title: Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

abstract: Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of extracellular vesicle inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed and high-throughput assays. We captured 4 subpopulations of EVs from colorectal cancer cell lines HT29 and SW403 based on EpCAM, CD9, CD63 and CD81 expression, and quantified the co-expression of 8 outer (integrins and tetraspanins) and 4 inner (heat shock, endosomal and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

category: bioengineering


title: CRISPRedict: The case for simple and interpretable efficiency prediction for CRISPR-Cas9 gene editing

abstract:
The development of the CRISPR-Cas9 technology has provided a simple yet powerful system for targeted genome editing. Compared with previous gene-editing tools, the CRISPR-Cas9 system identifies target sites by the complementarity between the guide RNA (gRNA) and the DNA sequence, which is less expensive and time-consuming, as well as more precise and scalable. To effectively apply the CRISPR-Cas9 system, researchers need to identify target sites that can be cleaved efficiently and for which the candidate gRNAs have little or no cleavage at other genomic locations. For this reason, numerous computational approaches have been developed to predict cleavage efficiency and exclude undesirable targets. However, current design tools cannot robustly predict experimental success as prediction accuracy depends on the assumptions of the underlying model and how closely the experimental setup matches the training data. Moreover, the most successful tools implement complex machine learning and deep learning models, leading to predictions that are not easily interpretable.

Here, we introduce CRISPRedict, a simple linear model that provides accurate and inter-pretable predictions for guide design. Comprehensive evaluation on twelve independent datasets demonstrated that CRISPRedict has an equivalent performance with the currently most accurate tools and outperforms the remaining ones. Moreover, it has the most robust performance for both U6 and T7 data, illustrating its applicability to tasks under different conditions. Therefore, our system can assist researchers in the gRNA design process by providing accurate and explainable predictions. These predictions can then be used to guide genome editing experiments and make plausible hypotheses for further investigation. The source code of CRISPRedict along with instructions for use is available at https://github.com/VKonstantakos/CRISPRedict.

category: bioinformatics


title: AGC: Compact representation of assembled genomes

abstract: High-quality sequence assembly is the ultimate representation of complete genetic information of an individual. Several ongoing pangenome projects are producing collections of high-quality assemblies of various species. Here, we show how to represent the sequenced genomes in 2-3 orders of magnitude smaller space, allowing easy and fast extraction of any contig or its part.


title: Streptococcus Pneumoniae Promotes Lung Tumorigenesis by Activating PI3K/AKT and NF-kB Pathways via Binding PspC to PAFR

abstract: Streptococcus pneumoniae (SP) is associated with lung cancer, yet its role in the tumorigenesis remains uncertain. Herein we find that SP attaches to lung cancer cells via binding pneumococcal surface protein C (PspC) to platelet-activating factor receptor (PAFR), a receptor overexpressed in lung tumors. Interaction between PspC and PAFR stimulates cell proliferation and activates PI3K/AKT and NF-kB signaling pathways, which triggers a pro-inflammatory response. Lung cancer cells infected with SP rapidly form larger tumors in BALB/C mice compared to untreated cells. Mice treated with tobacco carcinogen and SP develop more lung tumors and had shorter survival than mice treated with the carcinogen alone. Mutating PspC or deleting PAFR abolishes the tumor-promoting effects of SP. Overabundance of SP is found in lung tumors of patients with lung cancer and associated with the survival. SP plays a driving role in lung tumorigenesis by activating PI3K/AKT and NF-kB pathways via binding PspC to PAFR and provides a microbial target for diagnosis and treatment of the disease.

category: cancer biology


title: Extended functional connectivity of convergent structural alterations among individuals with PTSD: A neuroimaging meta-analysis

abstract:
BackgroundPost-traumatic stress disorder (PTSD) is a debilitating disorder defined by the onset of intrusive, avoidant, negative cognitive or affective, and/or hyperarousal symptoms after witnessing or experiencing a traumatic event. Previous voxel-based morphometry studies have provided insight into structural brain alterations associated with PTSD with notable heterogeneity across these studies. Furthermore, how structural alterations may be associated with brain function, as measured by task-free and task-based functional connectivity, remains to be elucidated.

MethodsUsing emergent meta-analytic techniques, we sought to first identify a consensus of structural alterations in PTSD using the anatomical likelihood estimation (ALE) approach. Next, we generated functional profiles of identified convergent structural regions utilizing resting-state functional connectivity (rsFC) and meta-analytic co-activation modeling (MACM) methods. Finally, we performed functional decoding to examine mental functions associated with our ALE, rsFC, and MACM brain characterizations.

ResultsWe observed convergent structural alterations in a single region located in the medial prefrontal cortex. The resultant rsFC and MACM maps identified functional connectivity across a widespread, whole-brain network that included frontoparietal and limbic regions.

ConclusionsConsensus-based functional connectivity was observed in regions of the default mode, salience, and central executive networks, which play a role in the tripartite model of psychopathology. Functional decoding revealed overlapping associations with attention, memory, and emotion processes. Taken together, these findings have important implications in understanding the neurobiological mechanisms associated with PTSD.

category: neuroscience


title: A transcriptional network required for Toxoplasma gondii tissue cyst formation is dispensable for long-term persistence

abstract: Cyst formation is a key feature of the T. gondii life cycle but the genetic networks that drive this process are not yet fully characterized. To identify new components of this network, we compared T. gondii to its nearest extant relative Hammondia hammondi given the critical differences between these species in the timing and efficiency of cyst formation. Using transcriptional data from critical developmental and pH exposure time points from both species, we identified the gene TGVEG_311100, which we named Regulator of Cystogenesis 1 (ROCY1), as being both necessary and sufficient for cyst formation in T. gondii. Compared to WT parasites, TGVEG{Delta}ROCY1 parasites formed significantly fewer tissue cysts in response to alkaline pH stress in vitro and cysts were nearly undetectable in mouse brains for up to 9 weeks post-infection. Overexpression of tagged ROCY1 in WT parasites was sufficient to induce cyst formation in vitro in both WT and ROCY1-deficient parasites, demonstrating that ROCY1 is both necessary and sufficient for cyst formation. Moreover this induction of cyst formation required at least 1 of 3 predicted CCCH Zinc finger domains. Mice chronically infected with {Delta}ROCY1 parasites had detectable tachyzoites in the brain for up to 37 days post-infection (while mice infected with WT parasites did not), and CNS transcriptional analyses at day 30 post-infection throughout the chronic phase of infection revealed inflammatory signatures consistent with acute infection in {Delta}ROCY1 parasites compared to WT. Despite our inability to detect brain cysts in infected mice, both WT and {Delta}ROCY1 knockout parasites reactivated after dexamethasone treatment with similar timing and magnitude for up to 5 months post infection, challenging the paradigm that long term parasite persistence in the CNS requires cyst formation. These data identify a new regulator of cyst formation in T. gondii that is both necessary and sufficient for cyst formation, and whose function relies on its conserved nucleic acid binding motif.

category: microbiology


title: Dissecting the conformational complexity and flipping mechanism of bacterial heme transport.

abstract: Iron-bound cyclic tetrapyrroles (hemes) are key redox-active cofactors in membrane-integrated oxygen reductases and other bioenergetic enzymes (1, 2). However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here we used a combination of cellular, biochemical, structural and computational methods to resolve ongoing controversies around the function of the heterodimeric bacterial ABC transporter CydDC (3). We provide multi-level evidence that CydDC is a highly specific heme transporter required for assembly and maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryo-EM approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and translocation. We found that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degree. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward open states.


title: The phosphatase Bph and peptidyl-prolyl isomerase PrsA are required for gelatinase expression and activity in Enterococcus faecalis

abstract:
Enterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. faecalis virulence factor that contributes to numerous cellular activities such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of gelE has been extensively studied and is regulated by the Fsr quorum-sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of gelE in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not fsr or gelE gene expression. Disrupting prsA also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in Enterococcus and also E. faecalis pathogenesis.

ImportanceIn Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of gelE. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of gelE through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the fsr locus coordinate expression of gelatinase.


title: Secreted dengue virus NS1 is predominantly dimeric and in complex with high-density lipoprotein

abstract:
Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and biotherapeutic target. To uncover the biologically relevant structure of sNS1, we extracted the native form of sNS1 from cells infected with either the DENV WT or T164S mutant whose appearance was associated with a dengue outbreak in Cuba. We determined the cryoEM structures of sNS1 and its complex with a monoclonal antibody/Fab and found that the major species of sNS1 is a 1:1 complex of the NS1 dimer embedded in a High Density Lipoprotein (HDL) particle. Cross-linking MS studies confirm NS1:ApoA1 dimer formation with most ApoA1 interaction sites mapped to the NS1 wing and hydrophobic domains. Our results shed fresh light on the molecular pathogenesis of dengue and may have broad implications for managing dengue infection.

SummaryCryoEM structures of secreted dengue virus NS1 protein reveal dimers in complex with high-density lipoprotein.


title: The Streptococcus pyogenes hyaluronic acid capsule promotes experimental nasal and skin infection by preventing neutrophil-mediated clearance

abstract:
Streptococcus pyogenes is a globally prominent human-specific pathogen responsible for an enormous burden of human illnesses, including >600 million pharyngeal and >100 million skin infections each year. Despite intensive efforts that focus on invasive indications, much remains unknown about this bacterium in its natural state during colonization of the nasopharynx and skin. Using acute experimental infection models in HLA-transgenic mice, we evaluated how the hyaluronic acid (HA) capsule contributes to S. pyogenes MGAS8232 infection within these limited biological niches. Herein, we demonstrate that HA capsule expression promotes bacterial burden in murine nasal turbinates and skin lesions by resisting neutrophil-mediated killing. HA capsule production is encoded by the hasABC operon and compared to wildtype S. pyogenes infections, mice infected with a {Delta}hasA mutant exhibited over a 1000-fold CFU reduction at 48-hours post{-}nasal challenge, and a 10,000-fold CFU reduction from skin lesions 72-hours post-skin challenge. HA capsule expression contributed substantially to skin lesion size development following subdermal inoculations. In the absence of capsule expression, S. pyogenes revealed drastically impeded growth in whole human blood and increased susceptibility to killing by isolated neutrophils ex vivo, highlighting its important role in resisting phagocytosis. Furthermore, we establish that neutrophil depletion in mice, but not macrophage depletion, recovered the reduced burden by the {Delta}hasA mutant in both the nasopharynx and skin. Together, this work confirms that the HA capsule is a key virulence determinant during acute infections by S. pyogenes and demonstrates that its predominant function is to protect S. pyogenes against neutrophil-mediated killing.

AUTHOR SUMMARYStreptococcus pyogenes is a globally disseminated and human-adapted bacterial pathogen that has evolved an arsenal of evasion strategies to overcome and escape host immune clearing mechanisms. Many strains of S. pyogenes are covered by a polysaccharide capsule composed of hyaluronic acid (HA) that is widely recognized to promote severe infections. In this study, we demonstrate using the encapsulated S. pyogenes MGAS8232 strain that the HA capsule is a key virulence factor that facilitates non-invasive infections of the nasopharynx and skin. Although bacterial adhesion and entry into host cells was impeded by HA capsule expression, we show that the key function for both nasal and skin infections is to protect S. pyogenes from neutrophil-mediated killing. Depletion of neutrophils, but not macrophages, recovered the low bacterial burden by unencapsulated S. pyogenes at both sites of infection. Our findings outline an important interaction between the HA capsule and neutrophils in the establishment of acute upper respiratory and skin infections by S. pyogenes.


title: Plant N-glycan breakdown by human gut Bacteroides

abstract: The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterised allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterise five CAZymes involved, including structures of the PNGase and GH92 -mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.

category: biochemistry


title: The Statistical Trends of Protein Evolution: A Lesson from AlphaFold Database

abstract: The recent development of artificial intelligence provides us with new and powerful tools for studying the mysterious relationship between organism evolution and protein evolution. In this work, based on the AlphaFold Protein Structure Database (AlphaFold DB), we perform comparative analyses of the protein structures of different species. The statistics of AlphaFold-predicted structures show that, as species evolve towards higher complexity (e.g., from prokaryotes to eukaryotes, from unicellular to multicellular organisms, etc.), the proteins within them evolve toward larger radii of gyration, higher coil fractions, higher modularity, and slower relaxations, indicating that the average flexibility of proteins gradually increases in the species evolution. With the scaling analyses, the size dependence of proteins shape, topology, and dynamics suggest a decreasing fractal dimension of the proteins in species evolution. This evolutionary trend is accompanied by the increasing eigengaps in the vibration spectra of proteins, implying that the proteins are statistically evolving toward higher functional specificity and lower dimensionality in the native dynamics. Furthermore, we also uncover the topology and sequence bases of this evolutionary trend. The residue contact networks of proteins evolve toward higher assortativity, and the hydrophobic and hydrophilic amino acid residues evolve to have increasing segregations in the sequences. This study provides new insights into how the diversity in the functionality of proteins increases and how the dimensionality of the manifold of protein dynamics reduces during evolution. The statistical evolutionary trends also shed light on the study of protein design.

category: biophysics


title: The Gene Knockout of Angiotensin II Type 1a Receptor Improves High-fat Diet-Induced Obesity in rat via Promoting Adipose Lipolysis

abstract:
AimsThe renin-angiotensin system (RAS) is over-activated and the serum angiotensin II (Ang II) level increased in obese patients, while their correlations were incompletely understood. This study aims to explore the role of Ang II in diet-induced obesity by focusing on adipose lipolysis.

MethodsRat model of AT1aR gene knockout were established to investigate the special role of Ang II. Wild-type (WT) and AT1aR gene knockout (AT1aR-/-) SD rats were fed with normal diet or high-fat diet for 12 weeks. Adipose morphology and adipose lipolysis were examined.

ResultsAT1aR deficiency activated lipolysis-related enzymes and increased the levels of NEFAs and glycerol released from adipose tissue in high-fat diet rats, while did not affect triglycerides synthesis. Besides, AT1aR knockout promoted energy expenditure and fatty acids oxidation in adipose tissue. cAMP levels and PKA phosphorylation in the adipose tissue were significantly increased in AT1aR-/- rats fed with high-fat. Activated PKA could promote adipose lipolysis and thus improved adipose histomorphology and insulin sensitivity in high-fat diet rats.

ConclusionsAT1aR deficiency alleviated adipocyte hypertrophy in high-fat diet rats by promoting adipose lipolysis via cAMP/PKA pathway, and thereby delayed the onset of obesity and related metabolic diseases.

category: physiology


title: The concordance of signals based on irregular incremental lines in the human tooth cementum with documented pregnancies: Results from a systematic approach

abstract:
Background and ObjectiveThere is evidence from previous studies that pregnancies and diseases are recorded in the tooth cementum. This study aims to assess the degree of concordance between signals based on irregular incremental lines (ILs) and reported pregnancies.

Material and Methods23 recent and 24 archaeological human teeth with known birth history were included in this investigation. 129 histological sections of tooth roots were assessed for irregularities in appearance and width using a standardized protocol. Similarity of observed irregularities at the section level allowed us to define signals at the tooth level. The sensitivity of signals to detect pregnancies was determined and related to the signal prevalence.

ResultsPregnancy signals were frequently visually observed. However, applying a standardized process we could only reach signal sensitivities to identify pregnancies up to 20 percentage points above chance level.

ConclusionsBased on a standardized and reproducible method it could be confirmed that some pregnancies leave visible signals in the tooth cementum. The results show the potential of the tooth cementum to support reconstruction of life courses in paleopathology. However, it seems that not all pregnancies affect the cementogenesis in such a way that irregular ILs are identifiable. Further research is needed to better understand which type of pregnancies and other conditions are recorded in the tooth cementum.


title: Modeling protected species distributions and habitats to inform siting and management of pioneering ocean industries: A case study for Gulf of Mexico aquaculture

abstract: Marine Spatial Planning (MSP) provides a process that uses spatial data and models to evaluate environmental, social, economic, cultural, and management trade-offs when siting ocean industries. Aquaculture is the fastest-growing food sector in the world. The U.S. has substantial opportunity for offshore aquaculture development given the size of its exclusive economic zone, habitat diversity, and variety of candidate species for cultivation. However, many protected species rely upon habitats that overlap with promising aquaculture areas. Siting surveys, farm construction, operations, and decommissioning can alter the habitat and behavior of animals in the vicinity of these activities. Vessel activity, underwater noise, and physical interactions between protected species and farms can potentially increase the risk of injury or cause direct mortality. In 2020, the U.S. Gulf of Mexico was identified as one of the first regions to be evaluated for offshore aquaculture opportunities as directed by a Presidential Executive Order. We developed a generalized scoring model for protected species data layers that captures vulnerability using species conservation status and demographic information. We applied this approach to data layers for eight species listed under the Endangered Species Act, including five species of sea turtles, Rices Whale, Smalltooth Sawfish, and Giant Manta Ray. We evaluated several methods for scoring (e.g., arithmetic mean, geometric mean, product, lowest scoring layer) and created a combined protected species data layer that was used within a multi-criteria decision-making modeling framework for MSP. The product approach for scoring provided the most logical ordering of and the greatest contrast in site suitability scores. This approach provides a transparent and repeatable method to identify aquaculture site alternatives with the least conflict with protected species. These modeling methods are transferable to other regions, to other sensitive or protected species, and for spatial planning for other ocean-uses.

category: scientific communication and education


title: Biogeography and taxonomy of the Hemileuca maia-nevadensis Stretch species complex (Lepidoptera: Saturniidae): insights to the midcontinental populations

abstract: An integrated assessment of geographic distribution, genetic variation and interpopulation reproductive barriers of the Great Plains and Great Lakes populations of the Hemileuca maia-nevadensis species complex is presented. The taxonomic circumscription of Hemileuca nevadensis Stretch, key to understanding the complex evolutionary history of the group, has previously been misinterpreted. Detailed distribution maps based on historic, literature, and contemporary occurrence records, combined with analysis of genetic variation using mtDNA-COI barcode sequences and pheromone compatibility trials, indicate that Great Plains H. "nevadensis" are disjunct and genetically divergent from nominate H. nevadensis, instead being more closely related to Great Lakes populations. Despite the genomic differentiation of the bog buckmoth established in recent studies, similar or identical mtDNA-COI haplotypes indicate past genetic linkages among now-isolated populations across the northern range periphery of the maia-complex. Based on the current evidence, northern Great Plains Hemileuca latifascia Barnes & McDunnough stat. nov. and eastern Great Lakes (bog buckmoth) Hemileuca menyanthevora Pavulaan stat. nov. (= Hemileuca iroquois Dirig & Cryan syn. nov.) should be considered as species distinct from others in the maia-group. Post-glacial dispersal hypotheses are evaluated in light of molecular variation and ecology, and key knowledge gaps are identified to guide future research.

category: zoology


title: Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.

abstract: Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switch between distinct cell death pathways and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite nucleolus. We also describe that TcPARP is present in a thread that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this wire during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids.


title: Intestinal microbiota dysbiosis and liver metabolomic changes during brain death

abstract:
BackgroundThe causative link between brain death and intestinal microbiota dysbiosis is unclear, and the distortion in liver metabolism caused by brain death requires further exploration.

Material and MethodsA rat model of brain death was constructed and sustained for 9 hours. Intestinal contents and portal vein plasma were collected for microbiota sequencing and microbial metabolite detection. Liver tissue was resected to investigate metabolic alterations, and the results were compared with those of a sham group.

ResultsMicrobiota dysbiosis occurred at the family and genus levels after 9 hours of brain death. Microbial metabolites remained unchanged in both the intestinal contents and portal vein plasma. Liver metabolic function deteriorated, and the levels of 80% of the differential metabolites decreased in the brain-dead rats. Most of the differential metabolites were related to energy metabolism.

ConclusionsBrain death resulted in microbiota dysbiosis in rats; however, this dysbiosis did not alter microbial metabolites. Deterioration in liver metabolic function during extended periods of brain death may reflect a continuous worsening in energy deficiency.


title: Experimental evolution of phosphomannomutase-deficient yeast reveals compensatory mutations in a phosphoglucomutase

abstract: The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene PMM2, which affect protein N-linked glycosylation. The yeast gene SEC53 encodes a nearly-identical homolog of human PMM2. We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, sec53-V238M and sec53-F126L, or wild-type SEC53. We find that after 1,000 generations, most populations compensate for the slow-growth phenotype associated with the sec53 human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known SEC53 genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including PGM1, which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved pgm1 mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the sec53-V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the sec53-V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease associated alleles.

category: evolutionary biology


title: A generic framework for hierarchical de novo protein design

abstract: De novo protein design enables the exploration of novel sequences and structures absent from the natural protein universe. De novo design also stands as a stringent test to our understanding of the underlying physical principles of protein folding and may lead to the development of proteins with unmatched functional characteristics. The first fundamental challenge of de novo design is to devise "designable" structural templates leading to sequences that will adopt the predicted fold. Here, we built on the TopoBuilder de novo design method to automatically assemble structural templates with native-like features starting from string descriptors that capture the overall topology of proteins. Our framework eliminates the dependency of hand-crafted and fold-specific rules through an iterative, data-driven approach that extracts geometrical parameters from structural tertiary motifs. We evaluated the TopoBuilder framework by designing sequences for a set of five protein folds and experimental characterization revealed that several sequences were folded and stable in solution. The TopoBuilder de novo design framework will be broadly useful to guide the generation of artificial proteins with customized geometries, enabling the exploration of the protein universe.


title: The impact of AlphaFold on experimental structure solution

abstract: AlphaFold2 is a machine-learning based program that predicts a protein structure based on the amino acid sequence. In this article, we report on the current usages of this new tool and give examples from our work in the Coronavirus Structural Task Force. With its unprecedented accuracy, it can be utilized for the design of expression constructs, de novo protein design and the interpretation of Cryo-EM data with an atomic model. However, these methods are limited by their training data and are of limited use to predict conformational variability and fold flexibility; they also lack co-factors, posttranslational modifications and multimeric complexes with oligonucleotides. They also are not always perfect in terms of chemical geometry. Nevertheless, machine learning based fold prediction are a game changer for structural bioinformatics and experimentalists alike, with exciting developments ahead.


title: Prediction of quality-control degradation signals in yeast proteins

abstract: Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin-proteasome system. This is achieved via recognition of so-called degradation motifs--degrons--that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the entire yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis.


title: Structural insights into sodium/iodide symporter (NIS) substrate binding and specificity

abstract: The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones--the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. For nearly 50 years before its molecular identification, NIS was already the molecule at the center of the single most effective internal radiation cancer therapy ever devised: radioiodide (131I-) treatment for thyroid cancer. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency. To date, the structure of NIS has been unknown. Here, we report three structures of rat NIS, determined by single-particle cryo-electron microscopy (cryo-EM): one with no substrates bound, one with 2 Na+ and 1 I- bound, and one with 1 Na+ and the oxyanion perrhenate bound. Structural analyses, functional characterization, and computational studies reveal the substrate binding sites and residues key for transport activity. Our results yield insights into how NIS selects, couples, and translocates anions--thereby establishing a framework for understanding NIS function--and into how it transports different substrates with different stoichiometries and releases substrates from its substrate-binding cavity into the cytosol.


title: Restricting α-Synuclein Transport into Mitochondria by Inhibition of α-Synuclein-VDAC Complexation as a Potential Therapeutic Target for Parkinson's Disease Treatment

abstract: Involvement of alpha-synuclein (Syn) in Parkinsons disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently we established that Syn can regulate mitochondrial function by voltage-activated complexation with the Voltage-Dependent Anion Channel (VDAC) of the outer mitochondrial membrane. When complexed with Syn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, Syn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of Syn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on the Syn membrane binding, and hence on Syn complex formation with VDAC and translocation through it. In electrophysiology experiments, addition of HK2p at micromolar concentrations to the same side of the membrane as Syn results in dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of Syn from lipid membranes. Experiments with live HeLa cells using proximity ligation assay confirmed that HK2p impedes Syn entry into mitochondria. Our results demonstrate that it is possible to regulate Syn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate Syn mitochondrial toxicity in PD and other synucleinopathies.


title: A consensus view on the folding mechanism of protein G, L and their mutants

abstract: Much of our understanding of folding mechanisms comes from interpretations of experimental {phi} and {psi} value analysis - relating the differences in stability of the transition state ensemble (TSE) and folded state. We introduce a unified approach combining simulations and Bayesian inference to provide atomistic detail for the folding mechanism of protein G, L and their mutants. Protein G and L fold to similar topologies despite low sequence similarity, but differ in their folding pathways. A fast folding redesign of protein G, NuG2, switches folding pathways and folds through a similar pathway with protein L. A redesign of protein L also leads to faster folding, respecting the original folding pathway. Our Bayesian inference approach starts from the same prior on all systems and correctly identifies the folding mechanism for each of the four proteins - a success of the force field and sampling strategy. The approach is computationally efficient and correctly identifies the TSE and intermediate structures along the folding pathway in good agreement with experiments. We complement our findings by using two orthogonal approaches that differ in computational cost and interpretability. Adaptive sampling MD combined with Markov State Model provide a kinetic model that confirms the more complex folding mechanism of protein G and its mutant. Finally, a novel fragment decomposition approach using AlphaFold identifies preferences for secondary structure element combinations that follows the order of events observed in the folding pathways.


title: Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

abstract: Insulin formulations are the hallmark of interventions for treatment of diabetes. Understanding the mechanism that governs insulin self assembly or disassembly --and the role of stabilizing additives--are essential for improving insulin formulations. We report here the real-time direct observation of single insulin self-assembly and disassembly events using single molecule fluorescence microscopy. Our direct observations revealed previously unaccounted monomeric additions to occur to all types of assemblies and allowed us to quantify the existence, abundance and kinetic characterization of diverse assembly pathways involving monomeric dimers or tetrameric insulin species. We proposed and experimentally validated a model where the insulin self-assembly pathway is rerouted favoring monomeric or oligomeric assembly events by solution concentration, additives and formulations. Our rate simulation predicted the abundance of each oligomeric species across a concentration range of 6 orders of magnitude. Besides providing fundamental new insights, the results and toolbox here can be universally applied contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for other proteins.


title: Neuronopathic GBA1 L444P mutation accelerates glucosylsphingosine levels and formation of hippocampal alpha-synuclein inclusions

abstract:
The most common genetic risk factor for Parkinsons disease (PD) is heterozygous mutations in the GBA1 gene which encodes for the lysosomal enzyme, glucocerebrosidase (GCase). GCase impairments are associated with an accumulation of abnormal -synuclein (-syn) called Lewy pathology, which characterizes PD. PD patients heterozygous for the GBA1 L444P mutation (GBA1+/L444P) have a 5.6-fold increased risk of cognitive impairments. In this study, we used GBA1+/L444P mice to determine the effects of this severe GBA1 mutation on lipid metabolism, expression of synaptic proteins, behavior, and -syn inclusion formation. GBA1+/L444P mice showed reduced GCase activity in limbic brain regions and expressed lower levels of hippocampal vGLUT1 compared to wildtype (GBA1+/+) mice. GBA+/L444P mice also demonstrated impaired fear conditioning, but no motor deficits. We show, using mass spectrometry, that mutant GCase and age increased levels of glucosylsphingosine (GlcSph), but not glucosylceramide (GlcCer), in the brains and serum of GBA1+/L444P mice. Aged GBA1+/+ mice also showed increased levels of GlcSph, and decreased GlcCer. To model disease pathology, templated -syn pathology was used. -Syn inclusions were increased in the hippocampus of GBA1+/L444P mice compared to GBA1+/+ mice, but not in the cortex, or substantia nigra pars compacta (SNc). Pathologic -syn did not cause a loss of dopamine neurons in the SNc. Treatment with a GlcCer synthase inhibitor prevented loss of cortical -syn inclusions, but not loss of dopamine neurons. Overall, these data suggest the critical importance to evaluate the contribution of hippocampal pathologic -syn and brain and serum glucosylsphingosine in synucleinopathies.

SIGNIFICANCE STATEMENTSynucleinopathies, such as Parkinsons disease (PD) and Dementia with Lewy bodies (DLB), are both pathologically characterized by abnormal -synuclein (-syn). Mutant GBA1 is a risk factor for both PD and DLB where a reduction of glucocerebrosidase (GCase) activity is seen. Collectively, this indicates the significance of evaluating mutant GCase in synucleinopathies. Our data suggest the critical importance to evaluate the contribution of hippocampal pathologic -syn and brain and serum glucosylsphingosine (GlcSph) accumulation in synucleinopathies. Moreover, these pathologic outcomes may contribute to the nonmotor symptoms clinically observed in PD and DLB. Our findings highlight the importance of GlcSph as a relevant biomarker for future therapeutics.


title: CyTOF-Lec: Single-cell glycomics analysis reveals glycan features defining cells differentially susceptible to HIV

abstract: High-parameter single-cell phenotyping has enabled in-depth classification and interrogation of immune cells, but to date has not allowed for glycan characterization. Here, we develop CyTOF-Lec as an approach to simultaneously characterize many protein and glycan features of human immune cells at the single-cell level. We implemented CyTOF-Lec to compare glycan features between different immune subsets from blood and multiple tissue compartments, and to characterize HIV-infected cell cultures. Using bioinformatics approaches to distinguish preferential infection of cellular subsets from viral-induced remodeling, we demonstrate that HIV upregulates the levels of cell surface fucose and sialic acid in a cell- intrinsic manner, and that memory CD4+ T cells co-expressing high levels of fucose and sialic acid are highly susceptible to HIV infection. Sialic acid levels were found to distinguish memory CD4+ T cell subsets expressing different amounts of viral entry receptors, pro-survival factors, homing receptors, and activation markers, and to play a direct role in memory CD4+ T cells susceptibility to HIV infection. The ability of sialic acid to distinguish memory CD4+ T cells with different susceptibilities to HIV infection was experimentally validated through sorting experiments. Together, these results suggest that HIV remodels not only cellular proteins but also glycans, and that glycan expression can differentiate memory CD4+ T cells with vastly different susceptibility to HIV infection.

category: immunology


title: Visualizing Molecular Interactions that Determine Assembly of a Bullet-Shaped Vesicular Stomatitis Virus Particle

abstract: Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with a non-segmented genome, closely related to rabies virus. Both have characteristic bullet-like shapes. We report the structure of intact, infectious VSV particles determined by cryogenic electron microscopy. By compensating for polymorphism among viral particles with computational classification, we obtained a reconstruction of the shaft ("trunk") at 3.5 [A] resolution, with lower resolution for the rounded tip. The ribonucleoprotein (RNP), genomic RNA complexed with nucleoprotein (N), curls into a dome-like structure with about eight gradually expanding turns before transitioning into the regular helical trunk. Two layers of matrix (M) protein link the RNP with the membrane. Radial inter-layer subunit contacts are fixed within single RNA-N-M1-M2 modules, but flexible lateral and axial interactions allow assembly of polymorphic virions. Together with published structures of recombinant N in various states, our results suggest a mechanism for membrane- coupled self-assembly of VSV and its relatives.

category: molecular biology


title: Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs

abstract:
The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular stress can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily post-mitotic and aids regeneration in mammals. In the present study we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of its target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Significance StatementIn humans and other mammals, many organs regenerate through the proliferation of cells that replace those that have succumbed to aging or injury. However, proliferation is largely absent in certain critical organs, including the heart, the central nervous system, and sensory organs such as the inner ear and retina. The Hippo-Yap biochemical signaling pathway, a cascade of proteins that--when active--inhibits cell division, constitutes one impediment to proliferation. We earlier identified a small molecule that interrupts Hippo-Yap signaling and thus relieves this block for some non-proliferating cells in vitro. In the present investigation, we have chemically modified the original substance to yield a more potent analog that is effective for several hours in mammalian tissues in vivo and initiates the proliferation of heart-muscle cells after cryolesioning. After further refinements, compounds of this family might prove useful in regenerative therapies.

category: cell biology


title: Rapid Rerouting of Myosin Traffic at the T Cell Immunological Synapse

abstract: Cytoskeletal motors travel in patterns set by the architecture of their tracks. Nevertheless, we have a limited understanding of how cells dynamically reorganize their traffic patterns in response to signaling events. To investigate cytoskeletal motor rerouting, we used T cells as a model system. Upon an encounter between a T cell and an antigen presenting cell, the T cell builds a specialized interface with spatially organized immunoreceptors and adhesion molecules called the immunological synapse (IS). The IS also constructs new actin networks within minutes that define the synaptic structure. Here we track the movements of single myosin motors along presynaptic and synaptic actin networks of the T cell. We find that both myosin-5 and myosin-6 reroute after IS construction. For example, most myosin-5 traffic moves inward at the IS, although most of the IS actin filaments have a barbed end out orientation. This anomalous myosin-5 traffic pattern indicates that the IS makes two types of actin networks: a structural network that controls IS shape, and a distinct trafficking network that supports myosin motility. We disrupt these trafficking networks with chemical probes against actin, which inhibits the appearance of cell surface markers of T cell activation. Our results highlight the importance of the sparse actin networks at the center of the IS in T cell function.


title: Circadian clock control of MRTF-SRF signaling suppresses beige adipocyte thermogenic recruitment

abstract: The morphological transformation of adipogenic progenitors into mature adipocytes requires dissolution of cytoskeleton driven by progressive loss of MRTF-SRF activity. Circadian clock confers temporal control in adipogenic differentiation, and MRTF-SRF inhibits beige adipocyte development. Here we identify that key components of the actin cytoskeleton-MRTF/SRF signaling cascade are direct transcriptional targets of circadian clock, and a clock-MRTF/SRF regulatory axis suppresses beige adipocyte thermogenic recruitment. Genetic loss- or gain-of-functions of the core clock regulator, Brain and Muscle Arnt-like 1 (Bmal1), altered actin cytoskeleton organization, cell shape and SRF activity. Genes involved in actin dynamic-MRTF-SRF pathway display diurnal expression profiles in beige adipose tissue, with identification of Bmal1 direct transcriptional regulation. Using beige adipocyte-selective genetic models together with pharmacological approaches, we demonstrate that Bmal1 inhibits beige adipogenesis and thermogenic capacity via the MRTF-SRF pathway. Selective ablation of Bmal1 induced beigeing, whereas its targeted overexpression attenuated the beige thermogenic program resulting in obesity. Collectively, our findings define a novel circadian clock control in actin cytoskeleton-MRTF-SRF signaling that suppresses beige adipogenesis to maintain metabolic homeostasis.


title: Systems modelling of TGF-β/Hippo signalling crosstalk uncovers molecular switches that coordinate YAP transcriptional complexes

abstract: The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to regulate cell fate and organ homeostasis. The central downstream pathway protein is the transcriptional co-activator Yes-associated protein (YAP). Although capable of inducing gene transcription, YAP cannot bind DNA directly. Instead, it mediates transcriptional activity through interaction with distinct DNA-binding transcriptional factors (TFs), including TEAD, SMAD, and p73, to form active and functionally opposing transcriptional complexes. Growing evidence in mammals demonstrates that YAP has a dual role and can either promote cell proliferation or apoptosis, which underpin its ability to function as both an oncogene or a tumour suppressor depending on the specific context. However, the mechanisms by which YAP coordinates its distinct transcriptional complexes and mediates context-dependent function remain poorly defined. This is in part due to the lack of systems-level studies that can decrypt the complexities of upstream signalling pathways and their crosstalk, which together dictate the transcriptional regulation at the YAP level. Here, we undertake an integrative systems-based approach combining computational network modelling and experimental studies to interrogate the dynamic formation of and transition between the YAP-SMAD and YAP-p73 transcriptional complexes, which control proliferative and apoptotic gene expression, respectively. We developed a new experimentally-validated mathematical model of the TGF-{beta}/Hippo signalling crosstalk and used this model to elucidate dynamic network behaviour. Our integrative studies uncovered previously unknown molecular switches that control the YAP-SMAD/p73 complexes in an on/off, switch-like manner. RASSF1A and ITCH were identified as major regulators of the switches, whereby a graded increase in ITCH expression can trigger YAP to abruptly switch from binding p73 to SMAD, swiftly promoting proliferative gene expression. Further, adjusting the model to reflect cell type-specific protein expression profiles using both in-house and publicly available experimental data enabled us to study the YAP switches under diverse and varied cellular contexts. Overall, our studies provide a new quantitative and systems-level understanding of the dynamic regulation of functionally opposing YAP transcriptional complexes in mammalian cells.


title: A method for stabilising the XX karyotype in female mESC cultures

abstract: Female mouse embryonic stem cells (mESCs) present differently to male mESCs in several fundamental ways, however complications with their in vitro culture have resulted in an underrepresentation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female mESCs apart from males. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state near indistinguishable from male. If female pluripotency is to be studied effectively in this system, it is critical that high quality cultures of XX mESCs are available. Here we report a method for better maintaining XX female mESCs in culture, which also stabilises the male karyotype, and makes study of female specific pluripotency more feasible.


title: A salt bridge mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a single step cell-based screen

abstract: Bacterial cell division proteins, especially the tubulin homolog FtsZ, have emerged as strong targets for developing new antibiotics. Several assays have been designed to screen for small molecules targeting FtsZ but rely upon a multitude of steps to validate the target and to ensure minimum toxicity to the eukaryotic cells. Here, we have utilized the fission yeast heterologous expression system to develop a single step cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ and are non-toxic to eukaryotic cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-sheet S10 and S7 mediate resistance to PC190723 in FtsZ. The single step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay functions as a powerful tool to rapidly identify new molecules that specifically target the bacterial cell division protein FtsZ, or other polymeric bacterial cytoskeletal proteins, understand how they affect polymerization dynamics and study resistance determinants in targets.


title: Probing the role of macromolecular crowding in cell volume regulation using fluorescence anisotropy

abstract: Cytoplasmic macromolecular crowding (MMC) influences multiple cellular functions. Using fluorescence anisotropy of EGFP, we explore the homeostasis of MMC in the cytoplasm and its role in cell volume regulation. Individual cells of different lineages maintain a distinctly unique average cytoplasmic MMC for considerably long durations. Despite diffusion, actin cytoskeleton facilitates spatially heterogeneous MMC in the cytoplasm. While hypertonic-stress triggers regulatory volume increase (RVI), other methods of increasing cytoplasmic MMC fail to start RVI, suggesting that cells lack capabilities for MMC homeostasis. During spreading or microtubule-depolymerized state, cells neglect changes in cytoplasmic MMC to undergo volume regulation. Inhibition of TNFR1 increases the membrane tension and deprives cells of their ability to undergo volume regulation. Current understanding contemplates that cytoplasmic MMC, the mechanical state of the plasma membrane or the actin cytoskeleton, could be sensors for cell volume regulation. Our observations establish the irrelevance of cytoplasmic MMC and the significance of plasma membrane tension in setting off the cell volume regulation machinery.


title: COP I and II dependent trafficking controls ER-associated degradation

abstract: Misfolded proteins and components of the endoplasmic reticulum (ER) quality control and ER associated degradation (ERAD) machineries concentrate in the pericentriolar ER-derived quality control compartment (ERQC), suggesting it as a staging ground for ERAD. By tracking the chaperone calreticulin and an established ERAD substrate, asialoglycoprotein receptor H2a, we have now determined that the trafficking to the ERQC is reversible and recycling back to the ER is slower than the movement in the ER periphery. The dynamics suggest vesicular trafficking rather than diffusion. Indeed, using dominant negative mutants of ARF1 and Sar1 or the drugs Brefeldin A and H89, we observed that COPI inhibition causes accumulation in the ERQC and increased retrotranslocation and ERAD, whereas COPII inhibition has the opposite effect. Our results suggest that targeting of misfolded proteins to ERAD involves COPII-dependent transport to the ERQC and that they can be retrieved to the peripheral ER in a COPI-dependent manner.


title: Dyslexia associated gene, KIAA0319, regulates cell cycle during human neuroepithelium development

abstract: Reading Disability (RD), also known as dyslexia, is defined as difficulty processing written language in individuals with normal intellectual capacity and educational opportunity. The prevalence of RD is between 5% and 17%, and the heritability ranges from 44% to 75%. Genetic linkage analysis and genome-wide association studies (GWAS) have identified several genes and regulatory elements linked to RD and reading ability. However, their functions and molecular mechanisms are not well understood. Prominent among these is KIAA0319, encoded in the DYX2 locus of human chromosome 6p22. Association of KIAA0319 has been independently replicated in multiple independent studies and languages. Rodent models suggest that KIAA0319 is involved in neuronal migration, but its precise function is unknown. This studies aim to determine the mechanisms by which KIAA0319 affects reading and language performance. We hypothesize that KIAA0319 plays a critical role in neuronal development. RT-qPCR and quantitative immunofluorescence in the cortical neurons differentiated from H7 hESC show regulatory effects on proliferation and differentiation of neuronal progenitor cells. Knockdown of KIAA0319 expression promotes early exit from the neuroepithelial cell stage and drives cells into cell cycle arrested neuronal progenitor cell stage. This suggests that KIAA0319 act by regulating neurogenesis in the reading related centers of the brain by targeting the cell cycle of proliferative cells. This demonstrates how subtle changes in expression could affect an isolated trait such as reading without global brain effects.

category: developmental biology


title: Development of the adult-like larval stomach of Lepidobatrachus laevis

abstract: Most frogs diets shift between the larval and adult phases, from a filter-feeding herbivore to a bulk-feeding carnivore. This change in diet corresponds to a biphasic mode of gut development that begins during embryogenesis and resumes at metamorphosis, when nearly the entire anatomy of the tadpole is reorganized into its adult morphology. The frog Lepidobatrachus laevis forgoes this metamorphic shift in feeding mode and instead consumes a bulk-feeding, carnivorous diet both as a larvae and as an adult. This unusual larval diet is enabled by the presence of an adult-like stomach in the tadpole. This study investigates the anatomy and embryonic development of the larval stomach of L. laevis and what, if any, further changes occur during metamorphosis. The histology of embryonic and metamorphic stomach development is compared to that of Xenopus tropicalis, a frog with a typical larval stomach. We find that L. laevis directly forms an adult-like stomach during embryonic development without first forming a larval-like configuration. Moreover, no additional major remodeling of the stomach occurs during metamorphosis, although the stomach does gradually and slightly increase in complexity, proliferating more glands and increasing connective tissue and muscle layers, between hatching and the end of metamorphosis. This developmental trajectory of the stomach in L. laevis corresponds with the megalophagous, carnivorous diet these frogs maintain from tadpole to adult, as well as the maintenance of active feeding throughout metamorphosis. These results will facilitate future investigations into the mechanisms underlying the evolution of this unusual larval anuran feeding strategy, as well as the broader study of how development mediates evolutionary change.


title: Comprehensive multiomic profiling of somatic mutations in malformations of cortical development

abstract: Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, and in promoting neuronal hyperexcitability after birth.

category: genetics


title: Pan-genome evolution and its association with divergence of metabolic functions in Bifidobacterium genus

abstract: Many works have been performed to characterize the genomic evolution and diversity among type species of the Bifidobacterium genus due to its health-promoting effect on their host. However, those studies were mainly based on species-level taxonomic resolution, adaptation, and characterization of carbohydrate metabolic features of the bifidobacterial species. Here, a comprehensive analysis of the type strain genome unveils the association of pan-genome evolution with the divergence of metabolic function of the Bifidobacterium genus. This study also has demonstrated that the horizontal gene transfer and genome expansion and reduction events in the evolutionary history influencing the diversity of metabolic functions of bifidobacterium genus. Furthermore, the genome-based search of probiotic traits among all the available bifidobacterial type strains gives hints on type species, that could confer health benefits to nutrient-deficient individuals. Altogether, the present study provides insight into the developments of genomic evolution, functional divergence, and potential probiotic type species of the Bifidobacterium genus.

category: genomics


title: A critical developmental interval of coupling axon branching to synaptic degradation during neural circuit formation

abstract: The emergence of neuronal wiring specificity requires stabilization of dynamic axonal branches at sites of selective synapse formation. Models that explain how axonal branching is coupled to synaptogenesis postulate molecular regulators acting in a spatiotemporally restricted fashion. We report that Epidermal Growth Factor Receptor (EGFR) activity is required in presynaptic axonal branches during two distinct temporal intervals to regulate circuit wiring in the developing Drosophila visual system. EGFR is required early to regulate primary axonal branching and independently again later to prevent autophagic degradation of the synaptic active zone protein Bruchpilot (Brp). The protection of synaptic material during this later interval of wiring ensures the stabilization of terminal branches, circuit connectivity and appropriate visual behavior. Phenotypes of EGFR inactivation were rescued by increasing Brp levels or downregulating autophagic genes. We identify a temporally restricted molecular mechanism required for coupling axonal branching and synaptic stabilization that contributes to the emergence of neuronal wiring specificity.


title: An overlapped natural antisense lncRNA FCER1A-AS controls anaphylactic reaction by promoting FcϵRIα expression

abstract: Functioned as -subunit of the high-affinity immunoglobulin E receptor (Fc{varepsilon} RI), Fc{varepsilon} RI plays a central role in the pathogenesis of Ig-E-mediated allergy and other IgE-related disorders. Fc{varepsilon} RI is normally expressed only in limited spectrum of cells like basophils and mast cells, but the mechanism of controlling Fc{varepsilon} RI expression in these cells is less well understood. In this study, we found fully overlapped natural antisense transcript (NATs) of Fc{varepsilon} RI (FCER1A-AS) is co-expressed with cognate sense transcript (FCER1A-S) in IL-3 induced Fc{varepsilon} RI-expressing cells or in high Fc{varepsilon} RI-expressing cell line MC/9. When FCER1A-AS is selectively knocked down by CRISPR/RfxCas13d (CasRx) approach in MC/9, expression of mRNA and proteins of FCER1A-S is also markedly decreased. Furthermore, deficiency of FCER1A-AS along with lack of FCER1A-S expression is found in two different lines of gene-targeted mice in which FCER1A locus is disturbed at different sites. More importantly, the FCER1A-AS-deficient homozygous mice display similarly diminished anaphylactic reaction as FCER1A gene knockout mice. Thus, we uncovered in this investigation that the expression of FCER1A-S is positively regulated by co-expressed fully overlapped antisense transcript.


title: Pharmacological inhibition of titan-like cells formation suggests that accumulation of endogenous free radicals that correlate with mitochondrial changes are required to induce this transition in Cryptococcus neoformans

abstract:
Cryptococcus neoformans is an encapsulated yeast able to cause disease (mainly meningoencephalitis) among immunosuppressed patients, mostly HIV+. This yeast can form the so-called titan cells in vivo, which are cells of an abnormal larger size due to an increase in both the capsule and the cell body size (total size reaching between 50-70 microns). This phenomenon can be partially reproduced in vitro to obtain cells of an intermediate size (25-30 um), which have been denominated titan-like cells. In this work, we have screened 1,520 compounds from the Prestwick Chemical Library and identified off-patent drugs that inhibited titan-like cell formation in vitro. We developed an automated fluorescence-based microscopy assay and identified 64 compounds as possible inhibitors of titan-like cells in vitro. We chose 10 of these compounds to confirm their inhibitory effect and confirmed them as inhibitors of titan-like cells with dose-response curves. Several of the compounds identified had antioxidant properties (i.e., retinoic acid), indicating a possible role of free radicals during titan cell formation. Using fluorescent probes, we found that there was an endogenous accumulation of ROS during cell growth, which was inhibited in the presence of retinoic acid. Furthermore, we found that during titanization, there were significant changes in the mitochondria, which is the main organelle where ROS are produced. We hypothesize that an intracellular increase of free radicals at the mitochondria might be a triggering signal to induce titanization.

ImportanceCryptococcus neoformans is an excellent model to investigate fungal pathogenesis. This yeast can produce "titan cells", which are cells of an abnormal larger size that contribute to the persistence of the yeast in the host. In this work, we have used a new approach to characterize them, which is identifying drugs that inhibit this process. We have used a repurposing off- patent drug library, combined with an automatic method to image and analyse fungal cell size. In this way, we have identified many compounds that inhibit this transition. Interestingly, several compounds were antioxidants, allowing us to confirm that endogenous ROS and mitochondrial changes are important for titan cell formation. This work provides new evidences of the mechanisms required for titanization. Furthermore, the future characterization of the inhibitory mechanisms of the identified compounds by the scientific community will contribute to better understand the role of titan cells in virulence.


title: ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants

abstract:
ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function.

Significance statementARP2/3 complex-positive dots associate exclusively with peroxisomes in plant cells, where it colocalizes with autophagosome marker ATG8f and several other proteins. Our experiments link ARP2/3 to pexophagy: colocalization between ARP2/3 dots and autophagosome increases when autophagy processes are induced or inhibited; ARP2/3 and ATG8f colocalize and co-immunoprecipitate, and finally, ARP2/3 mutants cells contain more peroxisomes than WT. Our results suggest a novel role of ARP2/3 in peroxisome structure and function regulation.


title: High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation

abstract: In recent years, the development of new image analysis approaches has highlighted the possibility of recovering super-resolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of [~] 1 volume/second.


title: Detection of Dugbe virus from ticks in Ghana

abstract:
Vector-borne pathogens historically have impacted U.S. warfighters and active-duty personnel stationed both domestically and globally during deployments or forward operations. Tick-borne diseases continue to spread to new geographical regions affecting both animal and human health. In Ghana, there is limited information on the circulating tick-borne pathogens and their risk of infections. This military-to-military vector surveillance study focused on seven sites in Ghana: Navrongo, Airforce Base, 6th Battalion Infantry, Air Borne Force, Army Recruit Training School, 1st Battalion Infantry and 5th Battalion Infantry for detection of tick-borne pathogens. Ticks from these sites were collected by hand-picking with a pair of forceps from domesticated animals including cattle, sheep, goats and dogs. A total of 2,016 ticks were collected from two main ecological zones; the northern Sahel savannah and the coastal savannah. Amblyomma variegatum was the predominant species, accounting for 59.5% of the collected ticks. Next-generation sequencing allowed for Dugbe virus whole genome detection. This study reports the second detection of Dugbe virus in Ghana, which is closely related to Dugbe virus strains from Kenya and Nigeria. This study (sponsored by the Armed Forces Health Surveillance Branch-Global Emerg-ing Infections Surveillance Section) aimed to better inform Force Health Protection (FHP) decisions within the U.S. AFRICOM area of responsibility, the Ghana Armed Forces, and enhance global health security countermeasures. Further surveillance needs to be conducted within the country to determine the distribution of tick-borne pathogens to formulate effective control measures.

Author summaryThe prevalence of Dugbe virus in tick species within Ghana was investigated. This study involved seven sampling sites that covered two ecological zones, the northern Sahel savannah and the coastal savannah. About 2,000 ticks were collected from cattle, sheep, goats and dogs and identified using a dissecting microscope. The most predominant tick species was Amblyomma variegatum (59.5%) also known as the tropical bont tick. Using Next-generation sequencing, the full genome of Dugbe virus was for the first time in Ghana identified in A. variegatum. This positive Amblyomma variegatum was collected from the Greater Accra region of Ghana within a military site. The virus was found to be related to Dugbe virus strains from Kenya and Nigeria. Findings from this study show that the trade of livestock across borders facilitates the spread of tick-borne pathogens hence there is a need to enforce measures that prevent the importation of potentially harmful ticks and tick-borne pathogens.


title: LILAC: Enhanced actin imaging with an optogenetic Lifeact

abstract: We have designed an improved Lifeact variant that binds to actin under the control of light using the LOV2 protein. Light control enables one to subtract the pre-illumination signal of the unbound label, yielding an enhanced view of F-actin dynamics in cells. Furthermore, the tool eliminates actin network perturbations and cell sickness caused by Lifeact overexpression.


title: Stabilizing brain-computer interfaces through alignment of latent dynamics

abstract: Intracortical brain-computer interfaces (iBCIs) restore motor function to people with paralysis by translating brain activity into control signals for external devices. In current iBCIs, instabilities at the neural interface result in a degradation of decoding performance, which necessitates frequent supervised recalibration using new labeled data. One potential solution is to use the latent manifold structure that underlies neural population activity to facilitate a stable mapping between brain activity and behavior. Recent efforts using unsupervised approaches have improved iBCI stability using this principle; however, existing methods treat each time step as an independent sample and do not account for latent dynamics. Dynamics have been used to enable high performance prediction of movement intention, and may also help improve stabilization. Here, we present a platform for Nonlinear Manifold Alignment with Dynamics (NoMAD), which stabilizes iBCI decoding using recurrent neural network models of dynamics. NoMAD uses unsupervised distribution alignment to update the mapping of nonstationary neural data to a consistent set of neural dynamics, thereby providing stable input to the iBCI decoder. In applications to data from monkey motor cortex collected during motor tasks, NoMAD enables accurate behavioral decoding with unparalleled stability over weeks- to months-long timescales without any supervised recalibration.


title: RNase H1 exists as phase-separated assemblies in association with elongating RNA Polymerase II during transcription

abstract: R-loops are three stranded nucleic acid structures consisting of a RNA/DNA hybrid and a single stranded displaced DNA. RNase H1 is an endonuclease which specifically degrades the RNA moiety in RNA-DNA hybrids. Here, we report that RNase H1 interacts with transcription elongation machinery during active transcription in a liquid-liquid phase separation (LLPS) dependent manner. We show that RNase H1 interacts with nascent RNA, and transcription elongation machinery in Hela cells, using in-situ nascent RNA labelling and Proximity ligation assay. Further, RNase H1 was found to exhibit properties of liquid-like condensates both in vitro and in vivo. Interestingly, RNase H1 interaction with elongating RNA Polymerase II can be disrupted by chemicals that perturb LLPS. Importantly, we show that LLPS is important for regulation of R-loop levels in the cell. Based on our results, we propose that RNase H1 exists as phase-separated assemblies in association with elongating RNA Polymerase II during active transcription.


title: Sus1 maintains a normal lifespan through regulation of TREX-2 complex-mediated mRNA export

abstract: Eukaryotic gene expression requires multiple cellular events, including transcription and RNA processing and transport. Sus1, a common subunit in both the Spt-Ada-Gcn5 acetyltransferase (SAGA) and transcription and export complex-2 (TREX-2) complexes, is a key factor in coupling transcription activation to mRNA nuclear export. Here we present that the SAGA DUB module and TREX-2 regulates distinctly yeast replicative lifespan in a Sir2-dependent and - independent manner, respectively. The growth and lifespan impaired by SUS1 loss depend on TREX-2 but not on the SAGA DUB module. Notably, an increased dosage of the mRNA export factors Mex67 and Dbp5 rescues the growth defect, shortened lifespan, and nuclear accumulation of poly(A)+ RNA in sus1{Delta} cells, suggesting that boosting the mRNA export process restores the mRNA transport defect and damage in the growth and lifespan of sus1{Delta} cells. Moreover, Sus1 is required for the proper association of Mex67 and Dbp5 with the nuclear rim. Together, these data suggest that Sus1 links transcription and mRNA nuclear export to the lifespan control pathway, indicating that prevention of an abnormal accumulation of nuclear RNA is necessary for maintaining a normal lifespan.


title: Inflammatory cytokines rewire the proinsulin interaction network in human islets

abstract: Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both Type I and Type II diabetes (T1D, T2D). Inflammatory cytokines are implicated in pancreatic islet stress in both forms of diabetes but the mechanisms remain unclear. Here we examined how the diabetes associated cytokines interleukin-1{beta} and interferon-{gamma} alter proinsulin interactions with proteins that regulate its folding, trafficking, and secretion. Human islets treated with cytokines exhibited secretion of proinsulin, IL6 and nitrite, as well as evidence of endoplasmic reticulum (ER) stress. Unbiased proinsulin Affinity Purification-Mass Spectrometry revealed a proinsulin interactome reshaped by cytokines relative to controls. Cytokine treatment increased proinsulin binding to multiple ER chaperones and oxidoreductases, including the major ER chaperone BiP. Moreover, increased BiP binding was an adaptive response required to maintain proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and T1D and T2D GWAS candidate proteins not previously known to interact with proinsulin (e.g., Ataxin-2) and these GWAS proteins formed a tight network with each other. Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins. Consistent with a role for these proteins in proinsulin trafficking and release, chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. Together, the data quantitatively map the proinsulin interactome rewired by cytokines, shedding new light on how human proinsulin biosynthesis is dysregulated by an inflammatory environment.


title: Long lifespan is maintained by a unique Heat Shock Factor in reproductive ants

abstract: Among eusocial insects, reproductive females show longer lifespan than non-reproductive female workers despite high genetic similarity. Using an ant species (Harpegnathos saltator, Hsal) featuring inducible worker reproduction and correlated extended lifespan, we find that long-lived reproductive individuals (called "gamergates") show elevated expression of heat shock response (HSR) genes specifically in the absence of heat stress. This HSR gene elevation is driven in part by gamergate-specific constitutive upregulation of a heat shock transcription factor gene most similar to mammalian HSF2 (called hsalHSF2). In sterile workers hsalHSF2 is bound to DNA only upon heat stress, but in gamergates hsalHSF2 binds to DNA in the absence of heat stress, and correlates with caste-biased gene expression of a subset of heat-inducible genes, thus showing natural bias to gamergates. Remarkably, ectopic expression in D. melanogaster of hsalHSF2 leads to enhanced survival compared to hsalHSF1 following heat stress, as well as extended lifespan. Molecular characterization of these longer-lived flies illustrates multiple parallels between long-lived flies and gamergates, underscoring the centrality of hsalHSF2 to extended lifespan in gamergates. Hence, ant caste-specific heat stress resilience and excessive longevity is, remarkably, transferrable to flies via a specialized ant heat shock factor, HSF2. These findings reinforce the critical role of proteostasis to health and to aging, and reveal novel mechanisms underlying facultative lifespan extension.


title: Differential interactions of resting, activated, and desensitized states of the α7 nicotinic acetylcholine receptor with lipidic modulators

abstract: The 7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that modulates neuronal excitability, largely by allowing Ca2+ permeation. Agonist binding promotes transition from a resting state to an activated state, and then rapidly to a desensitized state. Recently, cryo-EM structures of the human 7 receptor in nanodiscs were reported in multiple conformations. These were selectively stabilized by inhibitory, activating, or potentiating compounds. However, the functional annotation of these structures, and their differential interactions with unresolved lipids and ligands, remain incomplete. Here, we characterized their ion permeation, membrane interactions, and ligand binding using computational electrophysiology, free-energy calculations, and coarse-grained molecular dynamics. In contrast to non-conductive structures in apparent resting and desensitized states, the structure determined in the presence of the potentiator PNU-120596 was consistent with an activated state permeable to Ca2+. Transition to this state was associated with compression and rearrangement of the membrane, particularly in the vicinity of the peripheral MX helix. An intersubunit transmembrane site was implicated in selective binding of either PNU-120596 in the activated state, or cholesterol in the desensitized state. This substantiates functional assignment of all three lipid-embedded 7-receptor structures with ion permeation simulations. It also proposes testable models of their state-dependent interactions with lipophilic ligands, including a mechanism for allosteric modulation at the transmembrane subunit interface.


title: Identification and calibration of ultrabright localizations to eliminate quantification error in SMLM

abstract: Single molecule localization microscopy (SMLM) is irreplaceable among super-resolution microscopies in revealing biological ultra-structures, given its unmatched high resolution. However, its sub-optimal quantitative capability, which is critical for characterizing true biomolecular organization of ultra-structures in cells, has hindered its widest application in biomedical research. Here, in SMLM imaging of cellular structures such as lipid rafts and microtubules with saturation labelling, we identified ultra-bright localizations, each of which is contributed by simultaneous emission of multiple molecules within a diffraction-limit region and has been regarded before as a regular localization from single molecule. Consistently, ultra-bright localizations are also observed in simulated SMLM imaging of endoplasmic reticulum or microtubules from public resource. Furthermore, after calibrating each ultrabright localization into multiple single-molecule localizations using the photon-number-based models, the density of total localizations shows linear correlation with the true molecule density, presenting SMLM with new reconstruction method as a quantitative analysis approach. Therefore, identification and dissection of ultra-bright localizations in SMLM enable the close and quantitative estimate of the true biomolecular organization.


title: Amoeboid Cell Migration through Regular Arrays of Micropillars under Confinement

abstract: Migrating cells often encounter a wide variety of topographic features--including the presence of obstacles--when navigating through crowded biological environments. Unravelling the impact of topography and crowding on the dynamics of cells is key to better understand many essential physiological processes such as the immune response. We study how migration and search efficiency of HL-60 cells differentiated into neutrophils in quasi two-dimensional environments are influenced by the lateral and vertical confinement and spatial arrangement of obstacles. A microfluidic device is designed to track the cells in confining geometries between two parallel plates with distance h, in which identical micropillars are arranged in regular pillar forests. We find that at each cell-pillar contact event, the cell spends a finite time near the pillar surface, which is independent of the height h and the interpillar spacing e. At low pillar density regime, the directional persistence of cells reduces with decreasing h or e, influencing their diffusivity and first-passage properties. The dynamics is strikingly different at high pillar density regime, where the cells are in simultaneous contact with more than one pillar; the cell velocity and persistence are distinctly higher compared to dilute pillar configurations with the same h. Our simulations reveal that the interplay between cell persistence and cell-pillar interactions can dramatically affect cell diffusivity and, thus, its first-passage properties.


title: Bio-electric potentials in superior plants: electric collective behaviour

abstract: Electrical activity is used by plants in long term signalling and information transfer between the distant parts of the plant. Biopotential recordings from trees in a natural environment have been so far less discussed in scientific literature. Here we present our data about the open science experiment TRee-hUMAn iNterface (TRUMAN) located in Paneveggio forest (Valle di Fiemme, Trento, Italy), cultivated since one thousand years for the production of harmonic wood from Picea abies (red fir). We show that: i) biopotential features based on xylem can be correlated with the solar (and lunar) cycle ii) dead tree logs show an electrical activity that is correlated with that of neighbouring trees iii) statistical features of the spike-like peaks are evidenced, including amplitude, frequency, propagation speed, entropy iv) a quantum field theory is presented to highlight the collective behaviour of the forest, supported by preliminar correlation analyses between electrical signal Kolmogorov entropy and thermographies Shannon entropy.


title: Oncogenic signalling is coupled to colorectal cancer cell differentiation state

abstract: Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC/Wnt, KRAS, TP53 and SMAD4 stepwisely enable an oncogenic signalling network. Here, we developed a novel mass cytometry antibody panel to analyse colorectal cancer cell differentiation and signalling in human isogenic colorectal cancer organoids and in patient-derived cultures. We define a differentiation axis following EphrinB2 abundance in all tumour progression states from normal to cancer. We show that during colorectal cancer progression, oncogenes decrease dependence on external factors and shape distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell-promoting or restricting effects. Individual nodes of the signalling network remain coupled to the differentiation state, regardless of the presence of oncogenic signals. Our work underscores the key role of cell plasticity as a hallmark of cancer that is gradually unlocked during colorectal cancer progression.


title: Functional Contribution of Cancer-Associated Fibroblasts in Glioblastoma

abstract: The abundance and biological contribution of cancer associated fibroblasts (CAFs) in glioblastoma are poorly understood. Here, we applied single-cell RNA sequencing and spatial transcriptomics analyses to identify and characterize CAFs in human glioblastoma tumors and then performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant glioblastoma cells. We found that CAF abundance was significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling, using three large databases containing bulk RNA-sequencing data and clinical information. Proteomic analysis of the CAFs and their secretome revealed fibronectin (FN1) as a strong candidate mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF conditioned media and recombinant FN1 could facilitate the migration and invasion of glioblastoma cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of glioblastomas, while cell lines resembling the proneural-state responded to the CAF signaling better in terms of the migratory and invasive phenotypes. Investigating the in-situ expression of gene markers specifically associated with CAFs and mesenchymal malignant cells further indicated that CAFs were enriched in the perinecrotic and pseudopalisading zones of human tumors, where mesenchymal-like glioblastoma cells co-resided and thus likely interacted. Overall, this study characterized the molecular features and functional impacts of CAFs in glioblastoma, alluding to a novel cell-to-cell interaction axis mediated by CAFs in the glioblastoma microenvironment.


title: CMTM6 mediates cisplatin resistance in OSCC by regulating AKT/c-MYC driven ribosome biogenesis

abstract: CMTM6, a type 3 transmembrane protein, is known to stabilize the expression of programmed cell death ligand 1 (PD-L1) and hence facilitates the immune evasion of tumor cells. Recently, we demonstrated that CMTM6 is a major driver of cisplatin resistance in oral squamous cell carcinomas (OSCC). However, the detailed mechanism how CMTM6 rewires cisplatin resistance in OSCC is yet to be explored. RNA sequencing analysis of cisplatin resistant OSCC lines stably expressing NtShRNA and CMTM6 ShRNA revealed that CMTM6 might be a potential regulator of ribosome biogenesis network. Knocking down CMTM6 significantly inhibited transcription of 47S precursor rRNA and hindered the nucleolar structure, indicating reduced ribosome biogenesis. When CMTM6 was ectopically over expressed in CMTM6KD cells, almost all ribosomal machinery components were rescued. Mechanistically, CMTM6 induced the expression of C-Myc, which promotes RNA polymerase I mediated rDNA transcription. In addition to this, CMTM6 also found to regulate the AKT-mTORC1-dependent ribosome biogenesis and protein synthesis in cisplatin resistant lines. The nude mice and zebrafish xenograft experiments indicate that blocking ribosome synthesis either by genetic inhibitor (CMTM6KD) or by pharmacological inhibitor (CX-5461), significantly restores cisplatin medicated cell death in chemoresistant OSCC. Overall, our study suggests that CMTM6 is a major regulator of ribosome biogenesis network and targeting ribosome biogenesis network is a viable target to overcome chemoresistance in OSCC. The novel combination of CX-5461 and cisplatin deserves further clinical investigation in advanced OSCC.


title: Cancer-associated fibroblast-derived ROR2 induces WNT/PCP activation and polarized migration in receiving gastric cancer cells.

abstract: Bone marrow-derived mesenchymal stem cells and cancer-associated fibroblasts in the tumor-stromal environment have been linked to cancer progression in many studies. These fibroblasts provide signaling factors to the tumor cells that promote proliferation, survival, invasion, and metastasis. One signaling pathway influencing tumor cell behavior is the WNT/Planar Cell Polarity (PCP) signaling in gastric cancer. Here, we show that the gastric tumor cell line, AGS, can respond to the PCP ligand WNT5A, however, express a very low level of the bona-fide WNT/PCP receptor, ROR2. At the same time, we find that CAF display long filopodia and had significantly higher levels of ROR2 than normal gastric fibroblasts. By high-resolution imaging, we observe a direct, cytoneme-mediated transfer of a complex containing ROR2 and WNT5A from CAF to the gastric cancer cells. The amount of ROR2 transferred correlated with JNK signaling in receiving cells, showing a direct requirement for receptor transfer. Co-culture of AGS with CAF expressing a dominant-negative form of ROR2 exhibited reduced actin polarization and migration compared to wild-type CAF. Furthermore, induction of migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These unexpected findings demonstrate a fresh role in the direct transfer of a Wnt receptor from a signal-producing cell to a receiving cell and explain the mechanism by which gastric cancer cells expressing low levels of ROR2 can respond to a WNT5A-high tumor microenvironment.