TAP: Targeting and analysis pipeline for optimization and verification of coil placement in transcranial magnetic stimulation ObjectiveTranscranial magnetic stimulation (TMS) can modulate brain function via an electric field (E-field) induced in a region of interest (ROI) in the brain. The ROI E-field can be computationally maximized and set to match a specific reference using individualized head models to find the optimal coil placement and stimulus intensity. However, the available software lacks many practical features for prospective planning of TMS interventions and retrospective evaluation of the experimental targeting accuracy.

ApproachThe TMS targeting and analysis pipeline (TAP) software uses an MRI/fMRI-derived brain target to optimize coil placement considering experimental parameters such as the subjects hair thickness and coil placement restrictions. The coil placement optimization is implemented in SimNIBS 3.2, for which an additional graphical user interface (TargetingNavigator) is provided to visualize and adjust procedural parameters. The coil optimization process also computes the E-field at the target, allowing the selection of the TMS device intensity setting to achieve a specific E-field strength. The optimized coil placement information is prepared for neuronavigation software, which supports targeting during the TMS procedure. The neuronavigation system can record the coil placement during the experiment, and these data can be processed in TAP to quantify the accuracy of the experimental TMS coil placement and induced E-field.

Main resultsTAP was demonstrated in a study consisting of three repetitive TMS sessions in five subjects. TMS was delivered by an experienced operator under neuronavigation with the computationally optimized coil placement. Analysis of the experimental accuracy from the recorded neuronavigation data indicated coil location and orientation deviations up to about 2 mm and 2{degrees}, respectively, resulting in an 8% median decrease in the target E-field magnitude compared to the optimal placement.

SignificanceTAP supports navigated TMS with a variety of features for rigorous and reproducible stimulation delivery, including planning and evaluation of coil placement and intensity selection for E-field-based dosing.
Region-specific microRNA alterations in marmosets carrying SLC6A4 polymorphisms are associated with anxiety-like behavior. Psychiatric diseases such as depression and anxiety are multifactorial conditions, highly prevalent in western societies. Human studies have identified a number of high-risk genetic variants for these diseases. Among them, polymorphisms in the promoter region of the serotonin transporter gene (SLC6A4) have attracted much attention. However, due to the paucity of experimental models, molecular alterations induced by these genetic variants and how they correlate to behavioral deficits have not been examined. Marmosets have emerged as a powerful model in translational neuroscience to investigate molecular underpinnings of complex behaviors. Here, we took advantage of naturally occurring genetic polymorphisms in marmoset SLC6A4 gene that have been linked to anxiety-like behaviors. Using FACS-sorted cells from different brain regions, we revealed that marmosets bearing different SLC6A4 variants exhibit distinct microRNAs signatures in a region of the prefrontal cortex whose activity has been consistently altered in patients with depression/anxiety. We also identified DCC, a gene previously linked to these diseases, as a downstream target of the dysregulated microRNAs. Significantly, we showed that levels of both microRNAs and DCC in this region were highly correlated to anxiety-like behaviors as well as to the response to citalopram, a selective serotonin re-uptake inhibitor and widely prescribed anti-depressant. Our findings establish links between genetic variants, molecular modifications in specific cortical regions and complex behavioral/pharmacological responses, providing new insights into gene-behavior relationships underlying human psychopathology.
A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. 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. NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knockdown NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Further, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection. Phylogenomic analyses of the East Asian endemic Abelia (Caprifoliaceae) shed insights into the temporal and spatial diversification history with widespread hybridizations Background and AimsAbelia (Caprifoliaceae) is a small genus with five species, including one man-made hybrid and several natural hybrids. The genus has a discontinuous distribution in mainland China, Taiwan Island, and the Ryukyu islands, providing a model system to explore mechanisms of species dispersal in the East Asian flora. However, the current phylogenetic relationships within Abelia remain uncertain.

MethodsIn this study, we reconstructed phylogenetic relationships within Abelia using nuclear loci generated by target enrichment and plastomes from genome skimming. Divergence time estimation, ancestral area reconstruction, and ecological niche modelling (ENM) were used to examine the diversification history of Abelia.

Key ResultsWe found extensive cytonuclear discordance across the genus. By integrating lines of evidence from molecular phylogenies, divergence times, and morphology, we propose to merge A. macrotera var. zabelioides into A. uniflora. Network analyses suggested that there have been widespread and multiple hybridization events among Abelia species. These hybridization events may have contributed to the speciation mechanism and resulted in a high observed morphological diversity. The diversification of Abelia began in the early Eocene, followed by A. chinensis var. ionandra colonizing the island of Taiwan in the Middle Miocene. The ENM results suggested an expansion of climatically suitable areas during the Last Glacial Maximum and range contraction during the Last Interglacial. Disjunction between the Himalayan-Hengduan Mountain region (HHM) and the island of Taiwan is most likely the consequence of topographic isolation and postglacial contraction.

ConclusionsWe used genomic data to reconstruct the phylogeny of Abelia and found a clear pattern of reticulate evolution in the group. In addition, our results support shrinkage of postglacial range and the heterogeneity of the terrain have led to the disjunction of the mainland China-Taiwan island. This study provides important new insights into the speciation process and taxonomy of Abelia. SPOT-Contact-Single: Improving Single-Sequence-Based Prediction of Protein Contact Map using a Transformer Language Model MotivationAccurate prediction of protein contact-map is essential for accurate protein structure and function prediction. As a result, many methods have been developed for protein contact map prediction. However, most methods rely on protein-sequence-evolutionary information, which may not exist for many proteins due to lack of naturally occurring homologous sequences. Moreover, generating evolutionary profiles is computationally intensive. Here, we developed a contact-map predictor utilizing the output of a pre-trained language model ESM-1b as an input along with a large training set and an ensemble of residual neural networks.

ResultsWe showed that the proposed method makes a significant improvement over a single-sequence-based predictor SSCpred with 15% improvement in the F1-score for the independent CASP14-FM test set. It also outperforms evolutionary-profile-based methods TrRosetta and SPOT-Contact with 48.7% and 48.5% respective improvement in the F1-score on the proteins without homologs (Neff=1) in the independent SPOT-2018 set. The new method provides a much faster and reasonably accurate alternative to evolution-based methods, useful for large-scale prediction.

AvailabilityStand-alone-version of SPOT-Contact-Single is available at https://github.com/jas-preet/SPOT-Contact-Single. Direct prediction can also be made at https://sparks-lab.org/server/spot-contact-single. The datasets used in this research can also be downloaded from the GitHub.

Contactjaspreetsingh2@griffithuni.edu.au, k.paliwal@griffith.edu.au, and zhouyq@szbl.ac.cn

Supplementary informationSupplementary data are available at Bioinformatics online. 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. The maize gene maternal derepression of r1 (mdr1) encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in endosperm Demethylation of transposons can activate expression of nearby genes and cause imprinted gene expression in endosperm, and it is hypothesized to lead to expression of transposon siRNAs that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting are its main function in endosperm. 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. R7 photoreceptor axon targeting depends on the relative levels of lost and found expression in R7 and its synaptic partners As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections. Learning and motor inhibitory control in crows and domestic chickens Cognitive abilities allow animals to navigate through complex, fluctuating environments. In the present study, we tested the performance of a captive group of eight crows, Corvus corone, and ten domestic chickens, Gallus gallus domesticus, in the cylinder task, as a test of motor inhibitory control, and reversal learning as a measure of learning ability and behavioural flexibility. Four crows and nine chickens completed the cylinder task, eight crows and six chickens completed the reversal learning experiment. Crows performed better in the cylinder task compared to chickens. In the reversal learning experiment, species did not significantly differ in the number of trials until learning criterion was reached. Performance in the reversal learning experiment did not correlate with performance in the cylinder task in chickens. Our results suggest crows to possess better motor inhibitory control compared to chickens. In contrast, learning performance in a reversal learning task did not differ between the species, indicating similar levels of behavioural flexibility. Interestingly, we describe notable individual differences in performance. We stress the importance not only to compare cognitive performance between species but also between individuals of the same species when investigating the evolution of cognitive skills. Liberation from fibrogenesis or tumorigenesis via cellular senescence by a novel small molecule BackgroundUncontrolled proliferative diseases such as fibrosis or cancer can be fatal. We previously found that a compound containing a chromone scaffold, ONG41008, had potent anti-fibrotic effects in diseased human lung myofibroblasts but not normal human lung fibroblasts.

MethodsWe investigated the effects of ONG41008 on tumor cells, and compared these effects with those in pathogenic myofibrotic cells and normal fibroblasts cells.

FindingsStimulation of A549 lung carcinoma epithelial cells with ONG41008 resulted in cellular senescence, indicating that dysregulated cell proliferation is common to fibrotic cells and tumor cells. Replicative senescence of A549 cells resulted in multinucleation, which was followed by oncogene-induced senescence. There was significant upregulation of expression and nuclear translocation of p-TP53 and p16 in ONG41008-treated A549 cells, and all cells died after 72 hr. Similar effects occurred after ONG41008 treatment in several human aggressive cancer cell lines such as PANC1, MCF7, PC3, or primary non-small cell lung carcinoma cells. Unlike cisplatin, ONG41008 was not toxic to normal human lung fibroblasts or primary prostate epithelial cells, suggesting ONG41008 can distinguish the intracellular microenvironment between normal cells and aged or diseased cells. This effect might occur as a result of the increased NAD/NADH ratio or increased lactate dehydrogenase levels in aged or diseased cells.

InterpretationTo our best knowledge, this is the first study to show that a small molecule can arrest uncontrolled proliferation during fibrogenesis or tumorigenesis in vitro. ONG41008 could be a potential drug for a broad range of fibrotic or tumorigenic diseases.

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSThe notion that aging is a disease and that diseases occur as a consequence of aging was first put forward by David Sinclair and colleagues (Aging Cell;14(4):497-510). There are biological systems that provide evidence for this notion; for example, somatic cells can revert to embryonic cells, producing younger somatic cells. This phenomenon underlies induced pluripotent stem cells. Another example is that some types of jellyfish can live forever. These examples suggest that a counter-aging program exists in animals. Human diseases are the manifestations of cell aging generated by the accumulation of somatic mutations. Aged and pathogenic cells are senescent, so a drug that specifically targeted senescent cells might initiate a cellular program that could ameliorate age-associated disease. Indeed, the kinase inhibitor dasatinib induces cellular senescence (Clin Ther 2007 29:2289-2308). In 2017, two drugs that target senescent cells were identified: quercetin and fisetin. These drugs selectively kill senescent cells, and are referred to as senolytic drugs or senotherapeutics (Aging 2017 8;9(3):955-963). Although it is established that senescent cells accumulate in cancer and idiopathic pulmonary fibrosis (Nat Commun 2017 23;8 :14532), the effect of senolytic drugs in these diseases is largely unknown.

Added value of this studyThis study characterized a novel drug, termed ONG41008, which was found to have both senogenic and senolytic effects in cell-based assays. ONG41008 induced senescence in myofibroblasts and several cancer cell lines representative of aggressive human cancers, which was followed by cell death. Importantly, ONG41008 exhibited essentially no toxicity on normal human lung fibroblasts or primary prostate epithelial cells.

Implication of all the evidenceBased on our results, we believe that ONG41008 is a potent inducer of cellular senescence (replicative senescence and oncogene-induced senescence) and causes arrest of uncontrolled, pathogenic proliferation of myofibroblasts or cancer cells. Response of soil and vegetation in a warm-temperate Pine forest to intensive biomass harvests, phosphorus fertilisation, and wood ash application. Background and AimsConcerns about climate change and carbon economy have prompted the promotion of alternative energy sources, including forest-based bioenergy. An evaluation of the environmental consequences of intensive harvests (stumps and roots, and also branches and foliage) for energy wood supply, and use of wood-ash recycling as a compensatory practice, helps in the evaluation of the use of forest biomass for energy production.

MethodsWe made use of records from a split-plot experimental site crossing four different intensities of biomass harvesting (Stem-Only Harvest [SOH], Aboveground Additional Harvest [AAH], Belowground Additional Harvest [BAH], and Whole-Tree Harvest [WTH]) and three compensation methods (control [C], wood ash application [A] and phosphorus fertilisation [P]) to evaluate, in the 11-years-old stand (maritime pine: Pinus pinaster) that followed the biomass exports of the former stand, their effects on nutrient budgets, tree growth, soil fertility, chemical properties and soil carbon. This site is located in a forest on a poor soil, under a warm temperate climate (SW France).

Key resultsDespite their low additional biomass exports (+10% for AAH to +34% for WTH), the non-conventional harvest practices exported much higher quantities of nutrients than the conventional SOH technique (for example +145% for N and K in WTH). Consequently, these treatments had negative effects on the soil nutritive status. Additional biomass harvests impacted the soil organic matter content, with negative effects on P-organic, soil cation exchange capacity, exchangeable Ca, and most extractible nutrients. However, data suggested that tree growth and foliage nutrient content had not yet been significantly impacted by harvest treatments, whereas tree nutritional status was improved by P-fertiliser or wood ash. As expected, we observed a positive effect of wood ash application on soil pH and nutrient content but, like additional harvests, wood ash application decreased the pool of soil organic carbon (~10% of the initial stock with ~7% of N-total losses).

ConclusionsOverall, this factorial experiment showed that exporting more forest biomass due to the additional harvesting of tree canopies, stumps and roots had negative consequences on the ecosystem biogeochemistry. Additional harvests have aggravated the poverty of the already oligotrophic soil, and decreased the soil organic carbon content. Importantly, applying nutrients as fertiliser or wood ash did not compensate for all the negative impacts of biomass exports and the method of wood ash recycling in forests could even decrease the soil organic carbon. IRGM/Irgm1 Aggravates Progression of Atherosclerosis by Inducing Macrophage Apoptosis through the MAPK Signaling Pathway AimsAtherosclerosis underlies most cardiovascular diseases, among which acute coronary syndrome (ACS) caused by plaque rupture (PR) often leads to death. Immune-related GTPases (IRGM/Irgm1) have been extensively studied in inflammatory diseases, but their role in atherosclerosis is unclear. Determining how IRGM/Irgm1 promotes atherosclerotic plaque vulnerability will provide information for new biomarkers and/or therapeutic targets.

Methods and resultsWe identified ruptured and unruptured plaques by optical coherence tomography, and found that serum IRGM was highly expressed in patients with ST-segment elevation myocardial infarction in PR. We used ApoE-/-Irgm1+/+, ApoE-/-Irgm1+/- mice and chimeric mice to establish a model of advanced atherosclerosis. The results of pathological experiments showed that Irgm1 caused plaque necrosis. The ratio of neutral lipids and cholesterol crystals increases, while the content of collagen fibers decreases, aggravating the destabilization of atherosclerotic plaques. In vitro, we used multiple approaches to confirm that Irgm1 promotes macrophage apoptosis by promoting the production of reactive oxygen species and activating the MAPK signaling pathway.

ConclusionsIRGM may be a potential risk factor for PR. Mechanistic studies have shown that IRGM/Irgm1 contributes to the formation and rupture of fragile plaques. This is partly mediated by the induction of macrophage apoptosis via the MAPK signaling pathway. IRGM may offer new strategies for early treatment of ACS.

Translation viewOur findings indicate that IRGM/Irgm1 contributes to formation and rupture of vulnerable plaques. It suggests that IRGM may provide a new target for the early treatment of ACS. Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. The RAG1 Ubiquitin Ligase Domain Enhances Assembly and Selection of T Cell Receptor Genes to Restrain the Autoimmune Hazard of Generating T Cell Receptor Diversity RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte antigen receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. However, this process creates highly self-reactive cells that must be properly selected to suppress autoimmunity. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report that mice with a mutation that inactivates the RAG1 ubiquitin ligase in vitro exhibit modestly reduced thymic cellularity, decreased assembly and altered repertoires of T cell receptor (TCR) {beta} and  genes in thymocytes, and impaired thymocyte developmental transitions that require the assembly of TCR{beta} or  genes and signaling by their proteins. These RAG1 mutant mice also exhibit less efficient positive selection and superantigen-mediated negative selection of conventional {beta} T cells, 2) impaired differentiation of iNKT lineage {beta} T cells, and 3) CD4+ {beta} T cells with elevated autoimmune potential. Our findings demonstrate that the RAG1 ubiquitin ligase domain functions in vivo to stimulate the assembly and selection of TCR{beta} and TCR genes, thereby establishing replete diversity of {beta} TCRs and {beta} T cell lineages while restraining the inherent autoimmune hazard of generating diverse antigen specificities. An amino residue that guides the correct photoassembly the water-oxidation complex but is not required for high affinity Mn2+ binding The assembly of the Mn4O5Ca cluster of the photosystem II (PSII) starts from the initial binding and photooxidation of the first Mn2+ at a high affinity site (HAS). Recent cryo-EM apo-PSII structures reveal an altered geometry of amino ligands in this region and suggest the involvement of D1-Glu189 ligand in the formation of the HAS. We now find that Gln and Lys substitution mutants photoactivate with reduced quantum efficiency compared to the wild-type. However, the affinity of Mn2+ at the HAS in D1-E189K was very similar to the wild-type (~2.2 M). Thus, we conclude that D1-E189 does not form the HAS (~2.9 M) and that the reduced quantum efficiency of photoactivation in D1-E189K cannot be ascribed to the initial photooxidation of Mn2+ at the HAS. Besides reduced quantum efficiency, the D1-E189K mutant exhibits a large fraction of centers that fail to recover activity during photoactivation starting early in the assembly phase, becoming recalcitrant to further assembly. Fluorescence relaxation kinetics indicate on the presence of an alternative route for the charge recombination in Mn-depleted samples in all studied mutants and exclude damage to the photochemical reaction center as the cause for the recalcitrant centers failing to assemble and show that dark incubation of cells reverses some of the inactivation. This reversibility would explain the ability of these mutants to accumulate a significant fraction of active PSII during extended periods of cell growth. The failed recovery in the fraction of inactive centers appears to a reversible mis-assembly involving the accumulation of photooxidized, but non-catalytic high valence Mn at the donor side of photosystem II, and that a reductive mechanism exists for restoration of assembly capacity at sites incurring mis-assembly. Given the established role of Ca2+ in preventing misassembled Mn, we conclude that D1-E189K mutant impairs the ligation of Ca2+ at its effector site in all PSII centers that consequently leads to the mis-assembly resulting in accumulation of non-catalytic Mn at the donor side of PSII. Our data indicate that D1-E189 is not functionally involved in Mn2+ oxidation\binding at the HAS but rather involved in Ca2+ ligation and steps following the initial Mn2+ photooxidation. Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment. BackgroundMenthol, through its cooling and pleasant sensory effects, facilitates smoking and tobacco product initiation, resulting in the high popularity of mint/menthol-flavored E-cigarettes. More recently, E-cigarette vendors started marketing synthetic cooling agents as additives that impart a cooling effect but lack a characteristic minty odor. Knowledge about content of synthetic coolants in US-marketed E-cigarette products and associated health risks is limited.

MethodsE-liquid vendor sites were searched with the terms "koolada", "kool/cool", "ice", or WS-3/WS-23, denoting individual cooling agents, and relevant refill E-liquids were purchased. "Ice" flavor varieties of Puffbar, the most popular disposable E-cigarette brand, were compared with non-"Ice" varieties. E-liquids were characterized, and synthetic coolants quantified using GC/MS. Margin of exposure (MOE), a risk assessment parameter, was calculated to assess the risk associated with synthetic coolant exposure from E-cigarette use.

ResultsWS-3 was detected in 24/25 refill E-liquids analyzed. All Puffbar flavor varieties contained either WS-23 (13/14) or WS-3 (5/14), in both "Ice"- and non-"Ice" flavors. Modeling consumption of WS-3 from vaped E-liquids, resulted in MOEs below the safe margin of 100 for most daily use scenarios. MOEs for WS-23 were <100 for 10/13 Puffbar flavors in all use scenarios. Puffbar power specifications are identical to Juul devices.

ConclusionsSynthetic cooling agents (WS-3/WS-23) were present in US-marketed E-cigarettes, at levels that may result in consumer exposures exceeding safety thresholds set by regulatory agencies. Synthetic coolants are not only found in mint-or menthol-flavored products, but also in fruit- and candy-flavored products, including popular disposable E-cigarette products such as Puffbar.

ImplicationsSynthetic cooling agents are widely used in "kool/cool"- and "ice"-flavored E-liquids and in E-liquids without these labels, both as a potential replacement for menthol or to add cooling  notes to non-menthol flavors. These agents may be used to bypass current and future regulatory limits on menthol content in tobacco products, and not just E-cigarettes. Since synthetic cooling agents are odorless, they may not fall under the category of "characterizing flavor", potentially circumventing regulatory measures based on this concept. Regulators need to consider the additional health risks associated with exposure to synthetic cooling agents. 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. Modeling microRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses. Activation of the type 3 secretion system of enteropathogenic E. coli leads to remodeling of its membrane composition and function The cell envelope of gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterial-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic E. coli (EPEC). The EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in bacterial envelope upon attachment to host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism towards the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase pldA and the acyltransferase ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant to abnormal synthesis of O-antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacteria cell size and Zeta potential, and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild type EPEC to the antibiotic.

ImportanceThe characterization of EPEC membrane lipid metabolism upon attachment to host is an important step towards a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent life style. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and show that it is associated with increased cell permeability and higher sensitivity to vancomycin.

To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system. The coiled-coil domain of E. coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division The FtsLB complex is a key regulator of bacterial cell division, existing in either an off or on state which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a "detuned" structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction. Transcriptional regulation of HSCs in Aging and MDS reveals DDIT3 as a Potential Driver of Dyserythropoiesis Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in elderly individuals. In this work, we analyzed the transcriptome of human HSCs purified from young and elderly healthy donors, as well as MDS patients, identifying transcriptional alterations following eight different patterns of expression. While aging-associated lesions seemed to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detected the upregulation of the transcription factor DDIT3. Overexpression of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis. Such effect was associated with downregulation of transcription factors required for normal erythropoiesis, and with a failure in the activation of their transcriptional programs. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia was able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing MDS patients.

STATEMENT OF SIGNIFICANCEThis study defines how human aging and MDS development are characterized by transcriptional alterations in HSCs that follow different patterns, some of which may contribute to myeloid transformation. Among them, we demonstrate how MDS-specific upregulation of DDIT3 in HSCs induces dyserythropoiesis, while its knockdown in HSPCs from MDS patients restores proper erythroid differentiation. A coupled mechano-biochemical framework for root meristem morphogenesis Plants grow roots to adjust their bodies to dynamic changes in the surrounding environment. How do plant roots emerge from the stem cell reservoir during embryogenesis is however poorly understood. Here, we present a bottom-up strategy to address this challenge by combining empirical observations with advanced computer modeling techniques. We demonstrate that the anisotropy of root growth results from differential growth rates of adjacent tissues, whereas the root meristem development incorporates a multi-level feedback loop between complex transport network of phytohormone auxin, auxin-dependent cell growth and cytoskeleton rearrangements. In silico model predictions are in close agreement with in vivo patterns of anisotropic growth, auxin distribution, and cell polarity, as well as several root phenotypes caused by chemical, mechanical, or genetic perturbations. Our findings reveal a minimal set of design principles connecting tissue mechanics, cell anisotropy, and directional transport that are sufficient for self-organization of the root meristem shape. A mobile auxin signal transported through immobile cells orchestrates polarity and growth mechanics to instruct the morphogenesis of an independent organ. Increased early sodium current causes familial atrial fibrillation and dampens effect of flecainide Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the -subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF. Designing a new murine model carrying the Scn5a-M1875T mutation enabled us to study the effects of the Nav1.5 mutation in detail.

Atrial cardiomyocytes from newly generated Scn5a-M1875T+/- mice showed a selective increase in the early (peak) cardiac sodium current, measured by patch clamp. Atrial microelectrode recordings revealed larger action potential amplitudes and a faster peak upstroke velocity. Conduction was studied using optical mapping. Conduction slowing by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/- compared to wildtype atria. In vivo analysis using electrocardiograms and echocardiography, as well as cardiac histology, excluded overt hypertrophy or heart failure in young adult Scn5a-M1875T+/- mice.

The Scn5a-M1875T point mutation causes a cardiac sodium channel gain-of-function. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability and atrial fibrillation.

Statement of SignificanceThe observed changes induced by this point mutation suggest a selective increase in peak sodium current as a cause of familial AF. Our findings provide a possible explanation for the variable effectiveness of sodium channel blockers in patients with AF. Carriers of such sodium channel gain-of-function mutations may benefit more from tailored treatments.