Miracle-dz/UniFAR-FTUs huggingface dataset:
Deep learning tools and modeling to estimate the temporal expression of cell cycle proteins from 2D still images

Automatic characterization of fluorescent labeling in intact mammalian tissues remains a challenge due to the lack of quantifying techniques capable of segregating densely packed nuclei and intricate tissue patterns. Here, we describe a powerful deep learning-based approach that couples remarkably precise nuclear segmentation with quantitation of fluorescent labeling intensity within segmented nuclei, and then apply it to the analysis of cell cycle dependent protein concentration in mouse tissues using 2D fluorescent still images. First, several existing deep learning-based methods were evaluated to accurately segment nuclei using different imaging modalities with a small training dataset. Next, we developed a deep learning-based approach to identify and measure fluorescent labels within segmented nuclei, and created an ImageJ plugin to allow for efficient manual correction of nuclear segmentation and label identification. Lastly, using fluorescence intensity as a readout for protein concentration, a three-step global estimation method was applied to the characterization of the cell cycle dependent expression of E2F proteins in the developing mouse intestine.
Drug-driven reclassification of multiple tumour subtypes reveals intrinsic molecular concordance of therapy across histologically disparate cancers

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.
Slow, heterogeneous NFĸB single molecule conformational dynamics and implications for function

Single-molecule FRET was used to probe the interdomain motions of the transcription factor NFĸB. A continuum of conformations interconverting on hundreds of milliseconds to minutes were directly visualized. The widely different timescales of the motions suggest a hierarchical rugged energy landscape. Even when bound to DNA, the domains remain mobile with the conformational distribution shifted to two major populations; a high-FRET state expected from the NFκB-DNA x-ray structure and a low-FRET state consistent with one of the DNA-binding domains dissociated. When bound to the inhibitory protein IĸBα, which is known to accelerate DNA dissociation and block DNA binding, the interdomain motions were locked into a low-FRET state. Binding to the IĸBα mutant unable to accelerate DNA dissociation failed to lock the interdomain motions. Our results suggest that the slow, heterogeneous motions of NFĸB are essential in regulating the kinetics of DNA association and dissociation to achieve efficient transcription activation and termination.
A ciliopathy complex builds distal appendages to initiate ciliogenesis

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and super-resolved imaging, we identified a module which we term DISCO (DIStal centriole COmplex). DISCO components CEP90, MNR and OFD1 underlie human ciliopathies. This complex localized to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR did not generate cilia, failed to assemble distal appendages, and did not transduce Hedgehog signals. Disrupting the satellite pools did not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruited the most proximal known distal appendage component, CEP83, to root distal appendages formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricted centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies. eTOC summary Kumar et al. identifies a multi-protein complex called DISCO (DIStal centriole COmplex) required to nucleate distal appendages and restrain centriole elongation, essential for the initiation of cilium assembly. Without DISCO, cells fail to ciliate and transduce Hedgehog signals, critical for mammalian development.
Development of a chemiluminescence immunoassay to accurately detect African swine fever virus antibodies in serum

The outbreak of African swine fever (ASF) has caused significant economic losses to animal husbandry worldwide. Currently, there is no effective vaccine or treatment available to control the disease, and therefore, efficient disease control is dependent on early detection and diagnosis of ASF virus (ASFV). In this study, a chemiluminescent immunoassay (CLIA) was developed using the ASFV protein p54 as a serum diagnostic antigen and an anti-p54 monoclonal antibody. After optimizing the working parameters of the CLIA, the sensitivity of the established CLIA was 1:128, ASFV-specific serum antibody was identified, and there was no cross-reaction with other swine virus antibodies. After testing 49 clinical serum samples, the consistency rate between the CLIA and the World Organization for Animal Health (OIE) recommended commercial kit was 100%. Thus, this CLIA had a high degree of specificity, sensitivity, and reliability, and could be used as a rapid detection method for epidemiological investigations of ASFV infection.
ZFP207 controls pluripotency by multiple post-transcriptional mechanisms

The pluripotent state is not solely governed by the action of the core transcription factors Oct4, Sox2, and Nanog, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize the role of ZFP207 in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, ZFP207 does not transcriptionally regulate stem cell and neuronal-related genes but exerts its effects by control AS networks and acting as an RBP. Our study expands the role of ZFP207 to maintain ESC identity, and underscores ZFP207 functional versatility with key roles in neural fate commitment.
Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance

Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptotic programs. We isolated mitochondria from neuroblastoma cell lines obtained from children at diagnosis and after relapse following failed therapy, and profiled responses to tBid and Bim, death effectors activated by therapeutic stress. Mitochondria from post-relapse models had markedly attenuated cytochrome c release (surrogate for apoptotic commitment) in comparison with patient-matched diagnostic models. Mitochondrial DNA content, size, and shape did not differ consistently. However, we used electron microscopy to identify reduced endoplasmic reticulum-mitochondria contacts (ERMCs) as correlated with therapy resistance. ERMCs form microdomains for the transfer of Ca2+ to mitochondria. We confirmed reduced Ca2+ transfer in resistant cells, with restoration by re-opposing ERMCs via genetically-encoded linkers. However, reduced Ca2+ transfer was not present in all ERMC-reduced cancers with therapy resistance, supporting Ca2+-independent mechanisms. Genetically or biochemically reducing ERMCs in therapy sensitive tumors phenocopied resistance, validating these inter-organelle contacts as physiologic regulators of apoptosis. Our work confirms the importance of ERMCs in stress signaling and provides a previously unrecognized mechanism for cancer cell resistance that is not exclusive to other contributors.
A Comparison of Performance for Different SARS-Cov-2 Sequencing Protocols

SARS-Cov-2 genome sequencing has been identified as a fundamental tool for fighting the COVID-19 pandemic. It is used, for example, for identifying new variants of the virus and for elaborating phylogenetic trees that help to trace the spread of the virus. In the present study we provide a comprehensive comparison between the quality of the assemblies obtained from different sequencing protocols. We demonstrate how some protocols actively promoted by different high-level administrations are inefficient and how less-used alternative protocols show a significant increased performance. This increase of performance could lead to cheaper sequencing protocols and therefore to a more convenient escalation of the sequencing efforts around the world.
A role for Gcn5 in heterochromatin structure, gene silencing and NER at the HML locus in budding yeast

Histone acetyltransferase Gcn5 plays an important role in transcription activation, DNA replication-coupled nucleosome assembly and nucleotide excision repair (NER). However, its functions on the heterochromatin are unexplored. Here, we find that removal of Gcn5 leads to more condensed heterochromatin structure, as revealed by topology analysis of HML circles. Importantly, the altered heterochromatin structure is restored by re-expression of Gcn5 in the gcn5Δ cells. As a result of the more compact heterochromatin, gene silencing at the HML locus is increased and NER efficiency at HML is impaired in the absence of Gcn5. Interestingly, while the association of SIR complex with HML is enhanced in cells lacking Gcn5, the altered compaction of HML heterochromatin is also observed due to the deletion of Gcn5 from Sir− cells. These findings reveal a role of Gcn5 in the regulation of heterochromatin structure, gene silencing and NER efficiency at the heterochromatic HML locus in yeast.
Selective Deposition of Hydroxyapatite Nanoparticles by Electrophoretic Deposition

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

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 statement Different 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.
A new straightforward method for semi-automated segmentation of trabecular bone from cortical bone in diverse and challenging morphologies

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 (micro-CT) prior to analysis. Separation often involves manual subdivision of these two similarly radio-opaque compartments, which can be time-consuming and subjective. We have developed an objective, semi-automated protocol which reduces user bias and enables straightforward, user-friendly segmentation of trabecular from the 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 characterize 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 programmes. Our aim is that this method will be adopted widely to create a reproducible and time-efficient method of segmenting trabecular and cortical bone.
Delivering on Biden’s 2030 conservation commitment

On January 27, 2021, President Biden signed an executive order, Tackling the Climate Crisis at Home and Abroad, committing the United States to various goals within his campaign’s major climate policy, the Biden Plan for a Clean Energy Revolution and Environmental Justice. Included in this executive order is a commitment to “conserving at least 30 percent of [the United States’] lands and oceans by 2030. ” This ambitious conservation target signals a promising direction for biodiversity in the United States. However, while the executive order outlines several goals for climate mitigation, the ‘30×30’ target remains vague in its objectives, actions, and implementation strategies for protecting biodiversity. Biodiversity urgently needs effective conservation action, but it remains unclear where and what this 30% target will be applied to. Achieving different climate and biodiversity objectives will require different strategies and, in combination with the associated costs of implementation, will lead to different priority areas for conservation actions. Here, we illustrate what the 30% target could look like across four objectives reflective of the ambitious goals outlined in the executive order. We compile several variations of terrestrial protected area networks guided by these different objectives and examine the trade-offs in costs, ecosystem representation, and climate mitigation potential between each. We find little congruence in priority areas across objectives, emphasizing just how crucial it will be for the Biden administration to develop clear objectives and establish appropriate performance metrics from the outset to maximize both conservation and climate outcomes in support of the 30×30 target. We discuss important considerations that must guide the administration’s conservation strategies in order to ensure meaningful conservation outcomes can be achieved over the next decade.
Cryo-EM structure of lipid embedded human ABCA7 at 3.6Å resolution

Dysfunction of the ATP Binding Cassette (ABC) transporter ABCA7 alters cellular lipid homeostasis and is linked to Alzheimer’s Disease (AD) pathogenesis through poorly understood mechanisms. Here we determined the cryo-electron microscopy (cryo-EM) structure of human ABCA7 in a lipid environment at 3. 6Å resolution that reveals an open conformation, despite bound nucleotides, and bilayer lipids traversing the transmembrane domain (TMD). We show that ATP hydrolysis in ABCA7 is modulated by its lipid environment as well as apolipoprotein (apo) A1 and apoE, the latter in an isoform dependent manner, and that apoA1 can directly bind ABCA7. Structural similarities between ABCA and ABCG family transporters suggest that TMD-nucleotide binding domain (NBD) pairs in both sets move as single rigid bodies to affect conformational transitions. Our data suggest these transitions in ABCA7 are influenced by TMD cavity lipids and apolipoprotein binding in addition to being coupled to ATP hydrolysis.
State-Transition Analysis of Time-Sequential microRNA Expression Predicts Development of Acute Myeloid Leukemia

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in post-transcriptional regulation of gene expression and have been shown to hold prognostic value in a variety of settings, including acute myeloid leukemia (AML). However, the temporal dynamics of miRNA expression profiles as it relates to AML initiation and progression is poorly understood. Using serial samples from a mouse model of AML, we show that the miRNA transcriptome undergoes state-transition during AML initiation and progression. The AML state-transition was visualized and modeled by constructing an AML state-space from singular value decomposition of the time-series miRNA sequencing data. Within the AML state-space, we identified critical points of AML development characterized by unique differentially expressed miRNAs compared to healthy controls at critical points of leukemogenesis (early, transition, and late). Interestingly, we observed that changes in miRNA expression during leukemogenesis followed two patterns: 1) a monotonic pattern with continuously increasing or decreasing expression; and 2) a non-monotonic pattern with a local maximum or minimum at the transition critical point which was the “point of no-return” from health to AML. We validated the AML state-space and dynamics in an independent cohort of mice and demonstrated the state-transition model accurately predicted time to AML. Of note, we show that the miRNA-derived state-transition model produced a state-space and critical points that were strikingly similar, but not identical to that produced by the coding (i.e., messenger [m]RNA-based) transcriptome. This indicates that while both miRNA and mRNA expression may provide similar information, they also capture independent features of AML state-transition. Significance We show that the microRNA transcriptome undergoes a global state transition during the initiation and progression of acute myeloid leukemia, and accurately predicts time to disease development.
Widespread translational control regulates retinal development in mouse

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.