title: Dietary palmitic acid induces trained immunity that controls inflammation and infection

abstract: Trained immunity is an innate immune memory response that is induced by primary microbial or sterile stimuli that sensitizes monocytes and macrophages to a secondary pathogenic challenge, reprogramming the host response to infection and inflammatory disease. Nutritional components, such as dietary fatty acids, can act as inflammatory stimuli, but it is unknown if they can act as the primary stimuli in the context of innate immune memory. Here we find mice fed diets enriched in saturated fatty acids (SFAs) confer a hyper-inflammatory response to systemic lipopolysaccharide (LPS) and increased mortality, independent of diet-induced microbiome and glycemic modulation. Ex vivo, we show monocytes and splenocytes from mice fed enriched SFAs do not have altered baseline inflammation, but enhanced responses to a secondary inflammatory challenge. Lipidomics identified enhanced free palmitic acid (PA) and PA-associated lipids in SFA-fed mice serum. We found pre-treatment with physiologically relevant concentrations of PA alone reprograms macrophages to induce a hyper-inflammatory response to secondary challenge with LPS. This response was found to be dependent on the synthesis of ceramide, and reversible when treated with oleic acid, a mono-unsaturated FA that depletes intracellular ceramide. In vivo, we found systemic PA confers enhanced inflammation and mortality during an acute septic response to systemic LPS, which was not reversible for up to 7 days post-PA-exposure. While PA-treatment is harmful for acute septic shock outcome, we find PA exposure enhanced clearance of Candida albicans in RAG-/- mice. These are the first data to implicate enriched dietary SFAs, and specifically PA, in the induction of long-lived innate immune memory that is detrimental during an acute septic response, but beneficial for clearance of pathogens.


title: Dietary palmitic acid induces innate immune memory via ceramide production that enhances severity of acute septic shock and clearance of infection

category: immunology


title: In vivo augmentation of a complex gut bacterial community

abstract: Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex but not both, limiting their utility. In accompanying work, we constructed a complex synthetic community (104 strains, hCom1) containing the most common taxa in the human gut microbiome. Here, we used an iterative experimental process to improve hCom1 by filling open metabolic and/or anatomical niches. When we colonized germ-free mice with hCom1 and then challenged it with a human fecal sample, the consortium exhibited surprising stability; 89% of the cells and 58% of the taxa derive from the original community, and the pre- and post-challenge communities share a similar overall structure. We used these data to construct a second version of the community, adding 22 strains that engrafted following fecal challenge and omitting 7 that dropped out (119 strains, hCom2). In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by hCom2 versus human feces are similar in terms of microbiota-derived metabolites, immune cell profile, and bacterial density in the gut, suggesting that this consortium is a prototype of a model system for the human gut microbiome.

category: microbiology


title: Systematic dissection of a complex gut bacterial community

abstract: Efforts to model the gut microbiome have yielded important insights into the mechanisms of interspecies interactions, the impact of priority effects on ecosystem dynamics, and the role of diet and nutrient availability in determining community composition. However, the model communities studied to date have been defined or complex but not both, limiting their utility. Here, we construct a defined community of 104 bacterial strains composed of the most common taxa from the human gut microbiota. By propagating this community in growth media missing one amino acid at a time, we show that branched-chain amino acids have an outsize impact on community structure and identify a pathway in Clostridium sporogenes for generating ATP from arginine. We constructed and propagated the complete set of single-strain dropout communities, revealing a sparse network of strain-strain interactions including a novel interaction between C. sporogenes and Lactococcus lactis driven by metabolism. This work forms a foundation for studying strain-strain and strain-nutrient interactions in highly complex defined communities, and it provides a starting point for interrogating the rules of synthetic ecology at the 100+ strain scale.


title: Evolutionary History Impacts Phyllosphere Community Assembly on Forage Grasses

abstract: Benefits leaf bacterial communities provide to plant hosts are reduced by external stress. Understanding how plant hosts impact phyllosphere community assembly, how microbes influence plant traits, and how this interaction changes under stress will advance our insight into the evolutionary relationship between plants and their microbial communities. We investigated phyllosphere community assembly change over time, between host species, and under drought stress on three native temperate grasses and three non-native tropical grasses. By growing them together, effects of host geography and differences in environmental variables were eliminated allowing us to test evolutionary history on community assembly. We found evidence of phylosymbiosis which increased significantly under drought stress, indicating phyllosphere communities and their response to stress relate to grass species phylogeny. We also show native temperate grasses displayed stronger cophylogenetic relationships between grass hosts and their microbial communities and had increased selection by host species over time compared to non-native tropical hosts. Interestingly, the functional marker gene nifH, though differentially present on all host species was not susceptible to drought. The evidence of shared evolutionary history, presence of functionally important bacteria, and responses to drought suggest that microbial communities are important plant traits that coevolve alongside their plant hosts.


title: Evaluation of US state pollinators using 3 evidence-based policymaking frameworks

abstract: After the US federal government created a national pollinator protection plan in 2015, many states followed with their own. Since their goal is to promote pollinating insect conservation, we wanted to know whether the state plans are using best practices for evidence-based science policy. In early 2019 we found and downloaded every existing, publicly available US state pollinator protection plan. We then used content analysis to assess the goals, scope, and implementation of state-level pollinator protection plans across the US. This analysis was conducted using three distinct frameworks for evidence-based policymaking: US Department of Interior Adaptive Resources Management (ARM), US Environmental Protection Agency management pollinator protection plan (MP3) guidance, and Pew Trusts Pew-MacAthur Results First Project elements of evidence-based state policymaking (PEW) framework. Then we scored them using the framework criteria, to assess whether the plans were using known best practices for evidence based policymaking. Of the 31 states with a state pollinator plan, Connecticut was the state with the lowest total score across the three evaluation frameworks. The state with the highest overall scores, across the three frameworks, was Missouri. Most states did not score highly on the majority of the frameworks. Overall, many state plans were lacking policy elements that address monitoring, evaluation, and adjustment. These missing elements impact states ability to achieve their conservation goals. Our results indicate that states can improve their pollinator conservation policies to better match evidence-based science policy guidance, regardless of which framework is used.

category: ecology


title: Evaluation of US state pollinator plans using 3 evidence-based policymaking frameworks


title: Relative precision of the sibship and LD methods for estimating effective population size with genomics-scale datasets

abstract: Computer simulations were used to compare relative precision of two widely-used single-sample methods for estimating effective population size (Ne)--the sibship method and the linkage-disequilibrium (LD) method. Emphasis is on performance when thousands of gene loci are used, which now can easily be achieved even for non-model species. Results show that unless Ne is very small, if at least 500-2000 diallelic loci are used, precision of the LD method is higher than the maximum possible precision for the sibship method, which occurs when all sibling relationships have been correctly identified. Results also show that when precision is high for both methods, their estimates of effective population size are high and positively correlated, which limits additional gains in precision that might be obtained by combining information from the two estimators.

category: genetics


title: Human embryoid bodies as a novel system for genomic studies of functionally diverse cell types

abstract: Most disease-associated loci, though located in putatively regulatory regions, have not yet been confirmed to affect gene expression. One reason for this could be that we have not examined gene expression in the most relevant cell types or conditions. Indeed, even large-scale efforts to study gene expression broadly across tissues are limited by the necessity of obtaining human samples post-mortem, and almost exclusively from adults. Thus, there is an acute need to expand gene regulatory studies in humans to the most relevant cell types, tissues, and states. We propose that embryoid bodies (EBs), which are organoids that contain a multitude of cell types in dynamic states, can provide an answer. Single cell RNA-sequencing now provides a way to interrogate developmental trajectories in EBs and enhance the potential to uncover dynamic regulatory processes that would be missed in studies of static adult tissue. Here, we examined the properties of the EB model for the purpose mapping inter-individual regulatory differences in a large variety of cell types.

category: genomics


title: Convergence of case-specific epigenetic alterations identify a confluence of genetic vulnerabilities tied to opioid dependence

abstract: Opioid dependence is a highly heterogeneous disease driven by a variety of genetic and environmental risk factors which have yet to be fully elucidated. We interrogated the effects of opioid dependence on the brain using ChIP-seq to quantify patterns of H3K27 acetylation in dorsolateral prefrontal cortical neurons isolated from 51 opioid-overdose cases and 51 accidental death controls. Among opioid cases, we observed global hypoacetylation and identified 388 putative enhancers consistently depleted for H3K27ac. Machine learning on H3K27ac patterns predicts case-control status with high accuracy. We focus on case-specific regulatory alterations, revealing 81,399 hypoacetylation events, uncovering vast inter-patient heterogeneity. We developed a strategy to decode this heterogeneity based on convergence analysis, which leveraged promoter-capture Hi-C to identify five genes over-burdened by alterations in their regulatory network or "plexus": ASTN2, KCNMA1, DUSP4, GABBR2, ENOX1. These convergent loci are enriched for opioid use disorder risk genes and heritability for generalized anxiety, number of sexual partners, and years of education. Overall, our multi-pronged approach uncovers neurobiological aspects of opioid dependence and captures genetic and environmental factors perpetuating the opioid epidemic.


title: Deciphering Tumour Tissue Organization by 3D Electron Microscopy and machine learning

abstract: Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumoral tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also discovered that the blood capillary controls the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for new investigations in an emerging field that we call  onconanotomy.

category: cancer biology


title: Atlantic salmon farms are a likely source of Tenacibaculum maritimum infection in migratory Fraser River sockeye salmon

abstract: Infectious disease from domestic hosts, held for agriculture, can impact wild species that migrate in close proximity, potentially reversing selective advantages afforded by migration. For sockeye salmon in British Columbia, Canada, juveniles migrate past numerous Atlantic salmon farms from which they may acquire a number of infectious agents. We analyse patterns of molecular detection in juvenile sockeye salmon for one bacterial pathogen, Tenacibaculum maritimum, known to cause disease in fish species around the globe and to cause mouthrot disease in farmed Atlantic salmon in BC. Our data show a clear peak in T. maritimum detections in the Discovery Islands region of BC, where sockeye migrate close to salmon farms. Using well established differential-equation models to describe sockeye migration and T. maritimum infection spread, we fit models to our detection data to assess support for multiple hypotheses describing farm- and background-origin infection. Despite a data-constrained inability to resolve certain epidemiological features of the system, such as the relative roles of post infection mortality and recovery, our models clearly support the role of Discovery-Islands salmon farms in producing the observed patterns. Our best models (with 99.8% empirical model support) describe relatively constant (background) infection pressure, except around Discovery-Islands salmon farms, where farm-origin infection pressure peaked at 12.7 (approximate 95% CI: 4.5 to 31) times background levels. Given the evidence for farm-origin transfer of T. maritimum to Fraser-River sockeye salmon, the severity of associated disease in related species, and the imperilled nature of Fraser River sockeye generally, our results suggest the need for a more precautionary approach to managing farm/wild interactions in sockeye salmon.


title: Commonly used Hardy-Weinberg equilibrium filtering schemes impact population structure inferences using RADseq data

abstract: Reduced representation sequencing (RRS) is a widely used method to assay the diversity of genetic loci across the genome of an organism. The dominant class of RRS approaches assay loci associated with restriction sites within the genome (restriction site associated DNA sequencing, or RADseq). RADseq is frequently applied to non-model organisms since it enables population genetic studies without relying on well-characterized reference genomes. However, RADseq requires the use of many bioinformatic filters to ensure the quality of genotyping calls. These filters can have direct impacts on population genetic inference, and therefore require careful consideration. One widely used filtering approach is the removal of loci which do not conform to expectations of Hardy-Weinberg equilibrium (HWE). Despite being widely used, we show that this filtering approach is rarely described in sufficient detail to enable replication. Furthermore, through analyses of in silico and empirical datasets we show that some of the most widely used HWE filtering approaches dramatically impact inference of population structure. In particular, the removal of loci exhibiting departures from HWE after pooling across samples significantly reduces the degree of inferred population structure within a dataset (despite this approach being widely used). Based on these results, we provide recommendations for best practice regarding the implementation of HWE filtering for RADseq datasets.

category: evolutionary biology


title: A candidate for the sex-determination gene in tunas (Thunnus fishes)

abstract: Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex-determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies, and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex-determination gene in Thunnus fishes, or at least functions at a crucial point in the sex-differentiation cascade.


title: Prediction of the sex-determination gene in tunas (Thunnus fishes)


title: SARS-CoV-2 Viral Replication in a High Throughput Human Primary Epithelial Airway Organ Model

abstract: COVID-19 emerged as a worldwide pandemic early in 2020, and at this writing has caused over 170 million cases and 3.7 million deaths worldwide, and almost 600,000 deaths in the United States. The rapid development of several safe and highly efficacious vaccines stands as one of the most extraordinary achievements in modern medicine, but the identification and administration of efficacious therapeutics to treat patients suffering from COVID-19 has been far less successful. A major factor limiting progress in the development of effective treatments has been a lack of suitable preclinical models for the disease, currently reliant upon various animal models and in vitro culture of immortalized cell lines. Here we report the first successful demonstration of SARS-CoV-2 infection and viral replication in a human primary cell-based organ-on-chip, leveraging a recently developed tissue culture platform known as PREDICT96. This successful demonstration of SARS-CoV-2 infection in human primary airway epithelial cells derived from a living donor represents a powerful new pathway for disease modeling and an avenue for screening therapeutic candidates in a high throughput platform.

category: bioengineering


title: SARS-CoV-2 envelope-protein corruption of homeostatic signaling mechanisms in mammalian cells

abstract: During a SARS-CoV2 infection, host cells produce large amounts of the viral envelope protein (Ep-CoV2). Ep-CoV2 is partially inserted into the membrane of nascent viral particles and into cellular membranes. To mimic the pathophysiological impact of the cellular protein fraction, Ep-CoV2 was overexpressed in mammalian cells and effects on key signaling parameters were monitored. By tagging with green fluorescent protein (GFP), we found that Ep-CoV2 protein is mostly present in the endoplasmic reticulum with additional trace amounts in the plasma membrane. We observed that wild-type Ep-CoV2 and, to a lesser extent, its mutants (N15A, V25F) corrupted some of the most important homeostatic mechanisms in cells. The same was observed with isolated transmembrane domains of the protein. The Ep-CoV2-evoked elevation of intracellular Ca2+ and pH as well as the induced membrane depolarization produced by the presence of the protein interfere with major signal transduction cascades in host cells. These functions of Ep-CoV2, which likely contribute to the pathogenesis of the viral protein, result from the ion-channel activity of the viral protein. Two independent assays, a functional reconstitution of Ep-CoV2 protein in artificial membranes and a rescue of K+-deficient yeast mutants, confirm that Ep-CoV2 generates a cation-conducting channel with a low unitary conductance and a complex ion selectivity. The data presented here suggest that specific channel function inhibitors of Ep-CoV2 can provide cell protection and virostatic effects.

category: pathology


title: The Association Between Poor Sleep and Accelerated Brain Ageing

abstract: The ageing brain undergoes widespread gray (GM) and white matter (WM) degeneration. But numerous studies indicate large heterogeneity in the age-related brain changes, which can be attributed to modifiable lifestyle factors, including sleep. Inadequate sleep has been previously linked to GM atrophy and WM changes. However, the reported findings are highly inconsistent. By contrast to previous research independently characterizing patterns of either the GM or the WM changes, we used here linked independent component analysis (FLICA) to examine covariation in GM and WM in a group of older adults. Next, we employed a novel technique to estimate the brain age delta (i.e. difference between chronological and apparent brain age assessed using neuroimaging data) and study its associations with sleep quality and sleep fragmentation, hypothesizing that poor sleep accelerates brain ageing. FLICA revealed a number of multimodal (including both GM and WM) neuroimaging components, associated with age, but also with sleep quality and sleep fragmentation. Brain age delta estimates were highly sensitive in detecting the effects of sleep problems on the ageing brain. Specifically, we show significant associations between brain age delta and poor sleep quality, suggesting two years deviation above the chronological age. Our findings indicate that sleep problems in healthy older adults should be considered a risk factor for accelerated brain ageing.

category: neuroscience


title: Human motor cortical gamma activity relates to GABAergic signalling and to behaviour

abstract:
Gamma activity ({gamma}, >30 Hz) is universally demonstrated across brain regions and species. However, the physiological basis and functional role of {gamma} sub-bands (slow-{gamma}, mid-{gamma}, fast-{gamma}) have been predominantly studied in rodent hippocampus; {gamma} activity in the human neocortex is much less well understood.

Here we combined neuroimaging and non-invasive brain stimulation to examine the properties of {gamma} activity sub-bands in the primary motor cortex (M1), and their relationship to both local GABAergic activity and to motor learning. In 33 healthy individuals, we quantified movement-related {gamma} activity in M1 using magnetoencephalography, assessed GABAergic signaling using transcranial magnetic stimulation (TMS), and estimated motor learning via a serial reaction time task.

We characterised two distinct {gamma} sub-bands (slow-{gamma}, mid-{gamma}) which show movement-related increase in activity during unilateral index finger movements and are characterised by distinct temporal-spectral-spatial profiles. Bayesian correlation analysis revealed strong evidence for a positive relationship between slow-{gamma} ([~]30-60Hz) peak frequency and endogenous GABA signalling during movement preparation (as assessed using the TMS-metric short interval intracortical inhibition). There was also moderate evidence for a relationship between power of the movement-related mid-{gamma} activity (60-90Hz) and motor learning. These relationships were neurochemically- and frequency-specific.

These data provide new insights into the neurophysiological basis and functional roles of {gamma} activity in human M1 and allow the development of a new theoretical framework for {gamma} activity in the human neocortex.

Significance StatementGamma ({gamma}) activity is ubiquitous in the brain, yet our understanding of the mechanisms and function of {gamma} activity in the human neocortex, and particularly in the human motor cortex, is limited. Using a multimodal approach, we characterised two patterns of movement-related {gamma} activity in the human motor cortex (slow-{gamma} and mid-{gamma}), with different spatial, temporal and spectral properties. Slow-{gamma} peak frequency was correlated to local GABA-A activity, whereas mid-gamma power predicted performance in a subsequent motor learning task. Based on these findings and previous research, we propose a theoretical framework to explain how human motor cortical {gamma} activities may arise and their potential role in plasticity and motor learning, providing new hypotheses to be tested in future studies.

Key PointsO_LIWe combined neuroimaging (i.e. MEG) and non-invasive brain stimulation (i.e. TMS) to examine the properties of {gamma} activity sub-bands in the primary motor cortex.
C_LIO_LITwo distinct {gamma} sub-bands (slow-{gamma}, mid-{gamma}) show a movement-related increase in activity during finger movements and are characterised by distinct temporal-spectral-spatial profiles.
C_LIO_LIWe found strong evidence for a positive relationship between slow-{gamma} ([~]30-60Hz) peak frequency and endogenous GABA signalling during movement preparation (as assessed using the TMS-metric short interval intracortical inhibition).
C_LI


title: A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters

abstract: The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 g) or low (0.2 g) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.


title: Nafamostat-interferon-alpha combination suppresses SARS-CoV-2 infection in vitro and in vivo

abstract: SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNa) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNa and that both Serpin E1 and camostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.


title: Nafamostat-interferon-alpha combination suppresses SARS-CoV-2 infection by targeting cooperatively host TMPRSS2 in vitro and in vivo


title: The microtubule plus-end tracking protein Bik1 is required for chromosome congression

abstract: During mitosis, sister chromatids congress on both sides of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have a pivotal role in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are also involved. Here, we show that the microtubule plus-end tracking protein Bik1 - the budding yeast ortholog of CLIP-170 - is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell-cycle-dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to a slower cell cycle progression characterized by a delayed metaphase-anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in the Bik1-NES mutant and does not localize to the unclustered kinetochores. Thus, we propose that Bik1 functions with Cin8 to regulate kinetochore-microtubule dynamics for correct kinetochore positioning and chromosome congression.

category: cell biology


title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

abstract: Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a novel small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.

category: biochemistry


title: Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

abstract: In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.


title: Ligand Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase:Molecular Dynamic Simulations of Heptosyltransferase I Apo, Binary and Ternary Complexes

abstract: Understanding the dynamical motions and ligand recognition motifs of specific glycosyltransferase enzymes, like Heptosyltransferase I (HepI), is critical to discerning the behavior of other carbohydrate binding enzymes. Prior studies in our lab demonstrated that glycosyltransferases in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conservation of both structure and dynamical motions, despite low sequence conservation, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through a series of 100 nanosecond Molecular Dynamics simulations of HepI in apo enzyme state, and also in the binary and ternary complexes with the native substrates/products. Ligand free energy analysis allowed determination of an anticipated enzymatic path for ligand binding and release. Principle component, dynamic cross correlation and network analyses of the simulation trajectories revealed that there are not only correlated motions between the N- and C-termini, but also that residues within the N-terminal domain communicate via a path that includes substrate proximal residues of the C-terminal domain. Analysis of structural changes, energetics of substrate/products binding and changes in pKa have elucidated a variety of inter- and intradomain interactions that are critical for catalysis. These data corroborate and allow visualization of previous experimental observations of protein conformational changes of HepI. This study has provided valuable insights into the regions involved in HepI conformational rearrangement upon ligand binding, and are likely to enhance efforts to develop new dynamics disrupting enzyme inhibitors for GT-B structural enzymes in the future.


title: Cheater suppression and spite through quorum sensing

abstract: The evolutionary consequences of quorum sensing in regulating bacterial cooperation are not fully understood. In this study, we reveal unexpected consequences of regulating public good production through quorum sensing on bacterial population dynamics, showing that quorum sensing can be a collectively harmful alternative to unregulated production. We analyze a birth-death model of bacterial population dynamics accounting for public good production and the presence of non-producing cheaters. Our model demonstrates that when demographic noise is a factor, the consequences of controlling public good production according to quorum sensing depend on the cost of public good production and the presence of non-public fitness benefits. When public good production is inexpensive, quorum sensing is a destructive alternative to unconditional production, in terms of the mean population extinction time. When costs are higher, quorum sensing becomes a constructive strategy for the producing strain, both stabilizing cooperation and decreasing the risk of population extinction.

category: biophysics


title: Destructive and constructive cheater suppression through quorum sensing


title: A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states

abstract: Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human iPSC-derived microglia. We developed an efficient eight-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the "druggable genome". These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by SPP1 expression was selectively depleted by CSF1R inhibition. Thus, our platform can systematically uncover regulators of microglia states, enabling their functional characterization and therapeutic targeting.


title: NSun2 deficiency promotes tau hyperphosphorylation and neurodegeneration through epitranscriptomic regulation of miR-125b

abstract: Overproduction or suppression of certain microRNAs (miRNAs) in Alzheimers disease (AD) brains promote alterations in tau proteostasis and neurodegeneration. However, the mechanisms governing how specific miRNAs are dysregulated in AD brains are still under investigation. Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to modify mammalian non-coding RNAs and loss of function autosomal-recessive mutations in NSUN2 have been associated with neurological abnormalities in humans. Here, we provide evidence that NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. When we evaluated NSun2 protein expression in post-mortem brain tissue from AD patients we find is dysregulated which was also found in mice and human cellular AD models. To probe these observed alterations were unique to AD we further evaluated brain tissue from other tauopathies, observing NSun2 protein levels were similar between cases and controls. In a well-established Drosophila melanogaster model of tau-induced toxicity we investigated the pathological role of NSun2 observing that reduction of NSun2 protein levels exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. We further show using human induced pluripotent stem cell (iPSC) derived neuronal cultures that NSun2 deficiency results in tau hyperphosphorylation and we found in primary hippocampal neuronal cultures NSun2 levels decrease in response to amyloid-beta oligomers (A{beta}O). Furthermore, in mice, we observed that NSun2 deficiency promotes aberrant levels of m6A methylated miR-125b and tau hyperphosphorylation. Altogether, our study supports that neuronal NSun2 deficiency in AD promotes neurodegeneration by altering tau phosphorylation and tau toxicity through an epitranscriptomic regulatory mechanism and highlights a novel avenue for therapeutic targeting.


title: The E3 ligase Thin controls homeostatic plasticity through neurotransmitter release repression

abstract: Synaptic proteins and synaptic transmission are under homeostatic control, but the relationship between these two processes remains enigmatic. Here, we systematically investigated the role of E3 ligases, key regulators of protein degradation-mediated proteostasis, in presynaptic homeostatic plasticity (PHP). An electrophysiology-based genetic screen of 157 E3 ligase-encoding genes at the Drosophila neuromuscular junction identified thin, an ortholog of human tripartite motif-containing 32 (TRIM32), a gene implicated in several neural disorders, including Autism Spectrum Disorder and schizophrenia. We demonstrate that thin functions presynaptically during rapid and sustained PHP. Presynaptic thin negatively regulates neurotransmitter release under baseline conditions by limiting the number of release-ready vesicles, independent of gross morphological defects. We provide genetic evidence that thin controls release through dysbindin, a schizophrenia-susceptibility gene required for PHP. Thin and Dysbindin localize in close proximity within presynaptic boutons, and Thin degrades Dysbindin in vitro. Thus, the E3 ligase Thin links protein degradation-dependent proteostasis of Dysbindin to homeostatic regulation of neurotransmitter release.


title: Ceylon cinnamon and its major compound Cinnamaldehyde can limit overshooting inflammatory signaling and angiogenesis in vitro: implications for COVID-19 treatment

abstract: Overshooting immune reactions can occur during inflammatory responses that accompany severe infections, such as COVID-19. Cytokines, damage-associated molecular patterns (DAMPs), and reactive oxygen and nitrogen species can generate positive feedback loops of inflammation, leading to long-term complications such as vascular endothelialitis, thrombosis, endothelial dysfunction, neurological impairments, and chronic fatigue. Dexamethasone can limit inflammation by inhibiting the activation of pro-inflammatory transcription factors. High dose dexamethasone, however, has undesirable side effects. Here, we show that Ceylon cinnamon and its major compound cinnamaldehyde can mitigate inflammatory signaling in vitro. Cinnamaldehyde interferes with the dimerization of toll-like receptor 4 (TLR4), which can be activated by DAMPs like HSP60 and HMGB1. Our results suggest that supplementary treatment with Ceylon cinnamon may allow administration of lower doses of dexamethasone to avoid high dose steroid side effects. Moreover, preliminary results indicate that Ceylon cinnamon modulates angiogenesis, which is a reactive phenomenon in COVID-19.


title: Cellular heterogeneity and MTH1 play key roles in galactose mediated signaling of the GAL switch to utilize the disaccharide melibiose

abstract:
Yeast metabolizes the disaccharide melibiose by hydrolyzing it into equimolar concentrations of glucose and galactose by MEL1-encoded -galactosidase. Galactose metabolizing genes (including MEL1) are induced by galactose and repressed by glucose, which are the products of melibiose hydrolysis. Therefore, how melibiose catabolization and utilization take place by circumventing the glucose repression is an enigma. Other than the galactose metabolizing genes MTH1, a negative regulator of glucose signal pathway has Gal4p binding sites and is induced by galactose and repressed by high glucose concentration. But, at low or no glucose MTH1 along with its paralogue STD1 represses hexose transporters, that are involved in glucose transport. This sort of tuning of glucose and galactose regulation motivated us to delineate the role of MTH1 as a regulator of MEL1 expression and melibiose utilization. The deletion mutant of MTH1 shows growth defect on melibiose and this growth defect is enhanced upon the deletion of both MTH1 and its paralogue STD1. Microscopy and flowcytometry analysis, suggest, that even though MEL1 and GAL1 promoter are under Gal4p and Gal80p regulation, upon deletion of MTH1 it hampers only MEL1 expression, but not the GAL1 gene expression. By using 2-Deoxy galactose toxicity assay, we observed phenotypic heterogeneity in cells grown on melibiose i.e. after cleaving of melibiose a fraction of cell population utilizes glucose and another fraction utilizes galactose and coexist together. Understanding GAL/MEL gene expression patterns in melibiose will have great implication to understand various other complex sugar utilizations, tunable gene expressions and complex feedback gene regulations.

SignificanceSugar metabolism is an important phenomenon to understand the regulation of gene expression. Glucose is the most preferred carbon source. Yeast follows glycolytic pathways like cancer cells for metabolism of sugars and understanding this will throw more light to the metabolism of cancer cells. In this communication we observed cell-to-cell heterogeneity in yeast cells playing a key role in metabolism of a complex disaccharide melibiose, which gets cleaved into glucose and galactose by -galactosidase. Glucose represses -galactosidase and galactose induces it. Because of the heterogeneous population of cells one fraction consumes glucose liberated by melibiose hydrolysis, therefore it is not sufficient to repress -galactosidase and other GAL genes. Therefore, GAL genes are expressed and help in metabolizing melibiose and galactose.


title: The water-soluble chitosan derivative, N-methylene phosphonic chitosan, is an effective fungicide against the phytopathogen Fusarium eumartii

abstract: Chitosan has been considered an environmental-friendly polymer. However, its use in agriculture has not been extended yet due to its relatively low solubility in water. In an attempt to improve such chemical characteristics, a chitosan-derivative prepared by adding a phosphonic group to chitosan N-methylene phosphonic chitosan, NMPC, was obtained from shrimp fishing industry waste from Argentinean Patagonia. This study showed that NMPC had a fungicidal effect on the phytopathogenic fungus Fusarium solani f. sp. eumartii (F. eumartii). NMPC inhibited F. eumartti mycelial growth and spore germination with low IC50 values. In vivo studies showed that NMPC affected fungal membrane permeability, ROS production, and cell death. NMPC also exerted antifungal effects against two other phytopathogens, Botrytis cinerea, and Phytophthora infestans. NMPC did not affect tomato cell viability at the same doses applied to these phytopathogens. Furthermore, the selective cytotoxicity of NMPC could give it added value in its application as an antimicrobial agent in agriculture.


title: Crystal structure of the human NKR-P1 bound to its lymphocyte ligand LLT1 reveals receptor clustering in the immune synapse

abstract: Human NKR-P1 (CD161, KLRB1) and its ligand LLT1 (CLEC2D) are a prototypical inhibitory C-type lectin-like receptor:ligand pair of NK cells with a critical role in homing lymphocytes to immune-privileged sites, particularly in multiple sclerosis, rheumatoid arthritis, and Crohns disease. Furthermore, NKR-P1:LLT1 inhibitory signaling is associated with glioblastoma, non-Hodgkins lymphoma, breast, and prostate cancer. However, the lack of structural data on the formation of the NKR-P1:LLT1 complex limits our understanding of this signaling. We thus solved the crystal structures of NKR-P1 and the NKR-P1:LLT1 complex. NKR-P1 forms a homodimer with an unexpected arrangement that enables LLT1 binding in two modes, bridging two LLT1 molecules, thereby forming interaction clusters suggestive of an inhibitory immune synapse. Moreover, observing the formation of these clusters by SEC-SAXS analysis in solution and by dSTORM super-resolution microscopy on the cell surface, and following their role in receptor signaling using in vitro cytotoxicity assay with freshly isolated NK cells, we show how NKR-P1:LLT1 clustering allows these proteins to overcome the weak affinity of C-type lectin-like receptors to their ligands. Furthermore, only the ligation of both primary and secondary LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. Therefore, our findings show how inhibitory receptor cross-linking and clustering work together to trigger signal transduction upon cellular contact in the immune synapse.

category: molecular biology


title: Inflammation and Cellular Stress Induced Neurological Sequelae of Plasmodium falciparum Malaria

abstract:
BackgroundMalaria caused by Plasmodium falciparum results in severe complications including cerebral malaria (CM) especially in children. While the majority of falciparum malaria survivors make a full recovery, there are reports of some patients ending up with neurological sequelae.

MethodsWe performed an analysis of pooled transcriptome data of whole blood samples derived from two studies involving various Plasmodium falciparum infections, comprising mild malaria (MM), non-cerebral severe malaria (NCM) and CM. Pathways and gene ontologies (GOs) elevated in the distinct falciparum infections were identified.

ResultsContrary to other research findings, our analysis showed MM share similar biological processes with cancer and neurodegenerative diseases, NCM is associated with drug resistance and glutathione metabolism and CM is correlated with endocannabinoid signaling and non-alcoholic fatty liver disease (NAFLD). GO revealed the terms biogenesis, DNA damage response and IL-10 production in MM, down-regulation of cytoskeletal organization and amyloid-beta clearance in NCM and aberrant signaling, neutrophil degranulation and gene repression in CM. Differential gene expression analysis between CM and NCM showed the up-regulation of neutrophil activation and response to herbicides while regulation of axon diameter was down-regulated in CM.

ConclusionsThe results of this study have demonstrated that the deleterious effect of falciparum malaria on the brain may not be limited to CM and NCM alone but also MM. However, the severity of neurological deficit in CM might be due to the down-regulation of various genes involved in cellular function through transcriptional repression, axonal dysfunction, dysregulation of signaling pathways and neurodegeneration as a result of inflammation and cellular stress. We anticipate that our data might form the basis for future hypothesis-driven malaria research.


title: Age-related decline of the unfolded protein response in the heart promotes protein misfolding and cardiac pathology

abstract:
Cardiac myocyte death during heart failure is particularly detrimental, given that cardiac muscle exhibits limited regenerative potential. Protein aggregation was previously observed in end-stage heart failure, suggesting protein-misfolding in cardiac myocytes as a contributor to the disease process. However, the relationship between protein-misfolding, cardiac myocyte death, and myocardial dysfunction is yet to be clearly established. Here, we showed that protein synthesis and the unfolded protein response (UPR) declined as a function of mammalian postnatal development, especially in tissues with low mitotic activity, such as the heart. A deeper examination in animals models showed that compared to neonatal cardiac myocytes, adult cardiac myocytes expressed lower levels of the adaptive UPR transcription factor, ATF6, as well as lower levels of numerous ATF6-regulated genes, which was associated with susceptibility to ER stress-induced cell death. Further reduction of the ATF6-dependent gene program in ATF6 knock-out mice led to the accumulation of misfolded proteins in the myocardium and impaired myocardial function in response to cardiac stress, indicating that ATF6 plays a critical adaptive role in the setting of cardiac disease. Thus, strategies to increase ATF6 aimed at balancing proteostasis in cardiac myocytes might be a fruitful avenue for the development of novel therapies for heart disease and other age-associated diseases.

HighlightsO_LIThe unfolded protein response (UPR) declines as a function of age in adult mammalian tissues with low mitotic activity, such as the heart
C_LIO_LIDecreases in the UPR in adult cardiac myocytes is associated with impaired survival during ER stress
C_LIO_LIATF6 loss of function in adult hearts increases protein misfolding and cardiac disfunction during stress
C_LI


title: CHD4 conceals aberrant CTCF-binding sites at TAD interiors by regulating chromatin accessibility in mESCs

abstract: CTCF plays a critical role in the 3D chromatin organization by determining the TAD borders. Although CTCF primarily binds at the TAD borders, there also exist putative CTCF-binding sites within TADs, which are spread throughout the genome by retrotransposition. However, the detailed mechanism responsible for masking these putative CTCF-binding sites remains elusive. Here, we show that the ATP-dependent chromatin remodeler, CHD4, regulates chromatin accessibility to conceal aberrant CTCF-binding sites embedded in H3K9me3-enriched heterochromatic B2 SINEs in mouse embryonic stem cells (mESCs). Upon CHD4 depletion, these aberrant CTCF-binding sites become accessible, and aberrant CTCF recruitment occurs at the TAD interiors, resulting in disorganization of the local TADs. Furthermore, RNA-binding intrinsically disordered domains of CHD4 is required to prevent the aberrant CTCF bindings. Lastly, CHD4 is required for the repression of B2 SINE transcripts. These results highlight the CHD4-mediated mechanism that safeguards the appropriate CTCF bindings and associated TAD organizations in mESCs.


title: Blm Helicase Facilitates Rapid Replication of Repetitive DNA Sequences in early Drosophila Development

abstract: The absence of functional BLM DNA helicase, a member of the RecQ family of helicases, is responsible for the rare human disorder Bloom Syndrome, which results in developmental abnormalities, DNA repair defects, genomic instability, and a predisposition to cancer. In Drosophila melanogaster, the orthologous Blm protein is essential during early development when the embryo is under the control of maternal gene products. We show that lack of functional maternal Blm during the syncytial cell cycles of Drosophila embryonic development results in severe nuclear defects and lethality. Amongst the small fraction of embryos from Blm mutant mothers that survive to adulthood, a prominent sex-bias favors the class that inherits less repetitive DNA content, which serves as an endogenous source of replication stress. This selection against repetitive DNA content reflects a role for Blm in facilitating replication through repetitive sequences during the rapid S-phases of syncytial cell cycles. During these syncytial cycles, Blm is not required for complex DNA double-strand break repair; however, the progeny sex-bias resulting from the absence of maternal Blm is exacerbated by repetitive DNA sequences and by the slowing of replication fork progression, suggesting that the essential role for Blm during this stage is to manage replication fork stress brought about by impediments to fork progression. Additionally, our data suggest that Blm is only required to manage this replication stress during embryonic development, and likely only during the early, rapid syncytial cell cycles, and not at later developmental stages. These results provide novel insights into Blm function throughout development.


title: Systemic aneuploidization events drive phenotype switching in Saccharomyces cerevisiae

abstract: How cells leverage their phenotypic potential to adapt and survive in a changing environment is a complex biological problem, with important implications for pathogenesis and species evolution. One particularly fascinating adaptive approach is the bet hedging strategy known as phenotype switching, which introduces phenotypic variation into a population through stochastic processes. Phenotype switching has long been observed in species across the tree of life, yet the mechanistic causes of switching in these organisms have remained difficult to define. Here we describe the causative basis of colony morphology phenotype switching which occurs among cells of the pathogenic isolate of Saccharomyces cerevisiae, YJM311. From clonal populations of YJM311 cells grown in identical conditions, we identified colonies which displayed altered colony architectures, yet could revert to the wild-type morphology after passaging. Whole genome sequence analysis revealed that these variant clones had all acquired whole chromosome copy number alterations (i.e., aneuploidies). Cumulatively, the variant clones we characterized harbored an exceptional spectrum of karyotypic alterations, with individual variants carrying between 1 and 16 aneuploidies. Most variants harbored unique collections of aneuploidies, indicating that numerous distinct karyotypes could manifest in the same morphological variation. Intriguingly, the genomic stability of these newly aneuploid variant clones modulated how often cells reverted back to the wild-type phenotypic state. We found that such revertant switches were also driven by chromosome missegregation events, and in some cases occurred through a return to euploidy. Together, our results demonstrate that colony morphology switching in this yeast strain is driven by stochastic and systemic aneuploidization events. These findings add an important new perspective to our current understanding of phenotype switching and bet hedging strategies, as well as how environmental pressures perpetuate organismal adaption and genome evolution.


title: Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody

abstract: The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face and one to the cytosolic. Two are specific to VGLUT2 whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.


title: Tumor suppressor PLK2 may serve as a biomarker in triple-negative breast cancer for improved response to PLK1 therapeutics

abstract:
Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Paradoxically, PLK2 (chromosome 5q11.2) is frequently deleted in human breast cancers, preferentially in basal-like and triple-negative breast cancer subtypes. Here, we found that PLK2 was tumor suppressive in breast cancer and knockdown of PLK1 rescued phenotypes induced by PLK2-loss both in vitro and in vivo. We also demonstrated that PLK2 directly interacted with PLK1 at prometaphase and that mutations in the kinase domain of PLK2, but not polo-box binding domains, changed their interaction pattern. Furthermore, treatment of syngeneic transplantation mouse tumor models and patient-derived xenografts using the PLK1 inhibitor volasertib alone, or in combination with carboplatin, indicated that PLK2-low breast tumors had a significantly better response to these drugs. Re-expression of PLK2 in an inducible PLK2-null mouse model reduced the therapeutic efficacy of volasertib. Taken together, our data suggest PLK2 loss may serve as a biomarker to predict response to PLK1 therapeutics, alone and in combination with chemotherapy.

SignificanceThe tumor suppressive role of PLK2, and its relationship with the oncogene PLK1, provide a mechanistic rationalization to use PLK1 inhibitors in combination with chemotherapy to treat PLK2 low/deleted tumors. TNBC, and other cancers with low PLK2 expression, are such candidates to leverage precision medicine to identify patients who might benefit from treatment with these inhibitors.


title: The Human Tumor Atlas Network (HTAN) Breast PreCancer Atlas: A multi-omic integrative analysis of ductal carcinoma in situ with clinical outcomes

abstract:
Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We made observations on DNA, RNA, and protein expression, and generated a de novo clustering scheme for DCIS that represents a fundamental transcriptomic organization at this early stage of breast neoplasia. Distinct stromal expression patterns and immune cell compositions were identified. We found RNA expression patterns that correlate with later events. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/448585v2_ufig1.gif" ALT="Figure 1">
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C_LIO_LIFour stroma-specific signatures identified.
C_LIO_LIutcome analysis identifies pathways involved in DCIS progression.
C_LIO_LICNAs characterize high risk of distant relapse IBC subtypes observed in DCIS.
C_LI


title: DCIS genomic signatures define biology and correlate with clinical outcome: a Human Tumor Atlas Network (HTAN) analysis of TBCRC 038 and RAHBT cohorts


title: Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells

abstract: Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

category: synthetic biology


title: Three Isoforms of the Essential Translation Initiation Factor IF2 Differentially Modulate Homologous Recombination in Escherichia coli

abstract:
Homologous recombination (HR) is critically important for chromosomal replication as well as DNA damage repair in all life forms. In Escherichia coli, the process of HR is comprised of (i) two parallel pre-synaptic pathways that are mediated, respectively, by proteins RecB/C/D and RecF/O/R/Q; (ii) a synaptic step mediated by RecA that leads to generation of Holliday junctions (HJs); and (iii) post-synaptic steps mediated sequentially by HJ-acting proteins RuvA/B/C followed by proteins PriA/B/C of replication restart. Combined loss of RuvA/B/C and a DNA helicase UvrD is synthetically lethal, which is attributed to toxicity caused by accumulated HJs since viability in these double mutant strains is restored by removal of the pre-synaptic or synaptic proteins RecF/O/R/Q or RecA, respectively. Here we show that, as in {Delta}uvrD strains, ruv mutations confer synthetic lethality in cells deficient for transcription termination factor Rho, and that loss of RecFORQ pre-synaptic pathway proteins or of RecA suppresses this lethality. Furthermore, loss of IF2-1 (which is one of three isoforms [IF2-1, IF2-2, and IF2-3] of the essential translation initiation factor IF2 that are synthesized from three in-frame initiation codons in infB) also suppressed uvrD-ruv and rho-ruv lethalities, whereas deficiency of IF2-2 and IF2-3 exacerbated the synthetic defects. Our results suggest that Rho deficiency is associated with an increased frequency of HR that is mediated by the RecFORQ pathway along with RecA. They also lend support to earlier reports that IF2 isoforms participate in DNA transactions, and we propose that they do so by modulation of HR functions.

ImportanceThe process of homologous recombination (HR) is important for maintenance of genome integrity in all cells. In Escherichia coli, the RecA protein is a critical participant in HR, which acts at a step common to and downstream of two HR pathways mediated by the RecBCD and RecFOR proteins, respectively. In this study, an isoform (IF2-1) of the translation initiation factor IF2 has been identified as a novel facilitator of RecAs function in vivo during HR.


title: Modulation of RecFORQ- and RecA-mediated homologous recombination in Escherichia coli by isoforms of translation initiation factor IF2


title: Metabolic model of nitrogen-fixing obligate aerobe Azotobacter vinelandii demonstrates adaptation to oxygen concentration and metal availability

abstract:
There is considerable interest in promoting biological nitrogen fixation as a mechanism to reduce the inputs of nitrogenous fertilizers in agriculture, a problem of agronomic, economic, and environmental importance. For the potential impact of biological nitrogen fixation in agriculture to be realized, there are considerable fundamental knowledge gaps that need to be addressed. Biological nitrogen fixation or the reduction of N2 to NH3 is catalyzed by nitrogenase which requires a large amount of energy in the form of ATP and low potential electrons. Nitrogen-fixing organisms that respire aerobically have an advantage in meeting the energy demands of biological nitrogen fixation but face challenges of protecting nitrogenase from inactivation in the presence of oxygen. Here, we have constructed a genome-scale metabolic model of the aerobic metabolism of nitrogen-fixing bacteria Azotobacter vinelandii, which uses a complex electron transport system, termed respiratory protection, to consume oxygen at a high rate keeping intracellular conditions microaerobic. Our model accurately determines growth rate under high oxygen and high substrate concentration conditions, demonstrating the large flux of energy directed to respiratory protection. While respiratory protection mechanisms compensate the energy balance in high oxygen conditions, it does not account for all substrate intake, leading to increased maintenance rates. We have also shown how A. vinelandii can adapt under different oxygen concentrations and metal availability by rearranging flux through the electron transport system. Accurately determining the energy balance in a genome-scale metabolic model is required for future engineering approaches.

ImportanceThe worlds dependence on industrially produced nitrogenous fertilizers has created a dichotomy of issues. Some parts of the globe lack access to fertilizers and associated poor crop yields, significantly limiting nutrition, contributing to disease and starvation. In contrast, in other parts of the world, abundant nitrogenous fertilizers and associated overuse result in compromised soil quality and downstream environmental issues. There is considerable interest in expanding the impacts of biological nitrogen fixation to promote improved crop yields in places struggling with access to industrial fertilizers and reducing fertilizers inputs in areas where overuse is resulting in the degradation of soil health and other environmental problems. A more robust and fundamental understanding of biological nitrogen fixations biochemistry and microbial physiology will enable strategies to promote new and more robust associations between nitrogen-fixing microorganisms and crop plants.


title: A genetic shift in an escaped transmitted/founder virus guides combinatorial vaccine design against HIV-1

abstract:
A productive HIV-1 infection is often established by a single transmitted/founder (T/F) virus, which then evolves into a complex mixture of variants during the lifetime of infection. An effective vaccine should have sufficient breadth to block the entry of diverse T/F involved in different infections. Although the variable V1V2 domain of HIV-1 envelope protein (Env) is found to be a good target for vaccine design based on the correlates of protection in the modestly successful RV144 trial, the breadth of immune responses has to be substantially enhanced to improve vaccine efficacy and minimize the emergence of breakthrough infections. Here, we report a remarkable genetic shift in a T/F virus from a participant of an acute HIV infection cohort_RV217 study. It resulted in substitution of histidine at position 173 to tyrosine (H173Y) at week 24 (wk 24) after infection, coinciding with the disappearance of strictly H173-specific first wave antibodies. Intriguingly, a second wave antibodies emerged against the escaped Y173 variant that displayed increased breadth recognizing both the H173 and Y173 epitopes. This differential antibody responses towards variant epitopes were recapitulated in a mouse model. Structural analyses suggest distinct conformations for H173 and Y173 variants which might have led to antibody responses with different reactivity and breadth. Given the occurrence of conformational dynamism in the V2 region, combinatorial V2 vaccine candidates consisting of numerous conformational variants in the natural HIV-1 diversity were designed and tested as an immunogen. These libraries, especially the Y173 variant libraries that also contained a V1 loop deletion showed increased breadth and cross-reactivity to diverse HIV-1 Env proteins. This combinatorial design might be a powerful strategy in the future design of highly efficacious HIV-1 vaccine candidates.

Author summaryAfter numerous HIV-1 vaccine failures, only one human clinical study, RV144 has demonstrated protection with modest efficacy (31.2%) by virtue of antibody responses directed to V2-region of the Env that were correlated with reduced HIV acquisition. In natural infection, there occurs an evolutionary race between virus and the host immune system, as a result of which mutants of virus that escape the immune pressure are positively selected. Similarly, in vaccinated individuals, breakthrough infections occur when the infecting virus is able to escape from vaccine generated immune response compromising vaccine efficacy. Systematic learning of how these escape signatures are positively selected during the course of infection can have implications in designing a vaccine that can effectively counteract breakthrough infections. In the current study, we took an unprecedented approach of studying evolution of T/F viruses in acutely infected individuals to identify early V2-specific escape mutations and further study these mutations through biochemical, immunological and structural aspects. The knowledge obtained from these analyses was used to rationally design combinatorial vaccine libraries encompassing V2 variants mirroring natural HIV-1 population and assess their response in mice. The resultant vaccines generated antibodies were found to broadly cross-react diverse Env proteins. Such immune escape guided rationally designed vaccines have the potential to overcome breakthrough infections and improve vaccine efficacy.

Striking image

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title: Synergistic TOR and ERK inhibition mitigates the hereditary haemorrhagic telangiectasia-like phenotype and excess kugel formation in endoglin mutant zebrafish

abstract:
RationaleHereditary haemorrhagic telangiectasia (HHT) is an inherited bleeding disorder characterised by arteriovenous malformations (AVMs). Such AVMs affect lungs, liver and brain, whilst telangiectases in mucocutaneous tissues are prone to haemorrhage. HHT type I is caused by loss-of-function endoglin (ENG) mutations. Evidence suggests AVMs result from abnormal responses to VEGF signalling.

ObjectiveWe therefore characterised the vascular abnormalities in eng mutant zebrafish and investigated whether these are prevented by inhibiting different pathways downstream of VEGF signalling.

Methods and ResultsWe used light sheet fluorescence microscopy to visualise the vasculature in engmu130 mutant zebrafish. In addition to previously described significantly enlarged dorsal aorta and posterior cardinal vein at 3d post fertilisation, engmu130 embryos had an enlarged basilar artery (BA), and increased formation of endothelial "kugeln" on cerebral vessels. Adult engmu130 fish developed skin AVMs, retinal vascular abnormalities, and an enlarged heart. Tivozanib (AV951), a VEGF receptor tyrosine kinase inhibitor, prevented development of the abnormally enlarged major vessels and normalised the number of kugeln in engmu130 embryos. Inhibiting discrete signalling pathways downstream of VEGFR2 in engmu130 embryos gave further insights. Inhibiting TOR or MEK prevented the abnormal trunk and cerebral vasculature phenotype, whilst targeting NOS and MAPK had no effect. Combining subtherapeutic TOR and MEK inhibition prevented the vascular phenotype, suggesting synergy between TOR and MEK/ERK signalling pathways.

ConclusionsThese results indicate the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling, and implicate combination low dose ERK and TOR pathway inhibitors as a therapeutic strategy in HHT.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: developmental biology


title: Phase separation versus aggregation behavior for model disordered proteins

abstract: Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarse-grained model of disordered proteins to systematically investigate how sequence distribution, sticker fraction and chain length influence the phase behavior and regulate the formation of finite-size aggregates preempting macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a "soft" criterion for predicting the underlying phase transition of a model protein. Additionally, we find that this order parameter is strongly correlated to the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveals that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results predict that classical LLPS should be the dominant phase transition for disordered proteins and suggests a possible reason behind recent findings of widespread phase separation throughout living cells.


title: Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel

abstract: The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na+ and K+ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NMR to characterize how bound Na+ vs. K+ affects NaK SF structure and dynamics. We find that the extracellular end of the SF is flexible on the ps-ns timescale regardless of bound ion. On a slower timescale, we observe a structural change between the Na+ and K+-bound states, accompanied by increased structural heterogeneity in Na+. We also show direct evidence that the SF structure is communicated to the pore via I88 on the M2 helix. These results support a dynamic SF with multiple conformations involved in non-selective conduction. Our data also demonstrate allosteric coupling between the SF and pore-lining helices in a non-selective cation channel that is analogous to the allosteric coupling previously demonstrated for K+-selective channels, supporting the generality of this model.


title: Integrative genomic and epigenomic analyses identified IRAK1 as a novel target for chronic inflammation-driven prostate tumorigenesis

abstract: The impacts of many inflammatory genes in prostate tumorigenesis remain understudied despite the increasing evidence that associates chronic inflammation with prostate cancer (PCa) initiation, progression, and therapy resistance. The overarching goal of this study was to identify dysregulated inflammatory genes that correlate with PCa progression and decipher their molecular mechanisms as well as clinical significance in PCa using integrative genomics, transcriptomics, and epigenomics approach. Our Weighted Gene Co-expression Analysis (WGCNA) and multivariate analysis identified 10 inflammatory genes: IRAK1, PPIL5/LRR1, HMGB3, HMGB2, TRAIP, IL1F5/IL36RN, ILF2, TRIM59, NFKBIL2/TONSL, and TRAF7 that were significantly associated with PCa progression. We explored the potentials of IRAK1 and other inflammatory genes as diagnostic and/or prognostic biomarkers by performing both KM survival and AUROC curve analyses. Our results indicate the clinical significance of these inflammatory genes in predicting the development and progression of PCa. IRAK1 was found to be overexpressed and hypomethylated in most PCa samples. A significantly high percentage of castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC) samples display copy number variations, especially amplification of the IRAK1 gene compared to the indolent prostate adenocarcinoma (PRAD) samples. Furthermore, we identified missense and frameshift mutations of IRAK1 in a few PRAD samples with potential functional implications. In conclusion, the results from this study suggest that IRAK1 dysregulation may be an important contributor to chronic prostatitis (inflammation) and PCa progression.


title: HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160

abstract: HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM:LIGHT:CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knock-in mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.


title: Reciprocal EGFR signaling in the Anchor Cell ensures precise inter-organ connection during C. elegans vulval morphogenesis

abstract:
During C. elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades into the underlying vulval epithelium. Thereby, the AC establishes direct contact with the invaginating 1{degrees} vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood.

Using a conditional let-23 egf receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the 1{degrees} vulval cells, delayed AC invasion and disorganized adherens junctions at the newly forming contact site between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the 1{degrees} vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. EGFR signaling in the AC thus ensures the precise alignment of the two developing organs.


title: RosettaSurf - a surface-centric computational design approach

abstract:
Proteins are typically represented by discrete atomic coordinates providing an accessible framework to describe different conformations. However, in some fields proteins are more accurately represented as near-continuous surfaces, as these are imprinted with geometric (shape) and chemical (electrostatics) features of the underlying protein structure. Protein surfaces are dependent on their chemical composition and, ultimately determine protein function, acting as the interface that engages in interactions with other molecules. In the past, such representations were utilized to compare protein structures on global and local scales and have shed light on functional properties of proteins. Here we describe RosettaSurf, a surface-centric computational design protocol, that focuses on the molecular surface shape and electrostatic properties as means for protein engineering, offering a unique approach for the design of proteins and their functions. The RosettaSurf protocol combines the explicit optimization of molecular surface features with a global scoring function during the sequence design process, diverging from the typical design approaches that rely solely on an energy scoring function. With this computational approach, we attempt to address a fundamental problem in protein design related to the design of functional sites in proteins, even when structurally similar templates are absent in the characterized structural repertoire. Surface-centric design exploits the premise that molecular surfaces are, to a certain extent, independent of the underlying sequence and backbone configuration, meaning that different sequences in different proteins may present similar surfaces. We benchmarked RosettaSurf on various sequence recovery datasets and showcased its design capabilities by generating epitope mimics that were biochemically validated. Overall, our results indicate that the explicit optimization of surface features may lead to new routes for the design of functional proteins.

Author SummaryFinely orchestrated protein-protein interactions are at the heart of virtually all fundamental cellular processes. Altering these processes or encoding new functions in proteins has been a long-standing goal in computational protein design.

Protein design methods commonly rely on scoring functions that seek to identify amino acid sequences that optimize structural configurations of atoms while minimizing a variety of physics-based and statistical terms. The objectives of the large majority of computational design protocols have been focused on obtaining a predefined structural conformation. However, routinely introducing a functional aspect on designer proteins has been more challenging.

Our results suggest that the molecular surface features can be a useful optimization parameter to guide the design process towards functional surfaces that mimic known protein binding sites and interact with their intended targets. Specifically, we demonstrate that our design method can optimize experimental libraries through computational screening, creating a basis for highly specific protein binders, as well as design a potent immunogen that engages with site-specific antibodies. The ability to create proteins with novel functions will be transformative for biomedical applications, providing many opportunities for the design of novel immunogens, protein components for synthetic biology, and other protein-based biotechnologies.

category: bioinformatics


title: The role of epistasis in determining the fitness landscape of HIV proteins

abstract:
The rapid evolution of HIV is constrained by interactions between mutations which affect viral fitness. In this work, we explore the role of epistasis in determining the fitness landscape of HIV for multiple drug target proteins, including Protease, Reverse Transcriptase, and Integrase. Epistatic interactions between residues modulate the mutation patterns involved in drug resistance with unambiguous signatures of epistasis best seen in the comparison of a maximum entropy sequence co-variation (Potts) model predicted and experimental HIV sequence "prevalences" when expressed as higher-order marginals (beyond triplets) of the sequence probability distribution. In contrast, the evidence for epistasis based on experimental measures of fitness such as replicative capacity is weak; the correspondence with Potts model "prevalence"-based predictions is obscured by site conservation and limited precision. Double mutant cycles provide in principle one of the best ways to probe epistatic interactions experimentally without reference to a particular background, and we find they reveal that the most strongly interacting mutations in HIV involve correlated sets of drug-resistance-associated residues, however the analysis is complicated by the small dynamic range of measurements. The use of correlated models for the design of experiments to probe viral fitness can help identify the epistatic interactions involved in mutational escape, and lead to better inhibitor therapies.

Author summaryProtein covariation models provide an alternative to experimental measures for estimating the fitness of mutations in proteins from across a variety of organisms. Yet, for viral proteins, it has been shown that models including epistatic couplings between residues, or other machine learning models perform no better or even worse than a simpler independent model devoid of such epistatic couplings in estimating viral fitness measurements such as replicative capacities, providing weak or ambiguous evidence for epistasis. We show that the evidence for long-range epistasis is strong by the analysis of the high-order marginals of the MSA distribution (up to subsequences of length 14), which are accurately captured by a correlated Potts sequence-covariation model but not by an independent model. While double mutant cycles in principle provide well-established biophysical probes for epistatic interactions, we demonstrate that the analysis and comparison between model and experiment is difficult due to the much smaller dynamic range of the measurements, making them more susceptible to noise.


title: Electrical Potentials of Protoscoleces of the Cestode  Echinococcus granulosus from Bovine Origin

abstract:
Larval stages of the tenia Echinococcus granulosus are the infective forms of cystic echinococcosis or hydatidosis, a worldwide zoonosis. The protoscolex that develops into the adult form in the definitive host is enveloped by a complex cellular syncytial tegument, where all metabolic interchange takes place. Little information is available as to the electrical activity of the parasite in this developmental stage. To gain insight into the electrical activity of the parasite at the larval stage, here we conducted microelectrode impalements of bovine lung protoscoleces (PSCs) of Echinococcus granulosus in normal saline solution. We observed two distinct intra-parasitic potentials, a transient peak potential and a stable second potential, most likely representing tegumental and intra-parasitic extracellular space electrical potential differences, respectively. These values changed upon the developmental status of the parasite, its anatomical regions, or time course after harvesting. Changes in electrical potential differences of the parasite provide an accessible and useful parameter for the study of transport mechanisms and potential targets for the development of novel antiparasitic therapeutics.

Author summaryHydatid disease is a parasite-caused zoonosis that causes high morbidity and mortality and has a great impact on public health. The disease has no known cure, and the main lines of treatment include surgery and medical treatments which are not satisfactory, so new drug compounds are urgently needed. Genome sequencing of the parasite has identified different genes encoding ion channels in Echinococcus granulosus, making ion channel inhibitors a promising target for treating hydatidosis. However, no easy technical approaches are available to test the electrical contribution of ion channels to parasite physiology. In the present study, we used the microelectrode impalement technique to determine the electrical properties of the larval stages of the parasite harvested from infected cow lungs. We observed transient electrical potentials not previously reported for the parasite, and changes in these parameters associated with its developmental stage and aging. Our findings indicate that microelectrode impalement of protoscoleces of Echinococcus granulosus may be a strategy of choice to explore and test possible drugs suggested for their therapeutic potential against hydatid disease. Further evaluation of parasites coming from other animals and humans may help address important issues in the treatment and prevention of the hydatid disease.

category: zoology


title: Oxytocin receptor antagonism during song tutoring in zebra finches reduces preference for and learning of the tutor's song

abstract: In species with vocal learning, acquiring species-typical vocalizations relies on early social orienting. In zebra finches (Taeniopygia guttata), for example, learning song requires dynamic social interactions with a "tutor" during an early sensitive period. The oxytocin system plays a central role in social orienting across species, yet it is unknown whether this system participates in the attentional and motivational processes that support vocal learning. Here, we tested whether blocking oxytocin receptors during exposure to tutors would impact learning from those tutors. Juvenile, song-naive males were each tutored by two unfamiliar adults. During exposure to one tutor, juveniles were treated with oxytocin receptor antagonist (OTA) and during exposure to the other, saline (control). We found that OTA significantly reduced behaviors associated with approach and attention during tutoring sessions. Next, using an operant assay in which exposure to the two songs was balanced, we found that the juveniles preferred the control song over the OTA song. The developmental trajectory of preference for the control song resembled the pattern shown by father-reared birds choosing to hear their fathers song. Finally, the adult songs of the tutored birds more closely resembled control song than OTA song. The magnitude of this difference was significantly predicted by the early preference for the control song. Overall, oxytocin antagonism during exposure to a tutor seemed to bias juveniles against that tutor and his song. Our results suggest that oxytocin receptors play a role in socially-guided vocal learning in zebra finches, perhaps by affecting attention and motivation during tutoring.

category: animal behavior and cognition


title: Neural event segmentation of continuous experience in human infants

abstract:
How infants experience the world is fundamental to understanding their cognition and development. A key principle of adult experience is that, despite receiving continuous sensory input, we perceive this input as discrete events. Here we investigate such event segmentation in infants and how it differs from adults. Research on event cognition in infants often uses simplified tasks in which (adult) experimenters help solve the segmentation problem for infants by defining event boundaries or presenting discrete actions/vignettes. This presupposes which events are experienced by infants and leaves open questions about the principles governing infant segmentation. We take a different, data-driven approach by studying infant event segmentation of continuous input. We collected whole-brain fMRI data from awake infants (and adults, for comparison) watching a cartoon and used a hidden Markov model to identify event states in the brain. We quantified the existence, timescale, and organization of multiple event representations across brain regions. The adult brain exhibited a known hierarchical gradient of event timescales, from shorter events in early visual regions to longer events in later visual and associative regions. In contrast, the infant brain only represented longer events, even in early visual regions, with no timescale hierarchy. The boundaries defining these infant events only partially overlapped with boundaries defined from adult brain activity and behavioral judgments. These findings suggest that events are organized differently in infants, with longer timescales and more stable neural patterns, even in sensory regions. This may indicate greater temporal integration and reduced temporal precision during dynamic, naturalistic perception.

Significance StatementSensory input is continuous and yet humans perceive discrete events. This event segmentation has been studied in adults by asking them to indicate natural breaks in continuous input. This classic parsing task is impossible in infants who cannot understand or follow instructions. We circumvent this barrier by testing how the infant brain parses. We applied a computational model to rare awake fMRI data from infants to identify how their brains transitioned between stable states during a cartoon. Whereas adults showed a gradient in event timescales, from shorter events in sensory regions to longer events in associative regions, infants persistently segmented fewer, longer events across the cortical hierarchy. These findings provide new neuroscientific insight into how infants represent their environment.


title: Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

abstract:
Phenothiazines (PTZ) are well known as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they are used for decades as antipsychotic drugs. In addition, significant anti-cancer properties have been ascribed to them. Several attempts for their repurposing were made, however, their incompletely understood polypharmacology is challenging.

Here we examined the potential of PTZ to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras-MAPK pathway activity. Consistently, combinations of a CaM inhibitor and a PP2A activator synergistically inhibited cancer cells with KRAS or BRAF mutations. We identified the covalently reactive PTZ derivative fluphenazine mustard as an inhibitor of Ras driven proliferation and Ras membrane organization. We confirmed its anti-CaM activity in vitro and through a cellular CaM target engagement bioluminescence resonance energy transfer (BRET) assay. Our results suggest that improved PTZ derivatives retaining their synergistic CaM inhibitory and PP2A activating properties, but without neurological side-effects, may be interesting to pursue further as anti-cancer agents.