bluuebunny/crossref_metadata_2025 huggingface dataset:
Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics
Authors: Nonaka Hiroshi , Mino Takeharu , Sakamoto Seiji , Oh Jae Hoon , Watanabe Yu , Ishikawa Mamoru , Tsushima Akihiro , Amaike Kazuma , Kiyonaka Shigeki , Tamura Tomonori , Aricescu A. Radu , Kakegawa Wataru , Miura Eriko , Yuzaki Michisuke , Hamachi Itaru
Published: December 2021

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed “Fixation-driven chemical crosslinking of exogenous ligands (FixEL)” which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.
Application of a simple device for “attracting” and “trapping” glioma cells in situ
Authors: Song Haimin , Yang Runwei , Lai Runbin , Guo Jinglin , Li Kaishu , Ni Bowen , Liu Yawei
Published: December 2021

Glioblastoma multiforme (GBM) is the most malignant adult brain tumor. The current adjuvant therapies for GBM are disappointing, which are based on cytotoxicity strategy. Thus, other ways should be explored to improve the curative effect. According to the strong invasive ability of GBM cells, we assume a new treatment strategy for GBM by developing a new cell trap device (CTD) with some kind of “attractive” medium loaded in it to attract and capture the tumor cells. The in vitro experiment showed that Hepatocyte Growth Factor (HGF) presented stronger chemotaxis on C6 and U87 cell line than the Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF). A simple in vitro CTD loaded with HGF was made and in vivo experiments results showed that HGF successfully attracted tumor cells from tumor bed in situ into the CTD. This study proposes the new strategy for GBM treatment of “attract and trap” tumor cells is proved to be feasible.
Depriving mice of sleep also deprives of food
Authors: Đukanović Nina , La Spada Francesco , Emmenegger Yann , Niederhäuser Guy , Preitner Frédéric , Franken Paul
Published: December 2021

Both sleep-wake behavior and circadian rhythms are tightly coupled to energy metabolism and food intake. Altered feeding times in mice are known to entrain clock-gene rhythms in brain and liver and sleep-deprived humans tend to eat more and gain weight. Previous observations in mice showing that sleep deprivation (SD) changes clock-gene expression might thus relate to altered food intake and not to the loss of sleepper se. Whether SD affects food intake in the mouse and how this might affect clock-gene expression is, however, unknown. We therefore quantified i) the cortical expression of the clock genesPer1, Per2, Dbp, andCry1in mice that had access to food or not during a 6h SD, and ii) food intake during baseline, SD, and recovery sleep. We found that food deprivation did not modify the SD-incurred clock-gene changes in the cortex. Moreover, we discovered that although food intake during SD did not differ from baseline, mice lost weight and increased food intake during subsequent recovery. We conclude that SD is associated with food deprivation and that the resulting energy deficit might contribute to the effects of SD that are commonly interpreted as a response to sleep loss.
Intrinsic apoptosis is evolutionarily divergent among metazoans
Authors: Krasovec Gabriel , Quéinnec Éric , Chambon Jean-Philippe
Published: December 2021

Apoptosis is regulated cell death that depends on caspases. Upstream of each apoptotic signalling pathway is involved a specific initiator caspase. Characterised in nematode, fly and mammals, intrinsic apoptosis is considered to be ancestral and conserved among animals, and depends on shared initiators; caspase-9, Apaf-1 and Bcl-2. However, the biochemical role of mitochondria, the pivotal function of cytochrome c and the modality of caspase activation remain highly heterogeneous and hide profound molecular divergences among apoptotic pathways in animals. Uncovering the phylogenetic history of apoptotic actors, especially caspases, is crucial to shed light on intrinsic apoptosis evolutionary history. Here, we demonstrate by phylogenetic analyses, that caspase-9, the fundamental key of intrinsic apoptosis, is deuterostome-specific, while caspase-2 is ancestral to bilaterians. Our analysis of Bcl-2 and Apaf-1 confirm heterogeneity in functional organisation of apoptotic pathways in animals. Our results support emergence of distinct intrinsic apoptotic pathways during metazoan evolution.
Sex, amitosis, and evolvability in the ciliate Tetrahymena thermophila
Authors: Tarkington Jason , Zhang Hao , Azevedo Ricardo B. R. , Zufall Rebecca A.
Published: December 2021

Understanding the mechanisms that generate genetic variation, and thus contribute to the process of adaptation, is a major goal of evolutionary biology. Mutation and genetic exchange have been well studied as mechanisms to generate genetic variation. However, there are additional processes that may also generate substantial genetic variation in some populations and the extent to which these variation generating mechanisms are themselves shaped by natural selection is still an open question. Tetrahymena thermophila is a ciliate with an unusual mechanism of nuclear division, called amitosis, which can generate genetic variation among the asexual descendants of a newly produced sexual progeny. We hypothesize that amitosis thus increases the evolvability of newly produced sexual progeny relative to species that undergo mitosis. To test this hypothesis, we used experimental evolution and simulations to compare the rate of adaptation in T. thermophila populations founded by a single sexual progeny to parental populations that had not had sex in many generations. The populations founded by a sexual progeny adapted more quickly than parental populations in both laboratory populations and simulated populations. This suggests that the additional genetic variation generated by amitosis of a heterozygote can increase the rate of adaptation following sex and may help explain the evolutionary success of the unusual genetic architecture of Tetrahymena and ciliates more generally.
Explainable autoencoder-based representation learning for gene expression data
Authors: Yu Yang , Kossinna Pathum , Li Qing , Liao Wenyuan , Zhang Qingrun
Published: December 2021

Modern machine learning methods have been extensively utilized in gene expression data analysis. In particular, autoencoders (AE) have been employed in processing noisy and heterogenous RNA-Seq data. However, AEs usually lead to “black-box” hidden variables difficult to interpret, hindering downstream experimental validation and clinical translation. To bridge the gap between complicated models and biological interpretations, we developed a tool, XAE4Exp (eXplainable AutoEncoder for Expression data), which integrates AE and SHapley Additive exPlanations (SHAP), a flagship technique in the field of eXplainable AI (XAI). It quantitatively evaluates the contributions of each gene to the hidden structure learned by an AE, substantially improving the expandability of AE outcomes. By applying XAE4Exp to The Cancer Genome Atlas (TCGA) breast cancer gene expression data, we identified genes that are not differentially expressed, and pathways in various cancer-related classes. This tool will enable researchers and practitioners to analyze high-dimensional expression data intuitively, paving the way towards broader uses of deep learning.

Availability

Open source at https://github.com/QingrunZhangLab/Explainable-Deep-Autoencoder.

Contacts

qingrun.zhang@ucalgary.ca and wliao@ucalgary.ca.
Early exposure to UV radiation causes telomere shortening and poorer condition later in life
Authors: Lundsgaard Niclas U. , Cramp Rebecca L. , Franklin Craig E.
Published: December 2021

Determining the contribution of elevated ultraviolet-B radiation (UVBR; 280 – 315 nm) to amphibian population declines is being hindered by a lack of knowledge about how different acute UVBR exposure regimes during early life history stages might affect post-metamorphic stages via long-term carryover effects. We acutely exposed tadpoles of the Australian green tree frog (Litoria caerulea) to a combination of different UVBR irradiances and doses in a multi-factorial experiment, and then reared them to metamorphosis in the absence of UVBR to assess carryover effects in subsequent juvenile frogs. Dose and irradiance of acute UVBR exposure influenced carryover effects into metamorphosis in somewhat opposing manners. Higher doses of UVBR exposure in larvae yielded improved rates of metamorphosis. However, exposure at a high irradiance resulted in frogs metamorphosing smaller in size and in poorer condition than frogs exposed to low and medium irradiance UVBR as larvae. We also demonstrate some of the first empirical evidence of UVBR-induced telomere shortening in vivo, which is one possible mechanism for life-history trade-offs impacting condition post-metamorphosis. These findings contribute to our understanding of how acute UVBR exposure regimes in early life affect later life-history stages, which has implications for how this stressor may shape population dynamics.

SUMMARY STATEMENT

We demonstrate physiological carryover effects in amphibians that link larval UV exposure to detrimental impacts on juvenile frogs, including telomere shortening, which has implications for how UV shapes amphibian populations.
Plant growth promoting activity of bacteria isolated from Asian rice (Oryza sativa L.) are plant subspecies dependent
Authors: Balasjin Nasim Maghboli , Maki James S. , Schläppi Michael R. , Marshall Christopher W.
Published: December 2021

Asian rice is one of the most important crops because it is a staple food for almost half of the world’s population. Rice has two subspecies, JAPONICA and INDICA. To have production of rice keep pace with a growing world population, it is anticipated that the use of fertilizers will also need to increase, which may cause environmental damage through runoff impacts. An alternative strategy to increase crop yield is the use of plant growth promoting bacteria. Thousands of microbial species can exist in association with plant roots and shoots, and some are critical to the plant’s survival. We isolated 140 bacteria from rice and investigated whether JAPONICA and INDICA rice subspecies were positively influenced by these isolates. The bacterial isolates were screened for their ability to solubilize phosphate, a known plant growth promoting characteristic, and 25 isolates were selected for further analysis. These 25 phosphate solubilizing isolates were also able to produce other potentially growth-promoting factors. Five of the most promising bacterial isolates were chosen for whole genome sequencing. Four of these bacteria, isolates related to Pseudomonas mosselii, Microvirga sp., Paenibacillus rigui and Paenibacillus graminis, improved root and shoot growth, root to shoot ratio, and increased root dry weights of JAPONICA plants but had no effect on growth and development of INDICA plants. This indicates that while bacteria have several known plant growth promoting functions, their effects on growth parameters can be plant subspecies dependent and suggest close relationships between plants and their microbial partners.
Neural correlates of perceiving and interpreting engraved prehistoric patterns as human production: effect of archaeological expertise
Authors: Salagnon M , Cremona S , Joliot M , d’Errico F , Mellet E
Published: December 2021

It has been suggested that engraved abstract patterns dating from the Middle and Lower Palaeolithic served as means of representation and communication. Identifying the brain regions involved in visual processing of these engravings can provide insights into their function. In this study, brain activity was measured during perception of the earliest known Palaeolithic engraved patterns and compared to natural patterns mimicking human-made engravings. Participants were asked to categorise marks as being intentionally made by humans or due to natural processes (e.g. erosion, root etching). To simulate the putative familiarity of our ancestors with the marks, the responses of expert archaeologists and control participants were compared, allowing characterisation of the effect of previous knowledge on both behaviour and brain activity in perception of the marks. Besides a set of regions common to both groups and involved in visual analysis and decision-making, the experts exhibited greater activity in the inferior part of the lateral occipital cortex, ventral occipitotemporal cortex, and medial thalamic regions. These results are consistent with those reported in visual expertise studies, and confirm the importance of the integrative visual areas in the perception of the earliest abstract engravings. The attribution of a natural rather than human origin to the marks elicited greater activity in the salience network in both groups, reflecting the uncertainty and ambiguity in the perception of, and decision-making for, natural patterns. The activation of the salience network might also be related to the process at work in the attribution of an intention to the marks. The primary visual area was not specifically involved in the visual processing of engravings, which argued against its central role in the emergence of engraving production.
Continental-scale hyperspectral tree species classification in the United States National Ecological Observatory Network
Authors: Marconi Sergio , Weinstein Ben. G. , Zou Sheng , Bohlman Stephanie A. , Zare Alina , Singh Aditya , Stewart Dylan , Harmon Ira , Steinkraus Ashley , White Ethan P.
Published: December 2021

Advances in remote sensing imagery and machine learning applications unlock the potential for developing algorithms for species classification at the level of individual tree crowns at unprecedented scales. However, most approaches to date focus on site-specific applications and a small number of taxonomic groups. Little is known about how well these approaches generalize across broader geographic areas and ecosystems. Leveraging field surveys and hyperspectral remote sensing data from the National Ecological Observatory Network (NEON), we developed a continental-extent model for tree species classification that can be applied to the network, including a wide range of US terrestrial ecosystems. We compared the performance of a model trained with data from 27 NEON sites to models trained with data from each individual site, evaluating advantages and challenges posed by training species classifiers at the US scale. We evaluated the effect of geographic location, topography, and ecological conditions on the accuracy and precision of species predictions (72 out of 77 species). On average, the general model resulted in good overall classification accuracy (micro-F1 score), with better accuracy than site-specific classifiers (average individual tree level accuracy of 0.77 for the general model and 0.70 for site-specific models). Aggregating species to the genus-level increased accuracy to 0.83. Regions with more species exhibited lower classification accuracy. Predicted species were more likely to be confused with congeneric and co-occurring species and confusion was highest for trees with structural damage and in complex closed-canopy forests. The model produced accurate estimates of uncertainty, correctly identifying trees where confusion was likely. Using only data from NEON, this single integrated classifier can make predictions for 20% of all tree species found in forest ecosystems across the entire US, which make up to roughly 90% of the upper canopy of the studied ecosystems. This suggests the potential for integrating information from multiple datasets and locations to develop broad scale general models for species classification from hyperspectral imaging.
Multi-Pathway DNA Double-Strand Break Repair Reporters Reveal Extensive Cross-Talk Between End-Joining, Single Strand Annealing, and Homologous Recombination
Authors: van de Kooij Bert , Kruswick Alex , van Attikum Haico , Yaffe Michael B.
Published: December 2021

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to extensive mutagenesis and genomic loss. Repair pathway cross-talk and compensation within the DSB-repair network is incompletely understood, despite its importance for genomic stability, oncogenesis, and the outcome of genome editing by CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple distinct pathways to repair of a DSB. These reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibition of the essential c-NHEJ factor DNA-PKcs not only increases repair by HR, but also results in a substantial increase in mutagenic repair by SSA. We show that SSA-mediated repair of Cas9-generated DSBs can occur between Alu elements at endogenous genomic loci, and is enhanced by inhibition of DNA-PKcs. Finally, we demonstrate that the short-range end-resection factors CtIP and Mre11 promote both SSA and HR, whereas the long-range end-resection factors DNA2 and Exo1 promote SSA, but reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the rapid and comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.
ModelRevelator: Fast phylogenetic model estimation via deep learning
Authors: Burgstaller-Muehlbacher Sebastian , Crotty Stephen M. , Schmidt Heiko A , Drucks Tamara , Haeseler Arndt von
Published: December 2021

Selecting the best model of sequence evolution for a multiple-sequence-alignment (MSA) constitutes the first step of phylogenetic tree reconstruction. Common approaches for inferring nucleotide models typically apply maximum likelihood (ML) methods, with discrimination between models determined by one of several information criteria. This requires tree reconstruction and optimisation which can be computationally expensive. We demonstrate that neural networks can be used to perform model selection, without the need to reconstruct trees, optimise parameters, or calculate likelihoods.

We introduce ModelRevelator, a model selection tool underpinned by two deep neural networks. The first neural network, NNmodelfind, recommends one of six commonly used models of sequence evolution, ranging in complexity from Jukes and Cantor to General Time Reversible. The second, NNalphafind, recommends whether or not a Γ--distributed rate heterogeneous model should be incorporated, and if so, provides an estimate of the shape parameter, α. Users can simply input an MSA into ModelRevelator, and swiftly receive output recommending the evolutionary model, inclusive of the presence or absence of rate heterogeneity, and an estimate of α.

We show that ModelRevelator performs comparably with likelihood-based methods and the recently published machine learning method ModelTeller over a wide range of parameter settings, with significant potential savings in computational effort. Further, we show that this performance is not restricted to the alignments on which the networks were trained, but is maintained even on unseen empirical data. We expect that ModelRevelator will provide a valuable alternative for phylogeneticists, especially where traditional methods of model selection are computationally prohibitive.
Treated like dirt: Robust forensic and ecological inferences from soil eDNA after challenging sample storage
Authors: Frøslev Tobias Guldberg , Ejrnæs Rasmus , Hansen Anders J. , Bruun Hans Henrik , Nielsen Ida Broman , Ekelund Flemming , Vestergård Mette , Kjøller Rasmus
Published: December 2021

Biodiversity of soil microbiota is routinely assessed with environmental DNA-based methods, among which amplification and massive parallel sequencing of marker genes (eDNA metabarcoding) is the most common. Soil microbiota may for example be investigated in relation to biodiversity research or as a tool in forensic investigations.

After sampling, the taxonomic composition of soil biotic communities may change. In order to minimize community changes after sampling, it is desirable to reduce biological activity, e.g. by freezing immediately after sampling. However, this may be impossible due to remoteness of study sites or, in forensic cases, where soil has been attached to a questioned item for protracted periods of time.

Here we investigated the effect of storage duration and conditions on the assessment of the soil biota with eDNA metabarcoding. We extracted eDNA from freshly collected soil samples and again from the same samples after storage under contrasting temperature conditions.

We used five different primer sets targeting bacteria, fungi, protists (cercozoans), general eukaryotes, and plants. For these groups, we quantified differences in richness, evenness and community composition. Subsequently, we tested whether we could correctly infer habitat type and original sample identity after storage using a large reference dataset.

We found increased community composition differences with extended storage time and with higher storage temperature. However, for samples stored less than 28 days at a maximum of 20°C, changes were generally insignificant. Classification models could successfully assign most stored samples to their exact location of origin and correct habitat type even after weeks of storage. Even samples showing larger compositional changes generally retained the original sample as the best match (relative similarity).

Our results show that for most biodiversity and forensic applications, storage of samples for days and even several weeks may not be a problem, if storage temperature does not exceed 20°C. Even after suboptimal storage conditions, significant patterns can be reproduced.
Enhanced microvasculature formation and patterning in iPSC–derived kidney organoids cultured in physiological hypoxia
Authors: Schumacher A. , Roumans N. , Rademakers T. , Joris V. , Eischen-Loges M. , van Griensven M. , LaPointe V.L.S.
Published: December 2021

Functional kidney organoids have the potential to be used in implantable kidney grafts for patients with end-stage kidney disease, because they have been shown to self-organize from induced pluripotent stem cells into most important renal structures. To date, however, long-term kidney organoid culture has not succeeded, as nephrons lose their phenotype after approximately 25 days. Furthermore, the renal structures remain immature with diminishing endothelial networks with low connectivity and limited organoid invasion. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25 days and evaluated nephrogenesis, VEGF-A expression and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of proangiogenic VEGF-A189 mRNA and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids.

Significance statement

Culturing kidney organoids in a hypoxic environment improved patterning of the endothelial network and improved vascularization. These findings may help improve the quality of kidney organoids, and could eventually improve the kidney graft for transplantation in patients with end-stage kidney disease. Furthermore, the organoids will be more suitable for drug testing and in developmental biology. The findings might also be translatable to other organoid models containing endothelial cells.

Graphical abstract
Type I Interferon Signaling Drives Microglial Dysfunction and Senescence in Human iPSC Models of Down Syndrome and Alzheimer’s Disease
Authors: Jin Mengmeng , Xu Ranjie , Wang Le , Alam Mahabub Maraj , Ma Ziyuan , Zhu Sining , Martini Alessandra C. , Jadali Azadeh , Bernabucci Matteo , Xie Ping , Kwan Kelvin , Pang Zhiping P. , Head Elizabeth , Liu Ying , Hart Ronald P. , Jiang Peng
Published: December 2021

Microglia are critical for brain development and play a central role in Alzheimer’s disease (AD) etiology. Down syndrome (DS), also known as trisomy 21, is the most common genetic origin of intellectual disability and the most common risk factor for AD. Surprisingly, little information is available on the impact of trisomy of human chromosome 21 (Hsa21) on microglia in DS brain development and AD in DS (DSAD). Using our new induced pluripotent stem cell (iPSC)-based human microglia-containing cerebral organoid and chimeric mouse brain models, here we report that DS microglia exhibit enhanced synaptic pruning function during brain development. Consequently, electrophysiological recordings demonstrate that DS microglial mouse chimeras show impaired synaptic functions, as compared to control microglial chimeras. Upon being exposed to human brain tissue-derived soluble pathological tau, DS microglia display dystrophic phenotypes in chimeric mouse brains, recapitulating microglial responses seen in human AD and DSAD brain tissues. Further flow cytometry, single-cell RNA- sequencing, and immunohistological analyses of chimeric mouse brains demonstrate that DS microglia undergo cellular senescence and exhibit elevated type I interferon signaling after being challenged by pathological tau. Mechanistically, we find that shRNA-mediated knockdown of Hsa21encoded type I interferon receptor genes,IFNARs, rescues the defective DS microglial phenotypes both during brain development and in response to pathological tau. Our findings provide firstin vivoevidence supporting a paradigm shifting theory that human microglia respond to pathological tau by exhibiting accelerated senescence and dystrophic phenotypes. Our results further suggest that targeting IFNARs may improve microglial functions during DS brain development and prevent human microglial senescence in DS individuals with AD.
Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish
Authors: Uszczynska-Ratajczak Barbara , Sugunan Sreedevi , Kwiatkowska Monika , Migdal Maciej , Carbonell-Sala Silvia , Sokol Anna , Winata Cecilia L. , Chacinska Agnieszka
Published: December 2021

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.
Study of anFBXO7patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria
Authors: Al Rawi Sara , Simpson Lorna , McDonald Neil Q. , Chernuha Veronika , Elpeleg Orly , Zeviani Massimo , Barker Roger A. , Spiegel Ronen , Laman Heike
Published: December 2021

Mutations inFBXO7have been discovered associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerization domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, L250P patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts directly with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts as an adaptor enabling SCFFbxo7ligase to ubiquitinate MiD49. Thus, the L250P mutation changes the function of Fbxo7 by altering its substrate repertoire. Although MiD49/51 expression was reduced in L250P patient cells, there was no effect on the mitochondrial network. However, patient cells had higher levels of ROS and reduced viability under stress. Our study shows that Fbxo7 and PI31 affect each other’s functions in regulating both proteasomal and mitochondrial function and demonstrate a new function for PI31, as an adaptor for the SCFFbxo7E3 ubiquitin ligase.
Ureter single-cell and spatial mapping reveal cell types, architecture, and signaling networks
Authors: Fink Emily E. , Sona Surbhi , Tran Uyen , Desprez Pierre-Emmanuel , Bradley Matthew , Qiu Hong , Eltemamy Mohamed , Wee Alvin , Wolkov Madison , Nicolas Marlo , Min Booki , Haber Georges-Pascal , Wessely Oliver , Lee Byron H. , Ting Angela H.
Published: December 2021

Tissue engineering offers a promising treatment strategy for ureteral strictures, but its success requires an in-depth understanding of the architecture, cellular heterogeneity, and signaling pathways underlying tissue regeneration. Here we define and spatially map cell populations within the human ureter using single-cell RNA sequencing, spatial gene expression, and immunofluorescence approaches. We focused on the stromal and urothelial cell populations to enumerate distinct cell types composing the human ureter and inferred potential cell-cell communication networks underpinning the bi-directional crosstalk between these compartments. Furthermore, we analyzed and experimentally validated the importance of Sonic Hedgehog (SHH) signaling pathway in adult stem cell maintenance. The SHH-expressing basal cells supported organoid generation in vitro and accurately predicted the differentiation trajectory from basal stem cells to terminally differentiated umbrella cells. Our results highlight essential processes involved in adult ureter tissue homeostasis and provide a blueprint for guiding ureter tissue engineering.
The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans
Authors: Daiß Julia L. , Pilsl Michael , Straub Kristina , Bleckmann Andrea , Höcherl Mona , Heiss Florian B. , Abascal-Palacios Guillermo , Ramsay Ewan , Tlučková Katarina , Mars Jean-Clement , Bruckmann Astrid , Bernecky Carrie , Lamour Valérie , Panov Konstantin , Vannini Alessandro , Moss Tom , Engel Christoph
Published: December 2021

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This ‘dock II’ domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.
Heterogeneity and genomic loci of ubiquitous Cre reporter transgenes in zebrafish
Authors: Lalonde Robert L. , Kemmler Cassie L. , Riemslagh Fréderike W. , Aman Andrew J. , Kresoja-Rakic Jelena , Moran Hannah R. , Nieuwenhuize Susan , Parichy David M. , Burger Alexa , Mosimann Christian
Published: December 2021

The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed,lox-basedSwitchreporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of thelox-flanked recombination cassettes. Here, we compare commonly usedSwitchreporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. Using different experimental setups, we show thatubi:Switchandhsp70l:Switchoutperform current generations of two additionalSwitchreporters due to favorable transgene integration sites. Our comparisons also document preferential Cre-dependent recombination ofubi:Switchandhsp70l:Switchin distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility andloxrecombination efficiency in highly functionalSwitchlines, we mapped these transgenes and charted chromatin dynamics at their integration sites. Our data documents the heterogeneity amonglox-basedSwitchtransgenes towards informing suitable transgene selection for lineage labeling experiments. Our work further proposes thatubi:Switchandhsp70l:Switchdefine genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.
A dual-purpose real-time indicator and transcriptional integrator for calcium detection in living cells
Authors: Erdenee Elbegduuren , Ting Alice Y.
Published: December 2021

Calcium is a ubiquitous second messenger in eukaryotes, correlated with neuronal activity and T-cell activation among other processes. Real-time calcium indicators such as GCaMP have recently been complemented by newer calcium integrators that convert transient calcium activity into stable gene expression. Here we introduce LuCID, a dual-purpose real-time calcium indicator and transcriptional calcium integrator that combines the benefits of both calcium detection technologies. We show that the calcium-dependent split luciferase component of LuCID provides real-time bioluminescence readout of calcium dynamics in cells, while the GI/FKF1 split GAL4 component of LuCID converts calcium-generated bioluminescence into stable gene expression. We also show that LuCID’s modular design enables it to also read out other cellular events such as protein-protein interactions. LuCID adds to the arsenal of tools for studying cells and cell populations that utilize calcium for signaling.
An analytical solution simulating growth of Lewy bodies
Authors: Kuznetsov Ivan A. , Kuznetsov Andrey V.
Published: December 2021

This paper reports a minimal model simulating the growth of a Lewy body (LB). To the best of our knowledge, this is the first model simulating Lewy body growth. The LB is assumed to consist of a central spherical core, which is composed of membrane fragments and various dysfunctional intracellular organelles, and a halo, which is composed of alpha-synuclein fibrils. Membrane fragments and alpha-synuclein monomers are assumed to be produced in the soma at constant rates. The growth of the core and the halo are simulated by the Finke-Watzky model. Analytical (closed-form) solutions describing the growth of the core and the halo are obtained. A sensitivity analysis in terms of model parameters is performed.
A Lattice Model on the Rate of DNA Hybridization
Authors: Murugan R.
Published: December 2021

We develop a lattice model on the rate of hybridization of the complementary single-stranded DNAs (c-ssDNAs). Upon translational diffusion mediated collisions, c-ssDNAs interpenetrate each other to form correct (cc), incorrect (icc) and trap-correct contacts (tcc) inside the reaction volume. Correct contacts are those with exact registry matches which leads to nucleation and zipping. Incorrect contacts are the mismatch contacts which are less stable compared to tcc which can occur in the repetitive c-ssDNAs. Although tcc possess registry match within the repeating sequences, they are incorrect contacts in the view of the whole c-ssDNAs. The nucleation rate (kN) is directly proportional to the collision rate and the average number of correct-contacts () formed when both the c-ssDNAs interpenetrate each other. Detailed lattice model simulations suggest that 〈ncc〉 ∝L/VwhereLis the length of c-ssDNAs andVis the reaction volume. Further numerical analysis revealed the scaling for the average radius of gyration of c-ssDNAs (Rg) with their length as. Since the reaction space will be approximately a sphere with radius equals to 2RgandV∝L3/2, one obtains. When c-ssDNAs are nonrepetitive, then the overall renaturation rate becomes askR∝kNLand one finally obtainsin line with the experimental observations. When c-ssDNAs are repetitive with a complexity ofc, then earlier models suggested the scalingwhich breaks down atc=L. This clearly suggested the existence of at least two different pathways of renaturation in case of repetitive c-ssDNAs viz. via incorrect contacts and trap correct contacts. The trap correct contacts can lead to the formation of partial duplexes which can keep the complementary strands in the close vicinity for a prolonged timescale. This is essential for the extended 1D slithering, inchworm movements and internal displacement mechanisms which can accelerate the searching for the correct contacts. Clearly, the extent of slithering dynamics will be inversely proportional to the complexity. When the complexity is close to the length of c-ssDNAs, then the pathway via incorrect contacts will dominate. When the complexity is much lesser than the length of c-ssDNA, then pathway via trap correct contacts would be the dominating one.

PACS

87.10.-e; 87.14.gk; 82.39.Pj; 87.15.R-
External validation of six clinical models for prediction of unknown chronic kidney disease in a German population
Authors: Stolpe Susanne , Kowall Bernd , Zwanziger Denise , Frank Mirjam , Jöckel Karl-Heinz , Erbel Raimund , Stang Andreas
Published: December 2021

Background

Chronic kidney disease (CKD) is responsible for large personal health and societal burdens. Screening populations at higher risk for CKD is effective to initiate earlier treatment and decelerate disease progress. We externally validated clinical prediction models for unknown CKD that might be used in population screening.

Methods

We validated six risk models for prediction of unknown CKD using only non-invasive parameters. Validation data came from 4,185 participants of the German Heinz-Nixdorf-Recall study (HNR), drawn in 2000 from a general population aged 45-75 years. We estimated discrimination and calibration using the full model information, and calculated the diagnostic properties applying the published scoring algorithms of the models using various thresholds for the sum of scores.

Results

The risk models used four to nine parameters. Age and hypertension were included in all models. Five out of six c-values ranged from 0.71 to 0.73, indicating fair discrimination. Positive predictive values ranged from 15% to 19%, negative predictive values were >93% using score thresholds that resulted in values for sensitivity and specificity above 60%.

Conclusions

Most of the selected CKD prediction models show fair discrimination in a German general population. The estimated diagnostic properties indicate that the models are suitable for identifying persons at higher risk for unknown CKD without invasive procedures.
Infant malnutrition in low- and middle-income countries: assessment and prevalence of small and nutritionally at-risk infants aged under 6 months in 54 Demographic & Health Survey datasets
Authors: Kerac Marko , James Philip T , McGrath Marie G , Brennan Eilise , Opondo Charles , Frison Severine
Published: December 2021

Background

There is increasing global focus on malnutrition in infants aged under 6 months (u6m) but evidence on how best to identify and manage at-risk individuals is sparse. Our objectives were to: explore data quality of commonly used anthropometric indicators; describe prevalence and disease burden of infant u6m malnutrition; compare wasting and underweight as measures of malnutrition by determining the strength and consistency of associations with biologically plausible risk factors.

Methods

We performed a cross-sectional secondary analysis of Demographic and Health Survey (DHS) datasets, focussing on infants u6m. We calculated underweight (low weight-for-age), wasting (low weight-for-length), stunting (low length-for-age), and concurrent wasting and stunting. We explored data quality by recording extreme (flagged, as per standard criteria) or missing values. We calculated the population-weighted prevalence of each type of malnutrition and extrapolated the burden to all low- and middle-income countries (LMICs). We explored associations between infant, maternal and household risk factors with underweight and wasting using logistic regression models.

Results

We analysed 54 DHS surveys. Data quality in terms of refusals and missingness was similar for both weight and length. There were more extreme (flagged) values for length-based measures (6.1% flagged for weight-for-length, 4.8% for length-for-age) than for weight-for-age (1.0% flagged). Overall, 20.1% of infants (95% CI: 19.5, 20.7) were underweight, 21.3% (95% CI: 20.7, 22.3) were wasted, 17.6% (95% CI: 17.0, 18.2) were stunted, and 2.0% (95% CI: 1.8, 2.2) were concurrently wasted and stunted. This corresponds to an estimated burden in LMICs of 23.8m underweight infants, 24.5m wasted infants, 21.5m stunted infants and 2.2m concurrently wasted and stunted. Logistic regression models showed that numerous risk factors were associated with wasting and underweight. Effect sizes of risk factors tended to be stronger and more consistently associated with underweight compared to wasting.

Conclusion

Malnutrition in infants u6m is a major problem in LMICs. This is true whether assessed by underweight, wasting or stunting. Our data build on other evidence suggesting that underweight may be a better anthropometric case definition than wasting: data quality is better when length is not involved; biologically plausible risk factors are better reflected by an infant being underweight. Future research, ideally from intervention trials, should further explore how best to identify malnourished (small and nutritionally at-risk) infants u6m. For now, treatment programmes should note that many factors might underlie problems in this age group: services should thus consider how to address maternal health and wider social circumstances as well as caring for infants themselves.
Optimizing a High Performing Multiplex-CRISPRi P. putida Strain with Integrated Metabolomics and 13C-Metabolic Flux Analyses
Authors: Czajka Jeffrey J. , Banerjee Deepanwita , Eng Thomas , Menasalvas Javier , Yan Chunsheng , Munoz Nathalie Munoz , Poirier Brenton C. , Kim Young-Mo , Baker Scott E. , Tang Yinjie J. , Mukhopadhyay Aindrila
Published: December 2021

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C–metabolic flux analysis (13C–MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6–fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon (ΔphaAZC-IID) resulted in a 2.2–fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the ΔphaAZC-IID strain had 1.5–fold and 1.8–fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.
Limited, but potentially functional translation of non-coding transcripts in the HEK293T cellular cytosol
Authors: Bogaert Annelies , Fijalkowska Daria , Staes An , Van de Steene Tessa , Demol Hans , Gevaert Kris
Published: December 2021

Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins, while other studies failed to find evidence for such types of non-canonical translation products. Here, we attempted to discover translation products from non-coding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. Theoretically, we show that our strategy facilitates the detection of translation events of transcripts from non-coding regions, but experimentally only find 19 peptides (less than 1% of all identified peptides) that might originate from such translation events. Virotrap based interactome analysis of two N-terminal proteoforms originating from non-coding regions finally showed the functional potential of these novel proteins.