Spectral fiber-photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity Fiber-photometry is an emerging technique for recording fluorescent sensor activity in the brain. However, significant hemoglobin-absorption artifacts in fiber-photometry data may be misinterpreted as sensor activity changes. Because hemoglobin exists in nearly every location in the brain and its concentration varies over time, such artifacts could impede the accuracy of many photometry recording results. Here we present a novel use of spectral photometry technique and propose computational methods to quantify photon absorption effects by using activity-independent fluorescence signals, which can be used to derive oxy- and deoxy-hemoglobin concentration changes. Following time-locked neuronal activation in vivo, we observed that a 20% increase in CBV contributes to about a 4% decrease in green fluorescence signal and a 2% decrease in red fluorescence signal. While these hemoglobin concentration changes are often temporally delayed than the fast-responding fluorescence spikes, we found that erroneous interpretation may occur when examining pharmacology-induced sustained activity changes, and in some cases, hemoglobin-absorption could flip the GCaMP signal polarity. We provided hemoglobin-based correction methods to restore fluorescence signals across spectra and compare our results against the commonly used regression approach. We also demonstrated the utility of spectral fiber-photometry for delineating brain regional differences in hemodynamic response functions.

HighlightsO_LIHemoglobin-absorption compromises fiber-photometry recording in vivo
C_LIO_LISpectral photometry allows quantification of hemoglobin concentration changes for correction
C_LIO_LIThe proposed platform allows measuring regional differences in neurovascular transfer function
C_LI
Graph Ricci Curvatures Reveal Atypical Functional Connectivity in Autism Spectrum Disorder While standard graph-theoretic measures have been widely used to characterize atypical resting-state functional connectivity in autism spectrum disorder (ASD), geometry-inspired network measures have not been applied. In this study, we apply Forman-Ricci and Ollivier-Ricci curvatures to compare networks of ASD and typically developing individuals (N = 1112) from the Autism Brain Imaging Data Exchange I (ABIDE-I) dataset. We find brain-wide and region-specific ASD-related differences for both Forman-Ricci and Ollivier-Ricci curvatures, with region-specific differences concentrated in Default Mode, Somatomotor and Ventral Attention networks for Forman-Ricci curvature. We use meta-analysis decoding to demonstrate that brain regions with curvature differences are associated to those cognitive domains known to be impaired in ASD. Further, we show that brain regions with curvature differences overlap with those brain regions whose non-invasive stimulation improves ASD-related symptoms. These results suggest the utility of graph Ricci curvatures in characterizing atypical connectivity of clinically relevant regions in ASD and other neurodevelopmental disorders.
Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. Multivariate genome-wide association study on tissue-sensitive diffusion metrics identifies key molecular pathways for axonal growth, synaptogenesis, and astrocyte-mediated neuroinflammation The molecular determinants of tissue composition of the human brain remain largely unknown. Recent genome-wide association studies (GWAS) on this topic have had limited success due to methodological constraints. Here, we apply advanced whole-brain analyses on multi-shell diffusion imaging data and multivariate GWAS to two large scale imaging genetic datasets (UK Biobank and the Adolescent Brain Cognitive Development study) to identify and validate genetic association signals. We discovered 503 unique genetic loci that have impact on multiple regions of human brain. Among them, more than 79% were validated in either of two large-scale independent imaging datasets. Key molecular pathways involved in axonal growth, astrocyte-mediated neuroinflammation, and synaptogenesis during development were found to significantly impact the measured variations in tissue-specific imaging features. Our results shed new light on the biological determinants of brain tissue composition and their potential overlap with the genetic basis of neuropsychiatric disorders. Optimization of avian perching manoeuvres Perching at speed is amongst the most challenging flight behaviours that birds perform1,2, and beyond the capability of current autonomous vehicles. Smaller birds may touchdown by hovering3-8, but larger birds typically swoop upward to perch1,2 - presumably because the adverse scaling of their power margin prohibits slow flapping flight9, and because swooping transfers excess kinetic to potential energy1,2,10. Perching is risky in larger birds6,11, demanding precise control of velocity and pose12-15, but it is unknown how they optimize this challenging manoeuvre. More generally, whereas cruising flight behaviours such as migration and commuting are adapted to minimize cost-of-transport or time-of-flight16, the optimization of unsteady flight manoeuvres remains largely unexplored7,17. Here we show that swooping minimizes neither the time nor energy required to perch safely in Harris hawks Parabuteo unicinctus, but instead minimizes the distance flown under hazardous post-stall conditions. By combining motion capture data from 1,563 flights with flight dynamics modelling, we found that the birds choice of where to transition from powered dive to unpowered climb minimizes the distance from the landing perch over which very high lift coefficients are required. Time and energy are therefore invested to maintain the control authority needed to execute a safe landing, rather than being minimized continuously as in technical applications of autonomous perching under nonlinear feedback control13 and deep reinforcement learning18,19. Naive birds learn this behaviour on-the-fly, so our findings suggest an alternative reward function for reinforcement learning of autonomous perching in air vehicles. Endogenous retroviruses co-opted as divergently transcribed regulatory elements shape the regulatory landscape of embryonic stem cells Transposable elements are an abundant source of transcription factor binding sites and favorable genomic integration may lead to their recruitment by the host genome for gene regulatory functions. However, it is unclear how frequent co-option of transposable elements as regulatory elements is, to which regulatory programs they contribute and how they compare to regulatory elements devoid of transposable elements. Here, we report a transcription initiation-centric, in-depth characterization of the transposon-derived regulatory landscape of mouse embryonic stem cells. We demonstrate that a substantial number of transposable elements, in particular endogenous retroviral elements, carry open chromatin regions that are divergently transcribed into unstable RNAs in a cell-type specific manner, and that these elements contribute to a sizable proportion of active enhancers and gene promoters. We further show that transposon subfamilies contribute differently and distinctly to the pluripotency regulatory program through their repertoires of transcription factor binding sites, shedding light on the formation of regulatory programs and the origins of regulatory elements. Genome annotation of Caenorhabditis briggsae by TEC-RED identifies new exons, paralogs, and conserved and novel operons The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying the 5 ends of expressed genes, we have discovered operons and paralogs. In summary, our analysis yielded 10,243 unique 5 end sequence tags with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. These genes included 14 that are exclusively trans-spliced in C. briggsae when compared with C. elegans orthologs. A major contribution of this study is the identification of 493 operons, of which two-thirds are fully supported by tags. In addition, two SL1-type operons were discovered. Interestingly, comparisons with C. elegans showed that only 40% of operons are conserved. Of the remaining operons, 73 are novel, including 12 that entirely lack orthologs in C. elegans. Further analysis revealed that four of the 12 novel operons are conserved in C. nigoni. Altogether, the work described here has significantly advanced our understanding of the C. briggsae system and serves as a rich resource to aid biological studies involving this species. RNF213 loss of function reshapes vascular transcriptome and spliceosome leading to disrupted angiogenesis and aggravated vascular inflammatory responses. RationaleMoyamoya disease (MMD) is a rare cerebrovascular occlusive disease that affects Asian population more often. The pathogenesis of MMD is related to mutation in RNF213 gene. However, why, and how RNF213 mutation leads to MMD is still not fully understood.

ObjectiveAnalyze the impact of RNF213 loss of function on vascular cells and the observed changes correlate with MMD.

Methods and resultsRNF213 KD was conducted in HUVEC and vascular smooth muscle cells (vSMCs). First, HUVEC cells showed alteration of angiogenesis, migration under LPS stimulation, Leukocyte endothelial transmigration, and endothelial-to-vSMCs communication.

Transcriptome analysis revealed downregulation of genes regulating cell division and mitosis. Interestingly, Alternative splicing (AS) analysis revealed hundreds of AS events to occur after RNF213 KD in various types of AS. The alternatively spliced genes showed minimal overlap with transcriptome profiling. Pathway analysis revealed many processes and pathways to be regulated by AS events observed. Transcriptome profiling was also performed after LPS treatment and revealed the basis for increased sensitivity to LPS observed in our analysis. AS changes were also observed after LPS treatment in RNF213 KD HUVEC. Different types of AS showed different patterns of convergence or divergence in terms of the regulated pathways after LPS treatment. Finally, transcriptome and AS analysis was performed in vSMCs and showed various processes that impact vSMCs function and phenotype in RNF213 loss of function.

ConclusionOur data provide a wealth of information on RNF213 gene function in vascular tissues and shed important light on different processes that contribute to MMD pathogenesis. Our results signify an immune-driven and hemodynamically linked process of MMD initiation and progression. Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ-9-tetrahydrocannabinol and involving CB1R and CB2R in zebrafish In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. Correlation between speed and turning naturally arises for sparsely sampled cell movements Mechanisms regulating cell movement are not fully understood. One feature of cell movement that determines how far cells displace from an initial position is persistence, the ability to perform movements in a direction similar to the previous movement direction. Persistence is thus determined by turning angles between two sequential displacements and be characterized by an average turning angle or persistence time. Recent studies found that a cells average speed and turning are negatively correlated, suggesting a fundamental cell-intrinsic program whereby cells with a lower turning ability (i.e., larger persistence time) are intrinsically faster (or faster cells turn less). By simulating correlated or persistent random walks (PRWs) using two different frameworks (one based on von Mises-Fisher (vMF) distribution and another based on Ornstein-Uhlenbeck (OU) process) we show that the negative correlation between speed and turning naturally arises when cell trajectories are sub-sampled, i.e., when the frequency of sampling is lower than frequency at which cells make movements. This effect is strongest when the sampling frequency is on the order of magnitude with the typical cell persistence time and when cells vary in persistence time. Both conditions are observed for datasets of T cell movements in vivo that we have analyzed. In simulations the correlation arises due to randomness of cell movements resulting in highly variable persistence times for individual cells that with sub-sampling leads to large variability of average cell speeds. Interestingly, previously suggested methodology of calculating displacement of cell cohorts with different speeds resulted in similar results whether or not there is a cell-intrinsic correlation between cell speed and persistence. For both vMF- and OU-based simulations of PRWs we could find parameter values (distribution of persistence times, speeds, and sampling frequency) that matched experimentally measured correlations between speed and turning for two datasets of T cell movement in vivo suggesting that such simple correlations are not fully informative on the intrinsic link between speed and persistence. Our results thus suggest that sub-sampling may contribute to (and perhaps fully explains) the observed correlation between speed and turning at least for some cell trajectory data and emphasize the role of sampling frequency in inference of critical cellular parameters of cell motility such as speeds.

Secondary AbstractMeasurement of cell movements often results in a negative correlation between average speed and average turning angle suggesting an existence of a universal, cell-intrinsic movement program. We show that such a negative correlation may arise if cells in the population differ in their ability for persistent movement when the movement data are sub-sampled. We show that sub-sampling of cell trajectories generated using two different frameworks of persistent random walk can match the experimentally observed correlation between speed and turning for T cell movements in vivo. Muscle Activation Profile During Perturbed Walking is Modulated According to Body State During an unexpected loss of balance, avoiding a fall requires people to readjust their footing rapidly and effectively. We investigated the activation patterns of the ankle and knee muscles, and muscle fiber type recruitment resulting from unannounced, medio-lateral (i.e., right/left) horizontal-surface walking perturbations in twenty healthy adults (27.00{+/-}2.79 years, 10 females). Surface electromyography (sEMG) total spectral power for specific frequency bands (40-60Hz, 60-150Hz, 150-250Hz, 250-400Hz and 400-1000Hz), from tibialis anterior (TA) and vastus lateralis (VL) muscles were analyzed. Compared to non-perturbed walking, we found a significant increase in the total spectral power of lower-extremity muscles during the first 3 seconds after perturbation. When two feet were on the ground in time of perturbation we found a different muscle fiber type recruitment pattern between VL and TA muscles. This was not significant for perturbations implemented when one foot was on the ground. Our findings suggest that muscle operating frequency is modulated in real time to fit body state and somatosensory input in the context of functional goal requirements such as a rapid change of footing in response to unexpected loss of balance in single and double-support phases of gait.

New & NoteworthyTo study muscle spectral profiles in response to loss of balance, we investigated the dynamics of muscle spectral power changes, across different frequency bands after unannounced mechanical perturbations during walking. We showed increased activation of high-frequency motor units of the lower-limb muscles, subside 3 seconds after perturbation. Differences in power increase of specific frequency bands suggest that muscle activation is modulated in real time to fit body state in the context of functional goal requirements. The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. Lowland plant arrival in alpine ecosystems facilitates a decrease in soil carbon content under experimental climate warming Climate warming is releasing carbon from soils around the world1-3, constituting a positive climate feedback. Warming is also causing species to expand their ranges into new ecosystems4-9. Yet, in most ecosystems, whether range expanding species will amplify or buffer expected soil carbon loss is unknown10. Here we used two whole-community transplant experiments and a follow-up glasshouse experiment to determine whether the establishment of herbaceous lowland plants in alpine ecosystems influences soil carbon content under warming. We found that warming (transplantation to low elevation) led to a negligible decrease in alpine soil carbon content, but its effects became significant and 52% {+/-} 31% (mean {+/-} 95% CIs) larger after lowland plants were introduced at low density into the ecosystem. We present evidence that decreases in soil carbon content likely occurred via lowland plants increasing rates of root exudation, soil microbial respiration and CO2 release under warming. Our findings suggest that warming-induced range expansions of herbaceous plants have the potential to alter climate feedbacks from this system, and that plant range expansions among herbaceous communities may be an overlooked mediator of warming effects on carbon dynamics. Naming of human diseases on the wrong side of history BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

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

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

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

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. 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">
View larger version (34K):
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. Training-induced bioenergetic improvement in human skeletal muscle is associated with non-stoichiometric changes in the mitochondrial proteome without reorganization of respiratory chain content Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible and inexpensive therapeutic intervention improving mitochondrial bioenergetics and quality of life. By combining a multi-omics approach with biochemical and in silico normalization, we removed the bias arising from the training-induced increase in human skeletal muscle mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritized mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the increase in mitochondrial content. We demonstrate that enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and that training-induced supercomplex formation does not confer enhancements in mitochondrial bioenergetics. Our study provides a new analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding and calling for careful reinterpretation of previous findings. Feasibility of Administering Human Pancreatic Cancer Chemotherapy in a Spontaneous Pancreatic Cancer Mouse Model BackgroundBoth modified FOLFIRINOX (mFFX) and gemcitabine/nab-paclitaxel chemotherapy regimens have been shown to improve clinical outcomes in patients with pancreatic cancer, and are often used interchangeably as the standard of care. Preclinical studies often do not use these regimens, since administering these multiagent approaches can be difficult. In this study, we assessed the feasibility of administering these two chemotherapy regimens in spontaneous pancreatic tumors using KPC mice with the ultimate goal of advancing preclinical studies.

MethodsKPC mice were created by breeding KrasLSL-G12D/+ to Trp53fl/fl;Ptf1Cre/+, resulting in KrasLSL-G12D/+;p53fl/+;Ptf1Cre/+ mice. At 14 weeks of age, mice were palpated for spontaneous tumor growth that was verified using ultrasounds. Mice with tumors under 15 mm in diameter were used. The mice were assigned to one of seven treatment regimens: 1 cycle of mFOLFIRINOX (FFX X1), 2 cycles of mFOLFIRINOX (FFX X2), 1 cycle of mFOLFIRINOX with 40Gy SBRT (FFX SBRT), 1 cycle of gemcitabine/nab-paclitaxel (GEM/AB X1), 2 cycles of gemcitabine/nab-paclitaxel (GEM/AB X2), 2 cycles of gemcitabine/nab-paclitaxel with 40Gy SBRT (GEM/AB SBRT), or no chemotherapy treatment (control).

ResultsIn total, 92 mice were included. The median OS in the FFX X2 group was slightly longer that the median OS in the FFX X1 group (15 days vs 11 days, P=0.003). Mice in the GEM/AB X2 group had longer OS when compared to mice in the GEM/AB X1 group (33.5 vs 13 days, P=0.001). Mice treated with chemotherapy survived longer than untreated control animals (median OS: 6.5 days, P <0.001). Moreover, in mice treated with chemotherapy, mice that received 2 cycles of GEM/AB X2 had the longest survival, while the FFX X1 group had the poorest OS (P <0.001). Lastly, chemotherapy followed by consolidative SBRT trended towards increased local control and survival.

ConclusionsWe demonstrate the utility and feasibility of clinicall relevant mFOLFIRINOX and gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer. High-throughput submicron-resolution microscopyof entire C. elegans populations under strongimmobilization by cooling cultivation plates Despite its profound impact on biology, most high-resolution in vivo microscopy approaches remain low throughput because current immobilization techniques require significant manual effort. We greatly accelerate imaging of the nematode Caenorhabditis elegans by implementing a simple cooling approach to easily immobilize entire populations directly on their cultivation plates. We optimize and characterize cooling immobilization. Counterintuitively, relatively warmer temperatures immobilize animals significantly more effectively than colder temperatures utilized in prior studies. This enhanced immobilization enables clear submicron-resolution fluorescence imaging, which is challenging to achieve with most current immobilization techniques. We demonstrate 64x magnification 3D imaging and timelapse recording of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. By obviating individual animal manipulation, our approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows. Destructive and constructive cheater suppression through quorum sensing The evolutionary consequences of quorum sensing in regulating bacterial cooperation are not fully understood. In this study, we reveal unexpected consequences of regulating public good production through quorum sensing on bacterial population dynamics, showing that quorum sensing can be a collectively harmful alternative to unregulated production. We analyze a birth-death model of bacterial population dynamics accounting for public good production and the presence of non-producing cheaters. Our model demonstrates that when demographic noise is a factor, the consequences of controlling public good production according to quorum sensing depend on the cost of public good production and the presence of non-public fitness benefits. When public good production is inexpensive, quorum sensing is a destructive alternative to unconditional production, in terms of the mean population extinction time. When costs are higher, quorum sensing becomes a constructive strategy for the producing strain, both stabilizing cooperation and decreasing the risk of population extinction. scJoint: transfer learning for data integration of atlas-scale single-cell RNA-seq and ATAC-seq Single-cell multi-omics data continues to grow at an unprecedented pace, and effectively integrating different modalities holds the promise for better characterization of cell identities. Although a number of methods have demonstrated promising results in integrating multiple modalities from the same tissue, the complexity and scale of data compositions typically present in cell atlases still pose a significant challenge for existing methods. Here we present scJoint, a transfer learning method to integrate atlas-scale, heterogeneous collections of scRNA-seq and scATAC-seq data. scJoint leverages information from annotated scRNA-seq data in a semi-supervised framework and uses a neural network to simultaneously train labeled and unlabeled data, enabling label transfer and joint visualization in an integrative framework. Using multiple atlas data and a biologically varying multi-modal data, we demonstrate scJoint is computationally efficient and consistently achieves significantly higher cell type label accuracy than existing methods while providing meaningful joint visualizations. This suggests scJoint is effective in overcoming the heterogeneity in different modalities towards a more comprehensive understanding of cellular phenotypes. Comparative proteome signatures of trace samples by multiplexed Data-Independent Acquisition Single cell transcriptomics has revolutionized our understanding of basic biology and disease. Since transcript levels often do not correlate with protein expression, it is crucial to complement transcriptomics approaches with proteome analyses at single cell resolution. Despite continuous technological improvements in sensitivity, mass spectrometry-based single cell proteomics ultimately faces the challenge of reproducibly comparing the protein expression profiles of thousands of individual cells. Here, we combine two hitherto opposing analytical strategies, DIA and Tandem-Mass-Tag (TMT)-multiplexing, to generate highly reproducible, quantitative proteome signatures from ultra-low input samples. While conventional, data-dependent shotgun proteomics (DDA) of ultra-low input samples critically suffers from the accumulation of missing values with increasing sample-cohort size, data-independent acquisition (DIA) strategies do usually not take full advantage of isotope-encoded sample multiplexing. We developed a novel, identification-independent proteomics data-analysis pipeline that allows to quantitatively compare DIA-TMT proteome signatures across hundreds of samples independent of their biological origin, and to identify cell types and single protein knockouts. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. These data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultra-low input samples, such as single cells. Transcripts switched off at the stop of phloem unloading highlight the energy efficiency of sugar import in the ripening V. vinifera fruit. Transcriptomic changes at the cessation of sugar accumulation in the pericarp of Vitis vinifera were addressed on single berries re-synchronized according to their individual growth patterns. The net rates of water, sugars and K+ accumulation inferred from individual growth and solute concentration confirmed that these inflows stopped simultaneously in the ripe berry, while the small amount of malic acid remaining at this stage was still being oxidized at low rate. Re-synchronized individual berries displayed negligible variations in gene expression among triplicates. RNA-Seq studies revealed sharp reprogramming of cell wall enzymes and structural proteins at the stop of phloem unloading, associated with an 80% repression of multiple sugar transporters and aquaporins on the plasma or tonoplast membranes, with the noticeable exception of H+/sugar symporters, that were rather weakly and constitutively expressed. This was verified in three genotypes placed in contrasted thermo-hydric conditions. The prevalence of SWEET suggests that electrogenic transporters would play a minor role on the plasma membranes of SE/CC complex and the one of the flesh, while sucrose/H+ exchangers dominate on its tonoplast. Cis-regulatory elements present in their promoters allowed to sort these transporters in different groups, also including specific TIPs and PIPs paralogs, and cohorts of cell wall related genes. Together with simple thermodynamic considerations, these results lead to propose that H+/sugar exchangers at the tonoplast, associated with a considerably acidic vacuolar pH, may exhaust cytosolic sugars in the flesh and alleviate the need for supplementary energization of sugar transport at the plasma membrane. Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. 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. HYBRID REACTION-DIFFUSION AND CLOCK-AND-WAVEFRONT MODEL FOR THE ARREST OF OSCILLATIONS IN THE SOMITOGENESIS SEGMENTATION CLOCK AO_SCPLOWBSTRACTC_SCPLOWThe clock and wavefront paradigm is arguably the most widely accepted model for explaining the embryonic process of somitogenesis. According to this model, somitogenesis is based upon the interaction between a genetic oscillator, known as segmentation clock, and a differentiation wavefront, which provides the positional information indicating where each pair of somites is formed. Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation. Cotterell et al. called it a progressive oscillatory reaction-diffusion (PORD) model. In the Meinhardt-PORD model, somitogenesis is driven by short-range interactions and the posterior movement of the front is a local, emergent phenomenon, which is not controlled by global positional information. With this model, it is possible to explain some experimental observations that are incompatible with the clock and wavefront model. However the Meinhardt-PORD model has some important disadvantages of its own. Namely, it is quite sensitive to fluctuations and depends on very specific initial conditions (which are not biologically realistic). In this work, we propose an equivalent Meinhardt-PORD model, and then amend it to couple it with a wavefront consisting of a receding morphogen gradient. By doing so, we get a hybrid model between the Meinhardt-PORD and the clock-and-wavefront ones, which overcomes most of the deficiencies of the two originating models.

Somitogenesis, the process by which somites are formed, is an essential developmental stage in many vertebrates. This process occurs with a strikingly regular periodicity, that is preserved among embryos of a single species. The clock and wavefront paradigm is arguably the most widely accepted model for explaining somitogenesis. However, it is incapable of explaining some experimental facts, like the appearance of somites in the absence of an external wavefront (i.e. a receding morphogen gradient). Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation, and called it a progressive oscillatory reaction-diffusion (PORD) model. The Meinhardt-PORD model tackles some of the deficiencies of the clock and wave-front models, but it has some serious issues of its own. In the present work, we introduce an equivalent Meinhardt-PORD model, and then amend it to couple it with a receding morphogen gradient. By doing so, we get a hybrid model that incorporates characteristics of the Meinhardt-PORD and clock-and-wavefront models. We show that this hybrid model under-goes a bifurcation, from a stable to an unstable limit cycle, as the value of the parameter accounting for a background regulatory input (associated to the receding morphogen gradient) decreases. This bifurcation allows the model to explain why somites can form in the absence of an external wavefront, reassesses the role of the receding morphogen gradient as a conductor for somitogenesis, and makes the model behavior robust to random fluctuations, as well as independent from specific initial conditions (the latter, are two of the weak points of the Meinhardt-PORD model). We argue that this findings provide convincing evidence that reaction-diffusion and positional information (receding morphogen gradient) mechanisms could work together in somitogenesis. Circulating SARS-CoV-2 variants B.1.1.7, 501Y.V2, and P.1 have gained ability to utilize rat and mouse Ace2 and altered in vitro sensitivity to neutralizing antibodies and ACE2-Ig The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to [~]15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.