bluuebunny/crossref_metadata_2025 huggingface dataset:
SbCYP79A61 Produces Phenylacetaldoxime, a Precursor of Benzyl Cyanide and Phenylacetic Acid in Sorghum bicolor
Authors: Perez Veronica C. , Dai Ru , Tomiczek Breanna , Mendoza Jorrel , Wolf Emily S.A. , Grenning Alexander , Vermerris Wilfred , Block Anna K. , Kim Jeongim
Published: May 2022

Aldoximes are amino acid derivatives that serve as intermediates for numerous specialized metabolites including cyanogenic glycosides, glucosinolates, and auxins. Aldoxime formation is mainly catalyzed by cytochrome P450 monooxygenases of the 79 family (CYP79s) that can have broad or narrow substrate specificity. Aldoxime biosynthesis in the cereal sorghum (Sorghum bicolor) is not well characterized. This study identified nine CYP79-encoding genes in the genome of sorghum. A phylogenetic analysis of CYP79 showed that SbCYP79A61 formed a subclade with maize ZmCYP79A61, previously characterized to be involved in aldoxime biosynthesis. Functional characterization of this sorghum enzyme using transient expression in Nicotiana benthamiana and stable overexpression in Arabidopsis thaliana revealed that SbCYP79A61 catalyzes the production of phenylacetaldoxime (PAOx) from phenylalanine, but unlike the maize enzyme, displays no detectable activity against tryptophan. Additionally, targeted metabolite analysis after stable isotope feeding assays revealed that PAOx can serve as a precursor of phenylacetic acid (PAA) in sorghum and identified benzyl cyanide as an intermediate of PAOx-derived PAA biosynthesis in both sorghum and maize. Taken together, our results demonstrate that SbCYP79A61 produces PAOx in sorghum and may serve in the biosynthesis of other nitrogen-containing phenylalanine-derived metabolites involved in mediating biotic and abiotic stresses.

Highlight

Identification of SbCYP79A61, an enzyme catalyzing the production of phenylacetaldoxime (PAOx) in sorghum, and identification of benzyl cyanide as an intermediate of PAOx-derived phenylacetic acid (PAA) biosynthesis in monocots.
Geodesics to Characterize the Phylogenetic Landscape
Authors: Khodaei Marzieh , Owen Megan , Beerli Peter
Published: May 2022

Phylogenetic trees are fundamental for understanding evolutionary history. However, finding maximum likelihood trees is challenging due to the complexity of the likelihood landscape and the size of tree space. Based on the Billera-Holmes-Vogtmann (BHV) distance between trees, we describe a method to generate intermediate trees on the shortest path between two trees, called pathtrees. These pathtrees give a structured way to generate and visualize treespace in an area of interest. They allow investigating intermediate regions between trees of interest, exploring locally optimal trees in topological clusters of treespace, and potentially finding trees of high likelihood unexplored by tree search algorithms. We compared our approach against other tree search tools (Paup*, RAxML, and RevBayes) in terms of generated highest likelihood trees, new topology proportions, and consistency of generated treespace. We assess our method using two datasets. The first consists of 23 primate species (CytB, 1141 bp), leading to well-resolved relationships. The second is a dataset of 182 milksnakes (CytB, 1117 bp), containing many similar sequences and complex relationships among individuals. Our method visualizes the treespace using log likelihood as a fitness function. It finds similarly optimal trees as heuristic methods and presents the likelihood landscape at different scales. It revealed that we could find trees that were not found with MCMC methods. The validation measures indicated that our method performed well mapping treespace into lower dimensions. Our method complements heuristic search analyses, and the visualization allows the inspection of likelihood terraces and exploration of treespace areas not visited by heuristic searches.
Systemic coagulopathy drives host lethality in a new Drosophila tumor model
Authors: Hsi Tsai-Ching , Ong Katy L , Sepers Jorian J , Kim Jung , Bilder David
Published: May 2022

Malignant tumors trigger a complex network of inflammatory and wound repair responses, prompting Dvorak’s characterization of tumors as ‘wounds that never heal’ 1. Some of these responses lead to profound defects in blood clotting, such as Disseminated Intravascular Coagulopathy (DIC), which correlate with poor prognoses 2–4. Here, we demonstrate that a new tumor model in Drosophila provokes phenotypes that recapitulate coagulopathies observed in patients. Fly ovarian tumors overproduce multiple secreted components of the clotting cascade and trigger hypercoagulation of fly blood (hemolymph). Hypercoagulation occurs shortly after tumor induction and is transient; it is followed by a hypocoagulative state that is defective in wound healing. Cellular clotting regulators accumulate on the tumor over time and are depleted from the body, suggesting that hypocoagulation is caused by malignant growth exhaustion of host clotting components. Interestingly, clinical studies have suggested that lethality in patients with high serum levels of clotting components can be independent of thrombotic events 5,6. We show that rescuing coagulopathy improves survival of tumor-bearing flies, despite the fact that flies have an open circulatory system. Our work establishes a platform for identifying alternative mechanisms by which tumor-driven coagulopathy triggers early mortality, as well as exploring other conserved mechanisms of host responses to chronic wounds.
Potent activation of NAD+-dependent deacetylase Sirt7 by nucleosome binding
Authors: Kuznetsov Vyacheslav I. , Liu Wallace H. , Klein Mark A. , Denu John M.
Published: May 2022

Sirtuin-7 (Sirt7) is a nuclear NAD+-dependent deacetylase with a broad spectrum of biological functions. Sirt7 overexpression is linked to several pathological states and enhances anticancer drug resistance, making the enzyme a promising target for the development of novel therapeutics. Despite a plethora of reported in vivo functions the biochemical characterization of recombinant Sirt7 remains inadequate for the development of novel drug candidates. Here, we conduct an extensive biochemical analysis of Sirt7 using newly developed binding and kinetic assays to reveal that the enzyme preferentially interacts with and is activated by nucleosomes. Sirt7 activation by nucleic acids alone is effective towards long-chain acylated hydrophobic substrates while only nucleosome binding leads to 105-fold activation of deacetylase activity. Using endogenous chromatin and recombinant acetylated nucleosomes, we reveal that Sirt7 is one of the most efficient deacetylases in the sirtuin family and that its catalytic activity is limited by the rate of dissociation from deacetylated nucleosomes.
A Mathematical Model of the Within-Host Kinetics of SARS-CoV-2 Neutralizing Antibodies Following COVID-19 Vaccination
Authors: de Pillis Lisette , Caffrey Rebecca , Chen Ge , Dela Mark D. , Eldevik Leif , McConnell Joseph , Shabahang Shahrokh , Varvel Stephen A.
Published: May 2022

Compelling evidence continues to build to support the idea that SARS-CoV-2 Neutralizing Antibody (NAb) levels in an individual can serve as an important indicator of the strength of protective immunity against infection. It is not well understood why NAb levels in some individuals remain high over time, while in others levels decline rapidly. In this work, we present a two-population mathematical model of within-host NAb dynamics in response to vaccination. By fitting only four host-specific parameters, the model is able to capture individual-specific NAb levels over time as measured by the AditxtScore™ for NAbs. The model can serve as a foundation for predicting NAb levels in the long-term, understanding connections between NAb levels, protective immunity, and break-through infections, and potentially guiding decisions about whether and when a booster vaccination may be warranted.
Reconstruction and deconstruction of human somitogenesis in vitro
Authors: Miao Yuchuan , Djeffal Yannis , De Simone Alessandro , Zhu Kongju , Silberfeld Andrew , Lee Jong Gwan , Rao Jyoti , Tarazona Oscar A. , Mongera Alessandro , Rigoni Pietro , Diaz-Cuadros Margarete , Song Laura Min Sook , Di Talia Stefano , Pourquié Olivier
Published: May 2022

The body of vertebrates displays a segmental organization which is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm (PSM). Somites then become subdivided into anterior and posterior compartments essential for vertebral formation and segmental patterning of the peripheral nervous system1–4. How this key somitic subdivision is established remains poorly understood. Here we introduce novel tridimensional culture systems of human pluripotent stem cells (PSCs), called Somitoids and Segmentoids, which can recapitulate the formation of epithelial somite-like structures with antero-posterior (AP) identity. Using these systems, we identified a key organizing function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial salt-and-pepper expression pattern of the segmentation gene MESP2 in the newly formed segment is transformed into defined compartments of anterior and posterior identity via an active cell sorting mechanism. Moreover, we demonstrate a large degree of independence of the various patterning modules involved in somitogenesis including the segmentation clock, somite epithelialization and AP polarity patterning. Together we put forward a novel framework accounting for the symmetry breaking process initiating somite polarity patterning. Our work provides a valuable platform to decode general principles of somitogenesis and advance knowledge of human development.
Nuclear Ribosomal Internal Transcribed Spacer 1 (ITS1) variation in Lady Bird Beetles (Coccinellidae)
Authors: Aruggoda A. G. B. , Ren Shun-Xiang
Published: May 2022

The seventeen taxa of the family Coccinellidae were analyzed by sequencing the Internal Transcribed Spacer 1 region followed by analyzing the presence of significantly simple motifs (SSMs), to investigate the presence of functionally important areas in the gene if any and to find phylogenetic inference of the ITSI region between sequenced species. Length comparisons among the ITS 1 region indicated the absence of intraspecific variability. Further, length differences observed were among Adalia bipunctata showing a 3 bp maximum size difference and Psyllobora vigintiduopunctata with 1 bp size difference. The base composition showed highly similar values of A, T, G and C. Except Harmonia axyridis, Phyllobora vigintiduopunctata, Chilocorus renipustulatu, Lemnia duvauceli all other species showed significant number of simple repetitions. Adalia bipunctata showed the highest simple repetition with the RSF value of 1.4440. Thirteen ladybird beetles had higher SSM regions. Adalia bipunctata, Adalia decempunctata, Exochomus quadripustulatus shows high accumulation of AT repeats in 5 prime end flanking region and GC repeats in 3 prime end flanking region. Amida nigropectoralis, Amida quingquefasiata, Menochilus sexmaculata, Phaenochilus mesternalis, Coccidula rufa, Platynaspis luterubra shows a higher concentration of GC repeats towards the 3 prime end flanking regions. Phylogenetic analysis revealed that the tribe Psylloborini of the subfamily Coccinellidae was monophyletic. The members of the Subfamily Chilocorinae first associate with the tribes of subfamily Coccinellidae and then relate with the tribe Scymnini. Exochomus quadripustulatus which shows the highest length variably showed a relationship with Phaenochilus mesternalis 77 bootstrap and 84 posterior probabilities.
Dynamics of TCRβ repertoires from serial sampling of healthy individuals
Authors: Ayestaran Iñigo , Blundell Jamie R.
Published: May 2022

T-cell receptor (TCR) repertoires provide a historical record of antigen exposure. However, the dynamics of TCR repertoires in healthy individuals remain largely uncharacterised. How much of the repertoire is under immune selection in healthy individuals? Do groups of sequences under immune selection share similar dynamics due to convergent specificity? What is the relationship between dynamic similarity and sequence similarity of TCRs? Here we develop a statistical framework for identifying clonotypes under immune selection in time series repertoire data. Applying this framework to serially sampled repertoires collected over the course of a year from 3 healthy volunteers, we are able to detect hundreds of TCRs undergoing strong immune selection whereby clonotype frequencies can change by orders of magnitude over timescales as short as a month. Clonotypes under immune selection belong to a handful of distinct dynamic clusters each of which show highly coordinated temporal behaviour suggesting a common immunogenic stimulus. Whilst a subset of clonotypes within dynamic clusters show shared amino acid motif usage, most do not, suggesting the same immunogenic stimulus elicits a diverse TCR response. Conversely, shared amino acid motif usage alone identifies far fewer clonotypes under immune selection and these clonotypes do not routinely exhibit correlated temporal behaviour. These results highlight the potential of using information contained in the dynamics of TCR repertoires for identifying clonotypes responding to the same immunogenic stimulus in a sequence agnostic way.
Serotonin coordinates reproductive functions in Caenorhabditis elegans
Authors: Aprison Erin Z. , Dzitoyeva Svetlana , Ruvinsky Ilya
Published: May 2022

Reproduction alters animal behavior and physiology, but neuronal circuits that coordinate these changes remain largely unknown. Insights into mechanisms that regulate and possibly coordinate reproduction-related traits could be gleaned from the study of sex pheromones that manipulate potential mating partners to improve reproductive success. In C. elegans, the prominent male pheromone, ascr#10, modifies reproductive behavior and several aspects of reproductive physiology in hermaphrodite recipients, including improving oocyte quality. Here we show that a circuit that contains serotonin-producing and serotonin-uptaking neurons plays a key role in mediating these beneficial effects of ascr#10. We also demonstrate that increased serotonergic signaling promotes proliferation of germline progenitors in adult hermaphrodites. Our results establish a role for serotonin in maintaining germline quality and highlight a simple neuronal circuit that acts as a linchpin that couples food intake, mating behavior, reproductive output, and germline renewal and provisioning.
Paths to adaptation under fluctuating nitrogen starvation: The spectrum of adaptive mutations in Saccharomyces cerevisiae is shaped by transposons and microhomology-mediated recombination
Authors: Hays Michelle , Schwartz Katja , Schmidtke Danica T. , Aggeli Dimitra , Sherlock Gavin
Published: May 2022

There are many mechanisms that give rise to genomic change: while point mutations are often emphasized in genomic analyses, evolution acts upon many other types of genetic changes that can result in less subtle perturbations. Changes in chromosome structure, DNA copy number, and novel transposon insertions all create large genomic changes, which can have correspondingly large impacts on phenotypes and fitness. In this study we investigate the spectrum of adaptive mutations that arise in a population under consistently fluctuating nitrogen conditions. We specifically contrast these adaptive alleles and the mutational mechanisms that create them, with mechanisms of adaptation under batch glucose limitation and constant selection in low, non-fluctuating nitrogen conditions. We observe that retrotransposon activity accounts for a substantial number of adaptive events, along with microhomology-mediated mechanisms of insertion, deletion, and gene conversion. In addition to loss of function alleles, which are often exploited in genetic screens, we identify adaptive gain of function alleles and alleles acting through as-of-yet unclear mechanisms. Taken together, our findings emphasize that how selection (fluctuating vs. non-fluctuating) is applied also shapes adaptation, just as the selective pressure (nitrogen vs. glucose) does itself. Fluctuating environments can activate different mutational mechanisms, shaping adaptive events accordingly. Experimental evolution, which allows a wider array of adaptive events to be assessed, is thus a complementary approach to both classical genetic screens and natural variation studies to characterize the genotype-to-phenotype-to-fitness map.
MicroRNAs contribute to the host response to Coxiella burnetii
Authors: Sachan Madhur , Brann Katelynn R. , Voth Daniel E. , Raghavan Rahul
Published: May 2022

MicroRNAs (miRNAs), a class of small non-coding RNAs, are critical to gene regulation in eukaryotes. They are involved in modulating a variety of physiological processes, including the host response to intracellular infections. Little is known about miRNA functions during infection by Coxiella burnetii, the causative agent of human Q fever. This bacterial pathogen establishes a large replicative vacuole within macrophages by manipulating host processes such as apoptosis and autophagy. We investigated miRNA expression in C. burnetii-infected macrophages and identified several miRNAs that were down- or up-regulated during infection. We further explored the functions of miR-143-3p, an miRNA whose expression is down-regulated in macrophages infected with C. burnetii, and show that increasing the abundance of this miRNA in human cells results in increased apoptosis and reduced autophagy – conditions that are unfavorable to C. burnetii intracellular growth. In sum, this study demonstrates that C. burnetii infection elicits a robust miRNA-based host response, and because miR-143-3p promotes apoptosis and inhibits autophagy, down-regulation of miR-143-3p expression during C. burnetii infection likely benefits the pathogen.
A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern
Authors: Yuan Mengqi , Chen Xiangyu , Zhu Yanzhi , Dong Xiaoqing , Liu Yan , Qian Zhaohui , Ye Lilin , Liu Pinghuang
Published: May 2022

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a “single” agent to simultaneously target multiple distinct epitopes on the spike is desirable to overcome the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained the specificity and simultaneously bound to the two distinct epitopes on RBD and S2. Bi-Nab35B5-47D10showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs) and variants being monitored (VBMs) compared with its parental mAbs. Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10can efficiently neutralize VBMs including Alpha (B.1.1.7), Beta (B.1.351) and Kappa (B.1.617.1) and VOCs including Delta (B.1.617.2), Omicron BA.1 and Omicron BA.2. Crucially, Bi-Nab35B5-47D10substantially improved neutralizing activity against Omicron BA.1 (IC50= 27.3 ng/mL) and Omicron BA.2 (IC50= 121.1 ng/mL) compared with their parental mAbs. Therefore, Bi-Nab35B5-47D10represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern.

Importance

The new highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10,that simultaneously targets SARS-CoV-2 RBD and S2 and improved neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10is a feasible and potentially effective strategy to treat and prevent COVID-19.
MAP Kinase and mammalian target of rapamycin are main pathways of gallbladder carcinogenesis: Results from bioinformatic analysis of Next Generation Sequencing data from a hospital-based cohort
Authors: Rajput Monika , Chigurupati SatyaVjiay j , Purwar Roli , Shukla Mridula , Pandey Manoj
Published: May 2022

Background

Gallbladder Cancer (GBC) is one of the most common cancers of the biliary tract and the third commonest gastrointestinal (GI) malignancy worldwide. The disease is characterized by the late presentation and poor outcome despite treatment, and hence, newer therapies and targets need to be identified.

Methods

The current study investigated various functionally enriched pathways in GBC pathogenesis involving the genes identified through Next Generation Sequencing (NGS). The Pathway enrichment analysis and Gene Ontology (GO) were carried out after NGS, followed by the construction of the protein-protein interaction (PPI) network to discover associations among the genes.

Results

Of the thirty-three patients with GBC who were screened through next-generation sequencing (NGS), 27somatic mutations were identified. These mutations involved a total of 14 genes. The p53 and KRAS were commonly found to be mutated, while mutations in other genes were seen in one case each, the mean number of mutations were 1.2, and maximum mutation in a single case (eight) was seen in one case. The bioinformatics analysis identified MAP kinase, PI3K-AKT, EGF/EGFR, and Focal Adhesion PI3K-AKT-mTOR signaling pathways and cross-talk between these.

Conclusion

The results suggest that the complex crosstalk between the mTOR, MAPK, and multiple interacting cell signaling cascades can promote GBC progression, and hence, mTOR - MAPK targeted treatment will be an attractive option.
Myofiber reconstruction at micron scale reveals longitudinal bands in heart ventricular walls
Authors: Dileep Drisya , Syed Tabish A. , Sloan Tyler F. W. , Dhandapany Perundurai S. , Siddiqi Kaleem , Sirajuddin Minhajuddin
Published: May 2022

The coordinated contraction of myocytes drives the heart to beat and circulate blood. Due to the limited spatial resolution of whole heart imaging and the piecemeal nature of high-magnification studies, a confirmed model of myofiber geometry does not yet exist. Using microscopy and computer vision we report the first three-dimensional reconstruction of myofibers across entire mouse ventricular walls at the micron scale, representing a gain of three orders of magnitude in spatial resolution over the existing models. Our analysis reveals prominent longitudinal bands of fibers that are orthogonal to the well-known circumferential ones. Our discovery impacts present understanding of heart wall mechanics and electrical function, with fundamental implications for the study of diseases related to myofiber disorganization.
Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues
Authors: Tornaas Stian , Kleftogiannis Dimitrios , Fromreide Siren , Ytre-Hauge Smeland Hilde , Aarstad Hans Jørgen , Karsten Vintermyr Olav , Andreas Akslen Lars , Elena Costea Daniela , Dongre Harsh Nitin
Published: May 2022

To decipher the interactions between various components of the tumor microenvironment (TME) and tumor cells in a preserved spatial context, a multiparametric approach is essential. In this pursuit, imaging mass cytometry (IMC) emerges as a valuable tool, capable of concurrently analyzing up to 40 parameters at subcellular resolution. In this study, a set of antibodies was selected to spatially resolve multiple cell types and TME elements, including a comprehensive panel targeted at dissecting the heterogeneity of cancer-associated fibroblasts (CAF), a pivotal TME component. This antibody panel was standardized and optimized using formalin-fixed paraffin-embedded tissue (FFPE) samples from different organs/lesions known to express the markers of interest. The final composition of the antibody panel was determined based on the performance of conjugated antibodies in both immunohistochemistry (IHC) and IMC. Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R program. This paper provides a detailed description of the staining procedure and analysis workflow. Subsequently, the panel underwent validation on clinical FFPE samples from head and neck squamous cell carcinoma (HNSCC). The panel and bioinformatics pipeline established here proved to be robust in characterizing different TME components of HNSCC while maintaining a high degree of spatial detail. The platform we describe shows promise for understanding the clinical implications of TMA heterogeneity in large patient cohorts with FFPE tissues available in diagnostic biobanks worldwide.
Low immune response after 1.5 years of primary SARS-CoV-2 infection and Covishield vaccination lead to SARS-CoV-2 reinfection
Authors: Shete Anita M. , Patil Deepak Y. , Sahay Rima R. , Sapkal Gajanan N. , Deshpande Gururaj R. , Yadav Pragya D.
Published: May 2022

We have investigated six COVID recovered cases with two doses of Covishield vaccination followed by reinfection. The primary SARS-CoV-2 infection found to occur with B.1 and reinfection with Omicron BA.1 and BA.2 variants. The genomic characterization and duration between two infections confirms these cases as SARS-CoV-2 reinfection. The mutation analysis of the reinfection cases correlated with immune evasion potential of BA.1 and BA.2 sub lineages. The immune response determined at different time intervals demonstrated boost post two dose vaccination, decline in pre-reinfection sera post 7 months and rise post reinfection. Apparently, these cases suffered from SARS-CoV-2 reinfection with the declined hybrid immunity acquired from primary infection and two dose covishield vaccination. This suggests the need for booster dose of vaccination. Besides this, multiple non-pharmaceutical interventions should be used to cope up with SARS-CoV-2 infection.
Proliferation drives quorum sensing of microbial products in human macrophage populations
Authors: Rajab Nadia , Gearing Linden J. , Lyu Ruqian , Prawer Yair D.J. , Angel Paul W. , Grimmond Sean M. , Laslett Andrew L. , McCarthy Davis J. , Wells Christine A.
Published: May 2022

Macrophages coordinate the initial host inflammatory response to tissue infection, as well as mediating the reparative phase, by producing growth factors that promote tissue repair. One model of this functional dichotomy is that peripherally recruited monocyte-derived macrophages drive acute inflammatory responses to infection, whereas tissue-resident macrophages are responsible for tissue repair. Alternatively, inflammation and repair may be inter-dependent molecular programs, such that both recruited and resident cells have equivalent capacity to contribute. Repeated exposure to pathogenic challenge results in innate tolerance, which may also alter the contributions of discrete macrophage populations to inflammation or repair. In this study a village model of tissue resident and recruited macrophages was created using induced pluripotent stem cell-derived macrophages and peripheral blood monocyte-derived macrophages, respectively. Population responses to repeated exposure to lipopolysaccharide were assessed with single-cell RNA sequencing and donors demultiplexed with Vireo. A subset of genes escaped classical tolerance programs in the iPSC, but not monocyte-derived macrophages, and this was associated with differences in their proliferative capacity. This suggests that targeting the proliferative resident macrophages would be most effective to limit inflammatory signaling.
Tethering distinct molecular profiles of single cells by their lineage histories to investigate sources of cell state heterogeneity
Authors: Minkina Anna , Cao Junyue , Shendure Jay
Published: May 2022

Gene expression heterogeneity is ubiquitous within single cell datasets, even among cells of the same type. Heritable expression differences, defined here as those which persist over multiple cell divisions, are of particular interest, as they can underlie processes including cell differentiation during development as well as the clonal selection of drug-resistant cancer cells. However, heritable sources of variation are difficult to disentangle from non-heritable ones, such as cell cycle stage, asynchronous transcription, and measurement noise. Since heritable states should be shared by lineally related cells, we sought to leverage CRISPR-based lineage tracing, together with single cell molecular profiling, to discriminate between heritable and non-heritable variation in gene expression. We show that high efficiency capture of lineage profiles alongside single cell gene expression enables accurate lineage tree reconstruction and reveals an abundance of progressive, heritable gene expression changes. We find that a subset of these are likely mediated by structural genetic variation (copy number alterations, translocations), but that the stable attributes of others cannot be understood with expression data alone. Towards addressing this, we develop a method to capture cell lineage histories alongside single cell chromatin accessibility profiles, such that expression and chromatin accessibility of closely related cells can be linked via their lineage histories. We call this indirect “coassay” approach “THE LORAX” and leverage it to explore the genetic and epigenetic mechanisms underlying heritable gene expression changes. Using this approach, we show that we can discern between heritable gene expression differences mediated by large and small copy number changes, trans effects, and possible epigenetic variation.
Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing
Authors: Alfano Massimiliano , De Antoni Luca , Centofanti Federica , Visconti Virginia Veronica , Maestri Simone , Esposti Chiara Degli , Massa Roberto , D’Apice Maria Rosaria , Novelli Giuseppe , Delledonne Massimo , Botta Annalisa , Rossato Marzia
Published: May 2022

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully-expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ∼50-kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3′ end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype– phenotype correlations for the better stratification of DM2 patients in clinical trials.
RNA Methylome Reveals the m6A-Mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-Withering
Authors: Zhu Chen , Zhang Shuting , Zhou Chengzhe , Tian Caiyun , Shi Biying , Xu Kai , Huang Linjie , Sun Yun , Lin Yuling , Lai Zhongxiong , Guo Yuqiong
Published: May 2022

Epitranscriptomic mark N6-methyladenosine (m6A) is the most predominant internal modification in RNAs, which plays pivotal roles in response to diverse stresses. Multiple environmental stresses caused by withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, little is known about the effects of m6A-mediated regulatory mechanism on flavor-related metabolisms in tea leaves. Here, we explored m6A-mediated regulatory mechanism and its impacts on flavonoid and terpenoid metabolisms under solar-withering using integrated RNA methylome and transcriptome. Dynamic changes in global m6A levels of tea leaves are mainly controlled by two m6A erasers (CsALKBH4A and CsALKBH4B) under solar-withering. Differentially methylated peak (DMP)-associated genes under different shading rates of solar-withering were identified and found to be enriched in terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylation can not only directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related genes, but also indirectly influence the contents of flavonoids, catechins, and theaflavins via triggering the alternative splicing (AS)-mediated regulation. Our findings underscored a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a compelling link between m6A-mediated regulatory mechanism and the formation of high-quality flavor in tea leaves under solar-withering.
Contrasting reproductive strategies despite convergent traits for two dryland river-floodplain species
Authors: Higgisson Will , Broadhurst Linda , Shams Foyez , Gruber Bernd , Dyer Fiona
Published: May 2022

Aquatic plants share a range of convergent reproductive strategies, such as the ability to reproduce both sexually and asexually through vegetative growth. In dryland river systems, floodplain inundation is infrequent and irregular, and wetlands consist of discrete and unstable habitat patches. In these systems life-history strategies such as long-distance dispersal, seed longevity, self-fertilisation, and reproduction from vegetative propagules are important strategies which allow plants to persist. Using two aquatic plantsMarsilea drummondiiandEleocharis acuta, we investigated the proportions of sexual and asexual reproduction and self-fertilisation employing next generation sequencing approaches and used this information to understand population genetic structure in a large inland floodplain, in western New South Wales (NSW) Australia. Asexual vegetative reproduction and self-fertilisation was more common inM. drummondii, but both species used sexual reproduction as the main mode of reproduction. This resulted in highly differentiated genetic structure between wetlands and similar genetic structure within wetlands. The similarity in genetic structure was influenced by the wetland in the two species highlighting the influence of the floodplain landscape and hydrology in structuring population genetic structure. The high levels of genetic variation among wetlands and low variation within wetlands suggests that dispersal and pollination occur within close proximity and that gene flow is restricted. This suggests a reliance on locally sourced (persistent) seed, rather than asexual (clonal) reproduction or recolonisation via dispersal, for population maintenance in plants in dryland rivers. This highlights the importance of floodplain inundation to promote seed germination, establishment and reproduction in dryland regions.
Concurrent spinal and brain imaging with optically pumped magnetometers
Authors: Mardell Lydia C. , O’Neill George C. , Tierney Tim M. , Timms Ryan C. , Zich Catharina , Barnes Gareth R. , Bestmann Sven
Published: May 2022

The spinal cord and its interactions with the brain are fundamental for movement control and somatosensation. However, brain and spinal cord electrophysiology in humans have largely been treated as distinct enterprises, in part due to the relative inaccessibility of the spinal cord. Consequently, there is a dearth of knowledge on human spinal electrophysiology, including the multiple pathologies of the central nervous system that affect the spinal cord as well as the brain. Here we exploit recent advances in the development of wearable optically pumped magnetometers (OPMs) which can be flexibly arranged to provide coverage of both the spinal cord and the brain concurrently in unconstrained environments. Our system for magnetospinoencephalography (MSEG) measures both spinal and cortical signals simultaneously by employing a custom-made spinal scanning cast. We evidence the utility of such a system by recording simultaneous spinal and cortical evoked responses to median nerve stimulation, demonstrating the novel ability for concurrent non-invasive millisecond imaging of brain and spinal cord.
Simulating alternative forest management in a changing climate on aPinus nigrasubsp.laricioplantation in Southern Italy
Authors: Testolin Riccardo , Dalmonech Daniela , Marano Gina , Bagnara Maurizio , D’Andrea Ettore , Matteucci Giorgio , Noce Sergio , Collalti Alessio
Published: May 2022

Mediterranean pine plantations provide several ecosystem services but are particularly sensitive to climate change. Forest management practices might play a strategic role in the long-term adaptation of Mediterranean forests, but the joint effect of climate change and alternative management options in the near and far future have seldom been investigated together. Here, we developed a portfolio of management options and simulated the development of a Laricio pine (Pinus nigrasubsp.laricio) stand in the Bonis watershed (southern Italy) from its establishment in 1958 up to 2095 using a state-of-the-science process-based forest model. The model was run under three climate change scenarios corresponding to increasing levels of atmospheric CO2concentration, and seven management options with different goals, including post-disturbance management, wood production and renaturalization purposes. We analyzed the effect of climate change on annual carbon fluxes (i.e., gross and net primary production) and stocks (i.e., basal area and potential carbon woody stocks), as well as the impact of different management options compared to no management. Results show that, while climate change (i.e., warming and enriched atmospheric CO2concentration) seems to increase carbon fluxes and stocks in the first half of the century, both show a substantial decrease in the second half, along with higher temperatures (+3 to +5 °C) and lower precipitation (−20% to −22%). When compared to no management, alternative options had a moderate effect on carbon fluxes over the whole simulation (between −6% and +7%) but overall carbon stocks were maximized by thinning interventions and the shelterwood system (+54% to +55%). We demonstrate that the choice of management exerts greater effects on the features of Laricio pine plantations than climate change alone. Therefore, silvicultural strategies might enhance potential stocks and improve forest conditions, with cascading positive effects on the provision of ecosystem services in Mediterranean pine plantations.

Highlights

We simulated the development of a Laricio pine stand over 137 years under three different climatic scenarios and seven management options.

Carbon fluxes and stocks benefit from climate change (i.e., warming and enriched atmospheric CO2concentration) in the first half of the century but show a marked decrease in the longer-term.

Forest management exerts a much stronger effect on these features than climate change alone.

Silvicultural options aimed at reducing stand density preserve and enhance carbon fluxes and stocks over the simulated time period.
Evaluation of metagenomic, 16S rRNA gene and ultra-plexed PCR-based sequencing approaches for profiling antimicrobial resistance gene and bacterial taxonomic composition of polymicrobial samples
Authors: Chau KK , Matlock W , Constantinides B , Lipworth S , Newbold L , Tipper H , Goodall T , Brett H , Hughes J , Crook DW , Eyre DW , Read DS , Walker AS , Stoesser N
Published: May 2022

Background

Shotgun metagenomic sequencing is increasingly popular in taxonomic and resistome-profiling of polymicrobial samples due to its agnostic nature and data versatility. However, caveats include high- cost, sequencing depth/sensitivity trade-offs, and challenging bioinformatic deconvolution. Targeted PCR-based profiling optimises sensitivity and cost-effectiveness, but can only identify prespecified targets and may introduce amplification biases. Ultra-high multiplexed PCR is a potential compromise. As no comprehensive comparative evaluation exists, we evaluated performance of each method in taxonomic/resistome-profiling of a well-defined DNA mock sample and seven “real- world” wastewater samples.

Results

We tested three sequencing approaches (short-read shotgun metagenomics, Illumina Ampliseq™ ultra-plexed AMR Research Panel, 16S rRNA gene sequencing) with seven bioinformatic pipelines (ResPipe, Illumina DNA Amplicon App, One Codex Metagenomic-/Targeted Loci classification and Ampliseq™ Report, DADA2, and an in-house pipeline for AmpliSeq data [AmpliSeek]). Metagenomics outperformed 16S rRNA gene sequencing in accurately reconstituting a mock taxonomic profile and optimising the identification of diverse wastewater taxa, while 16S rRNA gene sequencing produced more even taxonomic outputs which may be quantitatively inaccurate but enhance detection of low abundance taxa. Shotgun metagenomic and AmpliSeq sequencing performed equally well in profiling AMR genes present in a mock sample, but AmpliSeq identified more genes in more complex, “real-world” samples, likely related to sensitivity of detection at the metagenomic sequencing depth used.

Conclusions

A complementary sequencing approach employing 16S rRNA gene or shallow-metagenomic sequencing for taxonomic profiling, and the AmpliSeq AMR panel for high-resolution resistome profiling represents a potential lower-cost alternative to deep shotgun sequencing and may also be more sensitive for the detection of low-prevalence AMR genes. However, our evaluation highlights that both the sequencing and bioinformatics approach used can significantly influence results; for AmpliSeq AMR gene profiling, we developed AmpliSeek which outperformed the other pipelines tested and is open source. Sequencing approach and bioinformatic pipeline should be considered in the context of study goals and sample type, with study-specific validation when feasible.
Modality specific roles for metabotropic GABAergic signaling and calcium induced calcium release mechanisms in regulating cold nociception
Authors: Patel Atit A. , Sakurai Akira , Himmel Nathaniel J. , Cox Daniel N.
Published: May 2022

Calcium (Ca2+) plays a pivotal role in modulating neuronal-mediated responses to multimodal sensory stimuli. Recent studies in Drosophila reveal class III (CIII) multidendritic (md) sensory neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Functional analyses indicate that CIII-mediated multimodal behavioral output is dependent upon activation levels with stimulus-evoked Ca2+ displaying relatively low vs. high intracellular levels in response to gentle touch vs. noxious cold, respectively. However, the mechanistic bases underlying modality-specific differential Ca2+ responses in CIII neurons remain incompletely understood. We hypothesized that noxious cold-evoked high intracellular Ca2+ responses in CIII neurons may rely upon Ca2+-induced Ca2+ release (CICR) mechanisms involving transient receptor potential (TRP) channels and/or metabotropic G-protein coupled receptor (GPCR) activation to promote cold nociceptive behaviors. GABAB receptor mutants and CIII-specific knockdown resulted in impaired noxious cold-evoked behaviors. Gαq and Phospholipase C signaling are likewise required for noxious cold sensing. Additionally, ER localized Ca2+ channels including the Ryanodine receptor (RyR) and Inositol trisphosphate receptor (IP3R) are required for cold nociceptive behaviors. GPCR mediated signaling, through GABAB-R2 and IP3R, is not required in CIII neurons for innocuous touch evoked behaviors. However, CICR via RyR is required in CIII neurons for innocuous touch-evoked behaviors. Disruptions in GABAB-R2, IP3R and RyR in CIII neurons leads to significantly lower levels of cold-evoked Ca2+ responses indicating GPCR and CICR signaling mechanisms function in regulating Ca2+ release. CIII neurons exhibit bipartite cold-evoked firing patterns, where CIII neurons burst during rapid temperature change and tonically fire during steady state cold temperatures. GABAB-R2 knockdown in CIII neurons resulted in disorganized firing patterns during cold exposure. Upon ryanodine pharmacological application, CIII neurons exhibit increased bursting activity and with CIII specific RyR knockdown, there is an increase in cold-evoked tonic firing and decrease in bursting. Lastly, our previous studies implicated the TRPP channel Pkd2 in cold nociception, and here, we show that Pkd2 and IP3R genetically interact in regulating cold-evoked behavior. Collectively, these analyses support novel, modality-specific roles for metabotropic GABAergic signaling and CICR mechanisms in regulating intracellular Ca2+ levels and cold-evoked behavioral output from multimodal CIII neurons.
Absorption changes in Photosystem II in the Soret band region upon the formation of the chlorophyll cation radical [PD1PD2]+
Authors: Boussac Alain , Sugiura Miwa , Nakamura Makoto , Nagao Ryo , Noguchi Takumi , Viola Stefania , Rutherford A. William , Sellés Julien
Published: May 2022

Flash-induced absorption changes in the Soret region arising from the [PD1PD2]+state, the chlorophyll cation radical formed upon light excitation of Photosystem II (PSII), were measured in Mn-depleted PSII cores at pH 8.6. Under these conditions, TyrDisi) reduced before the first flash, andii) oxidized before subsequent flashes. In wild-type PSII, when TyrD●is present, an additional signal in the [PD1PD2]+-minus-[PD1PD2] difference spectrum was observed when compared to the first flash when TyrDis not oxidized. The additional feature was “W-shaped” with troughs at 434 nm and 446 nm. This feature was absent when TyrDwas reduced, but was presenti) when TyrDwas physically absent (and replaced by phenylalanine) orii) when its H-bonding histidine (D2-His189) was physically absent (replaced by a Leucine). Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, seemed to require the native structural environment around the reduced TyrDand its H bonding partners to be present. We found no evidence of involvement of PD1, ChlD1, PheD1, PheD2, TyrZ, and the Cytb559heme in the W-shaped difference spectrum. However, the use of a mutant of the PD2axial His ligand, the D2-His197Ala, shows that the PD2environment seems involved in the formation of “W-shaped” signal.