Trypanosomal variant surface glycoprotein expression in human African trypanosomiasis patients Trypanosoma brucei gambiense, an extracellular protozoan parasite, is the primary causative agent of human African trypanosomiasis. T. b. gambiense is endemic to West and Central Africa, where it is transmitted by the bite of infected tsetse flies. In the bloodstream of an infected host, the parasite evades antibody recognition by altering the Variant Surface Glycoprotein (VSG) that forms a dense coat on its cell surface through a process known as antigenic variation. Each VSG has a variable N-terminal domain exposed to the host and a less variable C-terminal domain that is partially hidden from host antibodies. Our lab developed VSG-seq, a targeted RNA-seq method, to study VSG gene expression in T. brucei. Studies using VSG-seq to characterize antigenic variation in a mouse model have revealed marked diversity in VSG expression within parasite populations, but this finding has not yet been validated in a natural human infection. Here, we used VSG-seq to analyze VSGs expressed in the blood of 12 patients infected with T. b. gambiense. The number of VSGs identified per patient ranged from one to 14; notably, two VSGs were shared by more than one patient. Analysis of expressed VSG N-terminal domain types revealed that 82% of expressed VSGs encoded a type B N-terminus, a bias not seen in datasets from other T. brucei subspecies. C-terminal types in T. b. gambiense infection were also restricted. These results demonstrate a bias either in the underlying VSG repertoire of T. b. gambiense or in this subspecies expression of VSGs during infection. This work demonstrates the feasibility of using VSG-seq to study antigenic variation in human infections and highlights the importance of understanding VSG repertoires in the field.

Author SummaryHuman African Trypanosomiasis is a neglected tropical disease primarily caused by the extracellular parasite Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their dense surface coat of Variant Surface Glycoprotein (VSG). Despite the important role of VSGs in prolonging infection, VSG expression during natural human infections is poorly understood. A better understanding of natural VSG gene expression dynamics can clarify the mechanisms that T. brucei uses to alter its VSG coat and improve how trypanosomiasis is diagnosed in humans. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that parasites in both natural and experimental infections express diverse sets of VSGs.
Retraction ATPase motors from three orthologous type IVa pilus systems support promiscuous retraction of the Vibrio cholerae competence pilus Bacterial surface appendages called type IVa pili (T4aP) promote diverse activities including DNA uptake, twitching motility, and virulence. These activities rely on the ability of T4aP to dynamically extend and retract from the cell surface. Dynamic extension relies on a motor ATPase commonly called PilB. Most T4aP also rely on specific motor ATPases, commonly called PilT and PilU, to dynamically and forcefully retract. Here, we systematically assess whether motor ATPases from three orthologous T4aP can functionally complement Vibrio cholerae mutants that lack their endogenous motors. We found that the PilT and PilU retraction ATPases from the three T4aP systems tested are promiscuous and promote retraction of the V. cholerae competence T4aP despite a high degree of sequence divergence. In contrast, the orthologous extension ATPases from the same T4aP systems were not able to mediate extension of the V. cholerae competence T4aP despite exhibiting a similar degree of sequence divergence. Also, we show that one of the PilT orthologs characterized does not support PilU-dependent retraction and provide some data to indicate that the C-terminus of PilT is important for PilU-dependent retraction. Together, our data suggest that retraction ATPases may have maintained a high degree of promiscuity for promoting retraction of T4aP, while extension ATPases may have evolved to become specific for their cognate systems.

IMPORTANCEOne way that bacteria interact with their environments is via hair-like appendages called type IVa pili (T4aP). These appendages dynamically extend and retract from the cell surface via the action of distinct ATPase motors. T4aP are present in diverse bacterial species. Here, we demonstrate that retraction motors from three T4aP are promiscuous, and capable of promoting retraction of a heterologous T4aP system. By contrast, the extension ATPase motors from these same T4aP systems are specific and cannot promote extension of a heterologous T4aP. Thus, these results suggest that T4aP extension may be more tightly regulated compared to T4aP retraction.
FcγR responses to soluble immune complexes are governed by solubility and size Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings. 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 AMIGO1 adhesion protein activates Kv2.1 voltage sensors Kv2 voltage-gated potassium channels are modulated by AMIGO neuronal adhesion proteins. Here, we identify steps in the conductance activation pathway of Kv2.1 channels that are modulated by AMIGO1 using voltage clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines. AMIGO1 speeds early voltage sensor movements and shifts the gating charge-voltage relationship to more negative voltages. The gating charge-voltage relationship indicates that AMIGO1 exerts a larger energetic effect on voltage sensor movement than apparent from the midpoint of the conductance-voltage relationship. When voltage sensors are detained at rest by voltage sensor toxins, AMIGO1 has a greater impact on the conductance-voltage relationship. Fluorescence measurements from voltage sensor toxins bound to Kv2.1 indicate that with AMIGO1, the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. We conclude that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors.

Statement of SignificanceKv2 potassium channels activate a potassium conductance that shapes neuronal action potentials. The AMIGO family of adhesion proteins modulate activation of Kv2 conductances, yet, which activation steps are modified is unknown. This study finds that AMIGO1 destabilizes the earliest resting conformation of the Kv2.1 voltage sensors to promote activation of channel conductance. Multiscale mechanistic modelling of the host defense in invasiveaspergillosis reveals leukocyte activation and iron acquisition as driversof infection outcome Aspergillus species are ubiquitous environmental molds, the spores of which are inhaled daily by most humans. Immunocompromised hosts can develop an invasive infection resulting in high mortality. There is, therefore, a pressing need for host-centric therapeutics for this infection. To address this need, we created a multi-scale computational model of the infection, focused on its interaction with the innate immune system and iron, a critical nutrient for the pathogen. The model, parameterized using published data, was found to recapitulate a wide range of biological features and was experimentally validated in vivo. Conidial swelling was identified as critical in fungal strains with high growth, whereas the siderophore secretion rate is an essential prerequisite for establishment of the infection in low-growth strains. In immunocompetent hosts, high growth, high swelling probability, and impaired leukocyte activation lead to a high conidial germination rate. Similarly, in neutropenic mice, high fungal growth was achieved through synergy between high growth rate, high swelling probability, slow leukocyte activation, and high siderophore secretion. In summary, the model reveals a small set of parameters related to fungal growth, iron acquisition, and leukocyte activation as key determinants of the fate of the infection. G protein-coupled estrogen receptor activation by Bisphenol-A disrupts protection from apoptosis conferred by estrogen receptors ERα and ERβ in pancreatic beta cells 17{beta}-estradiol protects pancreatic {beta}-cells from apoptosis via the estrogen receptors ER, ER{beta} and GPER. Conversely, the endocrine disruptor Bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ER and ER{beta}, or in dispersed islet cells from ER{beta} knockout (BERKO) mice. Yet, the ER and ER{beta} agonists, PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ER and ER{beta} form homodimers and heterodimers. Molecular dynamic simulations together with proximity ligand assay and coimmunoprecipitation experiments indicated that the interaction of BPA with ER and ER{beta} as well as the GPER activation by G1 decreased ER{beta} heterodimers. We propose that ER{beta} heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decrease in ER{beta} heterodimers leads to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

HighlightsO_LIPharmacological blockade and gene silencing of estrogen receptors ER, ER{beta} and GPER indicate that they are antiapoptotic in basal conditions.
C_LIO_LIGPER activation by G1 and BPA triggered apoptosis via a crosstalk with ER and ER{beta}.
C_LIO_LIBPA interaction with ER and ER{beta} as well as GPER activation decreased ER{beta} heterodimers, which was associated to increased apoptosis.
C_LIO_LIThis pathway represents a novel molecular initiating event underlying the pro-apoptotic effect of BPA
C_LIO_LIThe EndoC-{beta}H1 cell line may be a valid model of human {beta}-cells for identifying diabetogenic pollutants.
C_LI Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as the model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation.

By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoids partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate. Cellular feedback to organotelluranes displays modulation of antioxidant proteins gene expression Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds.



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org.highwire.dtl.DTLVardef@52bb82org.highwire.dtl.DTLVardef@19476d9org.highwire.dtl.DTLVardef@6aa99org.highwire.dtl.DTLVardef@1aa605e_HPS_FORMAT_FIGEXP  M_FIG C_FIG A thermodynamic model for water activity and redox potential in evolution and development Reactions involving water and oxygen are basic features of geological and biological processes. To understand how life interacts with its environment requires monitoring interactions with H2O and O2 at timescales relevant to not only organismal growth but also billions of years of geobiological evolution. Here, chemical transformations intrinsic to evolution and development were characterized by analyzing data from recent phylostratigraphic and proteomic studies. This two-stage analysis involves obtaining chemical metrics (carbon oxidation state and stoichiometric hydration state) from the elemental compositions of proteins followed by modeling the relative stabilities of target proteins against a proteomic background to infer thermodynamic parameters (oxygen fugacity, water activity, and virtual redox potential (Eh)). The main results of this study are a rise in carbon oxidation state of proteins spanning the time of the Great Oxidation Event, a rise in virtual redox potential that coincides with the likely emergence of aerobic metabolism, a rise in carbon oxidation state of proteins inferred from the transcriptome in late stages of Bacillus subtilis biofilm growth, and a drop in stoichiometric hydration state of the fruit fly developmental proteome at the same time as a drop in organismal water content. Stoichiometric hydration state also decreases for proteins with more recent gene ages and through stages of biofilm development, leading to predictions of higher hydration potentials at earlier time points. By building chemical representations of protein evolution and developmental proteomes, exciting new types of geobiochemical proxies can be developed. Tradeoff breaking as model of evolutionary transitions in individuality and the limits of the fitness decoupling metaphor Evolutionary transitions in individuality (ETIs) involve the formation of Darwinian collectives from Darwinian particles. The transition from cells to multicellular life is a prime example. During an ETI, collectives become units of selection in their own right. However, the underlying processes are poorly understood. One observation used to identify the completion of an ETI is an increase in collective-level performance accompanied by a decrease in particle-level performance, for example measured by growth rate. This seemingly counterintuitive dynamic has been referred to as "fitness decoupling" and has been used to interpret both models and experimental data. Using a mathematical approach, we show this concept to be problematic in that the fitness of particles and collectives can never decouple--calculations of particle and collective fitness performed over appropriate and equivalent time intervals are necessarily the same. By way of solution, we draw attention to the value of mechanistic approaches that emphasise traits, and tradeoffs among traits, as opposed to fitness. This trait-based approach is sufficient to capture dynamics that underpin evolutionary transitions. In addition, drawing upon both experimental and theoretical studies, we show that while early stages of transitions might often involve tradeoffs among particle traits, later--and critical--stages are likely to involve the rupture of such tradeoffs. Thus, tradeoff-breaking stands as a useful marker for ETIs. Foraging with friends, moving with anyone: effects of the social environment on movement-integrated habitat selection O_LIMovement links the distribution of habitats with the social environment of animals using those habitats; yet integrating movement, habitat selection, and socioecology remains an opportunity for further study.
C_LIO_LIHere, our objective was to disentangle the roles of habitat selection and social association as drivers of collective movement in a gregarious ungulate. To accomplish this objective, we (1) assessed whether socially familiar individuals form discrete social communities and whether social communities have high spatial, but not necessarily temporal, overlap; and (2) we modelled the relationship between collective movement and selection of foraging habitats using socially informed integrated step selection analysis.
C_LIO_LIWe used social network analysis to assign individuals to social communities and determine short and long-term social preference among individuals. Using integrated step selection functions (iSSF), we then modelled the effect of social processes, i.e., nearest neighbour distance and social preference, and movement behaviour on patterns of habitat selection.
C_LIO_LIBased on assignment of individuals to social communities and home range overlap analyses, individuals assorted into discrete social communities, and these communities had high spatial overlap. By unifying social network analysis with iSSF, we identified movement-dependent social association, where individuals foraged with more familiar individuals, but moved collectively with any between foraging patches.
C_LIO_LIOur study demonstrates that social behaviour and space use are inter-related based on spatial overlap of social communities and movement-dependent habitat selection. Movement, habitat selection, and social behaviour are linked in theory. Here, we put these concepts into practice to demonstrate that movement is the glue connecting individual habitat selection to the social environment.
C_LI Effects of substrate on shrimp growth, water quality and bacterial community in the biofloc system nursing Penaeus vannamei postlarvae This study aimed to investigate the effects of substrate on water quality, shrimp growth and bacterial community in the biofloc system with a salinity of 5{per thousand}. Two treatments, biofloc system with (sB) or without (nB) addition of elastic solid packing filler (nylon) as substrate, were set up. Penaeus vannamei postlarvae (PL, [~] stage 15) were stocked at a density of 4000 PL m-3 in each treatment with triplicates for a 28-days culture experiment, taking glucose as carbon source (C:N 15:1). Results showed that the survival rate (96.3{+/-}3.6%), FCR (0.76{+/-}0.06) and productivity (1.54{+/-}0.12 kg m-3) in sB treatment were significantly better than those in nB treatment (81.0{+/-}7.1%, 0.98{+/-}0.08 and 1.14{+/-}0.09 kg m-3, P<0.05). All water parameters were in the recommended ranges. Substrate showed significant effect on TAN, TSS, turbidity, biofloc volume, pH and carbonate alkalinity (P < 0.05). Actinobacteria (4.0-22.7%), Bacteroidetes (10.4-33.5%), Firmicutes (0.2-11.2%), Planctomycetes (4.0-14.9%) and Proteobacteria (29.4-59.0%) were the most dominant phyla for both treatments. However, the bacterial community in sB treatment showed to be significantly different from that in nB treatment (Jaccard distance 0.94{+/-}0.01, P=0.001). Substrate showed significant effects on Shannon, Heip, Pielou and Simpson index, as well as relative abundances of Actinobacteria, Bacteroidetes and Planctomycetes (P < 0.05). The results suggested that addition of substrate affected the shrimp growth, water quality and bacterial community in the biofloc system nursing P. vannamei PL with a 5{per thousand} salinity. Natural variations in reproductive timing of brood size and X-chromosome nondisjunction in Caenorhabditis elegans Reproductive timing underlies self-sperm production and X-chromosome nondisjunction rate in Caenorhabditis elegans. These traits can be measured as brood size and male-production rate, and brood size decreases and male-production rate increases as worms age. These phenotypic changes occur simultaneously; however, whether these changes share common underlying genetic architectures still remains unclear. To enhance our understanding of reproductive timing in C. elegans, we measured and compared these two reproductive timing-related traits of 96 wild strains during early, late and total reproductive periods. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. The brood size and the male-production rate were not genetically correlated, implying that they might have different genetic architectures and that meiosis quality can be maintained despite increased progeny-production in the late reproductive period caused by more self-sperm. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life history traits in wild C. elegans strains. 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. Controllability reveals defining features of information seeking In uncertain environments, seeking information about alternative choice options is essential for adaptive learning and decision-making. However, information seeking is usually confounded with changes-of-mind about the reliability of the preferred option. Here, we exploited the fact that information seeking requires control over which option to sample to isolate its behavioral and neurophysiological signatures. We found that changes-of-mind occurring with control require more evidence against the current option, are associated with reduced confidence, but are nevertheless more likely to be confirmed on the next decision. Multimodal neurophysiological recordings showed that these changes-of-mind are preceded by stronger activation of the dorsal attention network in magnetoencephalography (MEG), and followed by increased pupil-linked arousal during the presentation of decision outcomes. Together, these findings indicate that information seeking increases the saliency of evidence perceived as the direct consequence of ones own actions.

HighlightsO_LIExploration-exploitation dilemmas confound information seeking with changes-of-mind.
C_LIO_LIUnlike changes-of-mind, information seeking requires control over information sampling.
C_LIO_LIInformation seeking is associated with reduced confidence and active hypothesis testing.
C_LIO_LIStronger neurophysiological correlates of attention and arousal during information seeking.
C_LI Tunable Cellular Localization and Extensive Cytoskeleton-Interplay of Reflectins Reflectin proteins are nature copolymers consist of repeated canonical domains, locating in a biophotonic system called Bragg lamellae and manipulating the dynamic structural coloration of iridocytes. Their biological functions are intriguing, but the underlying mechanism is not fully understood. In this study, reflectin A1, A2, B1 and C were found to present distinguished cyto-/nucleoplasmic localization preferences. Comparable intracellular localization can be reproduced by truncated reflectin variants, suggesting a conceivable evolutionary order among reflectin proteins. Secondly, the size-dependent access of reflectin variants into nucleus demonstrate a potential model of how reflectins get into Bragg lamellae. Thirdly, RfA1 was found to extensively interact with cytoskeleton, including its binding to actin and microtubule minus-end-directed movement. This implies cytoskeleton system plays fundamental role during the organization and transportation of reflectin proteins. Findings presented here recommend reflectins as programmable biomaterials which can be used to decipher their evolution processes, to delineate their biological mechanism, and to achieve tunable intracellular targeting as editable tags. HIF-dependent expression of creatine kinase brain isoform (CKB) promotes breast cancer metastasis, whereas cyclocreatine therapy impairs invasion and improves the efficacy of conventional chemotherapies The oxygen-responsive Hypoxia Inducible Factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple negative breast cancers (TNBCs) and correlates with poor patient survival. Since inhibiting the HIF transcription factors with small molecules is challenging, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss and gain of function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, formation of invadopodia, and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth. A nuclear import pathway exploited by pathogenic noncoding RNAs The prevailing view regarding intracellular RNA trafficking in eukaryotic cells describes that RNAs transcribed in the nucleus either stay in the nucleus or cross the nuclear envelope entering the cytoplasm for function. Interestingly, emerging evidence illustrates numerous functional RNAs trafficking in the reverse direction from the cytoplasm to the nucleus. However, the mechanism underlying the RNA nuclear import has not been well elucidated. Viroids are single-stranded circular noncoding RNAs that infect plants. Using nuclear-replicating viroids as a model, we showed that cellular Importin alpha-4 is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (Virp1) that binds both Importin alpha-4 and viroids. Furthermore, a conserved C-loop in nuclear-replicating viroids is critical for Virp1 binding. Disrupting C-loop impairs Virp1 binding, viroid nuclear accumulation and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on Virp1 for nuclear import. These results have significant implications for understanding the infection process of subviral agents. In addition, our data outline a cellular pathway responsible for the nuclear import of RNAs and uncover a 3-dimensional RNA motif-based regulation over RNA nuclear import. Intermittent KHz-frequency electrical stimulation selectively engages small unmyelinated vagal afferents Afferent and efferent vagal fibers mediate bidirectional communication between the brain and visceral organs. Small, unmyelinated C-afferents constitute the majority of vagal fibers, play critical roles in numerous interoceptive circuits and autonomic reflexes in health and disease and may contribute to the efficacy and safety of vagus nerve stimulation (VNS). Selective engagement of C-afferents with electrical stimuli has not been feasible, due to the default fiber recruitment order: larger fibers first, smaller fibers last. Here, we determine and optimize an electrical stimulus that selectively engages vagal C-afferents. Intermittent KHz-frequency electrical stimulation (KES) activates motor and, preferentially, sensory vagal neurons in the brainstem. During KES, asynchronous activity of C-afferents increases, while that of larger fibers remains largely unchanged. In parallel, KES effectively blocks excitability of larger fibers while moderately suppressing excitability of C-afferents. By compiling selectivity indices in individual animals, we find that optimal KES parameters for C-afferents are >5KHz frequency and 7-10 times engagement threshold (xT) intensity in rats, 15-25xT in mice. These effects can be explained in computational models by how sodium channel responses to KES are shaped by axonal size and myelin. Our results indicate that selective engagement of vagal C-afferents is attainable by intermittent KES. CLAMP and Zelda function together to promote Drosophila zygotic genome activation During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, we hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as CLAMP. Here, we demonstrate that Drosophila embryonic development requires maternal CLAMP to: 1) activate zygotic transcription; 2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; 3) enhance chromatin accessibility to facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, maternal CLAMP functions with ZLD in a pioneer-like role to drive zygotic genome activation. 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. Deep Learning Achieves Neuroradiologist-Level Performance in Detecting Hydrocephalus Requiring Treatment PurposeIn large clinical centers a small subset of patients present with hydrocephalus that requires surgical treatment. We aimed to develop a screening tool to detect such cases from the head MRI with performance comparable to neuroradiologists.

MethodsWe leveraged 496 clinical MRI exams collected retrospectively at a single clinical site from patients referred for any reason. This diagnostic dataset was enriched to have 259 hydrocephalus cases. A 3D convolutional neural network was trained on 16 manually segmented exams (ten hydrocephalus) and subsequently used to automatically segment the remaining 480 exams and extract volumetric anatomical features. A linear classifier of these features was trained on 240 exams to detect cases of hydrocephalus that required treatment with surgical intervention. Performance was compared to four neuroradiologists on the remaining 240 exams. Performance was also evaluated on a separate screening dataset of 451 exams collected from a routine clinical population to predict the consensus reading from four neuroradiologists using images alone. The pipeline was also tested on an external dataset of 31 exams from a 2nd clinical site.

ResultsThe most discriminant features were the Magnetic Resonance Hydrocephalic Index (MRHI), ventricle volume, and the ratio between ventricle and brain volume. At matching sensitivity, the specificity of the machine and the neuroradiologists did not show significant differences for detection of hydrocephalus on either dataset (proportions test, p > 0.05). ROC performance compared favorably with the state-of-the-art (AUC 0.90-0.96), and replicated in the external validation.

ConclusionHydrocephalus cases requiring treatment can be detected automatically from MRI in a heterogeneous patient population based on quantitative characterization of brain anatomy with performance comparable to that of neuroradiologists. Selective Src-Family B Kinase Inhibition Promotes Pulmonary Artery Endothelial Cell Dysfunction. Protein tyrosine kinases (PTKs) are essential for eukaryotic signaling. By targeting select PTKs, the group of drugs known as Tyrosine kinase inhibitors (TKIs) have proven to be effective for treating multiple diseases ranging from cancer to pulmonary fibrosis. However, some TKIs also paradoxically lead to the development of adverse conditions such as pulmonary arterial hypertension (PAH) by promoting endothelial cell dysfunction (ECD). We hypothesize that (1) subsets of PTKs may disproportionately modulate signaling pathways critical for endothelial homeostasis, such as BMPR2 signaling, and (2) inhibiting those pro-endothelial PTKs can promote the development of ECD. Herein we use an agnostic high-throughput siRNA screen to investigate how PTKs affect the canonical BMPR2 signaling pathway. Our major finding is that within the Src-family of non-receptor PTKs, the Src-B family promotes canonical BMPR2 signaling while the Src-A family suppresses it. We focus on two representative members of each family, Lck (for Src-B) and Fyn (for Src-A) that are the strongest activators or inhibitors of BMPR2 signaling in the screen. We confirm that Lck is expressed in the endothelium of pulmonary arteries and show that Lck knockout (termed si-Lck) in pulmonary artery endothelial cells (PAECs) suppresses canonical BMPR2 signaling while Fyn knockout (termed si-Fyn) promotes canonical BMPR2 signaling. Furthermore, Lck and Fyn are responsible for opposing functional behaviors in PAECs: si-Lck promotes apoptosis and interferes with tube formation while si-Fyn suppresses apoptosis and promotes tube formation. After analyzing the whole-transcriptome signature of si-Lck and si-Fyn PAECs we find that in addition to BMPR2 signaling suppression, si-Lck (and not si-Fyn) increases a broad number of ECD markers and increases canonical NF-{kappa}{beta} signaling. In summary, for the first time we show that Src-A and B Family of PTKs exert differential control over key endothelial cell signaling pathways resulting in direct phenotypic consequences. This knowledge may help to guide the design of more precise TKIs which avoid adverse drug reactions brought about through endothelial cell dysfunction. Assessing the impact of grassland management on landscape multifunctionality Land-use intensification has contrasting effects on different ecosystem services, often leading to land-use conflicts. While multiple studies have demonstrated how landscape-scale strategies can minimise the trade-off between agricultural production and biodiversity conservation, little is known about which land-use strategies maximise the landscape-level supply of multiple ecosystem services (landscape multifunctionality), a common goal of stakeholder communities.

We combine comprehensive data collected from 150 German grassland sites with a simulation approach to identify landscape compositions, with differing proportions of low-, medium-, and high-intensity grasslands, that minimise trade-offs between the six main grassland ecosystem services prioritised by local stakeholders: biodiversity conservation, aesthetic value, productivity, carbon storage, foraging, and regional identity. Results are made accessible through an online tool that provides information on which compositions best meet any combination of user-defined priorities (https://neyret.shinyapps.io/landscape_composition_for_multifunctionality/).

Results show that an optimal landscape composition can be identified for any pattern of ecosystem service priorities. However, multifunctionality was similar and low for all landscape compositions in cases where there are strong trade-offs between services (e.g. aesthetic value and fodder production), where many services were prioritised, and where drivers other than land use played an important role. We also found that if moderate service levels are deemed acceptable, then strategies in which both high and low intensity grasslands are present can deliver landscape multifunctionality. The tool presented can aid informed decision-making by predicting the impact of future changes in landscape composition, and by allowing for the relative roles of stakeholder priorities and biophysical trade-offs to be understood by scientists and practitioners alike.

HighlightsO_LIAn online tool identifies optimal landscape compositions for desired ecosystem services
C_LIO_LIWhen the desired services are synergic, the optimum is their common best landscape composition
C_LIO_LIWhen the desired services trade-off, a mix of grassland intensity is most multifunctional
C_LIO_LISuch tools could support decision-making processes and aid conflict resolution
C_LI

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/208199v5_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@3273f6org.highwire.dtl.DTLVardef@5b3a5aorg.highwire.dtl.DTLVardef@153f526org.highwire.dtl.DTLVardef@103cbea_HPS_FORMAT_FIGEXP  M_FIG C_FIG If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context.