title: Complement-associated loss of CA2 inhibitory synapses in the demyelinated hippocampus impairs memory

abstract: The complement system is implicated in synapse loss in the MS hippocampus, but the functional consequences of synapse loss remain poorly understood. Here, in post-mortem MS hippocampi with demyelination we find that deposits of the complement component C1q are enriched in the CA2 subfield, are linked to loss of inhibitory synapses and are significantly higher in MS patients with cognitive impairments compared to those with preserved cognitive functions. Using the cuprizone mouse model of demyelination, we corroborated that C1q deposits are highest within the demyelinated dorsal hippocampal CA2 pyramidal layer, and co-localized with inhibitory synapses engulfed by microglia/macrophages. In agreement with the loss of inhibitory perisomatic synapses, we further found that Schaffer collateral feedforward inhibition but not excitation was impaired in CA2 pyramidal neurons and accompanied by a reduced spike output. Ultimately, we show that these electrophysiological changes were associated with an impaired encoding of social memories. Together, our findings identify CA2 as a critical circuit in demyelinated intrahippocampal lesions and memory dysfunctions in MS.


title: Graded Visual Consciousness During the Attentional Blink

abstract: One of the ongoing debates about visual consciousness is whether it can be considered as an all-or-none or a graded phenomenon. This may depend on the experimental paradigm and the task used to investigate this question. The present event-related potential study (N = 32) focuses on the attentional blink paradigm for which so far only little and mixed evidence is available. Detection of T2 face targets during the attentional blink was assessed via an objective accuracy measure (reporting the faces gender), subjective visibility on a perceptual awareness scale (PAS) as well as event-related potentials time-locked to T2 onset (components P1, N1, N2, and P3). The behavioral results indicate a graded rather than an all-or-none pattern of visual awareness. Corresponding graded differences in the N1, N2, and P3 components were observed for the comparison of visibility levels. These findings suggest that conscious perception during the attentional blink can occur in a graded fashion.

category: neuroscience


title: 3D Mapping of Neurofibrillary Tangle Burden in the Human Medial Temporal Lobe

abstract: Tau protein neurofibrillary tangles (NFT) are closely linked to neuronal/synaptic loss and cognitive decline in Alzheimers disease (AD) and related dementias. Our knowledge of the pattern of NFT progression in the human brain, critical to the development of imaging biomarkers and interpretation of in vivo imaging studies in AD, is based on conventional 2D histology studies that only sample the brain sparsely. To address this limitation, ex vivo MRI and dense serial histological imaging in 18 human medial temporal lobe (MTL) specimens were used to construct 3D quantitative maps of NFT burden in the MTL at individual and group levels. These maps reveal significant variation in NFT burden along the anterior-posterior axis. While early NFT pathology is thought to be confined to the transentorhinal region, we find similar levels of NFT burden in this region and other MTL subregions, including amygdala, temporopolar cortex, and subiculum/CA1.


title: Discovering hidden brain network responses to naturalistic stimuli via tensor component analysis of multi-subject fMRI data

abstract: The study of brain network interactions during naturalistic stimuli facilitates a deeper understanding of human brain function. Intersubject correlation (ISC) analysis of functional magnetic resonance imaging (fMRI) data is a widely used method that can measure neural responses to naturalistic stimuli that are consistent across subjects. However, interdependent correlation values in ISC artificially inflated the degrees of freedom, which hinders the investigation of individual differences. Besides, the existing ISC model mainly focus on similarities between subjects but fails to distinguish neural responses to different stimuli features. To estimate large-scale brain networks evoked with naturalistic stimuli, we propose a novel analytic framework to characterize shared spatio-temporal patterns across subjects in a purely data-driven manner. In the framework, a third-order tensor is constructed from the timeseries extracted from all brain regions from a given parcellation, for all participants, with modes of the tensor corresponding to spatial distribution, time series and participants. Tensor component analysis (TCA) will then reveal spatially and temporally shared components, i.e., naturalistic stimuli evoked networks, their temporal courses of activity and subject loadings of each component. To enhance the reproducibility of the estimation with TCA, a novel spectral clustering method, tensor spectral clustering, was proposed and applied to evaluate the stability of TCA algorithm. We demonstrate the effectiveness of the proposed framework via simulations and real fMRI data collected during a motor task with a traditional fMRI study design. We also apply the proposed framework to fMRI data collected during passive movie watching to illustrate how reproducible brain networks are identified evoked by naturalistic movie viewing.


title: Topological Sholl Descriptors For Neuronal Clustering and Classification

abstract:
MotivationGiven that neuronal morphology can widely vary among cell classes, brain regions, and animal species, accurate quantitative descriptions allowing classification of large sets of neurons is essential for their structural and functional characterization. However, robust and unbiased computational methods currently used to characterize groups of neurons are scarce.

ResultsIn this work, we introduce a novel and powerful technique to study neuronal morphologies. We develop mathematical descriptors that quantitatively characterize structural differences among neuronal cell types and thus allow for their accurate classification. Each Sholl descriptor that is assigned to a neuron is a function of a distance from the soma with values in real numbers or more general metric spaces. To illustrate the use of Sholl descriptors, six datasets were retrieved from the large public repository http://neuromorpho.org/ comprising neuronal reconstructions from different species and brain regions. Sholl descriptors were subsequently computed, and standard clustering methods enhanced with detection and metric learning algorithms were then used to objectively cluster and classify each dataset. Importantly, our descriptors outperformed conventional techniques and thus provide a practical and effective approach to the classification of diverse neuronal cell types, with the potential for discovery of subclasses of neurons.


title: Metastatic Single Tumor Cells Evade NK Cell-mediated Killing by Thrombin-mediated Loss of the Activating Ligand CD155/PVR/Necl-5

abstract:
Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is not known. Here we have combined ultra-sensitive bioluminescence whole body imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that a disseminated tumor cell in a pulmonary capillary interacts with an NK cell every 2 hrs on average. In the first 4 hrs after tumor cell arrival, 50% of such encounters lead to tumor cell death but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. This evasion of NK cell surveillance is mediated by thrombin-dependent loss of cell surface CD155/PVR/Necl-5, a ligand for the NK cell activating receptor DNAM-1. This loss prevents the NK cell signaling needed for effective killing of tumor targets. By quantitatively visualizing the evasion of NK cell surveillance, we have uncovered a molecular mechanism for cancer evasion and provided an explanation for the anti-metastatic effect of anticoagulants.

SUMMARYIntravital functional two-photon microscopy reveals that metastatic tumor cells lodged in pulmonary capillaries acquire resistance to patrolling NK cells. Protease-mediated loss of the activating ligand CD155/PVR/Necl-5 on tumor cells prevents NK cells from ERK activation and tumor cell killing.

category: immunology


title: Impaired mRNA splicing and proteostasis in preadipocytes in obesity-related metabolic disease

abstract: Preadipocytes are crucial for healthy adipose tissue expansion. Preadipocyte differentiation is altered in obese individuals, which has been proposed to contribute to obesity-associated metabolic disturbances. Here, we demonstrate that impaired alternative splicing and dysregulated endoplasmic reticulum (ER)- associated protein degradation (ERAD) represent marker pathways of dysfunctional preadipocytes in obese individuals with insulin resistance (IR)/type 2 diabetes (T2D). Down-regulation of a key member of the major spliceosome, PRFP8/PRP8, as observed in IR/T2D preadipocytes from subcutaneous (SC) fat, prevented adipogenesis by altering both the expression and splicing patterns of adipogenic transcription factors and lipid droplet-related proteins, while adipocyte differentiation was restored upon recovery of PRFP8/PRP8 normal levels. Adipocyte differentiation was also compromised under conditions of ERAD hyperactivation, as occurs in SC and omental (OM) preadipocytes in IR/T2D obesity. Thus, targeting mRNA splicing and ER proteostasis in preadipocytes could improve adipose tissue function and thus contribute to metabolic health in obese individuals.

category: cell biology


title: Cladistic hypotheses as degree of equivalence relational structures: implications for three-itemstatements

abstract: Three-item statements, as minimal informative rooted binary phylogenetic trees on three items, are the minimal units of cladistic information. Their importance for phylogenetic reconstruction, consensus and supertree methods relies on both (i) the fact that any cladistic tree can always be decomposed into a set of three-item statements, and (ii) the possibility, at least under some conditions, to build a new cladistic tree by combining all or part of the three-item statements deduced from several prior cladistic trees. In order to formalise such procedures, several k-adic rules of inference, i.e., rules that allow us to deduce at least one new three-item statement from exactly k other ones, have been identified. However, no axiomatic background has been proposed, and it remains unknown if a particular k-adic rule of inference can be reduced to more basic rules. In order to solve this problem, we propose here to define three-item statements in terms of degree of equivalence relations. Given both the axiomatic definition of the latter and their strong connection to hierarchical classifications, we establish a list of the most basic properties for three-item statements. With such an approach, we show that it is possible to combine five three-item statements from basic rules although they are not combinable only from dyadic rules. Such a result suggests that all higher k-adic rules are well reducible to a finite set of simpler rules.

category: evolutionary biology


title: The dynamic ontogenetic patterns of adaptive divergence and sexual dimorphism in Arctic charr

abstract: Arctic charr (Salvelinus alpinus) in lake Thingvallavatn (Iceland) is one of the most iconic examples of post-glacial adaptive divergence, resulting in four ecomorphs that diverge along the ecological benthic-limnetic axis (bottom lake versus open water feeders), and are distinct both phenotypically and genotypically. Here, we used geometric morphometrics tools on a common garden setup to determine the factors responsible for genetically based shape variation during the post-embryonic ontogeny of two morphs that represent the benthic-limnetic axis: the small benthic (SB) and the planktivorous (PL). This experiment uses pure crosses and F1 reciprocal hybrids between the two morphs, and includes the onset of sexual maturation, offering an excellent opportunity to explore the genetic component of adaptive divergence and the role of sexual dimorphism in this scenario. We found that growth is the main driver of shape variation across time and provided evidence of a genetically-controlled ontogenetic shift that gives rise to the limnetic morph. Additionally, our results indicate that the onset of sexual maturation triggers differences both in sex ontogenetic trajectories and in static shape variation at different time points, likely dissipating the canalisation for traits traditionally associated with benthic-limnetic adaptations.


title: Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster

abstract: In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the bamBW hypomorphic mutant as a transheterozygote over a bam null. However, bamBW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bamBW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (bamL255F) and a new bam null disruption mutation into the w1118 isogenic background. We assess the fertility of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/ bamL255F mutant females, each infected individually with ten W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

category: genetics


title: Diverse wMel variants of Wolbachia pipientis differentially rescue fertility and cytological defects of the bag of marbles partial loss of function mutation in Drosophila melanogaster


title: RIPK3 promoter hypermethylation in hepatocytes protects from bile acid induced inflammation and necroptosis

abstract:
Background & AimsNecroptosis facilitates cell death in a controlled manner and is employed by many cell types following injury. It plays a major role in various liver diseases, albeit the cell type-specific regulation of necroptosis in the liver and especially hepatocytes has not yet been conceptualized.

Approaches & ResultsHere, we demonstrate that DNA methylation suppresses RIPK3 expression in human hepatocytes and HepG2 cells. In diseases leading to cholestasis the RIPK3 expression is induced in mice and humans in a cell-type specific manner. Over-expression of RIPK3 in HepG2 cells leads immediately to RIPK3 activation by phosphorylation that is further modulated by different bile acids.

ConclusionBile acids mediated RIPK3 activation facilitates the secretion and expression of IL-8 via the JNK-pathway, suggesting hepatocytes suppress RIPK3 expression to protect themselves from bile acid induced necroptosis and inflammation but in chronical liver diseases associated with cholestasis induction of RIPK3 expression may be an early event signaling danger and repair through release of IL-8.

Graphical abstract

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category: biochemistry


title: Metabolism Control in 3D Printed Living Materials

abstract: The three-dimensional printing of cells offers an attractive opportunity to design and develop innovative biotechnological applications, such as the fabrication of biosensors or modular bioreactors. Living materials (LMs) are cross-linked polymeric hydrogel matrices containing cells, and recently, one of the most deployed LMs consists of F127-bis-urethane methacrylate (F127-BUM). The material properties of F127-BUM allow reproducible 3D printing and stability of LMs in physiological environments. These materials are permissible for small molecules like glucose and ethanol. However, no information is available for oxygen, which is essential-- for example, towards the development of aerobic bioprocesses using microbial cell factories. To address this challenge, we investigated the role of oxygen as a terminal electron acceptor in the budding yeasts respiratory chain and determined its permissibility in LMs. We quantified the ability of cell-retaining LMs to utilize oxygen and compared it with cells in suspension culture. We found that the cells ability to consume oxygen was heavily impaired inside LMs, indicating that the metabolism mostly relied on fermentation instead of respiration. To demonstrate an application of these 3D printed LMs, we evaluated a comparative brewing process. The analysis showed a significantly higher (3.7%) ethanol production using 3D printed LMs than traditional brewing, indicating an efficient control of the metabolism. Towards molecular and systems biology studies using LMs, we developed a highly reliable method to isolate cells from LMs for flow cytometry and further purified macromolecules (proteins, RNA, and DNA). Our results show the application of F127-BUM-based LMs for microaerobic processes and envision the development of diverse bioprocesses using versatile LMs in the future.

category: bioengineering


title: Variational autoencoders learn universal latent representations of metabolomics data

abstract: Dimensionality reduction approaches are commonly used for the deconvolution of high-dimensional metabolomics datasets into underlying core metabolic processes. However, current state-of-the-art methods are widely incapable of detecting nonlinearities in metabolomics data. Variational Autoencoders (VAEs) are a deep learning method designed to learn nonlinear latent representations which generalize to unseen data. Here, we trained a VAE on a large-scale metabolomics population cohort of human blood samples consisting of over 4,500 individuals. We analyzed the pathway composition of the latent space using a global feature importance score, which showed that latent dimensions represent distinct cellular processes. To demonstrate model generalizability, we generated latent representations of unseen metabolomics datasets on type 2 diabetes, schizophrenia, and acute myeloid leukemia and found significant correlations with clinical patient groups. Taken together, we demonstrate for the first time that the VAE is a powerful method that learns biologically meaningful, nonlinear, and universal latent representations of metabolomics data.

category: bioinformatics


title: Chitinase 3-like-1 Stimulates PD-L1 and Other Immune Checkpoint Inhibitors

abstract: PD-1 and its ligand PD-L1 are major mediators of tumor-induced immunosuppression. Chitinase 3-like-1 (Chi3l1) is induced in many cancers where it portends a poor prognosis and contributes to tumor metastasis. Here we demonstrate that Chi3l1 regulates the expression of PD-L1, PD-L2, PD-1 and LAG3 in melanoma lung metastasis. Chi3l1 stimulates macrophage PD-L1 expression and mediates optimal IFN-{gamma}-stimulated PD-L1 expression via IL-13R2. We also demonstrate that RIG-like helicase innate immune activation suppresses Chi3l1, PD-L1, LAG3 and pulmonary metastasis. At least additive antitumor responses were seen in metastasis models treated simultaneously with individual antibodies against PD-1 and Chi3l1. At least additive cytotoxic T cell-induced tumor cell death was also seen in co-cultures of T and tumor cells treated with antibodies that target Chi3l1 and PD-1. Thus, Chi3l1 contributes to pulmonary metastasis by stimulating the PD1-PD-L1 axis and other checkpoint molecules. The simultaneous targeting of Chi3l1 and the PD-1-PD-L1 axis, represents a promising therapeutic strategy for pulmonary metastasis.

category: cancer biology


title: Porcine pancreatic ductal epithelial cells transformed with KRASG12D and SV40T are tumorigenic

abstract: We describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.


title: PECAM-1 preserves cardiac function in pressure overload-induced biomechanical stress

abstract:
BackgroundHaemodynamic forces play a critical role in proper development of the heart, however much less is known about the mechanisms that regulate cardiac remodelling and function in response to haemodynamic stress in the adult. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion and signalling molecule that has important roles in regulation of junctional integrity, transendothelial migration and mechanotransduction in response to fluid shear stress. Our previous work identified a role for PECAM-1 in regulating baseline cardiac function via regulation of endothelial-cardiomyocyte communication.

MethodsThis study investigates the role of PECAM-1 in cardiac remodelling in response to biomechanical stress due to pressure overload induced by transaortic constriction (TAC).

ResultsOur data reveal that loss of PECAM-1 is associated with systolic dysfunction that is further accentuated following TAC. Adaptive increases in cardiomyocyte cross-sectional area, capillary density and hypertrophic gene expression were all affected with loss of PECAM-1. In control mice, maintained cardiac function was associated with activation of the c-Jun NH(2)-terminal kinase (JNK) pathway, whereas PECAM-1 deletion significantly decreased JNK activation after pressure overload. Our data suggest that in the absence of PECAM-1 signalling, inadequate remodelling of the heart under increased mechanical strain leads to further deterioration of cardiac function, characterized by reduced cardiomyocyte hypertrophy, capillary density and defects in the JNK signalling pathway.

ConclusionsOur study reveals a role for PECAM-1 in preservation of cardiac function in response to biomechanical stress induced by pressure overload.

category: physiology


title: GAD65Cre drives reporter expression in multiple taste cell types

abstract: In taste buds, Type I cells represent the majority of cells (50-60%) and primarily have a glial-like function in taste buds. However, recent studies suggest that they have additional sensory and signaling functions including amiloride-sensitive salt transduction, oxytocin modulation of taste, and substance P mediated GABA release. Nonetheless, the overall function of Type I cells in transduction and signaling remains unclear, primarily because of the lack of a reliable reporter for this cell type. GAD65 expression is specific to Type I taste cells and GAD65 has been used as a Cre driver to study Type I cells in salt taste transduction. To test the specificity of transgene-driven expression, we crossed GAD65Cre mice with floxed tdTomato and Channelrhodopsin (ChR2) lines and examined the progeny with immunochemistry, chorda tympani recording, and calcium imaging. We report that while many tdTomato+ taste cells express NTPDase2, a specific marker of Type I cells, we see expression of tdTomato in both Gustducin and SNAP25 positive taste cells. We also see ChR2 in cells just outside the fungiform taste buds. Chorda tympani recordings in the GAD65Cre/ChR2 mice show large responses to blue light, larger than any response to standard taste stimuli. Further, several isolated tdTomato positive taste cells responded to KCl depolarization with increases in intracellular calcium, indicating the presence of voltage-gated calcium channels. Taken together, these data suggest that GAD65Cre mice drive expression in multiple taste cell types and thus cannot be considered a reliable reporter of Type I cell function.


title: Selective metal extraction by biologically produced siderophores during bioleaching from low-grade primary and secondary mineral resources

abstract: Siderophores are a class of biogenic macromolecules that have high affinities for metals in the environment, thus could be exploited for alternate sustainable metal recovery technologies. Here, we assess the role of siderophores in the extraction and complexation of metals from an iron oxide-rich metallurgical processing residue and a low-grade primary Ni ore. Evaluation of the biological siderophore bioproduction by three pseudomonads, P. fluorescens, P. azotoformans and P. putida identified that P. putida could generate the highest siderophore yield, which was characterized as a hydroxamate and catecholate mixed-type pyoverdine PyoPpC-3B. Key physiochemical parameters involved in raw siderophore mediated metal extraction were identified using a fractional factorial design of experiments (DOE) and subsequently employed in purified PyoPpC-3B leaching experiments. Further targeted experiments with hydroxamate and catecholate functional analogues of PyoPpC-3B confirmed their marked ability to competitively or selectively leach and chelate hard metal ions, including Al(OH)4-, Mn2+ and Zn2+. Interestingly, complexation of Mn and Zn ions exceeded the natural affinity of pyoverdine for Fe3+, thus despite the low metal recoveries from the materials tested in this study, this work provides important new insights in siderophore-metal interactions.


title: Reference-based error correction of amplicon sequencing data from synthetic communities

abstract:
SummarySynthetic microbial communities (SynComs) constitute an emerging and powerful tool in biological, biomedical, and biotechnological research. Despite recent advances in algorithms for the analysis of culture-independent amplicon sequencing data from microbial communities, there is a lack of tools specifically designed for analysing SynCom data, where reference sequences for each strain are available. Here we present Rbec, a tool designed for the analysis of SynCom data that accurately corrects PCR and sequencing errors in amplicon sequences and identifies intra-strain polymorphic variation. Extensive evaluation using mock bacterial and fungal communities show that our tool outperforms current methods for samples of varying complexity, diversity, and sequencing depth. Furthermore, Rbec also allows accurate detection of contaminants in SynCom experiments.

Availability and implementationRbec is freely available as an open-source multi-platform R package. Release versions can be obtained via Bioconductor. The developer version is maintained and can be downloaded at: https://github.com/PengfanZhang/Rbec.

Contactgarridoo@mpipz.mpg.de


title: Rbec: a tool for analysis of amplicon sequencing data from synthetic microbial communities


title: Association of a common genetic variant with Parkinson's disease is propagated through microglia

abstract: Studies of the genetic basis of Parkinsons disease (PD) have identified many disease-associated genetic variants, but the mechanisms linking variants to pathogenicity are largely unknown. PD risk is attributed to both coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene and to common non-coding variation upstream of the LRRK2 locus. Here we show that the influence of genotype at non-coding variant rs76904798 on LRRK2 expression is propagated specifically through microglia, in contrast to evaluations based on general rather than genotype-dependent expression. We find evidence of microglia-specific regulatory regions that may modulate LRRK2 expression using single nuclei sequencing analyses of human frontal cortex and confirm these results in a human induced pluripotent stem cell-derived microglia model. Our study demonstrates that cell type is an important consideration in interrogation of the role of non-coding variation in disease pathogenesis.


title: The sensitivity of ECG contamination to surgical implantation site in adaptive closed-loop neurostimulation systems

abstract:
BackgroundBrain sensing devices are approved today for Parkinsons, essential tremor, and epilepsy therapies. Clinical decisions for implants are often influenced by the premise that patients will benefit from using sensing technology. However, artifacts, such as ECG contamination, can render such treatments unreliable. Therefore, clinicians need to understand how surgical decisions may affect artifact probability.

ObjectivesInvestigate neural signal contamination with ECG activity in sensing enabled neurostimulation systems, and in particular clinical choices such as implant location that impact signal fidelity.

MethodsElectric field modelling and empirical signals from 85 patients were used to investigate the relationship between implant location and ECG contamination.a

ResultsThe impact on neural recordings depends on the difference between ECG signal and noise floor of the electrophysiological recording. Empirically, we demonstrate that severe ECG contamination was more than 3.2x higher in left-sided subclavicular implants (48.3%), when compared to right-sided implants (15.3%). Cranial implants did not show ECG contamination.

ConclusionsGiven the relative frequency of corrupted neural signals, we conclude that implant location will impact the ability of brain sensing devices to be used for "closed-loop" algorithms. Clinical adjustments such as implant location can significantly affect signal integrity and need consideration.

HighlightsO_LIChronic embedded brain sensing promises algorithm-based neurostimulation
C_LIO_LIAlgorithms for closed-loop stimulation can be impaired by artifacts
C_LIO_LIThe relationship of implant location to cardiac dipole has relevant impact on neural signal fidelity; simple models can provide guidance on the sensitivity
C_LIO_LIECG artifacts are present in up to 50% of neural signals from left subclavicular DBS systems
C_LIO_LIImplanting DBS in a right subclavicular location significantly reduces frequency of ECG artifacts
C_LIO_LICranial-mounted implants are relatively immune to artifacts
C_LI


title: Spontaneous neural synchrony links intrinsic spinal sensory and motor networks during unconsciousness

abstract: Non-random functional connectivity during unconsciousness is a defining feature of supraspinal networks. However, its generalizability to intrinsic spinal networks remains incompletely understood. Previously, Barry et al. (2014) used fMRI to reveal bilateral resting state functional connectivity within sensory-dominant and, separately, motor-dominant regions of the spinal cord. Here, we record spike trains from large populations of spinal interneurons in vivo in rats and demonstrate that spontaneous functional connectivity also links sensory- and motor-dominant regions during unconsciousness. The spatiotemporal patterns of connectivity could not be explained by latent afferent activity or by populations of interconnected neurons spiking randomly. We also document connection latencies compatible with mono- and di-synaptic interactions and putative excitatory and inhibitory connections. The observed activity is consistent with the hypothesis that salient, experience-dependent patterns of neural transmission introduced during behavior or by injury/disease are reactivated during unconsciousness. Such a spinal replay mechanism could shape circuit-level connectivity and ultimately behavior.


title: Human antibody immune responses are personalized by selective removal of MHC-II peptide epitopes

abstract:
Human antibody responses are established by the generation of combinatorial sequence diversity in antibody variable domains, followed by iterative rounds of mutation and selection via T cell recognition of antigen peptides presented on MHC-II. Here, we report that MHC-II peptide epitope deletion from B cell receptors (BCRs) correlates with antibody development in vivo. Large-scale antibody sequence analysis and experimental validation of peptide binding revealed that MHC-II epitope removal from BCRs is linked to genetic signatures of T cell help, and donor-specific antibody repertoire modeling demonstrated that somatic hypermutation selectively targets the personalized MHC-II epitopes in antibody variable regions. Mining of class-switched sequences and serum proteomic data revealed that MHC-II epitope deletion is associated with antibody class switching and long-term secretion into serum. These data suggest that the MHC-II peptide epitope content of a BCR is an important determinant of antibody maturation that shapes the composition and durability of humoral immunity.

HighlightsO_LIAntibody somatic hypermutation selectively removes MHC-II peptide epitopes from B cell receptors.
C_LIO_LIAntibodies with lower MHC-II epitope content show evidence of greater T cell help, including class-switching.
C_LIO_LIMHC-II peptide epitope removal from a BCR is linked to long-term antibody secretion in serum.
C_LIO_LIMHC-II genotype provides a personalized selection pressure on human antibody development.
C_LI


title: High trait variability in optimal polygenic prediction strategy within multiple-ancestry cohorts

abstract: Polygenic scores (PGSs) are individual-level measures that aggregate the genome-wide genetic predisposition to a given trait. As PGS have predominantly been developed using European-ancestry samples, trait prediction using such European ancestry-derived PGS is less accurate in non-European ancestry individuals. Although there has been recent progress in combining multiple PGS trained on distinct populations, the problem of how to maximize performance given a multiple-ancestry cohort is largely unexplored. Here, we investigate the effect of sample size and ancestry composition on PGS performance for fifteen traits in UK Biobank. For some traits, PGS estimated using a relatively small African-ancestry training set outperformed, on an African-ancestry test set, PGS estimated using a much larger European-ancestry only training set. We observe similar, but not identical, results when considering other minority-ancestry groups within UK Biobank. Our results emphasise the importance of targeted data collection from underrepresented groups in order to address existing disparities in PGS performance.

category: genomics


title: Optimal strategies for learning multi-ancestry polygenic scores vary across traits


title: Human Cytomegalovirus uses a Host Stress Response to Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very Long Chain Fatty Acids

abstract:
Stress and virus infection are known to regulate lipid metabolism in cells. Human cytomegalovirus infection induces fatty acid (FA) elongation and increases the cellular abundance of lipids with very long-chain FA tails (VLCFAs). While reprogramming of metabolism can be stress-related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knockout PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. We found that in HCMV-infected cells, PERK promotes the increase in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFAs tails. Consistent with the SFA/MUFA lipidome changes, PERK enhances the protein levels of FA elongase 7 (ELOVL7), which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection was independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Consistent with the PUFA lipidome changes, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK--and more broadly, ER stress--may be necessary for membrane biogenesis needed to generate infectious HCMV virions.

IMPORTANCEHCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator, PERK, controls fatty acid (FA) elongation and cellular abundance of several types of lipids following HCMV infection. Specifically, we found that PERK promotes FA elongase 7 synthesis of lipids with saturated/monounsaturated very long-chain FA tails which are important for building the viral membrane of infectious HCMV virions. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

category: microbiology


title: Regulation of bacterial Type III Secretion System export gate opening

abstract: Type III Secretion Systems (T3SS) transport proteins from the bacterial cytosol for assembly into cell surface nanomachines or for direct delivery into target eukaryotic cells. At the core of the flagellar T3SS, the FlhAB-FliPQR export gate regulates protein entry into the export channel whilst maintaining the integrity of the cell membrane. Here, we identify critical residues in the export gate FliR plug that stabilise the closed conformation, preserving the membrane permeability barrier, and we show that the gate opens and closes in response to export substrate availability. Our data indicate that FlhAB-FliPQR gate opening, which is triggered by substrate export signals, is energised by FlhA in a proton motive force-dependent manner. We present evidence that the export substrate and the FliJ stalk of the flagellar ATPase provide mechanistically distinct, non-redundant gate-activating signals that are critical for efficient export.


title: An updated structural model of the A domain of the Pseudomonas putida XylR regulator exposes a distinct interplay with aromatic effectors

abstract:
A revised model of the aromatic binding A domain of the {sigma}54-dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR, and DmpR. The resulting frame was instrumental for mapping the large number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic-binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc-binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Proteomic analyses of the thereby tagged peptides confirmed the predicted in/out distribution of residues but also suggested that the fully-folded protein is not accessible by externally added effectors. The data thus suggested that XylR inducers assist the folding and/or the structuring of the A domain in an intramolecular non-repressive form rather than interacting dynamically with the aromatic partner once a fully structured protein is shaped.

Originality-Significance StatementXylR is a transcriptional regulator of Pseudomonas putida strain mt-2 which activates the upper TOL pathway promoter Pu for catabolism of toluene and m-xylene upon binding of these aromatic effectors to its N-terminal A domain. While this feature has made XylR a popular platform for the development of whole-cell biosensors for aromatic compounds, the difficulty to crystallize the A domain --let alone the whole-length protein-- has made structural comprehension of the effector-regulator binding quite problematic. To overcome this impasse, we have combined homology-based structural predictions of the A domain of XylR with biochemical probing of exposed amino acids on the surface of the protein, both in vivo and in vitro. The results generally matched the effects of mutations known from previous genetic/phenotypic analyses of the protein. However, the data also suggested an intriguing mechanism of activation of XylR by effectors in which the inducer assists the shaping of the regulator in an active conformation rather than interacting a posteriori with an already formed protein invitro. This may in fact explain the longstanding failure to purify the protein in an effector-responsive form.


title: The dynamic trophic architecture of open-ocean protist communities revealed through machine- guided metatranscriptomics

abstract:
Intricate networks of single-celled eukaryotes (protists) dominate carbon flow in the ocean. Their growth, demise, and interactions with other microorganisms drive the fluxes of biogeochemical elements through marine ecosystems. Mixotrophic protists are capable of both photosynthesis and ingestion of prey and are dominant components of open-ocean planktonic communities. Yet, the role of mixotrophs in elemental cycling is obscured by their capacity to act as primary producers or heterotrophic consumers depending on factors that remain largely uncharacterized. Here we introduce a machine learning model that can predict the in situ nutritional mode of aquatic protists based on their patterns of gene expression. This approach leverages a public collection of protist transcriptomes as a training set to identify a subset of gene families whose transcriptional profiles predict trophic status. We applied our model to nearly 100 metatranscriptomes obtained during two oceanographic cruises in the North Pacific and found community-level and population-specific evidence that abundant open-ocean mixotrophic populations shift their predominant mode of nutrient and carbon acquisition in response to natural gradients in nutrient supply and sea surface temperature. In addition, metatranscriptomic data from ship-board incubation experiments revealed that abundant mixotrophic prymnesiophytes from the oligotrophic North Pacific subtropical gyre rapidly remodelled their transcriptome to enhance photosynthesis when supplied with limiting nutrients. Coupling the technique introduced here with experiments designed to reveal the mechanisms driving mixotroph physiology is a promising approach for understanding the ecology of mixotrophic populations in the natural environment.

Significance statementMixotrophy is a ubiquitous nutritional strategy in marine ecosystems. Although our understanding of the distribution and abundance of mixotrophic plankton has improved significantly, the functional roles of mixotrophs are difficult to pinpoint, as mixotroph nutritional strategies are flexible and form a continuum between heterotrophy and phototrophy. We employ a machine learning-driven metatranscriptomic technique to assess the nutritional strategies of abundant planktonic populations in situ and demonstrate that mixotrophic populations play varying functional roles along physico-chemical gradients in the North Pacific Ocean, revealing a degree of physiological plasticity unique to aquatic mixotrophs. Our results highlight mechanisms that may dictate the flow of biogeochemical elements and the ecology of the North Pacific Ocean, one of the largest biogeographical provinces on Earth.

category: ecology


title: Sporulation is dispensable for the vegetable-associated life cycle of the human pathogen Bacillus cereus

abstract: Bacillus cereus is a common food-borne pathogen that is responsible for important outbreaks of food poisoning in humans. Diseases caused by B. cereus usually exhibit two major symptoms, emetic or diarrheic, depending on the toxins produced. It is assumed that after the ingestion of contaminated vegetables or processed food, spores of enterotoxigenic B. cereus reach the intestine, where they germinate and produce the enterotoxins that are responsible for food poisoning. In our study, we observed that sporulation is required for the survival of B. cereus in leaves but is dispensable in ready-to-eat vegetables, such as endives. We demonstrate that vegetative cells of B. cereus that are originally impaired in sporulation but not biofilm formation are able to reach the intestine and cause severe disorders in a murine model. We propose that loss of part of the sporulation programme and reinforcement of structural factors related to adhesion, biofilm formation and pathogenic interaction with the host are adaptive traits of B. cereus with a life cycle primarily related to human hosts. Furthermore, our findings emphasise that the number of food poisoning cases associated with B. cereus is underestimated and suggest the need to revise the detection protocols, which are based primarily on spores and toxins.


title: Fractal triads efficiently sample ecological diversity and processes across spatial scales

abstract: The relative influence of ecological assembly processes, such as environmental filtering, competition, and dispersal, vary across spatial scales. Changes in phylogenetic and taxonomic diversity across environments provide insight into these processes, however, it is challenging to assess the effect of spatial scale on these metrics. Here, we outline a nested sampling design that fractally spaces sampling locations to concentrate statistical power across spatial scales in a study area. We test this design in northeast Utah, at a study site with distinct vegetation types (including sagebrush steppe and mixed conifer forest), that vary across environmental gradients. We demonstrate the power of this design to detect changes in community phylogenetic diversity across environmental gradients and assess the spatial scale at which the sampling design captures the most variation in empirical data. We find clear evidence of broad-scale changes in multiple features of phylogenetic and taxonomic diversity across aspect. At finer scales, we find additional variation in phylo-diversity, highlighting the power of our fractal sampling design to efficiently detect patterns across multiple spatial scales. Thus, our fractal sampling design and analysis effectively identify important environmental gradients and spatial scales that drive community phylogenetic structure. We discuss the insights this gives us into the ecological assembly processes that differentiate plant communities found in northeast Utah.


title: Chromosome size affects sequence divergence between species through the interplay of recombination and selection

abstract: The structure of the genome shapes the distribution of genetic diversity and sequence divergence. To investigate how the relationship between chromosome size and recombination rate affects sequence divergence between species, we combined empirical analyses and evolutionary simulations. We estimated pairwise sequence divergence among 15 species from three different Mammalian clades - Peromyscus rodents, Mus mice, and great apes - from chromosome-level genome assemblies. We found a strong significant negative correlation between chromosome size and sequence divergence in all species comparisons within the Peromyscus and great apes clades, but not the Mus clade, suggesting that the dramatic chromosomal rearrangements among Mus species may have masked the ancestral genomic landscape of divergence in many comparisons. Our evolutionary simulations showed that the main factor determining differences in divergence among chromosomes of different size is the interplay of recombination rate and selection, with greater variation in larger populations than in smaller ones. In ancestral populations, shorter chromosomes harbor greater nucleotide diversity. As ancestral populations diverge, diversity present at the onset of the split contributes to greater sequence divergence in shorter chromosomes among daughter species. The combination of empirical data and evolutionary simulations revealed that chromosomal rearrangements, demography, and divergence times may also affect the relationship between chromosome size and divergence, and deepen our understanding of the role of genome structure on the evolution of species divergence.


title: Multi Task deep learning for genomic predictions

abstract: Genomic predictions have been recognized as a new promising technique in animal and plant breeding. Linear mixed model is a widely used statistical technique, but it may not be desirable for large training sets and number of molecular markers, because it is intensive in computation. Deep learning is a subfield of machine learning and it can be used for complex predictions on a large scale. Multi task deep learning (MT-DL) incorporates related tasks(labels or traits) into one learning process to enable the learning model to perform better than single task deep learning (ST-DL). I applied MT-DL to genotype by environment genomic predictions to predict the performances of breeding lines at multiple environments. I compared MT-DL with linear mixed model-based Bayesian genotype x environment method (BGGE) and separate genomic predictions on single environments with widely used rrBLUP, ridge regression and ST-DL using cross validations. Compared with rrBLUP, MT-DL and non-linear BGGE showed a moderate increase of 9.4 and 7.6%, respectively, ST-DL has a small increase of 5.4%, ridge regression had a similar prediction accuracy and linear BGGE had a small decrease of -2.0% for prediction accuracy. I also found that all methods including rrBLUP had an overfitting, this is likely because yield genomic predictions are complex and the data set used in this study are small. rrBLUP, ridge regression, ST-DL and MT-DL has similar overfitting. Difference between training and test set prediction accuracies was between 0.344 and 0. 387. Linear and nonlinear BGGE methods seem to have much worse overfitting than other methods. Difference between training and test set prediction accuracies were 0.429 and 0.472, respectively. I also discussed the potential applications of ST-DL and MT-DL in genomic predictions of hybrid crops such as maize


title: Protective Effects of Matrine on Acute Myocardial Hypertrophy Induced by Isoproterenol via Akt/mTOR/p70S6K/eIF4E Signaling Pathway in Rats

abstract: This study aimed to investigate whether matrine (Ma) attenuates isoproterenol (ISO)-induced acute myocardial hypertrophy via activating Akt/mTOR/p70S6K/eIF4E signaling pathway in rats. We collected 42 male Sprague-Dawley rats weighing 300{+/-}20g, randomly divided into seven groups (n=6). The myocardial hypertrophy (MH) model was well established by 85 mg/kg ISO. Changes in hemodynamic parameters were recorded using electrocardiogram after 24h with ISO injection. Western blot and real-time polymerase chain reaction were used to evaluate the expression of Akt, mechanistic target of rapamycin (mTOR), p70S6K, and eIF4E. Tissue arrangement of the 200 and 100 mg/kg Ma+ISO groups was regularly smaller than that of the ISO group. For the protein expression, Akt values in the 200 and 100 mg/kg Ma+ISO groups were 1.57- and 1.81-fold higher than in the ISO group, respectively. Moreover, compared with the ISO group, the expression trends of mTOR in the 200, 100, and 50 mg/kg Ma+ISO groups significantly downregulated. The levels of p70S6K and eIF4E reduced in the 200, 100, and 50 mg/kg Ma+ISO groups according to the ISO group (P<0.05). MRNA expression of p70S6K and eIF4E in the ISO group were 1.90- and 6.38-fold higher compared with that in the 100 mg/kg Ma+ISO group. Ma exerted neuroprotective effects against pachyntic injury. Akt activity was accelerated, but activities of mTOR, p70S6K, and eIF4E were inhibited by Ma. Activation of the Akt/mTOR/p70S6K/eIF4E signaling pathway might be the targets for the protective effects of Ma on acute myocardial hypertrophy in rats.


title: Investigating the impact of reference assembly choice on genomic analyses in a cattle breed

abstract:
BackgroundReference-guided read alignment and variant genotyping are prone to reference allele bias, particularly for samples that are greatly divergent from the reference genome. A Hereford-based assembly is the widely accepted bovine reference genome. Haplotype-resolved genomes that exceed the current bovine reference genome in quality and continuity have been assembled for different breeds of cattle. Using whole genome sequencing data of 161 Brown Swiss cattle, we compared the accuracy of read mapping and sequence variant genotyping as well as downstream genomic analyses between the bovine reference genome (ARS-UCD1.2) and a highly continuous Angus-based assembly (UOA_Angus_1).

ResultsRead mapping accuracy did not differ notably between the ARS-UCD1.2 and UOA_Angus_1 assemblies. We discovered 22,744,517 and 22,559,675 high-quality variants from ARS-UCD1.2 and UOA_Angus_1, respectively. The concordance between sequence- and array-called genotypes was high and the number of variants deviating from Hardy-Weinberg proportions was low at segregating sites for both assemblies. More artefactual INDELs were genotyped from UOA_Angus_1 than ARS-UCD1.2 alignments. Using the composite likelihood ratio test, we detected 40 and 33 signatures of selection from ARS-UCD1.2 and UOA_Angus_1, respectively, but the overlap between both assemblies was low. Using the 161 sequenced Brown Swiss cattle as a reference panel, we imputed sequence variant genotypes into a mapping cohort of 30,499 cattle that had microarray-derived genotypes. The accuracy of imputation (Beagle R2) was very high (0.87) for both assemblies. Genome-wide association studies between imputed sequence variant genotypes and six dairy traits as well as stature produced almost identical results from both assemblies.

ConclusionsThe ARS-UCD1.2 and UOA_Angus_1 assemblies are suitable for reference-guided genome analyses in Brown Swiss cattle. Although differences in read mapping and genotyping accuracy between both assemblies are negligible, the choice of the reference genome has a large impact on detecting signatures of selection using the composite likelihood ratio test. We developed a workflow that can be adapted and reused to compare the impact of reference genomes on genome analyses in various breeds, populations and species.


title: Pathological turret mutations in the cardiac sodium channel cause long-range pore disruption

abstract: The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Germline mutations that disrupt Nav1.5 activity predispose affected individuals to inherited cardiopathologies. Some of these Nav1.5 mutations alter amino acids in extracellular turret domains DII and DIII. Yet the mechanism is unclear. In the rat Nav1.5 structure determined by cryogenic electron microscopy, the wild-type residues corresponding to these mutants form a complex salt-bridge between the DII and DIII turret interface. Furthermore, adjacent aromatic residues form cation-{pi} interactions with the complex salt-bridge. Here, we examine this region using site-directed mutagenesis, electrophysiology and in silico modeling. We confirm functional roles for the salt-bridges and the aromatic residues. We show that their disruption perturbs the geometry of both the DEKA selectivity ring and the inner pore vestibule that are crucial for sodium ion permeability. Our findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms too. Our work illustrates how the sodium channel structures now being reported can be used to formulate and guide novel functional hypotheses.


title: Circadian Alterations Increase with Progression in a Patient-Derived Cell Culture Model of Breast Cancer

abstract: Circadian rhythms are critical regulators of many physiological and behavioral functions; disruption of this time-tracking system can elicit the development of various diseases, including breast cancer. While multiple studies have used cell lines to study the correlation between altered circadian rhythms and cancer, these cells generally have different genetic backgrounds and do not mirror the changes that occur with disease development. Isogenic cell models can represent and recapitulate changes across cancer progression. Hence in the present study, a patient-derived breast cancer model, the 21T series, was used to evaluate changes to circadian oscillations of core clock protein transcription and translation as cells progress from normal to malignant states. Three cell lines from the series were used: H16N2, from normal breast epithelium; 21PT, from Atypical Ductal Hyperplasia; and 21MT, from Invasive Metastatic Carcinoma. Both of the cancerous cell lines are HER2 positive. We assessed the transcriptional profiles of two core circadian clock proteins, BMAL1 and PER2, which represent a positive and negative component of the molecular oscillator. In the normal H16N2 cells, BMAL1 and PER2 both possessed rhythmic mRNA oscillations with close to standard periods and the expected anti-phase relationship. However, in the cancerous cells, consistent changes were observed: both clock genes had periods that deviated farther from normal and did not have an anti-phase relationship. To provide a more complete understanding of circadian alterations in breast cancer, luciferase reporters and real-time luminometry should be used in future studies.


title: Analysis of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase

abstract:
Notwithstanding the "one-module-one-elongation-cycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all trans-configured triene moiety in the polyketide product. Here we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the "split-and-stuttering" module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.

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title: Evolution of the cytochrome-bd type oxygen reductase superfamily and the function of cydAA in Archaea

abstract: Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify 3 families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA is the first isoform of cytbd containing only b-type hemes shown to be active when isolated, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.


title: Phospholipase A2 inhibitor-loaded micellar nanoparticles attenuate inflammation and mitigate osteoarthritis progression

abstract: Treating osteoarthritis (OA) remains a major clinical challenge. Despite recent advances in drug discovery and development, no disease-modifying drug for knee OA has emerged with any significant clinical success, in part due to the lack of valid and responsive therapeutic targets and poor drug delivery within knee joints. In this work, we show that the amount of secretory phospholipase A2 (sPLA2) enzyme increases in articular cartilage in human and mouse OA cartilage tissues. We hypothesize that inhibition of sPLA2 activity may be an effective treatment strategy for OA. To develop a sPLA2-responsive and nanoparticle (NP)-based interventional platform for OA management, we incorporated a sPLA2 inhibitor (sPLA2i) into the phospholipid membrane of micelles. The engineered sPLA2i-loaded micellar nanoparticles (sPLA2i-NPs) were able to penetrate deep into the cartilage matrix, prolong retention in the joint space, and mitigate OA progression. These findings suggest that sPLA2i-NPs can be promising therapeutic agents for OA treatment.


title: Optimizing viral genome subsampling by genetic diversity and temporal distribution (TARDiS) for Phylogenetics

abstract: TARDiS for Philogenetics is a novel tool for optimal genetic sub-sampling. It optimizes both genetic diversity and temporal distribution through a genetic algorithm. TARDiS, along with example data sets and a user manual, is available at https://github.com/smarini/tardis-phylogenetics


title: The GA4GH Variation Representation Specification (VRS): a Computational Framework for the Precise Representation and Federated Identification of Molecular Variation

abstract: Maximizing the personal, public, research, and clinical value of genomic information will require that clinicians, researchers, and testing laboratories exchange genetic variation data reliably. Developed by a partnership among national information resource providers, public initiatives, and diagnostic testing laboratories under the auspices of the Global Alliance for Genomics and Health (GA4GH), the Variation Representation Specification (VRS, pronounced "verse") is an extensible framework for the semantically precise and computable representation of variation that complements contemporary human-readable and flat file standards for variation representation. VRS objects are designed to be semantically precise representations of variation, and leverage this design to enable unique, federated identification of molecular variation. We describe the components of this framework, including the terminology and information model, schema, data sharing conventions, and a reference implementation, each of which is intended to be broadly useful and freely available for community use. The specification, documentation, examples, and community links are available at https://vrs.ga4gh.org/.


title: The statistics of k-mers from a sequence undergoing a simple mutation process without spurious matches

abstract: K-mer-based methods are widely used in bioinformatics, but there are many gaps in our understanding of their statistical properties. Here, we consider the simple model where a sequence S (e.g. a genome or a read) undergoes a simple mutation process whereby each nucleotide is mutated independently with some probability r, under the assumption that there are no spurious k-mer matches. How does this process affect the k-mers of S? We derive the expectation and variance of the number of mutated k-mers and of the number of islands (a maximal interval of mutated k-mers) and oceans (a maximal interval of non-mutated k-mers). We then derive hypothesis tests and confidence intervals for r given an observed number of mutated k-mers, or, alternatively, given the Jaccard similarity (with or without minhash). We demonstrate the usefulness of our results using a few select applications: obtaining a confidence interval to supplement the Mash distance point estimate, filtering out reads during alignment by Minimap2, and rating long read alignments to a de Bruijn graph by Jabba.


title: Real-Time, Direct Classification of Nanopore Signals with SquiggleNet

abstract: Oxford Nanopore sequencers provide results in real time as DNA passes through a nanopore and can eject a molecule after it has been partly sequenced. However, the computational challenge of deciding whether to keep or reject a molecule in real time has limited the application of this capability. We present SquiggleNet, the first deep learning model that can classify nanopore reads directly from their electrical signals. SquiggleNet operates faster than the DNA passes through the pore, allowing real-time classification and read ejection. When given the amount of sequencing data generated in one second, the classifier achieves significantly higher accuracy than base calling followed by sequence alignment. Our approach is also faster and requires an order of magnitude less memory than approaches based on alignment. SquiggleNet distinguished human from bacterial DNA with over 90% accuracy, generalized to unseen species, identified bacterial species in a human respiratory meta genome sample, and accurately classified sequences containing human long interspersed repeat elements.


title: Traumatic Brain Injury Impairs Systemic Vascular Function Through Altered Lipid Metabolism and Disruption of Inward-Rectifier Potassium (Kir2.1) Channels

abstract:
BACKGROUND AND PURPOSETrauma can lead to widespread vascular endothelial dysfunction, but the underlying mechanisms remain largely unknown. Strong inward-rectifier potassium channels (Kir2.1) play a critical role in the dynamic regulation of regional perfusion and blood flow. Kir2.1 channel activity is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2), a minor membrane phospholipid that is degraded by phospholipase A2 (PLA2) in conditions of oxidative stress or severe inflammation. We hypothesized that PLA2-induced depletion of PIP2 impairs Kir2.1 channel function.

METHODSA fluid percussion injury model of traumatic brain injury (TBI) in rats was used to study mesenteric resistance arteries 24 hours after injury. Patch-clamp electrophysiology in freshly isolated endothelial and smooth muscle cells was performed to monitor Kir2.1 conductance, and the functional responses of intact arteries were assessed using pressure myography. We analyzed circulating PLA2, hydrogen peroxide (H2O2), and metabolites to identify alterations in signaling pathways associated with PIP2 in TBI.

RESULTSElectrophysiology analysis of endothelial and smooth muscle cells revealed a significant reduction of Ba2+-sensitive Kir2.1 currents after TBI. Additionally, dilations to elevated extracellular potassium and BaCl2- or ML 133-induced constrictions in pressurized arteries were significantly decreased following TBI, consistent with an impairment of Kir2.1 channel function. The addition of a PIP2 analog to the patch pipette successfully rescued endothelial Kir2.1 currents after TBI. Both H2O2 and PLA2 activity were increased after injury. Metabolomics analysis demonstrated altered lipid metabolism signaling pathways, including increased arachidonic acid, and fatty acid mobilization after TBI.

CONCLUSIONSOur findings support a model in which increased H2O2-induced PLA2 activity after trauma hydrolyzes endothelial PIP2, resulting in impaired Kir2.1 channel function.


title: Spatial metabolomics using imaging mass spectrometry to identify the localization of asparaptine in Asparagus officinalis

abstract: Spatial metabolomics uses imaging mass spectrometry (IMS) to localize metabolites within tissue section. Here, we performed matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance-IMS (MALDI-FTICR-IMS) to identify the localization of asparaptine, a naturally occurring inhibitor of angiotensin-converting enzyme, in green spears of asparagus (Asparagus officinalis). Spatial metabolome data were acquired with an untargeted manner. Segmentation analysis using the data characterized tissue-type-dependent and - independent distribution patterns in cross-sections of asparagus spears. Moreover, asparaptine accumulated at high levels in developing lateral shoot tissues. Quantification of asparaptine in lateral shoots using liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated the IMS analysis. These results provide valuable information for understanding the function of asparaptine in asparagus, and identify the lateral shoot as a potential region of interest for multiomic studies to examine gene-to-metabolite associations in asparaptine biosynthesis.

category: plant biology


title: Spatiotemporal dynamics of maximal and minimal EEG spectral power

abstract: Oscillatory neural activities are prevalent in the brain with their phase realignment contributing to the coordination of neural communication. Phase realignments may have especially strong (or weak) impact when neural activities are strongly synchronized (or desynchronized) within the interacting populations. We report that the spatiotemporal dynamics of strong regional synchronization measured as maximal EEG spectral power--referred to as activation--and strong regional desynchronization measured as minimal EEG spectral power--referred to as suppression--are characterized by the spatial segregation of small-scale and large-scale networks. Specifically, small-scale spectral-power activations and suppressions involving only 2-7% (1-4 of 60) of EEG scalp sites were prolonged (relative to stochastic dynamics) and consistently co-localized in a frequency specific manner. For example, the small-scale networks for{theta} , , {beta}1, and {beta}2 bands (4-30 Hz) consistently included frontal sites when the eyes were closed, whereas the small-scale network for{gamma} band (31-55 Hz) consistently clustered in medial-central-posterior sites whether the eyes were open or closed. Large-scale activations and suppressions involving over 17-30% (10-18 of 60) of EEG sites were also prolonged and generally clustered in regions complementary to where small-scale activations and suppressions clustered. In contrast, intermediate-scale activations and suppressions (involving 7-17% of EEG sites) tended to follow stochastic dynamics and were less consistently localized. These results suggest that strong synchronizations and desynchronizations tend to occur in small-scale and large-scale networks that are spatially segregated and frequency specific. These synchronization networks may broadly segregate the relatively independent and highly cooperative oscillatory processes while phase realignments fine-tune the network configurations based on behavioral demands.


title: Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

abstract: Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.


title: Fibrin is a critical regulator of neutrophil effector function at mucosal barrier sites

abstract: Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we document that the clotting factor fibrin is a critical regulator of neutrophil function at mucosal barriers. We demonstrate that fibrin engages neutrophils through the M{beta}2 integrin receptor and activates effector functions, including the production of reactive oxygen species and NET formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis. Indeed, the accumulation of fibrin due to Mendelian genetic defects in plasmin leads to severe oral mucosal immunopathology in mice and humans. Concordantly, genetic polymorphisms in the human PLG gene, encoding plasminogen, are associated with common forms of the oral mucosal disease periodontitis. Our work uncovers fibrin as a critical regulator of neutrophil effector function within the mucosal tissue microenvironment and suggests fibrin-neutrophil engagement as a pathogenic instigator and therapeutic target in common mucosal disease.


title: Towards a mathematical understanding of colonization resistance

abstract: Microbial community composition and dynamics are key to health and disease. Explaining the forces generating and shaping diversity in the microbial consortia making up our bodys defenses is a major aim of current research in microbiology. For this, tractable models are needed, that bridge the gap between observations of patterns and underlying mechanisms. While most microbial dynamics models are based on the Lotka-Volterra framework, we still do not have an analytic quantity for colonization resistance, by which a microbial systems fitness as a whole can be understood. Here, inspired by an epidemiological perspective, we propose a rather general modeling framework whereby colonization resistance can be clearly mathematically defined and studied. In our model, N similar species interact with each other through a co-colonization interaction network encompassing pairwise competition and cooperation, abstractly mirroring how organisms effectively modify their micro-scale environment in relation to others. This formulation relies on a generic notion of shared resources between members of a consortium, yielding explicit frequency-dependent dynamics among N species, in the form of a replicator equation, and offering a precise definition of colonization resistance. We demonstrate that colonization resistance arises and evolves naturally in a multispecies system as a collective quadratic term in a replicator equation, describing dynamic mean invasion fitness. Each pairwise invasion growth rate between two ecological partners, [Formula], is derived explicitly from species asymmetries and mean traits. This makes the systemic colonization resistance [Formula] also an emergent function of global mean-field parameters and trait variation architecture, weighted by the evolving relative abundances among species. In particular, if the underlying invasion fitness matrix {Lambda} displays species-specific  invasiveness or  invasibility structure, colonization resistance will be insensitive to mean micro-scale cooperation or competition. However, in general, colonization resistance depends on and may undergo critical transitions with changes in mean  environment, e.g. cooperation and growth level in a community. We illustrate several key links between our proposed measure of colonization resistance and invader success, including sensitivity to timing, and to the intrinsic pairwise invasion architecture of the resident community. Our simulations reveal that symmetric and invader-driven mutual invasion among resident species tend to maximize systemic colonization resistance to outsiders, when compared to resident-driven, anti-symmetric, almost anti-symmetric and random {Lambda} structures. We contend this modeling approach is a powerful new avenue to study, test and validate interaction networks and invasion topologies in diverse microbial consortia, and quantify analytically their role in colonization resistance, system function, and invasibility.


title: Towards a mathematical understanding of colonization resistance in multispecies microbial communities


title: Laboratory diagnostics of tomato spotted wilt virus by PCR

abstract: Tomato spotted wilt virus is a widespread and harmful virus. It affects a wide range of host plants. The outward signs of TSWV lesions are different on different cultures. For the early diagnosis of TSWV, molecular diagnostic methods such as PCR must be used. In the course of this work, primers were developed for the diagnosis of tomato bronzing virus by real-time PCR and classical PCR. We also compared the specificity and sensitivity of various test systems for the diagnosis of tomato bronzing virus. In the course of this comparison, it was found that the self-assembly test system and the Syntol test system can be recommended for laboratory diagnostics of tomato spotted wilt virus.

category: molecular biology


title: Dynamic molecular epidemiology reveals lineage-associated single-nucleotide variants that alter RNA structure in Chikungunya virus

abstract: Chikungunya virus (CHIKV) is an emerging Alphavirus which causes millions of human infections every year. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages: West African, East Central South African, and Asian Urban. As new outbreaks occurred in the Americas, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. Building on a recently updated phylogeny of CHIKV, we report here the availability of an interactive CHIKV phylodynamics dataset, which is based on more than 900 publicly available CHIKV genomes. We provide an interactive view of CHIKV molecular epidemiology built on Nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution. CHIKV molecular epidemiology reveals single nucleotide variants that change the stability and fold of locally stable RNA structures. We propose alternative RNA structure formation in different CHIKV lineages by predicting more than a dozen RNA elements that are subject to perturbation of the structure ensemble upon variation of a single nucleotide.


title: Wnt-responsive enhancer regulation in mammals by a CDX/TCF transcription factor collective and a novel DNA sequence motif

abstract: Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of {beta}-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. We characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal-related homeodomain (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, we found that WRE activity requires the formation of a TCF/CDX complex. Our systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Put together, our results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity.


title: Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif


title: DNA-based copy number analysis confirms genomic evolution of PDX models

abstract: We previously reported the genomic evolution of the copy number (CN) landscapes of patient-derived xenografts (PDXs) during their engraftment and passaging1. Woo et al. argue that the CN profiles of PDXs are highly conserved, and that the main conclusions of our paper are invalid due to our use of expression-based CN profiles2. Here, we reassess genomic evolution of PDXs using the DNA-based CN profiles reported by Woo et al. We find that the degree of genomic evolution in the DNA-based dataset of Woo et al. is similar to that which we had previously reported. While the overall Pearsons correlation of CN profiles between primary tumors (PTs) and their derived PDXs is high (as reported in our original paper as well), a median of ~10% of the genome is differentially altered between PTs and PDXs across cohorts (range, 0% to 73% across all models). In 24% of the matched PT-PDX samples, over a quarter of the genome is differentially affected by CN alterations. Moreover, in matched analyses of PTs and their derived PDXs at multiple passages, later-passage PDXs are significantly less similar to their parental PTs than earlier-passage PDXs, indicative of genomic divergence. We conclude that genomic evolution of PDX models during model generation and propagation should not be dismissed, and that the phenotypic consequences of this evolution ought to be assessed in order to optimize the application of these valuable cancer models.


title: FiCoS: a fine- and coarse-grained GPU-powered deterministic simulator for biochemical networks

abstract:
Mathematical models of biochemical networks can largely facilitate the comprehension of the mechanisms at the basis of cellular processes, as well as the formulation of hypotheses that can be tested by means of targeted laboratory experiments. However, two issues might hamper the achievement of fruitful outcomes. On the one hand, detailed mechanistic models can involve hundreds or thousands of molecular species and their intermediate complexes, as well as hundreds or thousands of chemical reactions, a situation generally occurring in rule-based modeling. On the other hand, the computational analysis of a model typically requires the execution of a large number of simulations for its calibration or to test the effect of perturbations. As a consequence, the computational capabilities of modern Central Processing Units can be easily overtaken, possibly making the modeling of biochemical networks a worthless or ineffective effort. To the aim of overcoming the limitations of the current state-of-the-art simulation approaches, we present in this paper FiCoS, a novel "black-box" deterministic simulator that effectively realizes both a fine-grained and a coarse-grained parallelization on Graphics Processing Units. In particular, FiCoS exploits two different integration methods, namely, the Dormand-Prince and the Radau IIA, to efficiently solve both non-stiff and stiff systems of coupled Ordinary Differential Equations. We tested the performance of FiCoS against different deterministic simulators, by considering models of increasing size and by running analyses with increasing computational demands. FiCoS was able to dramatically speedup the computations up to 855x, showing to be a promising solution for the simulation and analysis of large-scale models of complex biological processes.

Author summarySystems Biology is an interdisciplinary research area focusing on the integration of biological data with mathematical and computational methods in order to unravel and predict the emergent behavior of complex biological systems. The ultimate goal is the understanding of the complex mechanisms at the basis of biological processes, together with the formulation of novel hypotheses that can be then tested by means of laboratory experiments. In such a context, mechanistic models can be used to describe and investigate biochemical reaction networks by taking into account all the details related to their stoichiometry and kinetics. Unfortunately, these models can be characterized by hundreds or thousands of molecular species and biochemical reactions, making their simulation unfeasible with classic simulators running on Central Processing Units (CPUs). In addition, a large number of simulations might be required to calibrate the models and/or to test the effect of perturbations. In order to overcome the limitations imposed by CPUs, Graphics Processing Units (GPUs) can be effectively used to accelerate the simulations of these models. We thus designed and developed a novel GPU-based tool, called FiCoS, to speed-up the computational analyses typically required in Systems Biology.

category: systems biology


title: Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells

abstract: Brain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, the cerebellum is now recognized to contribute to human cognitive evolution having a broad range of non-motor cognitive functions in addition to motor control. Emerging studies highlight the complexity of human cerebellar histogenesis, compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. Here we report a rapid and simple protocol for the directed derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC), and strategies to decrease culture variability; a common limitation in hPSC studies. Upon transplantation into juvenile mice, early postmitotic hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification (TRAP)-seq, the ATOH1 lineage most closely resembled human cerebellar tissue in the second trimester. Unexpectedly, TRAP-seq identified a heterochronic shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, within granule cell progenitors (GCPs) in the human external granule layer. This molecular divergence may provide the mechanism by which the GCP pool persists into year two post birth in humans, but only lasts for two weeks in mice. Our approach provides a scalable in vitro model of the human ATOH1 lineage that yields cerebellar granule cells within 48 days as well as a strategy for identifying uniquely human cellular and molecular characteristics.

category: developmental biology


title: Idiosyncratic purifying selection on metabolic enzymes in the long-term evolution experiment with Escherichia coli

abstract:
Bacteria, Archaea, and Eukarya all share a common set of metabolic reactions. This implies that the function and topology of central metabolism has been evolving under purifying selection over deep time. Central metabolism may similarly evolve under purifying selection during longterm evolution experiments, although it is unclear how long such experiments would have to run (decades, centuries, millennia) before signs of purifying selection on metabolism appear. I hypothesized that central and superessential metabolic enzymes would show evidence of purifying selection in the long-term evolution experiment with Escherichia coli (LTEE). I also hypothesized that enzymes that specialize on single substrates would show stronger evidence of purifying selection in the LTEE than generalist enzymes that catalyze multiple reactions. I tested these hypotheses by analyzing metagenomic time series covering 62,750 generations of the LTEE. I find mixed support for these hypotheses, because the observed patterns of purifying selection are idiosyncratic and population-specific. To explain this finding, I propose the Jenga hypothesis, named after a childrens game in which blocks are removed from a tower until it falls. The Jenga hypothesis postulates that loss-of-function mutations degrade costly, redundant, and nonessential metabolic functions. Replicate populations can therefore follow idiosyncratic trajectories of lost redundancies, despite purifying selection on overall function. I tested the Jenga hypothesis by simulating the evolution of 1,000 minimal genomes under strong purifying selection. As predicted, the minimal genomes converge to different metabolic networks. Strikingly, the core genes common to all 1,000 minimal genomes show consistent signatures of purifying selection in the LTEE.

Significance StatementPurifying selection conserves organismal function over evolutionary time. However, few studies have examined the role of purifying selection during adaptation to novel environments. I tested metabolic enzymes for purifying selection in an ongoing long-term evolution experiment with Escherichia coli. While some populations show signs of purifying selection, the overall pattern is inconsistent. To explain these findings, I propose the Jenga hypothesis, in which loss-of-function mutations first degrade costly, redundant, and nonessential metabolic functions, after which purifying selection begins to dominate. I then tested several predictions of the Jenga hypothesis using computational simulations. On balance, the simulations confirm that we should find evidence of purifying selection on the metabolic pathways that sustain growth in a novel environment.


title: Cross-tissue single-cell transcriptomics reveals organizing principles of fibroblasts in health and disease

abstract: Fibroblasts are non-hematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and play key roles in fibrosis, cancer, autoimmunity and wound healing. Recent studies have described fibroblast heterogeneity within individual tissues. However, the field lacks a definition of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here, we integrated single-cell RNA transcriptomic data from ~150,000 fibroblast cells derived from 16 steady- and 11 perturbed-state mouse organs into fibroblast atlases. These data revealed two universal fibroblast cell subtypes, marked by expression of Pi16 or Col15a1, in all tissues; it also revealed discrete subsets of five specialized fibroblast subtypes in steady-state tissues and three activated fibroblast subtypes in perturbed or diseased tissues. These subsets were transcriptionally shaped by microenvironmental context rather than tissue-type alone. Inference of fibroblast lineage structure from the murine steady-state and perturbed-state fibroblast atlases suggested that specialized and activated subtypes are developmentally related to universal tissue-resident fibroblasts. Analysis of human samples revealed that fibroblast subtypes found in mice are conserved between species, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In sum, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution enabled us to define the organizing principles of the fibroblast lineage in health and disease.


title: Inhibition of mitochondrial dynamics preferentially targets pancreatic cancer cells with enhanced tumorigenic and invasive potential

abstract:
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to its intrinsic aggressiveness, metastatic potential, and chemoresistance of the contained cancer stem cells (CSCs). Pancreatic CSCs strongly rely on mitochondrial metabolism to maintain their stemness, therefore representing a putative target for their elimination. Since mitochondrial homeostasis depends on the tightly controlled balance between fusion and fission processes, namely mitochondrial dynamics, we aimed to study this mechanism in the context of stemness. In human PDAC tissues, the mitochondrial fission gene DNM1L (DRP1) was overexpressed and positively correlated with the stemness signature. Moreover, we observed that primary human CSCs display smaller mitochondria and a higher DRP1/MFN2 expression ratio, indicating activation of mitochondrial fission. Interestingly, treatment with the DRP1 inhibitor mDivi-1 induced dose-dependent apoptosis, especially in CD133+ CSCs, due to accumulation of dysfunctional mitochondria and subsequent energy crisis in this subpopulation. Mechanistically, mDivi-1 inhibited stemness-related features, such as self-renewal, tumorigenicity and invasiveness, and chemosensitized the cells to the cytotoxic effects of Gemcitabine. In summary, mitochondrial fission is an essential process for pancreatic CSCs and represents an attractive target for designing novel multimodal treatments that will more efficiently eliminate cells with high tumorigenic potential.

Simple SummaryDue to their intrinsic aggressiveness, cancer stem cells (CSCs) represent an essential target for the design of effective treatments against pancreatic cancer, one of the deadliest tumors. As pancreatic CSCs are particularly dependent on the activity of their mitochondria, we here focus on mitochondrial dynamics as a critical process in the homeostasis of these organelles. We found that pancreatic CSCs rely on mitochondrial fission, and its pharmacological inhibition by mDivi-1 resulted in the accumulation of dysfunctional mitochondria, provoking energy crisis and cell death in this subpopulation. Consequently, mDivi-1 blocked cellular functions related to cancer aggressiveness such as in vivo tumorigenicity, invasiveness and chemoresistance. Our data suggest that inhibition of mitochondrial fission represents a promising target for designing new multimodal therapies to fight pancreatic cancer.


title: Regulation of neurofilament length and transport by a dynamic cycle of polymer severing and annealing

abstract:
Neurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate during development to expand axon caliber. We previously described novel severing and end-to-end annealing mechanisms in neurons that alter neurofilament length. To explore the functional significance of neurofilament length, we developed a long-term multi-field time-lapse method to track the movement of fluorescently tagged neurofilaments in axons of cultured neurons for up to 30 minutes. All filaments moved rapidly, but long filaments paused and reversed more often, resulting in little net movement, whereas short filaments moved persistently for long distances, pausing and reversing less often. Long filaments severed more frequently, generating shorter filaments, and short filaments annealed more frequently, generating longer filaments. Thus, neurofilament length is regulated by a dynamic cycle of severing and annealing and this influences neurofilament transport. Site-directed mutagenesis to mimic phosphorylation at four known phosphorylation sites in the head domain of neurofilament protein L generated shorter neurofilaments that moved more frequently. A non-phosphorylatable mutant had the opposite effect. Treatment of cultured neurons with activators of protein kinase A, which phosphorylates three of these sites, increased neurofilament severing. This effect was blocked by the non-phosphorylatable mutant. We propose that focal destabilization of intermediate filaments by N-terminal phosphorylation of their constituent polypeptides at specific locations along their length may be a general enzymatic mechanism for severing this class of cytoskeletal polymers. Our data suggest a novel mechanism for the control of neurofilament transport and accumulation in axons based on regulation of neurofilament polymer length.

SUMMARYNeurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate to expand axon caliber, which is an important determinant of axonal conduction velocity. We reported previously that neurofilaments can lengthen and shorten by novel end-to-end annealing and severing mechanisms. Here, we show that neurofilament annealing and severing are robust phenomena in cultured neurons that act antagonistically to dynamically regulate neurofilament length, which in turn regulates their transport. In addition, we present evidence for a novel enzymatic mechanism of intermediate filament severing based on site-directed phosphorylation of the neurofilament subunit proteins. We propose that modulation of neurofilament length by annealing and severing may be a mechanism for the regulation of neurofilament transport and accumulation in axons.