bluuebunny/crossref_metadata_2025 huggingface dataset:
The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release
Authors: Jabbour Lea , Nguyen Trang , Gadet Rudy , Lohez Olivier , Mikaelian Ivan , Gonzalo Philippe , Luyten Thomas , Chalabi-Dcha Mounira , Bultynck Geert , Rimokh Ruth , Gillet Germain , Popgeorgiev Nikolay
Published: January 2021

Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localization and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localization is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we showed that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs demonstrated a reduced expression of the Unfolded Protein Response (UPR) markers upon ER stress and displayed reduced inositol trisphosphate receptor (IP3R)-mediated ER calcium release. Collectively, our data show that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the ER, apart from its cliché regulation of apoptosis.
PICASSO: Ultra-multiplexed fluorescence imaging of biomolecules through single-round imaging and blind source unmixing
Authors: Seo Junyoung , Sim Yeonbo , Kim Jeewon , Kim Hyunwoo , Cho In , Yoon Young-Gyu , Chang Jae-Byum
Published: January 2021

Ultra-multiplexed fluorescence imaging of biomolecules is essential to studying heterogeneous biological systems. However, this is challenging due to fluorophores’ spectral overlap and variation of the emission spectra. Here, we propose a strategy termed PICASSO, which enables more than 15-colour multiplexed imaging of thick tissue slices through a single imaging process and blind unmixing without reference spectra measurement. We show that PICASSO can be used to achieve a high multiplexing capability in diverse applications, such as 3D protein imaging, expansion microscopy, tissue clearing, imaging of clinical specimens, and cyclic immunofluorescence imaging. PICASSO only requires an equal number of images as the number of fluorophores, enabling such a high level of multiplexed imaging even with bandpass filter-based microscopy. As such, PICASSO would be a useful tool for the study of cancer, the immune system, and the brain, as well as for the diagnosis of cancer, as it enables ultra-multiplexed imaging of diverse specimens with minimum instrumental requirements and experimental processes.
Cooperativity boosts affinity and specificity of proteins with multiple RNA-binding domains
Authors: Stitzinger Simon H. , Sohrabi-Jahromi Salma , Söding Johannes
Published: January 2021

Numerous cellular processes rely on the binding of proteins with high affinity to specific sets of RNAs. Yet most RNA binding domains display low specificity and affinity, to the extent that for most RNA-binding domains, the enrichment of the best binding motif measured by high-throughput RNA SELEX or RNA bind-n-seq is usually below 10-fold, dramatically lower than that of DNA-binding domains. Here, we develop a thermodynamic model to predict the binding affinity for proteins with any number of RNA-binding domains given the affinities of their isolated domains. For the four proteins in which affinities for individual domains have been measured the model predictions are in good agreement with experimental values. The model gives insight into how proteins with multiple RNA-binding domains can reach affinities and specificities orders of magnitude higher than their individual domains. Our results contribute towards resolving the conundrum of missing specificity and affinity of RNA binding proteins and underscore the need for bioinformatic methods that can learn models for multi-domain RNA binding proteins from high-throughputin-vitroandin-vivoexperiments.
The MEMIC: Anex vivosystem to model the complexity of the tumor microenvironment
Authors: Janská Libuše , Anandi Libi , Kirchberger Nell C. , Marinkovic Zoran S. , Schachtner Logan T. , Guzelsoy Gizem , Carmona-Fontaine Carlos
Published: January 2021

There is an urgent need for accurate, scalable, and cost-efficient models of the complexity and heterogeneity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printedex vivomodel of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC can provide novel insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells can sense gradual changes in metabolite concentration, and tune intracellular cell signaling to form multicellular spatial patterns of cell proliferation. We also show that ischemic macrophages reduce epithelial features in neighboring tumor cells highlighting the power of this system to study cell-cell interactions and non-cell autonomous effects of the metabolic microenvironment. We propose that the MEMIC can be easily adapted to study early development, ischemic stroke, and other systems where multiple cell types interact within heterogeneous environments.
Leaf-to-whole plant spread bioassay for pepper andRalstonia solanacearuminteraction determines inheritance of resistance to bacterial wilt for further breeding
Authors: Kwon Ji-Su , Nam Jae-Young , Yeom Seon-In , Kang Won-Hee
Published: January 2021

Bacterial wilt (BW) disease byRalstonia solanacearumis a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance toR. solanacearumin peppers.Capsicum annuum‘MC4’ displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible ‘Subicho’. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F1and 90 F2progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.
Abolished miR158 activity leads to 21-nucleotide tertiary phasiRNA biogenesis that targetsNHX2inArabidopsis thaliana
Authors: Tripathi Abhinandan Mani , Singh Rajneesh , Singh Akanksha , Verma Ashwani Kumar , Mishra Parneeta , Narayan Shiv , Shirke Pramod Arvind , Roy Sribash
Published: January 2021

Small RNAs including microRNAs (miRNAs) are short 20-24-nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Some small RNAs, mostly 22-nucleotide long trigger biogenesis of secondary small interfering RNAs (siRNAs). Those siRNAs having distinctive phased configuration are known as phased siRNAs (phasiRNAs) and act either incisortransenhancing silencing cascade. Here, we report natural variants ofMIR158having deletions or insertions led to negligible or reduced expression of miR158. The deletion/insertion events affected processing of primary transcript of miR158 to precursor and to mature miR158. We show that miR158 targets a pseudo-pentatricopeptide gene and its abolished activity led to 21-nucleotide tertiary phasiRNA generation from its target. The biogenesis of these phasiRNAS is triggered byTAS2derived two siRNAs. Accordingly, small RNA analyses of these natural variants, mutants and over-expression lines ofMIR158or its target exhibited enhanced or reduced phasiRNA biogenesis. Finally, we functionally validated the highest expressed tertiary phasiRNA that targetsNHX2thereby regulating transpiration and stomatal conductance. Overall, we deciphered a new module of small RNA network, miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2, suggesting an additional layer of gene regulation and larger role of pseudogene in plants.
Improved spatio-temporal measurements of visually-evoked fields using optically-pumped magnetometers
Authors: Gialopsou Aikaterini , Abel Christopher , James Timothy M. , Coussens Thomas , Bason Mark G. , Puddy Reuben , Di Lorenzo Francesco , Rolfs Katharina , Voigt Jens , Sander Tilmann , Cercignani Mara , Krüger Peter
Published: January 2021

Recent developments in performance and practicality of optically pumped magnetometers have enabled new capabilities in non-invasive brain function mapping through magnetoencephalography. In particular the lack of need of cryogenic operating conditions allows for more flexible placement of the sensor heads closer to the brain surface, leading to improved spatial measurement resolution and increased source localisation capabilities. Through the recording of visually evoked brain fields (VEF), we demonstrate that the closer sensor proximity can be further exploited to improve the temporal resolution. We use optically pumped magnetometers (OPMs), and for reference superconducting quantum interference devices (SQUIDs), to measure brain responses to standard flash and pattern reversal stimuli. We find highly reproducible signals with consistency across multiple healthy participants, stimulus paradigms and sensor modalities. The temporal resolution advantage of OPMs is manifest in a fourfold improvement of the ratio of magnetic signal peak height to temporal width, compared to SQUIDs. The resulting capability of improved spatio-temporal signal tracing is illustrated by simultaneous vector recordings of VEFs in the primary (V1) and associative (V2) visual cortex, where a time lag on the order of 10-20 ms is consistently found. This paves the way for further studies of spatio-temporal neurophysiological signal tracking in visual stimulus processing and other brain responses with potentially far-reaching consequences for time-critical mapping of functionality in the healthy and pathological brains.
Natural diversity of honey bee (Apis mellifera) gut bacteriome in various climatic and seasonal states
Authors: Papp Márton , Békési László , Farkas Róbert , Makrai László , Maróti Gergely , Tőzsér Dóra , Solymosi Norbert
Published: January 2021

As pollinators and producers of numerous human consumed products, honey bees have great ecological, economic and health importance. The composition of their bacteriota, for which the available knowledge is limited, is essential for their body’s functioning. Based on our survey, we performed a metagenomic analysis of samples collected by repeated sampling. We used geolocations that represent the climatic types of the study area over two nutritionally extreme periods (March and May) of the collection season. In bacteriome composition, significant (p=0.002) difference was found between the samples from March and May. The samples’ bacteriome from March showed a significant (p=0.02) composition difference between cooler and the warmer regions. However, there were no significant bacteriome composition differences among the climatic classes of samples taken in May. Based on our results, one may conclude that the composition of healthy core bacteriome in honey bees varies depending on the climatic and seasonal conditions. This is likely due to climatic factors and vegetation states determining the availability and nutrient content of flowering plants. The results of our study prove that in order to gain a thorough understanding of a microbiome’s natural diversity, we need to obtain the necessary information from extreme ranges within the host’s health state.
DROSOPHILAMTOR COMPLEX 2 PRESERVES MITOCHONDRIAL AND CARDIAC FUNCTION UNDER HIGH FAT DIET TREATMENT
Authors: Chang Kai , Requejo Figueroa Guillermo A. , Bai Hua
Published: January 2021

High fat diet (HFD)-associated lipotoxicity is one of the major causes of cardiovascular diseases. The mechanistic target of rapamycin (mTOR) pathway, especially mTOR complex 1 (mTORC1), has been previously implicated in HFD-induced heart dysfunction. In the present study, we find that unlike mTORC1, mTOR complex 2 (mTORC2) protects hearts from HFD-induced cardiomyopathy and mitochondrial dysfunction inDrosophila. We show that HFD feeding induces contractile dysfunction along with altered mitochondrial morphology and function. Upon HFD feeding, the mitochondria of cardiomyocytes exhibit fragmentation, loss of membrane potential, and calcium overload. Interestingly, HFD feeding also reduces the activity of cardiac mTORC2. In line with this finding, the flies with cardiac-specific knockdown ofrictor, the key subunit of mTORC2, show cardiac and mitochondrial dysfunction similar to what is observed in HFD-fed wild-type flies. Conversely, cardiac-specific activation of mTORC2 by overexpressingrictorattenuates HFD-induced mitochondrial and cardiac dysfunction. Thus, our findings suggest that mTORC2 is a cardioprotective factor and regulates mitochondrial homeostasis upon HFD feeding.
Social and physical environment independently affect oviposition decisions inDrosophila melanogaster
Authors: Churchill Emily R. , Dytham Calvin , Bridle Jon R. , Thom Michael D.F.
Published: January 2021

In response to environmental stimuli, including variation in the presence of conspecifics, animals show highly plastic responses in behavioural and physiological traits influencing reproduction. These responses have been extensively documented in males, but equivalent study of females is so far lacking. We expect females to be highly responsive to environmental variation, with significant impacts on fitness given females’ direct impact on offspring number, size, and developmental conditions. UsingDrosophila melanogasteras a model, we manipulate (a) exposure to conspecific females, expected to influence their expectation of number of potential mates and larval density for their own offspring, and (b) test how prior consexual population density interacts with the spatial distribution of potential oviposition sites, with females expected to prefer clustered food resources that can support a larger number of eggs and larvae. After exposure to competition, females were slower to start copulating and reduced their copulation duration – the opposite effect to that observed in males previously exposed to rivals. There was a parallel and perhaps related effect on egg production, with females previously housed in groups laying fewer eggs than those that were housed in solitude. The spatial distribution of resources also influenced oviposition behaviour: females clearly preferred aggregated patches of substrate, being more likely to lay, and laying on more of the available patches, in the clustered environment. However, we found no significant interaction between prior housing conditions and resource patchiness, indicating that females did not perceive the value of different resource distributions differently when they were expecting either high or low levels of larval competition. While exposure to consexual competition influences copulatory behaviours, it is the distribution of oviposition resources that has a greater impact on oviposition decisions.
In vitroassessment of an engineered tBID-based safety switch system in human T lymphocytes
Authors: Lu Jiamiao , Collins Patrick , Lee Ki Jeong , Li Chi-Ming , Wang Songli
Published: January 2021

Cell therapy as a promising therapeutic modality to treat cancer has been intensively studied for decades. However, the clinical trials have indicated that patients under T cell therapy may develop severe cytokine release syndrome resulting in hospitalization or even death. Furthermore, genetic modifications to promote proliferation and persistence of T cells could result in high numbers of long-lived engineered cells in patients after treatment. In this study we developed a novel tBID-based safety switch regulated through a small molecule inducible on switch to control undesired toxicity or ablate engineered cells as needed. We compared several tBID-based safety switch constructs with the clinically validated safety switches, HSV-TK (human herpes simplex virus thymidine kinase) and iCasp9 (inducible Caspase 9), few tBID-based safety switch constructs were systematically tested and investigatedin vitroin Jurkat or human primary T cells. We demonstrated that our novel tBID based safety switch, with optimization, was able to eliminate up to ~90% of transduced human primary T cells within 72hr after activation, providing an alternative switch system to manage safety concerns for cell therapy.
Speed-related but not detrended gait variability increases with more sensitive self-paced treadmill controllers at multiple slopes
Authors: Castano Cesar R. , Huang Helen J.
Published: January 2021

Self-paced treadmills are being used more frequently to study humans walking with their self-selected gaits on a range of slopes. There are multiple options to purchase a treadmill with a built-in controller, or implement a custom written self-paced controller, which raises questions about how self-paced controller affect treadmill speed and gait biomechanics on multiple slopes. This study investigated how different self-paced treadmill controller sensitivities affected gait parameters and variability on decline, level, and incline slopes. We hypothesized that increasing self-paced controller sensitivity would increase gait variability on each slope. We also hypothesized that detrended variability could help mitigate differences in variability that arise from differences in speed fluctuations created by the self-paced controllers. Ten young adults walked on a self-paced treadmill using three controller sensitivities (low, medium, and high) and fixed speeds at three slopes (decline, −10°; level, 0°; incline, +10°). Within each slope, average walking speeds and spatiotemporal gait parameters were similar regardless of self-paced controller sensitivity. With higher controller sensitivities on each slope, speed fluctuations, speed variance, and step length variance increased whereas step frequency variance and step width variance were unaffected. Detrended variance was not affected by controller sensitivity suggesting that detrending variability helps mitigate differences associated with treadmill speed fluctuations. Speed-trend step length variances, however, increased with more sensitive controllers. Further, detrended step length variances were similar for self-paced and fixed speed walking, whereas self-paced walking included substantial speed-trend step length variance not present in fixed speed walking. In addition, regardless of the self-paced controller, subjects walked fastest on the level slope with the longest steps, narrowest steps, and least variance. Overall, our findings suggest that separating gait variability into speed-trend and detrended variability could be beneficial for interpreting gait variability among multiple self-paced treadmill studies and when comparing self-paced walking with fixed speed walking.
Caspase3-deficient cells require fibronectin for protection against autophagy-dependent death
Authors: Weir David B. , Boise Lawrence H.
Published: January 2021

Caspases are required for execution of apoptosis. However, in their absence, signals that typically induce apoptosis can still result in cell death. Our laboratory previously demonstrated that Casp3-deficient mouse embryonic fibroblasts (MEFs) have increased fibronectin (FN) secretion, and an adhesion-dependent survival advantage compared to wild type (WT) MEFs. Here, we show that FN is required for survival of Casp3-deficient MEFs following serum withdrawal. Furthermore, when FN is silenced, serum withdrawal-induced death is caspase-independent. However, procaspase-7 is cleaved, suggesting that MOMP is taking place. Indeed, in the absence of FN, cytochromecrelease is increased following serum withdrawal in Casp3-deficient MEFs. Yet death does not correspond to cytochromecrelease in Casp3-deficient MEFs. This is true both in the presence and absence of FN. Additionally, caspase-independent death is inhibited by Bcl-XLoverexpression. These findings suggest that Bcl-XLis not inhibiting death through regulation of Bax/Bak insertion into the mitochondria, but through a different mechanism. One such possibility is autophagy and induction of autophagy is associated with caspase-independent death in Casp3-deficient cells. Importantly, when ATG5 is ablated in Casp3-deficient cells, autophagy is blocked and death is largely inhibited. Taken together, our data indicate that Casp3-deficient cells incapable of undergoing canonical serum withdrawal-induced apoptosis, are protected from autophagy-dependent death by FN-mediated adhesion.
Genome and transcriptome of a pathogenic yeast,Candida nivariensis
Authors: Fan Yunfan , Gale Andrew N , Bailey Anna , Barnes Kali , Colotti Kiersten , Mass Michal , Morina Luke B , Robertson Bailey , Schwab Remy , Tselepidakis Niki , Timp Winston
Published: January 2021

We present a highly contiguous genome and transcriptome of the pathogenic yeast,Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes fromSaccharomyces cerevisiaeandCandida glabrata.
Modulating short-term auditory memory with focal transcranial direct current stimulation applied to the Supramarginal Gyrus
Authors: Lerud Karl D. , Vines Bradley W. , Shinde Anant B. , Schlaug Gottfried
Published: January 2021

Previous studies have shown that transcranial direct current stimulation (tDCS) can affect performance by decreasing regional excitability in a brain region that contributes to the task of interest. To our knowledge, no research to date has found both enhancing and diminishing effects on performance, depending upon which polarity of current is applied. The supramarginal gyrus (SMG) is an ideal brain region for testing tDCS effects because it is easy to identify using the 10-20 EEG coordinate system, and results of neuroimaging studies have implicated the left SMG in short-term memory for phonological and non-phonological sounds. In the present study, we found that applying tDCS to the left SMG affected pitch memory in a manner that depended upon the polarity of stimulation: cathodal tDCS had a negative impact on performance while anodal tDCS had a positive impact. These effects were significantly different from sham stimulation, which did not influence performance; they were also specific to the left hemisphere – no effect was found when applying cathodal stimulation to the right SMG – and were unique to pitch memory as opposed to memory for visual shapes. Our results provide further evidence that the left SMG is a nodal point for short-term auditory storage and demonstrate the potential of tDCS to influence cognitive performance, and to causally examine hypotheses derived from neuroimaging studies.
Sauna-like conditions or menthol treatment reduce tau phosphorylation through mild hyperthermia
Authors: Guisle Isabelle , Pétry Séréna , Morin Françoise , Kérauden Rémi , Whittington Robert A. , Calon Frédéric , Hébert Sébastien S. , Planel Emmanuel
Published: January 2021

In Alzheimer’s disease (AD), hyper-phosphorylation and aggregation of tau correlates with clinical progression and represents a valid therapeutic target. A recent 20-year prospective study revealed an association between moderate to high frequency of Finnish sauna bathing and a lower incidence of dementia and AD, but the molecular mechanisms underlying these benefits remain uncertain. Here, we tested the hypothesis that sauna-like conditions could lower tau phosphorylation by increasing body temperature. We observed a decrease in tau phosphorylation in wild-type and hTau mice as well as in neuron-like cells when exposed to higher temperatures. These effects were correlated with specific changes in phosphatase and kinase activities, but not with inflammatory or heat-shock responses. We also used a drug strategy to promote thermogenesis. Topical application of menthol led to a sustained increase in body temperature in hTau mice concomitant with a significant decrease in tau phosphorylation. Our results suggest that sauna-like conditions or menthol treatment could lower tau pathology through mild hyperthermia, and may provide promising therapeutic strategies for AD and other tauopathies.
Discovery of an IKK2 Site that Allosterically Controls Its Activation
Authors: Hotchkiss Sonjiala Jackson , Mulero Maria Carmen , Chan Garrett J. , Biswas Tapan , Polley Smarajit , Ohn Christine , Konduri Srihari , Siegel Dionicio , Demir Özlem , Amaro Rommie E. , Ghosh Gourisankar
Published: January 2021

IκB kinase 2/β (IKK2) is a critical regulator of inflammation which is inducibly activated by a host of stimuli. Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Efforts to prevent these diseases by small-molecule inhibitors of IKK2 activity have not been successful. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxicin vivodue to their off-target effects. Here we focused on identifying inhibitors to block IKK2 activity from an allosteric site, not the ATP-binding pocket. Using virtual screening, we first identified several candidate allosteric sites and screened for potential small-molecule binders, and then selected candidates inhibitory to IKK2 activity using cell-based functional assays. Hydrogen deuterium exchange coupled to mass-spectrometry (HDX-MS) and MS-MS assays revealed that a class of benzoyl conjugates of pyrrolidinedione covalently bound to a site located at the interface of the kinase domain (KD) and the helical domain (SDD), and inhibited IKK2 activation allosterically by preventing phosphorylation of its activation loop serines. Additionally, this class of inhibitor partially blocks IKK2’s catalytic activity by enhancing dynamics within the ATP binding pocket and likely the general active site. Hydrogen deuterium exchange (HDX) experiments further revealed that while binding of substrate ATP perturbs only the local structure surrounding its binding site, binding to ATP-competitive or allosteric inhibitors induces structural perturbations in an expansive area including the helical domain. We propose that these allosteric sites can act as specific targets for the development of novel potent IKK inhibitors.

SIGNIFICANCE

Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxicin vivodue to their off-target effects. By combination of virtual screening and cell-based functional assays, we identified small-molecule binders of the class of benzoyl conjugates of pyrrolidinedione that block IKK2 activity from an allosteric site through covalent attachment and could be specific only for IKK2. HDX-MS and MS-MS assays identified a binding pocket with a ‘Cys-Cys motif’ for these inhibitors, and revealed specific differences in IKK2 dynamics upon binding to substrate ATP vs ATP-competitive and allosteric inhibitors. Present work provides a framework for the development of allosteric inhibitors to combat IKK2-induced diseases inhibitors.
Revealing the human mucinome
Authors: Malaker Stacy A. , Riley Nicholas M. , Shon D. Judy , Pedram Kayvon , Krishnan Venkatesh , Dorigo Oliver , Bertozzi Carolyn R.
Published: January 2021

Mucin domains are densely O-glycosylated modular protein domains found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are key players in a host of human diseases, especially cancer, but the scope of the mucinome remains poorly defined. Recently, we characterized a bacterial mucinase, StcE, and demonstrated that an inactive point mutant retains binding selectivity for mucins. In this work, we leveraged inactive StcE to selectively enrich and identify mucins from complex samples like cell lysate and crude ovarian cancer patient ascites fluid. Our enrichment strategy was further aided by an algorithm to assign confidence to mucin-domain glycoprotein identifications. This mucinomics platform facilitated detection of hundreds of glycopeptides from mucin domains and highly overlapping populations of mucin-domain glycoproteins from ovarian cancer patients. Ultimately, we demonstrate our mucinomics approach can reveal key molecular signatures of cancer fromin vitroandex vivosources.
SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies elicited by ancestral Spike vaccines
Authors: Shen Xiaoying , Tang Haili , McDanal Charlene , Wagh Kshitij , Fischer Will , Theiler James , Yoon Hyejin , Li Dapeng , Haynes Barton F. , Sanders Kevin O. , Gnanakaran Sandrasegaram , Hengartner Nick , Pajon Rolando , Smith Gale , Dubovsky Filip , Glenn Gregory M. , Korber Bette , Montefiori David C.
Published: January 2021

The SARS-CoV-2 Spike glycoprotein mediates virus entry and is a major target for neutralizing antibodies. All current vaccines are based on the ancestral Spike with the goal of generating a protective neutralizing antibody response. Several novel SARS-CoV-2 variants with multiple Spike mutations have emerged, and their rapid spread and potential for immune escape have raised concerns. One of these variants, first identified in the United Kingdom, B.1.1.7 (also called VUI202012/01), contains eight Spike mutations with potential to impact antibody therapy, vaccine efficacy and risk of reinfection. Here we employed a lentivirus-based pseudovirus assay to show that variant B.1.1.7 remains sensitive to neutralization, albeit at moderately reduced levels (~2-fold), by serum samples from convalescent individuals and recipients of two different vaccines based on ancestral Spike: mRNA-1273 (Moderna), and protein nanoparticle NVX-CoV2373 (Novavax). Some monoclonal antibodies to the receptor binding domain (RBD) of Spike were less effective against the variant while others were largely unaffected. These findings indicate that B.1.1.7 is not a neutralization escape variant that would be a major concern for current vaccines, or for an increased risk of reinfection.
Cortical representation variability aligns with in-class variances and can help one-shot learning
Authors: Shang Jiaqi , Shea-Brown Eric , Mihalas Stefan
Published: January 2021

Learning invariance across a set of transformations is an important step in mapping high-dimensional inputs to a limited number of classes. After understanding the set of\ invariances, can a new class be learned from one element? We propose a representation which can facilitate such learning: if the variability in representing individual elements across trials aligns with the variability among different elements in a class, then class boundaries learned from the variable representations of one element should be representative of the entire class. In this study, we test whether such a representation occurs in mouse visual systems. We use Neuropixels probes recording single unit activity in mice observing 200 repeats of natural movies taken from a set of 9 continuous clips. We observe that the trial-by-trial variability in the representation of individual frames is well aligned to the variability in representation of multiple frames from the same clip, but not well aligned to the variability among frames from different clips. Thus, the variable representations of images in the mouse cortex can be efficiently used to classify images into their clips. We compare these representations to those in artificial neural networks. We find that, when introducing noise in networks trained for classification (both feed-forward and recurrent networks), the variability in the representation of elements aligns with the in-class variance. The networks which best reproduce the in-vivo observed directions of variability were those trained on a hierarchical classification task. Taken together, these results point to a solution which the cortex can use for one-shot learning of a class: by using noise as a mechanism for generalization. This is a potential computational explanation for the high level of noise observed in the cortex.
A Human Accelerated Region is a Leydig cellGLI2Enhancer that Affects Male-Typical Behavior
Authors: Norman Andrew R. , Ryu Ann H. , Jamieson Kirsty , Thomas Sean , Shen Yin , Ahituv Nadav , Pollard Katherine S. , Reiter Jeremy F.
Published: January 2021

Human accelerated regions (HARs) are sequences that have evolved at an accelerated rate in the human lineage. Some HARs are developmental enhancers. We used a massively parallel reporter assay (MPRA) to identify HARs with enhancer activity in a mammalian testis cell line. A subset of HARs exhibited differential activity between the human and chimpanzee orthologs, representing candidates for underlying unique human male reproductive biology. We further characterized one of these candidate testis enhancers, 2xHAR.238. CRISPR/Cas9-mediated deletion in a testis cell line and mice revealed that 2xHAR.238 enhances expression ofGli2, encoding a Hedgehog pathway effector, in testis Leydig cells. 4C-seq revealed that 2xHAR.238 contacts theGli2promoter, consistent with enhancer function. In adult male mice, deletion of 2xHAR.238 disrupted mouse male-typical behavior and male interest in female odor. Combined, our work identifies a HAR that promotes the expression ofGli2in Leydig cells and may have contributed to the evolution of human male reproductive biology.
Complexity and graded regulation of neuronal cell type-specific alternative splicing revealed by single-cell RNA sequencing
Authors: Feng Huijuan , Moakley Daniel F. , Chen Shuonan , McKenzie Melissa G. , Menon Vilas , Zhang Chaolin
Published: January 2021

The enormous neuronal cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell type-specific gene regulatory programs at the molecular level. Although prevalent in the brain, contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single cell RNA-sequencing (scRNA-seq) data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class, consisting of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2 and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs drive splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which provides a molecular mechanism orthogonal to, rather than downstream of, transcriptional regulation in specifying neuronal identity and function.

Significance

Alternative splicing (AS) is extensively used in the mammalian brain, but its contribution to the molecular and cellular diversity across neuronal cell types remains poorly understood. Through systematic and integrative analysis of AS regulation across over 100 transcriptomically defined cortical neuronal types, we found neuronal subclass-specific splicing regulatory programs consists of overlapping alternative exons showing differential splicing at multiple hierarchical levels. This graded AS regulation is controlled by unique combinations of RNA-binding proteins (RBPs). Importantly, these RBPs also drive splicing dynamics across neuronal cell types that do not conform to the hierarchical taxonomy established based on transcriptional profiles, suggesting that the graded AS regulation provides a molecular mechanism orthogonal to transcriptional regulation in specifying neuronal identity and function.
Proteomic portraits reveal evolutionarily conserved and divergent responses to spinal cord injury
Authors: Skinnider Michael A. , Rogalski Jason , Tigchelaar Seth , Manouchehri Neda , Prudova Anna , Jackson Angela M. , Nielsen Karina , Jeong Jaihyun , Chaudhary Shalini , Shortt Katelyn , Gallagher-Kurtzke Ylonna , So Kitty , Fong Allan , Gupta Rishab , Okon Elena B. , Rizzuto Michael A. , Dong Kevin , Streijger Femke , Belanger Lise , Ritchie Leanna , Tsang Angela , Christie Sean , Mac-Thiong Jean-Marc , Bailey Christopher , Ailon Tamir , Charest-Morin Raphaele , Dea Nicholas , Wilson Jefferson R. , Dhall Sanjay , Paquette Scott , Street John , Fisher Charles G. , Dvorak Marcel F. , Shannon Casey , Borchers Christoph , Balshaw Robert , Foster Leonard J. , Kwon Brian K.
Published: January 2021

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of CSF and serum samples from 111 acute SCI patients and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at six months with an AUC of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response, and establish the CSF abundance of glial fibrillary acidic protein (GFAP) as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.
Crop modeling defines opportunities and challenges for drought escape, water capture, and yield increase using chilling-tolerant sorghum
Authors: Raymundo Rubí , Sexton-Bowser Sarah , Ciampitti Ignacio A. , Morris Geoffrey
Published: January 2021

Many crop species, particularly those of tropical origin, are chilling sensitive so improved chilling tolerance can enhance production of these crops in temperate regions. For the cereal crop sorghum (Sorghum bicolor L.) early planting and chilling tolerance have been investigated for >50 years, but the potential value or tradeoffs of this genotype × management change has not been formally evaluated with modeling. To assess the potential of early-planted chilling-tolerant grain sorghum in the central US sorghum belt, we conducted CERES-Sorghum simulations and characterized scenarios under which this change would be expected to enhance (or diminish) drought escape, water capture, and yield. We conducted crop growth modeling for full- and short-season hybrids under rainfed systems that were simulated to be planted in very early (April), early (May 15), and normal (June 15) planting dates over 1986–2015 in four locations in Kansas representative of the central US sorghum belt. Simulations indicated that very early planting will generally lead to lower initial soil moisture, longer growing periods, and higher evapotranspiration. Very early planting is expected to extend the growing period by 20% for short- or full-season hybrids, reduce evaporation during fallow periods, and increase plant transpiration in the two-thirds of years with the highest precipitation (mean > 428 mm), leading to 11% and 7% increase grain yield for short- and full-season hybrids, respectively. Thus, in this major sorghum growing region very early and early planting could reduce risks of terminal droughts, extend seasons, and increase rotation options, suggesting that further development of chilling tolerant hybrids is warranted.
Nodal Modulator is required to sustain endoplasmic reticulum morphology
Authors: Amaya Catherine , Cameron Christopher JF , Devarkar Swapnil C. , Gerstein Mark B. , Xiong Yong , Schlieker Christian
Published: January 2021

Nodal Modulator (NOMO) is a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We identified NOMO1 in a proteomics approach aimed at the identification of proteins that support the structural integrity of the endoplasmic reticulum (ER). Overexpression of NOMO1 imposes a sheet morphology on the ER, while depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network. These structures are positive for the autophagy marker LAMP1, and LC3 is profoundly upregulated upon NOMO depletion.In vitroreconstitution of NOMO1 revealed a dimeric state that is mediated by the cytosolic tail domain, with each monomer featuring a “beads on a string” structure likely representing bacterial Ig-like folds. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.
Modeling mutational effects on biochemical phenotypes using convolutional neural networks: application to SARS-CoV-2
Authors: Wang Bo , Gamazon Eric R.
Published: January 2021

Biochemical phenotypes are major indexes for protein structure and function characterization. They are determined, at least in part, by the intrinsic physicochemical properties of amino acids and may be reflected in the protein three-dimensional structure. Modeling mutational effects on biochemical phenotypes is a critical step for understanding protein function and disease mechanism as well as enabling drug discovery. Deep Mutational Scanning (DMS) experiments have been performed on SARS-CoV-2’s spike receptor binding domain and the human ACE2 zinc-binding peptidase domain – both central players in viral infection and evolution and antibody evasion - quantifying how mutations impact binding affinity and protein expression. Here, we modeled biochemical phenotypes from massively parallel assays, using convolutional neural networks trained on protein sequence mutations in the virus and human host. We found that neural networks are significantly predictive of binding affinity, protein expression, and antibody escape, learning complex interactions and higher-order features that are difficult to capture with conventional methods from structural biology. Integrating the intrinsic physicochemical properties of amino acids, including hydrophobicity, solvent-accessible surface area, and long-range non-bonded energy per atom, significantly improved prediction (empirical p<0.01) though there was such a strong dependence on the sequence data alone to yield reasonably good prediction. We observed concordance of the DMS data and our neural network predictions with an independent study on intermolecular interactions from molecular dynamics (multiple 500 ns or 1 μs all-atom) simulations of the spike protein-ACE2 interface, with critical implications for the use of deep learning to dissect molecular mechanisms. The mutation- or genetically-determined component of a biochemical phenotype estimated from the neural networks has improved causal inference properties relative to the original phenotype and can facilitate crucial insights into disease pathophysiology and therapeutic design.
AnnoMiner: a new web-tool to integrate epigenetics, transcription factor occupancy and transcriptomics data to predict transcriptional regulators
Authors: Meiler Arno , Marchiano Fabio , Weikunat Michaela , Schnorrer Frank , Habermann Bianca H.
Published: January 2021

Gene expression regulation requires precise transcriptional programs, led by transcription factors in combination with epigenetic events. Recent advances in epigenomic and transcriptomic techniques provided insight into different gene regulation mechanisms. However, to date it remains challenging to understand how combinations of transcription factors together with epigenetic events control cell-type specific gene expression. We developed the AnnoMiner web-server and introduce an innovative and flexible way to annotate and integrate epigenetic, and transcription factor occupancy data. First, AnnoMiner annotates user-provided peaks with gene features. Second, AnnoMiner can integrate genome binding data from two different transcriptional regulators together with gene features. Third, AnnoMiner offers to explore the transcriptional deregulation of 10 genes nearby a user-provided peak. AnnoMiner’s fourth function performs transcription factor or histone mark enrichment analysis for user-provided gene lists by utilizing hundreds of public, high-quality datasets from ENCODE for the model organisms human, mouse,DrosophilaandC. elegans. Thus, AnnoMiner can predict transcriptional regulators for a studied process without the strict need for chromatin data from the same process. We compared AnnoMiner to existing tools and experimentally validated several transcriptional regulators predicted by AnnoMiner to indeed contribute to muscle morphogenesis inDrosophila. AnnoMiner is freely available athttp://chimborazo.ibdm.univ-mrs.fr/AnnoMiner/.