bluuebunny/crossref_metadata_2025 huggingface dataset:
Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering
Authors: Xie Keqiang , Starzyk Jakob , Majumdar Ishita , Rincones Katerina , Tran Thao , Lee Danna , Niemi Sarah , Famiglietti John , Suter Bernhard , Shan Richard , Wu Hao
Published: December 2022

The toolbox for genome editing in basic research and therapeutic applications is rapidly expanding. While efficient targeted gene ablation using nuclease editors has been demonstrated from bench to bedside, precise transgene integration remains a technical challenge. AAV6 has been a prevalent donor carrier for homology-directed repair (HDR) mediated genome engineering but has reported safety issues, manufacturing constraints, and restricted applications due to its 4.5 Kb packaging limit. Non-viral targeted genetic knock-ins rely primarily on double-stranded DNA (dsDNA) and linear single-stranded DNA (lssDNA) donors. Both dsDNA and lssDNA have been previously demonstrated to have low efficiency and cytotoxicity. Here, we developed a non-viralgenome writingcatalyst(GATALYST™) system which allows production of ultrapure, minicirclesingle-strandedDNAs(cssDNAs) up to ∼20 Kb as donor templates for highly efficient precision transgene integration. cssDNA donors enable knock-in efficiency of up to 70% in induced pluripotent stem cells (iPSCs), superior efficiency in multiple clinically relevant primary cell types, and at multiple genomic loci implicated for clinical applications with various nuclease editor systems. When applied to immune cell engineering, cssDNA engineered CAR-T cells exhibit more potent and durable anti-tumor efficacy than those engineered from AAV6 viral vectors. The exceptional precision and efficiency, improved safety, payload flexibility, and scalable manufacturability of cssDNA unlocks the full potential of genome engineering with broad applications in therapeutic development, disease modeling and other research areas.

Highlights

Scalable production of minicirclessDNA(cssDNA) with highly engineered phagemid system

Genome writingcatalyst(GATALYST™) system with cssDNA donor template demonstrates superior efficiency and safety in various cell types and genomic loci

GATALYST gene writing system enables ultra-large transgene integration

cssDNA engineered CAR-T outperforms AAV engineered CAR-T with superior anti-tumor function
Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation
Authors: Zhao Weisong , Huang Xiaoshuai , Yang Jianyu , Qiu Guohua , Qu Liying , Zhao Yue , Zhao Shiqun , Luo Ziying , Wang Xinwei , Jiu Yaming , Mao Heng , Ding Xumin , Tan Jiubin , Hu Ying , Pan Leiting , Chen Liangyi , Li Haoyu
Published: December 2022

In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions, which is especially intractable in emerging deep-learning ones. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) framework to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Beyond that, we provide a strategy for learning-based restorations, allowing a direct detection of both data and model uncertainties, and expect the representative cases can inspire further advances in this rapidly developing field.
Metacell projection for interpretable and quantitative use of transcriptional atlases
Authors: Ben-Kiki Oren , Bercovich Akhiad , Lifshitz Aviezer , Tanay Amos
Published: December 2022

We describe MCProj - an algorithm for analyzing query scRNA-seq data by projections over reference single cell atlases. We represent the reference as a manifold consisting of annotated metacell gene expression distributions. We then infer query metacells as mixtures of atlas distributions while correcting for technology-specific gene biases. This approach distinguishes and tags query cells that are consistent with existing atlas states from novel or artifactual behaviors that are not observed in the atlas. It also identifies significant expression differences observed in query states that are mapped coherently onto the atlas. We showcase MCProj functionality by analyzing blood gene expression from multiple sources and technologies, suggesting it as a method of choice for scRNA-seq analysis following extensive cell atlas projects.
An epitope-enriched immunogen increases site targeting in germinal centers
Authors: Caradonna Timothy M. , Windsor Ian W. , Roffler Anne A. , Song Shengli , Watanabe Akiko , Kelsoe Garnett , Kuraoka Masayuki , Schmidt Aaron G.
Published: December 2022

Antibody immunodominance is the asymmetric elicitation of responses against protein antigens. For influenza hemagglutinin (HA), antibody responses often target variable regions on HA and do not provide lasting protection. Next-generation influenza vaccines should elicit antibodies targeting conserved regions such as the receptor binding site (RBS). Understanding how presenting an epitope on a rationally-designed immunogen influences immune responses could help achieve this goal. Here, we compared an engineered RBS-enriched immunogen and its non-enriched counterparts to characterize RBS-directed responses. We found that enriching the RBS-epitope on a single immunogen preferentially expands RBS-directed responses relative to a cocktail of the non-epitope-enriched immunogens. Single B cell analyses showed a genetically diverse RBS-directed population that structural characterization showed engagement of the RBS with canonical features shared with both its receptor and human broadly neutralizing antibodies. These data show how epitope-enriched immunogens can expand responses to a conserved viral site, while maintaining genetic and structural diversity.
Chimeric U-Net – Modifying the standard U-Net towards Explainability
Authors: Schulze Kenrick , Peppert Felix , Schütte Christof , Sunkara Vikram
Published: December 2022

Healthcare guided by semantic segmentation has the potential to improve our quality of life through early and accurate disease detection. Convolutional Neural Networks, especially the U-Net-based architectures, are currently the state-of-the-art learningbased segmentation methods and have given unprecedented performances. However, their decision-making processes are still an active field of research. In order to reliably utilize such methods in healthcare, explainability of how the segmentation was performed is mandated. To date, explainability is studied and applied heavily in classification tasks. In this work, we propose the Chimeric U-Net, a U-Net architecture with an invertible decoder unit, that inherently brings explainability into semantic segmentation tasks. We find that having the restriction of an invertible decoder does not hinder the performance of the segmentation task. However, the invertible decoder helps to disentangle the class information in the latent space embedding and to construct meaningful saliency maps. Furthermore, we found that with a simple k-Nearest-Neighbours classifier, we could predict the Intersection over Union scores of unseen data, demonstrating that the latent space, constructed by the Chimeric U-Net, encodes an interpretable representation of the segmentation quality. Explainability is an emerging field, and in this work, we propose an alternative approach, that is, rather than building tools for explaining a generic architecture, we propose constraints on the architecture which induce explainability. With this approach, we could peer into the architecture to reveal its class correlations and local contextual dependencies, taking an insightful step towards trustworthy and reliable A.I.

Code to build and utilize the Chimeric U-Net is made available under:https://github.com/kenrickschulze/Chimeric-UNet---Half-invertible-UNet-in-Pytorch
The oncogenic fusion protein TAZ-CAMTA1 promotes genomic instability and senescence through hypertranscription
Authors: Neil Emily , Rubin Brian , Kouskoff Valerie
Published: December 2022

TAZ-CAMTA1 is a fusion protein found in over 90% of Epithelioid Hemangioendothelioma (EHE), a rare vascular sarcoma with an unpredictable disease course. To date, how TAZ-CAMTA1 initiates tumour formation remains unexplained. To study the oncogenic mechanism leading to EHE initiation, we developed a model system whereby TAZ-CAMTA1 expression is induced by doxycycline in primary endothelial cells. Using this model, we establish that upon TAZ-CAMTA1 expression endothelial cells rapidly enter a hypertranscription state, triggering considerable DNA damage. As a result, TC-expressing cells become trapped in S phase. Additionally, TAZ-CAMTA1-expressing endothelial cells have impaired homologous recombination, as shown by reduced BRCA1 and RAD51 foci formation. Consequently, the DNA damage remains unrepaired and TAZ-CAMTA1-expressing cells enter senescence. Knockout ofCdkn2a, the most common secondary mutation found in EHE, allows senescence bypass and uncontrolled growth. Together, this provides a mechanistic explanation for the clinical course of EHE and offers novel insight into therapeutic options.
Blochmanniaendosymbionts reduce brood rearing success in a carpenter ant (Camponotussp.)
Authors: Preuss Anika , Czuppon Peter , Ernst Ulrich R. , Gadau Jürgen
Published: December 2022

All ants of the species rich genusCamponotus(‘carpenter ants’) possess the obligate intracellular bacterial mutualistBlochmannia. We tested the relevance of the endosymbiontBlochmanniafor offspring rearing using cross-fostering experiments betweenCamponotussp. colonies and subcolonies (worker groups), which were either treated with antibiotics to removeBlochmanniaor untreated. Our antibiotic treatment reduced the level ofBlochmanniaendosymbionts in eggs, larvae and workers significantly. Corroborating previous results, we found that eggs from treated colonies had a significantly reduced probability to develop into larvae and almost zero probability to become adults. Surprisingly, subcolonies treated with antibiotics had a significantly higher success in raising their own and foreign eggs from treated and untreated colonies than untreated subcolonies. This might indicate that theBlochmanniasymbiosis entails significant costs for the host in terms of brood rearing, i.e., symbiont-free workers are more successful in brood rearing than untreated workers. If confirmed, this would be a rare case where the costs of a symbiosis can be empirically measured and quantified. Alternatively, the antibiotic treatment increased as a side effect the brood rearing effort of workers leading to the differences in brood rearing success of treated workers. But even if that would be the case, it still indicates that workers that have either lost or have a significantly reduced number of endosymbionts can still raise brood from antibiotic-treated and untreated colonies better than untreated workers. ThusBlochmannia, although crucial for brood development, may reduce the amount of brood a colony can raise.
Genome-wide Identification and Expression Profile Analysis of Laccase Family Genes in theHypsizygus marmoreus
Authors: Wang Gang , Wang Cheng , Wang HongBo , Zhu Ying , Wang Yuanyuan , chen Yu , Ma Lin , Sun Zijun , Liu Bobin , Liu Fang
Published: December 2022

Laccase exists widely in plants and fungi. It is a copper-containing polyphenol oxidase that can degrade lignin, oxidate, and phenolic substances, inhibit heterophytes, promote fruiting body formation, and improve the quality of mushrooms. In this study, 18 laccase genes were identified from the whole genome of a white strain (HM62) ofHypsizygus marmoreus, and the mapping, structure, and evolution of laccase genes were analyzed at the whole genome level, while the spatiotemporal expression was evaluated at different developmental stages. The laccase genes mainly distributed on chromosomes 1, 2, 3, 4, 6, 9, and 10, and 9 genes were clustered linearly on chromosome 6, indicating gene doubling. Phylogenetic tree analysis showed that the laccase gene family was divided into three subfamilies. The spatiotemporal expression analysis of the laccase gene family showed thatHmLac09andHmLac10were highly expressed in different periods and might be involved in lignin degradation and fruit body formation, respectively. The expression levels ofHmLac02, HmLac05, HmLac08, andHmLac17genes in gray or gray and white heterozygous strains were higher than those in white strains, which might be related to the difference in lignin decomposition in gray strains, and one of the factors leading to different growth rates. The present study investigated the characterization of theH. marmoreuslaccase gene family, extending our understanding of laccase mediated fruiting body development and growth rate mechanisms in this fungi.
YOUNG PLASMA REJUVENATES BLOOD DNA METHYLATION PROFILE, PROLONGS MEAN LIFESPAN AND IMPROVES HEALTH IN OLD RATS
Authors: Chiavellini Priscila , Canatelli Mallat Martina , Lehmann Marianne , Zoller Joseph A. , Gordevicius Juozas , Gallardo Maria D. , Pasquini Diana C. , Lacunza Ezequiel , Herenu Claudia B. , Morel Gustavo R. , Horvath Steve , Goya Rodolfo G.
Published: December 2022

There is converging evidence that young blood conveys cells, vesicles and molecules able to revitalize function and restore organ integrity in old individuals. Here, we assessed the effects of young rat plasma on the lifespan, epigenetic age and healthspan of old female rats. Beginning at 25.3 months of age, a group of 9 rats (group T) was intraperitoneally injected with plasma from young rats (2 months) until their natural death. A group of control rats of the same age, received no treatment. Blood samples were collected every other week. Survival curves showed that from age 26 to 30 months, none of the T animals died, whereas the survival curve of C rats began to decline at age 26 months. The external appearance of the T rats was healthier than that of the C counterparts. Blood DNA methylation (DNAm) was assessed using the HorvathMammalMethylChip320. Blood DNAm age versus chronological age showed that DNAm age in young animals increased faster than chronological age then slowed down progressively, entering a plateau after 27 months. Immediately after the start of the treatment, the DNAm age (i.e., epigenetic age) of the treated rats fell below the DNAm age of controls and remained consistently lower until the end of their lives. Assessment of each experimental group showed that the blood DNA methylation levels of 1638 CpGs were different between treated and control blood samples (false discovery rate q-value<0.05). Of these, 1007 CpGs exhibited increased methylation, with age while 631 CpGs showed decreased methylation levels. When rats were grouped according to the similarities in their differential blood DNA methylation profile, samples from the treated and control rats clustered in separate groups. Analysis of promoter differential methylation in genes involved in systemic regulatory activities revealed specific GO term enrichment related to the insulin-like factors (IGFs) pathways as well as to cytokines and chemokines associated with immune and homeostatic functions. We conclude that young plasma therapy may constitute a natural noninvasive intervention for epigenetic rejuvenation and health enhancement, readily translatable to the clinic.
The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking
Authors: Neumann Daniel P. , Pillman Katherine A. , Dredge B. Kate , Bert Andrew G. , Bracken Cameron P. , Hollier Brett G. , Selth Luke A. , Beilharz Traude H. , Goodall Gregory J. , Gregory Philip A.
Published: December 2022

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3’ end anchored RNA sequencing, we mapped the alternative polyadenylation landscape (APA) following TGF-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3’UTR lengthening during this cell state transition. Analysis of the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, revealed enrichment of its binding adjacent to cleavage and polyadenylation sites within 3’UTRs. Following QKI knockdown, APA of many transcripts are altered to produce predominantly shorter 3’UTRs associated with reduced gene expression. Among these, QKI binds to its own cleavage site to produce a transcript with a longer 3’UTR. These findings reveal extensive changes in APA occur during EMT and identify a novel function for QKI in this process.
Modulating the epigenetic state promotes the reprogramming of transformed cells to pluripotency in a line-specific manner
Authors: Fu Xiuling , Zhuang Qiang , Babarinde Isaac A. , Shi Liyang , Ma Gang , Hu Haoqing , Li Yuhao , Chen Jiao , Xiao Zhen , Deng Boping , Sun Li , Jauch Ralf , Hutchins Andrew P.
Published: December 2022

Somatic cell reprogramming and oncogenic transformation share surprisingly similar features, yet transformed cells are highly resistant to reprogramming. There must be barriers that block transformed cells from reprogramming, but the nature of those barriers is unclear. In this study, we generated a systematic panel of transformed mouse embryonic fibroblasts (MEFs) using a variety of oncogenic transgenes, and discovered transformed cell lines that remain compatible with reprogramming when transfected withOct4/Sox2/Klf4/Myc. By comparing the reprogramming-capable and incapable transformed lines we identified multiple stages of failure in the reprogramming process. Some transformed lines failed very early, whilst other lines seemed to progress through a normal-looking reprogramming process. Finally, we show that MEK inhibition overcomes one critical reprogramming barrier by indirectly suppressing a hyperactive epigenetic state in some of the transformed cells. This study reveals that the barriers underlying resistance to reprogramming vary between the different transformation methods.

Key findings

Somatic cell reprogramming of transformed cells is context-specific

Inhibition of MEK converts some cell lines to reprogramming-capable

Transformed cell lines are characterized by a hyperactive chromatin state

MEK inhibition indirectly affects chromatin to enable reprogramming
An ESCRT-dependent pathway coordinates Nuclear and Cytoplasmic Spatial Protein Quality Control at Nuclear Vacuolar Junctions
Authors: Sontag Emily M. , Morales-Polanco Fabián , Chen Jian-Hua , McDermott Gerry , Dolan Patrick T. , Gestaut Dan , Le Gros Mark A. , Larabell Carolyn , Frydman Judith
Published: December 2022

Effective Protein Quality Control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we elucidate the coordination of nuclear and cytoplasmic spatial PQC. While cytoplasmic misfolded proteins concentrate in a cytoplasmic, perinuclear Juxta Nuclear Quality control compartment (JUNQ), nuclear misfolded proteins sequester into a perinucleolar IntraNuclear Quality control compartment (INQ). Particle tracking reveals the INQ and JUNQ converge to face each other across the nuclear envelope at a site proximal to the Nuclear-Vacuolar Junction (NVJ) marked by perinuclear ESCRT-II/-III protein Chm7. Strikingly, this ESCRT-dependent convergence facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. We propose perinuclear ESCRT coordinates spatial PQC at nuclear-vacuolar contacts to facilitate vacuolar clearance of nuclear and cytoplasmic misfolded proteins.
Effect of mitochondrial circulation on mitochondrial age density distribution
Authors: Kuznetsov Ivan A. , Kuznetsov Andrey V.
Published: December 2022

Recent publications report that although the mitochondria population in an axon can be quickly replaced by a combination of retrograde and anterograde axonal transport (often within less than 24 hours), the axon contains much older mitochondria. This suggests that not all mitochondria that reach the soma are degraded and that some are recirculating back into the axon. To explain this, we developed a model that simulates mitochondria distribution when a portion of mitochondria that return to the soma are redirected back to the axon rather than being destroyed in somatic lysosomes. Utilizing the developed model, we studied how the percentage of returning mitochondria affects the mean age and age density distributions of mitochondria at different distances from the soma. We also investigated whether turning off the mitochondrial anchoring switch can reduce the mean age of mitochondria. For this purpose, we studied the effect of reducing the value of a parameter that characterizes the probability of mitochondria transition to the stationary (anchored) state. The reduction in mitochondria mean age observed when the anchoring probability is reduced suggests that some injured neurons may be saved if the percentage of stationary mitochondria is decreased. The replacement of possibly damaged stationary mitochondria with newly synthesized ones may restore the energy supply in an injured axon. We also performed a sensitivity study of the mean age of stationary mitochondria to the parameter that determines what portion of mitochondria re-enter the axon and the parameter that determines the probability of mitochondria transition to the stationary state.
Dietary sugar and protein exert opposing effects on key larval growth and metabolic regulators, theDrosophilainsulin-like peptides Dilp2 and Dilp6
Authors: Suzawa Miyuki , McPherson W. Kyle , Van Gorder Elizabeth E. , Reddy Shivani , Hilovsky Dalton L. , Keliinui Cami N. , Jamali Leila A. , Bland Michelle L.
Published: December 2022

Nutrient intake drives secretion of insulin and insulin-like peptides that stimulate glucose uptake, nutrient storage, protein synthesis and cell growth. TheDrosophilagenome encodes seven insulin-like peptides (Dilps) that bind to a single known insulin receptor to drive growth and nutrient storage. Whether Dilps respond uniformly to changes in dietary nutrients is unknown. Here we characterized the endocrine response to starvation and dietary sugar and protein in mid-third instarDrosophilalarvae, measuring circulating Dilp2, derived from insulin-producing cells in the brain, and Dilp6, produced by the fat body. Starvation led to a 90% reduction in circulating Dilp2 without affecting circulating Dilp6 levels. Dietary protein, but not sugar, restored hemolymph Dilp2 from starved levels, while elevated and imbalanced ratios of sugar to protein led to modest reductions in circulating Dilp2. In contrast, hemolymph Dilp6 was increased by a sugar-only diet. Surprisingly, dietary protein strongly reduced circulating Dilp6 levels. Dietary sugar drives glycogen and triglyceride storage, and levels of these stored nutrients positively correlate with Dilp6. Protein in the diet promotes whole-animal growth, which correlates strongly with circulating Dilp2. Our data show that Dilp2 and Dilp6 secretion are regulated in opposite ways by distinct dietary nutrients. These findings raise the question of how the single known insulin receptor integrates divergent signals from distinct Dilps to control growth and metabolism.
Characterization of the Human Immunodeficiency Virus (HIV-1) Envelope Glycoprotein Conformational States on Infectious Virus Particles
Authors: Nguyen Hanh T. , Wang Qian , Anang Saumya , Sodroski Joseph G.
Published: December 2022

Human immunodeficiency virus (HIV-1) entry into cells involves triggering of the viral envelope glycoprotein (Env) trimer ((gp120/gp41)3) by the primary receptor, CD4, and coreceptors, CCR5 or CXCR4. The pretriggered (State-1) conformation of the mature (cleaved) Env is targeted by broadly neutralizing antibodies (bNAbs), which are inefficiently elicited compared with poorly neutralizing antibodies (pNAbs). Here we characterize variants of the moderately triggerable HIV-1AD8Env on virions produced by an infectious molecular proviral clone; such virions contain more cleaved Env than pseudotyped viruses. We identified three types of cleaved wild-type AD8 Env trimers on virions: 1) State-1-like trimers preferentially recognized by bNAbs and exhibiting strong subunit association; 2) trimers recognized by pNAbs directed against the gp120 coreceptor-binding region and exhibiting weak, detergent-sensitive subunit association; and 3) a minor gp41-only population. The first Env population was enriched and the other Env populations reduced by introducing State-1-stabilizing changes in the AD8 Env or by treatment of the virions with crosslinker or the State-1-preferring entry inhibitor, BMS-806. These stabilized AD8 Envs were also more resistant to gp120 shedding induced by a CD4-mimetic compound or by incubation on ice. Conversely, a State-1-destabilized, CD4-independent AD8 Env variant exhibited weaker bNAb recognition and stronger pNAb recognition. Similar relationships between Env triggerability and antigenicity/shedding propensity on virions were observed for other HIV-1 strains. Our results show that State-1 Envs on virions can be significantly enriched by optimizing Env cleavage; stabilizing the pretriggered conformation by Env modification, crosslinking or BMS-806 treatment; strengthening Env subunit interactions; and using CD4-negative producer cells.

IMPORTANCE

Efforts to develop an effective HIV-1 vaccine have been frustrated by the inability to elicit broad neutralizing antibodies that recognize multiple virus strains. Such antibodies are able to bind a particular shape of the HIV-1 envelope glycoprotein trimer, as it exists on a viral membrane but before engaging receptors on the host cell. Here, we establish simple yet powerful assays to characterize the envelope glycoproteins in a natural context on virus particles. We find that, depending on the HIV-1 strain, some envelope glycoproteins change shape and fall apart, creating decoys that can potentially divert the host immune response. We identify requirements to keep the relevant envelope glycoprotein target for broad neutralizing antibodies intact on virus-like particles. These studies suggest strategies that should facilitate efforts to produce and use virus-like particles as vaccine immunogens.
Candida khanbhaisp. nov., a new clinically relevant yeast within theCandida haemuloniispecies complex
Authors: de Jong Auke W. , Al-Obaid Khaled , Tap Ratna Mohd , van den Ende Bert Gerrits , Groenewald Marizeth , Joseph Leena , Ahmad Suhail , Hagen Ferry
Published: December 2022

Invasive fungal infections caused by non-albicans Candidaspecies are increasingly reported. Recent advances in diagnostic and molecular tools enabled better identification and detection of these emerging pathogenic yeasts. Several of these emerging species belong to theCandida haemuloniispecies complex, which attracted much attention due to the rapid global emergence of its multi-drug resistant memberCandida auris. Here, we describe a new clinically relevant yeast isolated from geographically distinct regions, representing the proposed novel speciesCandida khanbhai, member of theC. haemuloniispecies complex. Moreover, several members of theC. haemuloniispecies complex were observed to be invalidly described, includingCandida aurisandCandida vulturna. Hence, the opportunity was taken to correct this here, formally validating the names of various yeast species.
N6-methyladenosine (m6A) and reader protein YTHDF2 enhance innate immune response by mediating DUSP1 mRNA degradation and activating mitogen-activated protein kinases during bacterial and viral infections
Authors: Feng Jian , Meng Wen , Chen Luping , Zhang Xinquan , Markazi Ashley , Yuan Weiming , Huang Yufei , Gao Shou-Jiang
Published: December 2022

Mitogen-activated protein kinases (MAPKs) play critical roles in the induction of numerous cytokines, chemokines, and inflammatory mediators that mobilize the immune system to counter pathogenic infections. Dual-specificity phosphatase-1 (DUSP1) is a member of dual-specificity phosphatases, which inactivates MAPKs through a negative feedback mechanism. Here we report that in response to viral and bacterial infections, not only DUSP1 transcript but also itsN6-methyladenosine (m6A) level rapidly increase together with the m6A reader protein YTHDF2, resulting in enhanced YTHDF2-mediated DUSP1 transcript degradation. Knockdown of DUSP1 promotes p38 and JNK phosphorylation and activation, thus increasing the expression of innate immune response genes including IL1β, CSF3, TGM2 and SRC. Similarly, knockdown of m6A eraser ALKBH5 increases DUSP1 transcript m6A level resulting in accelerated transcript degradation, activation of p38 and JNK, and enhanced expression of IL1β, CSF3, TGM2 and SRC. These results demonstrate that m6A and reader protein YTHDF2 orchestrate optimal innate immune response during viral and bacterial infections by downregulating the expression of a negative regulator DUSP1 of the p38 and JNK pathways that are central to innate immune response against pathogenic infections.

IMPORTANCE

Innate immunity is central for controlling pathogenic infections and maintaining the homeostasis of the host. In this study, we have revealed a novel mechanism regulating innate immune response during viral and bacterial infections. We have found thatN6-methyladenosine (m6A) and the reader protein YTHDF2 regulate dual-specificity phosphatase-1, a negative regulator of mitogen-activated protein kinases p38 and JNK, to maximize innate immune response during viral and bacterial infections. These results provide novel insights into the mechanism regulating innate immunity, which could help the development of novel approaches for controlling pathogenic infections.
Evaluating the use of social contact data to produce age-specific forecasts of SARS-CoV-2 incidence
Authors: Munday James D , Abbott Sam , Meakin Sophie , Funk Sebastian
Published: December 2022

Short-term forecasts can provide predictions of how an epidemic will change in the near future and form a central part of outbreak mitigation and control. Renewal-equation based models are increasingly popular. They infer key epidemiological parameters from historical epidemiological data and forecast future epidemic dynamics without requiring complex mechanistic assumptions. However, these models typically ignore interaction between age-groups, partly due to challenges in parameterising a time varying interaction matrix. Social contact data collected regularly by the CoMix survey during the COVID-19 epidemic in England, provide a means to inform interaction between age-groups in real-time.

We developed an age-specific forecasting framework and applied it to two age-stratified time-series: incidence of SARS-CoV-2 infection, estimated from a national infection and antibody prevalence survey; and, reported cases according to the UK national COVID-19 dashboard. Jointly fitting our model to social contact data from the CoMix study, we inferred a time-varying next generation matrix which we used to project infections and cases in the four weeks following each of 29 forecast dates between October 2021 and November 2022. We evaluated the forecasts using proper scoring rules and compared performance with three other models with alternative data and specifications alongside two naive baseline models.

Overall, incorporating age-interaction improved forecasts of infections and the CoMix-data-informed model was the best performing model at time horizons between two and four weeks. However, this was not true when forecasting cases. We found that age-group-interaction was most important for predicting cases in children and older adults. The contact-data-informed models performed best during the winter months of 2020 - 2021, but performed comparatively poorly in other periods. We highlight challenges regarding the incorporation of contact data in forecasting and offer proposals as to how to extend and adapt our approach, which may lead to more successful forecasts in future.
Back to the future: omnipresence of fetal influence on the human brain through the lifespan
Authors: Walhovd Kristine B. , Krogsrud Stine Kleppe , Amlien Inge K. , Sørensen Øystein , Wang Yunpeng , Bråthen Anne Cecilie Sjøli , Overbye Knut , Kransberg Jonas , Mowinckel Athanasia M. , Magnussen Fredrik , Herud Martine , Håberg Asta K. , Fjell Anders M. , Vidal-Piñeiro Didac
Published: December 2022

Human fetal development has been associated with brain health at later stages. It is unknown whether growth in utero, as indexed by birth weight (BW), relates consistently to lifespan brain characteristics and changes, and to what extent these influences are of a genetic or environmental nature. Here we show remarkably stable and life-long positive associations between BW and cortical surface area and volume across and within developmental, aging and lifespan longitudinal samples (N = 5794, 4-82 years of age, w/ 386 monozygotic twins, followed for up to 8.3 years w/12,088 brain MRIs). In contrast, no consistent effect of BW on brain changes was observed. Partly environmental effects were indicated by analysis of twin BW discordance. In conclusion, the influence of prenatal growth on cortical topography is stable and reliable through the lifespan. This early life factor appears to influence the brain by association of brain reserve, rather than brain maintenance. Thus, fetal influences appear omnipresent in the spacetime of the human brain throughout the human lifespan. Optimizing fetal growth may increase brain reserve for life, also in aging.
Investigating graph neural network for RNA structural embedding
Authors: Opuu Vaitea , Bret Hélène
Published: December 2022

The biological function of natural non-coding RNAs (ncRNA) is tightly bound to their molecular structure. Sequence analyses such as multiple sequence alignments (MSA) are the bread and butter of bio-molecules functional analysis; however, analyzing sequence and structure simultaneously is a difficult task. In this work, we propose CARNAGE (Clustering/Alignment of RNA with Graph-network Embedding), which leverages a graph neural network encoder to imprint structural information into a sequence-like embedding; therefore, downstream sequence analyses now account implicitly for structural constraints. In contrast to the traditional “supervised” alignment approaches, we trained our network on a masking problem, independent from the alignment or clustering problem. Our method is very versatile and has shown good performances in 1) designing RNAs sequences, 2) clustering sequences, and 3) aligning multiple sequences only using the simplest Needleman and Wunsch’s algorithm. Not only can this approach be readily extended to RNA tridimensional structures, but it can also be applied to proteins.
Effect of acute ultraviolet radiation onGalleria mellonellahealth and immunity
Authors: Sabockyte Ausrine , McAllister Samuel , Coates Christopher J. , Lim Jenson
Published: December 2022

For humans, acute and chronic overexposure to ultraviolet (UV) radiation can cause tissue damage in the form of sunburn and promote cancer(s). The immune-modulating properties of UV radiation and health-related consequences are not well known. Herein, we used the larvae of the wax moth (Galleria mellonella) to determine UV-driven changes in cellular components of innate immunity. From immune cell (haemocyte) reactivity and the production of antimicrobial factors, these insects share many functional similarities with mammalian innate immunity. After exposing insects to UVA or UVB, we monitored larval viability, susceptibility to infection, haemolymph (blood) physiology and faecal discharge. Prolonged exposure of larvae to UVB coincided with decreased survival, enhanced susceptibility to bacterial challenge, melanin synthesis in the haemolymph, compromised haemocyte functionality and changes in faecal (bacterial) content. We contendG. mellonellais a reliablein vivomodel for assessing the impact of UV exposure at the whole organism and cellular levels.
Quantification of bacterial DNA in blood using droplet digital PCR: a pilot study
Authors: Tedim Ana P. , Merino Irene , Ortega Alicia , Domínguez-Gil Marta , Eiros José Maria , Bermejo-Martín Jesús F.
Published: December 2022

Aim

To use genus/species-specific genes droplet digital PCR (ddPCR) assays to detect/quantify bacterial DNA fromEscherichia coli,Klebsiella pneumoniae,Staphylococcus aureusandEnterococcusspp in blood samples.

Methods and Results

Bacterial DNA from clinical strains (4<n<12) was extracted, quantified and diluted (10-0.0001ng/μL) and ddPCR assays were performed in triplicate. These ddPCR assays showed low replication variability, low detection limit (1–0.1pg/μL) and high genus/species specificity. ddPCR assays were also used to quantify bacterial DNA obtained from spiked blood (1×104-1CFU/mL) of each bacterial genus/species. Comparison between ddPCR assays and bacterial culture was performed by Pearson correlation. There was an almost perfect correlation (r≥0.997, p≤0.001) between the number of CFU/mL from bacterial culture and the number of gene copies/mL detected by ddPCR. The time from sample preparation to results was determined to be 3.5-4h.

Conclusions

The results demonstrated the quantification capacity and specificity of the ddPCR assays to detect/quantify four of the most important bloodstream infection (BSI) bacterial pathogens directly from blood.

Significance and Impact

This pilot study results reinforce the potential of ddPCR for the diagnosis and/or severity stratification of BSI. Applied to patients’ blood samples it can improve diagnosis and diminish sample-to-results time, improving patient care.
Chloroplast DNA methylation in the kelpSaccharina latissimais determined by origin and influenced by cultivation
Authors: Scheschonk Lydia , Nilsen Anne M.L. , Bischof Kai , Jueterbock Alexander
Published: December 2022

DNA cytosine methylation is an important epigenetic mechanism in genomic DNA, but absent in the chloroplast DNA of most land plants. We detected methylation in the chloroplast DNA of the kelpSaccharina latissima, a non-model macroalgal species of high ecological (wild populations) and economic importance. Since the functional role of the chloroplast methylome is yet largely unknown, we compared for the first time the chloroplast DNA cytosine methylation between wild and cultured kelp from different climatic origins (High- Arctic (79 °N) and temperate (54 °N), laboratory samples at 5 °C, 10 °C and 15 °C). Our results suggest genome-wide differences in methylated sites, and methylation level, between the origins, and a strong effect of cultivation. Functions related to photosynthesis showed differential methylation only between origins. Significant differences in methylation between cultivated and wild samples of genes related to transcription and translation were unique to the high-arctic samples. Both findings indicate that origin and cultivation strongly, but differently affect the chloroplast methylome. Similar methylomes for samples from the same origin – independent from whether they grow in the wild or in the lab – suggest that origin- specific methylation marks on the chloroplast genome are inherited. Given that DNA- methylation affects gene expression, our study suggests that lab-cultivation alters epigenetically determined kelp chloroplast characteristics at least to the same degree as ecotypic differentiation does, but likely on a much shorter timescale. This indicates the capacity for rapid non-genetic adaptation in the kelpSaccharina latissima.
CRMnet: a deep learning model for predicting gene expression from large regulatory sequence datasets
Authors: Ding Ke , Dixit Gunjan , Parker Brian J. , Wen Jiayu
Published: December 2022

Recent large datasets measuring the gene expression of millions of possible gene promoter sequences provide a resource to design and train optimised deep neural network architectures to predict expression from sequences. High predictive performance due to the modelling of dependencies within and between regulatory sequences is an enabler for biological discoveries in gene regulation through model interpretation techniques.

To understand the regulatory code that delineates gene expression, we have designed a novel deep-learning model (CRMnet) to predict gene expression inSaccharomyces cerevisiae. Our model outperforms the current benchmark models and achieves a Pearson correlation coefficient of 0.971. Interpretation of informative genomic regions determined from model saliency maps, and overlapping the saliency maps with known yeast motifs, support that our model can successfully locate the binding sites of transcription factors that actively modulate gene expression. We compare our model’s training times on a large compute cluster with GPUs and Google TPUs to indicate practical training times on similar datasets.
Effect of rectified gap junctional electrical coupling and spatial distribution of biologically engineered pacemaking cells on ventricular excitation
Authors: Li Yacong , Li Qince , Liu Jun , Ma Lei , Wang Kuanquan , Zhang Henggui
Published: December 2022

Aim

Biologically engineered pacemaker, or bio-pacemaker, is a promising replacement for electronic pacemakers for treating cardiac dysfunction. Previous animal experimental studies, however, have not been able to accurately demonstrate the stability and efficiency of the bio-pacemaker yet. This study aimed to elucidate the underlying factors that affect bio-pacemaker’s performance and to discover possible optimising solutions to enable the potential use of bio-pacemaker therapy.

Methods and results

The human ventricular myocytes model in this study followed the ten Tussucher’s model in 2006, and the bio-pacemaker single cell model was modified based on it as what has been expatiated in our previous work. In tissue model, two factors were primarily evaluated for their effects on bio-pacemakers to pace and drive surrounding cardiac tissue: gap junction between bio-pacemaker cells (PMs) and adjacent ventricular myocytes (VMs) and the spatial distribution of bio-pacemakers. A suppressed gap junctional electrical coupling between and heterotypic gap junctions were simulated and a combination of them led to the best performance of the bio-pacemaker. Then, the pacemaking behaviours of three kinds of idealised PM-VM slices were simulated, in which an electrically isolated distribution of bio-pacemaker showed optimal drive capacities. Finally, a real human ventricular slice model was used to verified the conclusions in idealized tissues.

Conclusion

This study develops a theory that weak-rectified electrical coupling and electrically isolated distribution can enhance the pacemaking efficiency of bio-pacemakers, which lays the groundwork for future research into therapeutic applications of bio-pacemakers.

Author summary

Biologically engineered pacemakers are expected to be a substitute for electronic pacemakers because of their physiological superiority, but how to transform them for practical application remains challenging. In this paper, we presented a theoretical perspective on optimising biological pacemaking capability based on a computational simulation approach. By manipulating the gap junctional electrical coupling among bio-pacemaking cells and between the pacemaker and their surrounding cells, and controlling spatial distribution of bio-pacemaker, we demonstrated that an enhanced capacity of a bio-pacemaker can be achieved. The results of this study may provide a theoretical basis for the further clinical development of bio-pacemakers.
Identification of a forkhead box protein transcriptional network induced in human neutrophils in response to inflammatory stimuli
Authors: Ismailova Aiten , Salehi-Tabar Reyhaneh , Dimitrov Vassil , Memari Babak , Barbier Camille , White John H.
Published: December 2022

Neutrophils represent the largest proportion of circulating leukocytes and, in response to inflammatory stimuli, are rapidly recruited to sites of infection where they neutralize pathogens. We have identified a novel neutrophil transcription network induced in response to inflammatory stimuli. We performed the first RNAseq analysis of human neutrophils exposed to lipopolysaccharide (LPS), followed by a meta-analysis of our dataset and previously published studies of LPS-challenged neutrophils. This revealed a robustly enhanced transcriptional network driven by forkhead box (FOX) transcription factors. The network is enriched in genes encoding proinflammatory cytokines and transcription factors, includingMAFFandATF3, which are implicated in responses to stress, survival and inflammation. Expression of transcription factors FOXP1 and FOXP4 is induced in neutrophils exposed to inflammatory stimuli, and potential FOXP1/FOXP4 binding sites were identified in several genes in the network, all located in chromatin regions consistent with neutrophil enhancer function. Chromatin immunoprecipitation (ChIP) assays in neutrophils confirmed enhanced binding of FOXP4, but not FOXP1, to multiple sites in response to LPS. Binding to numerous motifs and transactivation of network genes were also observed when FOXP proteins were transiently expressed in HEK293 cells. In addition to LPS, the transcriptional network is induced by other inflammatory stimuli, indicating it represents a general neutrophil response to inflammation. Collectively, these findings reveal a role for the FOXP4 transcription network as a regulator of responses to inflammatory stimuli in neutrophils.

Author Summary

In response to pathogens, neutrophils, the most abundant white blood cells in the body, are the first to be recruited to sites of infection. However, defects in neutrophil responses lead to common chronic inflammatory conditions such as atherosclerosis, chronic obstructive lung disease and autoimmune disorders. As such, it is critical to uncover the molecular players implicated in neutrophil responses to signals that induce inflammation. Here we profile how bacterial lipopolysaccharide (LPS), which is derived from the cell walls of bacteria and is a commonly used agent to mimic inflammation, alters gene transcription in isolated human neutrophils. We also combined our data with those of other published studies to identify conserved molecular pathways stimulated in LPS-exposed neutrophils. This analysis revealed a network of genes whose transcription is regulated by members of the so-called forkhead box (FOX) transcription factors. We provide evidence that FOXP4 regulates transcription of genes within the network in neutrophils. We also find that the same network of genes is induced by other inflammatory stimuli, suggesting it plays a role in neutrophil responses to inflammation.