signaling-Mediated Regulation of Fibrosis After Myocardial Infarction RATIONALEAppropriate fibrotic tissue formation after myocardial infarction (MI) is crucial to maintenance of the hearts structure. Reparative or M2-like macrophages play a vital role in post-MI fibrosis by activating cardiac fibroblasts. However, the mechanism by which post-MI cardiac fibrosis is regulated is not fully understood.

OBJECTIVEWe investigated the cellular and molecular mechanisms of post-MI fibrotic tissue formation, especially those related to regulation of cellular senescence and apoptosis.

METHODS AND RESULTSIn vivo and in vitro experiments were used to investigate the molecular and cellular mechanisms through which post-MI fibrosis occurs, with a focus on the role of M2-like macrophages. Microarray analysis revealed that CD206+F4/80+CD11b+ M2-like macrophages collected from mouse hearts on post-MI day 7 showed increased expression of neuregulin 1 (Nrg1). Nrg1 receptor epidermal growth factor receptor ErbB was expressed on cardiac fibroblasts in the infarct area. In cardiac fibroblasts in which hydrogen peroxide-induced senescence, M2-like macrophage-derived Nrg1 suppressed both senescence and apoptosis of the fibroblasts, whereas blockade of ErbB function significantly accelerated. M2-like macrophage-derived Nrg1/ErbB/PI3K/Akt signaling, which was shown to be related to anti-senescence, was activated in damaged cardiac fibroblasts. Interestingly, systemic blockade of ErbB function in MI model mice enhanced senescence and apoptosis of cardiac fibroblasts and exacerbated inflammation. Further, increased accumulation of M2-like macrophages resulted in excessive progression of fibrosis in post-MI murine hearts. The molecular mechanism underlying regulation of fibrotic tissue formation in the infarcted myocardium was shown in part to be attenuation of apoptosis and senescence of cardiac fibroblasts by activation of Nrg1/ErbB/PI3K/Akt signaling.

CONCLUSIONSM2-like macrophage-mediated regulation of Nrg1/ErbB signaling, have a substantial effect on fibrotic tissue formation in the infarcted adult mouse heart, is critical for suppressing the progression of senescence and apoptosis of cardiac fibroblasts. Human pluripotent stem cell-derived cardiomyocytes align under cyclic strain when guided by cardiac fibroblasts The human myocardium is a mechanically active tissue typified by anisotropic organization of the resident cells (cardiomyocytes (CMs) and cardiac fibroblasts (cFBs)) and the extracellular matrix (ECM). Upon ischemic injury, the anisotropic tissue is replaced by disorganized scar tissue, eventually resulting in loss of coordinated contraction. Efforts to re-establish tissue anisotropy in the injured myocardium are hampered by a lack of understanding on how CM and/or cFB structural organization is affected by the two major physical cues inherent in the myocardium: ECM organization and cyclic mechanical strain. Herein, we investigate the singular and combined effect of ECM (dis)organization and cyclic strain in a 2D human in vitro co-culture model of the myocardial microenvironment. We show that (an)isotropic ECM protein patterning can guide the orientation of CMs and cFBs, both in mono- and co-culture. Subsequent application of uniaxial cyclic strain - mimicking the local anisotropic deformation of beating myocardium - causes no effect when applied parallel to the anisotropic ECM. However, when cultured on isotropic substrates, cFBs, but not CMs, orient away from the direction of cyclic uniaxial strain (strain avoidance). In contrast, CMs show strain avoidance via active remodeling of their sarcomeres only when co-cultured with at least 30% cFBs. Paracrine signaling or N-cadherin-mediated communication between CMs and cFBs were no contributing factors, but our findings suggest that the mechanoresponsive cFBs provide structural guidance for CM orientation and elongation. Our study therefore highlights a synergistic mechanobiological interplay between CMs and cFBs in shaping tissue organization, which is of relevance for regenerating functionally organized myocardium. Generalization of Cortical MOSTest Genome-Wide Associations Within and Across Samples Genome-Wide Association studies have typically been limited to single phenotypes, given that high dimensional phenotypes incur a large multiple comparisons burden: ~1 million tests across the genome times the number of phenotypes. Recent work demonstrates that a Multivariate Omnibus Statistic Test (MOSTest) is well powered to discover genomic effects distributed across multiple phenotypes. Applied to cortical brain MRI morphology measures, MOSTest has resulted in a drastic improvement in power to discover loci when compared to established approaches (min-P). One question that arises is how well these discovered loci replicate in independent data. Here we perform 10 times cross validation within 35,644 individuals from UK Biobank for imaging measures of cortical area, thickness and sulcal depth (>1,000 dimensionality for each). By deploying a replication method that aggregates discovered effects distributed across multiple phenotypes, termed PolyVertex Score (PVS), we demonstrate a higher replication yield and comparable replication rate of discovered loci for MOSTest (# replicated loci: 348-845, replication rate: 94-95%) in independent data when compared with the established min-P approach (# replicated loci: 31-68, replication rate: 65-80%). An out-of-sample replication of discovered loci was conducted with a sample of 8,336 individuals from the Adolescent Brain Cognitive Development(R) (ABCD) study, who are on average 50 years younger than UK Biobank individuals. We observe a higher replication yield and comparable replication rate of MOSTest compared to min-P. This finding underscores the importance of using well-powered multivariate techniques for both discovery and replication of high dimensional phenotypes in Genome-Wide Association studies. 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 Distinct Neural Profiles of Frontoparietal Networks in Boys with ADHD and Boys with Persistent Depressive Disorder Working memory deficits are common in attention-deficit/hyperactivity disorder (ADHD) and depression, two common neurodevelopmental disorders with overlapping cognitive profiles but distinct clinical presentation. Multivariate techniques have previously been utilized to understand working memory processes in functional brain networks in healthy adults, but have not yet been applied to investigate how working memory processes within the same networks differ within typical and atypical developing populations. We used multivariate pattern analysis (MVPA) to identify whether brain networks discriminated between spatial vs. verbal working memory processes in ADHD and Persistent Depressive Disorder (PDD). 36 male clinical participants and 19 typically developing (TD) boys participated in a fMRI scan while completing a verbal and a spatial working memory task. Within a priori functional brain networks (frontoparietal, default mode, salience) the TD group demonstrated differential response patterns to verbal and spatial working memory. The PDD group showed weaker differentiation than TD, with lower classification accuracies observed in primarily the left frontoparietal network. The neural profiles of the ADHD and PDD differed specifically in the SN where the ADHD groups neural profile suggests significantly less specificity in neural representations of spatial and verbal working memory. We highlight within-group classification as an innovative tool for understanding the neural mechanisms of how cognitive processes may deviate in clinical disorders, an important intermediary step towards improving translational psychiatry. Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune circuits related to tissue injury Severe lung damage in COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways and genes present across the spectrum of histopathological damage in COVID-19 lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from areas of interest within well preserved post-mortem lung samples from three patients. Despite substantial inter-patient heterogeneity we discovered evidence for a common immune cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines including CXCL9, CXCL10 and CXCL11 which are known to promote the recruitment of CXCR3+ immune cells. The tumour necrosis factor (TNF) superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were found to be consistently upregulated in the areas with severe tissue damage. We used published spatial and single cell SARS-CoV-2 datasets to confirm our findings in the lung tissue from additional cohorts of COVID-19 patients. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.

One Sentence SummarySpatial analysis identifies IFN{gamma} response signatures as focal to severe alveolar damage in COVID-19 pneumonitis. 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. FGFR2 fusion protein-driven mouse models of intrahepatic cholangiocarcinoma unveil a necessary role for Erk signaling Background and aimsAbout 15% of intrahepatic cholangiocarcinoma (iCCA) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), most often in concert with mutationally inactivated TP53, CDKN2A or BAP1. FFs span residues 1-768 of FGFR2 fused to sequences encoded by any of a long list (>60) of partner genes, a configuration sufficient to ignite oncogenic FF activation. In line, FGFR-specific tyrosine kinase inhibitors (F-TKI) were shown to provide clinical benefit in FF+ iCCA, although responses were partial and/or limited by resistance mechanisms, including the FF V565F gatekeeper mutation. Herein we present an FF-driven murine iCCA model and exploit its potential for pre-clinical studies on FF therapeutic targeting.

MethodsFour iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by intrahepatic/subcutaneous transplantation in immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies.

ResultsTumors diagnosed as CCA were obtained upon transplantation of FF-expressing liver organoids. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or presence of V565F mutation. Double blockade of FF-Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo.

ConclusionsFF-driven iCCA pathogenesis was successfully modelled in murine Tp53-/- background. This model revealed biological heterogeneity among structurally different FFs. Double blockade of FF-Erk signaling deserves consideration for improving precision-based approaches against human FF+ iCCA.

Abbreviations used in this paper: ANOVA, analysis of variance; Bap1, BRCA1-Associated-Protein 1; Cdkn2a, cyclin-dependent kinase inhibitor 2A; Cftr, cystic fibrosis transmembrane conductance regulator; Ck19, cytokeratin 19; Cyp3A, cytochrome P450, family 3, subfamily A; EGF, Epidermal growth factor; EGFR, Epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; Erk, extracellular signal-regulated kinase; FGFR2, fibroblast growth factor receptor 2; FRS2, fibroblast growth factor receptor substrate 2; GRB2, growth factor receptor-bound 2; GSEA, gene set enrichment analysis; GSVA, gene set variation analysis; H&E, hematoxylin and eosin; HepPar1, hepatocyte Paraffin 1; Hnf4-7, hepatocyte nuclear factor 4 alpha; Hprt, hypoxanthine-guanine phosphoribosyl transferase; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; NTRK, neurotrophic Tyrosine Kinase; Parp, Poly (ADP-ribose) polymerase; RECIST, response evaluation criteria in solid tumors; SHP2, Src homology phosphatase 2; Ttr, transthyretin. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies.