bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic, metabolic, and molecular insights into the diverse outcomes of diet-induced obesity in mice
Authors: Bachmann Alexis Maximilien , Morel Jean-David , El Alam Gaby , Rodríguez-López Sandra , de lima Tanes Imamura , Goeminne Ludger J.E. , Benegiamo Giorgia , Bou Sleiman Maroun , Auwerx Johan
Published: September 2021

Overweight and obesity are increasingly common public health issues worldwide, leading to a wide range of diseases from metabolic syndrome to steatohepatitis and cardiovascular diseases. While the increase in the prevalence of obesity is partly attributable to changes in lifestyle (i.e. increased sedentarity and changes in eating behaviour), the metabolic and clinical impacts of these obesogenic conditions varies between sexes and genetic backgrounds. The conception of personalised treatments of obesity and its complications require a thorough understanding of the diversity of responses to conditions such as high-fat diet intake. By analysing nine genetically diverse mouse strains, we show that much like humans, mice respond to high-fat diet in a genetic- and sex-dependent manner. Physiological and molecular responses to high-fat diet are associated with expression of genes involved in immunity and mitochondrial function. Finally, we find that mitochondrial function may explain part of the diversity of physiological responses. By exploring the complex interactions between genetics and metabolic phenotypes via gene expression and molecular traits, we shed light on the importance of genetic background and sex in determining metabolic outcomes. In addition to providing the community with an extensive resource for optimizing future experiments, our work serves as an exemplary design for more generalizable translational studies.
Modeling CRISPR-Cas13d on-target and off-target effects using machine learning approaches
Authors: Cheng Xiaolong , Li Zexu , Shan Ruocheng , Li Zihan , Chao Lumen , Peng Jian , Fei Teng , Li Wei
Published: September 2021

A major challenge in the application of the CRISPR-Cas13d (RfxCas13d, or CasRx) RNA editing system is to accurately predict its guide RNA (gRNA) dependent on-target and off-target effect. Here, we performed CRISPR-Cas13d proliferation screens that target protein-coding genes and long non-coding RNAs (lncRNAs), followed by a systematic modeling of Cas13d on-target efficiency and off-target viability effect. We first designed a deep learning model, named DeepCas13, to predict the on-target activity of a gRNA with high accuracy from its sequence and secondary structure. DeepCas13 outperforms existing methods and accurately predicts the efficiency of guides targeting both protein-coding and non-coding RNAs (e.g., circRNAs and lncRNAs). Next, we systematically studied guides targeting non-essential genes, and found that the off-target viability effect, defined as the unintended effect of guides on cell viability, is closely related to their on-target RNA cleavage efficiency. This finding suggests that these gRNAs should be used as negative controls in proliferation screens to reduce false positives, possibly coming from the unwanted off-target viability effect of efficient guides. Finally, we applied these models to our screens that included guides targeting 234 lncRNAs, and identified lncRNAs that affect cell viability and proliferation in multiple cell lines. DeepCas13 is freely accessible via http://deepcas13.weililab.org.
MOSCATO: A Supervised Approach for Analyzing Multi-Omic Single-Cell Data
Authors: Towle-Miller Lorin M , Miecznikowski Jeffrey C
Published: September 2021

Advancements in genomic sequencing continually improve personalized medicine in complex diseases. Recent breakthroughs generate multiple types of signatures (or multi-omics) from each cell, producing different data ‘omic’ types per single-cell experiment. We introduce MOSCATO, a technique for selecting features across multi-omic single-cell datasets that relate to clinical outcomes. For example, we leverage penalization concepts often used in multi-omic network analytics to accommodate the high-dimensionality where multiple-testing is likely underpowered. We organize the data into multi-dimensional tensors where the dimensions correspond to the different ‘omic’ types. Using the outcome and the single-cell tensors, we perform regularized tensor regression to return a variable set for each ‘omic’ type that forms the clinically-associated network. Robustness is assessed over simulations based on available single-cell simulation methods. Real data comparing healthy subjects versus subjects with leukemia is also considered in order to identify genes associated with the disease. The flexibility of our approach enables future extensions on distributional assumptions and covariate adjustments. This algorithm may identify clinically-relevant genetic patterns on a cellular-level that span multiple layers of sequencing data and ultimately inform highly precise therapeutic targets in complex diseases. Code to perform MOSCATO and replicate the real data application is publicly available on GitHub athttps://github.com/lorinmil/MOSCATOandhttps://github.com/lorinmil/MOSCATOLeukemiaExample.
KIRCLE: An Analysis of Variations in KIR Genes in The Cancer Genome Atlas and UK Biobank
Authors: Gao Galen F. , Liu Dajiang , Zhan Xiaowei , Li Bo
Published: September 2021

Background

Natural killer (NK) cells represent a critical component of the innate immune system’s response against cancer and viral infections, among other diseases. To distinguish healthy host cells from infected or tumor cells, killer immunoglobulin receptors (KIR) on NK cells bind and recognize Human Leukocyte Antigen (HLA) complexes on their target cells. Just like the HLAs they bind, these KIRs exhibit high allelic diversity in the human population.

Results

In order to better understand these immunoreceptors, we have developed KIRCLE, a novel method for genotyping individual KIR genes from whole exome sequencing data, and used it to analyze approximately 60,000 patient samples in The Cancer Genome Atlas and UK Biobank. We were able to assess population frequencies for different KIR alleles and demonstrate that, similar to HLA alleles, individuals’ KIR alleles correlate strongly with their ethnicities. In addition, we observed associations between different KIR alleles and HLA alleles, including HLA-B*53 with KIR3DL2*013 (Fisher’s Exact FDR = 7.64e-51). Finally, we showcased statistically significant associations between KIR alleles and various clinical correlates, including peptic ulcer disease (Fisher’s Exact FDR = 0.0429) and age of onset of atopy and various KIR alleles (Mann-Whitney-U FDR = 0.0751).

Conclusions

KIR polymorphism and NK cells play a critical role in many diseases, often through their interactions with HLA complexes. Peptic ulcer disease and atopy are just two diseases in which NK cells may play a role beyond their “classical” realm of anti-tumor and anti-viral responses.
Dockground scoring benchmarks for protein docking
Authors: Kotthoff Ian , Kundrotas Petras J. , Vakser Ilya A.
Published: September 2021

Protein docking protocols typically involve global docking scan, followed by re-ranking of the scan predictions by more accurate scoring functions that are either computationally too expensive or algorithmically impossible to include in the global scan. Development and validation of scoring methodologies are often performed on scoring benchmark sets (docking decoys) which offer concise and nonredundant representation of the global docking scan output for a large and diverse set of protein-protein complexes. Two such protein-protein scoring benchmarks were built for the Dockground resource, which contains various datasets for the development and testing of protein docking methodologies. One set was generated based on the Dockground unbound docking benchmark 4, and the other based on protein models from the Dockground model-model benchmark 2. The docking decoys were designed to reflect the reality of the real-case docking applications (e.g., correct docking predictions defined as near-native rather than native structures), and to minimize applicability of approaches not directly related to the development of scoring functions (reducing clustering of predictions in the binding funnel and disparity in structural quality of the near-native and non-native matches). The sets were further characterized by the source organism and the function of the protein-protein complexes. The sets, freely available to the research community on the Dockground webpage, present a unique, user-friendly resource for the developing and testing of protein-protein scoring approaches.
Genomic characterisation of an mcr-1 and mcr-3-producing Escherichia coli strain isolated from pigs in France
Authors: Hamame Afaf , Davoust Bernard , Rolain Jean-Marc , Diene Seydina M.
Published: September 2021

The current study is about genomic characterisation of an atypical multidrug-resistant Escherichia coli harbouring two mobilised colistin resistance (mcr) genes isolated from pigs in France. Stool samples taken from a pig farm in Avignon in the department of the Vaucluse were subjected to a molecular screening for the detection of mcr gene variants. These samples were cultured on selective LBJMR medium. Growing bacteria were identified using MALDI-TOF, followed by antibiotic susceptibility testing (AST). Whole genome sequencing (WGS) and bioinformatic genome analysis was performed. The selective culture of stools revealed the presence of an E. coli strain named Q4552 which was simultaneously positive for the mcr-1.1 and mcr-3.5 genes. This strain exhibited resistance phenotype to fourteen antibiotics, including colistin. Genome sequencing revealed a circular chromosome and eight plasmids. Genomic analysis revealed a chromosomic integration of a mobile genetic element (MGE) harbouring the mcr-1.1 gene, while the mcr-3.5 gene was plasmidic (i.e., an IncFII plasmid). Its resistome exhibited twenty-two resistance genes, explaining its multidrug resistance phenotype. The Q4552 strain is an ST-843 clone belonging to the clonal complex Cplx-568 and is the only ST type of this cplx-568 which has been isolated from animals, humans, and the environment. Here, we report the first co-occurrence of the mcr-1 and mcr-3 genes in France from a pathogenic E. coli strain isolated from a pig farm. Since this clone (ST-843) has been reported in zoonotic transmissions, programs to monitor such colistin resistant bacterium are urgently required to avoid its spread and zoonotic transmission to humans.
A large-volume sputum dry storage and transportation device for molecular and culture-based diagnosis of tuberculosis
Authors: Dsouza Andrea , Jangam Saylee , Naik Vishwanath , Manjula J. , Nair Chandrasekhar B. , Toley Bhushan J.
Published: September 2021

Technologies for preservation of specimens in the absence of cold chains are essential for optimum utilization of existing laboratory services in the developing world. We present a prototype called specimen transportation tube (SPECTRA-tube) for the collection, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 mL) specimens. Specimens introduced into SPECTRA-tube are dried in glass fiber membranes, which are critical for efficient liquid-state sample recovery by rehydration and centrifugation. Mycobacterium smegmatis (Msm)-spiked mock sputum dried in native Standard 17 glass fiber was stable for molecular testing after 10-day storage at 45°C, and for culture testing after 10- and 5-day storage at 37°C and 45°C, respectively. Compatibility with human sputum storage was demonstrated by dry storing Mycobacterium bovis-spiked pooled human sputum in SPECTRA-tube for 5 days at room temperature followed by successful qPCR detection. By significantly increasing the volume of samples that can be transported in the dry state and enabling recovery of the entire sample in liquid state, SPECTRA-tube presents a potential universal solution for the preservation and transportation of liquid specimens.
Numb is required for optimal contraction of skeletal muscle
Authors: De Gasperi Rita , Mo Chenglin , Azulai Daniella , Wang Zhiying , Harlow Lauren M. , Du Yating , Graham Zachary , Pan Jiangping , Liu Xin-hua , Guo Lei , Zhang Bin , Ko Fred , Raczkowski Ashleigh M , Bauman William A. , Goulbourne Chris N , Zhao Wei , Brotto Marco , Cardozo Christopher P.
Published: September 2021

Background

The role of Numb, a protein that is important for cell fate and development was investigated in adult skeletal muscle in mice using a conditional, inducible knockout (cKO) model.

Methods

Numb expression was evaluated by Western blot. Numb localization was determined by confocal microscopy. The effects of cKO of Numb and the closely-related gene Numb-like in skeletal muscle fibers was evaluated by in-situ physiology; transmission and focused ion beam scanning electron microscopy; 3-dimensional reconstruction of mitochondrial; lipidomics; and bulk RNA-sequencing. Additional studies using primary mouse myotubes investigated the effects the effects of Numb knockdown on cell fusion, mitochondrial function and calcium transients.

Results

Numb protein expression was reduced by ∼70% (p < 0.01) at 24 as compared to 3 months of age. Numb was localized within muscle fibers as bands traversing fibers at regularly spaced intervals in close proximity to dihydropyridine receptors.

The cKO of Numb and Numb-like reduced specific tetanic force by 36%, p < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (p < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (p < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1 and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acid (PUFAs) through lipoxygenases were upregulated in Numb cKO skeletal muscle; 12-HEPE was increased by ∼250% (p < 0.05) and 17,18-EpETE by ∼240% (p < 0.05). In mouse primary myotubes, Numb knock-down reduced cell fusion (∼20%, p < 0.01) and mitochondrial function and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (p < 0.01).

Conclusions

These findings implicate Numb as a critical factor in skeletal muscle structure and function which appear to be critical for calcium release; we therefore speculate Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.
GLS1 is a protective factor rather than a molecular target in ARID1A-mutated ovarian clear cell carcinoma
Authors: Clemente Valentino , Nelson Andrew , Erickson Britt , Baker Ruth , Rubin Nathan , Khalifa Mahmoud , Hoshino Asumi , Shetty Mihir , Lou Emil , Bazzaro Martina
Published: September 2021

Targeting glutamine metabolism has emerged as a novel therapeutic strategy for several human cancers, including ovarian cancer. The primary target of this approach is the kidney isoform of glutaminase, glutaminase 1 (GLS1), a key enzyme in glutamine metabolism that is overexpressed in several human cancers. A first-in-class inhibitor of GLS1, called CB839 (Telaglenastat), has been investigated in several clinical trials, with promising results. The first clinical trial of CB839 in platinum-resistant ovarian cancer patients is forthcoming. ARID1A-mutated ovarian clear cell carcinoma (OCCC) is a relatively indolent and chemoresistant ovarian cancer histotype. In OCCC-derived cells in vitro and mouse models, loss of ARID1A leads to upregulation of GLS1. Thus, targeting of GLS1 with CB839 has been suggested as a targeted approach for OCCC patients with tumors harboring ARID1A-mutations. Here, we investigated whether GLS1 is differentially expressed between OCCC patients whose tumors are ARID1A positive and patients whose tumors are ARID1A negative. In clinical specimens of OCCC, we found that GLS1 overexpression was not correlated with ARID1A loss. In addition, GLS1 overexpression was associated with better clinical outcomes. Our findings suggest that GLS1 expression in OCCC may be a protective factor and that caution should be taken when considering the use of CB839 to treat OCCC patients.
A separation-of-functionZIP4wheat mutant allows crossover between related chromosomes and is meiotically stable
Authors: Martín Azahara C. , Alabdullah Abdul Kader , Moore Graham
Published: September 2021

Many species, including most flowering plants, are polyploid, possessing multiple genomes. During polyploidisation, fertility is preserved via the evolution of mechanisms to control the behaviour of these multiple genomes during meiosis. On the polyploidisation of wheat, the major meiotic geneZIP4duplicated and diverged, with the resulting new geneTaZIP4-B2being inserted into chromosome 5B. Previous studies showed that thisTaZIP4-B2promotes pairing and synapsis between wheat homologous chromosomes, whilst suppressing crossover between related (homoeologous) chromosomes. Moreover, in wheat, the presence ofTaZIP4-B2preserves up to 50% of grain number. The present study exploits a ‘separation-of-function’ wheatTazip4-B2mutant namedzip4-ph1d, in which theTazip4-B2copy still promotes correct pairing and synapsis between homologues (resulting in the same pollen profile and fertility normally found in wild type wheat), but which also allows crossover between the related chromosomes in wheat haploids of this mutant. This suggests an improved utility for the newzip4-ph1dmutant line during wheat breeding exploitation, compared to the previously described CRISPRTazip4-B2andph1mutant lines. The results also reveal that loss of suppression of homoeologous crossover between wheat chromosomes does not in itself reduce wheat fertility when promotion of homologous pairing and synapsis byTaZIP4-B2is preserved.
Production of Offspring from Azoospermic Mice with Meiotic Failure: Precise Biparental Meiosis within Halved Oocytes
Authors: Ogonuki Narumi , Kyogoku Hirohisa , Hino Toshiaki , Osawa Yuki , Fujiwara Yasuhiro , Inoue Kimiko , Kunieda Tetsuo , Mizuno Seiya , Tateno Hiroyuki , Sugiyama Fumihiro , Kitajima Tomoya S. , Ogura Atsuo
Published: September 2021

While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest.
Metabolic modeling of the International Space Station microbiome reveals key microbial interactions
Authors: Kumar Rachita K. , Singh Nitin K. , Balakrishnan Sanjaay , Parker Ceth W. , Raman Karthik , Venkateswaran Kasthuri
Published: September 2021

Background

Recent studies have provided insights into the persistence and succession of microbes aboard the International Space Station (ISS), notably the dominance of Klebsiella pneumoniae. However, the interactions between the various microbes aboard the ISS, and how it shapes the microbiome remain to be clearly understood. In this study, we apply a computational approach to predict possible metabolic interactions in the ISS microbiome and shed further light on its organization.

Results

Through a combination of a systems-based graph-theoretical approach, and a constraint-based community metabolic modelling approach, we demonstrated several key interactions in the ISS microbiome. These complementary approaches provided insights into the metabolic interactions and dependencies present amongst various microbes in a community, highlighting key interactions and keystone species. Our results showed that the presence of K. pneumoniae is beneficial to many other microorganisms it coexists with, notably those from the Pantoea genus. Species belonging to the Enterobacteriaceae family were often found to be the most beneficial for the survival of other microorganisms in the ISS microbiome. However, K. pneumoniae was found to exhibit parasitic and amensalistic interactions with Aspergillus and Penicillium species, respectively. To prove this metabolic prediction, K. pneumoniae and Aspergillus fumigatus were co-cultured under normal and simulated microgravity, where K. pneumoniae cells showed parasitic characteristics to the fungus. The electron micrography revealed that the presence of K. pneumoniae compromised the morphology of fungal conidia and its biofilm biofilm-forming structures.

Conclusions

Our study underscores the importance of K. pneumoniae in the ISS, and its potential contribution to the survival (mutualism) and eradication (parasitism) of other microbes, including potential pathogens. This integrated modelling approach, combined with experiments, demonstrates immense potential for understanding the organization of other such microbiomes, unravelling key organisms and their interdependencies.
Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ
Authors: Mensa Bruk , Polizzi Nicholas F. , Molnar Kathleen , Natale Andrew , Lemmin Thomas , DeGrado William F.
Published: September 2021

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutants distant from the sensor or catalytic site strongly influence PhoQ’s ligand-sensitivity as well as the magnitude and direction of the signal, endowing diverse signaling characteristics without need for epistasis. Data from 35 mutants are explained by a semi-empirical 3-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations, that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively ‘kinase-on’ conformation, while the HAMP favors the ‘off’ state; when coupled, they create a bistable system responsive to physiological [Mg2+]. Mutants alter signaling by locally modulating these intrinsic equilibrium constants and couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.
Plasma derived cell-free mitochondrial DNA originates mainly from circulating cell-free mitochondria
Authors: Roch Benoit , Pisareva Ekaterina , Sanchez Cynthia , Pastor Brice , Tanos Rita , Mirandola Alexia , Mazard Thibault , Amir Dache Zahra Al , Thierry Alain R.
Published: September 2021

Circulating mitochondrial DNA (cir-mtDNA) could have a potential comparable to circulating nuclear DNA (cir-nDNA), with numerous applications. However, research and development in this area have fallen behind, particularly considering its origin and structural features. To tackle this, we initially combined Q-PCR and low-pass whole genome sequencing in the same analytical strategy previously and successfully used for cir-nDNA. This revealed unexplained structural patterns and led us to correlate these data with observations made during physical examinations such as filtration, and differential centrifugation in various plasma preparations. Both the integrity index and number of reads revealed a very minor proportion of low size-ranged fragments (<1000 bp) in plasma obtained with a standard preparation (0.06%). Filtration and high speed second step centrifugation revealed that 98.7 and 99.4% corresponded to extracellular mitochondria either free or in large extracellular vesicles. When avoiding platelet activation during plasma preparation, the proportion of both types of entities was still preponderant (76-80%), but the amount of detected mitochondrial DNA decreased 67-fold. In correlation with our previous study on the presence of circulating cell-free mitochondria in blood, our differential centrifugation procedure suggested that cir-mtDNA is also associated with approximately 18% small extracellular vesicles, 1.7% exosomes and 4% protein complexes.
Protein Structure Prediction Using a Maximum Likelihood Formulation of a Recurrent Geometric Network
Authors: Qi Guowei , Tollefson Mallory R. , Gogal Rose A. , Smith Richard J. H. , AlQuraishi Mohammed , Schnieders Michael J.
Published: September 2021

Only ∼40% of the human proteome has structural coordinates available from experiment (i.e., X-ray crystallography, NMR spectroscopy, or cryo-EM) or homology modeling with quality templates (i.e., 30% sequence identity or greater), leaving most of the proteome structurally unsolved. Deep learning (DL) methods for predicting protein structure can help close knowledge gaps where experimental and homology models are difficult to obtain. Recent advances in these DL methods have shown promising results in expanding structural coverage to the scale of the entire human proteome, providing researchers with more complete protein structural information. Here, we improve upon an existing DL algorithm for protein structure prediction, the Recurrent Geometric Network (RGN). We first expand the training dataset to include experimental uncertainty data in the form of atomic displacement parameters, then derive a maximum likelihood loss function that incorporates this uncertainty data into model training. Compared to the original RGN, our novel maximum likelihood model improves the rate of convergence of initial model training and ultimately results in more accurate structure prediction according to the root mean square deviation (RMSD) of backbone atoms, the Global Distance Test (GDT), the Global Distance Test High Accuracy (GDT-HA), and the Template-Modeling Score (TM-Score). Our model also predicts structures with more favorable backbone torsions, which provide more accurate starting coordinates for downstream physics-based simulations. Based on these results, our maximum likelihood reformulation provides a framework for improving existing or future machine learning algorithms for protein structure prediction. The augmented dataset, data collection scripts, reformulated RGN source code, and a series of trained models are publicly available at https://github.com/SchniedersLab/likelihood-rgn.
Enzyme Aggregation and Fragmentation Induced by Catalysis Relevant Species
Authors: Gentile Kayla , Bhide Ashlesha , Kauffman Joshua , Ghosh Subhadip , Maiti Subhabrata , Adair James , Lee Tae Hee , Sen Ayusman
Published: September 2021

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-Glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.
Native Metabolomics Identifies the Rivulariapeptolide Family of Protease Inhibitors
Authors: Reher Raphael , Aron Allegra T , Fajtová Pavla , Stincone Paolo , Liu Chenxi , Ben Shalom Ido Y , Bittremieux Wout , Wang Mingxun , Matos-Hernandez Marie L , Alexander Kelsey L , Caro-Diaz Eduardo J , Naman C Benjamin , Hughes Chambers C. , Dorrestein Pieter C , O’Donoghue Anthony J , Gerwick William H , Petras Daniel
Published: September 2021

The identity and biological activity of most metabolites still remain unknown. A key bottleneck in the full exploration of this tremendous source of new structures and pharmaceutical activities is the compound purification needed for bioactivity assignments of individual compounds and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we developed a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry, and simultaneous detection of protein binding via native mass spectrometry. While screening for new protease inhibitors from an environmental cyanobacteria community, native metabolomics revealed 30 cyclodepsipeptides as chymotrypsin binders. Mass spectrometry-guided purification then allowed for the full structure elucidation of four new specialized metabolites via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy. Together with the evaluation of biological activities, our results identified the rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as promising approach for drug discovery, chemical ecology, and chemical biology studies.
Aberrant localization of CDC42 C-terminal variants to the Golgi apparatus drives pyrin inflammasome-dependent autoinflammation
Authors: Nishitani-Isa Masahiko , Mukai Kojiro , Honda Yoshitaka , Nihira Hiroshi , Tanaka Takayuki , Shibata Hirofumi , Kodama Kumi , Hiejima Eitaro , Izawa Kazushi , Kawasaki Yuri , Osawa Mitsujiro , Katata Yu , Onodera Sachiko , Watanabe Tatsuya , Kure Shigeo , Takita Junko , Ohara Osamu , Saito Megumu K. , Nishikomori Ryuta , Taguchi Tomohiko , Sasahara Yoji , Yasumi Takahiro
Published: September 2021

Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Elevated levels of serum IL-18 in patients and effectiveness of IL-1β-blocking therapy indicate that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but no that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave way for elucidating the mechanism of pyrin inflammasome formation.
Chromosomal-level genome assembly of the bioluminescent cardinalfishSiphamia tubifer, an emerging model for symbiosis research
Authors: Gould AL , Henderson JB , Lam AW
Published: September 2021

The bioluminescent symbiosis between the sea urchin cardinalfishSiphamia tubifer(Kurtiformes: Apogonidae) and the luminous bacteriumPhotobacterium mandapamensisis an emerging vertebrate-bacteria model for the study of microbial symbiosis. However, there is little genetic data available for the host fish, limiting the scope of potential research that can be carried out with this association. In this study, we present a chromosomal-level genome assembly ofS. tubiferusing a combination of PacBio HiFi sequencing and Hi-C technologies. The final genome assembly was 1.2 Gb distributed on 23 chromosomes and contained 32,365 protein coding genes with a BUSCO completeness score of 99%. A comparison of theS. tubifergenome to that of another non-luminous cardinalfish revealed a high degree of synteny, whereas a similar comparison to a more distant relative in the Gobiiformes order revealed a fusion of two chromosomes in the cardinalfish genomes. An additional comparison of orthologous clusters among these three genomes revealed a set of 710 clusters that were unique toS. tubiferin which 23 GO pathways were significantly enriched, including several relating to host-microbe interactions and one involved in visceral muscle development, which could be related to the musculature involved in the gut-associated light organ ofS. tubifer. We also assembled the complete mitogenome ofS. tubiferand discovered both an inversion in the WANCY tRNA gene region resulting in a WACNY gene order as well as heteroplasmy in the length of the control region for this individual. A phylogenetic analysis based on the whole mitochondrial genome indicated thatS. tubiferis divergent from the rest of the cardinalfish family, bringing up questions of the involvement of the bioluminescent symbiosis in the initial divergence of the ancestralSiphamiaspecies. This draft genome assembly ofS. tubiferwill enable future studies investigating the evolution of bioluminescence in fishes as well as candidate genes involved in the symbiosis and will provide novel opportunities to use this system as a vertebrate-bacteria model for symbiosis research.
BCN057, a Modulator of GSK3β, Induces KRAS G12D Mutant Pancreatic Cancer Cell Death
Authors: Singer Elizabeth M. , Chugh Rishi Man , Bhanja Payel , Gomez Adrian , Gao Lucy , Whitelegge Julian P. , McBride William H. , Saha Subhrajit , Norris Andrew J.
Published: September 2021

Effective treatment for Pancreatic Cancer remains a major challenge due to its resistance to radiation/chemotherapy and poor drug permeability. Moreover, treatment induced normal tissue toxicity, mainly to the duodenum and gastrointestinal epithelium, is common and is a dose limiting event, while toxicity to the pancreas is relatively rare1–3. Gastrointestinal toxicity, however, often results in interruption, reduction or premature withdrawal of anti-cancer therapy which is a very significant factor impacting the overall survival of patients being treated. Therefore, development of a therapeutic strategy to selectively sensitize tumor tissue without inducing normal tissue toxicity is important. In this manuscript, we show that the novel small molecule BCN057 can modulate chemo-sensitivity of oncogenic RAS pancreatic cancer cells while conversely protecting normal intestinal epithelium from off target toxicity. In particular, BCN 057 protects Lgr5 positive intestinal stem cells, thereby preserving barrier function. Further, it is demonstrated that BCN057 inhibits GSK3β and thereby induces a pro-apoptotic phosphorylation pattern on c-Jun in KRAS G12D mutant pancreatic cancer cells (Panc-1) leading to the restoration of PTEN expression and consequent apoptosis. This appears to be a new mechanistic observation for the oncogenic RAS phenotype. Lastly, concurrent with its GSK3β inhibition, BCN057 is a small molecule inhibitor of PD-1 expression on human T-lymphocytes co-cultured with human pancreatic cancer cells. In summary, BCN057 can promote synthetic lethality specifically to malignant cells and therefore should be considered to improve the therapeutic ratio in pancreatic and epithelial cancer treatment in conjunction with chemotherapy and radiation.
Structures of the human peroxisomal fatty acid transporter ABCD1 in a lipid environment
Authors: Le Le Thi My , Thompson James Robert , Dang Phuoc Xuan , Bhandari Janarjan , Alam Amer
Published: September 2021

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to cellular fatty acid catabolism and lipid biosynthesis. Its dysfunction underlies toxic cytosolic accumulation of VLCFAs, progressive nervous system demyelination, and neurological impairments including the potentially fatal disease X-linked adrenoleukodystrophy (X-ALD). Molecular details underlying substrate recognition and transport by ABCD1 are poorly understood. Here we determined cryo-EM structures of ABCD1 in phospholipid nanodiscs in a nucleotide bound conformation open to the peroxisomal lumen and an inward facing conformation open to the cytosol at up to 3.5 Å resolution that reveal key details of its transmembrane cavity and ATP dependent conformational transitions. We identify structural elements distinguishing ABCD1 from its closest homologs and show that coenzyme A (CoA) esters of VLCFAs modulate ABCD1 activity in a species dependent manner. Together, our data support a transport mechanism where only the CoA moieties of VLCFA-CoAs enter the hydrophilic transmembrane cavity while the acyl chains extend out into the surrounding membrane bilayer, help rationalize disease causing mutations, and provide a framework for ABCD1 targeted structure-based drug design.
Versatile, do-it-yourself, low-cost spinning disk confocal microscope
Authors: Halpern Aaron R. , Lee Min Yen , Howard Marco D. , Woodworth Marcus A. , Nicovich Philip R. , Vaughan Joshua C.
Published: September 2021

Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (~400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers.
When development is constantly but weakly perturbed - Canalization by microRNAs
Authors: Lu GuangAn , Zhao Yixin , Chen Qingjian , Lin Pei , Tang Tian , Tang Zhixiong , Liufu Zhongqi , Wu Chung-I
Published: September 2021

Recent studies have increasingly pointed to microRNAs (miRNAs) as the agent of GRN (gene regulatory network) stabilization as well as developmental canalization against constant but small environmental perturbations. Since the complete removal of miRNAs is lethal, we construct a Dicer-1 knockdown line (dcr-1KD) in Drosophila that modestly reduces all miRNAs. We hypothesize that flies with modest miRNA reductions will gradually deviate from the developmental norm, resulting in late-stage failures such as shortened longevity. In the optimal culture condition, the survival to adulthood is indeed normal in thedcr-1KD line but, importantly, adult longevity is reduced by ∼ 90%. When flies are stressed by high temperature,dcr-1KD induces lethality earlier in late pupation and, as the perturbations are shifted earlier, the affected stages are shifted correspondingly. We further show that the developmental failure is associated with GRN aberration in the larval stages even before phenotypic aberrations become observable. Hence, in late stages of development with deviations piling up, GRN would be increasingly in need of stabilization. In conclusion, miRNAs appear to be the genome’s solution to weak but constant environmental perturbations.
Robust de novo design of protein binding proteins from target structural information alone
Authors: Cao Longxing , Coventry Brian , Goreshnik Inna , Huang Buwei , Park Joon Sung , Jude Kevin M. , Marković Iva , Kadam Rameshwar U. , Verschueren Koen H.G. , Verstraete Kenneth , Walsh Scott Thomas Russell , Bennett Nathaniel , Phal Ashish , Yang Aerin , Kozodoy Lisa , DeWitt Michelle , Picton Lora , Miller Lauren , Strauch Eva-Maria , Halabiya Samer , Hammerson Bradley , Yang Wei , Benard Steffen , Stewart Lance , Wilson Ian A. , Ruohola-Baker Hannele , Schlessinger Joseph , Lee Sangwon , Savvides Savvas N. , Garcia K. Christopher , Baker David
Published: September 2021

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains an outstanding challenge. We describe a general solution to this problem which starts with a broad exploration of the very large space of possible binding modes and interactions, and then intensifies the search in the most promising regions. We demonstrate its very broad applicability by de novo design of binding proteins to 12 diverse protein targets with very different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of four of the binder-target complexes, and all four are very close to the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvement of both. Our approach now enables targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.
RNASeq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation
Authors: Richardson Louise , Wang Dapeng , Hughes Ruth , Johnson Colin A , Peckham Michelle
Published: September 2021

Skeletal muscle satellite cells cultured on soft surfaces (12kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of γ-interferon, 37°C). As expected, the largest change in overall gene expression occurred at day 1, as cells switch from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of RNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNASeq dataset will be a useful resource for understanding Pax3 expressing cells.
Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR
Authors: Basu Indrani , Bar Sudipta , Prasad Mohit , Datta Rupak
Published: September 2021

Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to β-glucuronidase (β-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or β-GUS-deficient mice still remains a mystery. We addressed this issue using our recently developed Drosophila model of MPS VII (the CG2135-/- fly), which also exhibited a significant loss of adiposity. We report here that the lean phenotype of the CG2135-/- fly is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. Our data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135-/- larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135-/- flies. Interestingly, treatment of the CG2135-/- larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. Our finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.
ELeFHAnt: A supervised machine learning approach for label harmonization and annotation of single cell RNA-seq data
Authors: Thorner Konrad , Zorn Aaron M. , Chaturvedi Praneet
Published: September 2021

Annotation of single cells has become an important step in the single cell analysis framework. With advances in sequencing technology thousands to millions of cells can be processed to understand the intricacies of the biological system in question. Annotation through manual curation of markers based on a priori knowledge is cumbersome given this exponential growth. There are currently ~200 computational tools available to help researchers automatically annotate single cells using supervised/unsupervised machine learning, cell type markers, or tissue-based markers from bulk RNA-seq. But with the expansion of publicly available data there is also a need for a tool which can help integrate multiple references into a unified atlas and understand how annotations between datasets compare. Here we present ELeFHAnt: Ensemble learning for harmonization and annotation of single cells. ELeFHAnt is an easy-to-use R package that employs support vector machine and random forest algorithms together to perform three main functions: 1) CelltypeAnnotation 2) LabelHarmonization 3) DeduceRelationship. CelltypeAnnotation is a function to annotate cells in a query Seurat object using a reference Seurat object with annotated cell types. LabelHarmonization can be utilized to integrate multiple cell atlases (references) into a unified cellular atlas with harmonized cell types. Finally, DeduceRelationship is a function that compares cell types between two scRNA-seq datasets. ELeFHAnt can be accessed from GitHub at https://github.com/praneet1988/ELeFHAnt.