bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans
Authors: Gervais Nicholas C. , La Bella Alyssa A. , Wensing Lauren F. , Sharma Jehoshua , Acquaviva Victoria , Best Madison , Cadena López Ricardo Omar , Fogal Meea , Uthayakumar Deeva , Chavez Alejandro , Santiago-Tirado Felipe , Flores-Mireles Ana L. , Shapiro Rebecca S. 
Published: August 2022

AbstractFor the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug resistance and contribute towards the identification of novel phenotypes. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.
Proteome-scale induced proximity screens reveal highly potent protein degraders and stabilizers
Authors: Poirson Juline , Dhillon Akashdeep , Cho Hanna , Lam Mandy Hiu Yi , Alerasool Nader , Lacoste Jessica , Mizan Lamisa , Taipale Mikko 
Published: August 2022

SUMMARYTargeted protein degradation and stabilization are promising therapeutic modalities due to their potency and versatility. However, only few E3 ligases and deubiquitinases have been harnessed for this purpose. Moreover, there may be other protein classes that could be exploited for protein stabilization or degradation. Here, we used a proteome-scale platform to identify hundreds of human proteins that can promote the degradation or stabilization of a target protein in a proximity-dependent manner. This allowed us to comprehensively compare the activities of human E3s and deubiquitinases, characterize non-canonical protein degraders and stabilizers, and establish that effectors have vastly different activities against diverse targets. Notably, the top degraders were more potent against multiple therapeutically relevant targets than the currently used E3s CBRN and VHL. Our study provides a functional catalogue of effectors for targeted protein degradation and stabilization and highlights the potential of induced proximity screens for discovery of novel proximity-dependent protein modulators.
Genomics of Natural Populations: Gene Conversion Events Reveal Selected Genes within the Inversions ofDrosophila pseudoobscura
Authors: Schaeffer Stephen W. , Richards Stephen , Fuller Zachary L. 
Published: August 2022

AbstractWhen adaptive phenotypic variation or QTLs map within an inverted segment of a chromosome, researchers often despair because it is thought that the suppression of crossing over will prevent the discovery of selective target genes that contribute to the establishment of the rearrangement. If an inversion polymorphism is old enough, then the accumulation of gene conversion tracts offers the promise that QTLs or selected loci within inversions can be mapped. This study uses the inversion polymorphism ofDrosophila pseudoobscuraas a model system to show that gene conversion analysis is a useful tool for mapping selected loci within inversions.D. pseudoobscurahas over 30 different chromosomal arrangements on the third chromosome (Muller C) in natural populations and their frequencies vary with changes in environmental habitats. Statistical tests of fiveD. pseudoobscuragene arrangements identified outlier genes within inverted regions based on local clusters of fixed SNP differences. These outlier genes also had potentially heritable variation, either fixed amino acid differences or differential expression patterns among arrangements. Here, we use genome sequences of the inverted third chromosome (Muller C) to infer 98,443 gene conversion tracts for a total coverage of 142 Mb or 7.2 x coverage of the 19.7 Mb chromosome. We estimated gene conversion tract coverage in the 2,668 genes on Muller C and tested whether the number of genes with significantly low coverage was the same for outlier versus non-outlier loci.. Genes with low gene conversion tract coverage were more frequent in the outlier class than the non-outlier class suggesting that selection removes exchanged DNA from the outlier genes more often than non-outlier genes. These data support the hypothesis that the pattern and organization of genetic diversity on the third chromosome inD. pseudoobscurais consistent with the capture of locally adapted combinations of alleles prior to inversion mutation events.
Açai (Euterpe precatoria Mart. Arecaceae) from the Northern Region of Bolivia is not contaminated with Trypanosoma cruzi
Authors: Rollano-Peñaloza OM , Roque-Marca N , Peñarrieta M , Mollinedo P , Rodriguez C , Larrea-Alcázar D.M. 
Published: August 2022

AbstractChagas disease is a very important public health problem in America. It is caused by the protozoan parasite Trypanosoma cruzi and transmitted by vectors such as Triatomine insects. However, oral transmission is generating more T. cruzi infections than vectorial transmission in Brazilian Amazonic regions, probably due to the increased consumption of tropical fruits such as açai. Açai palms have become very popular due to its good nutritional properties. Açai fruits have different sources depending of their geographical origin. Açai palms (Euterpe oleracea) in Brazil are cultivated, while in Bolivia grow in wild populations and belongs to a different species, the solitary açai (Euterpe precatoria). Only açai from Brazil has been involved in T. cruzi oral transmission, while Bolivian açai has been regarded as disease free. In order to verify the absence of T. cruzi on açai products from Bolivia, we developed a method to detect T. cruzi DNA by real-Time PCR with internal controls for solitary açai. In this study we show that açai good manufacturing process did not interfere with the detection of açai or T. cruzi DNA. Finally, we report that freshly collected açai fruits and açai frozen pulps from Bolivia were not contaminated with T. cruzi.
Three R2R3-MYB transcription factors from banana (Musaspp.) activate structural anthocyanin biosynthesis genes as part of an MBW complex
Authors: Busche Mareike , Pucker Boas , Weisshaar Bernd , Stracke Ralf 
Published: August 2022

AbstractBananas are among the most popular fruits in the world and provide food security and employment opportunities in several developing countries. An increased anthocyanin content could enhance the health promoting properties of banana fruits. The biosynthesis of anthocyanins is largely regulated at the transcriptional level. However, little is known about transcriptional activation of anthocyanin biosynthesis in banana. We analysed the regulatory activity of threeMusaMYBs predicted by bioinformatic analysis to transcriptionally regulate anthocyanin biosynthesis in banana.MusaMYBA1, MusaMYBA2andMusaMYBPA2did not complement the anthocyanin deficiency phenotype of theA. thaliana pap1/pap2mutant. However, co-transfection experiments inA. thalianaprotoplasts showed thatMusaMYBA1,MusaMYBA2 andMusaMYBPA2 function as components of a transcription factor complex with a bHLH and WD40 protein, called MBW complex, resulting in the activation of theanthocyanin synthaseanddihydroflavonol 4-reductasepromoters fromA. thaliana. The activation potential ofMusaMYBA1,MusaMYBA2 andMusaMYBPA2 increased when combined with the monocot bHLHZmR instead of the dicotAtEGL3. This work paves the path towards decoding the MBW complex-mediated transcriptional activation of anthocyanin biosynthesis in banana. Moreover, it facilitates research towards an elevated anthocyanin content in banana and other monocot crops.
ELI trifocal microscope: A precise cryogenic fabrication system to prepare target cryo-lamellae of cells for in situ cryo-ET study
Authors: Li Shuoguo , Wang Ziyan , Jia Xing , Niu Tongxin , Zhang Jianguo , Yin Guoliang , Zhang Xiaoyun , Zhu Yun , Ji Gang , Sun Fei 
Published: August 2022

AbstractCryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent in situ cryo-ET structural study. Here, we developed a precise cryogenic fabrication system, the ELI trifocal microscope (ELI-TriScope), by setting up an electron (E) beam, a light (L) beam and an ion (I) beam at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella without sacrificing the throughput. ELI-TriScope was developed starting from a commercial dual-beam scanning electron microscope (SEM) by incorporating a cryo-holder-based transfer system and embedding an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam (FIB) milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome, with an ∼100% success rate, and discovered new in situ structural features of the human centrosome through a subsequent cryo-ET structural study.
DNA methylation age studies of humpback whales
Authors: Horvath Steve , Haghani Amin , Zoller Joseph A. , Fei Zhe , Bérubé Martine , Robbins Jooke 
Published: August 2022

ABSTRACTSeveral previous studies have described epigenetic clocks for whale and dolphin species. Here we present a novel and highly robust epigenetic clock for the humpback whale based on methylation levels measured using the Mammalian Methylation Array platform. Skin samples were obtained from 76 individuals that had been studied since their birth year and known to range in age from <1 to 39.5 years. The humpback whale clock provided a highly accurate estimate of chronological age (R=0.96, median error 2.2 years) according to a leave-one-out cross validation analysis. We applied this clock to an independent set of samples from humpback whales of unknown exact age but with sighting histories that were as long as or longer than the upper 20% of the available known-age range. Although there was a strong correlation with minimum age (R=0.89), the clock underestimated age in these older animals by a median error of at least 7.8 years. Finally, we applied the humpback clock to publicly available methylation data from beluga whales. In this data set from a different species, the humpback clock provided an age correlation of R=0.78. While a DNAm age estimator has previously been described for humpback whales, this is the first such clock shown to apply to another cetacean species as well. Our humpback whale clock built from well-studied population lends itself for understanding humpback populations that otherwise lack age data.
Integrative Analysis of Histological Textures and Lymphocyte Infiltration in Renal Cell Carcinoma using Deep Learning
Authors: Brummer Otso , Pölönen Petri , Mustjoki Satu , Brück Oscar 
Published: August 2022

ABSTRACTEvaluating tissue architecture from routine hematoxylin and eosin-stained (H&E) slides is prone to subjectivity and sampling bias. Here, we extensively annotated ∼40,000 images of five tissue texture types and ∼25,000 images of lymphocyte quantity to train deep learning models. We defined histopathological patterns in over 400 clear-cell renal cell carcinoma H&E-stained slides of The Cancer Genome Atlas (TCGA) and resolved sampling and staining differences by harmonizing textural composition. By integrating multi-omic and imaging data, we profiled their clinical, immunological, genomic, and transcriptomic phenotypes. Histological grade, stage, adaptive immunity, the epithelial-to-mesenchymal transition signature and lower mutation burden were more common in stroma-rich samples. Histological proximity between the malignant and normal renal tissues was associated with poor survival, cellular proliferation, tumor heterogeneity, and wild-type PBRM1. This study highlights textural characterization to standardize sampling differences, quantify lymphocyte infiltration and discover novel histopathological associations both in the intratumoral and peritumoral regions.
Analysis of context-specific KRAS-effectors (sub)complexes in Caco-2 cells
Authors: Ternet Camille , Junk Philipp , Sevrin Thomas , Catozzi Simona , Oliviero Giorgio , Wynne Kieran , Kiel Christina 
Published: August 2022

AbstractRas is a key switch controlling cell behavior. In the GTP-bound form, Ras interacts with numerous effectors in a mutually exclusive manner, where individual Ras-effectors are likely part of larger cellular (sub)complexes. The molecular details of these (sub)complexes and their alteration in specific contexts is not understood. Focusing on KRAS, we performed affinity purification (AP)-mass spectrometry (MS) experiments of exogenous expressed FLAG-KRAS WT and three oncogenic mutants (‘genetic contexts’) in the human Caco-2 cell line, each exposed to 11 different culture media (‘culture contexts’) that mimic conditions relevant in the colon and colorectal cancer. We identified four effectors present in complex with KRAS in all genetic and growth contexts (‘context-general effectors’). Seven effectors are found in KRAS complexes in only some contexts (‘context-specific effectors’). Analyzing all interactors in complex with KRAS per condition, we find that the culture contexts had a larger impact on interaction rewiring than genetic contexts. We investigated how changes in the interactome impact functional outcomes and created a shiny app for interactive visualization. We validated some of the functional differences in metabolism and proliferation. Finally, we used networks to evaluate how KRAS effectors are involved in the modulation of functions by random walk analyses of effector-mediated (sub)complexes. Altogether, our work shows the impact of environmental contexts on network rewiring, which provides insights into tissue-specific signaling mechanisms. This may also explain why KRAS oncogenic mutants may be causing cancer only in specific tissues despite KRAS being expressed in most cells and tissues.
ERStruct: A Python Package for Inferring the Number of Top Principal Components from Whole Genome Sequencing Data
Authors: Yang Jinghan , Xu Yuyang , Yao Minhao , Wang Gao , Liu Zhonghua 
Published: August 2022

AbstractLarge-scale multi-ethnic DNA sequencing data is increasingly available owing to decreasing cost of modern sequencing technologies. Inference of the population structure with such sequencing data is fundamentally important. However, the ultra-dimensionality and complicated linkage disequilibrium patterns across the whole genome make it challenging to infer population structure using traditional principal component analysis (PCA) based methods and software. We present the ERStruct Python Package, which enables the inference of population structure using whole-genome sequencing data. By leveraging parallel computing and GPU acceleration, our package achieves significant improvements in the speed of matrix operations for large-scale data. Additionally, our package features adaptive data splitting capabilities to facilitate computation on GPUs with limited memory. Our Python package ERStruct is an efficient and user-friendly tool for estimating the number of top informative PCs that capture population structure from whole genome sequencing data.
Solid/liquid coexistence during aging of FUS condensates
Authors: Shen Yi , Chen Anqi , Wang Wenyun , Shen Yinan , Ruggeri Francesco Simone , Aime Stefano , Wang Zizhao , Qamar Seema , Espinosa Jorge R. , Garaizar Adiran , George-Hyslop Peter St , Collepardo-Guevara Rosana , Weitz David A. , Vigolo Daniele , Knowles Tuomas P. J. 
Published: August 2022

AbstractA wide range of macromolecules undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed in a reversible manner, and carry out important functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics and mechanics in time and space, that FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases co-exist simultaneously within the same condensate, resulting in highly inhomogeneous structures. We introduce two new optical techniques, dynamic spatial mapping and reflective confocal dynamic speckle microscopy, and use these to further show that the liquid-to-solid transition is initiated at the interface between the dense phase within condensates and the dilute phase. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a key element in driving pathological protein aggregation.
An exotic allele of barley EARLY FLOWERING 3 contributes to developmental plasticity at elevated temperatures
Authors: Zhu Zihao , Esche Finn , Babben Steve , Trenner Jana , Serfling Albrecht , Pillen Klaus , Maurer Andreas , Quint Marcel 
Published: August 2022

AbstractIncrease in ambient temperatures caused by climate change affects various morphological and developmental traits of plants, threatening crop yield stability. In the model plant Arabidopsis thaliana, EARLY FLOWERING 3 (ELF3) plays prominent roles in temperature sensing and thermomorphogenesis signal transduction. However, how crop species respond to elevated temperatures is poorly understood. Here, we show that the barley ortholog of AtELF3 interacts with high temperature to control growth and development. We used heterogeneous inbred family (HIF) pairs generated from a segregating mapping population and systematically studied the role of exotic ELF3 variants in barley temperature responses. An exotic ELF3 allele from Syrian origin promoted elongation growth in barley at elevated temperatures, whereas plant area and estimated biomass were drastically reduced, resulting in an open canopy architecture. The same allele accelerated inflorescence development at high temperature, which correlated with early transcriptional induction of MADS-box floral identity genes BM3 and BM8. Consequently, barley plants carrying the exotic ELF3 allele displayed stable total grain number and mitigated yield loss at elevated temperatures. Our findings therefore demonstrate that exotic ELF3 variants can contribute to phenotypic and developmental acclimation to elevated temperatures, providing a stimulus for breeding of climate resilient crops.HighlightWe demonstrate that an exotic allele of EARLY FLOWERING 3 (ELF3) contributes to plant architectural and developmental acclimation, and thereby improves yield stability at high ambient temperatures.
A Novel Framework for In-vivo Diffusion Tensor Distribution MRI of the Human Brain
Authors: Magdoom Kulam Najmudeen , Avram Alexandru V. , Sarlls Joelle E. , Dario Gasbarra , Basser Peter J. 
Published: August 2022

AbstractNeural tissue microstructure plays an important role in developmental, physiological and pathophysiological processes. Diffusion tensor distribution (DTD) MRI helps probe heterogeneity at the mesoscopic length scale, orders of magnitude smaller than the nominal MRI voxel size, by describing water diffusion within a voxel using an ensemble of non-exchanging compartments characterized by a probability density function of diffusion tensors. In this study, we provide a new framework for acquiring tensor encoded diffusion weighted images (DWIs) and estimating DTD from them for in-vivo human brain imaging. We interfused pulsed field gradients (iPFG) in a single spin echo to generate arbitrary b-tensors of rank one, two, or three without introducing concomitant gradient artifacts. Employing well-defined gradient pulse duration and mixing/diffusion times in our diffusion preparation, we show that iPFG retains salient features of traditional multiple-PFG (mPFG) sequence while overcoming some of its implementation issues thereby extending its applications beyond DTD MRI. We assume DTD is a maximum entropy tensor-variate normal distribution whose tensor random variables are constrained to be positive definite (CNTVD) to ensure their physicality. In each voxel, the second-order mean and fourth-order covariance tensors of the DTD are estimated using a Monte Carlo method that synthesizes micro-diffusion tensors with corresponding size, shape and orientation distributions to best fit the measured DWIs. From these tensors we obtain the mean diffusivity (MD) spectrum, spectrum of diffusion tensor shapes, microscopic orientation distribution function (µODF), and microscopic fractional anisotropy (µFA) which disentangle the underlying heterogeneity within a voxel. Using DTD derived µODF, we introduce a new method to perform fiber tractography capable of resolving complex fiber configurations. The results obtained in the live human brain showed microscopic anisotropy in various gray and white matter regions and skewed MD distribution in cerebellar gray matter not observed previously. DTD MRI tractography captured complex white matter fiber organization consistent with known anatomy. DTD MRI also resolved some degeneracies associated with diffusion tensor imaging (DTI) and identified the source of microscopic anisotropy which may help improve the diagnosis of various neurological diseases and disorders.