bluuebunny/crossref_metadata_2025 huggingface dataset:
The ADP-heptose biosynthesis enzyme GmhB is a conserved Gram-negative bacteremia fitness factor
Authors: Holmes Caitlyn L. , Smith Sara N. , Gurczynski Stephen J. , Severin Geoffrey B. , Unverdorben Lavinia V. , Vornhagen Jay , Mobley Harry L. T. , Bachman Michael A.
Published: March 2022

Klebsiella pneumoniae is a leading cause of Gram-negative bacteremia, which is a major source of morbidity and mortality worldwide. Gram-negative bacteremia requires three major steps: primary site infection, dissemination to the blood, and bloodstream survival. Since K. pneumoniae is a leading cause of healthcare-associated pneumonia, the lung is a common primary infection site leading to secondary bacteremia. K. pneumoniae factors essential for lung fitness have been characterized, but those required for subsequent bloodstream infection are unclear. To identify K. pneumoniae genes associated with dissemination and bloodstream survival, we performed insertion site sequencing (InSeq) using a pool of >25,000 transposon mutants in a murine model of bacteremic pneumonia. This analysis revealed the gene gmhB as important for either dissemination from the lung or bloodstream survival. In Escherichia coli, GmhB is a partially redundant enzyme in the synthesis of ADP-heptose for the lipopolysaccharide (LPS) core. To characterize its function in K. pneumoniae, an isogenic knockout strain (ΔgmhB) and complemented mutant were generated. During pneumonia, GmhB did not contribute to lung fitness and did not alter normal immune responses. However, GmhB enhanced bloodstream survival in a manner independent of serum susceptibility, specifically conveying resistance to spleen-mediated killing. In a tail-vein injection of murine bacteremia, GmhB was also required by K. pneumoniae, E. coli and Citrobacter freundii for optimal bloodstream survival. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance bloodstream survival.

IMPORTANCE

Klebsiella pneumoniae frequently causes healthcare-associated infections including pneumonia and bacteremia. This is particularly concerning due to emerging antimicrobial resistance and the propensity for bacteremia to initiate sepsis, which has high mortality and is the most expensive hospital-treated condition. Defining mechanisms of bloodstream survival is critical to understanding the pathology of bacteremia and identifying novel targets for future therapies. In this study, we identified the K. pneumoniae enzyme GmhB as a bloodstream-specific fitness factor that enables the bacteria to survive in the spleen but is dispensable in the lung. Furthermore, GmhB is also needed by the related bacterial pathogens Escherichia coli and Citrobacter freundii to cause bacteremia. Conserved bacteremia fitness factors such a GmhB could be the basis for future therapeutics that would alleviate significant disease caused by from multiple diverse pathogens.
Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells
Authors: Ohno Tomoyuki , Akase Taichi , Kono Shunya , Kurasawa Hikaru , Takashima Takuto , Aizawa Yasunori
Published: March 2022

Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we established a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ∼100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a new standard platform with which to pursue the design of noncoding regions for genome writing in human cells.
POPEYE directly regulates bHLH Ib genes and its own expression
Authors: Pu Meng Na , Liang Gang
Published: March 2022

Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency responsive genes. POPEYE (PYE) is a key bHLH transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive. We found that the over-expression ofPYEattenuates the expression of Fe deficiency responsive genes. PYE directly represses the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, andbHLH101) by associating with their promoters. Although PYE contains anEthylene response factor-associatedAmphiphilicRepression (EAR) motif, it does not interact with the transcriptional corepressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Subcellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains aNuclearExportSignal (NES) which is required for the cytoplasmic localization of PYE. The mutation of NES amplifies the repression function of PYE, resulting in downregulation of Fe deficiency responsive genes. Co-expression assays indicated that bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides insights into the complicated Fe deficiency response signaling pathway and enhances the understanding of PYE functions.

Short summary

PYE is a negative regulator of Fe homeostasis; however, it was still unclear how PYE integrates the Fe deficiency response signaling. Our study shows that conditional nuclear localization of PYE is crucial for Fe homeostasis. PYE not only negatively regulates FIT-dependent Fe uptake genes by directly targeting bHLH Ib genes, but also negatively regulates its own expression.
Comparative transcriptomics of the Drosophila olfactory subsystems identifies a support cell-expressed Osiris protein required for pheromone sensing
Authors: Scalzotto Marta , Ng Renny , Cruchet Steeve , Saina Michael , Armida Jan , Su Chih-Ying , Benton Richard
Published: March 2022

The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae comprise two morphologically and functionally distinct subsystems that express Odorant receptors (Ors) or Ionotropic receptors (Irs). Although these receptors have been thoroughly characterized in this species, the subsystem-specific expression and roles of other genes are much less well-understood. Here we generate subsystem-specific transcriptomic datasets to identify hundreds of genes, encoding diverse protein classes, that are selectively enriched in either Or or Ir subsystems. Using single-cell antennal transcriptomic data and RNA in situ hybridization, we find most neuronal genes – other than sensory receptor genes – are broadly expressed within the subsystems. By contrast, we identify many non-neuronal genes that exhibit highly selective cell-type expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of these olfactory subsystems. We characterize one Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large family of insect transmembrane proteins. Osi8 is expressed in tormogen support cells that are associated with pheromone sensing neurons. Loss of Osi8 abolishes high sensitivity neuronal responses to pheromone ligands. Together this work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in sensory responses, and provides a resource for future characterization of other olfactory subsystem-specific genes.
Novel pathogen introduction rapidly alters evolved movement strategies, restructuring animal societies
Authors: Gupte Pratik Rajan , Albery Gregory F. , Gismann Jakob , Sweeny Amy R. , Weissing Franz J.
Published: March 2022

Animal sociality emerges from individual decisions on how to balance the costs and benefits of being sociable. Movement strategies incorporating social information — the presence and status of neighbours — can modulate spatial associations, helping animals avoid infection while benefiting from indirect information about their environment. When a novel pathogen is introduced into a population, it should increase the costs of sociality, selecting against gregariousness. Yet current thinking about novel pathogen introductions into wildlife neglects hosts’ potential evolutionary responses. We built an individual-based model that captures essential features of the repeated introduction, and subsequent transmission of an infectious pathogen among social hosts. Examining movements in a foraging context, widely shared by many species, we show how introducing a novel pathogen to a population provokes a rapid evolutionary transition to a dynamic social distancing movement strategy. This evolutionary shift triggers a disease-dominated ecological cascade of increased individual movement, decreased resource harvesting, and fewer social encounters. Pathogen-risk adapted individuals form less clustered social networks than their pathogen-risk naive ancestors, which reduces the spread of disease. The mix of post-introduction social movement strategies is influenced by the usefulness of social information and disease cost. Our work demonstrates that evolutionary adaptation to pathogen introductions and re-introductions can be very rapid, comparable to ecological timescales. Our general modelling framework shows why evolutionary dynamics should be considered in movement-disease models, and offers initial predictions for the eco-evolutionary consequences of wildlife pathogen spillover scenarios.
GENESPACE: syntenic pan-genome annotations for eukaryotes
Authors: Lovell John T. , Sreedasyam Avinash , Schranz M. Eric , Wilson Melissa A. , Carlson Joseph W. , Harkess Alex , Emms David , Goodstein David , Schmutz Jeremy
Published: March 2022

The development of multiple high-quality reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple reference haplotypes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation in nature. Here, we address these challenges through the concept of a pan-genome annotation, where conserved gene order is used to restrict gene families and define the expected physical position of all genes that share a common ancestor among multiple genome annotations. By leveraging pan-genome annotations and exploring the underlying syntenic relationships among genomes, we dissect presence-absence and structural variation at four levels of biological organization: among three tetraploid cotton species, across 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic pan-genome annotations in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred and other complex genomes.
Observation of a single protein by ultrafast X-ray diffraction
Authors: Ekeberg Tomas , Assalauova Dameli , Bielecki Johan , Boll Rebecca , Daurer Benedikt J. , Eichacker Lutz A. , Franken Linda E. , Galli Davide E. , Gelisio Luca , Gumprecht Lars , Gunn Laura H. , Hajdu Janos , Hartmann Robert , Hasse Dirk , Ignatenko Alexandr , Koliyadu Jayanath , Kulyk Olena , Kurta Ruslan , Kuster Markus , Lugmayr Wolfgang , Lübke Jannik , Mancuso Adrian P. , Mazza Tommaso , Nettelblad Carl , Ovcharenko Yevheniy , Rivas Daniel E. , Samanta Amit K. , Schmidt Philipp , Sobolev Egor , Timneanu Nicusor , Usenko Sergej , Westphal Daniel , Wollweber Tamme , Worbs Lena , Xavier P. Lourdu , Yousef Hazem , Ayyer Kartik , Chapman Henry N. , Sellberg Jonas A. , Seuring Carolin , Vartanyants Ivan A. , Küpper Jochen , Meyer Michael , Maia Filipe R.N.C.
Published: March 2022

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory1, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes2. This was first demonstrated on biological samples a decade ago on the giant mimivirus3. Since then a large collaboration4has been pushing the limit of the smallest sample that can be imaged5,6. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that ofEscherichia coliGroEL which at 14 nm in diameter7is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.
A Trem2*R47H mouse model without cryptic splicing drives age- and disease-dependent tissue damage and synaptic loss in response to plaques
Authors: Tran Kristine M. , Kawauchi Shimako , Kramár Enikö A. , Rezaie Narges , Liang Heidi Yahan , Arreola Miguel , Da Cunha Celia , Phan Jimmy , Collins Sherilyn , Walker Amber , Neumann Jonathan , Milinkeviciute Giedre , Gomez-Arboledas Angela , Javonillo Dominic I. , Tran Katelynn , Gantuz Magdalena , Forner Stefania , Swarup Vivek , Tenner Andrea J. , LaFerla Frank , Wood Marcelo A. , Mortazavi Ali , MacGregor Grant R. , Green Kim N.
Published: March 2022

Genome-Wide Association Studies revealed the TREM2 R47H variant as one of the strongest genetic risk factors for late-onset Alzheimer’s Disease (AD). Unfortunately, many current TREM2*R47H mouse models are associated with cryptic mRNA splicing of the mutant allele that produces a confounding reduction in protein product. We have developed the Trem2R47H NSS (Normal Splice Site) mouse model where the Trem2 allele is expressed at a similar level to the wild-type Trem2 allele, without evidence of cryptic splicing products, and appropriate inflammatory responses to cuprizone challenge. Utilizing the 5xFAD mouse model, we report age- and disease-dependent changes in response to pathology. At an early disease stage (4 mo), homozygous Trem2R47H NSS; hemizygous 5xFAD (Trem2R47H NSS ; 5xFAD) mice have reduced size and number of microglia plus impaired interaction with plaques, that is associated with increased dystrophic neurites and axonal damage detected through plasma neurofilament light chain (NfL) level and suppressed inflammation. However, homozygosity for Trem2R47H NSS suppressed LTP deficits and presynaptic puncta loss caused by the 5xFAD transgene array. At a more advanced disease stage (12 mo,) Trem2R47H NSS ; 5xFAD mice no longer display impaired plaque-microglia interaction or suppressed inflammatory gene expression, although NfL levels remain elevated, and a unique interferon-related gene expression signature is seen. Furthermore, Trem2R47H NSS ; 5xFAD mice also display robust LTP deficits and exacerbated presynaptic loss. Collectively, we provide a Trem2R47H variant mouse without cryptic splicing, and demonstrate it has disease stage dependent effects when combined with a plaque bearing model, with an initial loss of function that ultimately resolves, giving rise to a unique interferon signature and associated tissue damage.
Resolving clonal substructure from single cell genomic data using CopyKit
Authors: Minussi Darlan Conterno , Sei Emi , Wang Junke , Schalck Aislyn , Yan Yun , Davis Alexander , Wu Hua-Jun , Bai Shanshan , Peng Cheng , Hu Min , Casasent Anna , Contreras Alejandro , Chen Hui , Hui David , Damodaran Senthil , Edgerton Mary E , Kopetz Scott , Lim Bora , Navin Nicholas
Published: March 2022

High-throughput methods for single cell copy number sequencing have enabled the profiling of thousands of cells in parallel, yet there remains a significant bottleneck for data analysis. Here we present CopyKit, a comprehensive set of computational methods for the pre-processing and analysis of single cell copy number data to resolve clonal substructure and reconstruct genetic lineages in tumors. We performed single cell DNA sequencing of 2977 cells from multiple spatial regions in two liver metastasis and 7365 cells from three primary tumors with matched metastatic tissues. In the liver metastases, CopyKit resolved clonal substructure in different spatial regions, which revealed both clonal intermixing and spatial segregation in the tumor mass. In the matched metastatic colorectal and breast cancers, CopyKit resolved metastatic lineages and identified subclones and genomic events that were associated with metastases. These applications show that CopyKit is comprehensive tool for resolving copy number substructure in tumors.
Identification and quantification of α- and β-amanitin in wild mushrooms by HPLC-UV-EC and HPLC-DAD-MS detection
Authors: Barbosa Isabel , Domingues Cátia , Ramos Fernando , Barbosa Rui M.
Published: March 2022

Amatoxins are a group of highly toxic peptides, which include α- and β-amanitin, found in several species of mushrooms (e.g.Amanita phalloides). Due to their high hepatotoxicity, they account for most deaths occurring after mushrooms ingestion. The determination of α- and β- amanitin content in wild mushrooms is invaluable for treating cases involving poisoning. In the present study, we have developed and validated an analytical method based on high-performance liquid chromatography, with in-line ultraviolet and electrochemical detection (HPLC-UV-EC), for the rapid quantification of α- and β-amanitin in wild mushroom samples collected from the Inner Center of Portugal. A reproducible and simple solid-phase extraction (SPE) using OASIS®PRIME HLB cartridges was used for sample pre-treatment, followed by chromatographic separation based on the RP-C18 column. The UV and EC chromatograms of α- and β-amanitin were recorded at 305 nm and +0.600 Vvs. Ag/AgCl, respectively. The linear quantification for both amanitins was in the range of 0.5–20.0 μg·mL-1(R2> 0.999). The LOD, calculated based on the calibration curve, was similar for UV and EC detection (0.12-0.33 μg ml.-1). Intra-day and inter-day precision were less than 13%, and the recovery ratios ranged from 89% to 117%. NineAmanita speciesand five edible mushrooms were analysed by HPLC-UV-EC, and HPLC-DAD-MS confirmed the identification of amatoxins. We find high α- and β-amanitin content inA. phalloidesand not in the other species analysed. In sum, the developed and validated method provides a simple and fast analysis of α- and β-amanitins contents in wild mushrooms and is suitable for screening and routine assessment of mushroom intoxication.

Highlights

New validated method using HPLC-UV-EC to determine α- and β-amanitin in wild mushrooms.

Reproducible and fast SPE procedure for small samples.

Effective sample pre-treatment with the OASIS®PRIME HLB SPE cartridge.

Identification and quantification of α- and β-amanitin in wild mushroom samples from Portugal.

HPLC-DAD-MS confirmation of amatoxins present in mushroom samples.

Graphical abstract
Oxytocin and Emotional Tear in Dogs
Authors: Murata K. , Nagasawa M. , Onaka T. , Kanemaki S. , Nakamura K. , Tsubota K. , Mogi K. , Kikusui T.
Published: March 2022

In humans, tear volume increases in emotional arousal. No studies have investigated the relationship of emotional arousal and tear volume in animals. We measured tear volume in dogs before and after reunion with their owners. Tear volume increased significantly during owner, but not familiar non-owner, reunions but not during reunions with familiar non-owners. When oxytocin instillation was applied to dogs, the tear volume increased, suggesting that oxytocin can mediate the emotion-induced tear secretion in dogs. When the photos of dog’s face in which artificial tear was applied to the dog’s eyes, the positive impression of these photo increased. These suggest that emotional tear can facilitate human-dog emotional connection.
Odors drive feeding through gustatory receptor neurons inDrosophila
Authors: Wei Hong-ping , Lam Thomas Ka Chung , Kazama Hokto
Published: March 2022

Odors are intimately tied to the taste system to aid food selection and determine the sensory experience of food. However, how smell and taste are integrated in the nervous system to drive feeding remains elusive. We show inDrosophilathat odors alone activate gustatory receptor neurons (GRNs), trigger proboscis extension reflex (PER), a canonical taste-evoked feeding behavior, and enhance food intake. Odor-evoked PER requires the function of sugar-sensing GRNs but not olfactory organs. Calcium imaging and electrophysiological recording show that GRNs directly respond to odors. Odor-evoked PER is mediated by the Gr5a receptor, and is bidirectionally modulated by olfactory binding proteins. Finally, odors and sucrose co-applied to GRNs synergistically enhance PER and food consumption. These results reveal a cell-intrinsic mechanism for odor-taste multimodal integration that takes place as early as in GRNs, indicating that unified chemosensory experience is a product of layered integration in peripheral neurons and in the brain.
Cortical structure of neural synchrony and information flow during transition from wakefulness to light non-rapid eye movement sleep
Authors: Fan Joline M. , Kudo Kiwamu , Verma Parul , Ranasinghe Kamalini G. , Morise Hirofumi , Findlay Anne M. , Vossel Keith , Kirsch Heidi E. , Raj Ashish , Krystal Andrew D. , Nagarajan Srikantan S.
Published: March 2022

Sleep is a highly stereotyped phenomenon, requiring robust spatial and temporal coordination of neural activity. How the brain coordinates neural activity with sleep onset can provide insight into the physiological functions subserved by sleep and pathologic phenomena associated with sleep onset. We quantified whole-brain network changes in synchrony and information flow during the transition from wake to non-rapid eye movement (NREM) sleep using magnetoencephalography imaging in healthy subjects. In addition, we performed computational modeling to infer excitatory and inhibitory properties of local neural activity. The sleep transition was identified to be encoded in spatially and temporally specific patterns of local and long-range neural synchrony. Patterns of information flow revealed that mesial frontal regions receive hierarchically organized inputs from broad cortical regions upon sleep onset. Finally, biophysical neural mass modeling demonstrated spatially heterogeneous properties of cortical excitation-to-inhibition from wake to NREM. Together, these findings reveal whole-brain corticocortical structure in the sleep-wake transition and demonstrate the orchestration of local and long-range, frequency-specific cortical interactions that are fundamental to sleep onset.
The Anti-Burkholderia Lasso Peptide Ubonodin Co-Opts the Siderophore Receptor PupB for Cellular Entry
Authors: Do Truc , Thokkadam Alina , Leach Robert , Link A. James
Published: March 2022

New antibiotics are needed as bacterial infections continue to be a leading cause of death. Notorious among antibiotic-resistant bacteria is the Burkholderia cepacia complex (Bcc), which infects cystic fibrosis patients, causing lung function decline. We recently discovered a novel ribosomally synthesized and post-translationally modified peptide (RiPP), ubonodin, with potent activity against several Burkholderia pathogens. Ubonodin inhibits RNA polymerase, but only select Bcc strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we speculate that cellular uptake of ubonodin determines susceptibility. Here, we report a new outer membrane siderophore receptor, PupB, that is required for ubonodin uptake in B. cepacia. Loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. Thus, outer membrane transport is the major determinant of ubonodin’s spectrum of activity. We also show that PupB is activated by a TonB protein and examine a transcriptional pathway that further regulates PupB. Finally, we elucidate the complete cellular uptake pathway for ubonodin by also identifying its inner membrane transporter in B. cepacia. Our work unravels central steps in the mechanism of action of ubonodin and establishes a general framework for dissecting RiPP function.
Parental age at conception on mouse lemur’s offspring longevity: sex-specific maternal effects
Authors: Martine Perret , Aude Anzereay
Published: March 2022

Parental age at conception often influences offspring’s longevity, a phenomenon referred as the “Lansing effect” described in large variety of organisms. But, the majority of the results refer to the survival of juveniles, mainly explained by an inadequate parental care by the elderly parents, mostly the mothers. Studies on the effect of parental age on offspring’s longevity in adulthood remain few, excepted in humans for whom effects of parental age are variable according to statistical models or socioeconomic environments. In a small primate which the longevity reaches up to 12 years, we investigated the effects of parental age at conception on the longevity of offspring (N = 278) issued from parents with known longevity. None of the postnatal parameters, including body mass, influenced offspring’s longevity. Mothers’ age at conception significantly affected offspring’s longevity in males but not females. By contrast, fathers’ age at conception did not influence offspring’s longevity. When considering the age of the breeding pairs at conception, the older the mother, the lower the longevity of male offspring with a minimum when the father was aged. No such correlation was observed for female offspring. Lastly, the longevity of female offspring was significantly positively related to the longevity of both parents. Compared with current studies, the surprisingly minor effect of fathers ‘age and the major impact of mothers’ age on male offspring survival, despite favourable conditions, were discussed in relation with the highly seasonal reproduction in mouse lemurs.
Activation of targetable inflammatory immune signaling is seen in Myelodysplastic Syndromes with SF3B1 mutations
Authors: Choudhary Gaurav S , Pellagatti Andrea , Agianian Bogos , Smith Molly A , Bhagat Tushar D , Gordon-Mitchell Shanisha , Pandey Sanjay , Shah Nishi , Aluri Srinivas , Schwartz Leya , Steeples Violetta , Booher Robert N , Ramachandra Murali , Samson Maria Elena , Carbajal Milagros , Pradhan Kith , Bowman Teresa V. , Pillai Manoj M , Will Britta , Wickrema Amittha , Shastri Aditi , Bradley Robert , Martell Robert , Steidl Ulrich G. , Gavathiotis Evripidis , Boultwood Jacqueline , Starczynowski Daniel T. , Verma Amit
Published: March 2022

Background

Mutations in the SF3B1 splicing factor are commonly seen in Myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML), yet the specific oncogenic pathways activated by missplicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood.

Methods

RNA-seq data from SF3B1 mutant samples was analyzed and functional roles of IRAK4 isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in pre-clinical models of MDS/AML

Results

RNA-seq splicing analysis of innate immune mediators in SF3B1 mutant MDS samples revealed retention of full-length exon 6 of interleukin-1 receptor-associated kinase 4 (IRAK4), a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-Long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteosomal degradation. Expression of IRAK4-Long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models.

Conclusions

SF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models.
Serotonin sensing by microglia conditions the proper development of neuronal circuits and of social and adaptive skills
Authors: Albertini Giulia , D’Andrea Ivana , Druart Mélanie , Béchade Catherine , Nieves-Rivera Nayadoleni , Etienne Fanny , Le Magueresse Corentin , Rebsam Alexandra , Heck Nicolas , Maroteaux Luc , Roumier Anne
Published: March 2022

The proper maturation of emotional and sensory circuits requires a fine tuning of serotonin (5-HT) level during early postnatal development. Consistently, dysfunctions of the serotonergic system have been associated with neurodevelopmental psychiatric diseases, including autism spectrum disorders (ASD). However, the mechanisms underlying the developmental effects of 5-HT remain partially unknown, one obstacle being the action of 5-HT on different cell types.

Here, we focused on microglia, which play a role in brain wiring refinement, and we investigated whether the control of these cells by 5-HT is relevant for neurodevelopment and spontaneous behaviors. Since the main 5-HT sensor in microglia is the 5-HT2B receptor subtype, we prevented 5-HT signaling specifically in microglia by conditionally invalidating Htr2b gene in these cells. We observed that abrogating the serotonergic control of microglia neonatally impacts the phagolysosomal compartment of these cells and their proximity to synapses, and perturbs neuronal circuits maturation. Furthermore, this early ablation of microglial 5-HT2B receptors leads to adult hyperactivity in a novel environment and behavioral defects in sociability and flexibility. Importantly, we show that these behavioral alterations result from a developmental effect, since they are not observed when microglial Htr2b invalidation is induced later, at P30 onward.

Thus, a primary alteration of 5-HT sensing in microglia, during a critical time window between birth and P30, is sufficient to impair social and flexibility skills. This link between 5-HT and microglia may explain the association between serotonergic dysfunctions and behavioral traits, like impaired sociability and inadaptability to novelty, which are prominent in several psychiatric disorders such as ASD.
Longitudinal dynamics of microvascular recovery after acquired cortical injury
Authors: Lin Xiaoxiao , Chen Lujia , Jullienne Amandine , Zhang Hai , Salehi Arjang , Hamer Mary , Holmes Todd , Obenaus Andre , Xu Xiangmin
Published: March 2022

Acquired brain injuries due to trauma damage cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury (TBI), but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14-21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (>200 μm), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.
Single-cell transcriptome profiles ofDrosophila fruitless-expressing neurons from both sexes
Authors: Palmateer Colleen M , Artikis Catherina , Brovero Savannah G , Friedman Benjamin , Gresham Alexis , Arbeitman Michelle N
Published: March 2022

Drosophila melanogasterreproductive behaviors are orchestrated byfruitlessneurons. We performed single-cell RNA-sequencing onfru P1pupal neurons. Uniform Manifold Approximation and Projection (UMAP) clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the UMAP clusters have sex-differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, we annotate clusters as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that expressdoublesex. Enriched marker gene analyses also shows that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters.In vivospatial gene-expression links to the UMAP clusters are provided. A functional analysis offru P1circadian neurons shows they have dimorphic roles in sleep/activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes havefru P1neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors.
Triosephosphate export from chloroplasts regulates flavonoid biosynthesis and permits high light acclimation through the inactivation of SnRK1
Authors: Zirngibl Max-Emanuel , Araguirang Galileo Estopare , Kitashova Anastasia , Jahnke Kathrin , Rolka Tobias , Kühn Christine , Nägele Thomas , Richter Andreas S.
Published: March 2022

Plants evolved multiple strategies to cope with rapid changes in the environment. During high light acclimation, biosynthesis of photoprotective flavonoids, such as anthocyanins, is induced. However, the exact nature of the signal and downstream factors for high light induction of flavonoid biosynthesis (FB) are still under debate. Here we show that carbon-fixation in chloroplasts, subsequent export of photosynthates by TRIOSEPHOSPHATE/PHOSPHATE TRANSLOCATOR (TPT), and the rapid increase in cellular sugar contents permit the transcriptional activation of FB during high light acclimation. In combination with genetic and physiological analysis, targeted and whole transcriptome gene expression studies showed that reactive oxygen species and phytohormones play only a minor role for rapid HL-induction of the anthocyanin branch of FB. In addition to FB, sugar-responsive genes were late-repressed or induced intpt-2in the course of the high light treatment and a significant overlap with transcripts regulated by SNF1-RELATED PROTEIN KINASE 1 (SnRK1) was found. Analysis of mutants with increased and repressed SnRK1 activity revealed that inactivation of SnRK1 is required for the rapid induction of FB during high light acclimation. Our study underlines the central role of chloroplasts as sensors for environmental changes and emphasizes the vital function of sugar-signalling in plant acclimation, even beyond the regulation of FB.
An easily modifiable conjugative plasmid for studying horizontal gene transfer
Authors: Wang Qinqin , Olesen Asmus Kalckar , Maccario Lorrie , Sørensen Søren J. , Madsen Jonas Stenløkke
Published: March 2022

Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e.g., fluorescent proteins) or antibiotic resistance genes would be efficient and convenient. For that, we introduced a single attTn7 site into the conjugative broad-host-range IncP-1 plasmid pKJK5 in a non-disruptive manner. Furthermore, a version with lower transfer rate and a non-conjugative version of pKJK5-attTn7 were also constructed. The advantage of having the attTn7 sites is that genes of interest can be introduced in a single step with very high success rate using the Tn7 transposition system. In addition, larger genetic fragments can be inserted. To illustrate the efficacy of the constructed pKJK5 plasmids, they were complimented with sfgfp in addition to seven different β-lactamase genes representing the four known classes of β-lactamases.
Clinical application of tumour-in-normal contamination assessment from whole genome sequencing
Authors: Mitchell Jonathan , Milite Salvatore , Bartram Jack , Walker Susan , Volkova Nadezda , Yavorska Olena , Zarowiecki Magdalena , Chalker Jane , Thomas Rebecca , Vago Luca , Sosinsky Alona , Caravagna Giulio
Published: March 2022

The unexpected contamination of normal samples with tumour cells reduces variant detection sensitivity, compromising downstream analyses in canonical tumour-normal analyses. Leveraging whole-genome sequencing data available at Genomics England, we develop a tool for normal sample contamination assessment, which we validate in silico and against minimal residual disease testing. From a systematic review of 771 patients with haematological malignancies and sarcomas, we find contamination across a range of cancer clinical indications and DNA sources, with highest prevalence in saliva samples from acute myeloid leukaemia patients, and sorted CD3+ T-cells from myeloproliferative neoplasms. Further exploration reveals 108 hotspot mutations in genes associated with haematological cancers at risk of being subtracted by standard variant calling pipelines. Our work highlights the importance of contamination assessment for accurate somatic variants detection in research and clinical settings, especially with large-scale sequencing projects being utilised to deliver accurate data from which to make clinical decisions for patient care.
The lateral intraparietal area preferentially supports stimulus selection in directed tasks compared to undirected free behavior
Authors: Johnston W. Jeffrey , Tetrick Stephanie M. , Freedman David J.
Published: March 2022

Our reactions to the sensory world depend on context. For instance, explicit directions to search for a particular object or feature will induce a different treatment of the sensory world than exploration without a fixed goal. To understand how we navigate the sensory world, it is necessary to understand how both directed search and undirected exploration are produced by the brain. The lateral intraparietal area (LIP) in the posterior parietal cortex has an established role in visual stimulus selection. However, most studies of LIP’s role in stimulus selection focus primarily on highly trained, directed tasks, in which animals are given explicit cues as to which stimulus they should select. Here, we compare neural activity in LIP across two tasks. In one task, the animal is given an explicit direction to select one of two natural images from an array; in the other, the animal is allowed to choose an image freely based on their innate preferences. We find that LIP reliably encodes the eye movement prior to its execution only in the directed task, while the eye movement encoding in the undirected task emerges significantly later. Further, LIP’s encoding of the image’s behavioral relevance emerges after the decision in both tasks. These results indicate that LIP preferentially supports stimulus selection in highly trained and directed behaviors, as opposed to free behavior.
Floating EMG Sensors and Stimulators Wirelessly Powered and Operated by Volume Conduction for Networked Neuroprosthetics
Authors: Becerra-Fajardo Laura , Krob Marc Oliver , Minguillon Jesus , Rodrigues Camila , Welsch Christine , Tudela-Pi Marc , Comerma Albert , Oliveira Barroso Filipe , Schneider Andreas , Ivorra Antoni
Published: March 2022

Background

Implantable neuroprostheses consisting of a central electronic unit wired to electrodes benefit thousands of patients worldwide. However, they present limitations that restrict their use. Those limitations, which are more adverse in motor neuroprostheses, mostly arise from their bulkiness and the need to perform complex surgical implantation procedures. Alternatively, it has been proposed the development of distributed networks of intramuscular wireless microsensors and microstimulators that communicate with external systems for analyzing neuromuscular activity and performing stimulation or controlling external devices. This paradigm requires the development of miniaturized implants that can be wirelessly powered and operated by an external system. To accomplish this, we propose a wireless power transfer (WPT) and communications approach based on volume conduction of innocuous high frequency (HF) current bursts. The currents are applied through external textile electrodes and are collected by the wireless devices through two electrodes for powering and bidirectional digital communications. As these devices do not require bulky components for obtaining power, they may have a flexible threadlike conformation, facilitating deep implantation by injection.

Methods

We report the design and evaluation of advanced prototypes based on the above approach. The system consists of an external unit, floating semi-implantable devices for sensing and stimulation, and a bidirectional communications protocol. The devices are intended for their future use in acute human trials to demonstrate the distributed paradigm. The technology is assayed in vitro using an agar phantom, and in vivo in hindlimbs of anesthetized rabbits.

Results

The semi-implantable devices were able to power and bidirectionally communicate with the external unit. Using 13 commands modulated in innocuous 3 MHz HF current bursts, the external unit configured the sensing and stimulation parameters, and controlled their execution. Raw EMG was successfully acquired by the wireless devices at 1 ksps.

Conclusions

The demonstrated approach overcomes key limitations of existing neuroprostheses, paving the way to the development of distributed flexible threadlike sensors and stimulators. To the best of our knowledge, these devices are the first based on WPT by volume conduction that can work as EMG sensors and as electrical stimulators in a network of wireless devices.
Nucleocapsid-specific humoral responses improve the control of SARS-CoV-2
Authors: Dangi Tanushree , Sanchez Sarah , Park Mincheol , Class Jacob , Richner Michelle , Richner Justin M. , Penaloza-MacMaster Pablo
Published: March 2022

The spike protein of SARS-CoV-2 is a critical antigen present in all approved SARS-CoV-2 vaccines. This surface viral protein is also the target for all monoclonal antibody therapies, but it is unclear whether antibodies targeting other viral proteins can also improve protection against COVID-19. Here, we interrogate whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine, and then transferred sera from these mice into naïve mice. On the next day, the recipient mice were challenged intranasally with SARS-CoV-2 to evaluate whether nucleocapsid-specific humoral responses affect viral control. Interestingly, mice that received nucleocapsid-specific sera exhibited enhanced control of a SARS-CoV-2 infection. These findings provide the first demonstration that humoral responses specific to an internal coronavirus protein can help clear infection, warranting the inclusion of other viral antigens in next-generation SARS-CoV-2 vaccines and providing a rationale for the clinical evaluation of nucleocapsid-specific monoclonals to treat COVID-19.

Highlights

A SARS-CoV-2 nucleocapsid vaccine elicits robust nucleocapsid-specific antibody responses.

This nucleocapsid vaccine generates memory B cells (MBC).

Nucleocapsid-specific humoral responses do not prevent SARS-CoV-2 infection.

Nucleocapsid-specific humoral responses help control a SARS-CoV-2 infection.
Profibrotic priming of airway cell types and drug responses in early-stage idiopathic pulmonary fibrosis
Authors: Chua Robert Lorenz , Veith Carmen , Schneider Marc A. , Jechow Katharina , Xu Elizabeth Chang , Kreuter Michael , Boots Agnes W. , Eils Roland , Kahn Nicolas C. , Conrad Christian
Published: March 2022

Early genetic studies hinted the role of airway epithelial cells in the development of idiopathic pulmonary fibrosis (IPF), while recent single-cell RNA sequencing (scRNA-seq) atlases utilized explant IPF lungs and therefore represent late-stage disease. Here, we used air liquid interface (ALI) cultures of primary cells taken from the subsegmental bronchi of newly diagnosed IPF patients, reflecting early-stage fibrosis, to interrogate the transcriptional landscape of the airway mucosa. Profiling of 129,986 cells identified a shared proinflammatory state in epithelial cells and an early activation state of fibroblasts. Moreover, IPF basal cells initiated awry repair mechanisms and primed the airway mucosa for TGF-β activation. Treatment with nintedanib, pirfenidone, both established antifibrotic drugs, and saracatinib, an Src kinase inhibitor that can limit IPF progression, only significantly affected certain IPF signatures. This study provides insight into the early disease mechanisms of IPF and may serve as a resource to further investigate pharmacological inhibition effects.