Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.
3D Reconstructions of Mouse Skeletal Muscle Reveal a Decrease in the MICOS Complex and Altered Mitochondrial Networks Background: Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. Studies of bioenergetics and protein turnover show that mitochondria mediate this decline in function. Mitochondria cristae are essential for the production of ATP. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging still requires further elucidation Further, it is not completely understood how 3D mitochondrial networks and the specialization of mitochondria are altered during aging. Methods: We measured changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. Nanotunnels were also measured through 3D reconstruction. CRISPR/Cas9 KO was performed to examine changes in mitochondria upon loss of the mitochondrial contact site and cristae organizing system (MICOS) complex and optic atrophy 1 (OPA-1). Metabolomics were used to find key metabolite pathways changed upon off of the MICOS complex. Results: We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle during aging. We also found an association of OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Further, the loss of the MICOS complex was linked with decreased oxidative capacity, and altered mitochondrial metabolism. Conclusions: To our knowledge, studies of mitochondrial changes in skeletal muscle throughout aging remains limited. 3D reconstructions of nanotunnels elucidated novel patterns of mitochondrial aging in skeletal muscle. MICOS proteins decreased with age and mitochondrial morphology was similar between aged skeletal muscle and that of young mice with MICOS protein loss. In tandem, our data suggest a relationship between the MICOS complex and aging, which could be potentially linked to disease states with additional 3D reconstruction.
Using Obviously-Related Instrumental Variables to Increase the Predictive Power of Polygenic Scores The conventional way of boosting the predictive power of polygenic scores is to increase the GWAS sample size by meta-analyzing GWAS results of multiple cohorts. In this paper, we challenge this convention. Through simulations, we show that Instrumental Variable (IV) regression using two polygenic scores constructed from independent GWAS summary statistics outperforms the typical Ordinary Least Squares (OLS) model employing a single meta-analysis based polygenic score in terms of bias, root mean squared error, and statistical power. We verify the empirical validity of the simulations by predicting educational attainment (EA) and height in a sample of siblings from the UK Biobank. We show that IV regression between-families approaches the SNP-based heritability, and improves the predictive power of polygenic scores by 12% (height) to 22% (EA). Furthermore, IV regression within-families provides the tightest lower bound for the direct genetic effect, increasing the lower bound for EA from 2.0% to 3.4%, and for height from 28.9% to 37.7%. Transcriptional and Functional Activity of Hemangiosarcoma Support Bone Marrow Nurse Cell Ontogeny Hemangiosarcoma and angiosarcoma are soft tissue sarcomas of malignant blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs that support the niche, enabling progression of canine hemangiosarcoma and human angiosarcoma. Here, we show that the transcriptional landscape of canine hemangiosarcoma and human angiosarcoma included comparable angiogenic and inflammatory programs. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells, a trait that was not observed in xenografts from canine osteosarcoma and lymphoma. In some cases, the xenografted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. We did not uncover a definitive transmissible etiology, but our data indicate that transcriptional programs of hemangiosarcoma cells resemble those of hematopoietic nurse cells, and these malignant cells support expansion and differentiation of human hematopoietic progenitors. We conclude that canine hemangiosarcoma, and possibly human angiosarcoma, originate from nurse cells that make up the stromal bone marrow niche, and that these cells may also support the growth of hematopoietic tumors. Metabarcoding faecal samples to investigate spatiotemporal variation in the diet of the endangered Westland petrel (Procellaria westlandica) As top predators, seabirds can be indirectly impacted by climate variability and commercial fishing activities through changes in marine communities. However, high mobility and foraging behaviour enables seabirds to exploit prey distributed patchily in time and space. This capacity to adapt to environmental change can be described through the study of their diet. Traditionally, the diet of seabirds is assessed through the morphological identification of prey remains in regurgitates. This sampling method is invasive for the bird and limited in terms of taxonomic resolution. However, the recent progress in DNA-based approaches is now providing a non-invasive means to more comprehensively and accurately characterize animal diets. Here, we used a non-invasive metabarcoding approach to characterize the diet of the Westland petrel (Procellaria westlandica), which is an endangered burrowing species, endemic to the South Island of New Zealand. We collected 99 fresh faecal samples at two different seasons and in two different sub-colonies. Our aims were to describe the diet of the Westland petrel, investigate seasonal and spatial variation in the petrels diet, and assess potential impacts of the New Zealand fishery industry. We found that amphipods were the most common prey, followed by cephalopods and fish. Our results could be the result of natural foraging behaviour, but also suggest a close link between the composition of prey items and New Zealands commercial fishing activities. In particular, the high abundance of amphipods could be the result of Westland petrels feeding on discarded fisheries waste (fish guts). Our results also showed significant differences in diet between seasons (before hatching vs chick-rearing season) and between sampling sites (two sub-colonies 1.5 km apart), indicating plasticity in the foraging strategy of the Westland petrel. Due to its non-invasive nature, metabarcoding of faecal samples can be applied to large numbers of samples to help describe dietary variation in seabirds and indicate their ecological requirements. In our example, dietary DNA (dDNA) provided valuable information regarding the dietary preferences of an iconic species in New Zealands unique biodiversity. dDNA can thus inform the conservation of endangered or at-risk species that have elusive foraging behaviours. 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. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Reliance of neuronal gene expression on cohesin scales with chromatin loop length Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in primary cortical neurons. Immediate early genes remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the Immediate early gene Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterised by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts. A new method for the sampling and preservation of placental specimens in low-resource settings for the identification of P. falciparum and analysis of nucleic acids Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope(R) hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater and RNA-seq. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Limited Phylogenetic Overlap Between Fluoroquinolone-Resistant Escherichia coli Isolated on Dairy Farms and those Causing Bacteriuria in Humans Living in the Same Geographical Region SynopsisO_ST_ABSBackgroundC_ST_ABSOur primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms were a significant cause of bacteriuria in humans living in the same 50 x 50 km geographical region located in South West England. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms.

MethodsFQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularisation approach.

ResultsOf 489 FQ-R human isolates, 255 were also 3rd generation cephalosporin-resistant (3GC-R), with strong genetic linkage between aac(6)Ib-cr and blaCTX-M-15. We identified possible farm-to-human sharing for pairs of ST744 and ST162 isolates, but core genome SNP distances (71 and 63, respectively) were smaller in pairs of ST744 and ST162 isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli while total dry cow therapy use showed a negative association.

ConclusionsThis work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population, however, this appears greater than observed for 3GC-R E. coli when studied in parallel. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli, and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm. 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. Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy As of October 2021, coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Due to high efficiency, remarkable stability, resilience to nebulization and low production cost, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status. Essential Regression - a generalizable framework for inferring causal latent factors from multi-omic human datasets High-dimensional cellular and molecular profiling of human samples highlights the need for analytical approaches that can integrate multi-omic datasets to generate predictive biomarkers and prioritized causal inferences. Current methods are limited by high dimensionality of the combined datasets, the differences in their data distributions and their integration to infer causal relationships. Here we present Essential Regression (ER), an interpretable machine learning approach for high-dimensional multi-omic datasets, that addresses these problems by identifying latent factors and their likely cause-effect relationships with the system-wide outcome/properties of interest. ER is a novel data-distribution-free latent-factor regression model that integrates multi-omic datasets and identifies latent factors significantly associated with an outcome. ER outperforms a range of state-of-the-art methods in terms of prediction performance on simulated datasets. ER can be coupled with probabilistic graphical modeling thereby strengthening the causal inferences. ER generates novel cellular and molecular predictions, using multi-omic human systems immunology datasets, pertaining to immunosenescence and immune dysregulation. State transitions in the statistically stable place cell population are determined by rate of perceptual change The hippocampus plays a central role in mammalian navigation and memory, yet an implementational understanding of the rules that govern the granularity of location encoding and the spatial-statistics of the population as a whole are lacking. We analysed large numbers of CA1 place fields recorded while rats foraged in environments up to 8.75 m2. We found that place cell propensities to form fields were proportional to open-field area, gamma-distributed, and conserved across environments. The properties of place fields varied positionally with a denser distribution of smaller fields near boundaries. Remarkably, field size and density were in a dynamic equilibrium, such that population-level activity statistics remained constant. We showed that the rate of transition through the statistically stable place cell population matched the rate of change in the animals visual scenes - demonstrating that the resolution of the spatial-memory system is bounded by perceptual information afforded by the environment. The N terminus of adhesion G protein-coupled receptor GPR126/ADGRG6 as allosteric force integrator The adhesion G protein-coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown.

In this study we provide this information by combining classic second messenger detection with single cell atomic force microscopy. We establish a monoclonal antibody targeting the N terminus to stimulate GPR126 and compare it to the activation through its known ECM ligands collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be viewed as an allosteric module that can fine-tune receptor activation in a context-specific manner. A multivariate view of cognitive performance reveals positive correlation in the Trinidadian Guppy (Poecilia reticulata) Cognitive variation is common among-individuals and can be consistent across time and context. From an evolutionary perspective, among-individual variation is important as a pre-requisite for natural selection and adaptive evolution. Selection is widely hypothesized to favor high cognitive performance but directional selection should erode variation over time, how then is cognitive variation maintained? As selection does not act on traits in isolation, covariance among specific cognitive traits and/or other aspects of phenotype (e.g. personality) could result in fitness trade-offs that are important in shaping evolutionary dynamics. Here we test this using Trinidadian guppies (Poecilia reticulata), using a multivariate approach by characterising the correlation structure among task-specific cognitive performance measures and a personality trait. We estimate the among-individual correlation matrix (ID) in performance across three cognitive tasks; colour association learning task; motor learning task; reversal learning task, and the personality trait  boldness measured as emergence time from a shelter. We found no support for trade-offs among performance in these tasks. Nor do we find evidence of hypothesised speed-accuracy trade-offs within the association learning task. Rather we find strong positive correlation structure in ID, with 57% of variation explained by the leading eigen vector. While noting that non-cognitive factors and assay composition may affect the structure of ID, we suggest our findings are consistent with the g-model of cognitive performance variation, in which a dominant axis of variation loads positively on all performance measures. Thus, we add to a growing body of support for general variation among individuals in animal cognitive ability. Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. Extensive transgressive gene expression in testis but not ovary in the homoploid hybrid Italian sparrow Hybridization can result in novel allelic combinations which can impact the hybrid phenotype through changes in gene expression. While mis-expression in F1 hybrids is well documented, how gene expression evolves in stabilized hybrid taxa remains an open question. As gene expression evolves in a stabilizing manner, break-up of co-evolved cis- and trans-regulatory elements could lead to transgressive patterns of gene expression in hybrids. Here, we address to what extent gonad gene expression has evolved in an established and stable homoploid hybrid, the Italian sparrow (Passer italiae). Through comparison of gene expression in gonads from individuals of the two parental species (i.e., house and Spanish sparrow) to that of Italian sparrows, we find evidence for strongly transgressive expression in male Italian sparrows - 22% of testis genes exhibit expression patterns outside the range of both parent species. In contrast, Italian sparrow ovary expression was similar to that of one of the parent species, the house sparrow (Passer domesticus). Moreover, the Italian sparrow testis transcriptome is 26 times as diverged from those of the parent species as the parental transcriptomes are from each other, despite being genetically intermediate. This highlights the potential for regulation of gene expression to produce novel variation following hybridization. Genes involved in mitochondrial respiratory chain complexes and protein synthesis are enriched in the subset that is over-dominantly expressed in Italian sparrow testis, suggesting that selection on key functions have molded the hybrid Italian sparrow transcriptome. 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. Hypertrophic Chondrocytes Serve as a Reservoir for Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage. Widespread redundancy in -omics profiles of cancer mutation states In studies of cellular function in cancer, researchers are increasingly able to choose from many -omics assays as functional readouts. Choosing the correct readout for a given study can be difficult, and which layer of cellular function is most suitable to capture the relevant signal may be unclear. In this study, we consider prediction of cancer mutation status (presence or absence) from functional -omics data as a representative problem. Since functional signatures of cancer mutation have been identified across many data types, this problem presents an opportunity to quantify and compare the ability of different -omics readouts to capture signals of dysregulation in cancer. The TCGA Pan-Cancer Atlas contains genetic alteration data including somatic mutations and copy number variants (CNVs), as well as several -omics data types. From TCGA, we focus on RNA sequencing, DNA methylation arrays, reverse phase protein arrays (RPPA), microRNA, and somatic mutational signatures as -omics readouts.

Across a collection of genes recurrently mutated in cancer, RNA sequencing tends to be the most effective predictor of mutation state. Surprisingly, we found that for many of the genes we considered, one or more other data types are approximately equally effective predictors. Performance was more variable between mutations than it was between data types for the same mutation, and there was often little difference between the top data types. We also found that combining data types into a single multi-omics model provided little or no improvement in predictive ability over the best individual data type. Based on our results, for the design of studies focused on the functional outcomes of cancer mutations, there are often multiple -omics types that can serve as effective readouts, although gene expression seems to be a reasonable default option. Beneficial effects of novel strains of Aureobasidium pullulans produced 1,3-1,6 β-glucans on non-esterified fatty acid levels in diabetic KKAy mice Obesity, metabolic syndrome, associated lipotoxicity and its cascade of events contribute to the majority of the burden related to non-communicable diseases globally. Preventive lifestyle changes aside, several beneficial effects have been reported in type II diabetes mellitus and dyslipidaemia patients with biological response modifier glucans (BRMG) produced as an exopolysaccharide by Aureobasidium pullulans. In this study, we compared two strains (AFO-202 and N-163) that produce beta glucans in alleviating lipotoxicity. This study was performed in obese diabetic mice model of KK-Ay mice, in four groups with six subjects in each group - Group 1: sacrificed on Day 0 for baseline values; Group 2: control (drinking water); Group 3: AFO-202 beta glucan--200 mg/kg/day; Group 4: N-163 beta glucan--300 mg/kg/day. The animals in groups 2-4 had the test solutions administered by gavage once daily for 28 consecutive days. Biochemical analyses were conducted of blood glucose, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol and non-esterified fatty acids (NEFA). Group 4 (N-163) had the lowest NEFA levels, as compared to the other groups, and marginally decreased triglyceride levels. The groups had no significant differences in blood glucose, HbA1c, triglycerides, or LDL and HDL cholesterol. N-163 produced by A. pullulans decreased NEFA in a diabetic mice model in 28 days. These results, although modest, warrant further in-depth research into lipotoxicity and associated inflammatory cascades in both healthy and disease affected subjects to develop novel strategies for prevention and management. Library-free BoxCarDIA solves the missing value problem in label-free quantitative proteomics The last decade has seen significant advances in the application of quantitative mass spectrometry-based proteomics technologies to tackle important questions in plant biology. The current standard for quantitative proteomics in plants is the use of data-dependent acquisition (DDA) analysis with or without the use of chemical labels. However, the DDA approach preferentially measures higher abundant proteins, and often requires data imputation due to quantification inconsistency between samples. In this study we systematically benchmarked a recently developed library-free data-independent acquisition (directDIA) method against a state-of-the-art DDA label-free quantitative proteomics workflow for plants. We next developed a novel acquisition approach combining MS1-level BoxCar acquisition with MS2-level directDIA analysis that we call BoxCarDIA. DirectDIA achieves a 33% increase in protein quantification over traditional DDA, and BoxCarDIA a further 8%, without any changes in instrumentation, offline fractionation, or increases in mass-spectrometer acquisition time. BoxCarDIA, especially, offers wholly reproducible quantification of proteins between replicate injections, thereby addressing the long-standing missing-value problem in label-free quantitative proteomics. Further, we find that the gains in dynamic range sampling by directDIA and BoxCarDIA translate to deeper quantification of key, low abundant, functional protein classes (e.g., protein kinases and transcription factors) that are underrepresented in data acquired using DDA. We applied these methods to perform a quantitative proteomic comparison of dark and light grown Arabidopsis cell cultures, providing a critical resource for future plant interactome studies. Our results establish BoxCarDIA as the new method of choice in quantitative proteomics using Orbitrap-type mass-spectrometers, particularly for proteomes with large dynamic range such as that of plants. Bioinformatic and mechanistic analysis of the palmerolide PKS-NRPS biosynthetic pathway from the microbiome of an Antarctic ascidian Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the [~]75 kb trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicohabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression. Visualization of loop extrusion by DNA nanoscale tracing in single human cells The spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensation and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random chromatin structures such as topologically associating domains2, 3. However, whether the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we developed LoopTrace, a fluorescence imaging workflow for high-resolution reconstruction of 3D genome architecture that conserves chromatin structure at the nanoscale and can resolve the 3D-fold of chromosomal DNA with better than 5-kb precision in single human cells. Our results show that the chromatin fibre behaves as a random coil up to the megabase scale and is further structured by contacts between sites that anchor loops. Our single cell folds reveal that such looping interactions are sparse and lead to a large heterogeneity of folds with one or two dynamically positioned loop bases as the main reproducible feature of megabase scale chromosomal regions. Clustering folds by their 3D conformations revealed a series of structures consistent with progressive loop extrusion between major anchor sites. Consistently, the looping interactions and their non-random positioning depend on the presence of the loop extrusion enzyme cohesin and its anchor protein CTCF, respectively. Our approach is scalable and will be instrumental to image the functional 3D architecture of the genome directly at the nanoscale.