title: Dual targeting of salt inducible kinases and CSF1R uncouples bone formation and bone resorption

abstract: Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.


title: Effects of L-Dopa, SKF-38393, and Quinpirole on exploratory, anxiety- and depressive-like behaviors in pubertal female and male mice

abstract: Adolescence is a phase of substantial changes in the brain, characterized by maturational remodeling of many systems. This remodeling allows functional plasticity to adapt in a changing environment but turns this period into a neurodevelopmental vulnerable window. The dopaminergic system is under morphological and physiological changes during this phase. The disruption of its balance can lead to molecular variation and abnormal behavior - representing a risk factor for neuropsychiatric disorders. In the present study, we investigated if changes in the dopaminergic tone alter mice behavior in a receptor and sex-specific manner, specifically in the beginning of puberty period. We administered L-Dopa, SKF-38393 (D1 dopamine receptor agonist) and Quinpirole (D2 dopamine receptor agonist) and tested male and female mice motor, anxiety- and depressive-like behavior. While females displayed an impaired exploratory drive, males presented an intense depressive-like response. Our results provide insights into the function of dopaminergic development in adolescent behavior and highlight the importance of studies in this time window with male and female subjects.

category: neuroscience


title: Marked differences in local bone remodeling in response to different marrow stimulation techniques in a large animal

abstract: Marrow stimulation, including subchondral drilling and microfracture, is the most commonly performed cartilage repair strategy, whereby the subchondral bone plate is perforated to release marrow-derived cells into a cartilage defect to initiate repair. Novel scaffolds and therapeutics are being designed to enhance and extend the positive short-term outcomes of this marrow stimulation. However, the translation of these newer treatments is hindered by bony abnormalities, including bone resorption, intralesional osteophytes, and bone cysts, that can arise after marrow stimulation. In this study, three different marrow stimulation approaches -- microfracture, subchondral drilling, and needle-puncture - were evaluated in a translationally relevant large animal model, the Yucatan minipig. The objective of this study was to determine which method of marrow access (malleted awl, drilled Kirschner wire, or spring-loaded needle) best preserved the underlying subchondral bone. Fluorochrome labels were injected at the time of surgery and 2 weeks post-surgery to capture bone remodeling over the first 4 weeks. Comprehensive outcome measures included cartilage indentation testing, histological grading, microcomputed tomography, and fluorochrome imaging. Our findings indicated that needle-puncture devices best preserved the underlying subchondral bone relative to other marrow access approaches. This may relate to the degree of bony compaction occurring with marrow access, as the Kirschner wire approach, which consolidated bone most, induced the most significant bone damage with marrow stimulation. This study provides basic science evidence in support of updated marrow stimulation techniques for preclinical and clinical practice.

category: bioengineering


title: Antiretroviral Theranostics Based on Intrinsic CEST Contrasts

abstract:
Human immunodeficiency virus type-1 (HIV-1) antiretroviral drug (ARV) theranostics facilitates biodistribution and efficacy of therapies designed to target viral reservoirs. To this end, we have now deployed intrinsic drug chemical exchange saturation transfer (CEST) contrast to detect ARV distribution within the central nervous system (CNS).

MethodsCEST effects for lamivudine (3TC) and emtricitabine (FTC) were measured by asymmetric magnetization transfer ratio analyses in solutions. CEST magnetic resonance imaging (MRI) was performed on 3TC-treated mice with analysis made by Lorentzian fitting.

ResultsCEST effects of 3TC and FTC hydroxyl and amino protons linearly correlated to drug concentrations. 3TC was successfully detected in brain sub-regions by MRI. The imaging results were validated by measurements of CNS drug concentrations.

ConclusionCEST contrasts can be used to detect ARVs using MRI. Such detection can be used to assess spatial-temporal drug biodistribution. This is most notable within the CNS where drug biodistribution may be more limited with the final goal of better understanding ARV-associated efficacy and potential toxicity.


title: Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

abstract:
Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 [A] resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of "gate" residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies.

IMPORTANCEPPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

category: biochemistry


title: Whole Genome and Exome Sequencing Reference Datasets from A Multi-center and Cross-platform Benchmark Study

abstract: With the rapid advancement of sequencing technologies in the past decade, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors or artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically benchmarking sequencing platforms, library protocols, bioinformatics pipelines and for measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples, a human triple-negative breast cancer cell line and a matched normal cell line derived from B lymphocytes. We generated whole-genome (WGS) and whole-exome sequencing (WES) data using 16 NGS library preparation protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the paired reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

category: bioinformatics


title: Flor yeasts rewire the central carbon metabolism during wine alcoholic fermentation

abstract: The identification of natural allelic variations controlling quantitative traits could contribute to decipher metabolic adaptation mechanisms within different populations of the same species. Such variations could result from man-mediated selection pressures and participate to the domestication. In this study, the genetic causes of the phenotypic variability of the central carbon metabolism Saccharomyces cerevisiae were investigated in the context of the enological fermentation. Carbon dioxide and glycerol production as well as malic acid consumption modulate the fermentation yield revealing a high level of genetic complexity. Their genetic determinism was found out by a multi environment QTL mapping approach allowing the identification of 14 quantitative trait loci from which 8 of them were validated down to the gene level by genetic engineering. Most of the validated genes had allelic variations involving flor yeast specific alleles. Those alleles were brought in the offspring by one parental strain that is closely related to the flor yeast genetic group while the second parental strain is part of the wine group. The causative genes identified are functionally linked to quantitative proteomic variations that would explain divergent metabolic features of wine and flor yeasts involving the tricarboxylic acid cycle (TCA), the glyoxylate shunt and the homeostasis of proton and redox cofactors. Overall, this work led to the identification of genetic factors that are hallmarks of adaptive divergence between flor yeast and wine yeast in the wine biotope. These alleles can also be used in the context of yeast selection to improve oenological traits linked to fermentation yield.

category: genetics


title: G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival

abstract: Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that a functional amalgamation of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and Sirt6 (H3K9 deacetylase) orchestrate cholesterol build-up in in-vitro and in-vivo models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while Sirt6 represses the genes involved in cholesterol efflux. The accumulated cholesterol promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and in vivo by pharmacological inhibition; or utilization of BMDMs derived from Sirt6 KO mice or in vivo infection in Sirt6 heterozygous mice; hampers host cholesterol accumulation and restricts Mtb burden. These findings shed light on the novel roles of G9a and Sirt6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

category: microbiology


title: Circumpolar peoples and their languages: lexical and genomic data suggest ancient Chukotko-Kamchatkan-Nivkh and Yukaghir-Samoyedic connections

abstract:
Relationships between universally recognized language families represent a hotly debated topic in historical linguistics, and the same is true for correlation between signals of genetic and linguistic relatedness. We developed a weighted permutation test and applied it on basic vocabularies for 31 pairs of languages and reconstructed proto-languages to show that three groups of circumpolar language families in the Northern Hemisphere show evidence of relationship though borrowing in the basic vocabulary or common descent: [Chukotko-Kamchatkan and Nivkh]; [Yukaghir and Samoyedic]; [Yeniseian, Na-Dene, and Burushaski]. The former two pairs showed the most significant signals of language relationship, and the same pairs demonstrated parallel signals of genetic relationship implying common descent or substantial gene flows. For finding the genetic signals we used genome-wide genetic data for present-day groups and a bootstrapping model comparison approach for admixture graphs or, alternatively, haplotype sharing statistics. Our findings further support some hypotheses on long-distance language relationship put forward based on the linguistic methods but lacking universal acceptance.

Significance statementIndigenous people inhabiting polar and sub-polar regions in the Northern Hemisphere speak diverse languages belonging to at least seven language families which are traditionally thought of as unrelated entities. We developed a weighted permutation test and applied it to basic vocabularies of a number of languages and reconstructed proto-languages to show that at least three groups of circumpolar language families show evidence of relationship though either borrowing in the basic vocabulary or common descent: Chukotko-Kamchatkan and Nivkh; Yukaghir and Samoyedic; Yeniseian, Na-Dene, and Burushaski. The former two pairs showed the most significant signals of language relationship, and the same pairs demonstrated parallel signals of genetic relationship implying common descent or substantial gene flows.

category: genomics


title: Whole-genome doubling-aware copy number phylogenies for cancer evolution with MEDICC2

abstract:
Chromosomal instability (CIN) and somatic copy-number alterations (SCNA) play a key role in the evolutionary process that shapes cancer genomes. SC-NAs comprise many classes of clinically relevant events, such as localised amplifications, gains, losses, loss-of-heterozygosity (LOH) events, and recently discovered parallel evolutionary events revealed by multi-sample phasing. These events frequently appear jointly with whole genome doubling (WGD), a transformative event in tumour evolution involving tetraploidization of genomes preceded or followed by individual chromosomal copy-number changes and associated with an overall increase in structural CIN.

While SCNAs have been leveraged for phylogeny reconstruction in the past, existing methods do not take WGD events into account and cannot model parallel evolution. They frequently make use of the infinite sites assumption, do not model horizontal dependencies between adjacent genomic loci and can not infer ancestral genomes. Here we present MEDICC2, a new phylogeny inference algorithm for allele-specific SCNA data that addresses these shortcomings. MEDICC2 dispenses with the infinite sites assumption, models parallel evolution and accurately identifies clonal and subclonal WGD events. It times SCNAs relative to each other, quantifies SCNA burden in single-sample studies and infers phylogenetic trees and ancestral genomes in multi-sample or single-cell sequencing scenarios with thousands of cells.

We demonstrate MEDICC2s ability on simulated data, real-world data of 2,778 single sample tumours from the Pan-cancer analysis of whole genomes (PCAWG), 10 bulk multi-region prostate cancer patients and two recent single-cell datasets of triple-negative breast cancer comprising several thousands of single cells.


title: MEDICC2: whole-genome doubling aware copy-number phylogenies for cancer evolution


title: Pathology dynamics in healthy-toxic protein interaction and the multiscale analysis of neurodegenerative diseases

abstract: Neurodegenerative diseases are frequently associated with aggregation and propagation of toxic proteins. In particular, it is well known that along with amyloid-beta, the tau protein is also driving Alzheimers disease. Multiscale reaction-diffusion models can assist in our better understanding of the evolution of the disease. We have modified the heterodimer model in such a way that it can now capture some of critical characteristics of this evolution such as the conversion time from healthy to toxic proteins. We have analyzed the modified model theoretically and validated the theoretical findings with numerical simulations.


title: Tetraploidy accelerates adaptation under drug-selection in a fungal pathogen

abstract: Baseline ploidy significantly impacts evolutionary trajectories, and in particular, tetraploidy has been associated with higher rates of adaptation compared to haploidy and diploidy. While the majority of experimental evolution studies investigating ploidy use Saccharomyces cerivisiae, the fungal pathogen Candida albicans is a powerful system to investigate ploidy dynamics, particularly in the context of antifungal drug resistance. C. albicans laboratory and clinical strains are predominantly diploid, but have also been isolated as haploid and polyploid. Here, we evolved diploid and tetraploid C. albicans for [~]60 days in the antifungal drug caspofungin. Tetraploid-evolved lines adapted faster than diploid-evolved lines and reached higher levels of caspofungin resistance. While diploid-evolved lines generally maintained their initial genome size, tetraploid-evolved lines rapidly underwent genome-size reductions and did so prior to caspofungin adaption. Furthermore, fitness costs in the absence of drug selection were significantly less in tetraploid-evolved lines compared to the diploid-evolved lines. Taken together, this work supports a model of adaptation in which the tetraploid state is transient but its ability to rapidly transition ploidy states improves adaptative outcomes and may drive drug resistance in fungal pathogens.

category: evolutionary biology


title: Episodic memory in aspects of brain information transfer by resting-state network topology

abstract: Studies suggest that resting-state functional connectivity conveys cognitive information; also, activity flow mediates cognitive information transfer. However, the exact mechanism of interregional interactions underlying episodic memory remains unclear. We performed a combined analysis of task-evoked activity and resting-state functional connectivity by activity flow mapping to estimate the information transfer mechanism of episodic memory. We found that the cognitive control and attentional networks were the most recruited structures in information transfers during both encoding and retrieval processes; these networks were correlated with task-evoked activation. Differences in information transfer intensity between encoding and retrieval mainly existed in the visual, somatomotor and hippocampal systems. Furthermore, information transfer showed high predictive power for episodic memory ability and mediated relationships between task-evoked activation and memory performance. Additional analysis indicated that structural connectivity had a transportive role in information transfer. Finally, our study presented the information transfer mechanism of episodic memory from multiple neural perspectives.


title: Host-derived Reactive Nitrogen Species mediate the Cryptococcus neoformans yeast-to-titan switch via fungal-derived superoxide

abstract:
In the host lung, the human fungal pathogen Cryptococcus neoformans undergoes a morphological switch from small haploid yeast to large polyploid titan cell, contributing to C. neoformans virulence. Titan cells are less readily phagocytosed and can survive host nitrosative and oxidative stresses. We and others previously showed that titanization is triggered by host-relevant signals including CO2 and lung-resident bacteria, and addition of these factor is sufficient to induce titan cells in vitro. Here we investigate the molecular mechanisms that drive this transition and demonstrate that host-derived immune signals can increase the degree and frequency of titanization. Specifically, host-relevant reactive nitrogen species increase the accumulation of endogenous superoxide within cryptococcal cells, particularly within nuclei, where it can cause genotoxic stress. Consistent with this, we observe the accumulation of Rad51 protein, a marker of the double strand break repair pathway, in titanizing cultures. Blocking superoxide accumulation inhibits titanization, yet titanization also requires superoxide detoxification through Superoxide Dismutase (SOD) activity. Loss of mitochondrial Sod2 activity locks cells in the yeast phase, while Sod1 is required for the production of viable titan daughter cells. We hypothesize that the redox responsive transcription factor Yap1 in part mediates this response by regulating SOD2/SOD1. In addition, we show that Sod1 translocates to the nucleus, where it is likely involved in the detoxification of genotoxic superoxide. Together, these findings reveal a major new regulatory mechanism for the yeast-to-titan transition.

Author SummaryDuring fungal infection, host phagocytes produce reactive oxygen and nitrogen species (ROS/RNS), major determinants of infection outcome. Fungal pathogens have developed numerous strategies to neutralize and detoxify ROS, but RNS remain important effectors for infection control. In the lung, the human fungal pathogen Cryptococcus neoformans can undergo a morphological switch from small haploid yeast to large highly polyploid titan cells with increased ROS/RNS stress resistance, and the capacity to produce haploid or aneuploid daughters. Here, we report that RNS are a major signal driving the frequency and degree of titanization and act by increasing endogenous ROS within the fungus. We show that the accumulation of endogenous ROS is required for the yeast-to-titan transition, and is associated with increased genotoxic stress leading to polyploidy. Yet, failure to detoxify this ROS, either in mutants defective in Superoxide Dismutase activity or the oxidative stress response protein Yap1, impairs titan cell budding and reduces progeny viability. Therefore, the interface of exogenous RNS and endogenous ROS regulation during host-pathogen interaction represents an Achilles heel for this major human fungal pathogen.


title: Reverse complementary matches simultaneously promote both back-splicing and exon-skipping

abstract: Circular RNAs (circRNAs) play diverse roles in different biological and physiological environments and are always expressed in a tissue-specific manner. Tissue-specific circRNA expression profile can help understand how circRNAs are regulated. Here, using large-scale neuron isolation from the first larval stage of Caenorhabditis elegans (C. elegans) followed by whole-transcriptome RNA sequencing, I provide the first neuronal circRNA data in C. elegans. I show that circRNAs are highly expressed in the neurons of C. elegans and are preferably derived from neuronal genes. More importantly, reverse complementary matches in circRNA-flanking introns are not only required for back-splicing but also promote the skipping of exon(s) to be circularized. Interestingly, one pair of RCM in zip-2 is highly conserved across five nematode ortholog genes, which show conserved exon-skipping patterns. Finally, through one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, I show that exon-skipping is not absolutely required for back-splicing, neither the other way. Instead, the coupled exon-skipping and back-splicing are happening at the same time.

category: molecular biology


title: Molecular Basis for Reduced Cleavage Activity and Drug Resistance in D30N HIV-1 protease

abstract: Nelfinavir is one of the FDA approved HIV-1 protease inhibitors and is a part of HAART therapy for the treatment of HIV-AIDS. Nelfinavir was the first HIV-1 protease inhibitor to be approved as a Paediatric formulation. The application of HAART had resulted into significant improvement in the life of AIDS patients. However, emergence of drug resistance in HIV-1 protease limited the use of many of these drugs including nelfinavir. A unique mutation observed frequently in patients treated with nelfinavir is D30N as it is selected exclusively by nelfinavir. It imparts very high resistance to nelfinavir but unlike other primary mutations does not give cross resistance to the majority of other drugs. D30N mutation also significantly reduces cleavage activity of HIV-1 protease and affects the viral fitness. Here, we have determined structures of D30N HIV-1 protease in unliganded form and in complex with the drug nelfinavir. These structures provide rationale for reduced cleavage activity and molecular basis of resistance induced by D30N mutation. The loss of coulombic interaction part of a crucial hydrogen bond between the drug and the enzyme, is a likely explanation for reduced affinity and drug resistance towards nelfinavir. The decreased catalytic activity of D30N HIV protease, due to altered interaction with substrates and reduced stability of folding core may be the reasons for reduced replicative capacity of the HIV harboring D30N HIV-1 protease.


title: Assessing heterogeneity in spatial data using the HTA index with applications to spatial transcriptomics and imaging

abstract:
MotivationTumour heterogeneity is being increasingly recognised as an important characteristic of cancer and as a determinant of prognosis and treatment outcome. Emerging spatial transcriptomics data hold the potential to further our understanding of tumour heterogeneity and its implications. However, existing statistical tools are not sufficiently powerful to capture heterogeneity in the complex setting of spatial molecular biology.

ResultsWe provide a statistical solution, the HeTerogeneity Average index (HTA), specifically designed to handle the multivariate nature of spatial transcriptomics. We prove that HTA has an approximately normal distribution, therefore lending itself to efficient statistical assessment and inference. We first demonstrate that HTA accurately reflects the level of heterogeneity in simulated data. We then use HTA to analyse heterogeneity in two cancer spatial transcriptomics datasets: spatial RNA sequencing by 10x Genomics and spatial transcriptomics inferred from H&E. Finally, we demonstrate that HTA also applies to 3D spatial data using brain MRI. In spatial RNA sequencing we use a known combination of molecular traits to assert that HTA aligns with the expected outcome for this combination. We also show that HTA captures immune-cell infiltration at multiple resolutions. In digital pathology we show how HTA can be used in survival analysis and demonstrate that high levels of heterogeneity may be linked to poor survival. In brain MRI we show that HTA differentiates between normal ageing, Alzheimers disease and two tumours. HTA also extends beyond molecular biology and medical imaging, and can be applied to many domains, including GIS.

Availability<attached; publicly available upon acceptance>.

Contactlevyalona@gmail.com zohar.yakhini@gmail.com

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Avalanches During Epithelial Tissue Growth; Uniform Growth and a Drosophila Eye Disc Model

abstract: In the physicists perspective, epithelial tissues constitute an exotic type of active matter with non-linear properties reminiscent of amorphous materials. In the context of a circular proliferating epithelium, modeled by the quasistatic vertex model, we identify novel discrete tissue scale rearrangements, i.e. cellular flow avalanches, which are a form of collective cell movement. During the avalanches, the cellular trajectories are radial in the periphery and form a vortex in the core. After the onset of these avalanches, the epithelial area grows discontinuously. The avalanches are found to be stochastic, and their strength is determined by the density of cells in the tissue. Overall, avalanches regularize the spatial tension distribution along tissue. Furthermore, the avalanche distribution is found to obey a power law, with an exponent consistent with sheer induced avalanches in amorphous materials. To decipher the role of avalanches in organ development, we simulate epithelial growth of the Drosophila eye disc during the third instar using a computational model, which includes both signaling and mechanistic signalling. During the third instar, the morphogenetic furrow (MF), a [~]10 cell wide wave of apical area constriction propagates through the epithelium, making it a system with interesting mechanical properties. These simulations are used to understand the details of the growth process, the effect of the MF on the growth dynamics on the tissue scale, and to make predictions. The avalanches are found to depend on the strength of the apical constriction of cells in the MF, with stronger apical constriction leading to less frequent and more pronounced avalanches. The results herein highlight the dependence of simulated tissue growth dynamics on relaxation timescales, and serve as a guide for in vitro experiments.

category: biophysics


title: Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice

abstract: Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.


title: Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

abstract:
Differences in gene expression, which can arise from divergence in cis-regulatory elements or alterations in transcription factors binding specificity, are one of the most important causes of phenotypic diversity during evolution. By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.

Author summaryOur study 1) represented a first systematic analysis of species-specific adaptation in DNA binding pattern of transcription factor. Although the mouse-specific amino acid changes did not manifest functional impact in our system, several explanations may account for it (See Discussion part for the detail); 2) represented a first study of cis-regulation between two reproductively isolated species by using a novel allodiploid system; 3) demonstrated a higher conservation of transcriptional output than that of DNA binding, suggesting the evolvability/plasticity of the latter; 4) finally provided a rich data resource for Cdx2 mediated regulation, including gene expression, chromatin accessibility and DNA binding etc.


title: Mapping Twenty Years of Antimicrobial Resistance Research Trends

abstract:
BackgroundAntimicrobial resistance (AMR) is a global threat to health and healthcare. In response to the growing AMR burden, research funding also increased. However, a comprehensive overview of the research output, including conceptual, temporal, and geographical trends, is missing. Therefore, this study uses topic modelling, a machine learning approach, to reveal the scientific evolution of AMR research and its trends, and provides an interactive user interface for further analyses.

MethodsStructural topic modelling (STM) was applied on a text corpus resulting from a PubMed query comprising AMR articles (1999-2018). A topic network was established and topic trends were analysed by frequency, proportion, and importance over time and space.

FindingsIn total, 88 topics were identified in 158616 articles from 166 countries. AMR publications increased by 450% between 1999 and 2018, emphasizing the vibrancy of the field. Prominent topics in 2018 were Strategies for emerging resistances and diseases, Nanoparticles, and Stewardship. Emerging topics included Water and environment, and Sequencing. Geographical trends showed prominence of Multidrug-resistant tuberculosis (MDR-TB) in the WHO African Region, corresponding with the MDR-TB burden. China and India were growing contributors in recent years, following the United States of America as overall lead contributor.

InterpretationThis study provides a comprehensive overview of the AMR research output thereby revealing the AMR research response to the increased AMR burden. Both the results and the publicly available interactive database serve as a base to inform and optimise future research.

FundingINTERREG-VA EurHealth-1Health (202085); European Commission Horizon 2020 Framework

Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrior to this study, PubMed, Web of Science, Scopus, and IEEE Xplore were queried to find studies providing a conceptual overview of antimicrobial resistance (AMR) research over time and space. The search string included keywords ("antimicrobial" OR antibiotic*) AND (resistan*) AND ("science mapping" OR bibliometric OR scientometric) in the title and abstract and focused on articles published before 2019 without language restrictions. Few studies were found relying on scientometric and bibliometric methods to assess either subfields of AMR research (e.g., AMR among uropathogens) or AMR-related fields (e.g., microbiology). No studies were found that focus on the entire AMR field. Therefore, this science mapping study using topic modelling was performed to provide an overview of the AMR field by identifying and assessing topics, trends, and geographical differences over time.

Added value of this studyTo the best of our knowledge, this study is the first to use a science mapping approach to provide a comprehensive overview of the entire AMR research field, covering over 150 thousand articles published between 1999 and 2018. Our findings revealed important (e.g., Strategies for emerging resistances and diseases, Nanoparticles, and Stewardship) and emerging (e.g., Water and environment, and Sequencing) topics in AMR research. Lastly, this study resulted in an interactive user interface where all data are presented for further analyses.

Implications of all the available evidenceOur comprehensive overview of the AMR field, including its conceptual structure, and temporal and geographical trends revealed the response of the research community to the AMR burden. The results and the openly available supporting data provide the base to guide future funding and research directions to tackle AMR.


title: Social behaviour and vocalizations of the tent-roosting Honduran White Bat

abstract: Bats are highly gregarious animals, displaying a large spectrum of social systems with different organizational structures. One important factor shaping sociality is group stability. To maintain group cohesion and stability, bats often rely on social vocal communication. The Honduran white bat, Ectophylla alba exhibits an unusual social structure compared to other tent-roosting species. This small white-furred bat lives in perennial stable mixed-sex groups. Tent construction requires several individuals and, as the only tent roosting species so far, involves both sexes. The bats social system and ecology render this species an interesting candidate to study social behaviour and social vocal communication. In our study, we investigated the social behaviour and vocalizations of E. alba in the tent by observing two stable groups, including pups, in the wild. We documented 16 different behaviours, among others, play and fur chewing, a behaviour presumably used for scent-marking. Moreover, we found 10 distinct social call types in addition to echolocation calls, and, for seven call types, we were able to identify the corresponding behavioural context. Most of the social call types were affiliative, including two types of contact calls, maternal directives, pup isolation calls and a call type related to the fur-chewing behaviour. In sum, this study entails an ethogram and describes the first vocal repertoire of a tent-roosting phyllostomid bat, providing the basis for further in-depth studies about the sociality and vocal communication in E. alba.

category: animal behavior and cognition


title: Transcription factor action orchestrates the complex expression pattern of CRABS CLAW, a gynoecium developmental regulator in Arabidopsis

abstract:
The flower of angiosperms is the most complex organ that plants generate and many transcription factors (TFs) are involved to regulate its morphogenesis in a coordinated way. In its center, the gynoecium develops consisting of specialized tissues such as secondary meristems, sites of postgenital fusion, ovules, pollen transmitting tract, all to assure successful sexual reproduction. Gynoecium development requires tight regulation of developmental regulators across time and tissues. However, while we know of several examples how simple on/off regulation of gene expression is achieved in plants, it remains unclear which regulatory processes generate complex expression patterns. Here, we use the gynoecium developmental regulator CRABS CLAW (CRC) from Arabidopsis to study regulatory mechanisms contributing to its sophisticated expression pattern. Using a combination of in silico promoter analyses, global TF-DNA interaction screens, co-expression and mutant analysis we find that miRNA action, DNA methylation, and chromatin remodeling do not contribute substantially to CRC regulation. We show that a plethora of TFs bind to the CRC promoter to fine-tune transcript abundance by activation of transcription, linking CRC to specific developmental processes but not biotic or abiotic stress. Interestingly, the temporal-spatial aspects of regulation of expression may be under the control of redundantly acting genes and may require higher order complex formation at TF binding sites. We conclude that endogenous regulation of complex expression pattern of Arabidopsis genes requires orchestrated transcription factor action on several conserved promotor sites over almost 4 kb in length.

Significance statementDifferent to genes that are simply switched on or off, depending on an environmental cue we find that genes directing development in plants often show complex expression pattern dependent on internal factors only. Here, we addressed the question how an complex expression pattern is achieved and use the CRABS CLAW (CRC) gene required for gynoecium development as an example. Combining wet lab experiments and in silico analysis we find that epigenetic regulation plays only a minor role and that a large number of transcription factors activates the transcription of CRC. Single regulators may have a profound effect on CRC transcript abundance but less so on the pattern of expression. Complex patterns most likely require the interplay of several transcription factors.


title: A breast cancer patient-derived xenograft and organoid platform for drug discovery and precision oncology

abstract:
Model systems that recapitulate the complexity of human tumors and the reality of variable treatment responses are urgently needed to better understand cancer biology and to develop more effective cancer therapies. Here we report development and characterization of a large bank of patient-derived xenografts (PDX) and matched organoid cultures from tumors that represent some of the greatest unmet needs in breast cancer research and treatment. These include endocrine-resistant, treatment-refractory, and metastatic breast cancers and, in some cases, multiple tumor collections from the same patients. The models can be grown long-term with high fidelity to the original tumors. We show that development of matched PDX and PDX-derived organoid (PDxO) models facilitates high-throughput drug screening that is feasible and cost-effective, while also allowing in vivo validation of results. Our data reveal consistency between drug screening results in organoids and drug responses in breast cancer PDX. Moreover, we demonstrate the feasibility of using these patient-derived models for precision oncology in real time with patient care, using a case of a triple negative breast cancer with early metastatic recurrence as an example. Our results uncovered an FDA-approved drug with high efficacy against the models. Treatment with the PDxO-directed therapy resulted in a complete response for the patient and a progression-free survival period more than three times longer than her previous therapies. This work provides valuable new methods and resources for functional precision medicine and drug development for human breast cancer.

Graphical Abstract

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category: cancer biology


title: Metacognitive Computations for Information Search: Confidence in Control

abstract: The metacognitive sense of confidence can play a critical role in regulating decisionmaking. In particular, a lack of confidence can justify the explicit, potentially costly, instrumental acquisition of extra information that might resolve the underlying uncertainty. Recent work has suggested a statistically sophisticated tapestry behind the information governing both the making and monitoring of choices. Here, we extend this tapestry to reveal extra richness in the use of confidence for controlling information seeking. We thereby highlight how different models of metacognition can generate diverse relationships between action, confidence, and information search. More broadly, our work shows how crucial it can be to treat metacognitive monitoring and control together.


title: Modeling gene x environment interactions in PTSD using glucocorticoid-induced transcriptomics in human neurons

abstract: Post-traumatic stress disorder (PTSD) results from severe trauma exposure, but the extent to which genetic and epigenetic risk factors impact individual clinical outcomes is unknown. We assessed the impact of genomic differences following glucocorticoid administration by examining the transcriptional profile of human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons and live cultured peripheral blood mononuclear cells from combat veterans with PTSD (n=5) and without PTSD (n=5). This parallel examination in baseline and glucocorticoid-treated conditions resolves cell-type specific and diagnosis-dependent elements of stress response, and permits discrimination of gene expression signals associated with PTSD risk from those induced by stress. Computational analyses revealed neuron-specific glucocorticoid-response expression patterns that were enriched for transcriptomic patterns observed in clinical PTSD samples. PTSD-specific signatures, albeit underpowered, accurately stratify veterans with PTSD relative to combat-exposed controls. Overall, in vitro PTSD and glucocorticoid response signatures in blood and brain cells represent exciting new platforms with which to test the genetic and epigenetic mechanisms underlying PTSD, identify biomarkers of PTSD risk and onset, and conduct drug-screening to identify novel therapeutics to prevent or ameliorate clinical phenotypes.

category: pathology


title: Employing connectome-based models to predict working memory in multiple sclerosis

abstract:
BackgroundIndividuals with multiple sclerosis (MS) are vulnerable to deficits in working memory, and the search for neural correlates of working memory in circumscribed areas has yielded inconclusive findings. Given the widespread neural alterations observed in MS, predictive modeling approaches that capitalize on whole-brain connectivity may better capture individual-level working memory in this population.

MethodsHere, we applied connectome-based predictive modeling to functional MRI data from working memory tasks in two independent samples with relapsing-remitting MS. In the internal validation sample (ninternal = 36), functional connectivity data were used to train a model through cross-validation to predict accuracy on the Paced Visual Serial Addition Test, a gold-standard measure of working memory in MS. We then tested its ability to predict performance on the N-back working memory task in the external validation sample (nexternal = 36).

ResultsThe resulting model successfully predicted working memory in the internal validation sample but did not extend to the external sample. We also tested the generalizability of an existing model of working memory derived in healthy young adults to people with MS. It showed successful prediction in both MS samples, demonstrating its translational potential. We qualitatively explored differences between the healthy and MS models in intra- and inter-network connectivity amongst canonical networks.

DiscussionThese findings suggest that connectome-based predictive models derived in people with MS may have limited generalizability. Instead, models identified in healthy individuals may offer superior generalizability to clinical samples, such as MS, and may serve as more useful targets for intervention.

Impact StatementWorking memory deficits in people with multiple sclerosis have important consequence for employment, leisure, and daily living activities. Identifying a functional connectivity-based marker that accurately captures individual differences in working memory may offer a useful target for cognitive rehabilitation. Manglani et al. demonstrate machine learning can be applied to whole-brain functional connectivity data to identify networks that predict individual-level working memory in people with multiple sclerosis. However, existing network-based models of working memory derived in healthy adults outperform those identified in multiple sclerosis, suggesting translational potential of brain networks derived in large, healthy samples for predicting cognition in multiple sclerosis.


title: Distinct sub-cellular autophagy impairments occur independently of protein aggregation in induced neurons from patients with Huntington's disease

abstract: Huntingtons disease (HD) is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling HD has remained challenging, as rodent and cellular models poorly recapitulate the disease. To address this, we generated induced neurons (iNs) through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. HD-iNs displayed profound deficits in autophagy, characterised by reduced transport of late autophagic structures from the neurites to the soma. The neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites presented by HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in ctrl-iNs, highlighting the importance of wild type HTT in neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in aged patient derived HD-neurons and provides a new rational for future development of autophagy activation therapies.


title: The perception of odor pleasantness is shared across cultures

abstract: Human sensory experience varies across the globe. Nonetheless, all humans share sensory systems with a common anatomical blueprint. In olfaction, it is unknown to what degree sensory perception, in particular the perception of odor pleasantness, is dictated by universal biological principles versus sculpted by culture. To address this issue, we asked 235 individuals from 9 diverse non-western cultures to rank the hedonic value of monomolecular odorants. We observed substantial global consistency, with molecular identity explaining 41% of the variance in individual pleasantness rankings, while culture explained only 6%. These rankings were predicted by the physicochemical properties of out-of-sample molecules and out-of-sample pleasantness ratings given by a separate group of industrialized western urbanites, indicating human olfactory perception is strongly constrained by universal principles.


title: Top-down control of hippocampal signal-to-noise by prefrontal long-range inhibition

abstract: The prefrontal cortex (PFC) is postulated to exert  top-down control by modulating information processing throughout the brain to promote specific actions based on current goals. However, the pathways mediating top-down control remain poorly understood. In particular, knowledge about direct prefrontal connections that might facilitate top-down prefrontal control of information processing in the hippocampus remains sparse. Here we describe novel monosynaptic long-range GABAergic projections from PFC to hippocampus. These preferentially inhibit vasoactive intestinal polypeptide expressing interneurons, which are known to disinhibit hippocampal microcircuits. Indeed, stimulating prefrontal-hippocampal GABAergic projections increases hippocampal feedforward inhibition and reduces hippocampal activity in vivo. The net effect of these actions is to specifically enhance the signal-to-noise ratio for hippocampal representations of objects. Correspondingly, stimulation of PFC-to-hippocampus GABAergic projections promotes object exploration. Together, these results elucidate a novel top-down pathway in which long-range GABAergic projections target disinhibitory microcircuits, thereby enhancing signals and network dynamics underlying exploratory behavior.


title: Gallic Acid, a Methyl 3,4-Dihydroxybenzoate Derivative, Induces Neural Stem Cells to Differentiate and Proliferate

abstract: Differentiation and proliferation of neural stem cells (NSCs) are both important biological processes in cerebral neural network. However, these two capacities of NSCs are limited. Thus, the induction of differentiation and/or proliferation via administration of small molecules derived from natural plants can be considered as a potential approach to repair damaged neural networks. This paper reports that gallic acid, a catechol compound selected from derivatives of methyl 3,4-dihydroxybenzoate (MDHB), selectively induces NSCs to differentiate into immature neurons and promotes proliferation by activating phosphorylation of key proteins in the MAPK-ERK pathway. In addition, we found that 3,4-dihydroxybenzoic acid was the main active structure which could show neurotrophic activity. The substitution of carboxyl group on the benzene ring into ester group may promote differentiation on the basis of the structure of 3,4-dihydroxybenzoic acid. Meanwhile, the introduction of 5-hydroxyl group may promote proliferation. Generally, this study identified a natural catechol compound that promotes differentiation and proliferation of NSCs in vitro.


title: Synthetic algal-bacteria consortia for space-efficient microalgal growth in a simple hydrogel system

abstract: Photosynthetic microalgae are an attractive source of food, fuel or nutraceuticals, but commercial production of microalgae is limited by low spatial efficiency. In the present study, we developed a simple photosynthetic hydrogel system that cultivates the green microalga, Marinichlorella kaistiae KAS603, together with a novel strain of the bacteria Erythrobacter sp.. We tested the performance of the co-culture in the hydrogel using a combination of chlorophyll-a fluorimetry, microsensing and bio-optical measurements. Our results showed that growth rates in algal-bacterial hydrogels were about 3-fold enhanced compared to hydrogels with algae alone. Chlorophyll-a fluorimetry based light curves found that electron transport rates were enhanced about 20% for algal-bacterial hydrogels compared to algal hydrogels for intermediate irradiance levels. We also show that the living hydrogel is stable under different environmental conditions and when exposed to natural seawater. Our study provides a potential bio-inspired solution for problems that limit the space-efficient cultivation of microalgae for biotechnological applications.


title: The Hob proteins are novel and conserved lipid binding proteins at ER-PM contact site

abstract: Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit/Hob proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in higher animals, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

category: cell biology


title: Pericentromeric heterochromatin impacts genome compartmentalization and sex chromosome evolution in a fish

abstract: Compartmentalization is one of the principles of chromosome 3D organization and has been suggested to be driven by the attraction of heterochromatin. The extent to which the pericentromeric heterochromatin (PCH) impacts chromosome compartmentalization is yet unclear. Here we produced a chromosome-level and fully phased diploid genome of an aquaculture fish, zig-zag eel (Mastacembelus armatus), and identified the centromeric and pericentromeric regions in the majority of chromosomes of both haploid genomes. The PCH is on average 4.2 Mb long, covering 17.7% of the chromosomes, and is the major target of histone 3 lysine 9 trimethylation (H3K9me3). In nearly half of the chromosomes, the PCH drives the chromosomes into two or three megascale chromatin domains with the PCH being a single one. We further demonstrate that PCH has a major impact in submetacentric, metacentric and small telocentric chromosomes in which the PCH drives the distribution of active and inactive compartments along the chromosomes. Additionally, we identified the young and homomorphic XY sex chromosomes that are submetacentric with the entire short-arm heterochromatinized. Interestingly, the sex-determining region seems to arise within the PCH that has been in place prior to the X-Y divergence and recombination suppression. Together, we demonstrate that the PCH can cover a considerably large portion of the chromosomes, and when it does so, it drives chromosome compartmentalization; and we propose a new model for the origin and evolution of homomorphic sex chromosomes in fish.


title: A pan-ontology view of machine-derived knowledge representations and feedback mechanisms for curation

abstract: Biomedical ontologies are established tools that organize knowledge in specialized research areas. They can also be used to train machine-learning models. However, it is unclear to what extent representations of ontology concepts learned by machine-learning models capture the relationships intended by ontology curators. It is also unclear whether the representations can provide insights to improve the curation process. Here, we investigate ontologies from across the spectrum of biological research and assess the concordance of formal ontology hierarchies with representations based on plain-text definitions. By comparing the internal properties of each ontology, we describe general patterns across the pan-ontology landscape and pinpoint areas with discrepancies in individual domains. We suggest specific mechanisms through which machine-learning approaches can lead to clarifications of ontology definitions. Synchronizing patterns in machine-derived representations with those intended by the ontology curators will likely streamline the use of ontologies in downstream applications.


title: Distinct forms of synaptic plasticity during ascending vs. descending control of medial olivocochlear efferent neurons

abstract: Activity in each brain region is shaped by the convergence of ascending and descending axonal pathways, and the balance and characteristics of these determine neural output. The medial olivocochlear (MOC) efferent system is part of a reflex arc that critically controls auditory sensitivity. Multiple central pathways contact MOC neurons, raising the question of how a reflex arc could be engaged by diverse inputs. We examined functional properties of synapses onto brainstem MOC neurons from ascending (ventral cochlear nucleus, VCN), and descending (inferior colliculus, IC) sources in mice using an optogenetic approach. We found that these pathways exhibited opposing forms of short-term plasticity, with VCN input showing depression and IC input showing marked facilitation. By using a conductance clamp approach, we found that combinations of facilitating and depressing inputs enabled firing of MOC neurons over a surprisingly wide dynamic range, suggesting an essential role for descending signaling to a brainstem nucleus.


title: Antigen and Checkpoint Receptor Recalibration of T Cell Receptor Signal Strength

abstract:
How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice as a digital read-out of NFAT pathway activity, we identify the rapid quantitative and qualitative changes that occur in CD4+ T cells in response to a range of TCR signalling strengths. We demonstrate that the time and dose dependent programming of distinct co-inhibitory receptors rapidly re-calibrates T cell activation thresholds. By developing a new in vivo model, we analyse the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 but not anti-Lag3 immunotherapy leads to an increased TCR signal strength. We define a strong TCR signal metric of five genes specifically upregulated by anti-PD1 in T cells (TCR.strong), which can stratify clinical outcomes during anti-PD1 monotherapy in melanoma patients. Our study therefore reveals how analysis of TCR signal strength - and its manipulation - can provide powerful metrics for monitoring outcomes to immunotherapy.

Key PointsO_LITCR signal strength-dependent programming of CD4+ T cells revealed over time in vivo
C_LIO_LIInhibitory receptor expression is dynamic, TCR signal strength dependent, and rapidly re-calibrates T cell activation thresholds
C_LIO_LIPD1 but not Lag3 blockade leads to a unique and increased TCR signal strength signature (coined TCR.strong)
C_LIO_LITCR.strong metric stratifies melanoma patient survival in response to Nivolumab (anti-PD1) therapy
C_LI

category: immunology


title: Drug-driven reclassification of multiple tumour subtypes reveals intrinsic molecular concordance of therapy across histologically disparate cancers

abstract:
Cancers that are histologically defined as the same type of cancer often need a distinct therapy based on underlying heterogeneity; likewise, histologically disparate cancers can require similar treatment approaches due to intrinsic similarities. A comprehensive analysis integrated with drug response data and genomic alterations, particularly to reveal therapeutic concordance mechanisms across histologically disparate tumour subtypes, has not yet been fully exploited.

In this study, we used pharmacogenomic profiling data provided from the Cancer Genome Project (CGP) in a systematic in silico investigation of the pharmacological subtypes of cancers and the intrinsic concordance of molecular mechanisms leading to similar therapeutic responses across histologically disparate tumour subtypes. We further developed a novel approach to redefine cell-to-cell similarity and drug-to-drug similarity from the therapeutic concordance, providing a new point of view to study cancer heterogeneity.

Our study identified that histologically different tumours, such as malignant melanoma and colorectal adenocarcinoma, could belong to the same pharmacological subtype regarding drug sensitivity to MEK inhibitors, which was determined by their genomic alterations, high occurrence of BRAF or KRAS mutations. Therapeutic concordance for chemotherapy drugs was identified across histologically disparate hematological tumors mainly due to the extraordinary activation of the cell cycle in blood cancers. A subcluster of SCLC had a more similar profile with hematological tumors, and was associated with the malignant phenotype, with a higher level of MYC expression. We developed a website to store and visualize the pharmacological subtypes of drugs, as well as their connected genomic and expression alterations.


title: Panakeia - A universal tool for bacterial pangenome analysis

abstract:
AO_SCPLOWBSTRACTC_SCPLOWWe present Panakeia, a tool which strives to be easy to use and providing a detailed view of the pangenome structure which can efficiently be utilised for discovery, or further in-depth analysis, of features of interest. It analyses synteny and multiple structural patterns of the pangenome, giving insights into the biological diversity and evolution of the studied taxon. Panakeia hence provides both broad and detailed information on the structure of a pangenome, for diverse and highly clonal populations of bacteria. Development of new pan-genome analysis tools is important, as the pangenome of a microbial species has become an important method to define the diversity of a selected taxon, most commonly a species, in the last years. This enables comparison of strains from different ecological niches and can be used to define the functional potential in a bacterial population. It gives us a much better view of microbial genomics than can be gained from singular genomes which after all are just single representatives of a much more varied population.

Previously published pangenome tools often reduce the information to a presence/absence matrix of unconnected genes or generate massive hard to interpret output graphs. However, Panakeia includes synteny and structural information and presents it in a way that can readily be used for further analysis. Panakeia can be downloaded at https://github.com/BioSina/Panakeia.


title: A New Straightforward Method for Automated Segmentation of Trabecular Bone from Cortical Bone in Diverse and Challenging Morphologies

abstract: Many physiological, biomechanical, evolutionary and clinical studies that explore skeletal structure and function require successful separation of trabecular from cortical compartments of a bone that has been imaged by X-ray micro-computed tomography (microCT) prior to analysis. Separation is often time-consuming, involves user bias and needs manual sub-division of these two similarly radio-opaque compartments. We have developed an objective, automated protocol which reduces user bias and enables straightforward, user-friendly segmentation of trabecular from cortical bone without requiring sophisticated programming expertise. This method can conveniently be used as a "recipe" in commercial programmes (Avizo herein) and applied to a variety of datasets. Here, we characterise and share this recipe, and demonstrate its application to a range of murine and human bone types, including normal and osteoarthritic specimens, and bones with distinct embryonic origins and spanning a range of ages. We validate the method by testing inter-user bias during the scan preparation steps and confirm utility in the architecturally challenging analysis of growing murine epiphyses. We also report details of the recipe, so that other groups can readily re-create a similar method in open access programs. Our aim is that this method will be adopted widely to create a more standardized and time efficient method of segmenting trabecular and cortical bone.

category: physiology


title: The impact of real-time fMRI denoising on online evaluation of brain activity and functional connectivity

abstract:
ObjectiveComprehensive denoising is imperative in fMRI analysis to reliably evaluate neural activity from the blood oxygenation level dependent signal. In real-time fMRI, however, only a minimal denoising process has been applied and the impact of insufficient denoising on online brain activity estimation has not been assessed comprehensively. This study evaluated the noise reduction performance of online fMRI processes in a real-time estimation of regional brain activity and functional connectivity.

ApproachWe performed a series of real-time processing simulations of online fMRI processing, including slice-timing correction, motion correction, spatial smoothing, signal scaling, and noise regression with high-pass filtering, motion parameters, motion derivatives, global signal, white matter/ventricle average signals, and physiological noise models with image-based retrospective correction of physiological motion effects (RETROICOR) and respiration volume per time (RVT).

Main resultsAll the processing was completed in less than 400 ms for whole-brain voxels. Most processing had a benefit for noise reduction except for RVT that did not work due to the limitation of the online peak detection. The global signal regression, white matter/ventricle signal regression, and RETORICOR had a distinctive noise reduction effect, depending on the target signal, and could not substitute for each other. Global signal regression could eliminate the noise-associated bias in the mean dynamic functional connectivity across time.

SignificanceThe results indicate that extensive real-time denoising is possible and highly recommended for real-time fMRI applications.


title: Increased Resistance of SARS-CoV-2 Variant P.1 to Antibody Neutralization

abstract: The relative resistance of SARS-CoV-2 variants B.1.1.7 and B.1.351 to antibody neutralization has been described recently. We now report that another emergent variant from Brazil, P.1, is not only refractory to multiple neutralizing monoclonal antibodies, but also more resistant to neutralization by convalescent plasma (3.4 fold) and vaccinee sera (3.8-4.8 fold). The cryo-electron microscopy structure of a soluble prefusion-stabilized spike reveals the P.1 trimer to adopt exclusively a conformation in which one of the receptor-binding domains is in the "up" position, with the functional impact of mutations appearing to arise from local changes instead of global conformational alterations. The P.1 variant threatens current antibody therapies but less so the protective efficacy of our vaccines.


title: Desmosomal dualism: the core is stable while plakophilin is dynamic.

abstract: Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise 5 major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with FRAP and FLAP we show that desmosomes consist of two contrasting protein fractions or modules: a very stable desmosomal core of desmosomal cadherins and plakoglobin, and a highly mobile plakophilin. As desmosomes mature from calcium-dependence to calciumindependent hyper-adhesion, core stability increases, but Pkp2a remains highly mobile. Desmoplakin is initially mobile but stabilises with hyper-adhesion. We show that desmosome down-regulation during growth factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable core and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in the desmosome.


title: Widespread translational control regulates retinal development in mouse

abstract: Retinal development is tightly regulated to ensure the generation of appropriate cell types and the assembly of functional neuronal circuitry. Despite remarkable advances that have been made in understanding the regulation of gene expression during retinal development, how translational regulation guides retinogenesis is less understood. Here, we conduct a comprehensive translatome and transcriptome survey to the mouse retinogenesis from the embryonic to the adult stages. We discover thousands of genes that have dynamic changes at the translational level and pervasive translational regulation in a developmental stage-specific manner with specific biological functions. We further identify genes whose translational efficiencies are frequently controlled by changing usage in the upstream open reading frames during retinal development. These genes are enriched for biological functions highly important to neurons, such as neuron projection organization and microtubule-based protein transport. Surprisingly, we discover hundreds of previously uncharacterized micropeptides, translated from putative long non-coding RNAs and circular RNAs. We validate their protein products in vitro and in vivo and demonstrate their potentials in regulating retinal development. Together, our study presents a rich and complex landscape of translational regulation and provides novel insights into their roles during retinogenesis.

category: developmental biology


title: On the capacity of putative plant odorant-binding proteins to bind volatile plant isoprenoids

abstract: Plants use odors not only to recruit other organisms for symbioses, but to  talk to each other. Volatile organic compounds (VOCs) from "emitting" plants inform the "receiving" (listening) plants of impending stresses or simply of their presence. However, the receptors that allow receivers to perceive the volatile cue are elusive. Most likely, plants (as animals) have odorant bind proteins (OBPs), and in fact few OBPs are known to bind "stress-induced" plant VOCs. We investigated whether OBPs may bind volatile constitutive and stress-induced isoprenoids, the most emitted plant VOCs, with well-established roles in plant communication. First, we performed a data base search that generated a list of candidate plant OBPs. Second, we investigated in silico the ability of the identified candidate plant OBPs to bind VOCs by molecular simulation experiments. Our results show that monoterpenes can bind the same OBPs that were described to bind other stress-induced VOCs. Whereas, the constitutive hemiterpene isoprene does not bind any investigated OBP and may not have an info-chemical role. We conclude that, as for animal, plant OBPs may bind different VOCs. Despite being generalist and not specialized, plant OBPs may play an important role in allowing plants to eavesdrop messages sent by neighboring plants.

category: plant biology


title: Independent and distinct patterns of abnormal lateral orbitofrontal cortex activity during compulsive grooming and reversal learning normalize after fluoxetine

abstract:
BackgroundPatients with obsessive-compulsive disorder (OCD) display disrupted performance and abnormal lateral orbitofrontal cortex (LOFC) activity during reversal learning tasks, yet it is unknown whether compulsions and reversal learning deficits share a common neural substrate. To answer this question, we measured neural activity with in vivo calcium imaging in LOFC during compulsive grooming and reversal learning before and after fluoxetine treatment.

MethodsSapap3-knockout (KO) mice were used as a model for OCD-relevant behaviors. Sapap3-KOs and control littermates were injected with virus encoding GCaMP6f and implanted with gradient-index lenses to visualize LOFC activity using miniature microscopes. Grooming, reversal learning, and neural activity were measured pre- and post-fluoxetine treatment (18mg/kg, 4 weeks).

ResultsBaseline compulsive grooming and reversal learning impairments in KOs improved after fluoxetine treatment. Additionally, KOs display distinct patterns of abnormal LOFC activity during grooming and reversal learning, both of which normalize after fluoxetine. Finally, modulation in response to reversal learning and compulsive behavior are independent, as reversal learning-associated neurons are distributed randomly amongst grooming-associated neurons (i.e. overlap is what would be expected by chance).

ConclusionsIn OCD, the LOFC is disrupted during both compulsive behaviors and reversal learning, yet whether these behaviors share common neural underpinnings is unknown. We find that the LOFC plays distinct and independent roles in compulsive grooming and impaired reversal learning and their improvement with fluoxetine. These findings suggest that LOFC plays separate roles in pathophysiology and treatment of different perseverative behaviors in OCD.

category: neuroscience


title: Novel anti-repression mechanism of H-NS proteins by a phage "early protein".

abstract: H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the early protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 modulates MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an "electrostatic zipper" between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their "half-open" conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

category: biochemistry


title: Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell epitope mapping

abstract: The current COVID-19 pandemic represents a global challenge. A better understanding of the immune response against SARS-CoV-2 is key to unveil the differences in disease severity and to develop future vaccines targeting novel SARS-CoV-2 variants. Feature barcode technology combined with CITE-seq antibodies and DNA-barcoded peptide-MHC I Dextramer reagents enabled us to identify relevant SARS-CoV-2-derived epitopes and compare epitope-specific CD8+ T cell populations between mild and severe COVID-19. We identified a strong CD8+ T cell response against an S protein-derived epitope. CD8+ effector cells in severe COVID-19 displayed hyperactivation, T cell exhaustion and were missing characteristics of long-lived memory T cells. We identify A*0101 WTAGAAAYY as an immunogenic CD8+ T cell epitope with the ability to drive clonal expansion. We provide an in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection which will be relevant for the development of molecular and targeted therapies and potential adjustments of vaccination strategies.


title: Interstitial leukocyte navigation through a search and run response to gradients

abstract: Migrating cells must interpret chemical gradients to guide themselves within tissues. A general premise has been that gradients influence the direction of leading-edge protrusions. However, recent evidence indicates that actin flows correlate better with directed motion than protrusions. A unified chemotaxis model that integrates the role of protrusions and actin flows and that accounts for in vivo cell motion patterns is lacking. Here, we provide direct experimental evidence of how neutrophils sense gradients in real-time in vivo and demonstrate a two-stage process: first a  search phase, that requires actin network expansion by Arp2/3, whereby cells slow down, explore the environment and execute path corrections. This is followed by a  run phase, that requires Myosin-II-driven contractility and fast actin flows, whereby cells accelerate and persist in the source direction. Thus, protrusive forces support chemotaxis by enhancing signal detection, while actin flows act as the ultimate output of gradient processing.


title: SUMOylation of GMFB regulates the stability and function of GMF in RPE cells under oxidative stress and inflammation

abstract: Glia maturation factor beta (GMFB) is a growth and differentiation factor that act as an intracellular regulator of signal transduction pathways. The SUMOylation is a post-translational modification (PTM) that plays a key role in protein subcellular localization, stability, transcription, and enzymatic activity. Recent studies have highlighted the importance of SUMOylation in the inflammation and progression of numerous diseases. But little is known about the relationship between GMFB and SUMOylation. Here we first report that GMFB can be mono-SUMOylated at multiple sites by the covalent addition of a single SUMO1 protein, and identified K20, K35, K58, and K97 as major SUMO acceptor sites. We also found that SUMOylation leading to increased stability and trans-localization of GMFB. Furthermore, RNA-seq data and Real-time quantitative polymerase chain reaction (rt-qPCR) also indicated that the SUMOylated GMFB upregulated multiple pathways, including the cytokine-cytokin receptor interaction, NOD-like receptor signaling pathway, TNF signaling pathway, RIG-I-like receptor signaling pathway, and NF-kappa B signaling pathway. Our studies intend to provide a novel direction for the study into the biofunction of GMFB, SUMOylated GMFB and the mechanism, clinical therapy, and prognosis of inflammation-related RPE disorders like age-related macular degeneration (AMD) and diabetic retinopathy (DR).


title: Evidence of post-domestication hybridization and adaptive introgression in Western European grapevine varieties

abstract: Grapevine (Vitis vinifera L.) is one of the most significant crops in the world. Todays richness in grapevine diversity results from a complex domestication history over multiple historical periods. Here, we employed whole genome resequencing to elucidate different aspects of the recent evolutionary history of this crop. Our results support a model in which a central domestication event in grapevine was followed by post-domestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties, which show large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by geneflow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.


title: Recapitulate Human Cardio-pulmonary Co-development Using Simultaneous Multilineage Differentiation of Pluripotent Stem Cells

abstract: The extensive crosstalk between the developing heart and lung is pivotal for their proper morphogenesis and maturation. However, there remains a lack of model systems for investigating the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human pluripotent stem cells (hPSCs) via temporal specific tuning of WNT and TGF-{beta} signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (Ts), and observed expedited alveolar maturation in the presence of cardiac accompany. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage T effectively segregated from each other with concurrent initiation of cardiac contraction. We expect our multilineage differentiation model to offer an experimentally tractable system for investigating human cardio-pulmonary interplay and tissue boundary formation during embryogenesis.


title: A Cultured Sensorimotor Organoid Model Forms Human Neuromuscular Junctions

abstract: Human induced pluripotent stem cells (iPSCs) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) within a cultured organoid system and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from ALS subject iPSC lines and isogenic lines edited to harbor familial ALS mutations all show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable for more comprehensive disease modeling.


title: Analysis of coding variants in the human FTO gene from the ExAC (gnomAD) Database

abstract: Single nucleotide polymorphisms (SNPs) in the first intron of the FTO gene (alpha-ketoglutarate-dependent dioxygenase) identified by a genome-wide association study (GWAS) in 2007 continue to be the known variants with the greatest effect on adiposity in different human populations. Currently available data reveal a total of 61 different intronic SNPs associated with adiposity. Coding variants in the FTO gene, on the other hand, have been little explored, but data from complete sequencing of the exomes of various populations are available in public databases and provide an excellent opportunity to investigate potential functional variants in FTO. This study aimed to track nonsynonymous variants in the exons of the FTO gene in different population groups using the ExAC database (gnomAD) (http://exac.broadinstitute.org/) and to analyze the potential functional impact of these variants on the FTO protein. Variants were analyzed using five publicly available pathogenicity prediction programs. Of the 158 mutations identified (152 missense and 6 stop-gain), 64 (40.5%) were classified as pathogenic, 67 (42.4%) were classified as benign, and 27 (17%) were classified as inconclusive. Thirty variants were classified as pathogenic by all five predictors used in this study, and 16 mutations were classified as pathogenic by only one predictor. The largest number of mutations was found in Europeans (non-Finnish) (85/158), all with very low frequencies, and half (32/64) of the variants classified as pathogenic by the five predictors used were also found in this population. The data obtained in this analysis show that a large number of rare coding variants classified as pathogenic or potentially pathogenic by different in silico pathogenicity prediction programs are not detected by GWAS due to the low linkage disequilibrium as well as the limitations of GWAS in capturing rare variants present in less than 1.0% of the population.


title: Closer appendage spacing augments metachronal swimming speed by promoting tip vortex interactions

abstract: Numerous species of aquatic invertebrates, including crustaceans, swim by oscillating multiple closely spaced appendages. The coordinated, out-of-phase motion of these appendages, known as "metachronal paddling", has been well-established to improve swimming performance relative to synchronous paddling. Invertebrates employing this propulsion strategy cover a wide range of body sizes and shapes, but the ratio of appendage spacing (G) to the appendage length (L) has been reported to lie in a comparatively narrow range of 0.2 < G/L [≤] 0.65. The functional role of G/L on metachronal swimming performance is unknown. We hypothesized that for a given Reynolds number and stroke amplitude, hydrodynamic interactions promoted by metachronal stroke kinematics with small G/L can increase forward swimming speed. We used a dynamically scaled self-propelling robot to comparatively examine swimming performance and wake development of metachronal and synchronous paddling under varying G/L, phase lag, and stroke amplitude. G/L was varied from 0.4 to 1.5, with the expectation that when G/L is large, there should be no performance difference between metachronal and synchronous paddling due to a lack of interaction between vortices that form on the appendages. Metachronal stroking at non-zero phase lag with G/L in the biological range produced faster swimming speeds than synchronous stroking. As G/L increased and as stroke amplitude decreased, the influence of phase lag on the swimming speed of the robot was reduced. For smaller G/L, vortex interactions between adjacent appendages generated a horizontally-oriented wake and increased momentum fluxes relative to larger G/L, which contributed to increasing swimming speed. We find that while metachronal motion augments swimming performance for closely spaced appendages (G/L < 1), moderately spaced appendages (1.0 [≤] G/L [≤] 1.5) can benefit from metachronal motion only when the stroke amplitude is large.

category: bioengineering


title: An NCCN-IPI based immune-related gene prognostic model for diffuse large B-cell lymphoma

abstract:
BackgroundInternational Prognostic Index (IPI) was widely used to better discriminate prognosis of patients with diffuse large B-cell lymphoma (DLBCL). However, there is a significant need to identify novel valuable biomarkers in the context of targeted therapies, such as immune checkpoint blockade (ICB) therapy.

MethodsGene expression data and clinical information of DLBCL were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. 371 immune-related hub genes in DLBCL patients with different IPI levels were identified by weighted gene co-expression network analysis (WGCNA), and 8 genes were selected to construct an IPI-based immune prognostic model (IPI-IPM). Afterward, the genetic, somatic mutational and molecular profiles of IPI-IPM subgroups were analyzed, as well as the potential clinical response of ICB in different IPI-IPM subgroups.

ResultsThe IPI-IPM was constructed based on the expression of CMBL, TLCD3B, SYNDIG1, ESM1, EPHA3, HUNK, PTX3 and IL12A, where high-risk patients had shorter overall survival (OS) than low-risk patients, consistent with the results in the GEO cohorts. The comprehensive results showed that high IPI-IPM risk scores were correlated with immune-related signaling pathways, high KMT2D and CD79B mutation rates, as well as up-regulation of inhibitory immune checkpoints including PD-L1, BTLA and SIGLEC7, indicating more potential response to ICB therapy.

ConclusionThe IPI-IPM has independent prognostic significance for DLBCL patients, which provides an immunological perspective to elucidate the mechanisms on tumor progression, also sheds a light on developing immunotherapy for DLBCL.

category: bioinformatics


title: An IPI based immune prognostic model for diffuse large B-cell lymphoma


title: Phenotyping heart failure using model-based analysis and physiology-informed machine learning

abstract: To determine the underlying mechanistic differences between diagnoses of Heart Failure (HF) and specifically heart failure with reduced and preserved ejection fraction (HFrEF & HFpEF), a closed loop model of the cardiovascular system coupled with patient specific transthoracic echocardiography (TTE) and right heart catheterization (RHC) measures was used to identify key parameters representing cardiovascular hemodynamics. Thirty-one patient records (10 HFrEF, 21 HFpEF) were obtained from the Cardiovascular Health Improvement Project (CHIP) database at the University of Michigan. Model simulations were tuned to match RHC and TTE pressure, volume and cardiac output measures in each patient with average error between data and model of 4.87 {+/-} 2%. The underlying physiological model parameters were then plotted against model-based norms and compared between the HFrEF and HFpEF group. Our results confirm that the main mechanistic parameter driving HFrEF is reduced left ventricular contractility, while for HFpEF a much wider underlying phenotype is presented. Conducting principal component analysis (PCA), k-means, and hierarchical clustering on the optimized model parameters, but not on clinical measures, shows a distinct group of HFpEF patients sharing characteristics with the HFrEF cohort, a second group that is distinct as HFpEF and a group that exhibits characteristics of both. Significant differences are observed (p-value<.001) in left ventricular active contractility and left ventricular relaxation, when comparing HFpEF patients to those grouped as similar to HFrEF. These results suggest that cardiovascular system modeling of standard clinical data is able to phenotype and group HFpEF as different subdiagnoses, possibly elucidating patient-specific treatment strategies.


title: Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis

abstract:
BackgroundExperimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades.

ResultsWe sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10% of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days.

ConclusionLong read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.


title: Suppression of PIK3CA-driven epileptiform activity by acute pathway control

abstract: Patients harboring mutations in the PI3K-AKT-MTOR signaling pathway often develop a spectrum of neurodevelopmental disorders including epilepsy. A significant proportion of them remain unresponsive to conventional anti-seizure medications. Understanding mutation-specific pathophysiology is thus critical for molecularly targeted therapies. We previously determined that mouse models expressing patient-related activating mutation in PIK3CA are epileptic and acutely treatable with PI3K inhibition, irrespective of dysmorphology (Roy et al. 2015). Using the same mutant model, we have now identified physiological mechanisms underlying the dysregulated neuronal excitability and its acute attenuation. We show that Pik3ca-driven hyperexcitability in hippocampal pyramidal neurons is mediated by changes in multiple non-synaptic, cell-intrinsic properties. These are distinct from mechanisms driving epilepsy in TSC/RHEB models. Further, we report that acute inhibition of PI3K or AKT, but not MTOR, suppresses the intrinsic epileptiform nature of the mutant neurons. These data represent an important step towards precision therapeutics against intractable epilepsy, using pathway drugs originally developed as anti-cancer agents.


title: Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

abstract:
PurposeEwing sarcoma (EwS) is a highly aggressive bone- or soft tissue-associated malignancy mostly affecting children, adolescents, and young adults. Although multimodal therapies have strongly improved patients overall survival over the past decades, the development of prognostic biomarkers for risk-based patient stratification and more effective therapies with less adverse effects is stagnating. Thus, new personalized medicine approaches are urgently required.

Experimental designGene expression data of EwS and normal tissues were crossed with survival data to identify highly overexpressed, prognostically relevant, and actionable potential targets. RNA-interference and dose-response assays as well as tissue-microarray analyses were carried out to explore the functional role and druggability of a prominent candidate gene in vitro and in vivo, and to validate its suitability as a prognostic biomarker.

ResultsEmploying a multilayered screening approach, we discover ribonucleotide reductase regulatory subunit M2 (RRM2) as a promising therapeutic target and prognostic biomarker in EwS. Through analysis of two independent EwS patient cohorts, we show that RRM2 mRNA and protein overexpression is associated with an aggressive clinical phenotype and poor patients overall survival. In agreement, RRM2 silencing as well as pharmacological inhibition by the specific inhibitor triapine (3-AP) significantly reduces EwS growth in vitro and in vivo. Furthermore, we present evidence that pharmacological RRM2 inhibition by triapine can overcome chemoresistance against doxorubicin or gemcitabine, and synergize with cell cycle checkpoint inhibitors (CHEK1 or WEE1).

ConclusionsBased on the aggressive phenotype mediated by and the druggability of RRM2 our results provide a translational rationale for exploiting RRM2 as a novel therapeutic target in EwS and prompt further clinical investigations.


title: Identifying Dihydropyrimidine Dehydrogenase as a Novel Regulator of Hepatic Steatosis

abstract: Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine Dehydrogenase (DPYD) is an enzyme responsible for uracil and thymine catabolism, and DPYD human genetic variability affects clinically observed toxicity following 5-Fluorouracil (5-FU) administration. In an in vitro model of diet-induced steatosis, the pharmacologic inhibition of DPYD resulted in protection from lipid accumulation. Additionally, a gain-of-function mutation of DPYD, created through clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) engineering, led to an increased lipid burden, which was associated with altered mitochondrial functionality in a hepatocarcionma cell line. The studies presented herein describe a novel role for DPYD in hepatocyte metabolic regulation as a modulator of hepatic steatosis.