bluuebunny/crossref_metadata_2025 huggingface dataset:
A programmable system to methylate and demethylate m6A on specific mRNAs
Authors: Chang Chen , Ma Gang , Cheung Edwin , Hutchins Andrew P.
Published: April 2021

RNA N6-Methyladenosine (m6A) is the most abundant mRNA modification, and forms part of an epitranscriptomic system that modulates RNA function. RNA modifications can be reversibly catalyzed by several specific enzymes, and those modifications can be recognized by RNA binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m6A participation in critical biological functions, this exploration has mainly been conducted through the global knockout or knockdown of the writers, erasers, or readers of m6A. Consequently, there is a lack of information about the role of m6A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m6A. Here, we demonstrate a CRISPR/dCas13a-based RNA m6A-editor which can target mRNAs using single crRNA or multiple crRNAs array to methylate or demethylate m6A. We systematically assay its capabilities to enable the targeted rewriting of m6A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we demonstrate the utility of the system by specifically modulating XIST m6A levels, which can control X chromosome silencing and activation. Based on our editors, m6A on single and multiple transcripts can be modified to allow the exploration of the role of m6A on in biological processes.
Homo-harringtonine (HHT) – A highly effective drug against coronaviruses and the potential for large-scale clinical applications
Authors: Wen Hai-Jun , Lin Pei , Zhong Gong-Xun , Xu Zhi-Chao , Shuai Lei , Wen Zhi-Yuan , Wang Chong , Cao Xue , He Wen-Bin , Feng Jing , Cai Qi-Chun , Ma Hua-Juan , Wu Si-Jin , Wang Guo-Dong , Lyu Xue-Mei , Liu Feng-Liang , Zheng Yong-Tang , Zeng Hui , He Xiong-Lei , Chen Hualan , Zhang Fu-Jie , Wu Chung-I
Published: April 2021

In the search for treatment schemes of COVID-19, we start by examining the general weakness of coronaviruses and then identify approved drugs attacking that weakness. The approach, if successful, should identify drugs with a specific mechanism that is at least as effective as the best drugs proposed and are ready for clinical trials. All coronaviruses translate their non-structural proteins (∼16) in concatenation, resulting in a very large super-protein. Homo-harringtonine (HHT), which has been approved for the treatment of leukemia, blocks protein elongation very effectively. Hence, HHT can repress the replication of many coronaviruses at the nano-molar concentration. In two mouse models, HHT clears SARS-CoV-2 in 3 days, especially by nasal dripping of 40 ug per day. We also use dogs to confirm the safety of HHT delivered by nebulization. The nebulization scheme could be ready for large-scale applications at the onset of the next epidemics. For the current COVID-19, a clinical trial has been approved by the Ditan hospital of Beijing but could not be implemented for want of patients. The protocol is available to qualified medical facilities.
Dynamics of switching at stall reveals non-equilibrium mechanism in the allosteric regulation of the bacterial flagellar switch
Authors: Wang Bin , Niu Yuhui , Zhang Rongjing , Yuan Junhua
Published: April 2021

Behavior of the bacterial flagellar motor depends sensitively on the external loads it drives. Motor switching, which provides the basis for the run-and-tumble behavior of flagellated bacteria, has been studied for motors under zero to high loads, revealing a non-equilibrium effect that is proportional to the motor torque. However, behavior of the motor switching at stall (with maximum torque) remains unclear. An extrapolation from previous studies would suggest maximum non-equilibrium effect for motor switching at stall. Here, we stalled the motor using optical tweezers and studied the motor switching with a high time resolution of about 2 ms. Surprisingly, our results showed exponentially distributed counterclockwise (CCW) and clockwise (CW) intervals, indicating that motor switching at stall is probably an equilibrium process. Combined with previous experiments at other loads, our result suggested that the non-equilibrium effect in motor switching arises from the asymmetry of the torque generation in the CCW and CW directions. By including this non-equilibrium effect in the general Ising-type conformation spread model of the flagellar switch, we consistently explained the motor switching over the whole range of load conditions. We expect to see similar mechanism of non-equilibrium regulation in other molecular machines.
Improving statistical power in severe malaria genetic association studies by augmenting phenotypic precision
Authors: Watson James A , Ndila Carolyne M , Uyoga Sophie , Macharia Alexander W , Nyutu Gideon , Shebe Mohammed , Ngetsa Caroline , Mturi Neema , Peshu Norbert , Tsofa Benjamin , Rockett Kirk , Leopold Stije , Kingston Hugh , George Elizabeth C , Maitland Kathryn , Day Nicholas PJ , Dondorp Arjen , Bejon Philip , Williams Thomas N , Holmes Chris C , White Nicholas J
Published: April 2021

Severe falciparum malaria has substantially affected human evolution. Genetic association studies of patients with clinically defined severe malaria and matched population controls have helped characterise human genetic susceptibility to severe malaria, but phenotypic imprecision compromises discovered associations. In areas of high malaria transmission the diagnosis of severe malaria in young children and, in particular, the distinction from bacterial sepsis, is imprecise. We developed a probabilistic diagnostic model of severe malaria using platelet and white count data. Under this model we re-analysed clinical and genetic data from 2,220 Kenyan children with clinically defined severe malaria and 3,940 population controls, adjusting for phenotype mis-labelling. Our model, validated by the distribution of sickle trait, estimated that approximately one third of cases did not have severe malaria. We propose a data-tilting approach for case-control studies with phenotype mis-labelling and show that this reduces false discovery rates and improves statistical power in genome-wide association studies.
Protein content and lipid profiling of isolated native autophagosomes
Authors: Schmitt Daniel , Bozkurt Süleyman , Henning-Domres Pascale , Huesmann Heike , Eimer Stefan , Bindila Laura , Tascher Georg , Münch Christian , Behl Christian , Kern Andreas
Published: April 2021

Autophagy is a central eukaryotic catabolic pathway responsible for clearance and recycling of an extensive portfolio of cargoes, which are packed in vesicles, called autophagosomes, and are delivered to lysosomes for degradation. Besides basal autophagy, which constantly degrades cellular material, the pathway is highly responsive to several stress conditions. However, the exact protein content and phospholipid composition of autophagosomes under changing autophagy conditions remain elusive so far. Here, we introduce a FACS-based approach for isolation of native unmanipulated autophagosomes and ensure the quality of the preparations. Employing quantitative proteomics and phospholipidomics, we obtained a profound cargo and lipid profile of autophagosomes purified upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. Indeed, starvation only mildly affected the content profile, while interference with proteasome activity showed stronger effects and specifically altered autophagosome cargoes. Interestingly, the phospholipid composition of autophagosomes was unaffected by the different treatments. Thus, the novel isolation method enables purification of intact autophagosomes in large quantities and allows protein content and phospholipid profiling without the requirement of exhaustive cellular fractionation or genetic manipulation.
Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells
Authors: Nguyen Vy N. , Brunon Salomé , Pavlova Maria N. , Lazarchuk Pavlo , Sharifian Roya D. , Sidorova Julia M.
Published: April 2021

The cGAS/STING pathway, part of the innate immune response to foreign DNA, is known to be activated by cell’s own DNA arising from the processing of the genome, including the excision of nascent DNA at arrested replication forks. We found STING activation to affect nascent DNA processing, suggesting a novel, unexpected feedback connection between the two events. Depletion of STING suppressed and re-expression of the protein in STING-deficient cells upregulated degradation of nascent DNA. Fork arrest was accompanied by the STING pathway activation, and a STING mutant that does not activate the pathway failed to upregulate nascent strand degradation. Consistent with this, cells expressing the STING mutant had a reduced level of RPA on parental and nascent DNA of arrested forks as well as a reduced CHK1 activation compared to the cells with wild type STING. Together our findings reveal a novel connection between replication stress and innate immunity.
Decellularized Articular Cartilage Microparticles for Expansion of Mesenchymal Stem Cells and Zonal Regeneration of Articular Cartilage
Authors: Sepahvandi Azadeh , Kader Safaa Ibrahim , Monavarian Mehri , Madormo Victor Anthony , Jabbari Esmaiel
Published: April 2021

Introduction

The objective was to create multilayer cellular constructs using fetal or adult, decellularized articular cartilage in particulate form as microcarriers for expansion and fusion of mesenchymal stem cells (MSCs) to regenerate the stratified structure of articular cartilage.

Methods

Porous microparticles (CMPs) generated from decellularized fetal or adult bovine articular cartilage were used as microcarriers for expansion of human MSCs. The CMP expanded MSCs (CMP-MSCs) were used to generate injectable hydrogels or preformed multilayer constructs for articular cartilage regeneration. In the injectable approach, CMP-MSCs were suspended in alginate gel, crosslinked with calcium chloride, and incubated in chondrogenic medium to generate an injectable regenerative construct. In the preformed approach, fetal or adult CMP-MSCs were suspended in a culture medium, allowed to settle sequentially by the force of gravity, and fused by incubation in chondrogenic medium to generate multilayer cell sheets. The constructs were characterized with respect to compressive modulus, cellularity, and expression of chondrogenic markers.

Results

Human MSCs expanded on fetal or adult CMPs in basal medium maintained the expression of mesenchymal markers. The injectable CMP-MSCs hydrogels had significantly higher expression of chondrogenic markers and compressive modulus after four weeks incubation in chondrogenic medium compared to MSCs directly encapsulated in alginate gel; preformed CMP-MSCs cell sheets had significantly higher compressive modulus and expression of chondrogenic markers compared to MSCs in the pellet culture.

Conclusion

The preformed cell sheet approach is potentially useful for creating multilayer constructs by sequential gravitational settling of CMP-MSCs to mimic the stratified structure of articular cartilage.

Insight, Innovation, Integration

This work described a novel approach to recreate the zonal structure of articular cartilage. Human MSCs were expanded on porous microcarrier beads generated from decellularized fetal or adult bovine articular cartilage. The cell-seeded microbeads were fused by gravitational settling to form mono- or bi-layer cell sheets. The cell sheets were cultured in chondrogenic medium to regenerate the articular cartilage tissue. Thein vitroregenerated tissue had higher compressive modulus and expression of chondrogenic markers compared to the MSC pellet culture.
Drosophila fabpis a retinoid-inducible gene required for Rhodopsin-1 homeostasis and photoreceptor survival
Authors: Huang Huai-Wei , Ryoo Hyung Don
Published: April 2021

Retinoids act as chromophore co-factors for light-detecting rhodopsin proteins. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate gene expression inDrosophilafor a specific biological function remains unclear. Here, we report thatDrosophila fatty acid binding protein(fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we performed a photoreceptor-specific gene expression profiling study in flies bearing a misfolding-prone Rhodopsin-1 (Rh1) mutant,ninaEG69D, which serves as aDrosophilamodel for Retinitis Pigmentosa.ninaEG69Dphotoreceptors showed increased expression of genes that control Rh1 protein levels, along with a poorly characterized gene,fabp. We found that in vivofabpexpression was reduced when the retinoids were deprived through independent methods. Conversely,fabpmRNA was induced when we challenged culturedDrosophilacells with retinoic acid. In flies reared under light, loss offabpcaused an accumulation of Rh1 proteins in cytoplasmic vesicles.fabpmutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-activated Rh1 degradation. These observations indicate that a retinoid-inducible gene expression program regulatesfabpthat is required for Rh1 proteostasis and photoreceptor survival.

Author Summary

Rhodopsins are light-detecting proteins that use retinoids as chromophore co-factors. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate Rhodopsin function aside from its role as a chromophore remains unclear. Here, we report thatDrosophila fatty acid binding protein(fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we found thatfabpis among the genes induced by a misfolding-prone Rhodopsin-1 (Rh1) mutant,ninaEG69D, which serves as aDrosophilamodel for Retinitis Pigmentosa. We further found thatfabpinduction inninaEG69Dphotoreceptors required retinoids.fabpwas required in photoreceptors to help degrade light-activated Rh1. In the absence offabp, Rh1 accumulated in cytoplasmic vesicles in a light-dependent manner, and exhibited light-dependent retinal degeneration. These observations indicate that a retinoid-inducible gene expression program regulatesfabpthat is required for Rh1 proteostasis and photoreceptor survival.
The UBAP2L ortholog PQN-59 contributes to stress granule assembly and development inC. elegans
Authors: Abbatemarco Simona , Bondaz Alexandra , Schwager Francoise , Wang Jing , Hammell Christopher M , Gotta Monica
Published: April 2021

When exposed to stressful conditions, eukaryotic cells respond by inducing the formation of cytoplasmic ribonucleoprotein complexes called stress granules. Stress granules are thought to have a protective function but their exact role is still unclear. Here we useC. elegansto study two proteins that have been shown to be important for stress granule assembly in human cells: PQN-59, the ortholog of human UBAP2L, and GTBP-1, the ortholog of the human G3BP1 and G3BP2 proteins. Both proteins fall into stress granules in the embryo and in the germline whenC. elegansis exposed to stressful conditions. None of the two proteins is essential for the assembly of stress induced granules, but the granules formed in absence of PQN-59 or GTBP-1 are less numerous and dissolve faster than the ones formed in control embryos. Despite these differences,pqn-59orgtbp-1mutant embryos do not show a higher sensitivity to stress than control embryos.pqn-59mutants display reduced progeny and a high percentage of embryonic lethality, phenotypes that are not dependent on stress exposure and that are not shared withgtbp-1mutants. Our data indicate that both GTBP-1 and PQN-59 contribute to stress granule formation but that PQN-59 is, in addition, required forC. elegansdevelopment.

Author summary

The formation of so-called stress granules is an adaptive response that cells and organisms put into action to cope with changes in internal and environmental conditions and thus to survive to stressful conditions. Although it is generally thought that stress granule formation protects cells from stress-related damage, the exact role of stress granules in cells and organisms is not well understood. Moreover, the mechanisms governing stress granule assembly, and if and how the ability to form stress granules is important forC. elegansdevelopment is still unclear.

Our work focuses on two conserved proteins, known to be involved in stress granule assembly in mammalian cells, and investigates their role inC. elegansembryos. We find that these proteins are important but not essential to assemble stress-induced granules inC. elegans. We moreover did not observe a different sensitivity to stress exposure between wild-type and mutant developing embryos, suggesting that at least in these conditions these proteins do not exert a protective role.
SARS-CoV-2 proteins bind heme and hemoglobin
Authors: Lechuga Guilherme Curty , Souza-Silva Franklin , de Queiroz Sacramento Carolina , de Oliveira Trugilho Monique Ramos , Valente Richard Hemmi , Napoleão-Pêgo Paloma , da Silva Gomes Dias Suelen , Fintelman-Rodrigues Natalia , Temerozzo Jairo Ramos , Carels Nicolas , Alves Carlos Roberto , de Souza Pereira Mirian Claudia , Provance David William , Lopez Souza Thiago Moreno , De-Simone Salvatore Giovanni
Published: April 2021

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins’ possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach.In silicoanalysis indicated binding motifs between a cavity in the viral nucleoprotein and hemoglobin’s porphyrin coordination region. Different hemin binding capacities of mock and SARS-CoV-2-infected culture extracts were noticed using gel electrophoresis and TMB staining. Hemin-binding proteins were isolated from SARS-CoV-2-infected cells by affinity chromatography and identified by shotgun proteomics, indicating that structural (nucleoprotein, spike, and membrane protein) and non-structural (Nsp3 and Nsp7) viral proteins interact with hemin.In vitroanalyses of virus adsorption to host cells and viral replication studies in Vero cells demonstrated inhibitory activities - at different levels - by hemin, protoporphyrin IX (PpIX) Hb. Strikingly, free Hb at 1μM suppressed viral replication (99 %), and its interaction with SARS-CoV-2 was localized to the RBD region of the Spike protein. The findings showed clear evidence of new avenues to disrupt viral replication and understand virus physiopathology that warrants further investigation.
Photon-free (s)CMOS camera characterization for artifact reduction in high- and super-resolution microscopy
Authors: Diekmann Robin , Deschamps Joran , Li Yiming , Tschanz Aline , Kahnwald Maurice , Matti Ulf , Ries Jonas
Published: April 2021

Modern implementations of widefield fluorescence microscopy often rely on sCMOS cameras, but this camera architecture inherently features pixel-to-pixel variations. Such variations lead to image artifacts and render quantitative image interpretation difficult. Although a variety of algorithmic corrections exists, they require a thorough characterization of the camera, which typically is not easy to access or perform. Here, we developed a fully automated pipeline for camera characterization based solely on thermally generated signal, and implemented it in the popular open-source software Micro-Manager and ImageJ/Fiji. Besides supplying the conventional camera maps of noise, offset and gain, our pipeline also gives access to dark current and thermal noise as functions of the exposure time. This allowed us to avoid structural bias in single-molecule localization microscopy (SMLM), which without correction is substantial even for scientific-grade, cooled cameras. In addition, our approach enables high-quality 3D super-resolution as well as live-cell time-lapse microscopy with cheap, industry-grade cameras. As our approach for camera characterization does not require any user interventions or additional hardware implementations, numerous correction algorithms demanding camera characterization become easily applicable.
Assessing the efficacy of antibiotic treatment for the creation of axenic earthworms
Authors: Omosigho Henny O. , Swart Elmer , Sizmur Tom P. , Spurgeon Dave J. , Svendsen Claus , Shaw Liz J.
Published: April 2021

Earthworms are an integral part of soil ecosystems, especially for their role in soil functions such as organic matter (OM) decomposition and nutrient cycling. Earthworms and microorganisms are interdependent, and a considerable portion of the contribution earthworms make to influencing OM fate is through interactions with microorganisms. However, the importance of the earthworm-associated microbiome is not fully understood, because it is difficult to separate the direct influence of the earthworms from the indirect influence of their microbiome. Here, we evaluated an antibiotic-based procedure for producing axenic ecologically-contrasting earthworm species (E. fetida, L. terrestris, A. chlorotica) as the first step towards soil studies aimed at understanding the importance of the earthworm microbiome for host health and function. Individual earthworms were exposed to antibiotics: cycloheximide (150 μg ml−1), ampicillin (100 μg ml−1), ciprofloxacin (50 μg ml−1), nalidixic acid (50 μg ml−1), and gentamicin (50 μg ml−1) either singly or in a cocktail via culture (96 h) in a semi-solid agar carrier. Compared to the non-antibiotic treated control, the cocktail (for all three species) and ciprofloxacin (forE. fetidaandA. chlorotica) treatments significantly reduced (P<0.05) culturable microbial abundance on nutrient agar and potato dextrose agar. The microbial counts were reduced to below detection (<50 CFU individual−1) forE. fetidaandA. chloroticareceiving the cocktail. Illumina 16S rDNA amplicon sequence analysis of culturableL. terrestris-associated bacteria showed that antibiotic treatment influenced community composition revealing putative sensitive (Comomonas, KosakoniaandSphingobacterium) and insensitive (Aeromonas, Pseudochrobactrum) taxa. Overall, we report a rapid, with minimal earthworm-handling, process of creating ‘axenic’E. fetidaandA. chloroticaindividuals orL. terrestriswith a suppressed microbiome as a tool to be used in future ecological studies of earthworm microbial interactions affecting host health and function.
Neuroprotection in early stages of Alzheimer’s Disease is promoted by Transthyretin angiogenic properties
Authors: Gião Tiago , Saavedra Joana , Vieira José Ricardo , Pinto Marta Teixeira , Arsequell Gemma , Cardoso Isabel
Published: April 2021

While still controversial, it has been demonstrated that vascular defects can precede the onset of the other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. AD transgenic mice with TTR genetic reduction, AD/TTR+/−, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Furtherin vivoinvestigation, using the chick chorioallantoic membrane (CAM) assay, revealed that TTR is a pro-angiogenic molecule. Also, TTR increased the expression of key angiogenic molecules, by endothelial cells under tube formation conditions. We showed that TTR reduction leads to a thicker BM in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length when compared to non-treated littermates. Ourin vivoresults show the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.
Cholesterol promotes both head group visibility and clustering of PI(4,5)P2driving unconventional secretion of Fibroblast Growth Factor 2
Authors: Lolicato Fabio , Saleppico Roberto , Griffo Alessandra , Pokrandt Bianca , Müller Hans-Michael , Ewers Helge , Hähl Hendrik , Fleury Jean-Baptiste , Seemann Ralf , Brügger Britta , Jacobs Karin , Vattulainen Ilpo , Nickel Walter
Published: April 2021

Fibroblast Growth Factor 2 (FGF2) is a cell survival factor involved in tumor-induced angiogenesis. FGF2 is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further reveal cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers in a fully reconstituted system. Based on extensive atomistic molecular dynamics simulations and membrane tension experiments, we propose cholesterol to modulate FGF2 binding to PI(4,5)P2by (i) increasing head group visibility of PI(4,5)P2on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2molecules triggering FGF2 oligomerization and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 towards the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.
Bioelectricity generation from live marine photosynthetic macroalgae
Authors: Shlosberg Yaniv , Krupnik Nimrod , Tóth Tünde N. , Eichenbaum Ben , Meirovich Matan , Meiri David , Yehezkeli Omer , Schuster Gadi , Israel Álvaro , Adir Noam
Published: April 2021

Conversion of solar energy into electrical current by photosynthetic organisms has the potential to produce clean energy. Previously reported bio-photoelectrochemical cells (BPECs) have utilized unicellular photosynthetic microorganisms. In this study, we describe for the first time BPECs that utilize intact live marine macroalgae (seaweeds) in natural seawater or saline buffer or natural seawater. The BPECs produce electrical currents from of >50mA/cm2, from both light-dependent (photosynthesis) and light independent processes. These values are significantly greater than the current densities that have been reported for single-cell microorganisms. The photocurrent is inhibited by the Photosystem II inhibitor DCMU, indicating that the source of light-driven electrons is from water oxidation via NADPH and other reduced molecules. We show here that intact seaweed cultures can be used in a large-scale BPEC containing seawater that produces bias-free photocurrent. The ability to produce bioelectricity from intact seaweeds may pave the way to future development of a low-cost energy technology using BPECs.
Subgroup-specific gene expression profiles and mixed epistasis in chronic lymphocytic leukemia
Authors: Lütge Almut , Lu Junyan , Hüllein Jennifer , Walther Tatjana , Sellner Leopold , Wu Bian , Rosenquist Richard , Oakes Christopher C. , Dietrich Sascha , Huber Wolfgang , Zenz Thorsten
Published: April 2021

Despite the extensive catalogue of recurrent mutations in chronic lymphocytic leukaemia (CLL), the diverse molecular driving events and the resulting range of disease phenotypes remain incompletely understood. To study the molecular heterogeneity of CLL, we performed RNA-sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major independent axes of gene expression variation: the first one aligned with the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, with the three-group stratification of CLL by global DNA methylation pattern, and affected biological functions including B- and T-cell receptor signaling. The second one aligned with trisomy 12 status and affected chemokine signaling. Furthermore, we searched for differentially expressed genes associated with gene mutations and copy-number aberrations and detected strong signatures forTP53, BRAFandSF3B1, as well as for del(11)(q22.3), del(17)(p13) and del(13)(q14) beyond the dosage effect. We discovered strong non-additive effects (i.e., genetic interactions, or epistasis) of IGHV mutation status and trisomy 12 on multiple phenotypes, including the expression of 893 genes. Multiple types of epistasis were observed, including synergy, buffering, suppression and inversion. Our study reveals previously underappreciated gene expression signatures for (epi)genomic variants in CLL and the presence of epistasis between them. The findings will serve as a reference for a functional resolution of CLL molecular heterogeneity.
Major effect loci for plant size before onset of nitrogen fixation allow accurate prediction of yield in white clover
Authors: Moeskjær Sara , Skovbjerg Cathrine Kiel , Tausen Marni , Wind Rune , Roulund Niels , Janss Luc , Andersen Stig U.
Published: April 2021

White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover-grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover and 169 rhizobium genotypes. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog ofArabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.
Controlling gene expression in mammalian cells using multiplexed conditional guide RNAs for Cas12a
Authors: Oesinghaus Lukas , Simmel Friedrich C.
Published: April 2021

Spatiotemporal control of the activity of Cas proteins is of considerable interest for both basic research and therapeutics. Only few mechanisms have been demonstrated for regulating the activity of guide RNAs (gRNAs) for Cas12a in mammalian cells, however, and combining and compactly integrating multiple control instances on single transcripts has not been possible so far. Here, we show that conditional processing of the 3’ tail is a viable general approach towards switchable Pol II-transcribed Cas12a gRNAs that can activate gene expression in mammalian cells in an input-dependent manner. Processing of the 3’ tail can be achieved using microRNA and short hairpin RNA as inputs, via a guanine-responsive ribozyme, and also using an RNA strand displacement mechanism. We further show that Cas12a along with several independently switchable gRNAs can be integrated on a single transcript using stabilizing RNA triplexes, providing a route towards compact Cas12a-based gene regulation constructs with multi-input switching capabilities.
Chronic toxicity of three neonicotinoid insecticides and their mixture on two daphniid species:Daphnia magnaandCeriodaphnia dubia
Authors: Duchet Claire , Mitchell Chelsea J. , McIntyre Jenifer K. , Stark John D.
Published: April 2021

Neonicotinoid insecticides represent nearly a quarter of the global market and are widely used in agriculture but also for lawn, garden care, and pest control. They are highly water-soluble, persistent in soil, and may enter the aquatic compartment via spray drift, runoff, or leaching, and contribute to downstream aquatic toxicity. Although insects appear to be the most sensitive group to neonicotinoids, other groups, such as crustaceans and birds, may also be affected. Furthermore, most studies focus on single-insecticide exposure and very little is known concerning the impact of neonicotinoid mixtures on aquatic invertebrates. The present study was designed to test potential toxicological effects of an environmentally relevant mixture of imidacloprid, clothianidin, and thiamethoxam on populations ofCeriodaphnia dubiaandDaphnia magnaunder controlled conditions. Chronic toxicity tests were conducted in the laboratory, and survival and reproduction were measured for both species under exposure to nominal concentrations of imidacloprid (0.256 µg/L), clothianidin (3.11 µg/L), thiamethoxam (1.49 µg/L), and a mixture of the three compounds at the same concentrations of the individual compounds. The neonicotinoids did not affect the survival ofC. dubiaandD. magnafounders. Reproduction ofC. dubiawas affected only by the mixture. All three individual insecticides as well as the mixture caused a significant reduction in the reproduction ofD. magna. Our results highlight the complexity of pesticide toxicity and show that traditional toxicological approaches such as acute mortality studies, especially tests with single compounds, can underestimate negative impacts that occur in the environment.

Highlights

Neonicotinoids are currently the most frequently used insecticides worldwide.

An environmentally relevant mixture of three neonicotinoids was evaluated on two daphniid species.

The mixture negatively affected the reproduction ofC. dubiaandDaphnia magna.

Traditional toxicological approaches with single compounds may underestimate the effects occurring in the environment at low concentrations.
Transmission of vibrations in buzz-pollinated plant species with disparate floral morphologies
Authors: Nevard Lucy , Russell Avery L. , Foord Karl , Vallejo-Marin Mario
Published: April 2021

In buzz-pollinated plants, bees apply vibrations produced by their thoracic muscles to the flower, causing pollen release from anthers, often through small apical pores. During floral buzzing, bees grasp one or more anthers with their mandibles, and vibrations are transmitted to the focal anther(s), adjacent anthers, and the whole flower. Because pollen release depends on the vibrations experienced by the anther, the transmission of vibrations through flowers with different morphologies may determine patterns of release, affecting both bee foraging and plant fitness. Anther morphology and intra-floral arrangement varies widely among buzz-pollinated plants. Here, we compare the transmission of vibrations among focal and non-focal anthers in four species with contrasting anther morphologies:Cyclamen persicum(Primulaceae),Exacum affine(Gentianaceae),Solanum dulcamaraandS. houstonii(Solanaceae). We used a mechanical transducer to apply bee-like artificial vibrations to focal anthers, and simultaneously measured the vibration frequency and displacement amplitude at the tips of focal and non-focal anthers using high-speed video analysis (6,000 frames per second). In flowers in which anthers are tightly held together (C. persicumandS. dulcamara), vibrations in focal and non-focal anthers are indistinguishable in both frequency and displacement amplitude. In contrast, flowers with loosely arranged anthers (E. affine) including those in which stamens are morphologically differentiated within the same flower (heterantherousS. houstonii), show the same frequency but higher displacement amplitude in non-focal anthers compared to focal anthers. Our results suggest that stamen arrangement affects vibration transmission with potential consequences for pollen release and bee behaviour.
Extracellular Hsp90α Detoxifies β-Amyloid Fibrils Through an NRF2 and Autophagy Dependent Pathway
Authors: Murshid Ayesha , Lang Benjamin J. , Borges Thiago J. , Okusha Yuka , Doshi Sachin P. , Yasmine Suraya , Clark-Matott Joanne , Choudhury Reeham , Ang Lay-Hong , Woodbury Maya , Ikezu Tsuneya , Calderwood Stuart K.
Published: April 2021

We have investigated the role ofextracellularHeatshockprotein90alpha(eHsp90α) in conferring protection of neuronal cells against fibrillary amyloid beta (f-Aβ1-42) toxicity mediated by microglial cells. Formation of f-Aβ1-42plaques leads to neurotoxic inflammation, a critical pathological feature of Alzheimer’s Disease. We observed increased uptake and clearance of internalized f-Aβ1-42by microglial cells treated with eHsp90α, an effect associated with activation of NRF2 (NF-E2-related factor 2) - mediated autophagy. eHsp90α thus mitigated the neuronal toxicity of f-Aβ1-42-activated microglia. In addition, eHsp90α facilitated f-Aβ1-42engulfment by microglial cellsin vitro. In summary, eHsp90α triggers NRF2-mediated autophagy in microglia and thus protects against the neurotoxic effects of f-Aβ1-42.
Whole Chloroplast Genomes reveals the uniqueness of Bolivian native cacao (Theobroma cacao) from the northern part of Bolivia
Authors: Gumiel M , Rollano-Peñaloza OM , Peralta-Rivero C , Tejeda L , Palma V , Cartagena P , Mollinedo P , Peñarrieta JM
Published: April 2021

We report the complete chloroplast sequences of two varieties ofTheobroma cacaocollected in the Bolivian Amazonia using Next-Generation Sequencing. Comparisons made between these two chloroplast genomes and the Belizean reference plastid genome identified 19 and 22 nucleotide variants. The phylogenetic analysis reported three mainT. cacaoclades belonging to the Forastero, Criollo and Trinitario groups. The Bolivian Native Cacao varieties were located inside the Trinitario group forming their unique branch. The Bolivian Native Cacao branch reveals a possible new subpopulation different from the well-characterizedT. cacaosubpopulations. The phylogenetic trees showed that the relationships among theT. cacaovarieties were consistent with their geographical locations placing the Cacao Center of Origin in Western Amazon. The data presented here will contribute to the usage of ultrabarcoding to distinguish differentT. cacaovarieties and to identify native cacaos from introduced cacaos. Thus helping in the conservation of local native varieties ofT. cacao.
A competitive activity-based protein profiling platform yields cell wall synthesis inhibitors active against replicating and non-replicatingMycobacterium tuberculosis
Authors: Li Michael , Patel Hiren V. , Cognetta Armand B. , Smith Trever C. , Mallick Ivy , Cavalier Jean-François , Canaan Stephane , Aldridge Bree B. , Cravatt Benjamin F. , Seeliger Jessica C.
Published: April 2021

The identification and validation of a small molecule’s targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule’s targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here we studied the effects of 1,2,3-triazole ureas onMycobacterium tuberculosis(Mtb). After screening ~200 compounds, we focused on two inhibitors active against both exponentially replicating and hypoxia-induced drug-tolerantMtbthat form part of a four-compound structure-activity series. The compound with negligible activity revealed potential false positive targets not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirmed that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings showed that ABPP identifies the targets most likely relevant to a compound’s antibacterial activity.
TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila brains
Authors: Romano Giulia , Holodkov Nikola , Klima Raffaella , Feiguin Fabian
Published: April 2021

Alterations in the function of the RNA-binding protein TDP-43 is largely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease of the human motor system that leads to motoneurons degeneration and reduced life expectancy by molecular mechanisms not well known. Regarding to that, we found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible to convert glutamate to γ-aminobutyric acid (GABA), were downregulated in TBPH-null flies and motoneurons derived from ALS patients carrying mutations in TDP-43 suggesting that defects in the regulation of GAD1 may lead to neurodegeneration by affecting neurotransmitter balance. In this study, we observed that TBPH was required to regulate GAD1 pre-mRNA splicing and GABA levels in Drosophila brains. Interestingly, we discovered that pharmacological treatments aimed to modulate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies revealing novel mechanisms and therapeutic strategies in ALS.
A gene regulatory network for neural induction
Authors: Trevers Katherine E. , Lu Hui-Chun , Yang Youwen , Thiery Alexandre , Strobl Anna C. , Pálinkášová Božena , de Oliveira Nidia M. M. , de Almeida Irene M. , Khan Mohsin A. F. , Moncaut Natalia , Luscombe Nicholas M. , Dale Leslie , Streit Andrea , Stern Claudio D.
Published: April 2021

During early vertebrate development, signals from a special region of the embryo, the organizer, can re-direct the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here we undertake a comprehensive analysis, in very fine time-course, of the events following exposure of ectoderm to the organizer. Using transcriptomics and epigenomics we generate a Gene Regulatory Network comprising 175 transcriptional regulators and 5,614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing and reporter assays we show that neural induction by a grafted organizer closely resembles normal neural plate development. The study is accompanied by a comprehensive resource including information about conservation of the predicted enhancers in different vertebrate systems.
Defective satellite DNA clustering into chromocenters underlies hybrid incompatibility inDrosophila
Authors: Jagannathan Madhav , Yamashita Yukiko M.
Published: April 2021

Although rapid evolution of pericentromeric satellite DNA repeats is theorized to promote hybrid incompatibility (HI)(1–4), how divergent repeats affect hybrid cells remains poorly understood. Recently, we demonstrated that sequence-specific DNA-binding proteins cluster satellite DNA from multiple chromosomes into ‘chromocenters’, thereby bundling chromosomes to maintain the entire genome in a single nucleus(5, 6). Here we show that ineffective clustering of divergent satellite DNA in the cells ofDrosophilahybrids results in chromocenter disruption, associated micronuclei formation and tissue atrophy. We further demonstrate that previously identified HI factors trigger chromocenter disruption and micronuclei in hybrids, linking their function to a conserved cellular process. Together, we propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation.
MHC-matched allogeneic bone marrow transplants fail to eliminate SHIV-infected cells from ART-suppressed Mauritian cynomolgus macaques
Authors: Weinfurter Jason T. , D’souza Saritha S. , Matschke Lea M. , Bennett Sarah , Kelnhofer-Millevolte Laurel E. , Suknuntha Kran , Kumar Akhilesh , Coonen Jennifer , Capitini Christian M. , Hematti Peiman , Golos Thaddeus G. , Slukvin Igor I. , Reynolds Matthew R.
Published: April 2021

Background

Allogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood but may include pre-transplant conditioning regimens, ART-mediated protection of donor cells, and graft-versus-host (GvH) responses. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay.

Results

We infected four MCMs with CCR5-tropic SHIV162P3 and started ART 6-16 weeks post-infection (p.i.) to establish robust viral reservoirs. We maintained the MCMs on continuous ART during myeloablative conditioning with total body irradiation (TBI) and while transplanting allogeneic MHC-matched α/β T cell-depleted bone marrow cells. Post-transplant, we prophylactically treated the MCMs with cyclophosphamide and tacrolimus to prevent GvH disease (GvHD). The transplants produced ~85% whole blood donor chimerism without causing high-grade GvHD. Consequently, three MCMs had undetectable SHIV DNA in their peripheral blood mononuclear cells post-transplant. However, SHIV-harboring cells persisted in various tissues. We detected viral DNA in lymph node biopsies and terminal analyses of tissues between 38 and 62 days post-transplant. Further, we removed ART from one MCM at 63 days post-transplant, resulting in viral rebound within seven days and viral loads nearing 1×108SHIV RNA copies/ml of plasma after treatment interruption.

Conclusions

Our results indicate that myeloablative conditioning and maintaining ART through the peri-transplant period alone are insufficient for eradicating latent viral reservoirs early after allo-HSCTs. Furthermore, our findings suggest that extended ART and GvH responses may be necessary to substantially deplete viral reservoirs after allo-HSCTs.
Stronger net selection on males across animals
Authors: Winkler Lennart , Moiron Maria , Morrow Edward H. , Janicke Tim
Published: April 2021

Sexual selection is considered the major driver for the evolution of manifold sex differences. However, the eco-evolutionary dynamics of sexual selection and their role for a population’s adaptive potential to respond to environmental change have only recently been explored. Theory predicts that sexual selection promotes adaptation at a low demographic cost only if net selection is stronger on males compared to females. We used a comparative approach to show that net selection is indeed stronger in males in species prone to intense sexual selection. Given that both sexes share the vast majority of their genes, our findings corroborate the notion that the genome is often confronted with a more stressful environment when expressed in males. Collectively, our study supports a long-standing key assumption required for sexual selection to bolster adaptation, and intense sexual selection may therefore enable some species to track environmental change more efficiently.

One sentence summary

Comparative study finds support for stronger net selection in males.