Miracle-dz/UniFAR-FTUs huggingface dataset:
CIP2A interacts with TopBP1 and is selectively essential for DNA damage-induced basal-like breast cancer tumorigenesis

Despite saturated genetic profiling of breast cancers, oncogenic drivers for the clinically challenging basal-like breast cancer (BLBC) subtype are still poorly understood. Here, we demonstrate that CIP2A is selectively essential for DNA damage-induced initiation of mouse BLBC tumors, but not of other cancer types. Mechanistically, CIP2A was discovered genome-widely the closest functional homologue for DNA-damage proteins TopBP1, RHNO, POLQ, NBN and PARP1. CIP2A directly interacts with the ATR-activation domain of TopBP1, and dampens both, chromatin binding of TopBP1 and RAD51, and G2/M checkpoint in DNA-damaged cells. CIP2A also drives BLBC-associated proliferative MYC and E2F1 signaling. Consistently with high DNA-damage response activity BLBCs, and CIP2A’s novel role in checkpoint signaling, CIP2A was found essential for DNA-damaged, and BRCA-mutant BLBC cells. Selective role for CIP2A as BLBC driver was supported by association of high CIP2A expression with poor patient prognosis only in BLBC, but not in other breast cancer types. Therapeutically, small molecule reactivators of PP2A (SMAPs) phenocopy CIP2A-dependent DNA damage response, and inhibit in vivo growth of patient-derived BLBC xenograft. In summary, we discover sub-type selective essential role for CIP2A in BLBC initiation and maintenance that can be explained by its newly discovered association with DNA-damage response, coordinated with regulation of proliferative signaling. The results also identify therapeutic strategy for CIP2A-dependent BLBCs.
A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art SHAPE reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.
Spatially-targeted proteomics of the host-pathogen interface during staphylococcal abscess formation

Staphylococcus aureus is a common cause of invasive and life-threatening infections that are often multi-drug resistant. To develop novel treatment approaches, a detailed understanding of the complex host-pathogen interactions during infection is essential. This is particularly true for the molecular processes that govern the formation of tissue abscesses, as these heterogeneous structures are important contributors to staphylococcal pathogenicity. To fully characterize the developmental process leading to mature abscesses, temporal and spatial analytical approaches are required. Spatially targeted proteomic technologies, such as micro liquid extraction surface analysis, offer insight into complex biological systems including detection of bacterial proteins, and their abundance in the host environment. By analyzing the proteomic constituents of different abscess regions across the course of infection, we defined the immune response and bacterial contribution to abscess development through spatial and temporal proteomic assessment. The information gathered was mapped to biochemical pathways to characterize the metabolic processes and immune strategies employed by the host. These data provide insights into the physiological state of bacteria within abscesses and elucidate pathogenic processes at the host-pathogen interface.
ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA binding protein that in human erythroid cells directly and measurably activates only one gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplant settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells whose perturbation might offer new therapeutic opportunities in the treatment of hemoglobinopathies. Highlights A CRISPR screen implicates ZNF410 in fetal globin gene repression The CHD4 gene is the singular direct ZNF410 target in erythroid cells The fetal globin genes are exquisitely sensitive to CHD4 levels Five C2H2 zinc fingers of ZNF410 recognize the major groove of a 14 base pair sequence
White matter integrity requires continuous myelin synthesis at the inner tongue

Myelin, the electrically insulating axonal sheath, is composed of lipids and proteins with exceptionally long lifetime. This raises the question how myelin function is affected by myelin turnover. We have studied the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survived recombination, continued expressing myelin genes, but failed to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualized myelin-like membranes that failed to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicated degradation of existing myelin at the abaxonal side and at the inner tongue of the sheath. Compacted myelin thinning and shortening of internodes, with about 50% myelin lost after 20 weeks (=5 months), ultimately led to axonal pathology and neurological disease. These data reveal that functional axon-myelin units require the continuous incorporation of new myelin membranes.
Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila

The spatial configuration of the eukaryotic genome is organized and dynamic, providing the structural basis for regulated gene expression in living cells. In Drosophila melanogaster, 3D genome organization is characterized by the phenomenon of somatic homolog pairing, where homologous chromosomes are intimately paired from end to end. While this organizational principle has been recognized for over 100 years, the process by which homologs identify one another and pair has remained mysterious. Recently, a model was proposed wherein specifically-interacting “buttons” encoded along the lengths of homologous chromosomes drive somatic homolog pairing. Here, we turn this hypothesis into a precise biophysical model to demonstrate that a button-based mechanism can lead to chromosome-wide pairing. We test our model and constrain its free parameters using live-imaging measurements of chromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. Our analysis shows strong agreement between model predictions and experiments in the separation dynamics of tagged homologous loci as they transition from unpaired to paired states, and in the percentage of nuclei that become paired as embryonic development proceeds. In sum, our data strongly support a button-based mechanism of somatic homolog pairing in Drosophila and provide a theoretical framework for revealing the molecular identity and regulation of buttons.
Breathing new life into the critical oxygen partial pressure (Pcrit): a new definition, interpretation and method of determination

The critical oxygen partial pressure (Pcrit) is most commonly defined as the oxygen partial pressure below which an animal’s standard metabolic rate can no longer be maintained. It is widely interpreted as measure of hypoxia tolerance, which influences a species’ aerobic scope and, thus, constrains biogeography. However, both the physiology underlying that interpretation and the methodology used to determine Pcrit remain topics of active debate. The debate remains unresolved in part because Pcrit, as defined above, is a purely descriptive metric that lacks a clear mechanistic basis. Here we redefine Pcrit as the PO2 at which physiological oxygen supply is maximized and refer to these values, thus determined, as Pcrit-α. The oxygen supply capacity (α) is a species- and temperature-specific coefficient that describes the slope of the relationship between the maximum achievable metabolic rate and PO2. This α is easily determined using respirometry and provides a precise and robust estimate of the minimum oxygen pressure required to sustain any metabolic rate. To determine α, it is not necessary for an individual animal to maintain a consistent metabolic rate throughout a trial (i.e. regulation) nor for the metabolic rate to show a clear break-point at low PO2. We show that Pcrit-α can be determined at any metabolic rate as long as the organisms’ oxygen supply machinery reaches its maximum capacity at some point during the trial. We reanalyze published representative Pcrit trials for 40 species across five phyla, as well as complete datasets from six additional species, five of which have not previously been published. Values determined using the Pcrit-α method are strongly correlated with Pcrit values reported in the literature. Advantages of Pcrit-α include: 1) Pcrit-α is directly measured without the need for complex statistics that hinder measurement and interpretation; 2) it makes clear that Pcrit is a measure of oxygen supply, which does not necessarily reflect hypoxia tolerance; 3) it alleviates many of the methodological constraints inherent in existing methods; 4) it provides a means of predicting the maximum metabolic rate achievable at any PO2, 5) Pcrit-α sheds light on the temperature- and size-dependence of oxygen supply and metabolic rate and 6) Pcrit-α can be determined with greater precision than traditional Pcrit.
Flagellar energetics from high-resolution imaging of beating patterns in tethered mouse sperm

While much is known about the microstructure of sperm flagella, the mechanisms behind the generation of flagellar beating patterns by the axoneme are still not fully understood. We demonstrate a technique for investigating the energetics of flagella or cilia. We record the planar beating of tethered wildtype and Crisp2-knockout mouse sperm at high-speed and high-resolution and extract centerlines using digital image processing techniques. We accurately reconstruct beating waveforms using a Chebyshev-polynomial based Proper Orthogonal Decomposition of the centerline tangent-angle profiles. External hydrodynamic forces and the internal resistance from the passive flagellar material are calculated from the observed kinematics of the beating patterns using a Soft, Internally-Driven Kirchhoff-Rod (SIDKR) model. Energy conservation is employed to further compute the flagellar energetics. We thus obtain the distribution of mechanical power exerted by the dynein motors without any further assumptions about mechanisms regulating axonemal function. We find that, in both the mouse genotypes studied, a large proportion of the mechanical power exerted by the dynein motors is dissipated internally, within the passive structures of the flagellum and by the motors themselves. This internal dissipation is considerably greater than the hydrodynamic dissipation in the aqueous medium outside. The net power input from the dynein motors in sperm from Crisp2-knockout mice is significantly smaller than in corresponding wildtype samples. The reduced power is correlated with slower beating and smaller amplitudes. These measurements of flagellar energetics indicate that the ion-channel regulating cysteine-rich secretory proteins (CRISPs) may also be involved in regulating mammalian sperm motility.
Two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis thaliana

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling ‘Structure Establishment Phase’ followed by a ‘Chloroplast Proliferation Phase’ during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.
Severe childhood and adulthood stress associates with neocortical layer-specific reductions of mature spines in psychiatric disorders

Severe stress exposure causes the loss of dendritic spines on cortical pyramidal neurons and induces psychiatric-like symptoms in rodent models. These effects are strongest following early-life stress and are most persistent on apical dendrites. However, the long-term impacts and temporal effects of stress exposure on the human brain remain poorly understood. Using a novel postmortem cohort of psychiatric cases with severe stress experienced in childhood, adulthood, or no severe stress, and matched controls, we aimed to determine the impact of stress timing on pyramidal neuron structure in the human orbitofrontal cortex (OFC). We performed Golgi Cox staining and manually measured the morphology and density of over 22,000 dendritic spines on layer-specific pyramidal neuron apical dendrites. We also quantified glucocorticoid receptor mRNA and protein as a marker of stress dysregulation. Both childhood and adulthood stress were associated with large reductions in mature mushroom spine density (up to 56% loss) in both the superficial (II/III) and deeper layers (V) of the OFC. However, childhood stress caused more substantial reductions to both total and mature mushroom spines. No difference in glucocorticoid receptor mRNA and protein were seen between groups, although both negatively correlated with total spine density within the whole cohort. These findings indicate that severe stress, especially when experienced during childhood, persistently affects the fine morphological properties of neurons in the human OFC. This may impact on cell connectivity in this brain area, and at least partly explain the social and emotional symptoms that originate in the OFC in psychiatric disorders.
Unraveling Endothelial Cell Phenotypic Regulation By Spatial Hemodynamic Flows With Microfluidics

Human endothelial cells (hECs) experience complex spatiotemporal hemodynamic flows and that directly regulate hEC function and susceptibility to cardiovascular disease. Recent medical imaging studies reveal that helical flows strongly correlate with lowered disease susceptibility, as contrasted to multidirectional disturbed flows. However, a lack of platforms to replicate these spatial profiles of flow (SPF) has prevented biological studies to investigate the role hECs play in tuning the observed SPF-correlated disease susceptibility. Here, we utilize microfluidic devices to apply varying SPF upon hECs for the first time, and discover that these flows can differentially impact hEC morphology, transcription, and polarization. Collectively, our platform and studies significantly advance our ability to delineate flow-regulated hEC function and disease susceptibility. Significance Statement In vivo, hECs experience complex hemodynamic flows, including those that are spatially helical or disturbed, which is in stark contrast to the unidirectional flows typically used to study hECs in vitro. Understanding the impact of SPF on hEC function informs our understanding of the pathophysiology of hEC dysfunction and can lead to interventional solutions that specifically perturb SPF to lower disease risk. Here, we leverage microfluidics to apply and discover the specific impact of SPF on hECs for the first time. Broadly, our platform bridges the mutual interests of the vascular biology and interventional cardiology communities to collectively understand how cardiovascular health is tied to the way blood flows upon the endothelium.
In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during three stages of intrahemocoelic infection in Galleria mellonella. A total of 1,285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection and 167 transcripts responded throughout all three stages of infection. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2 and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.
Molecular underpinnings of ssDNA specificity by Rep HUH endonucleases and implications for HUH-tag multiplexing and engineering

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay termed HUH-seq to interrogate subtleties in Rep specificity, and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed us to engineer a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.
Bayesian Connective Field Modeling: a Markov Chain Monte Carlo approach

The majority of neurons in the human brain process signals from neurons elsewhere in the brain. Connective Field (CF) modeling is a biologically-grounded method to describe this essential aspect of the brain’s circuitry. It allows characterizing the response of a population of neurons in terms of the activity in another part of the brain. CF modeling translates the concept of the receptive field (RF) into the domain of connectivity by assessing the spatial dependency between signals in distinct cortical visual field areas. Standard CF model estimation has some intrinsic limitations in that it cannot estimate the uncertainty associated with each of its parameters. Obtaining the uncertainty will allow identification of model biases, e.g. related to an over- or under-fitting or a co-dependence of parameters, thereby improving the CF prediction. To enable this, here we present a Bayesian framework for the CF model. Using a Markov Chain Monte Carlo (MCMC) approach, we estimate the underlying posterior distribution of the CF parameters and consequently, quantify the uncertainty associated with each estimate. We applied the method and its new Bayesian features to characterize the cortical circuitry of the early human visual cortex of 12 healthy participants that were assessed using 3T fMRI. In addition, we show how the MCMC approach enables the use of effect size (beta) as a data-driven parameter to retain relevant voxels for further analysis. Finally, we demonstrate how our new method can be used to compare different CF models. Our results show that single Gaussian models are favoured over differences of Gaussians (i.e. center-surround) models, suggesting that the cortico-cortical connections of the early visual system do not possess center-surround organisation. We conclude that our new Bayesian CF framework provides a comprehensive tool to improve our fundamental understanding of the human cortical circuitry in health and disease. Highlights □ We present and validate a Bayesian CF framework based on a MCMC approach. □ The MCMC CF approach quantifies the model uncertainty associated with each CF parameter. □ We show how to use effect size beta as a data-driven threshold to retain relevant voxels. □ The cortical connective fields of the human early visual system are best described by a single, circular symmetric, Gaussian.