bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual-axis Volta phase plate cryo-electron tomography of Ebola virus-like particles reveals actin-VP40 interactions
Authors: Winter Sophie L. , Chlanda Petr
Published: April 2021

Cryo-electron tomography (cryo-ET) is a pivotal imaging technique for studying the structure of pleomorphic enveloped viruses and their interactions with the host at native conditions. Owing to the limited tilting range of samples with a slab geometry, electron tomograms suffer from so-called missing wedge information in Fourier space. In dual-axis cryo-ET, two tomograms reconstructed from orthogonally oriented tilt series are combined into a tomogram with improved resolution as the missing wedge information is reduced to a pyramid. Volta phase plate (VPP) allows to perform in-focus cryo-ET with high contrast transfer at low-resolution frequencies and thus its application may improve the quality of dual-axis tomograms. Here, we compare dual-axis cryo-ET with and without VPP on Ebola virus-like particles to visualize and segment viral and host cell proteins within the membrane-enveloped filamentous particles. Dual-axis VPP cryo-ET reduces the missing wedge information and ray artifacts arising from the weighted back-projection during tomogram reconstruction, thereby minimizing ambiguity in the analysis of crowded environments and facilitating 3D segmentation. We show that dual-axis VPP tomograms provide a comprehensive description of macromolecular organizations such as nucleocapsid assembly states, the distribution of glycoproteins on the viral envelope and asymmetric arrangements of the VP40 layer in non-filamentous regions of virus-like particles. Our data reveal actin filaments within virus-like particles in close proximity to the viral VP40 scaffold, suggesting a direct interaction between VP40 and actin filaments. Dual-axis VPP cryo-ET provides more complete 3D information at high contrast and allows for better interpretation of macromolecule interactions and pleomorphic organizations.

Highlights

Volta phase plate dual-axis cryo-electron tomography provides high contrast tomography data with reduced back-projection ray artifacts and missing wedge information in Fourier space

Volta phase plate dual-axis cryo-electron tomography facilitates interpretation of protein-membrane interactions

Volta phase plate dual-axis cryo-electron tomography reduces ambiguity in manual 3D rendering and markedly improves 3D isosurface modeling

Ebola virus-like particles contain actin filaments in close proximity to the VP40 layer
IL-7 is essential for accumulation of antigen-specific CD8 T cells and to generate clonotype-specific effector responses during airway influenza/A infection
Authors: Sheikh Abdalla , Jackson Jennie , Shim Hanjoo Brian , Yau Clement , Seo Jung Hee , Abraham Ninan
Published: April 2021

Airborne diseases are the leading cause of infectious disease-related deaths in the world. In particular, the influenza virus activates a network of immune cells that leads to clearance or an overzealous response that can be fatal. Tight regulation of the cytokines that enable proper activation and function of immune cells is necessary to clear infections efficiently while minimizing damage to the host. Interleukin-7 (IL-7) is a cytokine known for its importance in T cell development and survival. How IL-7 shapes CD8 T cell responses during an acute viral infection is less understood. We had previously shown that IL-7 signaling deficient mice have reduced accumulation of influenza-specific CD8 T cells following infection. We sought to determine whether IL-7 affects early CD8 T cell expansion in the mediastinal lymph node and effector function in the lungs. Using IL-7Rα signaling deficient mice, we show that IL-7 is required for a normal sized mediastinal lymph node and the early clonal expansion of antigen-specific CD8 T cells therein. Bone marrow chimeric models and adoptive transfer of transgenic TCR CD8 T cells reveal a cell-intrinsic role for IL-7 in the accumulation of NP366–374and PA224–233-specific CD8 T cells. We also found that IL-7 dictates terminal differentiation, degranulation and cytokine production in PA224–233-specific but not NP366–374-specific CD8 T cells. We further demonstrate that IL-7 is induced in the lung tissue by viral infection and we characterize multiple cellular sources that contribute to IL-7 production. Drugs that manipulate IL-7 signaling are currently under clinical trial for multiple conditions. Our findings on IL-7 and its effects on lower respiratory diseases will be important for expanding the utility of these therapeutics.

Author Summary

Interleukin-7 plays an important role in development of immune cells such as lymphocytes. In recent years, its role in the immune system has been expanded beyond the development of immune cells to include revitalizing of lymphocytes during tumor and chronic viral response. We show here that IL-7 is required for accumulation and function of specialized lymphocytes in the lungs during an acute influenza infection.
Defining function of wild-type and patient specificTP53mutations in a zebrafish model of embryonal rhabdomyosarcoma
Authors: Chen Jiangfei , Baxi Kunal , Lipsitt Amanda E. , Hensch Nicole R. , Wang Long , Baudin Antoine , Robledo Daniel G. , Bandyopadhyay Abhik , Sugalski Aaron , Challa Anil K. , Gilbert Andrea R. , Tomlinson Gail E. , Houghton Peter , Chen Eleanor Y. , Libich David S. , Ignatius Myron S.
Published: April 2021

In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss or gain of functionTP53mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understandingTP53effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-typeTP53, yetTP53mutations when present in tumors are associated with poor prognosis. Employing akRASG12D-driven ERMS tumor model and newly generated tp53 null (tp53-/-) zebrafish, we define both wild-type and patient-specificTP53mutant effects on tumorigenesis. We demonstrate thattp53is a major suppressor of tumor initiation, wheretp53loss expands tumors initiation from <35% to >97% of animals. Next, characterizing three patient-specific mutants finds thatTP53C176Fpartially retains wild-type p53 apoptotic activity that can be exploited, while theTP53P153Δand TP53Y220Cmutants define two structural mutations that predispose to head musculature ERMS.
Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow
Authors: Engel Martin , Belfiore Lisa , Aghaei Behnaz , Sutija Margareta
Published: April 2021

Advanced three-dimensional cell culture techniques have been adopted in many laboratories to better modelin vivotissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the workflow in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3β phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability toin vivodrug response.
AGO104 is an RdDM effector of paramutation at the maizeb1locus
Authors: Aubert Juliette , Bellegarde Fanny , Oltehua-Lopez Omar , Leblanc Olivier , Arteaga-Vazquez Mario A. , Martienssen Robert A. , Grimanelli Daniel
Published: April 2021

Paramutation is an exception among eukaryotes, in which epigenetic information is conserved through mitosis and meiosis. It has been studied for over 70 years in maize, but the mechanisms involved are largely unknown. Previously described actors of paramutation encode components of the RNA-dependent DNA-methylation (RdDM) pathway all involved in the biogenesis of 24-nt small RNAs. However, no actor of paramutation have been identified in the effector complex of RdDM. Here, through a combination of reverse genetics, immunolocalization and immunoprecipitation (siRNA-IP) we found that ARGONAUTE104 (AGO104), AGO105 and AGO119 are members of the RdDM effector complex in maize and bind siRNAs produced from the tandem repeats required for paramutation at theb1locus. We also showed that AGO104 is an effector of theb1paramutation in maize.

Author summary

Reprogramming of epigenetic information has been described in both plants and mammals. Here, we show that maizeARGONAUTE (AGO) AGO104andAGO105/AGO119, respectively the close homologs ofA. thaliana AGO9andAGO4, are required to enable paramutation at theb1locus in maize. Paramutation is an epigenetic phenomenon that is stable over many generations (both mitotically and meiotically). A classic example is thebooster1(b1) gene in maize, where the weakly expressedBooster’(B’) allele stably decreases the expression of theBooster-Intense(B-I) allele, and changes it into a newB’allele. This newB’allele will in turn changeB-Iinto newB’in subsequent crosses. Previous research demonstrated that paramutation requires several proteins involved in the biosynthesis of small interfering RNAs (siRNAs) all related to the RNA-dependent DNA-methylation (RdDM) pathway. Yet, few members of the RdDM were functionally identified in maize. Here, we identify two new members of the maize RdDM pathway, and provide evidence that they are also involved in paramutation at theb1locus.
Assessment of dependency and consumption pattern of different forest products by the forest fringe villages of Shivalik Himalaya, Uttarakhand, India
Authors: Jiju J.S. , Dasgupta Soumya , Kumar Amit , Gera Mohit
Published: April 2021

Forest are essential for human beings for the enormous services it gives for livelihood and subsistence in the developing countries. We estimated the consumption and extraction levels of three major forest products viz., timber, fuelwood, and fodder in 20 forest fringe villages of the Timli forest range of Uttarakhand, India. We used a questionnaire-based household-level survey to collect information on the household economy and dependence of 380 households selected through stratified random sampling. We estimated that 69% of the overall yearly timber consumption of 20 villages, which comes around 2750 cubic meters (cum), was extracted from the nearby forest. The average timber consumption was 0.52 ± 0.22 cum household-1year-1. We estimated the total annual fuelwood and fodder consumption to be 298913.89, and 204475 Quintal (Qt). The average fuelwood and fodder consumption were estimated to be 417.6 Quintal household-1year-1and 49 ± 9.1 Qt. household-1year-1. We did general linear regression analysis to assess major biophysical and socio-economic determinants of villages and households for dependency on timber, fuelwood, and fodder. We found that the population of the village, distance from forest, distance from market, and annual average income are the major determining factors for timber, fodder, and fuelwood demand of the villages. Extraction of timber and non-timber forest products was the primary cause of depletion of forest biomass and forest carbon emission. Conservation effective management strategies in collaboration with all the stakeholder departments are needed to conserve forest resources with minimum extraction pressure from forest fringe villages of the study area.

Highlights

Questionnaire based household level survey was done to assess extraction and consumption patter of fuelwood, fodder and timber in Shivalik range of Uttarakhand, India.

Fuelwood and Timber consumption was found high compared to other studies in the low altitude areas.

69% of total timber and 62% of the total fodder requirement met from the adjacent forest areas.

Population of the village, distance from the forest and nearby market place, and annual average income were the major determinants of dependency of different forest products.
The genetic architecture underlying body-size traits plasticity over different temperatures and developmental stages inCaenorhabditis elegans
Authors: Maulana Muhammad I. , Riksen Joost A.G. , Snoek Basten L. , Kammenga Jan E. , Sterken Mark G.
Published: April 2021

Most ectotherms obey the temperature-size rule, meaning they grow larger in a colder environment. This raises the question of how the interplay between genes and temperature affect the body size of ectotherms. Despite the growing body of literature on the physiological life-history and molecular genetic mechanism underlying the temperature-size rule, the overall genetic architecture orchestrating this complex phenotype is not yet fully understood. One approach to identify genetic regulators of complex phenotypes is Quantitative Trait Locus (QTL) mapping. Here, we explore the genetic architecture of body size phenotypes, and plasticity of body-size phenotypes in different temperatures usingCaenorhabditis elegansas a model ectotherm. We used 40 recombinant inbred lines (RILs) derived from N2 and CB4856, which were reared at four different temperatures (16°C, 20°C, 24°C, and 26°C) and measured at two developmental stages (L4 and adult). The animals were measured for body length, width at vulva, body volume, length/width ratio, and seven other body-size traits. The genetically diverse RILs varied in their body-size phenotypes with heritabilities ranging from 0.0 to 0.99. We detected 18 QTL underlying the body-size traits across all treatment combinations, with the majority clustering on Chromosome X. We hypothesize that the Chromosome X QTL could result from a known pleiotropic regulator –npr-1 –known to affect the body size ofC. elegansthrough behavioral changes. We also found five plasticity QTL of body-size which three of them colocalized with some body-size QTL at certain temperature. In conclusion, our findings shed more light on multiple loci affecting body size plasticity and the possibility of co-regulation of traits and traits plasticity by the same loci under different environment.
Homologs of circadian clock proteins impact the metabolic switch between light and dark growth in the cyanobacteriumSynechocystissp. PCC 6803
Authors: Scheurer Nina M. , Rajarathinam Yogeswari , Timm Stefan , Köbler Christin , Kopka Joachim , Hagemann Martin , Wilde Annegret
Published: April 2021

The putative circadian clock system of the facultative heterotrophic cyanobacterial strainSynechocystissp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and nontargeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaAand, to a lesser extent, in the ΔkaiAB1C1mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaAmutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulatorrpaAhad a more pronounced effect on metabolism than the deletion of thekaigenes. The larger impact of therpaAmutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins inSynechocystisfor balanced carbon and nitrogen metabolism during light-to-dark transitions.
LIN37-DREAM Prevents DNA End Resection and Homologous Recombination at DNA Double Strand Breaks in Quiescent Cells
Authors: Chen Bo-Ruei , Wang Yinan , Tubbs Anthony T. , Zong Dali , Fowler Faith C. , Zolnerowich Nicholas , Wu Wei , Bennett Amelia , Chen Chun-Chin , Feng Wendy , Nussenzweig Andre , Tyler Jessica K. , Sleckman Barry P.
Published: April 2021

DNA double strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0cells. Loss of LIN37 leads to expression of HR proteins, including BRCA1, BRCA2, PALB2 and RAD51, and DNA end resection in G0cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.
Human-derived alleles inSOSTandRUNX23′UTRs cause differential regulation in a bone cell-line model
Authors: Moriano Juan , Martínez-Gil Núria , Andirkó Alejandro , Balcells Susana , Grinberg Daniel , Boeckx Cedric
Published: April 2021

The inquiry into the phenotypic features that set apart human species, such as a light, gracile skeleton and a rounded skull characteristic ofHomo sapiens, can now benefit from the examination of ancient genomes. These have added a new layer of analysis allowing the identification of genetic differences between species like ours and our closest extinct relatives. Most of these genetic differences are non-coding changes with unknown functional consequences, and dissecting their putative regulatory effect remains challenging. Here we focus on the three prime untranslated regions (3’UTR), known to play a critical role in messenger RNA regulation and a plausible locus for divergent regulation betweenHomospecies. We report a set of genes with derived 3’UTR changes in either theHomo sapiensor the Neanderthal/Denisovan lineages and experimentally evaluate the impact of 3’UTR variants in four genes:E2F6,GLI3,RUNX2andSOST. We performed a luciferase reporter assay in a bone cell-line model and found a statistically significant difference for the 3’UTR variants ofSOST(Homo sapiens-derived) andRUNX2(Neanderthal/Denisovan-derived). The differential expression caused by these variants in our experimental model points to species differences in bone mineral density. Thus, this study adds insights into the functional effects of regulatory variants that emerged in recent human evolution.
Direct observation of aggregate-triggered selective autophagy
Authors: Janssen Anne F.J. , Korsten Giel , Nijenhuis Wilco , Katrukha Eugene A. , Kapitein Lukas C.
Published: April 2021

Degradation of aggregates by selective autophagy is important as damaged proteins may impose a threat to cellular homeostasis. Although the core components of the autophagy machinery are well-characterized, the spatiotemporal regulation of many selective autophagy processes, including aggrephagy, remains largely unexplored. Furthermore, because most live-cell imaging studies have so far focused on starvation-induced autophagy, little is known about the dynamics of aggrephagy. Here, we describe the development and application of the mKeima-PIM assay, which enables live-cell observation of autophagic turnover and degradation of inducible protein aggregates in conjunction with key autophagy players. This allowed us to quantify the relative timing and duration of different steps of aggrephagy and revealed the short-lived nature of the autophagosome. The assay furthermore showed the spatial distribution of omegasome formation, highlighting that autophagy initiation is directly instructed by the cargo. Moreover, we found that nascent autophagosomes mostly remain immobile until acidification occurs. Thus, our assay provides new insights into the spatiotemporal regulation and dynamics of aggrephagy.
Single Cell Chemical Proteomics (SCCP) Interrogates the Timing and Heterogeneity of Cancer Cell Commitment to Death
Authors: Végvári Ákos , Rodriguez Jimmy E , Zubarev Roman A
Published: April 2021

Chemical proteomics studies the effects of drugs upon cellular proteome. Due to the complexity and diversity of tumors, the response of cancer cells to drugs is also heterogeneous, and thus proteome analysis at single cell level is needed. Here we demonstrate that single cell proteomics techniques have become quantitative enough to tackle the drug effects on the target proteins, enabling single cell chemical proteomics (SCCP). Using SCCP, we studied here the time-resolved response of individual adenocarcinoma A549 cells to anticancer drugs methotrexate, camptothecin and tomudex, revealing the early emergence of cellular subpopulations committed and uncommitted to death. As a novel and useful approach to exploring the heterogeneous response to drugs of cancer cells, SCCP may prove to be a “killer application” for single cell proteomics.
Absolute quantification and single-cell dose-response of cytosolic siRNA delivery
Authors: Hedlund Hampus , Du Rietz Hampus , Johansson Johanna , Zedan Wahed , Huang Linfeng , Wallin Jonas , Wittrup Anders
Published: April 2021

Endosomal escape and subsequent cytosolic delivery of small inhibitory RNA (siRNA) therapeutics is believed to be highly inefficient. Since, it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determination of delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to detect cytosolic delivery of fluorescently labelled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.
Host regulator PARP1 contributes to sex differences and immune responses in a mouse model of tuberculosis
Authors: Krug Stefanie , Ordonez Alvaro A. , Klunk Mariah , Kang Bong Gu , Jain Sanjay K. , Dawson Ted M. , Dawson Valina L. , Bishai William R.
Published: April 2021

Tuberculosis (TB) is a devastating infectious disease responsible for nearly 2 million deaths annually that has a poorly understood male bias. Elucidating the basis of this male bias may enable precision medicine interventions for TB treatment and prevention. Here, we identify the master regulator Poly(ADP-ribose) Polymerase 1 (PARP1) as a driver of TB sex differences. We found that infection withM. tuberculosis(M. tb) triggers robust PARP activation in mouse lungs, suggesting that PARP1 activation is a fundamental host response to TB. Remarkably, PARP1 deletion abolished known sex differences in TB cytokine responses and blunted the early induction of TNFα, IL-1ß, IFNγ, MCP-1, and IL-6, particularly in male mice. In contrast, PARP1 was required for IL-10 induction in male or female mice. PARP1 deletion was protective against TB in female mice, resulting in significantly prolonged survival and reduced bacterial burden, but impaired TB containment in male mice. Our findings indicate that PARP1 contributes to TB sex differences via sexually divergent immune regulation and uniquely enhances early proinflammatory responses in males that prove beneficial for TB containment.
Stem cell quiescence requires PRC2/PRC1-mediated mitochondrial checkpoint
Authors: Ishibashi JR , Taslim TH , Hussein AM , Brewer D , Liu S , Harper S , Nguyen B , Dang J , Chen A , Del Castillo D , Mathieu J , Ruohola-Baker H
Published: April 2021

Both normal and tumorous stem cells can arrest cell division, avoid apoptosis, and then regenerate lost daughter cells following acute genotoxic insult. This protective, reversible proliferative arrest, known as “quiescence,” is still poorly understood. Here, we show that mTOR-regulated mitophagy is required for radiation insult-induced quiescence inDrosophilagermline stem cells (GSCs). In GSCs, depletion of mito-fission (Drp1) or mitophagy (Pink1 and Parkin) eliminates entry into quiescence, while depletion of mitochondrial biogenesis (PGC1α) or fusion (Mfn2) eliminates exit from quiescence. We also find that mitophagy-dependent quiescence is under epigenetic control; knockdown of Jarid2 (PRC2) or Pc or Sce (PRC1) stabilizes the mitochondria and locks GSCs out of quiescence, while knockdown of PRC2-specific demethylase, Utx, prevents re-accumulation of the mitochondria and locks GSCs in quiescence. These data suggest that mitochondrial number coordinates reversible quiescence. We further identify that the mechanism of quiescence in both GSCs and human induced pluripotent stem cells (iPSCs) relies on mitophagy to deplete the mitochondrial pool of CycE and limit cell cycle progression. This alternative method of G1/S regulation may present new opportunities for therapeutic purposes.
GC-biased gene conversion in X-Chromosome palindromes conserved in human, chimpanzee, and rhesus macaque
Authors: Jackson Emily K. , Bellott Daniel W. , Skaletsky Helen , Page David C.
Published: April 2021

Gene conversion is GC-biased across a wide range of taxa. Large palindromes on mammalian sex chromosomes undergo frequent gene conversion that maintains arm-to-arm sequence identity greater than 99%, which may increase their susceptibility to the effects of GC-biased gene conversion. Here, we demonstrate a striking history of GC-biased gene conversion in 12 palindromes conserved on the X chromosomes of human, chimpanzee, and rhesus macaque. Primate X-chromosome palindrome arms have significantly higher GC content than flanking single-copy sequences. Nucleotide replacements that occurred in human and chimpanzee palindrome arms over the past 7 million years are one-and-a-half times as GC-rich than the ancestral bases they replaced. Using simulations, we show that our observed pattern of nucleotide replacements is consistent with GC-biased gene conversion with a magnitude of 70%, similar to previously reported values based on analyses of human meioses. However, GC-biased gene conversion explains only a fraction of the observed difference in GC content between palindrome arms and flanking sequence, suggesting that additional factors are required to explain elevated GC content in palindrome arms. This work supports a greater than 2:1 preference for GC bases over AT bases during gene conversion, and demonstrates that the evolution and composition of mammalian sex chromosome palindromes is strongly influenced by GC-biased gene conversion.
Dynamic conformational states of apo and cabozantinib bound TAM kinases to differentiate active-inactive kinetic models
Authors: Naresh Gatta K R S , Guruprasad Lalitha
Published: April 2021

Tyro3, Axl, Mer (TAM) receptor tyrosine kinases (RTKs) are overexpressed in several human cancers. Cabozantinib, a small molecule inhibitor constrains the activity of TAM kinases at nanomolar concentrations. The dynamic active and inactive conformations of kinases play a crucial role in inhibitor binding and the activation of intracellular downstream signalling pathways. The all atom molecular dynamics (MD) simulations at microsecond timescale and longer provide robust insights into the structural details of conformational alterations of proteins due to their role cellular metabolic activities and signaling pathways. In this current study we report microsecond molecular dynamics (MD) simulations of apo, cabozantinib complexed active and inactive TAM RTKs and analysed the post-MD trajectories using the principal component analysis (PCA). Markov State Models (MSM) and transition pathways from Perron-cluster cluster analysis. For consensus, the 1µs atomistic simulations with enhanced computational algorithms indicated us to treat tyrosine kinase family by overwhelming dynamic states existence when bound to kinase inhibitors. The dynamic mechanistic pathways intrinsic to the kinase activity and protein conformational landscape in the TAM kinases are revealed due to the alterations in the P-loop, αC-helix, activation loop and αF-helix that result in breaking the regulatory and catalytic spines. We deciphered the long lived kinetic transition states of distinct active and inactive structural models from MD simulations trajectories of TAM RTKs bound inhibitor complex that have not been revealed so far.
NrnA is a linear dinucleotide phosphodiesterase with limited function in cyclic dinucleotide metabolism inListeria monocytogenes
Authors: Gall Aaron R. , Siletti Cheta , Huynh TuAnh N.
Published: April 2021

Listeria monocytogenesproduces both c-di-AMP and c-di-GMP to mediate many important cellular processes, but the levels of both nucleotides must be regulated. C-di-AMP accumulation attenuates virulence and diminishes stress response, and c-di-GMP accumulation impairs bacterial motility. An important regulatory mechanism to maintain c-di-AMP and c-di-GMP homeostasis is to hydrolyze them to the linear dinucleotides pApA and pGpG, respectively, but the fates of these hydrolytic products have not been examined inL. monocytogenes. We found that NrnA, a stand-alone DHH-DHHA1 phosphodiesterase, has a broad substrate range, but with a strong preference for linear dinucleotides over cyclic dinucleotides. Although NrnA exhibited detectable cyclic dinucleotide hydrolytic activities in vitro, NrnA had negligible effects on their levels in the bacterial cell, even in the absence of the c-di-AMP phosphodiesterases PdeA and PgpH. The ΔnrnAmutant had a mammalian cell infection defect that was fully restored byE. coliOrn. Together, our data indicate thatL. monocytogenesNrnA is functionally orthologous to Orn, and its preferred physiological substrates are most likely linear dinucleotides. Furthermore, our findings revealed that, unlike some other c-di-AMP and c-di-GMP-producing bacteria,L. monocytogenesdoes not employ their hydrolytic products to regulate their phosphodiesterases, at least at the pApA and pGpG levels in the ΔnrnAmutant. Finally, the ΔnrnAinfection defect was overcome by constitutive activation of PrfA, the master virulence regulator, suggesting that accumulated linear dinucleotides might inhibit the expression, stability, or function of PrfA-regulated virulence factors.

IMPORTANCE

Listeria monocytogenesproduces both c-di-AMP and c-di-GMP, and encodes specific phosphodiesterases that degrade them into pApA and pGpG, respectively, but the metabolism of these products has not been characterized in this bacterium. We found thatL. monocytogenesharbors an NrnA homolog that degrades a broad range of nucleotides, but exhibits a strong biochemical and physiological preference for linear dinucleotides, including pApA and pGpG. Unlike in some other bacteria, these oligoribonucleotides do not appear to interfere with cyclic dinucleotide hydrolysis. The absence of NrnA is well tolerated byL. monocytogenesin broth cultures but impairs its ability to infect mammalian cells. These findings indicate a separation of cyclic dinucleotide signaling and oligoribonucleotide metabolism inL. monocytogenes.
3’ untranslated regions of Marburg and Ebola virus mRNAs possess negative regulators of translation that are modulated by ADAR1 editing
Authors: Khadka Sudip , Williams Caroline G. , Sweeney-Gibbons Joyce , Basler Christopher F.
Published: April 2021

The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5’ and 3’ untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3’UTRs are prone to editing by ADAR1. A reporter mRNA approach, in which different 5’ or 3’UTRs were inserted into luciferase encoding mRNAs, demonstrates that MARV 3’UTRs yield different levels of reporter gene expression suggesting modulation of translation. The modulation occurs in cells unable to produce miRNAs and can be recapitulated in a minigenome assay. Deletion mutants identified negative regulatory regions at end of the MARV NP and L 3’UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3’UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3’UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3’UTR, but not the edited or deletion mutant NP 3’UTRs, induce a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the VP40 3’UTR, VP40 3’UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3’UTR edits also enhanced translation but neither the wildtype nor the edited 3’UTRs induced IFN. These findings implicate filoviral mRNA 3’UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1.

Importance

UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and of ADAR1 editing have been uncertain. This study demonstrates that MARV and EBOV 3’UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3’UTRs, relieves the negative effects of the UTRs. These data indicate that filovirus 3’UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.
WzcandWcaJmutations in hypervirulentKlebsiella pneumoniaelead to phage resistance at the cost of reduced virulence
Authors: Song Lingjie , Yang Xianggui , Huang Jinwei , Zhu Xiaokui , Han Guohui , Wan Yan , Xu Ying , Luan Guangxin , Jia Xu
Published: April 2021

HypervirulentKlebsiella pneumoniae(hvKp) is one of the major community-acquired pathogens, which can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment ofKlebsiella pneumoniae, but the characteristics of the phage resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. Invitroand invivoexperiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations ofwzcandwcaJgenes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule, meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. The findings can contribute to a better understanding of that bacteriophage therapy can not only kill bacteria directly, but also reduce the virulence of bacteria by phage screening.

Importance

Bacteriophages are considered potential therapeutic alternative to antibiotics; however host-evolved phage resistance has accounted for the resurgences of pathogens, meaning further measures are need to improve the efficacy of phage therapy. This study showed two phages capable of infecting hypervirulentK. pneumoniaeand identified phage-resistant mutants whose virulence was significantly reduced. Gene sequencing analysis revealed that mutations ofwzcandwcaJgene, related to capsule synthesis, recovered phage resistance but reduced the virulence of hypervirulentK. pneumoniae.
Efficient prediction of a spatial transcriptomics profile better characterizes breast cancer tissue sections without costly experimentation
Authors: Monjo Taku , Koido Masaru , Nagasawa Satoi , Suzuki Yutaka , Kamatani Yoichiro
Published: April 2021

Spatial transcriptomics is an emerging technology requiring costly reagents and considerable skills, limiting the identification of transcriptional markers related to histology. Here, we show that predicted spatial gene-expressions in unmeasured regions and tissues can enhance biologists’ histological interpretations. We developed theDeeplearning model forSpatial geneClusters andExpression, DeepSpaCE and confirmed its performance using the spatial-transcriptome profiles and immunohistochemistry images of consecutive human breast cancer tissue sections. For example, the predicted expression patterns ofSPARC, an invasion marker, highlighted a small tumor-invasion region that is difficult to identify using raw data of spatial transcriptome alone because of a lack of measurements. We further developed semi-supervised DeepSpaCE using unlabeled histology images and increased the imputation accuracy of consecutive sections, enhancing applicability for a small sample size. Our method enables users to derive hidden histological characters via spatial transcriptome and gene annotations, leading to accelerated biological discoveries without additional experiments.
Brain-Wide Inferiority and Equivalence Tests in fMRI Group Analyses: Selected Applications
Authors: Gerchen Martin Fungisai , Kirsch Peter , Feld Gordon Benedikt
Published: April 2021

Null hypothesis significance testing is the major statistical procedure in the field of fMRI, but provides only a rather limited picture of the effects in a data set. When sample size and power is low relying only on strict significance testing may lead to a host of false negative findings. In contrast, with very large data sets virtually every voxel might become significant.

It is thus desirable to complement significance testing with procedures like inferiority and equivalence tests that allow to formally compare effect sizes within and between data sets and offer novel approaches to obtain insight into fMRI data. The major component of these tests are estimates of standardized effect sizes and their confidence intervals. Here we show how Hedge’s g, the bias corrected version of Cohen’s d, and its confidence interval can be obtained from SPM t maps. We then demonstrate how these values can be used to evaluate whether non-significant effects are really statistically smaller than significant effects to obtain “regions of undecidability” within a data set, and to test for the replicability and lateralization of effects.

This method allows the analysis of fMRI data beyond point estimates enabling researchers to take measurement uncertainty into account when interpreting their findings.
Genome-wide association uncovers the genetic architecture of tradeoff between flowering date and yield components in sesame
Authors: Sabag Idan , Morota Gota , Peleg Zvi
Published: April 2021

Unrevealing the genetic makeup of crop morpho-agronomic traits is essential for improving yield quality and sustainability. Sesame (Sesamum indicumL.), one of the oldest oil-crops in the world, which despite its economical and agricultural importance, is an ‘orphan crop-plant’ that undergone limited modern selection, thus, preserving wide genetic diversity. Here we harnessed this natural variation in a newly developed sesame panel (SCHUJI) to perform genome-wide association studies for morpho-agronomic traits under the Mediterranean climate conditions. Field-based phenotyping of the SCHUJI panel across two seasons exposed wide phenotypic variation for all traits. Using 20,294 single-nucleotide polymorphism markers, we detected 50 genomic signals associated with these traits. Major genomic region on LG2 was associated with flowering date and yield-related traits, exemplified the key role of the flowering date on productivity. Our results shed light on the genetic architecture of flowering date and its interaction with yield components in sesame and may serve as a basis for future sesame breeding programs in the Mediterranean basin.
Prevalence and genotyping of Trichomonas gallinae in Riyadh, Saudi
Arabia
Authors: Albeshr Mohammed Fahad , Alrefaei Abdulwahed Fahad
Published: April 2021

Trichomonas gallinae is a single-celled protozoan
parasite and causative agent of trichomonosis. Trichomonosis is a disease with a
worldwide distribution, and has recently been highlighted as a pandemic threat
to several wild bird species. The aim of this study is to investigate the
prevalence and genotypic diversity of Trichomonas
gallinae in Riyadh, Saudi Arabia. A total of 273 oral swab
samples from birds were collected and tested for T.
gallinae infection, and the overall prevalence of
Trichomonas gallinae in these samples was 26.4%
(72 of 273). We compared the rates of infections with T.
gallinae between wild and domestic pigeons and found that the
infection was significantly higher in domestic pigeons; a finding which should
be considered by the Ministry of Environment, Water and Agriculture of Saudi
Arabia when evaluating the role of poultry markets in the transmission of
trichomonosis. Sequence analyses of the internal transcribed spacer (ITS) region
indicated genetic variation in these parasite strains, as found in the samples
collected. Among 48 sequences 15 different ribotypes were found, 12 of which
were novel, and 3 of which were previously-described ribotypes: ribotype A, C,
and II. This study demonstrates the diversity of T.
gallinae strains in Saudi Arabian birds for the first time,
and revealed that ribotypes A and C are predominant among Riyadh birds.
Interfaces between cellular responses to DNA damage and cancer immunotherapy
Authors: Pilger Domenic , Seymour Leonard W. , Jackson Stephen P.
Published: April 2021

The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications.
Modeling transcriptional regulation of model species with deep learning
Authors: Cofer Evan M. , Raimundo João , Tadych Alicja , Yamazaki Yuji , Wong Aaron K. , Theesfeld Chandra L. , Levine Michael S. , Troyanskaya Olga G.
Published: April 2021

To enable large-scale analyses of transcription regulation in model species, we developed DeepArk, a set of deep learning models of the cis-regulatory activities for four widely studied species: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, and Mus musculus. DeepArk accurately predicts the presence of thousands of different context-specific regulatory features, including chromatin states, histone marks, and transcription factors. In vivo studies show that DeepArk can predict the regulatory impact of any genomic variant (including rare or not previously observed) and enables the regulatory annotation of understudied model species.
Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes
Authors: Durham Timothy J. , Daza Riza M. , Gevirtzman Louis , Cusanovich Darren A. , Bolonduro Olubusayo , Noble William Stafford , Shendure Jay , Waterston Robert H.
Published: April 2021

Recently developed single-cell technologies allow researchers to characterize cell states at ever greater resolution and scale. Caenorhabditis elegans is a particularly tractable system for studying development, and recent single-cell RNA-seq studies characterized the gene expression patterns for nearly every cell type in the embryo and at the second larval stage (L2). Gene expression patterns give insight about gene function and into the biochemical state of different cell types; recent advances in other single-cell genomics technologies can now also characterize the regulatory context of the genome that gives rise to these gene expression levels at a single-cell resolution. To explore the regulatory DNA of individual cell types in C. elegans, we collected single-cell chromatin accessibility data using the sci-ATAC-seq assay in L2 larvae to match the available single-cell RNA-seq data set. By using a novel implementation of the latent Dirichlet allocation algorithm, we identify 37 clusters of cells that correspond to different cell types in the worm, providing new maps of putative cell type–specific gene regulatory sites, with promise for better understanding of cellular differentiation and gene regulation.
Financial innovation regulations and firm performance: Evidence from Chinese listed firms
Authors: Yang Minhua
Published: April 2021

This study investigates the impacts of financial innovation regulations on Chinese listed firms in the period of 2008 to 2020, which comes to the theoretical contribution of this paper. These regulations introduced in 2016 mainly focus on the peer‐to‐peer (P2P) lending platforms in this country and tend to be negatively associated with firm performance, especially for those firms investing in the P2P lending platforms, as a large amount of P2P lending platforms with unfavourable performance disappear in recent years, which may end up negatively affecting those firms investing in them. Survived P2P lending platforms tend to be stronger, then become a type of substitute of traditional banks and are able to carve up the market share of bank. The latter therefore has to increase credit supply to maximise profits, which leads to the access to more credits and therefore worse firm performance, especially for those firms with financial constraints in terms of the cash–cash flow sensitivity. The empirical results are robust to the potential endogeneity issue of financial constraints. Larger firms and state‐owned enterprises (SOEs) tend to have better performance, implying that authorities may take actions for smaller firms (non‐SOEs) to offset the negative impacts of financial innovation regulations they suffer and achieve better implementation of financial innovation regulations, which comes to the practical contribution of this paper.