bluuebunny/crossref_metadata_2025 huggingface dataset:
Unique functions of two overlappingPAX6retinal enhancers
Authors: Uttley Kirsty , Papanastasiou Andrew S. , Lahne Manuela , Brisbane Jennifer M. , MacDonald Ryan B. , Bickmore Wendy A. , Bhatia Shipra
Published: November 2022

Enhancers play a critical role in development by precisely modulating spatial, temporal, and cell type-specific gene expression. Sequence variants in enhancers have been implicated in disease, however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active.PAX6is the master regulator of eye development, and has a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive, redundant, or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of twoPAX6retinal enhancers, HS5 and NRE. Using a unique combination of live imaging and single-cell RNA sequencing in dual enhancer-reporter zebrafish embryos, we find significant differences in the spatiotemporal activity of these enhancers, and show that HS5 and NRE are active in distinct cell types of the developing retina. Our results show that although overlapping, these enhancers have distinct activities in different cell types and therefore likely non-redundant functions. This work demonstrates that unique cell type-specific functions can be uncovered for apparently similar enhancers when investigated at high resolution in vivo.
Resilient CD8+T cells maintain a high cytotoxic capacity by balancing ROS via ME1 upregulation
Authors: Gicobi Joanina K. , Mao Zhiming , DeFranco Grace , Li Ying , Liu Xin , Hirdler Jacob B. , Vianzon Vianca V. , Dellacecca Emilia R. , Hsu Michelle A. , Barham Whitney , Kim Yohan , Abraha Feven , Harmsen William S. , Yan Yiyi , Dronca Roxana S. , Zhu Mojun , Markovic Svetomir N. , Mansfield Aaron S. , Lin Yi , Wu Xiaosheng , Owen Dawn , Grams Michael P. , Orme Jacob J. , Lucien Fabrice , Zeng Hu , Park Sean S. , Dong Haidong
Published: November 2022

Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignances. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+CD8+T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+CD8+T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysisindependent manner while concurrently curtailing excessive ROS in activated CD8+T cells; and expands CX3CR1+NKG7+effector CD8+T cells with enhanced cytotoxicity. Importantly, transfection ofME1mRNA promotes tumoricidal activity in CD8+T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion.

Graphical abstract

Highlights

CX3CR1+and low Dy m identify functional resilient CD8+T cells.

Resilient CD8+T cells are highly cytotoxic and have less ROS.

Resilient CD8+T cells express more ME1 that can balance extra ROS.

ME1 overexpression can promote CTL function of CD8+T cells.
Evolution of Secondary Cell Number and Position In the Drosophila Accessory Gland
Authors: Takashima Yoko A. , Majane Alex C. , Begun David J.
Published: November 2022

In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. InDrosophilaand other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. TheD. melanogasterAG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in theDrosophilaAG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. TheDrosophilaAG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of theD. melanogasterAG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies ofDrosophilaAG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. Here, we investigate the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of theDrosophilagenus. We document a heretofore underappreciated rapid evolutionary rate for both number and position of these specialized AG cells, raising many interesting questions about the developmental, functional, and evolutionary significance of this variation.
Analysis of hormonal regulation of promoter activities ofCannabis sativaprenyltransferase 1 and 4 and salicylic acid mediated regulation of cannabinoid biosynthesis
Authors: Sands Lauren B. , Haiden Samuel R. , Ma Yi , Berkowitz Gerald A.
Published: November 2022

Cannabis sativaprenyltransferase 4 (CsPT4) and prenyltransferase 1 (CsPT1) have been shown to catalyze the step in the cannabinoid biosynthetic pathway that generates cannabigerolic acid (CBGA), the substrate for the end-point enzymes that generate cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA). Prior studies from our lab suggest that CBGA production rate-limits the pathway. There is a lack of understanding concerning how important cannabinoid biosynthetic genes are regulated as cannabinoid synthesis increases during female flower development. BothCsPTgenes were shown to be highly expressed in flowers. The genes were also found to be present in leaves and roots. GUS staining also detected the promoter activities in leaves of seedlings, and the promoter activities were drastically stronger in the section of the sugar leaves where glandular trichomes are formed.In silicoanalysis of the twoCsPTgenes revealed several hormone and transcription factor responsive elements. Dual luciferase assays were conducted to determine whether a hormone could alter the promoter activities ofCsPT1andCsPT4. The results showed thatCsPT4pro was activated following treatment from salicylic acid (SA), gibberellic acid (GA), ethylene, ABA, and cytokinin, while theCsPT1promoter was activated following SA, ethylene, ABA, and auxin treatment. In parallel studies, a correlation was observed between multiple cannabinoid biosynthetic pathway genes and SA application to the cannabis growing medium, along with a correlation between MeSA floral application and an increase in cannabinoid content. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis.
Topological transitions, turbulent-like motion and long-time-tails driven by cell division in biological tissues
Authors: Li Xin , Sinha Sumit , Kirkpatrick T. R. , Thirumalai D.
Published: November 2022

The complex spatiotemporal flow patterns in living tissues, driven by active forces, have many of the characteristics associated with inertial turbulence even though the Reynolds number is extremely low. Analyses of experimental data from two-dimensional epithelial monolayers in combination with agent-based simulations show that cell division and apoptosis lead to directed cell motion for hours, resulting in rapid topological transitions in neighboring cells. These transitions in turn generate both long ranged and long lived clockwise and anticlockwise vortices, which gives rise to turbulent-like flows. Both experiments and simulations show that at long wavelengths the wave vector (k) dependent energy spectrumE(k) ≈k−5/3, coinciding with the Kolmogorov scaling in fully developed inertial turbulence. Using theoretical arguments and simulations, we show that long-lived vortices lead to long-time tails in the velocity auto-correlation function,Cv(t) ∼t−1/2, which has the same structure as in classical 2D fluids but with a different scaling exponent.
A primary microcephaly-associatedsas-6mutation perturbs centrosome duplication, dendrite morphogenesis, and ciliogenesis inCaenorhabditis elegans
Authors: Bergwell Mary , Smith Amy , Smith Ellie , Dierlam Carter , Duran Ramon , Haastrup Erin , Napier-Jameson Rebekah , Seidel Rory , Potter William , Norris Adam , Iyer Jyoti
Published: November 2022

The human SASS6(I62T) missense mutation has been linked with the incidence of primary microcephaly in a Pakistani family, although the mechanisms by which this mutation causes disease remain unclear. The SASS6(I62T) mutation corresponds to SAS-6(L69T) inC. elegans. Given that SAS-6 is highly conserved, we modeled this mutation inC. elegansand examinedsas-6(L69T)effect on centrosome duplication, ciliogenesis and dendrite morphogenesis. Our studies revealed that all the above processes are perturbed by thesas-6(L69T)mutation. Specifically,C. eleganscarrying thesas-6(L69T)mutation exhibit an increased failure of centrosome duplication in a sensitized genetic background. Further, worms carrying this mutation also display shortened phasmid cilia, an abnormal phasmid cilia morphology, shorter phasmid dendrites and chemotaxis defects. Our data show that the centrosome duplication defects caused by this mutation are only uncovered in a sensitized genetic background, indicating that these defects are mild. However, the ciliogenesis and dendritic defects caused by this mutation are evident in an otherwise wild-type background, indicating that they are stronger defects. Thus, our studies shed light on the novel mechanisms by which thesas-6(L69T)mutation could contribute to the incidence of primary microcephaly in humans.
Centromere-associated retroelement evolution inDrosophila melanogasterreveals an underlying conflict
Authors: Hemmer Lucas W. , Negm Sherif , Geng Xuewen , Courret Cécile , Navarro-Domínguez Beatriz , Speece Iain , Wei Xiaolu , Altidor Eddyson , Chaffer James , Sproul John S. , Larracuente Amanda M.
Published: November 2022

Centromeres are chromosomal regions essential for coordinating chromosome segregation during cell division. While centromeres are defined by the presence of a centromere-specific histone H3 variant rather than a particular DNA sequence, they are typically embedded in repeat-dense chromosomal genome regions. In many species, centromeres are associated with transposable elements, but it is unclear if these elements are selfish or if they play a role in centromere specification or function. Here we useDrosophila melanogasteras a model to understand the evolution of centromere-associated transposable elements.G2/Jockey-3is a non-LTR retroelement in theJockeyclade and the only sequence shared by all centromeres. We study the evolution ofG2/Jockey-3using short and long read population genomic data to infer insertion polymorphisms across the genome. We combine estimates of the age, frequency, and location of insertions to infer the evolutionary processes shapingG2/Jockey-3and its association with the centromeres. We find thatG2/Jockey-3is an active retroelement targeted by the piRNA pathway that is enriched in centromeres at least in part due to an insertion bias. We do not detect signatures of positive selection on anyG2/Jockey-3insertions that would suggest than individual copies are favored by natural selection. Instead, we infer that most insertions are neutral or weakly deleterious both inside and outside of the centromeres. Therefore,G2/Jockey-3evolution is consistent with it being a selfish genetic element that targets centromeres. We propose that targeting centromeres helps active retroelements escape host defenses, as the unique centromeric chromatin may prevent targeting by the host silencing machinery. At the same time, centromeric TEs insertions may be tolerated or even beneficial if they also contribute to the transcriptional and chromatin environment. Thus, we suspect centromere-associated retroelements likeG2/Jockey-3reflect a balance between conflict and cooperation at the centromeres.
Quantitative assessment of angioplasty induced vascular inflammation with19F cardiovascular magnetic resonance imaging
Authors: Nienhaus Fabian , Walz Moritz , Rothe Maik , Jahn Annika , Pfeiler Susanne , Busch Lucas , Stern Manuel , Heiss Christian , Vornholz Lilian , Cames Sandra , Cramer Mareike , Schrauwen-Hinderling Vera , Gerdes Norbert , Temme Sebastian , Roden Michael , Flögel Ulrich , Kelm Malte , Bönner Florian
Published: November 2022

Early macrophage rich vascular inflammation is a key feature in the pathophysiology of restenosis after angioplasty.19F MRI with intravenously applied perfluorooctyl bromide-nanoemulsion (PFOB-NE) could offer ideal features for serial imaging of the inflammatory response after angioplasty. We aimed to non-invasively image monocyte/macrophage infiltration in response to angioplasty in pig carotid arteries using Fluorine-19 magnetic resonance imaging (19F MRI) to assess early inflammatory response to mechanical injury. Early macrophage rich vascular inflammation is a key feature in the pathophysiology of restenosis after angioplasty.19F MRI with intravenously applied perfluorooctyl bromide-nanoemulsion (PFOB-NE) could offer ideal features for serial imaging of the inflammatory response after angioplasty. In eight minipigs, injury of the right carotid artery was induced by either balloon oversize angioplasty only (BA, n=4) or in combination with endothelial denudation (BA + ECDN, n=4). PFOB-NE was administered intravenously three days after injury followed by1H and19F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using immunohistology. Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and adventitial infiltration of monocytes/macrophages.19F signal could be detectedin vivoin four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 ± 4.8.Ex vivoanalysis revealed a linear correlation of19F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was as about 400 cells/mm2. Therefore,19F MRI enables quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This might open an avenue to non-invasively monitor inflammatory response to mechanical injury after angioplasty and thus to identify individuals with distinct patterns of vascular inflammation promoting restenosis.

One Sentence Summary

19F MRI enables radiation-free quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity.
Proteomic Landscape of Human Spermatozoa: Optimized Extraction Method and Application
Authors: Luo Mengqi , Su Tao , Wang Shisheng , Chen Jianhai , Lin Tianhai , Cheng Qingyuan , Chen Younan , Gong Meng , Yang Hao , Li Fuping , Zhang Yong
Published: November 2022

Human spermatozoa proteomics exposed to some physical, biological or chemical stressors is being explored. However, there is a lack of optimized sample preparation methods to achieve in-depth protein coverage for sperm cells. Meanwhile, it is not clear whether antibiotics can regulate proteins to affect sperm quality. Here, we systematically compared a total of six different protein extraction methods based the combination of three commonly used lysis buffers and physical lysis strategies. The urea buffer combined with ultrasonication (UA_ultrasonication) produced the highest protein extraction rate, leading to the deepest coverage of human sperm proteome (5685 protein groups) from healthy human sperm samples. Since the antibiotics, amoxicillin and clarithromycin, have been widely used againstH. pyloriinfection, we conduct a longitudinal study of sperm proteome via data-independent acquisition tandem mass spectrometry (DIA-MS/MS) on an infected patient during on and off therapy with these two drugs. The semen examination and morphological analysis were performed combined with proteomics analysis. Our results indicated that antibiotics may cause an increase in the sperm concentration and the rate of malformed sperm and disrupt proteome expression in sperm. This work provides an optimized extraction method to characterize the in-depth human sperm proteome and to extend its clinical applications.
A conditional histone H3.3 mutation in mice orthologous to a driver of paediatric diffuse instrinsic pontine glioma
Authors: Mann Jeffrey R. , Tang Michelle C. W. , Wong Lee H.
Published: November 2022

Most paediatric diffuse intrinsic pontine gliomas carry a lysine- to methionine-27 (K27M) mutation in the histone variant H3.3 encoded by the ‘H3.3 histone A’ (H3F3A) gene. To establish a pre-clinical model of the disease we made a Cre/loxPconditional H3.3K27M mutation at the orthologousH3f3alocus in mice. Importantly, expression of the mutant transcript is under endogenousH3f3aregulatory control. This system is distinct from others in which H3.3M27 is ectopically expressed, thereby providing a resource for the development of pDIPG models with orthologous regulation of mutant H3.3. Mice in which expression of the mutant transcript was induced with a nestin-cretransgene developed as dwarfs in the presence of intact growth hormone signaling.
Structural and dynamical basis for the interaction of HSP70-EEVD with JDP Sis1
Authors: Matos Carolina O. , Pinheiro Glaucia M.S. , Caruso Icaro P. , Amorim Gisele C. , Almeida Fabio C. L. , Ramos Carlos H. I.
Published: November 2022

We employed NMR spectroscopy to investigate the structure and dynamics of the class B J domain protein (JDP) of S. cerevisiae (Sis1) complexed with an EEVD peptide of HSP70. It is widely recognized that the interactions between the EEVD motif and Sis1 play a crucial role in the chaperone activity. Notably, the deletion of the EEVD impairs the ability of Sis1 to bind with HSP70, while leaving the interaction between the class A JDP Ydj1 and HSP70 unaffected. Leveraging the advantages of NMR, which is particularly suitable for studying transient interactions, we provide compelling evidence that the EEVD motif transiently engages multiple sites on Sis1. Our findings revealed that EEVD binds to two distinct sites within the C-terminal domain I (CTDI) of Sis1. The interaction at these sites plays a crucial role in anchoring HSP70 to Sis1 at site I, as well as displacing the client protein at site II. Notably, site II is also the binding site for the client protein, and its displacement occurs through competition with the binding to site II. In addition to these interactions, we observed that EEVD, as a transient electrostatic binder, also interacts with the J domain and the GF-rich loop located between the J domain and α-helix 6. We propose that the interaction between EEVD and Sis1 facilitates the dissociation of α-helix 6, promoting a conformational state that is more favorable for interaction with HSP70 at the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) interface. We also employed α-synuclein as a substrate to investigate the competitive nature between EEVD and the client protein. Our experimental findings provided evidence supporting the interaction of EEVD with the client protein at multiple sites. Our findings contribute essential insights into the mechanistic cycle of class B JDPs, paving the way toward a more complete understanding of the primary function of Sis1, which is the transfer of the client protein to HSP70, where multiple site transient interactions play a collective role.
Effects of Plastic Ingestion on Blood Chemistry, Gene Expression and Body Condition in Wedge-Tailed Shearwaters (Ardenna Pacifica)
Authors: Mejia Nicole , Garcia Flavia Termignoni , Learned Jennifer , Penniman Jay , Edwards Scott V.
Published: November 2022

Plastic pollution is a global threat and affects almost every marine ecosystem. The amount of plastic in the ocean has increased substantially over the past decade, posing a mounting threat to biodiversity. Seabirds, typically top predators in marine food chains, have been negatively affected by plastic pollution. Here we focused on documenting the sublethal effects of plastic in Wedge-tailed Shearwaters (Ardenna pacifica, WTSH) on the island of Maui, Hawai’i. Through analyses of blood chemistry, gene expression, morphometrics and stomach contents, we documented the effects of plastic ingestion on adult WTHS from 3 established colonies. We detected a negative relationship between body weight and the presence of plastic in regurgitated stomach contents. Genes associated with metabolic, biosynthetic pathways, inflammatory responses and ribosome function were upregulated in lighter birds. Birds that had ingested plastic tended to be lighter in weight, in comparison to birds that did not have plastic and tended to weight more. Furthermore, there were 43 genes differentiating males and females that did not have plastic compared to only 11 genes differentiating males and females that had ingested plastic. There was also a marginal negative relationship between lighter birds and blood urea nitrogen levels. We also hope that the morphometric measurements, blood parameters and gene expression data we collected contributes to a database that will be used for future studies on understanding anthropogenic effects on seabird body condition.
The endocytic recycling pathway is controlled by the ADP-ribosylated GTPase Rab14
Authors: Corteggio Annunziata , Lo Monte Matteo , Schembri Laura , Dathan Nina , Di Paola Simone , Grimaldi Giovanna , Corda Daniela
Published: November 2022

The GTPase Rab14 is localized at the trans-Golgi network and at the intermediate compartment associated to sorting/recycling endosomes-like structures of the transferrin-recycling pathway: as other Rab family members, it is involved in the regulation of intracellular vesicle trafficking, though its role and functional relationship with effector/endosomal proteins is still incomplete.

We have analysed whether post-translational modifications could affect Rab14 activity: the results obtained define mono-ADP-ribosylation (MARylation) as the yet-unknown Rab14 modification, catalysed by the ADP-ribosyltransferase PARP12, which specifically modifies glutamic acid residues in position 159/162. This modification is essential for the Rab14-dependent endosome progression. Accordingly, recycling of the transferrin receptor is inhibited when MARylation of Rab14 is prevented by PARP12 knocking-down or inhibition, or by overexpression of Rab14 ADP-ribosylation-defective mutant. Under these conditions, Rab14 and transferrin receptors are withheld at the cell periphery at the level of the Rab4-RUFY1-positive sorting endosomes, indicating that the interaction of Rab14 with the dual effectors RUFY and then FIP1c (which specifically binds both Rab11 and Rab14) determines the progression between the Rab4-RUFY- and Rab11-FIP1c-specific vesicles. Therefore Rab14-MARylation determines the sequential binding of this GTPase to RUFY and FIP1c, thus controlling endosome progression (i.e., transferrin receptors recycling) through the Rab4-, Rab14- and Rab11-specific vesicles. This identifies a Rab14-specific compartment of the recycling pathway and a crucial enzymatic reaction amenable to pharmacological control.
A deep neural network provides an ultraprecise multi-tissue transcriptomic clock for the short-lived fishNothobranchius furzeriand identifies predicitive genes translatable to human aging
Authors: Ferrari Elisa , Reichwald Kathrin , Koch Philipp , Groth Marco , Baumgart Mario , Cellerino Alessandro
Published: November 2022

A key and unresolved question in aging research is how to quantify aging at the individual level that led to development of ”aging clocks”, machine learning algorhythms trained to predict individual age from high-dimensional molecular data under the the assumption that individual deviations of the predicted age from the chronological age contain information on the individual condition (often referred to as ”biological age”). A full validation of such clocks as biomarkers for clinical studies of ageing would require a comparison of their predictions with information on actual lifespan and long-term health. Such studies take decades in humans, but could be conducted in a much shorter time-frame in animal models. We developed a transcriptomic clock in the turquoise killifishNothobranchius furzeri. This species is the shortest-lived vertebrate that can be cultured in captivity and is an emerging model organism for genetic and experimental studies on aging. We developed a proprietary deep learning architecture that autonomously selects a customizable number of input genes to use for its predictions in order to reduce overfitting and increase interpretability, and adopts an adversarial learning framework to identify tissue-independent transcriptional patterns. We called this architecture the Selective Adversarial Deep Neural Network (SA-DNN) and trained it on a multi-tissue transcriptomic dataset ofN. furzeri. This SA-DNN predicted age of the test set with an accuracy of 1 day, i.e. less than 1% of the total species’ lifespan and detected genetic, pharmacological and environmental interventions that are known to influence lifespan in this species. Finally, a human transcriptomic multi-tissue clock that uses as input the orthologs of the genes selected by our SA-DNN inN. furzerireaches an average error of∼3 years rivalling epigenetic clocks. Our SA-DNN represents the prototype of a new class of aging clocks that provide biomarkers applicable to intervention studies in model organisms and humans.
CRISPR correction ofGBAmutation in hiPSCs restores normal function to Gaucher macrophages and increases their susceptibility toMycobacterium tuberculosis
Authors: Ramalingam Sivaprakash , Kumar Amit , Krug Stefanie , Mohan Harikrishnan , Rao Desirazu N , Bishai William R , Chandrasegaran Srinivasan
Published: November 2022

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase (GCase)GBAgene, which result in macrophage dysfunction. To investigate whether correction ofGBAmutations restores normal function to Gaucher macrophages, we performed CRISPR editing of homozygous L444P (1448T→C)GBAmutation in Type 2 GD (GBA-/-) hiPSCs, which yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived fromGBA-/-,GBA+/- andGBA+/+ hiPSCs, were compared for GCase enzymatic activity, motility, and phagocytosis, all of which showed thatGBAmutation correction restores normal macrophage functions. Furthermore, we investigated whether lysosomal disorders drive susceptibility toMycobacterium tuberculosis, by infectingGBA-/-,GBA+/- andGBA+/+ macrophages with the virulent H37Rv lab strain. The results showed that impaired mobility and phagocytic activity of Gaucher macrophages, correlated with reduced levels of TB engulfment and TB multiplication, supporting the hypothesis that GD may be protective against tuberculosis.
Kinetochore-fiber lengths are maintained locally but coordinated globally by poles in the mammalian spindle
Authors: Richter Manuela , Neahring Lila , Tao Jinghui , Sutanto Renaldo , Cho Nathan H. , Dumont Sophie
Published: November 2022

At each cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers attach to chromosomes and focus into spindle poles. Despite evidence suggesting that poles can set spindle length, their role remains poorly understood. In fact, many species do not have spindle poles. Here, we probe the pole’s contribution to mammalian spindle length, dynamics, and function by inhibiting dynein to generate spindles whose kinetochore-fibers do not focus into poles, yet maintain a metaphase steady-state length. We find that unfocused kinetochore-fibers have a mean length indistinguishable from control, but a broader length distribution, and reduced length coordination between sisters and neighbors. Further, we show that unfocused kinetochore-fibers, like control, can grow back to their steady-state length if acutely shortened by drug treatment or laser ablation: they recover their length by tuning their end dynamics, albeit slower due to their reduced baseline dynamics. Thus, kinetochore-fiber dynamics are regulated by their length, not just pole-focusing forces. Finally, we show that spindles with unfocused kinetochore-fibers can segregate chromosomes but fail to correctly do so. We propose that mammalian spindle length emerges locally from individual k-fibers while spindle poles globally coordinate k-fibers across space and time.
Novel regulators of islet function identified from genetic variation in mouse islet Ca2+oscillations
Authors: Emfinger Christopher H. , Clark Lauren E. , Yandell Brian , Schueler Kathryn L. , Simonett Shane P. , Stapleton Donnie S. , Mitok Kelly A. , Merrins Matthew J. , Keller Mark P. , Attie Alan D.
Published: November 2022

Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca2+into β-cells triggers insulin release. Since genetics strongly influences variation in islet secretory responses, we surveyed islet Ca2+dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: 1) 8 mM glucose; 2) 8 mM glucose plus amino acids; 3) 8 mM glucose, amino acids, plus 10 nM GIP; and 4) 2 mM glucose. These stimuli interrogate β-cell function, α-cell to β-cell signaling, and incretin responses. We then correlated components of the Ca2+waveforms to islet protein abundances in the same strains used for the Ca2+measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated SNPs in human GWAS. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominated novel regulators of islet Ca2+oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.
[SNG2], a prion-form of Cut4/ Apc1, enforces non-Mendelian inheritance of heterochromatin silencing defect in Fission Yeast
Authors: Srivastava Suchita , Dubey Rudra Narayan , Shukla Poonam , Singh Jagmohan
Published: November 2022

Prions represent epigenetic regulator proteins that can self-propagate their structure and confer their misfolded structure and function on normally folded proteins. Like the mammalian prion PrPSc, prions also occur in fungi. While a few prions, like Swi1, affect gene expression, none are shown to affect heterochromatin structure and function. In fission yeast and metazoans, histone methyltransferase Clr4/Suv39 causes H3-Lys9 methylation, which is bound by the chromodomain protein Swi6/HP1 to assemble heterochromatin. Earlier, we showed thatsng2-1mutation in the Cut4 subunit of Anaphase Promoting Complex abrogates heterochromatin structure due to defective binding and recruitment of Swi6. Here, we demonstrate that the Cut4p forms a non-canonical prion form, designated as [SNG2], which abrogates heterochromatin silencing. [SNG2] exhibits various prion-like properties, e.g., non-Mendelian inheritance, requirement of Hsp proteins for its propagation,de novogeneration uponcut4overexpression, reversible curing by guanidine, cytoplasmic inheritance and formation of infectious protein aggregates, which are converted into monomers upon overexpression ofhspgenes. Interestingly, [SNG2] prion imparts an enhanced tolerance to stress conditions, supporting its role in promoting cell survival under environmental stress during evolution.
scRNAseq of healthy and irradiated mouse parotid glands highlights crosstalk between immune and secretory cells during chronic injury
Authors: Rheinheimer Brenna A. , Pasquale Mary C. ,   NIDCD/NIDCR Genomics and Computational Biology Core, Limesand Kirsten H. , Hoffman Matthew P. , Chibly Alejandro M
Published: November 2022

Translational frameworks to understand the chronic loss of salivary dysfunction that follows after clinical irradiation, and the development of regenerative therapies remain an unmet clinical need. Understanding the transcriptional landscape long after irradiation treatment that results in chronic salivary hypofunction will help identify injury mechanisms and develop regenerative therapies to address this need. Advances in single cell (sc)RNAseq have made it possible to identify previously uncharacterized cell types within tissues and to uncover gene regulatory networks that mediate cell-cell communication and drive specific cell states. scRNAseq studies have been performed for virtually all major tissues including salivary glands; however, there are currently no scRNAseq studies evaluating the long-term chronic effects of irradiation on salivary glands. Here, we present scRNAseq from control and irradiated murine parotid glands collected 10 months post-irradiation. We identify a population of epithelial cells in the gland defined by expression ofEtv1, which may be an acinar cell precursor. These Etv1+ cells also expressNtrk2andErbb3and thus may respond to myoepithelial cell-derived growth factor ligands. Furthermore, our data suggests that CD4+CD8+ T-cells and secretory cells are the most transcriptionally affected during chronic injury with radiation, suggesting active immune involvement during chronic injury post-irradiation. Thus, our study provides a resource to understand the transcriptional landscape in a chronic post-irradiation microenvironment and identifies cell-specific pathways that may be targeted to repair chronic damage.

Highlights

We generated a scRNAseq dataset of chronic irradiation injury in parotid glands

A newly identifiedEtv1+ epithelial population may be acinar precursors

Ntrk2 and Erbb3 are highly specific Etv1+ cell receptors that may mediate cell-cell communication with myoepithelial cells

CD8+ T-cells and secretory acinar cells have the greatest transcriptional changes post-IR
Cell type-specific attenuation of brassinosteroid signalling drives stomatal asymmetric cell division
Authors: Kim Eun-Ji , Zhang Cheng , Guo Boyu , Eekhout Thomas , Houbaert Anaxi , Wendrich Jos R. , Vandamme Niels , Tiwari Manish , Simon--Vezo Claire , Vanhoutte Isabelle , Saeys Yvan , Wang Kun , Zhu Yuxian , De Rybel Bert , Russinova Eugenia
Published: November 2022

InArabidopsis thaliana, the negative brassinosteroid (BR) signalling regulator, BR INSENSITIVE2 (BIN2) promotes and restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffolding protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages with either exogenous BRs or the specific BIN2 inhibitor revealed that the two modes of BR activation triggered spatiotemporally distinct transcriptional responses. We established that when in a complex with POLAR and its closest homolog POLAR-LIKE1, BIN2 is insulated from BR-mediated inactivation, nevertheless, it remains sensitive to the inhibitor. Subsequently, BR signalling is attenuated in ACD precursors, whereas it remains active in epidermal cells that would undergo differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.
Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein during translation regulation
Authors: Hollmann Nele Merret , Ankush Jagtap Pravin Kumar , Linse Johanna-Barbara , Ullmann Philip , Payr Marco , Murciano Brice , Simon Bernd , Hub Jochen S. , Hennig Janosch
Published: November 2022

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical π-π-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). This provides first atomistic details towards understanding regulation of translation initiation that is mediated by the interplay of these two proteins with each other and RNA.
Inhibitory effect of lactobacilli supernatants on biofilm and filamentation ofC. albicans, C. tropicalis, andC. parapsilosis
Authors: Poon Yeuklan , Hui Mamie
Published: November 2022

ProbioticLactobacillusstrains had been investigated for the potential to protect against infection caused by the major fungal pathogen of human,Candida albicans. Besides antifungal activity, lactobacilli demonstrated a promising inhibitory effect on biofilm formation and filamentation ofC. albicans. On the other hand, two commonly isolated non-albicansCandidaspecies,C. tropicalisandC. parapsilosis, have similar characteristics in filamentation and biofilm formation withC. albicans. However, there is scant information of the effect of lactobacilli on the two species. In this study, biofilm inhibitory effects ofL. rhamnosusATCC 53103,L. plantarumATCC 8014, andL. acidophilusATCC 4356 were tested on the reference strainC. albicansSC5314 and six bloodstream isolated clinical strains, two each ofC. albicans, C. tropicalis, andC. parapsilosis. Cell-free culture supernatants (CFSs) ofL. rhamnosusandL. plantarumsignificantly inhibitedin vitrobiofilm growth ofC. albicansandC. tropicalis. L. acidophilus, conversely, had little effect onC. albicansandC. tropicalisbut was more effective on inhibitingC. parapsilosisbiofilms. NeutralizedL. rhamnosusCFS at pH 7 retained the inhibitory effect, suggesting that exometabolites other than lactic acid produced by theLactobacillusstrain might be accounted for the effect. Furthermore, we evaluated the inhibitory effects ofL. rhamnosusandL. plantarumCFSs on the filamentation ofC. albicansandC. tropicalisstrains. Significantly lessCandidafilaments were observed after co-incubating with CFSs under hyphae-inducing conditions. Expressions of six biofilm-related genes (ALS1, ALS3, BCR1, EFG1, TEC1, andUME6inC. albicansand corresponding orthologs inC. tropicalis) in biofilms co-incubated with CFSs were analyzed using quantitative real-time PCR. When compared to untreated control, the expressions ofALS1, ALS3, EFG1, andTEC1genes were downregulated inC. albicansbiofilm. InC. tropicalisbiofilms,ALS3andUME6were downregulated whileTEC1was upregulated. Taken together, theL. rhamnosusandL. plantarumstrains demonstrated an inhibitory effect, which is likely mediated by the metabolites secreted into culture medium, on filamentation and biofilm formation ofC. albicansandC. tropicalis. Our finding suggested an alternative to antifungals for controllingCandidabiofilm.
Transcriptional analysis in multiple barley varieties identifies signatures of waterlogging response
Authors: Miricescu Alexandra , Brazel Ailbhe Jane , Beegan Joseph , Wellmer Frank , Graciet Emmanuelle
Published: November 2022

Waterlogging leads to major crop losses globally, particularly for waterlogging sensitive crops such as barley. Waterlogging reduces oxygen availability and results in additional stresses, leading to the activation of hypoxia and stress response pathways that promote plant survival. Although certain barley varieties have been shown to be more tolerant to waterlogging than others and some tolerance-related QTLs have been identified, the molecular mechanisms underlying this trait are mostly unknown. Transcriptomics approaches can provide very valuable information for our understanding of waterlogging tolerance. Here, we surveyed 21 barley varieties for the differential transcriptional activation of conserved hypoxia-response genes under waterlogging, and selected five varieties with different levels of induction of core hypoxia-response genes. We further characterized their phenotypic response to waterlogging in terms of shoot and root traits. RNA-sequencing to evaluate the genome-wide transcriptional responses to waterlogging of these selected varieties led to the identification of a set of 98 waterlogging-response genes common to the different datasets. Many of these genes are orthologs of the so-called ‘core hypoxia response genes’, thus highlighting the conservation of plant responses to waterlogging. Hierarchical clustering analysis also identified groups of genes with intrinsic differential expression between varieties prior to waterlogging stress. These genes could constitute interesting candidates to study ‘predisposition’ to waterlogging tolerance or sensitivity in barley.
Evaluating restriction enzyme selection for genome reduction in conservation genomics
Authors: López Ainhoa , Carreras Carlos , Pascual Marta , Pegueroles Cinta
Published: November 2022

Conservation genomic studies in non-model organisms generally rely on genome reduction techniques based on restriction enzymes to identify population structure as well as candidate loci for local adaptation. These reduced libraries ensure a high density of SNP loci and high coverage for accurate genotyping. Despite the fraction of the genome that is sequenced is expected to be randomly located, the reduction of the genome might depend on the recognition site of the restriction enzyme used. Here, we evaluate the distribution and functional composition of loci obtained after Genotyping-by-sequencing (GBS) genome reduction with two widely used restriction enzymes (EcoT22I and ApeKI). To do so, we compared data from two endemic fish species (Symphodus ocellatusandSymphodus tinca, EcoT22I enzyme) and two ecosystem engineer sea urchins (Paracentrotus lividusandArbacia lixula, ApeKI enzyme). In brief, we mapped the sequenced loci to the phylogenetically closest reference genome available (Labrus bergyltafor fish andStrongylocentrotus purpuratusfor sea urchins), classified them as exonic, intronic, and intergenic, and studied their functionality by using GO terms. We detected an enrichment towards exonic or intergenic regions depending on the restriction enzyme used, and we did not detect differences between total loci and candidate loci for adaptation. Despite most GO terms being shared between species, the analysis of their abundance showed differences between taxonomic groups, which may be attributed to differences of the targeted loci. Our results highlight the importance of restriction enzyme selection and the need for high-quality annotated genomes in conservation genomic studies.
The virome of the invasive Asian bush mosquitoAedes japonicusin Europe
Authors: Abbo Sandra R. , de Almeida João P. P. , Olmo Roenick P. , Balvers Carlijn , Griep Jet S. , Linthout Charlotte , Koenraadt Constantianus J. M. , Silva Bruno M. , Fros Jelke J. , Aguiar Eric R. G. R. , Marois Eric , Pijlman Gorben P. , Marques João T.
Published: November 2022

Background

The Asian bush mosquitoAedes japonicusis rapidly invading North America and Europe. Due to its potential to transmit multiple pathogenic arthropod-borne (arbo)viruses including Zika virus, West Nile virus and chikungunya virus, it is important to understand the biology of this vector mosquito in more detail. In addition to arboviruses, mosquitoes can also carry insect-specific viruses that receive increasing attention due to their potential effects on host physiology and arbovirus transmission. In this study, we characterized the collection of viruses, referred to as the virome, circulating inAe. japonicuspopulations in the Netherlands and France.

Results

Applying a small RNA-based metagenomic approach toAe. japonicus, we uncovered a distinct group of viruses present in samples from both the Netherlands and France. These included one known virus,Ae. japonicusnarnavirus 1 (AejapNV1), and three new virus species that we namedAe. japonicustotivirus 1 (AejapTV1),Ae. japonicusanphevirus 1 (AejapAV1) andAe. japonicusbunyavirus 1 (AejapBV1). We also discovered sequences that were presumably derived from two additional novel viruses:Ae. japonicusbunyavirus 2 (AejapBV2) andAe. japonicusrhabdovirus 1 (AejapRV1). All six viruses induced strong RNA interference responses, including the production of 21 nucleotide sized small interfering RNAs, a signature of active replication in the host. Notably, AejapBV1 and AejapBV2 belong to different viral families, however, no RNA-dependent RNA polymerase sequence has been found for AejapBV2. Intriguingly, our small RNA-based approach identified a ∼1 kb long ambigrammatic RNA that is associated with AejapNV1 as a secondary segment but showed no similarity to any sequence in public databases. We confirmed the presence of AejapNV1 primary and secondary segments, AejapTV1, AejapAV1 and AejapBV1 by reverse-transcriptase PCR in wild-caughtAe. japonicusmosquitoes. AejapNV1 and AejapTV1 were found at high prevalence (87-100%) in adult females, adult males and larvae.

Conclusions

Using a small RNA-based, sequence-independent metagenomic strategy, we uncovered a conserved and prevalent virome amongAe. japonicusmosquito populations. The high prevalence of AejapNV1 and AejapTV1 across all tested mosquito life stages suggests that these viruses are intimately associated withAe. japonicusand may affect different aspects of the physiology of this vector mosquito.
Thermodynamic Architecture and Conformational Plasticity of GPCRs
Authors: Anantakrishnan Sathvik , Naganathan Athi N.
Published: November 2022

G-protein-coupled receptors (GPCRs) are ubiquitous integral membrane proteins involved in diverse cellular signaling processes and consequently serve as crucial drug targets. Here, we carry out the first large-scale ensemble thermodynamic study of 45 different ligand-free GPCRs employing a structure-based statistical mechanical framework and identify extensive conformational plasticity encompassing the seven transmembrane (TM) helices. Multiple partially structured states or intermediates co-exist in equilibrium in the native ensemble, with the TM helices 1, 6 and 7 displaying varied degrees of structure, and TM3 exhibiting the maximal stability. Active state GPCRs are characterized by reduced conformational heterogeneity with altered coupling-patterns distributed not just locally but throughout the structural scaffold. Strongly coupled residues are distributed across the structure in an anisotropic manner accounting for only 13% of the residues, highlighting that a large number of residues in GPCRs are inherently dynamic to enable structural motions critical for function. Our work thus uncovers the thermodynamic hallmarks of GPCR structure and activation, and how differences quantifiable only via higher-order coupling free energies provide insights into their exquisite structural specialization and the fluid nature of the intramolecular interaction network. The intricate landscapes and perturbation methodologies presented here lay the foundation for understanding allosteric mechanisms in GPCRs, location of structural-functional hot-spots, and effects of disease-causing mutations.