bluuebunny/crossref_metadata_2025 huggingface dataset:
Is there a role of phase partitioning in coordinating DNA damage response?
Authors: Tosolini D. , Antoniali G. , Dalla E. , Tell G.
Published: August 2020

DNA repair pathways are critical processes that need both spatial and temporal fine regulation. Liquid-liquid phase separation (LLPS) is a way to concentrate biochemical reactions, while excluding non-interacting components. Protein’s disordered domains, as well as RNA, favor condensation to modulate this process. Recent insights about phase-separation mechanisms pointed to new fascinating models that could explain how cells could cope with DNA damage responses. In this context, it is emerging that RNA-processing pathways and PARylation events, through the addition of an ADP-ribose moiety to both proteins and DNA, participate in different aspects of the DNA Damage Response (DDR). Remarkably, defects in these regulatory connections are associated with genomic instability and human pathologies. In addition, it has been recently noticed that several DNA repair enzymes, such as 53BP1 and APE1, are endowed with RNA binding abilities. APE1 is a multifunctional protein belonging to the Base Excision Repair (BER) pathway of non-distorting DNA lesions, bearing additional ‘non-canonical’ DNA-repair functions associated with processes coping with RNA metabolism. In this work, after reviewing the recent literature supporting a role of LLPS in DDR, we analyze, as a proof of principle, the interactome of APE1 using a bioinformatics approach to look for clues of LLPS in BER. Some of the APE1 interactors are associated with cellular processes in which LLPS has been either proved or proposed and are involved in several tumorigenic and amyloidogenic events. This work represents a paradigmatical pipeline for evaluating the relevance of LLPS in DDR.

Statement of significance

In this work, we aimed to test the hypothesis of an involvement of phase-separation in regulating the molecular mechanisms of the multifunctional enzyme APE1 starting from the analysis of its recently-characterized protein-protein interactome (PPI). We compared APE1-PPI to phase-separation databases and we performed functional enrichment analysis, uncovering links between APE1 and already known demixing factors, establishing an association with liquidliquid phase separation. This analysis could represent a starting point for implementing downstream experimental validations, using in vitro and in vivo approaches, to assess actual demixing.
DNA methylation inVerticillium dahliaerequires only one of three putative DNA methyltransferases, yet is dispensable for growth, development and virulence
Authors: Kramer H. Martin , Cook David E. , van den Berg Grardy C.M. , Seidl Michael F. , Thomma Bart P.H.J.
Published: August 2020

DNA methylation is an important epigenetic control mechanism that in many fungi is restricted to genomic regions containing transposons. Two DNA methyltransferases, Dim2 and Dnmt5, are known to perform methylation at cytosines in fungi. While most ascomycete fungi encode both Dim2 and Dnmt5, only few functional studies have been performed in species containing both. In this study, we report functional analysis of bothDim2andDnmt5in the plant pathogenic fungusVerticillium dahliae. Our results show that Dim2, but not Dnmt5 or the putative sexual-cycle related DNA methyltransferase Rid, is responsible for nearly all DNA methylation. Single or double DNA methyltransferase mutants did not show altered development, virulence, or transcription of genes or transposons. In contrast,Hp1andDim5mutants that are impacted in chromatin-associated processes upstream of DNA methylation are severely affected in development and virulence and display extensive transcriptional reprogramming in specific hypervariable genomic regions (so-called lineage-specific (LS) regions) that contain genes associated with host colonization. As these LS regions are largely devoid of DNA methylation and of Hp1- and Dim5-associated heterochromatin, the differential transcription is likely caused by pleiotropic effects rather than by differential DNA methylation. Overall, our study suggests that Dim2 is the main DNA methyltransferase inV. dahliaeand, in conjunction with work on other fungi, is likely the main active DNMT in ascomycetes, irrespective ofDnmt5presence. We speculate that Dnmt5 acts under specific, presently enigmatic, conditions or, alternatively, acts in DNA-associated processes other than DNA methylation.
CREB-binding protein gene,HAC701, negatively regulates WRKY45-dependent immunity in rice
Authors: Espinas Nino A. , Le Tu Ngoc , Saori Miura , Shimajiri Yasuka , Shirasu Ken , Saze Hidetoshi
Published: August 2020

CREB-binding protein (CBP) is a known transcriptional coactivator and an acetyltransferase that functions in several cellular processes by regulating gene expression. However, how it functions in plant immunity remains unexplored. By characterizinghac701, we demonstrate thatHAC701negatively regulates the immune responses in rice.hac701shows enhanced disease resistance against a bacterial pathogen,Pseudomonas syringaepv.oryzae(Pso), which causes bacterial halo blight of rice. Our transcriptomic analysis revealed that riceWRKY45, one of the main regulators of rice immunity, is upregulated inhac701and possibly conferring the resistance phenotype againstPso. The morphological phenotypes ofhac701single mutants were highly similar toWRKY45overexpression transgenic lines reported in previous studies. In addition, we also compared the list of genes in these studies whenWRKY45is overexpressed and chemically induced transiently with the differentially expressed genes (DEGs) inhac701, and found that they largely overlap. When we investigated forcis-elements found 1kb upstream ofWRKY45gene and WRKY45-dependent DEGs, we found thatWRKY45promoter contains the CRE motif, a possible target of HAC701-mediated regulation. Genome-wide H3K9 acetylation profiling showed depletion of acetylation at large set of genes inhac701. However, consistent with the upregulation ofWRKY45gene expression, our ChIP-sequencing analysis demonstrated that regions ofWRKY45promoter are enriched in H3K9 acetylation inhac701compared to the segregated wild type control in the mock condition.WRKY45promoter might be on the receiving end for possible genome-wide compensatory effects when a global regulator likeHAC701is mutated. Finally, we show thatHAC701may have roles in systemic immune signaling. We therefore propose that wild typeHAC701negatively regulatesWRKY45gene expression, thereby suppressing immune responses.

SIGNIFICANCE

HAC701 is a member of CREB-binding protein (CBP) family that acts as transcriptional coactivator and acetyltransferase. However, little is known how it regulates innate immunity in plants. Herein we reported that riceHAC701suppresses WRKY45-dependent defense pathway. Our study showed thatHAC701seemingly interacts genetically withWRKY45in rice to modulate immune responses against pathogens.
Heterochromatin-dependent transcription of satellite DNAs in theDrosophila melanogasterfemale germline
Authors: Wei Xiaolu , Eickbush Danna G. , Speece Iain , Larracuente Amanda M.
Published: August 2020

Large blocks of tandemly repeated DNAs—satellite DNAs (satDNAs)—play important roles in heterochromatin formation and chromosome segregation. We know little about how satDNAs are regulated, however their misregulation is associated with genomic instability and human diseases. We use theDrosophila melanogastergermline as a model to study the regulation of satDNA transcription and chromatin. Here we show that complex satDNAs (>100-bp repeat units) are transcribed into long noncoding RNAs and processed into piRNAs (PIWI interacting RNAs). This satDNA piRNA production depends on the Rhino-Deadlock-Cutoff complex and the transcription factor Moonshiner—a previously-described non-canonical pathway that licenses heterochromatin-dependent transcription of dual-strand piRNA clusters. We show that this pathway is important for establishing heterochromatin at satDNAs. Therefore, satDNAs are regulated by piRNAs originating from their own genomic loci. This novel mechanism of satDNA regulation provides insight into the role of piRNA pathways in heterochromatin formation and genome stability.
A molecular taxonomy of tumors independent of tissue-of-origin
Authors: Nguyen Peter T. , Coetzee Simon G. , Lakeland Daniel L. , Hazelett Dennis J.
Published: August 2020

Cancer is a complex disease involving disrupted cellular metabolism, basic biochemical processes, and the microenvironment. However, despite some generally agreed upon unifying principles (Hanahan and Weinberg 2000, 2011), molecular signatures remain largely indistinguishable from tissue-of-origin, presenting a major barrier for precision health and individualized medicine. To address this challenge, we reduce mutation data to disruptions in a select set of pathways relevant to basic cell biology, from DNA replication to cellular communication. Using dimensionality reduction techniques, we assign tumor samples into ten clusters distinct from tissue-of-origin and largely free of bias from mutational burden or clinical stage. We show that the clusters vary in prognosis by modeling relative risk of death by cancer type and cluster. We identify cluster-specific mutations in different tissues, demonstrating that tissue-specific signatures contribute to common cellular phenotypes. Moreover, germline risk genes involved in replication fidelity and genome stability are equally distributed among clusters, contrary to the expectation that such genes are avatars of molecular subtype. We investigate metastatic and non-metastatic pathways, and show that most differences are cluster-specific. Some metastatic pathways from one cluster are cluster-specific pathways from non-metastatic tumors of another cluster, suggesting phenotypic convergence. Taken as a whole, our observations suggest that common driver genes combine with tissue-specific disruptions in tumor-promoting pathways to produce a limited number of distinct molecular phenotypes. Thus, we present a coherent view of global tumor biology, and explain how common cellular dysfunction might arise from tissue-specific mutations.
Effects of hyperbaric environment on endurance and metabolism are exposure time-dependent in well-trained mice
Authors: Suzuki Junichi
Published: August 2020

Hyperbaric exposure (1.3 atmospheres absolute with 20.9% O2) for 1 h a day was shown to improve exercise capacity. The present study was designed to reveal whether the daily exposure time affects exercise performance and metabolism in skeletal and cardiac muscles. Male mice in the training group were housed in a cage with a wheel activity device for 7 weeks from 5 weeks old. Trained mice were then subjected to hybrid training (HT, endurance exercise for 30 min followed by sprint interval exercise for 30 min). Hyperbaric exposure was applied following daily HT for 15 min (15HT), 30 min (30HT), or 60 min (60HT) for 4 weeks (each group, n = 10). In the endurance capacity test, maximal work values were significantly increased by 30HT and 60HT. In the left ventricle (LV), activity levels of 3-hydroxyacyl-CoA-dehydrogenase, citrate synthase, and carnitine palmitoyl transferase (CPT) 2 were significantly increased by 60HT. CPT2 activity levels were markedly increased by 60HT in the plantaris muscle (PL). Pyruvate dehydrogenase complex (PDHc) activity values in PL were enhanced more by 30HT and 60HT than by HT. In both 30HT and 60HT groups, lactate dehydrogenase activity levels were significantly increased and decreased, respectively, in the gastrocnemius muscle and LV. These results indicate that hyperbaric exposure for 30 min or longer has beneficial effects on endurance, and 60-min exposure has the potential to further increase performance by facilitating fatty acid metabolism in skeletal and cardiac muscles in highly-trained mice.
Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands
Authors: Roberts Brian S. , Partridge E. Christopher , Moyers Bryan A. , Agarwal Vikram , Newberry Kimberly M. , Martin Beth K. , Shendure Jay , Myers Richard M. , Cooper Gregory M.
Published: August 2020

Massively parallel reporter assays (MPRAs) are useful tools to discover and characterize regulatory elements in human genomes. Partly because enhancer function is assumed to be orientation independent with respect to each strand of the DNA helix, most reported MPRA results ignore stranded information. However, we find pervasive strand asymmetry of MPRA signals in datasets from multiple reporter configurations and in both published and newly reported data. These effects are reproducible across different cell types and in different treatments within a cell type, and are observed both within and outside of annotated regulatory elements. From elements in gene bodies, MPRA strand asymmetry favors the sense strand, suggesting that biological function related to endogenous transcription is driving the phenomenon. Similarly, within Alu mobile element insertions, we find that strand asymmetry favors the transcribed strand of the ancestral retrotransposon. The effect is consistent across the multiplicity of Alu elements in human genomes, and is more pronounced in younger, less diverged Alu elements. We find sequence features driving MPRA strand asymmetry and demonstrate its prediction from sequence alone. We see some evidence for both RNA stabilization and transcriptional activation mechanisms, and hypothesize that the effect is driven by natural selection favoring efficient transcription. Our results indicate that strand asymmetry, as a pervasive and reproducible feature, should be accounted for in analysis of MRPA data. More importantly, the fact that MPRA asymmetry favors naturally transcribed strands suggests that it stems from preserved biological functions that have a substantial, global impact on gene and genome evolution.
SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans
Authors: Supekar Nitin T. , Shajahan Asif , Gleinich Anne S. , Rouhani Daniel , Heiss Christian , Azadi Parastoo
Published: August 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.
Deep learning to decipher the progression and morphology of axonal degeneration
Authors: Palumbo Alex , Grüning Philipp , Landt Svenja Kim , Heckmann Lara Eleen , Bartram Luisa , Pabst Alessa , Flory Charlotte , Ikhsan Maulana , Pietsch Sören , Schulz Reinhard , Kren Christopher , Koop Norbert , Boltze Johannes , Mamlouk Amir Madany , Zille Marietta
Published: August 2020

Background

Axonal degeneration (AxD) is a pathological hallmark of many neurodegenerative diseases. Deciphering the morphological patterns of AxD will help to understand the underlying mechanisms and to develop effective therapeutic interventions. Here, we evaluated the progression of AxD in cortical neurons using a novel microfluidic device in combination with a deep learning tool, the EntireAxon, that we developed for the enhanced-throughput analysis of AxD on microscopic images.

Results

The EntireAxon convolutional neural network sensitively and specifically segmented the features of AxD, including axons, axonal swellings, and axonal fragments, and its performance exceeded that of human expert raters. In anin vitromodel of AxD in hemorrhagic stroke induced by the hemolysis product hemin, we detected the concentration- and time-dependent degeneration of axons leading to a decrease in axon area, while the axonal swelling and axonal fragment area increased. Time course analysis revealed that axonal swellings preceded axon fragmentation, suggesting that swellings may be reliable predictors of AxD. Using a recurrent neural network, we further identified four morphological patterns of AxD (granular, retraction, swelling, and transport degeneration) in cortical axons subjected to hemin.

Conclusions

These findings indicate a morphological heterogeneity of AxD under pathophysiologic conditions. The combination of the microfluidic device with the EntireAxon deep learning tool enable the systematic analysis of AxD but also unravel a so far unknown intricacy in which AxD can occur in a disease context.
Boosting L-type Ca2+channel activity in tuberous sclerosis mitigates excess glutamate receptors
Authors: Niere Farr , Cacheaux Luisa P. , Egido-Betancourt Hailey X. , Taylor William C. , Raab-Graham Kimberly F.
Published: August 2020

Tuberous sclerosis complex (TS) is a dominant, multisystem disorder with devastating neurological symptoms. Approximately 85% of TS patients suffer from epilepsy over their lifespan and roughly 25-50% of those patients develop Autism Spectrum Disorder (1, 2). Current seizure therapies are effective in some, but not all, and often have significant risk factors associated with their use (1, 3). Thus, there is a critical need for new medication development or drug repositioning. Herein, we leveraged proteomic signatures of epilepsy and ASD, often comorbid in TS, to utilize anin silcoapproach to identify new drug therapies for TS-related seizures. We have discovered that activation of L-type voltage dependent calcium channels (L-VDCC) by Bay-K8644 (BayK) in a preclinical mouse model of TS rescues the excess expression of ionotropic, AMPA-subtype glutamate (GluA) receptors. As added proof of BayK working through L-VDCC to regulate GluA levels, we found that increasing expression of alpha2delta2 (α2δ2), an auxiliary calcium channel subunit that boosts L-VDCC surface expression, similarly lowers the surface expression of dendritic GluA in TS. These BayK-induced molecular alterations may potentially improve the quality of life of humans suffering from TS.

Significance Statement

Causal mechanisms of Tuberous Sclerosis (TS)-associated neurological disorders are under-characterized and treatment options are lacking. Using a computational approach of mTOR/DJ-1 target mRNAs to predict new medications, we report that boosting L-type voltage-dependent Ca2+channel (L-VDCC) activity in a preclinical TS mouse model that exhibits a deficit in dendritic L-VDCC activity ameliorates key molecular pathologies that are predicted to underlie seizures. Restoring the mTOR/DJ-1 pathway upstream of L-VDCC, therefore, may serve as a new therapeutic avenue to mitigate seizures and mortality in TS.
Modern, archaeological, and paleontological DNA analysis of a human-harvested marine gastropod (Strombus pugilis) from Caribbean Panama
Authors: Sullivan Alexis P. , Marciniak Stephanie , O’Dea Aaron , Wake Thomas A. , Perry George H.
Published: August 2020

Although protocols exist for the recovery of ancient DNA from land snail and marine bivalve shells, marine conch shells have yet to be studied from a paleogenomic perspective. We first present reference assemblies for both a 623.7 Mbp nuclear genome and a 15.4 kbp mitochondrial genome forStrombus pugilis, the West Indian fighting conch. We next detail a method to extract and sequence DNA from conch shells and apply it to conch from Bocas del Toro, Panama across three time periods: recently-eaten and discarded (n=3), Late Holocene (984-1258 BP) archaeological midden (n=5), and a mid-Holocene (5711-7187 BP) paleontological fossil coral reef (n=5). These results are compared to control DNA extracted from live-caught tissue and fresh shells (n=5). Using high-throughput sequencing, we were able to obtainS. pugilisnuclear sequence reads from shells across all age periods: up to 92.5 thousand filtered reads per sample in live-caught shell material, 4.57 thousand for modern discarded shells, 12.1 thousand reads for archaeological shells, and 114 reads in paleontological shells. We confirmed authenticity of the ancient DNA recovered from the archaeological and paleontological shells based on 5.7x higher average frequency of deamination-driven misincorporations and 15% shorter average read lengths compared to the modern shells. Reads also mapped to theS. pugilismitochondrial genome for all but the paleontological shells, with consistent ratios of mitochondrial to nuclear mapped reads across sample types. Our methods can be applied to diverse archaeological sites to facilitate reconstructions of the long-term impacts of human behavior on mollusc evolutionary biology.
Prostate Epithelial Genes Define Docetaxel-Sensitive Prostate Cancer Molecular Subtype
Authors: Han Hyunho , Choi Kwibok , Moon Young Jun , Heo Ji Eun , Ham Won Sik , Jang Won Sik , Rha Koon Ho , Cho Nam Hoon , Giancotti Filippo G. , Choi Young-Deuk
Published: August 2020

BACKGROUND & OBJECTIVES

Analysis of the transcriptomic landscape of prostate adenocarcinoma shows multidimensional gene expression variability. Understanding cancer transcriptome complexity can provide biological insight and therapeutic guidance. To avoid potential confounding factors, such as stromal contamination and stress-related material degradation, we utilized a set of genes expressed by prostate epithelial cells from single-cell transcriptome data of the human prostate gland.

MATERIALS & METHODS

Analyzing publicly available bulk and single-cell RNA sequencing data, we defined 1,629 genes expressed by prostate epithelial cells. Consensus clustering and CIBERSORT deconvolution were used for class discovery and proportion estimate analysis. The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) dataset served as a training set. The resulting clusters were analyzed in association with clinical, pathologic, and genomic characteristics and impact on survival.

RESULTS

TCGA-PRAD tumors were separated into four subtypes: A (30.0%), B (26.0%), C (14.7%), D (4.2%), and mixed (25.0%). Subtype A was characterized by low frequency of ETS-family fusions and high expression ofKLK3, which encodes prostate-specific antigen (PSA). Subtype B showed the highest expression ofACP3, encoding PAP (prostatic acid phosphatase). Subtypes C and D were commonly associated with advanced T/N stages, high Gleason grades, and p53 or PIK3CA mutations. In silico drug-sensitivity screening suggested that subtype B is likely sensitive to docetaxel and paclitaxel. Serum PSA/PAP ratio was predictive of a radiographic response to docetaxel in metastatic castration-resistant prostate cancer patients.

CONCLUSION

We propose four prostate adenocarcinoma subtypes with distinct transcriptomic, genomic, and pathologic characteristics. PSA/PAP ratio in advanced cancer may aid in determining which patients would benefit from maximized androgen receptor inhibition or early use of antimicrotubule agents. Molecular subtypes and biomarkers must be validated in a prospective cohort study.
REAP supplemental fertilizer improves greenhouse crop yield
Authors: Backer Rachel , Solomon Damian
Published: August 2020

Background

Mine tailings contain rare earth elements, including lanthanum and cerium, and plant micronutrients including iron. Previous studies have demonstrated that fertilizers containing rare earth elements and/or micronutrients can influence plant physiology, nutrient uptake and crop yield. However, applying the right dose of these fertilizers is critical since the concentration range associated with benefits is often narrow, and overapplication can lead to crop yield reductions. This study aimed to quantify the effects of a water-soluble fertilizer, REAP, on the yield of greenhouse crops.

Methods

In the first experiment, the effects of three concentrations of REAP (100, 250 or 500 ppm) were compared to a control (0 ppm REAP) on growth of lettuce, tomato and pepper growing in soilless media. In the second experiments, the effects of REAP applied at higher rates (500, 1000 and 2000 ppm) were compared to a control (0 ppm REAP) on the growth of lettuce, peppers, tomato and cantaloupe.

Results

In the first experiment, there were no significant differences in yield between treatments, REAP appeared to promote root development. In the second experiment, there were significant yield increases for all crops treated with REAP. Gas exchange rates and nutrient concentration of tomato plants receiving REAP were not significantly different from the control. These results demonstrated that nutrient elements in REAP, including lanthanum, cerium, and micronutrients, improved the growth and yield of vegetable crops when applied at rates ranging from 500 to 2000 ppm.
Gene expression profiles of inflammatory breast cancer reveal high heterogeneity across the epithelial-hybrid-mesenchymal spectrum
Authors: Chakraborty Priyanka , George Jason T , Woodward Wendy A , Levine Herbert , Jolly Mohit Kumar
Published: August 2020

Inflammatory breast cancer (IBC) is a highly aggressive breast cancer that metastasizes largely via tumor emboli, and has a 5-year survival rate of less than 30%. No unique genomic signature has yet been identified for IBC nor has any specific molecular therapeutic been developed to manage the disease. Thus, identifying gene expression signatures specific to IBC remains crucial. Here, we compare various gene lists that have been proposed as molecular footprints of IBC using different clinical samples as training and validation sets and using independent training algorithms, and determine their accuracy in identifying IBC samples in three independent datasets. We show that these gene lists have little to no mutual overlap, and have limited predictive accuracy in identifying IBC samples. Despite this inconsistency, single-sample gene set enrichment analysis (ssGSEA) of IBC samples correlate with their position on the epithelial-hybrid-mesenchymal spectrum. This positioning, together with ssGSEA scores, improves the accuracy of IBC identification across the three independent datasets. Finally, we observed that IBC samples robustly displayed a higher coefficient of variation in terms of EMT scores, as compared to non-IBC samples. Pending verification that this patient-to-patient variability extends to intratumor heterogeneity within a single patient, these results suggest that higher heterogeneity along the epithelial-hybrid-mesenchymal spectrum can be regarded to be a hallmark of IBC and a possibly useful biomarker.
Imbalance of flight-freeze responses and their cellular correlates in theNlgn3-/yrat model of autism
Authors: Anstey Natasha J , Kapgal Vijayakumar , Tiwari Shashank , Watson Thomas C , Toft Anna KH , Dando Owen R , Inkpen Felicity H , Baxter Paul S , Kozić Zrinko , Jackson Adam D , He Xin , Nawaz Mohammad Sarfaraz , Kayenaat Aiman , Bhattacharya Aditi , Wyllie David JA , Chattarji Sumantra , Wood Emma R , Hardt Oliver , Kind Peter C
Published: August 2020

Mutations in the postsynaptic transmembrane protein neuroligin-3 are highly correlative with autism spectrum disorders (ASDs) and intellectual disabilities (IDs). Fear learning is well studied in models of these disorders, however differences in fear response behaviours are often overlooked. Whilst examining fear in a rat model of ASD/ID lackingNlgn3, we observed that they display a greater propensity to exhibit flight responses in contrast to classic freezing seen in wildtypes during fearful situations. Consequently, we examined the physiological underpinnings of this in neurons of the periaqueductal grey (PAG), a midbrain area involved in flight-or-freeze responses. Inex vivoslices fromNlgn3-/y, rats, dorsal PAG (dPAG) neurons showed intrinsic hyperexcitability. Further analysis of this revealed lower magnitudein vivodPAG stimulation evoked flight behaviour inNlgn3-/y, rats, indicating the functional impact of the increased cellular excitability. This study provides new insight into potential pathophysiologies leading to emotional disorders in individuals with ASD.
A rendezvous of two second messengers: The c-di-AMP receptor protein DarB controls (p)ppGpp synthesis inBacillus subtilis
Authors: Krüger Larissa , Herzberg Christina , Wicke Dennis , Bähre Heike , Heidemann Jana L. , Dickmanns Achim , Schmitt Kerstin , Ficner Ralf , Stülke Jörg
Published: August 2020

Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. InBacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Most likely these proteins transduce their signal by regulatory interactions with other proteins. Here, we have investigated the function of the c-di-AMP-binding protein DarB inB. subtilis, a protein consisting of two CBS (cystathionine-beta synthase) domains. We have used an unbiased search for DarB interaction partners and identified the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. Indeed, c-di-AMP inhibits the binding of DarB to Rel. The interaction results in the Rel-dependent accumulation of pppGpp. Our results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.
Cell-type, single-cell, and spatial signatures of brain-region specific splicing in postnatal development
Authors: Joglekar Anoushka , Prjibelski Andrey , Mahfouz Ahmed , Collier Paul , Lin Susan , Schlusche Anna Katharina , Marrocco Jordan , Williams Stephen R. , Haase Bettina , Hayes Ashley , Chew Jennifer G. , Weisenfeld Neil I , Wong Man Ying , Stein Alexander N. , Hardwick Simon , Hunt Toby , Bent Zachary , Fedrigo Olivier , Sloan Steven A. , Risso Davide , Jarvis Erich D. , Flicek Paul , Luo Wenjie , Pitt Geoffrey S. , Frankish Adam , Smit August B. , Ross M. Elizabeth , Tilgner Hagen U.
Published: August 2020

Alternative RNA splicing varies across brain regions, but the single-cell resolution of such regional variation is unknown. Here we present the first single-cell investigation of differential isoform expression (DIE) between brain regions, by performing single cell long-read transcriptome sequencing in the mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 (www.isoformAtlas.com). Using isoform tests for brain-region specific DIE, which outperform exon-based tests, we detect hundreds of brain-region specific DIE events traceable to specific cell-types. Many DIE events correspond to functionally distinct protein isoforms, some with just a 6-nucleotide exon variant. In most instances, one cell type is responsible for brain-region specific DIE. Cell types indigenous to only one anatomic structure display distinctive DIE, where for example, the choroid plexus epithelium manifest unique transcription start sites. However, for some genes, multiple cell-types are responsible for DIE in bulk data, indicating that regional identity can, although less frequently, override cell-type specificity. We validated our findings with spatial transcriptomics and long-read sequencing, yielding the first spatially resolved splicing map in the postnatal mouse brain (www.isoformAtlas.com). Our methods are highly generalizable. They provide a robust means of quantifying isoform expression with cell-type and spatial resolution, and reveal how the brain integrates molecular and cellular complexity to serve function.
PP2A-Cdc55 is responsible for mitotic arrest in DNA re-replicating cells inS. cerevisiae
Authors: Khondker Shoily , Ikui Amy E.
Published: August 2020

The cell cycle is an ordered process in which cells replicate their DNA in S-phase and divide them into two identical daughter cells in mitosis. DNA replication takes place only once per cell cycle to preserve genome integrity, which is tightly regulated by Cyclin Dependent Kinase (CDK). Formation of the pre-replicative complex, a platform for origin licensing, is inhibited through CDK-dependent phosphorylation. Failure of this control leads to re-licensing, re-replication and DNA damage. Eukaryotic cells have evolved surveillance mechanisms to maintain genome integrity, termed cell cycle checkpoints. It has been shown that the DNA damage checkpoint is activated upon the induction of DNA re-replication and arrests cell cycle in mitosis inS. cerevisiae. In this study, we show that PP2A-Cdc55 is responsible for the metaphase arrest induced by DNA re-replication, leading to dephosphorylation of APC component, Exclusion of Cdc55 from the nucleus bypassed the mitotic arrest and resulted in enhanced cell lethality in re-replicating cells. The metaphase arrest in re-replication cells was retained in the absence of Mad2, a key component of the spindle assembly checkpoint. Moreover, re-replicating cells showed the same rate of DNA damage induction in the presence or absence of Cdc55. These results indicate that PP2A-Cdc55 maintains metaphase arrest upon DNA re-replication and DNA damage through APC inhibition.
Disproportionate presence of adenosine in mitochondrial and chloroplast DNA ofChlamydomonas reinhardtii
Authors: El-Sayed Waleed M. M. , Gombolay Alli L. , Xu Penghao , Yang Taehwan , Jeon Youngkyu , Balachander Sathya , Newnam Gary , Tao Sijia , Bowen Nicole E. , Schinazi Raymond F. , Kim Baek , Chen Yongsheng , Storici Francesca
Published: August 2020

Ribonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genomic DNA of cells. The distribution of rNMPs in DNA has been studied only in limited genomes, such as yeast nuclear and mitochondrial DNA, as well as human mitochondrial DNA. In this study, we used the ribose-seq protocol and the Ribose-Map bioinformatics toolkit to reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga,Chlamydomonas reinhardtii. The study presents the discovery of a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the nucleotide content of theseC. reinhardtiigenomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. In addition, we reveal specific rNMP biases and patterns in the mitochondrial, chloroplast and nuclear DNA ofC. reinhardtii.
Gene co-expression analysis of humanRNASEH2Areveals functional networks associated with DNA replication, DNA damage response, and cell cycle regulation
Authors: Marsili Stefania , Tichon Ailone , Storici Francesca
Published: August 2020

Ribonuclease H2 (RNase H2) is a key enzyme for the removal of RNA found in DNA-RNA hybrids, playing a fundamental role in biological processes such as DNA replication, telomere maintenance and DNA damage repair. RNase H2 is a trimer composed of three subunits, being RNASEH2A the catalytic subunit.RNASEH2Aexpression levels have been shown to be upregulated in transformed and cancer cells. In this study we used a bioinformatics approach to identifyRNASEH2Aco-expressed genes in different human tissues to uncover biological processes in whichRNASEH2Ais involved. By implementing this approach, we identified functional networks forRNASEH2Athat are not only involved in the processes of DNA replication and DNA damage response, but also in cell cycle regulation. Additional examination of protein-protein networks for RNASEH2A by the STRING database analysis, revealed a high co-expression correlation betweenRNASEH2Aand the genes of the protein networks identified. Mass spectrometry analysis of RNASEH2A-bound proteins highlighted players functioning in cell cycle regulation. Further bioinformatics investigation showed increased gene expression ofRNASEH2Ain different types of actively cycling cells and tissues, and particularly in several cancers, supporting a biological role for RNASEH2A, but not the other two subunits of RNase H2, in cell proliferation.
Organic nanoelectronics inside us: charge transport and localization in RNA could orchestrate ribosome operation
Authors: Sosorev Andrey , Kharlanov Oleg
Published: August 2020

Translation—protein synthesis at the ribonucleic acid (RNA) based molecular machine, ribosome, — proceeds in a similar manner in all life forms. However, despite several decades of research, the physics underlying this process remains enigmatic. Specifically, during translation, a ribosome undergoes large-scale conformational changes of its distant parts, and these motions are precisely synchronized by an unknown mechanism. In this study, we suggest that such a mechanism could be related to charge (electron hole) transport along and between the RNA molecules, localization of these charges at certain sites and successive relaxation of molecular geometry. Thus, we suppose that RNA-based molecular machines, e.g., ribosome, are electronically controlled, having “wires”, “actuators”, “battery”, and other “circuitry”. Taking transfer RNA as an example, we justify the reasonability of our suggestion using ab initio and atomistic simulations. We anticipate that our findings will qualitatively advance the understanding of the key biological processes and could inspire novel approaches in medicine.
Divergence in alternative polyadenylation contributes to gene regulatory differences between humans and chimpanzees
Authors: Mittleman Briana , Pott Sebastian , Warland Shane , Barr Kenneth , Cuevas Claudia , Gilad Yoav
Published: August 2020

Comparative functional genomic studies have shown that differences in gene expression between species can often be explained by corresponding inter-species differences in genetic and epigenetic regulatory mechanisms. In the quest to understand gene regulatory evolution in primates, the role of co-transcriptional regulatory mechanisms, such as alternative polyadenylation (APA), have so far received little attention. To begin addressing this gap, we studied APA in lymphoblastoid cell lines from six humans and six chimpanzees, and estimated usage for 44,432 polyadenylation sites (PAS) in 9,518 genes in both species. While APA is largely conserved in humans and chimpanzees, we identified 1,705 genes with significantly different PAS usage (FDR of 0.05) between the two species. We found that genes with divergent APA patterns are enriched among differentially expressed genes, as well as among genes that show differences in protein translation between species. In particular, differences in APA between humans and chimpanzees can explain a subset of observed inter-species protein expression differences that do not display corresponding differences at the transcript level. Finally, we focused on genes that have a dominant PAS, namely a PAS that is used more often than all others. Dominant PAS are highly conserved, and inter-species differences in dominant PAS are particularly enriched for genes that also show expression differences between the species. This study establishes APA as another key mechanism underlying the genetic regulation of transcript and protein expression levels in primates.
The initial gut microbiota and response to antibiotic perturbation influenceClostridioides difficilecolonization in mice
Authors: Tomkovich Sarah , Stough Joshua M.A. , Bishop Lucas , Schloss Patrick D.
Published: August 2020

The gut microbiota has a key role in determining susceptibility toClostridioides difficileinfections (CDIs). However, much of the mechanistic work examining CDIs in mouse models use animals obtained from a single source. We treated mice from 6 sources (2 University of Michigan colonies and 4 commercial vendors) with clindamycin, followed by aC. difficilechallenge and then measuredC. difficilecolonization levels throughout the infection. The microbiota were profiled via 16S rRNA gene sequencing to examine the variation across sources and alterations due to clindamycin treatment andC. difficilechallenge. While all mice were colonized 1-day post-infection, variation emerged from days 3-7 post-infection with animals from some sources colonized withC. difficilefor longer and at higher levels. We identified bacteria that varied in relative abundance across sources and throughout the experiment. Some bacteria were consistently impacted by clindamycin treatment in all sources of mice includingLachnospiraceae, Ruminococcaceae, andEnterobacteriaceae. To identify bacteria that were most important to colonization regardless of the source, we created logistic regression models that successfully classified mice based on whether they clearedC. difficileby 7 days post-infection using community composition data at baseline, post-clindamycin, and 1-day post-infection. With these models, we identified 4 bacteria that were predictive of whetherC. difficilecleared. They varied across sources (Bacteroides), were altered by clindamycin (Porphyromonadaceae), or both (EnterobacteriaceaeandEnterococcus). Allowing for microbiota variation across sources better emulates human inter-individual variation and can help identify bacterial drivers of phenotypic variation in the context of CDIs.

Importance

Clostridioides difficileis a leading nosocomial infection. Although perturbation to the gut microbiota is an established risk, there is variation in who becomes asymptomatically colonized, develops an infection, or has adverse infection outcomes. Mouse models ofC. difficileinfection (CDI) are widely used to answer a variety ofC. difficilepathogenesis questions. However, the inter-individual variation between mice from the same breeding facility is less than what is observed in humans. Therefore, we challenged mice from 6 different breeding colonies withC. difficile. We found that the starting microbial community structures andC. difficilepersistence varied by the source of mice. Interestingly, a subset of the bacteria that varied across sources were associated with how longC. difficilewas able to colonize. By increasing the inter-individual diversity of the starting communities, we were able to better model human diversity. This provided a more nuanced perspective ofC. difficilepathogenesis.
Projection of gut microbiome pre and post-bariatric surgery to predict surgery outcome
Authors: Izhak Meirav Ben , Eshel Adi , Cohen Ruti , Shapiro Liora Madar , Meiri Hamutal , Wachtel Chaim , Leung Conrad , Messick Edward , Jongkam Narisra , Mavor Eli , Sapozhnikov Shimon , Maharshak Nitsan , Abu-Abeid Subhi , Alis Avishai , Mahler Ilanit , Meoded Aviel , Eldar Shai Meron , Koren Omry , Louzoun Yoram
Published: August 2020

Background

Bariatric surgery is often the preferred method to resolve obesity and diabetes, with ~800,000 cases worldwide yearly and high outcome variability. The ability to predict the long-term Body Mass Index (BMI) change following surgery has important implications on individuals and the health care system in general. Given the tight connection between eating habits, sugar consumption, BMI, and the gut microbiome, we tested whether the microbiome before any treatment is associated with different treatment outcomes, as well as other intakes (high-density lipoproteins (HDL), Triglycerides, etc.).

Results

A projection of the gut microbiome composition of obese (sampled before and after bariatric surgery) and slim patients into principal components was performed and the relation between this projection and surgery outcome was studied. The projection reveals 3 different microbiome profiles belonging to slim, obese, and obese who underwent bariatric surgery, with post-surgery more different from the slim than the obese. The same projection allowed for a prediction of BMI loss following bariatric surgery, using only the pre-surgery microbiome.

Conclusions

The gut microbiome can be decomposed into main components depicting the patient’s development and predicting in advance the outcome. Those may be translated into better clinical management of obese individuals planning to undergo metabolic surgery.

Importance

BMI and diabetes can affect the gut microbiome composition.

Bariatric surgery has large variabilities in outcome.

The microbiome was previously shown to be a good predictor for multiple diseases.

We analyzed here the gut microbiome before and after bariatric surgery and show that:

The microbiome before surgery can be used to predict surgery outcome.

Post-surgery microbiome drifts further away from the slim microbiome than pre-surgery obese patients.

These results can lead to a microbiome-based pre-surgery decision whether to perform surgery.
Detecting the nature and solving the crystal structure of a contaminant protein from an opportunistic pathogen
Authors: Pederzoli Riccardo , Tarantino Delia , Gourlay Louise J. , Chaves-Sanjuan Antonio , Bolognesi Martino
Published: August 2020

The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is presented. The contamination led to the unintended crystallization and structure analysis of a cyanate hydratase from a bacterial strain of the Serratia genus, an opportunistic enterobacterium that grows under conditions similar to those of E. coli and that is found in a variety of habitats, including the laboratory environment. In this context, the procedures that were adopted to identify the contaminant based on crystallographic data only are presented and the crystal structure of Serrata spp. cyanate hydratase is briefly discussed.