title: Mesenchymal stem cells carry and transport clusters of cancer cells

abstract:
Cell clusters that collectively migrate from primary tumors appear to be far more potent in forming distant metastases than single cancer cells. A better understanding of collective cell migration phenomenon and the involvement of different cell types during this process is needed. Here, we utilize a micropatterned surface composed of a thousand of low-adhesive microwells to screen motility of spheroids containing different cell types by analyzing their ability to move from the bottom to the top of the microwells. Mesenchymal stem cells (MSCs) spheroid migration was efficient in contrast to cancer cell only spheroids. In spheroids with both cell types mixed together, MSCs were able to carry the low-motile cancer cells during migration. As the percentage of MSCs increased in the spheroids, more migrating spheroids were detected. Extracellular vesicles secreted by MSCs also contributed to the pro-migratory effect exerted by MSCs. However, the transport of cancer cells was more efficient when MSCs were physically present in the cluster. Similar results were obtained when cell clusters were encapsulated within a micropatterned hydrogel, where collective migration was guided by micropatterned matrix stiffness. These results suggest that stromal cells facilitate the migration of cancer cell clusters, which is contrary to the general belief that malignant cells metastasize independently.

SignificanceDuring metastasis, tumor cells may migrate as a cluster, which exhibit higher metastatic capacity compared to single cells. However, whether and how non-cancer cells contained in tumor cluster regulate its migration is not clear. Here, we utilize two unique approaches to study collective tumor cell migration in vitro: first, in low-adhesive microwells and second, in micropatterned hydrogels to analyze migration in 3D microenvironment. Our results indicate that MSCs in tumor cell clusters could play an important role in the dissemination of cancer cells by actively transporting low-motile cancer cells. In addition, MSC-released paracrine factors also increase the motility of tumor cells. These findings reveal a new mechanism of cancer cell migration and may lead to new approaches to suppress metastases.


title: Comprehensive metabolic engineering for fermenting glycerol efficiently in Saccharomyces cerevisiae

abstract:
Glycerol is an eco-friendly solvent enhancing plant-biomass decomposition through a glycerolysis process in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae restrains many of these scenarios. Here we outline the complete strategy for the generation of efficient glycerol fermenting yeast by rewriting the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by O2-dependent dynamic shuttle while abolishing both glycerol phosphorylation and biosynthesis pathways. By following a vigorous glycerol oxidative pathway, the engineered strain demonstrated augmentation in conversion efficiency (CE) reach up to 0.49g-ethanol/g-glycerol--98% of theoretical conversion--with production rate >1 g/L-1h-1 when supplementing glycerol as a single fed-batch on a rich-medium. Furthermore, the engineered strain showed a new capability toward ferment a mixture of glycerol and glucose with producing >86 g/L of bioethanol with 92.8% of the CE. To our knowledge, this is the highest ever reported titer in this regard. Notably, this strategy flipped our ancestral yeast from non-growth on glycerol, on the minimal medium, to a fermenting strain with productivities 0.25-0.5 g/L-1h-1 and 84-78% of CE, respectively and 90% of total conversions to the products. The findings in metabolic engineering here may release the limitations of utilizing glycerol in several eco-friendly biorefinery approaches.

IMPORTANCEWith the avenues for achieving efficient lignocellulosic biorefinery scenarios, glycerol gained keen attention as an eco-friendly biomass-derived solvent for enhancing the dissociation of lignin and cell wall polysaccharides during pretreatment process. Co-fermentation of glycerol with the released sugars from biomass after the glycerolysis expands the resource for ethanol production and release from the burden of component separation. Titer productivities are one of the main obstacles for industrial applications of this process. Therefore, the generation of highly efficient glycerol fermenting yeast significantly promotes the applicability of the integrated biorefineries scenario. Besides, the glycerol is an important carbon resource for producing chemicals. Hence, the metabolic flux control of yeast from glycerol contributes to generation of cell factory producing chemicals from glycerol, promoting the association between biodiesel and bioethanol industries. Thus, this study will shed light on solving the problems of global warming and agricultural wastes, leading to establishment of the sustainable society.

category: bioengineering


title: Aggregation-induced emission dots assisted non-invasive fluorescence hysterography in near-infrared IIb window

abstract: Uterine diseases seriously threaten the physical and mental health of women. The main principle, when clinicians adopt examinations, is to achieve efficient diagnosis without negative effect on the physical function including fertility. Hysterography in near-infrared (NIR) IIb window (1500-1700 nm) presents perceptibly enhanced signal to background ratio (SBR) and higher penetration capability compared with those beyond 1000 nm and 1300 nm, but lays down high requirements for the biosafety of fluorophores at the same time. Assisted by the biologically excretable aggregation-induced emission (AIE) dots, non-invasive NIR-IIb fluorescence hysterography visualized typical Y-shaped uteruses, real-time uterine peristalsis or the uterine lesions (mimetic disease statuses in clinic) in mouse models. Significantly, after intrauterine perfusion, the reproductive capacity was unimpaired via fertility assessment and histological analysis. This work could inspire some new ideas for non-invasive clinical diagnosis of uterine diseases and effectively promote the clinical translation of AIE dots.


title: ParticleChromo3D: A Particle Swarm Optimization Algorithm for Chromosome and Genome 3D Structure Prediction from Hi-C Data

abstract: The three-dimensional (3D) structure of chromatin has a massive effect on its function. Because of this, it is desirable to have an understanding of the 3D structural organization of chromatin. To gain greater insight into the spatial organization of chromosomes and genomes and the functions they perform, chromosome conformation capture techniques, particularly Hi-C, have been developed. The Hi-C technology is widely used and well-known because of its ability to profile interactions for all read pairs in an entire genome. The advent of Hi-C has greatly expanded our understanding of the 3D genome, genome folding, gene regulation and has enabled the development of many 3D chromosome structure reconstruction methods. Here, we propose a novel approach for 3D chromosome and genome structure reconstruction from Hi-C data using Particle Swarm Optimization approach called ParticleChromo3D. This algorithm begins with a grouping of candidate solution locations for each chromosome bin, according to the particle swarm algorithm, and then iterates its position towards a global best candidate solution. While moving towards the optimal global solution, each candidate solution or particle uses its own local best information and a randomizer to choose its path. Using several metrics to validate our results, we show that ParticleChromo3D produces a robust and rigorous representation of the 3D structure for input Hi-C data. We evaluated our algorithm on simulated and real Hi-C data in this work. Our results show that ParticleChromo3D is more accurate than most of the existing algorithms for 3D structure reconstruction. Our results also show that constructed ParticleChromo3D structures are very consistent, hence indicating that it will always arrive at the global solution at every iteration. The source code for ParticleChromo3D, the simulated and real Hi-C datasets, and the models generated for these datasets are available here: https://github.com/OluwadareLab/ParticleChromo3D

category: bioinformatics


title: SARS-CoV-2 lineage B.1.526 emerging in the New York region detected by software utility created to query the spike mutational landscape

abstract: Wide-scale SARS-CoV-2 genome sequencing is critical to tracking viral evolution during the ongoing pandemic. Variants first detected in the United Kingdom, South Africa, and Brazil have spread to multiple countries. We developed the software tool, Variant Database (VDB), for quickly examining the changing landscape of spike mutations. Using VDB, we detected an emerging lineage of SARS-CoV-2 in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage (now designated as B.1.526) are L5F, T95I, D253G, E484K or S477N, D614G, and A701V. This lineage was first sequenced in late November 2020 when it represented <1% of sequenced coronavirus genomes that were collected in New York City (NYC). By February 2021, genomes from this lineage accounted for ~32% of 3288 sequenced genomes from NYC specimens. Phylodynamic inference confirmed the rapid growth of the B.1.526 lineage in NYC, notably the sub-clade defined by the spike mutation E484K, which has outpaced the growth of other variants in NYC. Pseudovirus neutralization experiments demonstrated that B.1.526 spike mutations adversely affect the neutralization titer of convalescent and vaccinee plasma, indicating the public health importance of this lineage.


title: Detection and characterization of the SARS-CoV-2 lineage B.1.526 in New York


title: Mutations in bdcA and valS correlate with quinolone resistance in wastewater Escherichia Coli

abstract:
Single mutations can confer resistance to antibiotics. Identifying such mutations can help to develop and improve drugs. Here, we systematically screen for candidate quinolone resistance-conferring mutations. We sequenced highly diverse wastewater E. coli and performed a genome-wide association study (GWAS) correlating over 200,000 mutations against quinolone resistance phenotypes. We uncovered 13 statistically significant mutations including one located at the active site of the biofilm dispersal genes bdcA and six silent mutations in the aminoacyl-tRNA synthetase valS. The study also recovered the known mutations in the topoisomerases gyrA and parC.

In summary, we demonstrate that GWAS effectively and comprehensively identifies resistance mutations without a priori knowledge of targets and mode of action. The results suggest that bdcA and valS may be novel resistance genes with biofilm dispersal and translation as novel resistance mechanisms.


title: Streamlining differential exon and 3 UTR usage with diffUTR

abstract:
BackgroundDespite the importance of alternative poly-adenylation and 3 UTR length for a variety of biological phenomena, there are limited means of detecting UTR changes from standard transcriptomic data.

ResultsWe present the diffUTR Bioconductor package which streamlines and improves upon differential exon usage (DEU) analyses, and leverages existing DEU tools and alternative polyadenylation site databases to enable differential 3 UTR usage analysis. We demonstrate the diffUTR features and show that it is more flexible and more accurate than state-of-the-art alternatives, both in simulations and in real data.

ConclusionsdiffUTR enables differential 3 UTR analysis and more generally facilitates DEU and the exploration of their results.


title: A fully automated approach for quality control of cancer mutations in the era of high-resolution whole genome sequencing

abstract: Cancer genomes contain thousands of somatic point mutations, chromosome copy alterations and more complex structural variants, which contribute to tumour growth and therapy response. Whole genome sequencing is a well established approach for somatic variant identification, but its broad application comes with complications, particularly in how proposed calls are quality assessed. To address this issue, we present CNAqc, a quantitative framework to quality control somatic mutations and allele-specific copy numbers, both in clonal and subclonal settings while accounting for variations in tumour purity, as commonly seen in bulk sampling. We test the model via extensive simulations, validate it using low-pass single-cell data, and apply it to 2778 single-sample PCAWG whole-genomes, 10 in-house multi-region whole-genomes and 48 TCGA whole-exomes. CNAqc is compatible with common bioinformatic pipelines and designed to support automated parameterization processes that are crucial in the era of large-scale whole genome sequencing.


title: Integrated quality control of allele-specific copy numbers, mutations and tumour purity from cancer whole genome sequencing assays


title: HaVoC, a bioinformatic pipeline for reference-based consensus assembly and lineage assignment for SARS-CoV-2 sequences

abstract:
BackgroundSARS-CoV-2 related research has increased in importance worldwide since December 2019. Several new variants of SARS-CoV-2 have emerged globally, of which the most notable and concerning currently are the UK variant B.1.1.7, the South African variant B1.351 and the Brazilian variant P.1. Detecting and monitoring novel variants is essential in SARS-CoV-2 surveillance. While there are several tools for assembling virus genomes and performing lineage analyses to investigate SARS-CoV-2, each is limited to performing singular or a few functions separately.

ResultsDue to the lack of publicly available pipelines, which could perform fast reference-based assemblies on raw SARS-CoV-2 sequences in addition to identifying lineages to detect variants of concern, we have developed an open source bioinformatic pipeline called HaVoC (Helsinki university Analyzer for Variants Of Concern). HaVoC can reference assemble raw sequence reads and assign the corresponding lineages to SARS-CoV-2 sequences.

ConclusionsHaVoC is a pipeline utilizing several bioinformatic tools to perform multiple necessary analyses for investigating genetic variance among SARS-CoV-2 samples. The pipeline is particularly useful for those who need a more accessible and fast tool to detect and monitor the spread of SARS-CoV-2 variants of concern during local outbreaks. HaVoC is currently being used in Finland for monitoring the spread of SARS-CoV-2 variants. HaVoC user manual and source code are available at https://www.helsinki.fi/en/projects/havoc and https://bitbucket.org/auto_cov_pipeline/havoc, respectively.


title: SearcHPV: a novel approach to identify and assemble human papillomavirus-host genomic integration events in cancer

abstract:
BackgroundHuman papillomavirus (HPV) is a well-established driver of malignant transformation in a number of sites including head and neck, cervical, vulvar, anorectal and penile squamous cell carcinomas; however, the impact of HPV integration into the host human genome on this process remains largely unresolved. This is due to the technical challenge of identifying HPV integration sites, which includes limitations of existing informatics approaches to discover viral-host breakpoints from low read coverage sequencing data.

MethodsTo overcome this limitation, we developed a new HPV detection pipeline called SearcHPV based on targeted capture technology and applied the algorithm to targeted capture data. We performed an integrated analysis of SearcHPV-defined breakpoints with genome-wide linked read sequencing to identify potential HPV-related structural variations.

ResultsThrough analysis of HPV+ models, we show that SearcHPV detects HPV-host integration sites with a higher sensitivity and specificity than two other commonly used HPV detection callers. SearcHPV uncovered HPV integration sites adjacent to known cancer-related genes including TP63 and MYC, as well as near regions of large structural variation. We further validated the junction contig assembly feature of SearcHPV, which helped to accurately identify viral-host junction breakpoint sequences. We found that viral integration occurred through a variety of DNA repair mechanisms including non-homologous end joining, alternative end joining and microhomology mediated repair.

ConclusionsIn summary, we show that SearcHPV is a new optimized tool for the accurate detection of HPV-human integration sites from targeted capture DNA sequencing data.


title: A computational approach to identify possible symbiotic mechanisms between Klebsiella and the Mediterranean fly (Ceratitis capitata)

abstract: Nowadays, scientists develop multiple methods to control pests, like chemical control as the insecticides and biological control as the Sterile Insect Technique (SIT) to release infertile males produced by irradiation. This technique is complemented with research in the medflys (Ceratitis capitata) microbiota, this approach is based on the identification of molecular mechanisms from three Klebsiella strains using the reconstruction of metabolic pathways technique. We focus on the reconstruction of metabolic pathways involved in nitrogen metabolization because the bacteria are in charge of metabolizing the nitrogen compounds and make available for the medfly. We propose a pipeline to process the genome sequences the bacteria, reconstruct metabolic pathways and identify possible symbiont molecular mechanisms. The result of processing the biological data were ten pathways, six pathways produce L-Glutamate as final product, three pathways with ammonia product and one pathway was discarded because it was an internal pathway and dont generate a metabolic product. Those pathways corresponding to L-Glutamate and ammonia metabolic products are part of nitrogen metabolism on Klebsiella, which shows that this metabolic process is one of the molecular mechanisms involved in symbiosis process between Klebsiella and the medfly. Finally, by the results of the sequences analysis, in the medflys metamorphose the L-Glutamate are used in the synthesis of new proteins and the production of energy. The final consideration suggest that the pipeline propose can be used as first step to identify molecular mechanisms that can improve the production of industrial the medfly.


title: APPLES-2: Faster and More Accurate Distance-based Phylogenetic Placement using Divide and Conquer

abstract: Phylogenetic placement of query samples on an existing phylogeny is increasingly used in molecular ecology, including sample identification and microbiome environmental sampling. As the size of available reference trees used in these analyses continues to grow, there is a growing need for methods that place sequences on ultra-large trees with high accuracy. Distance-based placement methods have recently emerged as a path to provide such scalability while allowing flexibility to analyze both assembled and unassembled environmental samples. In this paper, we introduce a distance-based phylogenetic placement method, APPLES-2, that is more accurate and scalable than existing distance-based methods and even some of the leading maximum likelihood methods. This scalability is owed to a divide-and-conquer technique that limits distance calculation and phylogenetic placement to parts of the tree most relevant to each query. The increased scalability and accuracy enables us to study the effectiveness of APPLES-2 for placing microbial genomes on a data set of 10,575 microbial species using subsets of 381 marker genes. APPLES-2 has very high accuracy in this setting, placing 97% of query genomes within three branches of the optimal position in the species tree using 50 marker genes. Our proof of concept results show that APPLES-2 can quickly place metagenomic scaffolds on ultra-large backbone trees with high accuracy as long as a scaffold includes tens of marker genes. These results pave the path for a more scalable and widespread use of distance-based placement in various areas of molecular ecology.


title: Fast and Accurate Distance-based Phylogenetic Placement using Divide and Conquer


title: Domain adaptive neural networks improvecross-species prediction of transcription factor binding

abstract: The intrinsic DNA sequence preferences and cell-type specific cooperative partners of transcription factors (TFs) are typically highly conserved. Hence, despite the rapid evolutionary turnover of individual TF binding sites, predictive sequence models of cell-type specific genomic occupancy of a TF in one species should generalize to closely matched cell types in a related species. To assess the viability of cross-species TF binding prediction, we train neural networks to discriminate ChIP-seq peak locations from genomic background and evaluate their performance within and across species. Cross-species predictive performance is consistently worse than within-species performance, which we show is caused in part by species-specific repeats. To account for this domain shift, we use an augmented network architecture to automatically discourage learning of training species-specific sequence features. This domain adaptation approach corrects for prediction errors on species-specific repeats and improves overall cross-species model performance. Our results demonstrate that cross-species TF binding prediction is feasible when models account for domain shifts driven by species-specific repeats.


title: VisVariant: A java program to visualise genetic variants in next-generation sequencing data

abstract:
SummaryExtremely large datasets are impossible or very difficult for humans to comprehend by standard mental approaches. Intuitive visualization of genetic variants in genomic sequencing data could help in the review and confirmation process of variants called by automated variant calling programs. To help facilitate interpretation of genetic variant next-generation sequencing (NGS) data we developed VisVariant, a customizable visualization tool that creates a figure showing the overlapping sequence information of thousands of individual reads including the variant and flanking regions.

Availability and implementationDetailed information on how to download, install and run VisVariant together with an example is available on our github website [https://github.com/hugging-biorxiv/visvariant].


title: PDBrenum: a webserver and program providing Protein Data Bank files renumbered according to their UniProt sequences

abstract:
The Protein Data Bank (PDB) was established at Brookhaven National Laboratories in 1971 as an archive for biological macromolecular crystal structures. In early 2021, the database has more than 175,000 structures solved by X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy, and other methods. Many proteins have been studied under different conditions, including binding partners such as ligands, nucleic acids, or other proteins; mutations, and post-translational modifications, thus enabling extensive comparative structure-function studies. However, these studies are made more difficult because authors are allowed by the PDB to number the amino acids in each protein sequence in any manner they wish. This results in the same protein being numbered differently in the available PDB entries. For instance, some authors may include N-terminal signal peptides or the N-terminal methionine in the sequence numbering and others may not. In addition to the coordinates, there are many fields that contain information regarding specific residues in the sequence of each protein in the entry. Here we provide a webserver and Python3 application that fixes the PDB sequence numbering problem by replacing the author numbering with numbering derived from the corresponding UniProt sequences. We obtain this correspondence from the SIFTS database from PDBe. The server and program can take a list of PDB entries or a list of UniProt identifiers (e.g., "P04637" or "P53_HUMAN") and provide renumbered files in mmCIF format and the legacy PDB format for both asymmetric unit files and biological assembly files provided by PDBe.

AvailabilitySource code is freely available at https://github.com/Faezov/PDBrenum. The webserver is located at: http://dunbrack3.fccc.edu/PDBrenum.

Contactbulat.faezov@fccc.edu or roland.dunbrack@fccc.edu.


title: Overexpression of ELOVL6 has been associated with poor prognosis in patients with head and neck squamous cell carcinoma

abstract: Head and neck squamous cell carcinoma (HNSCC) is a high mortality disease. Extension of long-chain fatty acid family member 6 (ELOVL6) is a key enzyme involved in fat formation that catalyzes the elongation of saturated and monounsaturated fatty acids. Overexpression of ELOVL6 has been associated with obesity-related malignancies, including hepatocellular carcinoma, breast, colon, prostate, and pancreatic cancer. The following study investigated the role of ELOVL6 in HNSCC patients. Gene expression and clinicopathological analysis, enrichment analysis, and immune infiltration analysis were based on the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA), with additional bioinformatics analyses. The statistical analysis was conducted in R, and TIMER was used to analyze the immune response of ELOVL6 expression in HNSCC. The expression of ELOVL6 was related to tumor grade. Survival analysis showed that patients with high expression of ELOVL6 had a poor prognosis. Moreover, the results of GSEA enrichment analysis showed that ELOVL6 affects the occurrence of HNSCC through fatty acid metabolism, biosynthesis of unsaturated fatty acids, and other pathways. Finally, ELOVL6 verified by the Human Protein Atlas (HPA) database were consistent with the mRNA levels in HNSCC samples. ELOVL6 is a new biomarker for HNSCC that may be used as a potential predictor of the prognosis of human HNSCC.


title: Sparcle: assigning transcripts to cells in multiplexed images

abstract:
BackgroundImaging-based spatial transcriptomics has the power to reveal patterns of single-cell gene expression by detecting mRNA transcripts as individually resolved spots in multiplexed images. However, molecular quantification has been severely limited by the computational challenges of segmenting poorly outlined, overlapping cells, and of overcoming technical noise; the majority of transcripts are routinely discarded because they fall outside the segmentation boundaries. This lost information leads to less accurate gene count matrices and weakens downstream analyses, such as cell type or gene program identification.

ResultsHere, we present Sparcle, a probabilistic model that reassigns transcripts to cells based on gene covariation patterns and incorporates spatial features such as distance to nucleus. We demonstrate its utility on both multiplexed error-robust fluorescence in situ hybridization (MERFISH) and single-molecule FISH (smFISH) data.

ConclusionsSparcle improves transcript assignment, providing more realistic per-cell quantification of each gene, better delineation of cell boundaries, and improved cluster assignments. Critically, our approach does not require an accurate segmentation and is agnostic to technological platform.


title: Expanding N-Glycopeptide Identifications by Fragmentation Prediction and Glycome Network Smoothing

abstract: Accurate glycopeptide identification in mass spectrometry-based glycoproteomics is a challenging problem at scale. Recent innovation has been made in increasing the scope and accuracy of glycopeptide identifications, with more precise uncertainty estimates for each part of the structure. We present a layered approach to glycopeptide fragmentation modeling that improves N-glycopeptide identification in samples without compromising identification quality, and a site-specific method to increase the depth of the glycoproteome confidently identifiable even further. We demonstrate our techniques on a pair of previously published datasets, showing the performance gains at each stage of optimization, as well as its flexibility in glycome definition and search space complexity. These techniques are provided in the open-source glycomics and glycoproteomics platform GlycReSoft available at https://github.com/mobiusklein/glycresoft.


title: Mutation N501Y in RBD of Spike Protein Strengthens the Interaction between COVID-19 and its Receptor ACE2

abstract: SARS-CoV-2 is spreading around the world for the past year. Enormous efforts have been taken to understand its mechanism of transmission. It is well established now that the receptor-binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) as its first step of entry. Being a single-stranded RNA virus, SARS-CoV-2 is evolving rapidly. Recently, several variants such as B.1.1.7, B.1.351, and P.1, with a key mutation N501Y on the RBD, appear to be more infectious to humans. To understand its mechanism, we combined cell surface binding assay, kinetics study, single-molecule technique, and computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction characterized from all these methodologies, while the other two mutations from B.1.351 contributed to a less effect. Fluorescence-activated cell scan (FACS) assays found that RBD N501Y mutations are of higher binding affinity to ACE2 than the wild type. Surface plasmon resonance further indicated that N501Y mutation had a faster association rate and slower dissociation rate. Consistent with the kinetics study, atomic force microscopy-based single-molecule force microscopy quantify their strength on living cells, showing a higher binding probability and unbinding force for the mutation. Finally, Steered Molecular Dynamics (SMD) simulations on the dissociation of RBD-ACE2 complexes revealed that the N501Y introduced additional {pi}-{pi} and {pi}-cation interaction for the higher force/interaction. Taken together, we suggested that the reinforced interaction from N501Y mutation in RBD should play an essential role in the higher transmission of COVID-19 variants.

category: biophysics


title: Experimental Control of a Reaction Occurring during the Interaction between Chicken Anemia Virus (CAV) and Its Corresponding Antibodies

abstract: In our ongoing investigations, we have studied a specific interaction between electromagnetic fields and matter - the so-called Electromagnetic echo effect (EMEE). It enables rapid and contactless investigations of gases, liquids and solids to be performed, since the signal generated as a result of the effect is quite sensitive to all kinds of changes occurring within the studied samples. The effect can be considered universal for all matter and provides analysis in real time. We use this phenomenon to demonstrate the practical possibility to control reactions, occurring between Chicken anemia virus (CAV) and the corresponding antibodies. This methodology can be used for simple but reliable control of similar, otherwise hard to detect, antigen-antibody reactions, in order to confirm the presence of a certain viral species. The approach offers a high level of safety, since it enables measurements to be taken remotely, thus limiting exposure to contagion. We further discuss the possibility to register the presence of SARS-nCoV-2 in an attempt to address current global pandemic.


title: Tyrosine 121 moves revealing a druggable pocket that couples catalysis to ATP-binding in serine racemase

abstract: Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a  closed hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the ATP a-phosphate. In contrast, in  open hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzymefragment structures show that Tyr121 is flipped out of its pocket, suggesting that this pocket is druggable.


title: A Multiscale Model for Quantitative Prediction of Insulin Aggregation Nucleation Kinetics

abstract:
We combined kinetic, thermodynamic, and structural information from single molecule (protein folding) and two molecule (association) explicit-solvent simulations for determination of kinetic parameters in protein aggregation nucleation with insulin as model protein. A structural bioinformatics approach was developed to account for heterogeneity of aggregation-prone species with the transition complex theory found applicable in modeling association kinetics involving non-native species. We show that a key simplification arises from presence of only a few relevant modes for non-native association kinetics. The kinetic parameters thus obtained were used in a population balance model and accurate predictions for aggregation nucleation time varying over two orders of magnitude with changes in concentration of insulin or an aggregation-inhibitor ligand were obtained while an empirical parameter set was not found to be transferable for prediction of ligand effects. This physically determined kinetic parameter set also allowed identification of the rate-limiting step in aggregation nucleation.

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title: Structural and thermodynamic analyses of human TMED1 (p24γ1) Golgi dynamics

abstract: The transmembrane emp24 domain-containing proteins (TMED), also called p24 proteins, are members of a family of sorting receptors present in all representatives of the domain Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMEDs structure, oligomerization propensity, and biophysics of their interactions with the transport cargo. Here, we describe the first high-resolution structure of the Golgi dynamics (GOLD) domain of a TMED1 representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane are also presented.


title: The Kinetic Landscape of Nucleosome Assembly: A Coarse-Grained Molecular Dynamics Study

abstract: The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. We find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences.


title: Close to optimal cell sensing ensures the robustness of tissue differentiation process: the avian photoreceptor mosaic case

abstract: The way that progenitor cell fate decisions and the associated environmental sensing are regulated to ensure the robustness of the spatial and temporal order in which cells are generated towards a fully differentiating tissue still remains elusive. Here, we investigate how cells regulate their sensing intensity and radius to guarantee the required thermodynamic robustness of a differentiated tissue. In particular, we are interested in finding the conditions where dedifferentiation at cell level is possible (microscopic reversibility) but tissue maintains its spatial order and differentiation integrity (macroscopic irreversibility). In order to tackle this, we exploit the recently postulated Least microEnvironmental Uncertainty Principle (LEUP) to develop a theory of stochastic thermodynamics for cell differentiation. To assess the predictive and explanatory power of our theory, we challenge it against the avian photoreceptor mosaic data. By calibrating a single parameter, the LEUP can predict the cone color spatial distribution in the avian retina and, at the same time, suggest that such a spatial pattern is associated with quasi-optimal cell sensing. By means of the stochastic thermodynamics formalism, we find out that thermodynamic robustness of differentiated tissues depends on cell metabolism and cell sensing properties. In turn, we calculate the limits of the cell sensing radius that ensure the robustness of differentiated tissue spatial order. Finally, we further constrain our model predictions to the avian photoreceptor mosaic.


title: Inactivation of Aβ42 Protomer-dimer Dissociation Reaction via Increasing Protomer Size

abstract: Multimeric protein complexes are molecular apparatuses to regulate biological systems and often determine their fate. Among proteins forming such molecular assemblies, amyloid proteins have drawn attention over a half-century since amyloid fibril formation of these proteins is supposed to be a common pathogenic cause for neurodegenerative diseases. This process is triggered by the accumulation of fibril-like aggregates, while the microscopic mechanisms are mostly elusive due to technical limitation of experimental methodologies in individually observing each of diverse aggregate species in the aqueous solution. We then addressed this problem by employing atomistic molecular dynamics simulations for the paradigmatic amyloid protein, amyloid-{beta} (1-42) (A{beta}42). Seven different dimeric forms of oligomeric A{beta}42 fibril-like aggregate in aqueous solution, ranging from tetramer to decamer, were considered. We found additive effects of the size of these fibril-like aggregates on their thermodynamic stability and have clarified kinetic suppression of protomer-protomer dissociation reactions at and beyond the point of pentamer dimer formation. This observation was obtained from the specific combination of the A{beta}42 protomer structure and the physicochemical condition that we here examined, while it is worthwhile to recall that several amyloid fibrils take dimeric forms of their protomers. We could thus conclude that the stable formation of fibril-like protomer dimer should be involved in a turning point where rapid growth of amyloid fibrils is triggered.


title: Inactivation of Aβ42 Protomer-protomer Dissociation Reaction by Pentamer Dimer Formation


title: Remarked Suppression of Aβ42 Protomer-protomer Dissociation Reaction via Pentamer Dimerization


title: Remarked Suppression of Aβ42 Protomer-protomer Dissociation Reaction via Pentamer Dimerization Elucidated by Molecular Dynamics Simulation


title: MEMS enabled miniaturized light-sheet microscopy with all optical control

abstract: We have designed and implemented a compact, cost-efficient miniaturised light-sheet microscopy system based on optical Microelectromechanical Systems (MEMS) scanners and tunable lenses. The system occupies a footprint of 20 x 28 x 13 cm3 and combines off-the-shelf optics and optomechanics with 3D-printed structural and optical elements, and an economically costed objective lens, excitation laser and camera. All-optical volume scanning enables imaging of 340 x 190 x 60 {micro}m3 volumes with 0.25 vps and minimum lateral and axial resolution of 0.9 {micro}m and 2.95 {micro}m respectively. An open-top geometry allows imaging of samples on flat bottomed holders, allowing integration with microfluidic devices, multi-well plates and slide mounted samples, with applications envisaged in biomedical research and pre-clinical settings.


title: Effect of cellular rearrangement time delays on the rheology of vertex models for confluent tissues

abstract: Large-scale tissue deformation during biological processes such as morphogenesis requires cellular rearrangements. The simplest rearrangement in confluent cellular monolayers involves neighbor exchanges among four cells, called a T1 transition, in analogy to foams. But unlike foams, cells must execute a sequence of molecular processes, such as endocytosis of adhesion molecules, to complete a T1 transition. Such processes could take a long time compared to other timescales in the tissue. In this work, we incorporate this idea by augmenting vertex models to require a fixed, finite time for T1 transitions, which we call the "T1 delay time". We study how variations in T1 delay time affect tissue mechanics, by quantifying the relaxation time of tissues in the presence of T1 delays and comparing that to the cell-shape based timescale that characterizes fluidity in the absence of any T1 delays. We show that the molecular-scale T1 delay timescale dominates over the cell shape-scale collective response timescale when the T1 delay time is the larger of the two. We extend this analysis to tissues that become anisotropic under convergent extension, finding similar results. Moreover, we find that increasing the T1 delay time increases the percentage of higher-fold coordinated vertices and rosettes, and decreases the overall number of successful T1s, contributing to a more elastic-like - and less fluid-like - tissue response. Our work suggests that molecular mechanisms that act as a brake on T1 transitions could stiffen global tissue mechanics and enhance rosette formation during morphogenesis.


title: Structural origins of protein conformational entropy

abstract: The thermodynamics of molecular recognition by proteins is a central determinant of complex biochemistry. For over a half-century detailed cryogenic structures have provided deep insight into the energetic contributions to ligand binding by proteins1. More recently, a dynamical proxy based on NMR-relaxation methods has revealed an unexpected richness in the contributions of conformational entropy to the thermodynamics of ligand binding2,3,4,5. There remains, however, a discomforting absence of an understanding of the structural origins of fast internal motion and the conformational entropy that this motion represents. Here we report the pressure-dependence of fast internal motion within the ribonuclease barnase and its complex with the protein barstar. Distinctive clustering of the pressure sensitivity correlates with the presence of small packing defects or voids surrounding affected side chains. Prompted by this observation, we performed an analysis of the voids surrounding over 2,500 methyl-bearing side chains having experimentally determined order parameters. We find that changes in unoccupied volume as small as a single water molecule surrounding buried side chains greatly affects motion on the subnanosecond timescale. The discovered relationship begins to permit construction of a united view of the relationship between changes in the internal energy, as exposed by detailed structural analysis, and the conformational entropy, as represented by fast internal motion, in the thermodynamics of protein function.


title: A Brownian ratchet model for DNA loop extrusion by the cohesin complex

abstract: The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities, supported by biochemical experiments. ATP and DNA binding to cohesin promote conformational changes that guide DNA through a kleisin gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesins heads and hinge, are now juxtaposed. ATP hydrolysis disassembles the gripping state, allowing unidirectional hinge module movement to complete topological DNA entry. Without initial kleisin gate passage, biased hinge module motion during gripping state resolution creates a Brownian ratchet that drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian fluctuations has important implications for chromosomal cohesin function.


title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

abstract: While molecular targeted therapies have improved prognoses of advanced stage lung adenocarcinoma expressing oncogenic driver mutations, acquired therapeutic resistance continues to be a major problem. Epidermal growth factor receptor (EGFR) activating mutations are among the most common targetable genetic alterations in lung adenocarcinoma, and EGFR tyrosine kinase inhibitors (TKIs) are recommended first-line therapy for EGFR mutation positive cancer patients. Unfortunately, most patients develop resistance to EGFR TKIs and rapid disease progression occurs. A better mechanistic understanding of therapy refractory cancer progression is necessary to develop new therapeutic approaches to predict and prevent acquired resistance to EGFR TKIs. Here, we identify a new mechanism of ERBB3-mediated resistance to EGFR TKIs in human lung adenocarcinoma. Specifically, we show that dopamine and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32) physically recruits ERBB3 to EGFR to mediate a switch from EGFR homodimers to EGFR:ERBB3 heterodimers to bypass EGFR TKI-mediated inhibition to potentiate ERBB3-dependent activation of oncogenic AKT and ERK signaling that drives therapy refractory tumor cell survival. In a cohort of paired tumor specimens derived from 30 lung adenocarcinoma patients before and after the development of EGFR TKI refractory disease progression, we reveal that DARPP-32 as well as kinase-activated EGFR and ERBB3 proteins are overexpressed upon acquired EGFR TKI resistance. In vivo studies suggest that ablation of DARPP-32 protein activity sensitizes gefitinib-resistant lung tumor xenografts to EGFR TKI treatment, while DARPP-32 overexpression increases gefitinib-refractory lung cancer progression in gefitinib-sensitive lung tumors orthotopically xenografted into mice. Taken together, our findings introduce a DARPP-32-mediated, ERBB3-dependent mechanism used by lung tumor cells to evade EGFR TKI-induced cell death, potentially paving the way for the development of new therapies to prevent or overcome therapy-refractory lung adenocarcinoma progression.

category: cancer biology


title: Tumor expressed CD95 causes suppression of anti-tumor activity of NK cells in a model of triple negative breast cancer

abstract: The apoptosis inducing receptor CD95/Fas has multiple tumorigenic activities. Stimulation by its cognate ligand CD95L on many cancer cells increases their growth, motility, ability to invade and/or their cancer stemness. Using genetically engineered mouse models of ovarian and liver cancer, we previously reported that deletion of CD95 in the tumor cells strongly reduced their ability to grow in vivo [1, 2]. Using a combination of immune-deficient and immune-competent mouse models, we now establish that loss of CD95 in metastatic triple negative breast cancer cells prevents tumor growth by modulating the immune landscape. CD95 deficient but not wild-type tumors barely grow in an immune-competent environment and show an increase in immune infiltrates into the tumor. This growth reduction is caused by NK cells and does not involve CD8+ T cells. On the other hand, in immune compromised mice CD95 k.o. cells are not growth inhibited, but they fail to form metastases. In summary, we demonstrate that in addition to its tumor and metastasis promoting activities, CD95 expression by tumor cells can exert immune suppressive activities providing a new target for immune therapy.


title: BET Inhibition Enhances TNF Mediated Anti-Tumor Immunity

abstract:
Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance anti-tumor immunity and augment cancer immunotherapies. By screening a small molecule library of epigenetics-based therapeutics, BET bromodomain inhibitors (BETi) were identified as agents that promote the anti-tumor activity of CD8+ T-cells. BETi sensitized diverse tumor types to the cytotoxic effects of the pro-inflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-{kappa}B target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of pro-survival NF-{kappa}B signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific (TCB) antibodies increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune oncology agents.

STATEMENT OF SIGNIFICANCEManipulating the epigenome is an evolving strategy to enhance anti-tumor immunity. We demonstrate that BET bromodomain inhibitors potently sensitize solid tumors to CD8+ T-cell killing in a TNF-dependent manner. This immunomodulatory mechanism can be therapeutically leveraged to augment immuno-oncology therapies, including TCB antibodies and immune checkpoint blockade.


title: Synthesis, Physiochemical and Biological evaluation of Inclusion Complex of Benzyl Isothiocyanate encapsulated in cyclodextrins for triple negative breast cancer

abstract: Benzyl isothiocyanate (BITC), an organic dietary compound, is allied with a major role in the potential chemopreventive effects. BITC has acknowledged rising attention as a therapeutic compound to be used in medicine because of its high potency and characteristic biopharmaceutical properties, like high permeability with marginal aqueous solubility. The highly volatile and hydrophobic nature brought a need to provide a suitable delivery-matrix to BITC to exploit its pharmacological potential to the fullest. It has been successfully incorporated in {beta}-CD and HP-{beta}-CD using acoustic forces and thoroughly characterized using UV-vis spectroscopy, FTIR, DSC, TEM, and SAXS. The complexation helped in masking the acute odour, achieving a controlled release of BITC, and made its use viable by prolonging the retention time and thereby sustaining the biological effects. Different models like Higuchi, first-order kinetic decay, Korsmeyer-Peppas model were applied, suggesting a diffusion-controlled mechanism of release. Also, the bioaccessibility and stability of BITC in an in vitro digestion model was evaluated. The main objective of the present work was to systemically study the credibility of BITC-CD complexes in well-established tumor mimicking 2D cell culture models and produce a conclusive report on its chemotherapeutic activity. The in vitro anti-cancer activity of BITC and the formed sonochemical complexes was confirmed by MTT assay and further evaluated using apoptosis assay and production of ROS like moieties. Cell cycle analysis was done to evaluate the growth inhibitory mechanism of BITC. Strikingly, BITC and its complexes showcased ROS generation and lysosome-mediated cell death. Effect on cell migration was assessed using wound healing assay. The results promptly suggest the functional efficacy of the CDs in releasing BITC and attest the ability of the complexes to provide alternate to otherwise remedially sparse triple-negative breast cancer.


title: Metabolomics, Transcriptomics and Functional Glycomics Reveals Bladder Cancer Cells Plasticity and Enhanced Aggressiveness Facing Hypoxia and Glucose Deprivation

abstract: Bladder cancer constitutes one of the deadliest genitourinary diseases, especially when diagnosed at late stages. These tumours harbour microenvironmental niches characterized by low levels of oxygen (hypoxia) and limited glucose supply due to poor vascularization. However, the synergic contribution of these features to disease development is poorly understood. Here, we demonstrated that cells with distinct histopathological and molecular backgrounds responded similarly to such stimuli. Cancer cells arrested proliferation, significantly increased invasive capacity in vitro and enhanced tolerance to cisplatin-based chemotherapy. Reoxygenation and access to glucose restored basal proliferation and invasion levels without triggering stress-induced apoptosis, denoting significant cellular plasticity in adapting to microenvironmental cues. Whole transcriptomics showed major molecular reprogramming, supporting main functional alterations. Metabolomics evidenced fatty acids {beta}-oxidation as main bioenergetic pathway rather than anaerobic glycolysis generally adopted by hypoxic cells. Joint pathway analysis also suggested relevant alterations in mucin-type O-glycan biosynthesis. Glycomics confirmed a major antagonization of O-glycosylation pathways, leading to simple cell glycophenotypes characterized by the accumulation of immature short-chain O-glycans such as Tn and STn antigens at the cell surface. Glycoengineered models reflecting simple cell glycophenotypes were developed and functional studies in vitro and in vivo showed that Tn and STn overexpression decreased proliferation and promoted chemoresistance, reinforcing their close link with tumour aggressiveness. Collectively, we have demonstrated that hypoxia and glucose deprivation trigger more aggressive cell behaviours, in what appears to be an escape mechanism from microenvironmental stress. We propose that, altered glycosylation may be used to target these subpopulations, paving the way for precision oncology.


title: Medicago Sativa Defensin 1 (MsDef1), A Natural Tumor Targeted Sensitizer for Improving Chemotherapy: Translation from Anti-Fungal Agent to Potential Anti-Cancer Agent

abstract:
IntroductionMsDef1, a 45-amino acid cysteine-rich peptide from the seed of Medicago sativa is an antifungal defensin small protein. It exhibits broad-spectrum antifungal activity against fungal pathogens of plants at low micromolar concentrations. The common vulnerability of fungal and cancer cells determines the utility of MsDef1 as a potential anti-tumor agent.

ResultsThe solution dynamics of 15N-labeled MsDef1, 15N longitudinal relaxation (T1) and 15N-1H Nuclear Overhauser Effect (NOE) shows that GlcCer binds at two sites on the peptide molecule, i.e., Asp36-Cys39 and amino acids between 12-20 and 33-40. MsDef1 interacts with drug resistant breast cancer MCF-7R cells, permeates GlcCer-rich plasma membrane and releases apoptotic ceramide. This results in the activation of ceramide pathway involving interaction of the peptide with intracellular thioredoxin (Trx), another tumor specific biomarker. MsDef1 oxidizes Trx through four S-S bonds and in the process, gets reduced to thiols. Oxidation of Trx is correlated with the activation of Apoptosis Stimulating Kinase 1 (ASK1) which is known to sensitize cancer cells to chemotherapeutics including front-line drug Doxorubicin. A combination of MsDef1 and Doxorubicin exhibits 5-10-fold greater apoptosis in vitro in MDR triple negative breast cancer (TNBC) cells compared to either MsDef1 or Doxorubicin alone.

ConclusionAn antifungal plant defensin MsDef1 is shown to be a cell permeating peptide (CPP) for MDR cancer cells targeted to two tumor specific targets activating two cell death pathways. That makes MsDef1, potentially a tumor targeted sensitizer neoadjuvant to cancer therapy.


title: The APOBEC3A deaminase drives episodic mutagenesis in cancer cells

abstract: The APOBEC3 family of cytidine deaminases is widely speculated to be a major source of somatic mutations in cancer1-3. However, causal links between APOBEC3 enzymes and mutations in human cancer cells have not been established. The identity of the APOBEC3 paralog(s) that may act as prime drivers of mutagenesis and the mechanisms underlying different APOBEC3-associated mutational signatures are unknown. To directly investigate the roles of APOBEC3 enzymes in cancer mutagenesis, candidate APOBEC3 genes were deleted from cancer cell lines recently found to naturally generate APOBEC3-associated mutations in episodic bursts4. Deletion of the APOBEC3A paralog severely diminished the acquisition of mutations of speculative APOBEC3 origins in breast cancer and lymphoma cell lines. APOBEC3 mutational burdens were undiminished in APOBEC3B knockout cell lines. APOBEC3A deletion reduced the appearance of the clustered mutation types kataegis and omikli, which are frequently found in cancer genomes. The uracil glycosylase UNG and the translesion polymerase REV1 were found to play critical roles in the generation of mutations induced by APOBEC3A. These data represent the first evidence for a long-postulated hypothesis that APOBEC3 deaminases generate prevalent clustered and non-clustered mutational signatures in human cancer cells, identify APOBEC3A as a driver of episodic mutational bursts, and dissect the roles of the relevant enzymes in generating the associated mutations in breast cancer and B cell lymphoma cell lines.


title: APOBEC mutagenesis inhibits breast cancer growth through induction of a T cell-mediated antitumor immune response

abstract:
The APOBEC family of cytidine deaminases is one of the most common endogenous sources of mutations in human cancer. Genomic studies of tumors have found that APOBEC mutational signatures are particularly enriched in the HER2 subtype of breast cancer and have been associated with immunotherapy response in diverse cancer types. However, the direct consequences of APOBEC mutagenesis on the tumor immune microenvironment have not been thoroughly investigated. To address this, we developed syngeneic murine mammary tumor models with inducible expression of APOBEC3B. We found that APOBEC activity induces an antitumor adaptive immune response and CD4+ T cell-mediated tumor growth inhibition. While polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected by the immune system, suggesting that APOBEC-mediated genetic heterogeneity limits the antitumor adaptive immune response. Consistent with the observed immune infiltration in APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to checkpoint inhibition. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures as a biomarker predicting immunotherapy response in HER2-positive breast cancers.

SIGNIFICANCEAPOBEC mutational signatures are observed in many cancers, yet the consequences of these mutations on the tumor immune microenvironment are not well understood. Using a novel mouse model, we show that APOBEC activity sensitizes HER2-driven mammary tumors to checkpoint inhibition and could inform immunotherapy treatment strategies for HER2-positive breast cancer patients.


title: EZH2 Engages TGFβ Signaling to Promote Breast Cancer Bone Metastasis via Integrin β1-FAK Activation

abstract: Bone metastasis is a frequent complication of breast cancer, occurring in about 50-70% of breast cancer patients with late-stage disease. The lack of effective therapy suggests that the precise molecular mechanisms underlying bone metastasis are still unclear. Enhancer of zeste homolog 2 (EZH2) is considered a breast cancer oncogene and its expression is correlated with metastasis of breast cancer, but its function in bone metastasis has not been well explored. Herein we report that EZH2 promotes osteolytic metastasis of breast cancer through regulating transforming growth factor beta (TGF{beta}) signaling, a key pathway in bone metastasis. Knocking down EZH2 decreases bone metastasis incidence and outgrowth in vivo. EZH2 induces cancer cell proliferation and osteoclast maturation, when breast cancer cells are co-cultured with osteoblasts and osteoclasts together in vitro. Mechanistically, EZH2 increases transcription of ITGB1, which encodes for integrin {beta}1. Integrin {beta}1 activates focal adhesion kinase (FAK), which phosphorylates TGF{beta} receptor type I (TGF{beta}RI) at tyrosine 182, thus enhances the binding of TGF{beta}RI to TGF{beta} receptor type II (TGF{beta}RII), therefore activates Smad2 and increases parathyroid hormone-like hormone (PTHLH) expression. Clinically applicable FAK inhibitors but not EZH2 methyltransferase inhibitor effectively inhibits breast cancer bone metastasis in vivo. Overall, our data signify integrin {beta}1-FAK as a new downstream effector of EZH2 in breast cancer cells, and EZH2-integrin {beta}1-FAK axis cooperates with TGF{beta} signaling pathway to promote bone metastasis of breast cancer.


title: Invadopodia enable cooperative invasion and metastasis of breast cancer cells

abstract: Invasive and non-invasive cancer cells can invade together during the cooperative invasion. However, the events leading to it, role of EMT and the consequences this may have on metastasis are unknown. In this study, we demonstrate that the isogenic 4T1 and 67NR breast cancer cells sort from each other in 3D spheroids, followed by cooperative invasion. By time-lapse microscopy, we show that the invasive 4T1 cells move more persistently compared to non-invasive 67NR, sorting and accumulating at the spheroid-ECM interface, a process dependent on cell-ECM adhesions and independent from E-cadherin cell-cell adhesions. Elimination of invadopodia in 4T1 cells blocks invasion, demonstrating that invadopodia requirement is limited to leader cells. Importantly, we demonstrate that cells with and without invadopodia can also engage in cooperative metastasis in preclinical mouse models. Altogether, our results suggest that a small number of cells with invadopodia can drive the metastasis of heterogeneous cell clusters.


title: Stomach serosal arteries distinguish gastric regions of the rat

abstract: Because the stomach in situ has few distinctive surface features and changes shape dramatically with food intake, we have used microCT imaging to (1) characterize the pattern of arteries, potential landmarks, on the stomach wall and (2) evaluate how meal-related shape changes affect the size of the different regions. The stomach receives its blood supply primarily from two pairs of vessels, the gastric and gastroepiploic arteries. Each of the three regions of the stomach is delineated by a distinctive combination of arterial fields: The corpus, consistent with its dynamic secretory activity and extensive mucosa, is supplied by extensive arterial trees formed by the left and right gastric arteries. These major arteries course circularly from the lesser towards the greater curvature, distally along both left and right walls of the corpus, and branch rostrally to supply the region. The muscular antrum is characterized by smaller arterial branches arising primarily from the right gastroepiploic artery that follows the distal greater curvature and secondarily from small, distally directed arteries supplied by the large vessels of the left and right gastric arteries. The forestomach, essentially devoid of mucosal tissue and separated from the corpus by the limiting ridge, is vascularized predominantly by a network of small arteries issued from the left gastroepiploic artery coursing around the proximal greater curvature, as well as from higher order and smaller branches issued by the gastric and celiac arteries. The regions of the stomach empty at different rates, thus changing the dimensions of the organ regions non-linearly.

category: physiology


title: Impact of an oral amino acid provision on Achilles peritendinous amino acid concentrations in young and older adults

abstract: Recent studies have indicated that consumption of amino acid-rich compounds can increase tendon collagen content and enhance biomechanical function. Still, it is not clear as to what extent oral consumption of amino acids alters peritendinous amino acid concentrations. Whether aging alters the delivery of amino acids to tendon tissue after oral consumption is also not known. Using microdialysis, we determined the impact of a single oral essential amino acid bolus on Achilles peritendinous amino acid concentrations in younger (n=7; 27{+/-}1 yr.) and older adults (n=6; 68{+/-}2 yrs.) over four hours. The peritendinous concentration of all amino acids in the beverage except methionine (p=0.136) and glycine (p=0.087) increased with time (p<0.05). Additionally, the concentrations of glycine and arginine were greater in older adults (p[≤]0.05). We also accessed the impact of amino acid consumption on peritendinous concentrations of pro-collagen I1, a marker of collagen synthesis. Pro-collagen I1 tended to change with time (p=0.071) but was not altered age (p=0.226). We demonstrate that an oral amino acid bolus leads to modest increases in Achilles peritendinous amino acid concentrations in young and older adults. The concentration of some amino acids was also greater in older adults. However, the amino acid bolus did not significantly impact peritendinous pro-collagen concentrations.


title: Endothelial alpha globin is a nitrite reductase

abstract: Small artery vasodilation in response to hypoxia is essential for matching oxygen supply to tissue oxygen demand. One source of hypoxic dilation via nitric oxide (NO) signaling is nitrite reduction by erythrocytic hemoglobin (2{beta}2). However, the alpha subunit of hemoglobin is also expressed in resistance artery endothelium and localized to myoendothelial junctions, a subcellular domain that contacts underlying vascular smooth muscle cells. We hypothesized that nitrite reduction mediated by endothelial alpha globin may occur at myoendothelial junctions to regulate hypoxic vasodilation. To test this concept, we created two novel mouse strains: one lacking alpha globin specifically in endothelium (EC Hba1{Delta}/{Delta}) and one where alpha globin is mutated such that its inhibitory association with endothelial NO synthase (eNOS) is prevented (Hba1WT/{Delta}36-39). In EC Hba1{Delta}/{Delta} or Hba1WT/{Delta}36-39 mice hemoglobin levels, hematocrit and erythrocyte counts were unchanged from littermate controls. Loss of the full alpha globin protein from the endothelium in the EC Hba1{Delta}/{Delta} model was associated with decreased exercise capacity and decreased intracellular nitrite utilization in hypoxic conditions. These effects were not seen in Hba1WT/{Delta}36-39 animals. Hypoxia induced vasodilation was decreased by 60% in isolated thoracodorsal arteries from EC Hba1{Delta}/{Delta}, while infusion of erythrocytes only partially rescued the dilatory response. Lastly, unlike other models where blood pressure is decreased, EC Hba1{Delta}/{Delta} blood pressure was not altered in response to hypoxia. Overall, we conclude that alpha globin in the resistance artery endothelium can act as a nitrite reductase to provide a local vasodilatory response to hypoxia.


title: Subcellular dynamics studies reveal how tissue-specific distribution patterns of iron are established in developing wheat grains

abstract: Understanding iron trafficking in plants is key to enhancing the nutritional quality of crops. Due to the difficulty of imaging iron in transit, little is known about iron translocation and distribution in developing seeds. A novel approach, combining 57Fe isotope labelling and NanoSIMS, was used to visualize iron translocation dynamics at the subcellular level in wheat grain, Triticum aestivum L. We were able to track the main route of iron from maternal tissues to the embryo through different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, as confirmed by histological staining and TEM-EDS. Virtually all iron was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Aleurone cells contained a new type of iron body, highly enriched in 57Fe, and most likely represents iron-nicotianamine being delivered to phytate globoids. Correlation with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification efforts.

category: plant biology


title: GABA plays a key role in plant acclimation to a combination of high light and heat stress

abstract:
Plants are frequently subjected to different combinations of abiotic stresses, such as high light intensity and elevated temperatures. These environmental conditions pose an important threat to agriculture production, affecting photosynthesis and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources towards stress responses and suppressing growth. Here we show that the primary metabolic response of Arabidopsis thaliana plants to high light or heat stress is different than that of plants subjected to a combination of high light and heat stress. We further demonstrate that a combination of high light and heat stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids, coupled with decreased levels of metabolites participating in the tricarboxylic acid (TCA) cycle. Among the amino acids exclusively accumulated during a combination of high light and heat stress, we identified the non-proteinogenic amino acid {gamma}-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis, in particular two independent alleles of glutamate decarboxylase 3 (gad3), reveal that GABA plays a key role in the acclimation of plants to a combination of high light and heat stress. Taken together, our findings identify a new role for GABA in regulating plant responses to stress combination.

One sentence summaryThe non-proteinogenic amino acid {gamma}-aminobutyric acid (GABA) is required for plant acclimation to a combination of high light and heat stress in Arabidopsis.


title: MSA Transformer

abstract: Unsupervised protein language models trained across millions of diverse sequences learn structure and function of proteins. Protein language models studied to date have been trained to perform inference from individual sequences. The longstanding approach in computational biology has been to make inferences from a family of evo lutionarily related sequences by fitting a model to each family independently. In this work we combine the two paradigms. We introduce a protein language model which takes as input a set of sequences in the form of a multiple sequence alignment. The model interleaves row and column attention across the input sequences and is trained with a variant of the masked language modeling objective across many protein families. The performance of the model surpasses current state-of-the-art unsupervised structure learning methods by a wide margin, with far greater parameter efficiency than prior state-of-the-art protein language models.

category: synthetic biology


title: Protocell Arrays for Simultaneous Detection of Diverse Analytes

abstract: Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexing still typically faces the critical limitation of measuring only one type of molecule per assay platform - for example, only small molecules or only nucleic acids. In this work, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS) to produce membrane-less "protocells" containing transcription and translation machinery used for analyte detection. Multiple protocells are arrayed in microwells where each protocell droplet performs distinct reactions to detect chemically diverse targets including small molecules, minerals, and nucleic acid sequences, all from the same sample. We demonstrate that these protocell arrays can measure analytes in a human biofluid matrix, maintain function after lyophilization and rehydration, and produce visually interpretable readouts, illustrating its potential for application as a minimal-equipment, field-deployable, multi-analyte detection tool.


title: picCASO: A Cellular Automaton for Spatial Organisation

abstract:
Microbial consortia exhibit spatial patterning across diverse environments. Since probing the self-organization of natural microbial communities is limited by their inherent complexity, synthetic models have emerged as attractive alternatives. In this study, we develop novel frameworks of bacterial communication and explore the emergent spatiotemporal organization of microbes. Specifically, we build quorum sensing-mediated models of microbial growth that are utilized to characterize the dynamics of communities from arbitrary initial configurations and establish the effectiveness of our communication strategies in coupling the growth rates of microbes. Our simulations indicate that the behavior of quorum sensing-coupled consortia can be most effectively modulated by the rates of secretion of AHLs. Such a mechanism of control enables the construction of desired relative populations of constituent species in spatially organized populations. Our models accurately recapitulate previous experiments that have investigated pattern formation in synthetic multi-cellular systems. Additionally, our software tool enables the easy implementation and analysis of our frameworks for a variety of initial configurations and simplifies the development of sophisticated gene circuits facilitating distributed computing. Overall, we demonstrate the potential of spatial organization as a tunable parameter in synthetic biology by introducing a communication paradigm based on the location and strength of coupling of microbial strains.

Author SummaryInteracting microorganisms that coexist in a given environment tend to have well-defined spatial arrangements. While the emergence of such organization is seen across different microbiomes in nature, it is hitherto not well understood. Decoding the inherent spatial patterning of microbes is constrained by the complexity of their natural habitats. Here, we take advantage of synthetic models of microbial communities to study the dynamics of emergent spatial organization. Our framework of bacterial communication utilizes modular synthetic devices to couple growth rates. In addition to uncovering potential principles of spatial organization, this work enables the construction of complex genetic circuits distributed across communicating strains. Additionally, we have developed a software tool, picCASO, that streamlines the investigation of microbial systems communicating through such frameworks.

category: systems biology


title: Probing Patterning in Microbial Consortia with picCASO: a Cellular Automaton for Spatial Organisation


title: Integument transcriptome profile of the European sea cucumber Holothuria forskali (Holothuroidea, Echinodermata)

abstract: In non-model organisms, Next Generation Sequencing (NGS) technology improve our ability to analyze gene expression and identify new genes or transcripts of interest. In this research, paired-end Illumina HiSeq sequencing has been used to describe a composite transcriptome based on two libraries generated from dorsal and ventral integuments of the European sea cucumber Holothuria forskali (Holothuroidea, Echinodermata). A total of 43,044,977 million HQ reads were initially generated. After de novo assembly, a total of 111,194 unigenes were predicted. On all predicted unigenes, 32,569 show significant matches with genes/proteins present in the reference databases. Around 50% of annotated unigenes were significantly similar to sequences from the purple sea urchin Strongylocentrotus purpuratus genome. Annotation analyses were performed on predicted unigenes using public reference databases. These RNA-seq data provide an interesting resource for researchers with a broad interest in sea cucumber biology.

category: zoology


title: Impact of Binge Drinking During College on Resting State Functional Connectivity

abstract:
AimThe current study aimed to examine the longitudinal effects of standard binge drinking (4+/5+ drinks for females/males in 2 hours) and extreme binge drinking (8+/10+ drinks for females/males in 2 hours) on resting state functional connectivity.

Method119 college students with distinct alcohol bingeing patterns (35 non-bingeing controls, 44 standard bingers, and 40 extreme bingers) were recruited to ensure variability in bingeing frequency. Resting state fMRI scans were obtained at time 1 when participants were college freshmen and sophomores and again approximately two years later. On four occasions during the 2-year period between scans, participants reported monthly standard and extreme binge drinking for the past 6 months. Association between bingeing and change in functional connectivity was studied using both network-level and edge-level analysis. Network connectivity was calculated by aggregating multiple edges (a functional connection between any two brain regions) affiliated with the same network. The network-level analysis used mixed-effects models to assess the association between standard/extreme binge drinking and change in network connectivity, focusing on canonical networks often implicated in substance misuse. On the other hand, the edge-level analysis tested the relationship between bingeing and change in whole-brain connectivity edges using connectome-based predictive modeling (CPM).

ResultsFor network-level analysis, higher standard bingeing was associated with a decrease in connectivity between Default Mode Network-Ventral Attention Network (DMN-VAN) from time 1 to time 2, controlling for the initial binge groups at time 1, longitudinal network changes, in-scanner motion and other demographic covariates. For edge-level analysis, the CPM failed to identify a generalizable predictive model of cumulative standard/extreme bingeing from change in connectivity edges.

ConclusionsOur findings suggest that binge drinking is associated with abnormality in networks implicated in attention allocation and self-focused processes, which, in turn, have been implicated in rumination, craving, and relapse. More extensive alterations in functional connectivity might be observed with heavier or longer binge drinking pattern.

category: neuroscience


title: Direct activation of endothelial cells by SARS-CoV-2 nucleocapsid protein is blocked by Simvastatin

abstract: Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multi-organ failure in patients with COVID-19. However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the SARS-CoV-2 viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved with endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through TLR2/NF-{kappa}B and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkablely, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, MERS-CoV, HUB1-CoV and influenza virus H1N1 did not affect endothelial activation. These findings are well consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.

category: immunology


title: Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

abstract: Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 {micro}M). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a "BL2 groove" that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

category: microbiology


title: Pulsed broad-spectrum UV light effectively inactivates SARS-CoV-2 on multiple surfaces

abstract: The ongoing SARS-CoV-2 pandemic has resulted in an increased need for technologies capable of efficiently disinfecting public spaces as well as personal protective equipment. UV light disinfection is a well-established method for inactivating respiratory viruses. Here, we have determined that broad-spectrum, pulsed UV light is effective at inactivating SARS-CoV-2 on multiple surfaces. For hard, non-porous surfaces we observed that SARS-CoV-2 was inactivated to undetectable levels on plastic and glass with a UV dose of 34.9 mJ/cm2 and stainless steel with a dose of 52.5 mJ/cm2. We also observed that broad-spectrum, pulsed UV light is effective at reducing SARS-CoV-2 on N95 respirator material to undetectable levels with a dose of 103 mJ/cm2. We included UV dosimeter cards that provide a colorimetric readout of UV dose and demonstrated their utility as a means to confirm desired levels of exposure were reached. Together, the results present here demonstrate that broad-spectrum, pulsed UV light is an effective technology for the inactivation of SARS-CoV-2 on multiple surfaces.


title: ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model

abstract:
O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 M). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.


title: An allosteric ligand stabilizes distinct conformations in the M2 muscarinic acetylcholine receptor

abstract: Allosteric modulators provide therapeutic advantages over orthosteric drugs. A plethora of allosteric modulators have been identified for several GPCRs, particularly for muscarinic receptors (mAChRs)1,2. To study the molecular mechanisms governing allosteric modulation, we utilized a recently developed NMR system to investigate the conformational changes in the M2 muscarinic receptor (M2R) in response to the positive allosteric modulator (PAM) LY2119620. Our studies provide the first biophysical data showing that LY2119620 can substantially change the structure and dynamics of M2R in both the extracellular and G-protein coupling domains during the activation process. These NMR data suggest that LY2119620 may function by stabilizing distinct sets of conformations not observed in the presence of orthosteric agonists alone, which may account for the different signaling behaviors of the M2R when bound to LY2119620. Our studies provide new structural information for understanding the mechanism of GPCR allostery, and may facilitate the rational design of allosteric therapeutics targeting muscarinic receptors.


title: The Progenetix oncogenomic resource in 2021

abstract:
In cancer, copy number aberrations (CNA) represent a type of nearly ubiquitous and frequently extensive structural genome variations. To disentangle the molecular mechanisms underlying tumorigenesis as well as identify and characterize molecular subtypes, the comparative and meta-analysis of large genomic variant collections can be of immense importance. Over the last decades, cancer genomic profiling projects have resulted in a large amount of somatic genome variation profiles, however segregated in a multitude of individual studies and datasets. The Progenetix project, initiated in 2001, curates individual cancer CNA profiles and associated metadata from published oncogenomic studies and data repositories with the aim to empower integrative analyses spanning all different cancer biologies.

During the last few years, the fields of genomics and cancer research have seen significant advancement in terms of molecular genetics technology, disease concepts, data standard harmonization as well as data availability, in an increasingly structured and systematic manner. For the Progenetix resource, continuous data integration, curation and maintenance have resulted in the most comprehensive representation of cancer genome CNA profiling data with 138663 (including 115357 tumor) CNV profiles. In this article, we report a 4.5-fold increase in sample number since 2013, improvements in data quality, ontology representation with a CNV landscape summary over 51 distinctive NCIt cancer terms as well as updates in database schemas, and data access including new web front-end and programmatic data access. Database URL: progenetix.org


title: Jumper Enables Discontinuous Transcript Assembly in Coronaviruses

abstract:
Genes in SARS-CoV-2 and, more generally, in viruses in the order of Nidovirales are expressed by a process of discontinuous transcription mediated by the viral RNA-dependent RNA polymerase. This process is distinct from alternative splicing in eukaryotes, rendering current transcript assembly methods unsuitable to Nidovirales sequencing samples. Here, we introduce the DO_SCPLOWISCONTINUOUSC_SCPLOW TO_SCPLOWRANSCRIPTC_SCPLOW AO_SCPLOWSSEMBLYC_SCPLOW problem of finding transcripts [Formula] and their abundances c given an alignment [Formula] under a maximum likelihood model that accounts for varying transcript lengths. Underpinning our approach is the concept of a segment graph, a directed acyclic graph that, distinct from the splice graph used to characterize alternative splicing, has a unique Hamiltonian path. We provide a compact characterization of solutions as subsets of non-overlapping edges in this graph, enabling the formulation of an efficient mixed integer linear program. We show using simulations that our method, JO_SCPLOWUMPERC_SCPLOW, drastically outperforms existing methods for classical transcript assembly. On short-read data of SARS-CoV-1 and SARS-CoV-2 samples, we find that JO_SCPLOWUMPERC_SCPLOW not only identifies canonical transcripts that are part of the reference transcriptome, but also predicts expression of non-canonical transcripts that are well supported by direct evidence from long-read data, presence in multiple, independent samples or a conserved core sequence. JO_SCPLOWUMPERC_SCPLOW enables detailed analyses of Nidovirales transcriptomes.

Code availabilitySoftware is available at https://github.com/elkebir-group/Jumper


title: Quantifying and Mitigating Motor Phenotypes Induced by Antisense Oligonucleotides in the Central Nervous System

abstract: Antisense oligonucleotides (ASOs) are emerging as a promising class of therapeutics for neurological diseases. When injected directly into the cerebrospinal fluid, ASOs distribute broadly across brain regions and exert long-lasting therapeutic effects. However, many phosphorothioate (PS)-modified gapmer ASOs show transient motor phenotypes when injected into the cerebrospinal fluid, ranging from reduced motor activity to ataxia or acute seizure-like phenotypes. The effect of sugar and phosphate modifications on these phenotypes has not previously been systematically studied. Using a behavioral scoring assay customized to reflect the timing and nature of these effects, we show that both sugar and phosphate modifications influence acute motor phenotypes. Among sugar analogues, PS-DNA induces the strongest motor phenotype while 2-substituted RNA modifications improve the tolerability of PS-ASOs. This helps explain why gapmer ASOs have been more challenging to develop clinically relative to steric blocker ASOs, which have a reduced tendency to induce these effects. Reducing the PS content of gapmer ASOs, which contain a stretch of PS-DNA, improves their toxicity profile, but in some cases also reduces their efficacy or duration of effect. Reducing PS content improved the acute tolerability of ASOs in both mice and sheep. We show that this acute toxicity is not mediated by the major nucleic acid sensing innate immune pathways. Formulating ASOs with calcium ions before injecting into the CNS further improved their tolerability, but through a mechanism at least partially distinct from the reduction of PS content. Overall, our work identifies and quantifies an understudied aspect of oligonucleotide toxicology in the CNS, explores its mechanism, and presents platform-level medicinal chemistry approaches that improve tolerability of this class of compounds.

category: pharmacology and toxicology


title: Ongoing habenular activity is driven by forebrain networks and modulated by olfactory stimuli

abstract: Ongoing neural activity, which represents internal brain states, is constantly modulated by the sensory information that is generated by the environment. In this study, we show that the habenular circuits act as a major brain hub integrating the structured ongoing activity of the limbic forebrain circuitry and the olfactory information. We demonstrate that ancestral homologs of amygdala and hippocampus in zebrafish forebrain are the major drivers of ongoing habenular activity. We also reveal that odor stimuli can modulate the activity of specific habenular neurons that are driven by this forebrain circuitry. Our results highlight a major role for the olfactory system in regulating the ongoing activity of the habenula and the forebrain, thereby altering brains internal states.


title: Sphingosine kinase 2 is essential for remyelination following cuprizone intoxication

abstract:
Therapeutics that promote oligodendrocyte survival and remyelination are needed to restore neurological function in demyelinating diseases. Sphingosine 1-phosphate (S1P) is an essential lipid metabolite that signals through five G-protein coupled receptors. S1P receptor agonists such as Fingolimod are valuable immunosuppressants used to treat multiple sclerosis, and promote oligodendrocyte survival. However, the role for endogenous S1P, synthesized by the enzyme sphingosine kinase 2 (SphK2), in oligodendrocyte survival and myelination has not been established. This study investigated the requirement for SphK2 in oligodendrocyte survival and remyelination using the cuprizone mouse model of acute demyelination, followed by spontaneous remyelination. Oligodendrocyte density did not differ between untreated wild- type (WT) and SphK2 knockout (SphK2-/-) mice. However, cuprizone treatment caused significantly greater loss of mature oligodendrocytes in SphK2-/- compared to WT mice. Following cuprizone withdrawal, spontaneous remyelination occurred in WT but not SphK2-/- mice, even though progenitor and mature oligodendrocyte density increased in both genotypes. Levels of cytotoxic sphingosine and ceramide were higher in the corpus callosum of SphK2-/- mice, and in contrast to WT mice, did not decline following cuprizone withdrawal in SphK2-/- mice. We also observed a significant reduction in myelin thickness with ageing in SphK2-/- compared to WT mice. These results provide the first evidence that SphK2, the dominant enzyme catalysing S1P synthesis in the adult brain, is essential for remyelination following a demyelinating insult and myelin maintenance with ageing. We propose that persistently high levels of sphingosine and ceramide, a direct consequence of SphK2 deficiency, may block remyelination.

Table of Contents Image

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/431183v2_ufig1.gif" ALT="Figure 1">
View larger version (25K):
org.highwire.dtl.DTLVardef@e01edcorg.highwire.dtl.DTLVardef@9fdf6org.highwire.dtl.DTLVardef@6012org.highwire.dtl.DTLVardef@a3513b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Main PointsO_LIThe lipid kinase sphingosine kinase 2 (SphK2) is essential for remyelination following a demyelinating insult (cuprizone).
C_LIO_LISphK2 protects against cuprizone-mediated oligodendrocyte loss.
C_LIO_LISphK2 deficiency leads to thinner myelin with ageing.
C_LI


title: The dynamic transition of persistence towards the VBNC state during stationary phase is driven by protein aggregation

abstract: Decades of research into bacterial persistence has been unable to fully characterize this antibiotic-tolerant phenotype, thereby hampering the development of therapies effective against chronic infections. Although some active persister mechanisms have been identified, the prevailing view is that cells become persistent because they enter a dormant state. We therefore characterized starvation-induced dormancy in Escherichia coli. Our findings indicate that dormancy develops gradually; persistence strongly increases during stationary phase and decreases again as persisters enter the viable but nonculturable (VBNC) state. Importantly, we show that dormancy development is tightly associated with progressive protein aggregation, which occurs concomitantly with ATP depletion during starvation. Persisters contain protein aggregates in an early developmental stage while VBNC cells carry more mature aggregates. Finally, we show that at least one persister protein, ObgE, works by triggering aggregation and thereby changing the dynamics of persistence and dormancy development. These findings provide evidence for a genetically-controlled, gradual development of persisters and VBNC cells through protein aggregation.