bluuebunny/crossref_metadata_2025 huggingface dataset:
Eliminating Malaria Vectors with Precision Guided Sterile Males
Authors: Smidler Andrea L. , Apte Reema A. , Pai James J. , Chow Martha L. , Chen Sanle , Mondal Agastya , Sánchez C. Héctor M. , Antoshechkin Igor , Marshall John M. , Akbari Omar S.
Published: July 2023

Controlling the principal African malaria vector, the mosquitoAnopheles gambiae, is considered essential to curtail malaria transmission. However existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet theA. gambiaetoolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass-releases of non-biting, non-driving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here we develop precision guided Sterile Insect Technique (pgSIT) in the mosquitoA. gambiaefor inducible, programmed male-sterilization and female-elimination for wide scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male-sterility and >99.9% female-lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce population suppression in cage trials, and are predicted to eliminate wildA. gambiaepopulations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, for the first time enabling scalable SIT-like confinable suppression in the species.
The timing of cellular events: a stochastic vs deterministic perspective
Authors: Ham Lucy , Coomer Megan A. , Öcal Kaan , Grima Ramon , Stumpf Michael P.H.
Published: July 2023

Changes in cell state are driven by key molecular events whose timing can often be measured experimentally. Of particular interest is the time taken for the levels of RNA or protein molecules to reach a critical threshold defining the triggering of a cellular event. While this mean trigger time can be estimated by numerical integration of deterministic models, these ignore intrinsic noise and hence their predictions may be inaccurate. Here we study the differences between deterministic and stochastic model predictions for the mean trigger times using simple models of gene expression, post-transcriptional feedback control, and enzyme-mediated catalysis. By comparison of the two predictions, we show that when promoter switching is present there exists a transition from a parameter regime where deterministic models predict a longer trigger time than stochastic models to a regime where the opposite occurs. Furthermore, the ratio of the trigger times of the two models can be large, particularly for auto-regulatory genetic feedback loops. Our theory provides intuitive insight into the origin of these effects and shows that deterministic predictions for cellular event timing can be highly inaccurate when molecule numbers are within the range known for many cells.
Membrane mechanics dictate axonal morphology and function
Authors: Griswold Jacqueline M. , Bonilla-Quintana Mayte , Pepper Renee , Lee Christopher T. , Raychaudhuri Sumana , Ma Siyi , Gan Quan , Syed Sarah , Zhu Cuncheng , Bell Miriam , Suga Mitsuo , Yamaguchi Yuuki , Chéreau Ronan , Nägerl U. Valentin , Knott Graham , Rangamani Padmini , Watanabe Shigeki
Published: July 2023

Axons are thought to be ultrathin membrane cables of a relatively uniform diameter, designed to conduct electrical signals, or action potentials. Here, we demonstrate that unmyelinated axons are not simple cylindrical tubes. Rather, axons have nanoscopic boutons repeatedly along their length interspersed with a thin cable with a diameter of ∼60 nm like pearls-on-a-string. These boutons are only ∼200 nm in diameter and do not have synaptic contacts or a cluster of synaptic vesicles, hence non-synaptic. Ourin silicomodeling suggests that axon pearling can be explained by the mechanical properties of the membrane including the bending modulus and tension. Consistent with modeling predictions, treatments that disrupt these parameters like hyper- or hypo-tonic solutions, cholesterol removal, and non-muscle myosin II inhibition all alter the degree of axon pearling, suggesting that axon morphology is indeed determined by the membrane mechanics. Intriguingly, neuronal activity modulates the cholesterol level of plasma membrane, leading to shrinkage of axon pearls. Consequently, the conduction velocity of action potentials becomes slower. These data reveal that biophysical forces dictate axon morphology and function and that modulation of membrane mechanics likely underlies plasticity of unmyelinated axons.
Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos
Authors: Kiyomitsu Ai , Nishimura Toshiya , Hwang Shiang Jyi , Ansai Satoshi , Kanemaki Masato T. , Tanaka Minoru , Kiyomitsu Tomomi
Published: July 2023

Despite drastic cellular changes during cleavage divisions, a mitotic spindle is assembled in each blastomere to accurately segregate duplicated chromosomes. Recent studies indicate that early embryonic divisions are highly error-prone in bovines and humans. However, processes and mechanisms of embryonic spindle assembly remain little understood in vertebrates. Here, we established live functional assay systems in medaka fish (Oryzias latipes) embryos by combining CRISPR knock-in with an auxin-inducible degron technology. In contrast to mammals, mitoses during cleavage divisions are very rapid (<12 min), but segregation errors are rarely observed. Importantly, we found that the Ran-GTP gradient assembles a specialized, dense microtubule network at the spindle midplane during metaphase, which is essential for faithful chromosome segregation in early embryos. In contrast, Ran-GTP becomes dispensable for chromosome segregation in later stages, where spindles are morphologically remodeled into short, somatic-like spindles lacking the dense microtubule network. We propose that the specialized Ran-based spindle structure ensures high fidelity of chromosome segregation in large, vertebrate early embryos.
Insight into the structures of unusual base pairs in RNA complexes containing primer/template/adenosine ligand
Authors: Dantsu Yuliya , Zhang Ying , Zhang Wen
Published: July 2023

In prebiotic RNA world, the self-replication of RNA without enzymes can be achieved through the utilization of 2-aminoimidazole activated nucleotides as efficient substrates. The mechanism of RNA nonenzymatic polymerization has been extensively investigated biophysically and structurally by using the model of RNA primer/template complex which is bound by the imidazolium-bridged or triphosphate-bridged diguanosine intermediate. However, beyond the realm of guanosine substrate, the structural insight into how alternative activated nucleotides bind and interact with RNA primer/template complex remains unexplored, which is important for understanding the low reactivity of adenosine and uridine substrates in RNA primer extension, as well as its relationship with the structures. Here we use crystallography as method and determine a series of high-resolution structures of RNA primer/template complexes bound by ApppG, the close analog of dinucleotide intermediate containing adenosine and guanosine. The structures show that ApppG ligands bind to RNA template through both Watson-Crick and noncanonical base pairs, with the primer 3′-OH group far from the adjacent phosphorus atom of the incoming substrate. The structures indicate that, when adenosine is included in the imidazolium-bridged intermediate, the complexes are likely preorganized in a suboptimal conformation, making it difficult for the primer to in-line attack the substrate. Moreover, by cocrystallizing the RNA primer/template with chemically activated adenosine and guanosine monomers, we successfully observe the slow formation of the imidazolium-bridged intermediate (Ap-AI-pG) and the preorganized structure for RNA primer extension. Overall, our studies offer a structural explanation for the slow rate of RNA primer extension when using adenosine-5’-phosphoro-2-aminoimidazolide as a substrate during nonenzymatic polymerization.
Chromosome-scale genome assembly ofTinospora sagittata(Oliv.) Gagnep. enhances identifying genes involved in the biosynthesis of jatrorrhizine
Authors: Alami Mohammad Murtaza , Shu Shaohua , Liu Sanbo , Ouyang Zhen , Zhang Yipeng , Lv Meijia , Sang Yonghui , Gong Dalin , Yang Guozheng , Feng Shengqiu , Mei Zhinan , Xie De-Yu , Wang Xuekui
Published: July 2023

Tinospora sagittata(Oliv.) Gagnep. is an important medicinal tetraploid plant in the Menispermaceae family. Its tuber, namely “Radix Tinosporae” used in Traditional Chinese Medicine, is rich in medicinal terpenoids and benzylisoquinoline alkaloids (BIAs), To enhance understanding the biosynthesis of medicinal compounds, we, herein, report the assembly of a high quality chromosome-scale genome with both PacBio HiFi and Illumina sequencing technologies. The size of assembled genome was 2.33 Gb consisting of 4070 scaffolds (N50=42.06Mb), of which 92.05% were assigned to 26 pseudochromosomes in A and B sub-genomes. A phylogenetic analysis with theT. sagittataand other 16 plant genomes estimated the evolutionary placement ofT. sagittataand its divergence time in Ranunculales. Further genome evolution analysis characterized one round tandem duplication about 1.5 million years ago (MYA) and one whole-genome duplication (WGD) about 86.9 MYA. WGD contributed to the duplication of clade-specific cytochrome P450 gene family in Ranunculales. Moreover, sequencing mining obtained genome-wide genes involved in the biosynthesis of alkaloids and terpenoids.TsA02G014550, one candidate, was functionally characterized to catalyze the formation of (S)-canadine in the jatrorrhizine biosynthetic pathway. Taken together, the assembled genome ofT. sagittataprovides useful sequences to understand the biosynthesis of jatrorrhizine and other BIAs in plants.
Large effects and the infinitesimal model
Authors: Parsons Todd L. , Ralph Peter L.
Published: July 2023

The infinitesimal model of quantitative genetics relies on the Central Limit Theorem to stipulate that under additive models of quantitative traits determined by many loci having similar effect size, the difference between an offspring’s genetic trait component and the average of their two parents’ genetic trait components is Normally distributed and independent of the parents’ values. Here, we investigate how the assumption of similar effect sizes affects the model: in particular, if the tail of the effect size distribution is polynomial with exponentα <2, then sums of effects should be well-approximated by a “stable distribution”, and find tail exponents between 1 and 2 in effect sizes estimated by genome-wide association studies of many human disease-related traits. We show that the independence of offspring trait deviations from parental averages in many cases implies a Gaussian distribution, suggesting that non-Gaussian models of trait evolution must explicitly track the underlying genetics, at least for loci of large effect. We also characterize possible limiting trait distributions of the infinitesimal model with infinitely divisible noise distributions, and compare our results to simulations.
Structural characterisation of the complete cycle of sliding clamp loading inEscherichia coli
Authors: Xu Zhi-Qiang , Jergic Slobodan , Lo Allen T.Y. , Pradhan Alok C. , Brown Simon H.J. , Bouwer James C. , Ghodke Harshad , Lewis Peter J. , Tolun Gökhan , Oakley Aaron J. , Dixon Nicholas E.
Published: July 2023

Ring-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened or trapped open and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place clamps around DNA remains limited. Here, we present a series of six structures of theEscherichia coliCLC bound to an open or closed clamp on and off a primer-template DNA that represent all intermediates in the clamp loading process. We show that the ATP-bound CLC first binds to a clamp, then constricts to hold onto it. The CLC then expands to open the clamp with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing ATP hydrolysis and clamp closing around DNA. Although both yeast andE. coliCLCs open clamps by crab claw-like motions, they do it by the CLC expanding in opposite directions. These structures provide critical high-resolution snapshots of clamp loading by theE. coliCLC, revealing how the molecular machine works.
Effect of ‘spent’ nucleotides on nonenzymatic RNA replication
Authors: Patki Gauri M. , Rajamani Sudha
Published: July 2023

Nonenzymatic template-directed replication would have been affected by co-solutes in a heterogenous prebiotic soup due to lack of enzymatic machinery. Unlike in contemporary biology, these reactions use chemically-activated nucleotides, which undergo rapid hydrolysis forming nucleoside monophosphates (‘spent’ monomers). These co-solutes cannot extend the primer but continue to base pair with the template, thereby interfering with replication. We therefore aimed to understand how a mixture of ‘spent’ ribonucleotides would affect nonenzymatic replication. We observed inhibition of replication in presence of the mixture, wherein predominant contribution came from the cognate Watson-Crick monomer, showing potential sequence dependence. Our study highlights how nonenzymatic RNA replication would have been directly affected by co-solutes, with ramifications for the emergence of functional polymers in an RNA World.
An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E
Authors: Lie Laurensius Kevin , Synowiec Aleksandra , Mazur Jedrzej , Pyrć Krzysztof
Published: July 2023

The lack of suitablein vitroculture model has hampered research on wild-type (WT) human coronaviruses. While 3D tissue or organ cultures have been instrumental for this purpose, such models are challenging, time-consuming, expensive and require extensive cell culture adaptation and directed evolution. Consequently, high-throughput applications are beyond reach in most cases. Here we developed a robust A549 cell line permissive to a human coronavirus 229E (HCoV-229E) clinical isolate by transducing CD13 and transmembrane serine protease 2 (TMPRSS2), henceforth referred to as A549++cells. This modification allowed for productive infection, and a more detailed analysis showed that the virus might use the TMPRSS2-dependent pathway but can still bypass this pathway using cathepsin-mediated endocytosis. Overall, our data showed that A549++cells are permissive to HCoV-229E clinical isolate, and applicable for further studies on HCoV-229E infectiology. Moreover, this line constitutes a uniform platform for studies on multiple members of theCoronaviridaefamily.

Graphical abstract
Distinct composition of plasma virome in HIV-infected subjects on antiretroviral therapy compared to controls
Authors: Bhagchandani Tannu , Ul Haque Mohd. Maksuf , Malik Md Zubbair , Ray Ashwini Kumar , Kaur S Urvinder , Rai Ankita , Verma Anjali , Sawlani Kamal Kumar , Chaturvedi Rupesh , Himanshu D , Tandon Ravi
Published: July 2023

Microbiome study during HIV infection is widely documented with major emphasis on bacteriome, while virome studies are few. The alteration of plasma virome is reported in HIV patients who are either treatment naïve or have a weak immune system. Less is known about the plasma virome in HIV-infected patients on ART with preserved CD4 counts. In our pilot study, viral DNA isolated from plasma was sequenced on Illumina Nextseq500 platform. With the help of VIROMATCH pipeline, we observed that the plasma virome of HIV patients were significantly distinct from controls on the basis of Bray-Curtis dissimilarity index. The species,Human gammaherpesvirus 4and families,HerpesviridaeandSiphoviridaewere found to be significantly enriched and differentially abundant in HIV patients. Hence, plasma virome is an important component for future study that might influence disease progression among HIV patients during therapy.
Cell division angle regulates the tissue mechanics and tunes the amount of cerebellar folding
Authors: Cook Amber G. , Bishop Taylor V. , Crowe Hannah R. , Stevens Daniel , Reine Lauren , Joyner Alexandra L , Lawton Andrew K
Published: July 2023

Modeling has proposed that the amount of neural tissue folding is set by the level of differential-expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a critical period where the folding amount diverges between the strains. In this period, regional changes in the level of differential-expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL is regionally adjusted during the critical period alongside corresponding changes in wavelength. While the number of SHH-expressing Purkinje cells predicts the folding amount, the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential-expansion and thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential-expansion and thickness of the EGL are regionally tuned to set the amount and wavelength of folding.
Autophagy maintains the homeostatic environment in the male reproductive accessory organs playing a key role in fertility
Authors: Jaulim Adil , Cassidy Liam D , Young Andrew RJ , Chan Adelyne SL , Warren Anne Y , Taylor Angela E , Arlt Wiebke , Lan Guochen , Blayney Martyn L , Davidson Olivia , Barratt Christopher LR , Pacey Simon , Narita Masashi
Published: July 2023

Autophagy has been implicated in male fertility but its specific role in the post-testicular organs remains unclear. Here, we investigate this in mice expressing a doxycycline-inducible RNAi against Atg5 (Atg5i). Systemic autophagy inhibition in Atg5i mice resulted in the morphological and functional abrogation of the male accessory sex organs, leading to male subfertility. However, the testis was largely protected, likely due to the limited permeability of doxycycline through the blood-testis barrier. Interestingly, restoration of autophagy by doxycycline withdrawal in Atg5i mice led to substantial recovery of the phenotype in the accessory organs. This model offers a unique opportunity to dissect the pre- and post-testicular roles of autophagy, highlighting the non-autonomous impact of autophagy on male fertility.
The Type VI secretion systemof Stenotrophomonas rhizophilaCFBP13503 limits the transmission ofXanthomonas campestrispv. campestris8004 from radish seeds to seedlings
Authors: Garin Tiffany , Brin Chrystelle , Préveaux Anne , Brault Agathe , Briand Martial , Simonin Marie , Barret Matthieu , Journet Laure , Sarniguet Alain
Published: July 2023

Stenotrophomonas rhizophilaCFBP13503 is a seed-borne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacteriaXanthomonas campestrispv.campestris(Xcc8004). The type VI Secretion System (T6SS), an interference contact-dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS ofS. rhizophilaCFBP13503 in the inhibition of Xcc8004 growth and seed-to-seedling transmission was assessed. The T6SS cluster ofS. rhizophilaCFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes,hcpandtssB, abolished the competitive advantage ofS. rhizophilaagainst Xcc8004 invitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and eitherS. rhizophilawild type (wt) strain or isogenichcpmutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with theS. rhizophilawt in comparison to T6SS- deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of theStenotrophomonas genus is scarce. In contrast, in all availableS. rhizophilagenomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S.rhizophilaCFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in theS. rhizophilacomplex could make it possible to exploit these strains as biocontrol agents againstX. campestrispv.campestris.
ISGylation-independent protection of cell growth by USP18 following interferon stimulation
Authors: Clancy Anne , Rusilowicz-Jones Emma V. , Wallace Iona , Swatek Kirby N. , Urbé Sylvie , Clague Michael J.
Published: July 2023

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signaling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.
Non-invasive Analysis of Peptidoglycan from Living Animals
Authors: Ocius Karl L. , Kolli Sree H. , Ahmad Saadman S. , Dressler Jules M. , Chordia Mahendra D. , Jutras Brandon L. , Rutkowski Melanie R. , Pires Marcos M.
Published: July 2023

The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activate peptidoglycan sensors in human cells, including the Nucleotide-binding oligomerization domain containing protein 2 (NOD2). When present in the gastrointestinal tract, both the polymeric form (sacculi) and de-polymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To leverage this growing area of biology towards therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and non-invasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a non-invasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for non-invasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.
Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature
Authors: Horisawa Kenichi , Miura Shizuka , Araki Hiromitsu , Miura Fumihito , Ito Takashi , Suzuki Atsushi
Published: July 2023

Direct reprogramming is a technique for inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors. Cell differentiation is generally determined by specific gene expression profiles based on distinct genome-wide epigenetic signatures. Although the CpG methylation of genomic DNA is an essential epigenetic factor that affects the transcriptional state of genes, little is known about how DNA methylation changes and what roles it plays in direct reprogramming. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs to investigate the impact of DNA methylation dynamics on direct reprogramming. We found that the methylation state of CpG was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, as they show that the DNA methylation state transitions to a state close to that of ISCs during direct reprogramming from MEFs to iISCs.
Spatiotemporal and single-cell atlases to dissect cell lineage differentiation and regional specific cell types in mouse ovary morphogenesis
Authors: Zhao Zheng-Hui , Meng Tie-Gang , Gao Fei , Schatten Heide , Sun Qing-Yuan
Published: July 2023

Characterization of cell heterogeneity and molecular diversity using single-cell RNA sequencing has greatly enhanced our understanding of the ovary’s dynamic differentiation processes. However, regional specification of ovarian cells for certain physiological functions remains largely unexplored in the physical space. Here, we combine spatial transcriptomics with single-cell RNA sequencing technologies to build a spatiotemporal and single-cell atlas of ovaries from fetal to adult stages. We construct the pseudotime trajectories of female germ cells and bipotential pregranulosa cells and define key regulatory transcription factors responsible for their differentiation processes. Specifically, we dissect the relationships between two waves of meiosis initiation, oogenesis processes and folliculogenesis. Moreover, we characterize the region-specific subtypes of granulosa cells and luteal cells and construct pseudo-space-time trajectories from granulosa cells to luteal cells. Notably, we identify small luteal cells, a novel cell type, which highly expressOnecut2and exclusively locate at the corpus luteum. Altogether, this study comprehensively delineates ovary development and regional specific ovarian cell types.
Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics
Authors: de Boer Eddy N. , Vroom Vincent , Scheper Arjen J. , Johansson Lennart F. , Bosscher Laura , Rietema Nettie , Commandeur Sabrina Z. , Knoers Nine V.A.M. , Sikkema-Raddatz Birgit , van den Berg Eva , van Diemen Cleo C.
Published: July 2023

Leukemias are genetically heterogeneous, and knowing the type of genetic aberration is clinically important for progression prognosis and choice of treatment. The standard-of-care (SOC) methods in leukemia diagnostics are karyotyping, SNP-array and FISH. Optical genome mapping (OGM) has emerged as their replacement as it can detect different types of structural aberrations simultaneously. However, although OGM can additionally detect much smaller aberrations (500 bp vs. 5–10 Mb with karyotyping), its resolution is too low to define the location of aberrations between two labels and the breakpoints are disputable when labels are not distinct enough. Here, we test whether Cas9-directed long-read sequencing (LRS) can fill this gap.

From an internal Bionano implementation study we selected ten OGM calls either located in low resolution areas or with disputable breakpoints that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION / GridION device.

We could confirm all the OGM aberrations with Cas9-directed LRS and the actual breakpoints of the OGM calls were located between 0.2–5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance.

We show that Cas9-directed LRS can fill the gap of low resolution OGM areas thereby improving the prediction of clinical significance. We envisage that this technique can be an relevant secondary technique in diagnostic workflows including OGM.
Expanding the Substrate Scope ofN- andO-Methyltransferases from Plants for Chemoselective Alkylation
Authors: Jockmann Emely , Subrizi Fabiana , Mohr Michael K. F. , Carter Eve M. , Hebecker Pia M. , Popadić Désirée , Hailes Helen C. , Andexer Jennifer N.
Published: July 2023

Methylation reactions are of significant interest when generating pharmaceutically active molecules and building blocks for other applications. Synthetic methylating reagents are often toxic and unselective due to their high reactivity.S-Adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) present a chemoselective and environmentally friendly alternative. The anthranilateN-MT fromRuta graveolens(RgANMT) is involved in acridone alkaloid biosynthesis, methylating anthranilate. Although it is known to methylate substrates only at theN-position, the closest relatives with respect to amino acid sequence similarities of over 60% areO-MTs catalysing the methylation reaction of caffeate and derivatives containing only hydroxyl groups (CaOMTs). In this study, we investigated the substrate range ofRgANMT and a CaOMT fromPrunus persica(PpCaOMT) using compounds with both, an amino- and hydroxyl group (aminophenols) as possible methyl group acceptors. For both enzymes, the reaction was highly chemoselective. Furthermore, generating cofactor derivatives in situ enabled the transfer of other alkyl chains onto the aminophenols, leading to an enlarged pool of products. Selected MT reactions were performed at a preparative biocatalytic scale in in vitro and in vivo experiments resulting in yields of up to 62%.
Parallel evolution and enhanced virulence uponin vivopassage of an RNA virus inDrosophila melanogaster
Authors: Lezcano Oscar M. , Fuhrmann Lara , Ramakrishnan Gayatri , Beerenwinkel Niko , Huynen Martijn A. , van Rij Ronald P.
Published: July 2023

Virus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit flyDrosophila melanogasterand its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease geneDicer-2was either overexpressed or inactivated. All evolved virus populations replicated more efficientlyin vivoand were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced inDicer-2knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position 95 of VP3 was substituted with either one of five different amino acids in 14 out of 15 lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.
Autophagy restricts fungal accommodation in the roots ofArabidopsis thaliana
Authors: Zecua-Ramirez Patricia , Llamas Ernesto , Charura Nyasha M. , Dunken Nick , De Quattro Concetta , Mandel Alexander , Langen Gregor , Dagdas Yasin , Zuccaro Alga
Published: July 2023

Endophytic colonization ofArabidopsis thalianaby the beneficial root endophyteSerendipita indicais characterized by an initial biotrophic phase followed by a restricted host cell death-associated phase. This latter phase involves regulated cell death (RCD) for fungal accommodation. However, the host molecular pathways that limitS. indicacolonization and govern symbiosis remain largely unknown. Our study demonstrates that autophagy, a major cellular degradation pathway, is activated duringS. indicacolonization and is required to restrict fungal colonization in Arabidopsis. Independent Arabidopsis knockout (KO) mutants deficient in autophagosome formation are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced by two secretedS. indicaeffectors at the onset of the cell death-associated phase. In theatg5autophagy mutant background, impaired dAdo uptake prevents dAdo-induced and symbiosis-mediated cell death. Based on our data, we propose that autophagy-mediated pro-survival responses in the host are crucial for maintaining a balanced symbiotic interaction betweenS. indicaand Arabidopsis.

In a Nutshell

Our study reveals that during colonization ofArabidopsis thalianaroots by the beneficial root endophyteSerendipita indica, autophagy, a key cellular degradation pathway, is activated to limit fungal colonization. Autophagy-deficient Arabidopsis mutants are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced byS. indica. We propose that autophagy-mediated pro-survival responses are essential for maintaining a balanced symbiotic interaction betweenS. indicaand Arabidopsis.
The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking
Authors: Georgiou Xenia , Dimou Sofia , Diallinas George , Samiotaki Martina
Published: July 2023

Neosynthesized plasma membrane (PM) proteins co-translationally translocate to the ER, concentrate at regions called ER-exit sites (ERes) and pack into COPII secretory vesicles which are sorted to the early-Golgi through membrane fusion. Following Golgi maturation, membrane cargoes reach the late-Golgi, from where they exit in clathrin-coated vesicles destined to the PM, directly or through endosomes. Post-Golgi membrane cargo trafficking also involves the cytoskeleton and the exocyst. The Golgi-dependent secretory pathway is thought to be responsible for the trafficking of all major membrane proteins. However, our recent findings inAspergillus nidulansshowed that several plasma membrane cargoes, such as transporters and receptors, follow a sorting route that seems to bypass Golgi functioning. To gain insight on membrane trafficking and specifically Golgi-bypass, here we used proximity dependent biotinylation (PDB) coupled with data-independent acquisition mass spectrometry (DIA-MS) for identifying transient interactors of the UapA transporter. Our assays, which included proteomes of wild-type and mutant strains affecting ER-exit or endocytosis, identified both expected and novel interactions that might be physiologically relevant to UapA trafficking. Among those, we validated, using reverse genetics and fluorescence microscopy, that COPI coatomer is essential for ER-exit and anterograde trafficking of UapA and other membrane cargoes. We also showed that ArfAArf1GTPase activating protein (GAP) Glo3 contributes to UapA trafficking at increased temperature. This is the first report addressing the identification of transient interactions during membrane cargo biogenesis using PDB and proteomics coupled with fungal genetics. Our work provides a basis for dissecting dynamic membrane cargo trafficking via PDB assays.

Highlights

A PDB-proteomics system for identifying transient interactions inA.nidulans

UapA interactomes in wild-type and specific mutants

Analysis and validation of UapA interacting proteins

COPI and Arf1 are essential for cargo anterograde trafficking

Graphical Abstract
BMP-dependent patterning of ectoderm tissue material properties modulates lateral mesendoderm cell migration during early zebrafish gastrulation
Authors: Tavano Stefania , Brückner David B. , Tasciyan Saren , Tong Xin , Kardos Roland , Schauer Alexandra , Hauschild Robert , Heisenberg Carl-Philipp
Published: July 2023

Cell migration is a fundamental process during embryonic development. Most studies on cell migrationin vivohave focussed on the migration of cells using the extracellular matrix (ECM) as their substrate for migration. In contrast, much less is known about how cells migrate on other cells, as found in early embryos when the ECM has not yet formed. Here, we show that lateral mesendoderm (LME) cells in the early zebrafish gastrula use the overlying ectoderm as their substrate for migration. We show that the lateral ectoderm is permissive for the animal pole-directed migration of LME cells, while the ectoderm at the animal pole of the gastrula halts animal pole-directed LME migration. These differences in the permissive properties of the ectoderm for LME migration are due to the lateral ectoderm being more cohesive and viscous than the animal ectoderm. Consistently, tuning cell contractility in lateral and animal ectoderm via modulation of the actomyosin cytoskeleton is sufficient to change their permissiveness for LME migration. Finally, we found that BMP signalling is critical for reducing animal ectoderm cohesion and, thus, its capacity to halt LME migration towards the animal pole. Collectively, these findings identify a critical role of ectoderm tissue cohesion and viscosity in guiding LME migration during zebrafish gastrulation.
Nucleotide-protocell interactions: A reciprocal relationship in prebiotically pertinent environments
Authors: Deshpande Kshitij , Mulewar Sahil Sunil , Rajamani Sudha
Published: July 2023

Spontaneous interactions between nucleotides and lipid membranes are likely to have played a prominent role in the emergence of life on Earth. However, the effect of nucleotides on the physicochemical properties of model protocellular membranes is relatively less understood. To this end, we aimed to discern the effect of canonical nucleotides on the properties of single-chain amphiphile membranes under prebiotically relevant conditions of multiple wet-dry cycles. Furthermore, the change in critical aggregation concentration of the membranes, and their stability in the presence of nucleotides, was also investigated in Astrobiologically analogue environments. We report that different nucleotides, lipid headgroups, and the ionic makeup of the system affect lipid-nucleotide interactions, which in turn can modulate the effect of nucleotides on the membranes. Specifically, oleic acid membranes seemingly showed an increase in the critical aggregation concentration, and an increase in the stability against prebiotically relevant selection pressures, in the presence of certain nucleotides. Our work has implications for understanding how nucleotides might have shaped the protocellular landscape of the prebiotic Earth.
Genome Sequence and Annotation the B3 Mycobacteriophage Phayeta
Authors: Bishop Emily , Earlely Warren , Greco Alexandra , Hofseth Emma , Kinerson Emma , Lafayette Brandon , Llanot-Arocho Nestor , Mazen Brittney , Cevasco Megan , Williams Daniel C.
Published: July 2023

Phayeta is a siphovirus extracted from soil near Myrtle Beach, South Carolina which infectsMycobacterium smegmatis. Annotation of the 68,700 base-pair circularly permuted genome identified 104 predicted protein-encoding genes, 36 of which have functional assignments.
Cdc42 mobility and membrane flows regulate fission yeast cell shape and survival
Authors: Rutkowski David M. , Vincenzetti Vincent , Vavylonis Dimitrios , Martin Sophie G.
Published: July 2023

Local Cdc42 GTPase activation promotes polarized exocytosis, resulting in membrane flows that deplete low-mobility membrane-associated proteins from the growth region. To investigate the self-organizing properties of the Cdc42 secretion-polarization system under membrane flow, we developed a reaction-diffusion particle model. The model includes positive feedback activation of Cdc42, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis. Simulations show how polarization relies on flow-induced depletion of low mobility GAPs. To probe the role of Cdc42 mobility in the fission yeastSchizosaccharomyces pombe, we changed its membrane binding properties by replacing its prenylation site with 1, 2 or 3 repeats of the Rit1 C terminal membrane binding domain (ritC), yielding alleles with progressively lower unbinding and diffusion rates. Concordant modelling predictions and experimental observations show that lower Cdc42 mobility results in lower Cdc42 activation level and wider patches. Indeed, while Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC is inviable. The model further predicts that GAP depletion increases Cdc42 activity at the expense of loss of polarization. Experiments confirm this prediction, as deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells. Our combined experimental and modelling studies demonstrate how membrane flows are an integral part of Cdc42-driven pattern formation.

Significance Statement

The delivery of new membrane from internal pools at zones of polarized secretion induces in-plane plasma membrane flows that displace slowly mobile membrane-associated proteins from the zone of secretion. However, zones of polarized secretion are themselves specified by the activity of membrane-associated polarity factors, such as the small GTPase Cdc42. Through combined modelling and experimental approaches, this work demonstrates that the fast mobility of the Cdc42 GTPase is critical to allow the establishment and maintenance of a polarity patch, which is reinforced by flow-mediated displacement of a negative regulator.
Structure of a tripartite protein complex that targets toxins to the type VII secretion system
Authors: Klein Timothy A. , Shah Prakhar Y. , Gkragkopoulou Polyniki , Grebenc Dirk W. , Kim Youngchang , Whitney John C.
Published: July 2023

Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6Å structure of the LXG domain of the TelA toxin from the opportunistic pathogenStreptococcus intermediusin complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.
Macrophage Xanthine Oxidoreductase Links LPS Induced Lung Inflammatory Injury to NLRP3 Inflammasome Expression and Mitochondrial Respiration
Authors: Fini Mehdi A. , Monks Jenifer A. , Li Min , Gerasimovskaya Evgenia , Paucek Petr , Wang Kepeng , Frid Maria G. , Pugliese Steven C. , Bratton Donna , Yu Yen-Rei , Irwin David , Karin Michael , Wright Richard M. , Stenmark Kurt R.
Published: July 2023

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) remain poorly treated inflammatory lung disorders. Both reactive oxygen species (ROS) and macrophages are involved in the pathogenesis of ALI/ARDS. Xanthine oxidoreductase (XOR) is an ROS generator that plays a central role in the inflammation that contributes to ALI. To elucidate the role of macrophage-specific XOR in endotoxin induced ALI, we developed a conditional myeloid specific XOR knockout in mice. Myeloid specific ablation of XOR in LPS insufflated mice markedly attenuated lung injury demonstrating the essential role of XOR in this response. Macrophages from myeloid specific XOR knockout exhibited loss of inflammatory activation and increased expression of anti-inflammatory genes/proteins. Transcriptional profiling of whole lung tissue of LPS insufflated XORfl/fl//LysM-Cremice demonstrated an important role for XOR in expression and activation of the NLRP3 inflammasome and acquisition of a glycolytic phenotype by inflammatory macrophages. These results identify XOR as an unexpected link between macrophage redox status, mitochondrial respiration and inflammatory activation.
Generation of an induced pluripotent stem cell line from a healthy adult indigenous Nigerian participant
Authors: Muhammad Zaid , Brown Phoebe W. , Babazau Larema , Alkhamis Abdulrahman I. , Goni Baba W. , Nggada Haruna A. , Mbaya Kefas M. , Wray Selina , Marte Isa H. , Karch Celeste M. , Serpell Louise C. , Maina Mahmoud B.
Published: July 2023

Genetic backgrounds contribute to cellular phenotypes, drug responsiveness, and health outcomes. However, the majority of human induced pluripotent stem cell (iPSC) lines are derived from individuals of European descent. Thus, there is a major, unmet need in the generation, characterisation, and distribution of iPSCs from diverse ancestries. To begin to address this need, we have generated iPSCs from dermal fibroblasts isolated from a healthy 60-year-old indigenous Nigerian male belonging to the Babur ethnic group. The iPSCs were generated using Sendai virus, and copy number variation (CNV) analysis revealed no new major abnormalities compared to the parental fibroblasts. The iPSCs have been characterised for pluripotency markers and morphology and successfully differentiated into neural progenitor cells and astrocytes. This iPSC line could serve as a healthy control in comparative studies and can be used in disease modelling, toxicity assessments, genetic analyses, and drug discovery processes within an African genetic background. To bolster the inclusion of African models in biomedical research, this iPSC line will be made available to the broader scientific community. Ongoing efforts focus on generating more lines from diverse indigenous populations towards creating a dedicated open-access African iPSC biobank.