Epitope-tagging of the endogenous murine BiP/GRP78/Hspa5 locus allows direct analysis of the BiP interactome and protein misfolding in vivo BiP/GRP78, encoded by the Hspa5 gene, is the major HSP70 family member in the endoplasmic reticulum (ER) lumen, and controls ER protein folding. The essential functions of BiP in facilitating proper protein folding are mainly mediated through its dynamic interaction with unfolded or misfolded client proteins, and by serving as a negative regulator of the Unfolded Protein Response. A mechanistic understanding of the dynamics of BiP interaction with its protein partners is essential to understand ER biology, and therefore, we have sought to develop a tractable model to study misfolded protein interaction with BiP. For this purpose, we have used homologous recombination to insert a 3xFLAG epitope tag into the endogenous murine Hspa5 gene, just upstream from the essential KDEL signal necessary for ER localization of BiP. Tagging BiP in this way did not alter Hspa5 expression under basal or ER-stress induced conditions in hepatocytes ex vivo or in fibroblasts. Furthermore, the tag did not alter the cellular localization of BiP or its functionality. All of these findings in primary tissue culture were also confirmed in vivo in livers of heterozygous mice harboring one WT and one FLAG-tagged Hspa5 allele. Hepatocyte-specific BiP-FLAG modification did not alter liver function or UPR signaling. Importantly, immunoprecipitation with anti-FLAG antibody completely pulled down FLAG-tagged BiP from lysates of BiP-FLAG expressing livers. Affinity purification-mass spectrometry (AP-MS) of BiP-FLAG protein complexes isolated from the BiP-FLAG-expressing livers of tunicamycin (TM)- and vehicle-treated mice revealed a marked increase in interaction of glycoproteins with BiP-FLAG in response to inhibition of N-glycosylation due to TM-treatment, validating utility of our BiP-Flag mice as a tool to identify ER misfolded proteins in vivo. Significantly, our AP-MS analysis also provided in vivo evidence demonstrating that BiP-FLAG binds to UPR transducers IRE1 and PERK under basal conditions but is released upon TM-treatment to activate UPR. We have also employed this mouse model to demonstrate that proinsulin in pancreatic {beta} cells misfolds and interacts with BiP-FLAG in healthy mice. In summary, we generated a novel model that can be used to investigate the BiP interactome in vivo under physiological and pathophysiological conditions in a cell type-specific manner. This tool provides, for the first time, an unbiased approach to identify unfolded and misfolded BiP-client proteins, and a new approach to study ER protein misfolding in a cell-type and temporal manner to uncover the role of BiP in many essential ER processes.
Loss of Acta2 in cardiac fibroblasts does not prevent myofibroblast differentiation or affect cardiac repair after myocardial infarction In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair in the infarcted area. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMA) that is assembled into stress fibers. However, the requirement of Acta2 in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair were still not known. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal survival rate after MI. Moreover, Acta2 deletion did not affect the function or overall histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT cardiac fibroblasts and Acta2-null cardiac myofibroblasts. Additional analysis identified that Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a unique compensatory increase in the transcription level of Actg2 and a possible increase in the protein abundance of cytoplasmic actin isoforms. In conclusion, the deletion of Acta2 does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increase in the expression of non-SMA actin isoforms and the functional redundancy between actin isoforms are likely able to compensate for the loss of Acta2 in cardiac myofibroblasts.
Force profile of the two-handed hardstyle kettlebell swing in novice older adults: an exploratory profile BackgroundUnderstanding the force profile of an exercise increases clinical confidence when assessing the benefits and potential risks of a prescribed exercise. This exploratory study presents the force profile of the hardstyle kettlebell swing in novice older adults and compares peak force with kettlebell deadlifts. These data will help inform healthcare providers and coaches who are considering prescribing kettlebell exercises for older adults.

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

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

ConclusionDuring kettlebell swings, there is negligible difference in normalised net peak force between novice males and females using the same absolute loads. Where ground reaction force is a therapeutic target, kettlebell swings with an 8 kg kettlebell could have similar effects to much heavier deadlifts (>24 kg). Kettlebell swings performed with lighter loads, could provider similar therapeutic value to much heavier deadlifts, and may be a more appealing, affordable, and convenient option for older adults. First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs Human in vitro models of neural tissue with controllable cellular identity, tunable microenvironment, and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing in jammed microgel supports (i.e., granular gels) holds great promise as it allows precise and programmable patterning of extremely soft and compliant tissue constructs. However, in contrast to the vast material landscape available for bulk hydrogels, granular printing support formulations are restricted to a handful of materials without the ability for facile adjustment of biofunctional properties of the cellular microenvironment. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, we present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise freeform patterning of human stem cells and consequent generation and long-term maintenance of mature subtype-specific neurons that extend projections within the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular channels. Due to its modularity, SHAPE biomanufacturing toolbox not only offers a solution for functional modeling of mechanically sensitive neural constructs, but also has potential to be applied to a wide range of biomaterials with different crosslinking mechanisms to model tissues and diseases where recapitulation of complex architectural features and topological cues is essential. 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. Bamboos flower after the return of almost the same sun-moon phasing as at seedling emergence All Asian woody bamboo species of economic importance are semelparous. They remain vegetative during time intervals that are specific to each species and range from three to 120 years, with notable concentrations around a series of values (3, 7-8, 14-17, 29-36, 42-48, 61-64, and 120 years). Then, they flower gregariously within a short period. Entire forests temporarily disappear during these periods, and the dates of these dramatic events have been recorded over the last 200 years.

While searching for a correlated environmental cue, I have found that the concentrations of flowering cycles were highly correlated with the series of successive returns of almost the same sun-moon phasing as at seedling emergence. On basis of knowledge on plant photoperiod sensitivity, I hypothesize that bamboo plants i) run a lunar cellular clock that is set at the full moon, ii) retain in their cellular memory the exact sun-moon phasing of the year of their emergence as seedlings, and iii) inhibit flowering until the occurrence of a unique, species-specific sun-moon phasing that is shifted by a precise amount from the sun-moon phasing at their emergence. Recent evidence of plant responses to lunar cycles supports this hypothesis, for which experimental evidence is now anticipated.

HighlightThe long-standing enigma of the triggering of the gregarious flowering of bamboos is possibly solved. Flowering would be inhibited until the occurrence of the species-specific sun-moon phasing. A biochemically-realisable relational model of the self-manufacturing cell As shown by Hofmeyr, the processes in the living cell can be divided into three classes of efficient causes that produce each other, so making the cell closed to efficient causation, the hallmark of an organism. They are the enzyme catalysts of covalent metabolic chemistry, the intracellular milieu that drives the supramolecular processes of chaperone-assisted folding and self-assembly of polypeptides and nucleic acids into functional catalysts and transporters, and the membrane transporters that maintain the intracellular milieu, in particular its electrolyte composition. Each class of efficient cause can be modelled as a relational diagram in the form of a mapping in graph-theoretic form, and a minimal model of a self-manufacturing system that is closed to efficient causation can be constructed from these three mappings using the formalism of relational biology. This Fabrication-Assembly or (F,A)-system serves as an alternative to Robert Rosens replicative Metabolism-Repair or (M,R)-system, which has been notoriously problematic to realise in terms of real biochemical processes. A key feature of the model is the explicit incorporation of formal cause, which arrests the infinite regress that plagues all relational models of the cell. The (F,A)-system is extended into a detailed relational model of the self-manufacturing cell that has a clear biochemical realisation. This (F,A) cell model allows the interpretation and visualisation of concepts such as the metabolism and repair components of Rosens (M,R)-system, John von Neumanns universal constructor, Howard Pattees symbol-function split via the symbol-folding transformation, Marcello Barbieris genotype-ribotype-phenotype ontology, and Tibor Gantis chemoton. Novel MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine -ketoglutarate (KG) is a central metabolic node with a broad influence on cellular physiology. The KG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other KG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG. TeamTree analysis: a new approach to evaluate scientific production Advances in science and technology depend on the work of research teams and the publication of results through peer-reviewed articles representing a growing socio-economic resource. Current methods to mine the scientific literature regarding a field of interest focus on content, but the workforce credited by authorship remains largely unexplored, and appropriate measures of scientific production are debated. Here, a new bibliometric approach named TeamTree analysis is introduced that visualizes the development and composition of the workforce driving a field. A new citation-independent measure that scales with the H index estimates impact based on publication record, genealogical ties and collaborative connections. This author-centered approach complements existing tools to mine the scientific literature and to evaluate research across disciplines. Acetyltransferases GCN5 and PCAF are required for B lymphocyte maturation in mice B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an open chromatin state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacks both GCN5 and PCAF in B cells. We found that double-deficient mice possess low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We conclude that both GCN5 and PCAF are required for B cell development in vivo. An hidden Markov model to estimate homozygous-by-descent probabilities associated with nested layers of ancestors Inbreeding results from the mating of related individuals and has negative consequences because it brings together deleterious variants in one individual. Genomic estimates of the inbreeding coefficients are preferred to pedigree-based estimators as they measure the realized inbreeding levels and they are more robust to pedigree errors. Several methods identifying homozygous-by-descent (HBD) segments with hidden Markov models (HMM) have been recently developed and are particularly valuable when the information is degraded or heterogeneous (e.g., low-fold sequencing, low marker density, heterogeneous genotype quality or variable marker spacing). We previously developed a multiple HBD class HMM where HBD segments are classified in different groups based on their length (e.g., recent versus old HBD segments) but we recently observed that for high inbreeding levels with many HBD segments, the estimated contributions might be biased towards more recent classes (i.e., associated with large HBD segments) although the overall estimated level of inbreeding remained unbiased. We herein propose a new model in which the HBD classification is modeled in successive nested levels with decreasing expected HBD segment lengths, the underlying exponential rates being directly related to the number of generations to the common ancestor. The non-HBD classes are now modeled as a mixture of HBD segments from later generations and shorter non-HBD segments (i.e., both with higher rates). The new model has improved statistical properties and performs better on simulated data compared to our previous version. We also show that the parameters of the model are easier to interpret and that the model is more robust to the choice of the number of classes. Overall, the new model results in an improved partitioning of inbreeding in different HBD classes and should be preferred. Dynamic and stationary brain connectivity during movie watching as revealed by functional MRI Spatially remote brain regions show synchronized activity as typically revealed by correlated functional MRI (fMRI) signals. An emerging line of research has focused on the temporal fluctuations of connectivity; however, its relationships with stationary connectivity have not been clearly illustrated. We examined dynamic and stationary connectivity when the participants watched four different movie clips. We calculated point-by-point multiplication between two regional time series to estimate the time-resolved dynamic connectivity, and estimated the inter-individual consistency of the dynamic connectivity time series. Widespread consistent dynamic connectivity was observed for each movie clip, which also showed differences between the clips. For example, a cartoon movie clip, Wall-E, showed more consistent of dynamic connectivity with the posterior cingulate cortex and supramarginal gyrus, while a court drama clip, A Few Good Men, showed more consistent of dynamic connectivity with the auditory cortex and temporoparietal junction, which might suggest the involvement of specific brain processing for different movie contents. In contrast, the stationary connectivity as measured by the correlations between regional time series was highly similar among the movie clips, and showed fewer statistically significant differences. The patterns of consistent dynamic connectivity could be used to classify different movie clips with higher accuracy than the stationary connectivity and regional activity. These results support the functional significance of dynamic connectivity in reflecting functional brain changes, which could provide more functionally relevant information than stationary connectivity. Feline tooth resorption findings from a questionnaire survey on 8115 Finnish cats and a case-control study for a more defined population drawn from the survey Tooth resorption (TR) is one of the most common dental diseases in cats. It is a painful disease characterised by progressive dental destruction, which eventually results in loss of teeth. The aetiology of the disease remains unclear, but associations with old age, breed, other oral and dental diseases, and certain environmental factors have been suspected. We collected health and environmental information of 8115 Finnish cats from 41 breeds through an online survey for cat owners to study TR in Finland with an observational study. The frequency of TR that the owner reported as veterinarian-diagnosed was 3.9% in the entire data (316/8115) and 15% in cats that the owner reported as having veterinarian-diagnosed oral or dental disease (316/2070). We determined the risk factors and breed variation of feline TR by logistic regression with the entire data and a case-control study in a more defined population drawn from the entire data. In the entire data, we defined those cats that the owners had reported as being diagnosed with TR by a veterinarian as TR-cats and compared them to all other cats. In the case-control study, we included only cats with owner-reported veterinarian-diagnosed oral and dental diseases. We defined cats with TR-diagnosis made under sedation as TR-cases. Other veterinarian-diagnosed TR-cats were excluded. Cats with other oral and dental diseases were used as controls. The model with the entire population included 308 TR-cats compared with 7508 cats, and the case-control study included 198 TR-cats and 1683 controls. Although the diagnoses were reportedly made by a veterinarian, the owner answered the survey and might have understood, remembered, or interpreted the diagnoses in a way that could affect the results. The results indicated an increasing risk of TR with age. Dental calculus, gingivitis, and periodontitis were potential risk factors for TR. These findings and the interaction between dental calculus and gingivitis suggest that inflammatory changes caused by dental calculus increase the risk of TR. Keeping food available constantly was a potential protective factor. We found that Cornish Rex, European, and Ragdoll breeds were at higher risk for TR. The observed differences between breeds highlight a genetic contribution. Glycosylated Antibiotics: New Promising Bacterial Efflux Pumps Inhibitors BackgroundAntimicrobial resistance is considered a major concern problem; bacteria have evolved mechanisms to overcome antibiotics action through evolutionary process. One main resistance mechanism that bacteria developed is the pumping of the antibiotics out of bacterial cells by transmembrane transporter proteins known as efflux pumps.

Materials and methodsTo overcome bacterial resistance guided by efflux pumps, efflux pumps inhibitors (EPIs) are small molecules that obstruct efflux pumps binding sites and its structural assembly leading to disability in the efflux pumps normal function, new EPIs which under the current study are created by modifying the chemical structure of most common antibiotics including Ampicillin, Penicillin, Chloramphenicol, Ciprofloxacin and Tetracycline, such antibiotics are modified by adding N-acetyl glucose amine moiety to acceptor OH group of the respective antibiotic, the newly modified antibiotics are glycosylated EPIs. To test the effectiveness of the new EPIs in inhibiting AcrB-TolC and MexA-OprM efflux pumps functions, ADME properties for all of glycosylated antibiotics have been measured through applying Lipinskis role of 5, docking and simulation studies have been included as well.

ResultsDocked glycosylated tetracycline has given the highest binding energy in the active sites of both pumps, with -9.4 against AcrB and -8.8 against MexA. The simulation study has confirmed the binding of the glycosylated tetracycline in the active sites of both pumps, as well as its stability during the biological dynamicity of both pumps (opening and closing channels).

ConclusionThe results validation requires a long simulation time about 50 ns or more which was un applicable due to cost limitation, however, the newly glycosylated antibiotics have promising results that might make it eligible as drug candidates to overcome bacterial resistance. The impact of housing conditions on mesenchymal stromal/stem cell populations differ between adipose tissue and skeletal muscle BackgroundIn pigs, the ratio between lean mass and fat mass in the carcass determines production efficiency and is strongly influenced by the number and size of cells in tissues. During growth, the increase in the number of cells results from the recruitment of different populations of multipotent mesenchymal stromal/stem cells (MSCs) residing in the tissues. We hypothesized that the impact of hygiene of housing conditions during growth on the proportions of MSCs in adipose tissue and skeletal muscle may differ between pigs with different residual feed intake (RFI), a measure of feed efficiency.

MethodsAt the age of 12 weeks, Large White pigs from two lines divergently selected for low and high RFI were housed in two contrasting hygiene conditions (good vs poor). After six weeks, pigs were slaughtered (n = 30; 5-9/group). Samples of subcutaneous adipose tissue and longissimus skeletal muscle were collected, and cells from the stromal vascular fraction (SVF), which includes mesenchymal stromal/stem cell populations, were isolated from each tissue. Adipose and muscle cell populations from the SVF were phenotyped by flow cytometry using antibodies that targeted different cell surface markers (CD45 to separate hematopoietic cells from MSCs; CD34, CD38, CD56 and CD140a to identify MSC populations with adipogenic and/or myogenic potential).

ResultsAdipose tissue and muscle shared some common MSC populations although MSC diversity was higher in muscle than in adipose tissue. In muscle, the CD45-CD56+CD34-CD140a+ and CD45-CD56+CD34+CD140a+ cell populations were abundant. Of these two cell populations, only the proportions of CD45-CD56+CD34+CD140a+ cells increased (P < 0.05) in pigs housed in poor hygiene as compared with pigs in good hygiene conditions. For the CD45-CD56-CD34- cell population, present in low proportion, there was an interaction between hygiene condition and genetic line (P < 0.05) with a decrease in low RFI pigs housed in poor hygiene conditions. In adipose tissue, the two abundant MSC populations were CD45-CD56-CD34- and CD45-CD56+CD34-. The proportion of CD45-CD56-CD34- cells increased (P < 0.05) whereas the proportion of CD45-CD56+CD34- tended to decrease (P < 0.1) in pigs housed in poor conditions. This study shows that the proportions of some MSC populations were affected by hygiene of housing conditions in a tissue-dependent manner in pigs of both RFI lines. It suggests that MSCs may play a significant role in adipose tissue and skeletal muscle homeostasis and may influence later growth and body composition in growing animals. Epigenetic scores for the circulating proteome as tools for disease prediction Protein biomarkers have been identified across many age-related morbidities. However, characterising epigenetic influences could further inform disease predictions. Here, we leverage epigenome-wide data to study links between the DNAm signatures of the circulating proteome and incident diseases. Using data from four cohorts, we trained and tested epigenetic scores (EpiScores) for 953 plasma proteins, identifying 109 scores that explained between 1% and 58% of the variance in protein levels after adjusting for known protein quantitative trait loci (pQTL) genetic effects. By projecting these EpiScores into an independent sample, (Generation Scotland; n=9,537) and relating them to incident morbidities over a follow-up of 14 years, we uncovered 137 EpiScore - disease associations. These associations were largely independent of immune cell proportions, common lifestyle and health factors and biological aging. Notably, we found that our diabetes-associated EpiScores highlighted previous top biomarker associations from proteome-wide assessments of diabetes. These EpiScores for protein levels can therefore be a valuable resource for disease prediction and risk stratification. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. The evolution of mechanisms to produce phenotypic heterogeneity in microorganisms In bacteria and other microorganisms, the cells within a population often show extreme phenotypic variation. Different species use different mechanisms to determine how distinct phenotypes are allocated between individuals, including coordinated, random, and genetic determination. However, it is not clear if this diversity in mechanisms is adaptive--arising because different mechanisms are favoured in different environments--or is merely the result of non-adaptive artifacts of evolution. We use theoretical models to analyse the relative advantages of the two dominant mechanisms to divide labour between reproductives and helpers in microorganisms. We show that coordinated specialisation is more likely to evolve over random specialisation in well-mixed groups when: (i) social groups are small; (ii) helping is more "essential"; and (iii) there is a low metabolic cost to coordination. We find analogous results when we allow for spatial structure with a more detailed model of cellular filaments. More generally, this work shows how diversity in the mechanisms to produce phenotypic heterogeneity could have arisen as adaptations to different environments. A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in five hours. The screen tested 1280 FDA-approved drugs and identified Pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacologic and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with Pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt-signaling. In contrast, HTR2C induced epithelial to mesenchymal transition (EMT) through activation of Wnt-signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs. Cell Cycle Delay, Pro-metaphase Arrest and C-metaphase Inducing Effects of Petroleum Ether Fraction of Leaf Aqueous Extract of Clerodendrum viscosum Vent. Clerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze cell cycle delay, pro-metaphase arrest, and c-metaphase inducing effects of the petroleum ether fraction (AQPEF) of leaf aqueous extract of C. Viscosum (LAECV). The LAECV was fractionated with petroleum ether and its metaphase arrest, cell cycle delay, and c-metaphase inducing activities were tested on A. cepa root tip cells. The AQPEF induced cell cycle delay, and colchicine like metaphase, c-metaphase, in A. cepa root tip cells. Thus, the present study explores AQPEF as an active fraction of LAECV having metaphase arresting, cell cycle delay, and c-metaphase inducing potentials. Damaraland mole-rats are not obligate cooperative breeders Damaraland mole-rats (Fukomys damarensis) are usually viewed as a eusocial or obligate cooperative breeder in which successful reproduction is dependent on help from closely related group members. However, because longitudinal studies of mole-rats in their natural environment are uncommon, the extent to which successful reproduction by breeders relies on assistance from other group members is unclear, and for non-breeders, the immediate and delayed fitness consequences of staying and helping are poorly understood. Using data from a 7-year field study of marked individuals, we investigate whether increasing group size confers reproductive and survival benefits to breeders and non-breeders, and explore the different routes through which individuals acquire breeding positions. We show that solitary natal dispersal was the most common route to breeding for both sexes and that the inheritance of dominant breeding positions was uncommon in both sexes. After dispersing, females typically settled alone in new burrow systems where they enjoyed high survival rates and remained in good body condition - often for several years - before being joined by males. In contrast to most obligately cooperative species, pairs of potential breeders reproduced successfully without helpers and experimentally formed pairs had the same reproductive success as larger established groups. Though larger breeding groups recruited slightly more pups on average, our data suggest that neither survival nor reproduction depend on the presence of non-breeding helpers, indicating that Damaraland mole-rats are not obligate cooperative breeders. We suggest that extended philopatry and group living in Damaraland mole-rats have evolved because of the high costs and constraints of dispersal rather than because of strong indirect benefits accrued through cooperative behaviour and that similarities between their breeding systems and those of obligatorily eusocial insects have been over-emphasized.

Significance StatementThe social mole-rats are often considered eusocial mammals in which successful reproduction depends on assistance from non-breeding helpers. In this study we show that in wild Damaraland mole-rats, the presence of non-breeders is associated with both costs and benefits and that nascent breeding pairs show high reproductive success despite the lack of non-breeding helpers. These findings indicate that Damaraland mole-rats are not obligate cooperative breeders and suggest that similarities between their breeding systems and those of obligatorily eusocial insects have been over-emphasized. A platform of patient-derived microtumors identifies therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment. Agonist efficiency from concentration-response curves: structural implications and applications Agonists are evaluated by a concentration-response curve (CRC), with a midpoint (EC50) that indicates potency, a high-concentration asymptote that indicates efficacy and a low-concentration asymptote that indicates constitutive activity. A third agonist attribute, efficiency ({eta}), is the fraction of binding energy that is applied to the conformational change that activates the receptor. We show that {eta} can be calculated from EC50 and the asymptotes of a CRC derived from either single-channel or whole-cell responses. For 20 agonists of skeletal muscle nicotinic receptors, the distribution of {eta} values is bimodal with population means at 51% (including ACh, nornicotine and DMPP) and 40% (including epibatidine, varenicline and cytisine). The value of {eta} is related inversely to the size of the agonists head-group, with high-versus low-efficiency ligands having an average volume of 70 [A]3 versus 102 [A]3. Most binding site mutations have only a small effect on ACh efficiency except for Y190A (35%), W149A (60%) and those at G153 (42%). If {eta} is known, the midpoint and high-concentration asymptote can be calculated from each other. Hence, an entire CRC can be estimated from the response to a single agonist concentration, and efficacy can be estimated from EC50 of a CRC that has been normalized to 1. Given {eta}, the level of constitutive activity can be estimated from a single CRC.

Statement of significanceReceptors are molecular machines that convert chemical energy from agonist binding into mechanical energy of a global conformational change that generates a cell response. Agonists are distinguished by their potency (proportional to affinity) and efficacy, but also by the efficiency at which their binding energy is applied to receptor activation. Here we show that agonist efficiency can be estimated from a single concentrationresponse curve, and estimate efficiencies of 20 nicotinic receptor agonists. These have a bimodal distribution with larger agonists belonging to the lower-efficiency population. We further show that mutations of some binding site residues alter efficiency, and that knowledge of agonist efficiency simplifies and extends dose-response curve analysis. Moonlighting proteins activate transformation in epigenetically-differentiated phase variants of multidrug-resistant Streptococcus pneumoniae Transformation of Streptococcus pneumoniae GPSC1 has enabled vaccine evasion and the acquisition of antibiotic resistance. Epigenetic phase variants of GPSC1 isolate RMV7, differentiated by rearrangements at the tvr restriction-modification locus, differed [~]100-fold in their transformation efficiency. This variation was recapitulated in knock-in mutants of the relevant tvr alleles. RNA-seq showed the difference was due to blocking of the early competence regulatory cascade. The more transformable variant upregulated expression of manLMN, encoding a carbon source importer. This was shown to be necessary for efficient competence induction, despite being dispensable for growth in rich media. Transformation was promoted by import of N-acetylglucosamine, which activated competence through an orthologue of the gram-negative competence regulator TfoX, and an enzyme likely involved in nucleotide-mediated signalling. A mobile genetic element was more active in the less transformable variant, which limited competence induction through inhibiting CIRCE motif binding by the chaperone regulator HrcA. Correspondingly, both heat shock and decreased Ca2+ concentrations reduced competence by limiting HrcA binding regulatory sequence motifs. Hence both ManLMN and HrcA moonlighted as activators of competence. Such proteins may be important in potentiating horizontal DNA exchange, as selection on their primary function likely constrains them from mutating into alleles that selfishly downregulate transformation. Variation in female songbird state determines signal strength needed to evoke copulation It is the female response to male signals that determines courtship success. In most songbirds, females control reproduction via the copulation solicitation display (CSD), an innate, stereotyped posture produced in direct response to male displays. Because CSD can be elicited in the absence of males by the presentation of recorded song, CSD production enables investigations into the effects of underlying signal features and behavioral state on female mating preferences. Using computer vision to quantify CSD trajectory in female brown-headed cowbirds (Molothrus ater), we show that both song quality and a females internal state predict CSD production, as well as the onset latency and duration of the display. We also show that CSD can be produced in a graded fashion based on both signal strength and internal state. These results emphasize the importance of underlying receiver state in determining behavioral responses and suggest that female responsiveness acts in conjunction with male signal strength to determine the efficacy of male courtship.