bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacillusendospore appendages form a novel family of disulfide-linked pili
Authors: Pradhan Brajabandhu , Liedtke Janine , Sleutel Mike , Lindbäck Toril , Llarena Ann-Katrin , Brynildsrud Ola , Aspholm Marina , Remaut Han
Published: August 2020

Bacillus cereus sensu latois a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here we isolate endospore appendages (Enas) from the food poisoning outbreak strainB. cereusNVH 0075-95 and find proteinaceous fibers of two main morphologies. By using cryo-EM and 3D helical reconstruction we show thatBacillusEnas form a novel class of Gram-positive pili. Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought and chemical damage. Phylogenomic analysis reveals the presence of definedenaclades amongst different eco- and pathotypes. We propose Enas to represent a novel class of pili specifically adapted to the harsh conditions encountered by bacterial spores.
Histidine Protonation Controls Structural Heterogeneity in the Cyanobacteriochrome AnPixJg2
Authors: Rao Aditya G. , Wiebeler Christian , Sen Saumik , Cerutti David S. , Schapiro Igor
Published: August 2020

Cyanobacteriochromes are compact and spectrally diverse photoreceptor proteins that bind a linear tetrapyrrole as a chromophore. They show photochromicity by having two stable states that can be interconverted by the photoisomerization of the chromophore. Hence, these photochemical properties make them an attractive target for biotechnological applications. However, their application is impeded by structural heterogeneity that reduces the yield of the photoconversion. The heterogeneity can originate either from the chromophore structure or the protein environment. Here, we study the origin of the heterogeneity in AnPixJg2, a representative member of the red/green cyanobacteriochrome family, that has a red absorbing parental state and a green absorbing photoproduct state. Using molecular dynamics simulations and umbrella sampling we have identified the protonation state of a conserved histidine residue as a trigger for structural heterogeneity. When the histidine is in a neutral form, the chromophore structure is homogenous, while in a positively charged form, the chromophore is heterogeneous with two different conformations. We have identified a correlation between the protonation of the histidine and the structural heterogeneity of the chromophore by detailed characterization of the interactions in the protein binding site. Our findings reconcile seemingly contradicting spectroscopic studies that attribute the heterogeneity to different sources. Furthermore, we predict that circular dichroism can be used as a diagnostic tool to distinguish different substates.

Significance statement

Cyanobacteriochromes are photoreceptor proteins that have attracted attention for their immense potential in bioimaging and optogenetics applications. This is due to their desirable properties such as compactness, photochromicity and diverse spectral tuning. Despite these advantages, nature has set a limitation in the form of structural heterogeneity that presents a drawback for its application in biotechnology. We have identified a histidine residue in the vicinity of the chromophore as the origin of the heterogeneity in red/green CBCRs. The protonation state of this conserved histidine alters an extended network of protein-chromophore interactions and induces heterogeneity. Furthermore, theoretical CD spectroscopy has revealed easy identification of heterogeneity. Hence, our study paves the way for rational design and optimization of protein properties.
Selective dephosphorylation by PP2A-B55 directs the meiosis I - meiosis II transition in oocytes
Authors: Swartz S. Zachary , Nguyen Hieu T. , McEwan Brennan C. , Adamo Mark E. , Cheeseman Iain M. , Kettenbach Arminja N.
Published: August 2020

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in sea star oocytes from prophase I through the first embryonic cleavage. Although protein levels are broadly stable, dynamic waves of phosphorylation underlie each meiotic stage. We find that the phosphatase PP2A-B55 is reactivated at the Meiosis I/II transition resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI / MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after meiosis I. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 rewires the cell division apparatus to direct the MI / MII transition.
Sourdoughs fermented by autochthonousLactobacillusstrains improve the quality of gluten-free bread
Authors: Nami Yousef , Gharekhani Mehdi , Aalami Mehran , Hejazi Mohammad Amin
Published: August 2020

Sourdoughs based on fermentation by lactobacilli have the potential to produce gluten-free maize-based bread with acceptable technological and rheological characteristics, nutritional quality and a more prolonged shelf life. Of the 17 treatments compared (with or without sourdough, and involving single and multiple LAB species), treatments 12C (Lactobacillus brevis, L. sanfranciscensis+L. plantarum) and 8C (L. brevis+L. paralimentarius) showed the lowest rate of complex modulus, while treatments 11C (L. sanfranciscensis+L. brevis+L. paralimentarius) and 2C (L. brevis) led to the greatest reduction in baking loss. The crumb moisture content of all of the formulations decreased with storage. Breads produced with treatment 2C (L. brevis) had the highest crumb moisture content when freshly baked; while loaves produced with treatment 3C (L. paralimentarius) had the highest crumb moisture content after four days of storage. A sensory evaluation indicated that sourdough-based maize breads were superior to both control and chemically acidified breads. The optimal treatments were to use sourdough seeded with treatment 2C (L. brevis), with treatment 4C (L. plantarum), with treatment 8C (L. brevis+L. paralimentarius) or with treatment 11C (L. sanfranciscensis+L. brevis+L. paralimentarius).

Significance Statement

Of the 17 treatments compared, treatments 12C and 8C showed the lowest rate of complex modulus, while treatments 11C and 2C led to the greatest reduction in baking loss. The optimal treatments were to use sourdough seeded with treatment 2C, with treatment 4C, with treatment 8C or with treatment 11C.
Dysregulation of thede novoproteome accompanies pathology progression in the APP/PS1 mouse model of Alzheimer’s disease
Authors: Elder Megan K. , Erdjument-Bromage Hediye , Oliveira Mauricio M. , Mamcarz Maggie , Neubert Thomas A. , Klann Eric
Published: August 2020

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder, but neuropathological changes in AD begin years before memory impairment. Investigation of the early molecular abnormalities in AD might offer innovative opportunities to target memory impairment prior to its onset. Decreased protein synthesis plays a fundamental role in AD, yet the consequences for cellular function remain unknown. We hypothesize that alterations in thede novoproteome drive early metabolic changes in the hippocampus that persist throughout AD progression. Using a combinatorial amino acid tagging approach to selectively label newly synthesized proteins, we found that thede novoproteome is disturbed in APP/PS1 mice prior to pathology development, affecting the synthesis of multiple components of synaptic, lysosomal and mitochondrial pathways. Furthermore, the synthesis of large clusters of ribosomal subunits were affected throughout neuropathology development in these mice. Our data suggest that large-scale changes in protein synthesis could underlie cellular dysfunction in AD.
Routine sub-2.5 Å cryo-EM structure determination of B-family G protein-coupled receptors
Authors: Danev Radostin , Belousoff Matthew , Liang Yi-Lynn , Zhang Xin , Wootten Denise , Sexton Patrick M.
Published: August 2020

Cryo-electron microscopy (cryo-EM) experienced game-changing hardware and software advances about a decade ago. Since then, there have been gradual and steady improvements in experimental and data analysis methods. Nonetheless, structural analysis of nonsymmetric membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Their relatively low molecular weight and obstruction by the micelle/nanodisc result in marginal signal levels, which combined with the intrinsic flexibility of such complexes creates difficult structural study scenarios. Pushing the performance limits of cryo-EM requires careful optimization of all experimental aspects. To this end, it is necessary to build quantitative knowledge of the effect each parameter has on the outcome. Here, we present in-depth analysis of the influence of the main cryo-EM experimental factors on the performance for GPCR structure determination. We used a tandem experiment approach that combined real-world structural studies with parameter testing. We quantified the effects of using a Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. Through such systematic optimization of the experimental conditions, it has been possible to routinely determine class B1 GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps helps to build higher confidence atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines drafted here are not limited to GPCRs and can be applied directly for the study of other challenging membrane protein targets.
Evolution of dynamical networks enhances catalysis in a designer enzyme
Authors: Bunzel H. Adrian , Anderson J. L. Ross , Hilvert Donald , Arcus Vickery L. , van der Kamp Marc W. , Mulholland Adrian J.
Published: August 2020

Activation heat capacity is emerging as a crucial factor in enzyme thermoadaptation, as shown by non-Arrhenius behaviour of many natural enzymes1,2. However, its physical origin and relationship to evolution of catalytic activity remain uncertain. Here, we show that directed evolution of a computationally designed Kemp eliminase introduces dynamical changes that give rise to an activation heat capacity absent in the original design3. Extensive molecular dynamics simulations show that evolution results in the closure of solvent exposed loops and better packing of the active site with transition state stabilising residues. Remarkably, these changes give rise to a correlated dynamical network involving the transition state and large parts of the protein. This network tightens the transition state ensemble, which induces an activation heat capacity and thereby nonlinearity in the temperature dependence. Our results have implications for understanding enzyme evolution (e.g. in explaining the role of distal mutations and evolutionary tuning of dynamical responses) and suggest that integrating dynamics with design and evolution will accelerate the development of efficient novel enzymes.
Automated analysis of rabbit knee calcified cartilage morphology using micro-computed tomography and deep learning segmentation
Authors: Rytky Santeri J. O. , Huang Lingwei , Tanska Petri , Tiulpin Aleksei , Panfilov Egor , Herzog Walter , Korhonen Rami K. , Saarakkala Simo , Finnilä Mikko A. J.
Published: August 2020

Purpose

Only little is known how calcified cartilage (CC) structure changes during exercise, aging and disease. CC thickness (CC.Th) can be analyzed using conventional histological sections. Micro-computed tomography (μCT) allows for three-dimensional (3D) imaging of mineralized tissues, however, the segmentation between bone and CC is challenging. Here, we present state-of-the-art deep learning segmentation for μCT images to enable assessment of CC morphology.

Methods

Sixteen knees from twelve New Zealand White rabbits were dissected into osteochondral samples from six anatomical regions: lateral and medial femoral condyles, lateral and medial tibial plateaus, femoral groove and patella (n = 96). Samples were imaged with μCT and processed for conventional histology. Manually segmented CC from the histology and reconstructed μCT images was used as the gold standard to train segmentation models with different encoder-decoder architectures. The models with the greatest out-of-fold evaluation Dice score were used for automated CC.Th analysis. Subsequently, the automated CC.Th analysis was compared across a total of 24 regions, co-registered between the imaging modalities, using Pearson correlation and Bland-Altman analyses. Finally, the anatomical variation in CC.Th was assessed via a Linear Mixed Model analysis.

Results

The best segmentation models yielded average Dice scores of 0.891 and 0.807 for histology and μCT segmentation, respectively. The correlation between the co-registered regions across the modalities was strong (r = 0.897). The Bland-Altman analysis yielded a bias of 21.9 μm and a standard deviation of 21.5 μm between the methods. Finally, both methods could separate the CC morphology between the patella, femoral, and tibial regions (p < 0.001).

Conclusion

The presented method allows for ex vivo 3D assessment of CC.Th in an automated and non-destructive manner. We demonstrated its utility by quantifying CC.Th in different anatomical regions. CC.Th was the thickest in the patella and the thinnest in the tibial plateau.

Graphical abstract

We present a μCT-based method with deep learning segmentation for analyzing calcified cartilage thickness (CC.Th). The method is compared throughout the study against conventional histology. The comparison against co-registered regions yielded a strong Pearson correlation (r = 0.90). Both methods were able to separate the CC.Th properties between tibia, femur, and patella.
Recurrent human papillomavirus-related head and neck cancer undergoes metabolic re-programming and is driven by oxidative phosphorylation
Authors: Vyas Avani , Harbison R. Alex , Faden Daniel , Kubik Mark , Palmer Drake , Zhang Qing , Osmanbeyoglu Hatice U. , Méndez Eduardo , Duvvuri Umamaheswar
Published: August 2020

Human papillomavirus (HPV) infection drives the development of some head and neck cancer squamous cell carcinomas (HNSC). This disease is rapidly increasing in incidence worldwide. Although these tumors are sensitive to treatment, ~10% of patients fail therapy. However, the mechanisms that underlie treatment failure remain unclear. Here, we show that the oxidative phosphorylation (OXPHOS) pathway is enriched in recurrent HPV-associated HNSC and may contribute to treatment failure. Nrf2-enriched HNSC samples from the Cancer Genome Atlas with enrichment in OXPHOS, fatty acid metabolism, Myc, Mtor, ROS, and glycolytic signaling networks exhibited worse survival. HPV-positive HNSC cells demonstrated sensitivity to the OXPHOS inhibitor, IACS-010759, in a Nrf2-dependent manner. Further, using murine xenograft models, we identified Nrf2 as a driver of tumor growth. Mechanistically, Nrf2 drives ROS and mitochondrial respiration, and Nrf2 is a critical regulator of redox homeostasis that can be crippled by disruption of OXPHOS. Nrf2 also mediated cisplatin sensitivity in endogenously overexpressing primary HPV-related HNSC cells. Cisplatin treatment demonstrated Nrf2-dependent synergy with OXPHOS inhibition. These results unveil a paradigm shifting translational target harnessing Nrf2-mediated metabolic reprogramming in HPV-related HNSC.
Hemodynamic responses link individual differences in informational masking to the vicinity of superior temporal gyrus
Authors: Zhang Min , Alamatsaz Nima , Ihlefeld Antje
Published: August 2020

Suppressing unwanted background sound is crucial for aural communication. A particularly disruptive type of background sound, informational masking (IM), often interferes in social settings. However, IM mechanisms are incompletely understood. At present, IM is identified operationally: when a target should be audible, based on suprathreshold target/masker energy ratios, yet cannot be heard because perceptually similar background sound interferes. Here, functional near infrared spectroscopy recordings show that task-evoked blood oxygenation changes near the superior temporal gyrus (STG) covary with behavioral speech detection performance for high-IM but not low-IM background sound, suggesting that the STG is part of an IM-dependent network. Moreover, listeners who are more vulnerable to IM show increased hemodynamic recruitment near STG. In contrast, task-evoked responses near another auditory region of cortex, the caudal inferior frontal sulcus (cIFS), do not predict behavioral sensitivity, suggesting that the cIFS belongs to an IM-independent network. Results are consistent with the idea that cortical gating shapes individual vulnerability to IM.
Extensive age-dependent loss of antibody diversity in naturally short-lived turquoise killifish
Authors: Bradshaw William J , Poeschla Michael , Placzek Aleksandra , Kean Samuel , Valenzano Dario Riccardo
Published: August 2020

Aging individuals exhibit a pervasive decline in adaptive immune function, with important implications for health and lifespan. Previous studies have found a pervasive loss of immune-repertoire diversity in human peripheral blood during aging; however, little is known about repertoire aging in other immune compartments, or in species other than humans. Here, we perform the first study of immune-repertoire aging in an emerging model of vertebrate aging, the African turquoise killifish (Nothobranchius furzeri). Despite their extremely short lifespans, these killifish exhibit complex and individualized heavy-chain repertoires, with a generative process capable of producing millions of distinct productive sequences. Whole-body killifish repertoires decline rapidly in within-individual diversity with age, while between-individual variability increases. Large, expanded B-cell clones exhibit far greater diversity loss with age than small clones, suggesting important differences in how age affects different B cell populations. The immune repertoires of isolated intestinal samples exhibit especially dramatic age-related diversity loss, related to an elevated prevalence of expanded clones. Lower intestinal repertoire diversity was also associated with transcriptomic signatures of reduced B-cell activity, supporting a functional role for diversity changes in killifish immunosenescence. Our results highlight important differences in systemic vs. organ-specific aging dynamics in the adaptive immune system.

Significance Statement

Immune function declines dramatically with age, with important consequences for health in the elderly. Changes in the diversity and composition of the body’s antibody repertoire are an important contributor to this age-related dysfunction. However, studying repertoire aging at high spatial and temporal resolution is hampered by the long lifespans of humans and other vertebrate models, and the difficulty of acquiring human samples other than peripheral blood. Here, we characterize the aging of the antibody repertoire in a naturally short-lived vertebrate, the turquoise killifish(Nothobranchius furzeri).We found that aging killifish undergo rapid loss of repertoire diversity in both whole-body and intestinal samples within four months of hatching. Loss of repertoire diversity in the gut was also associated with broader gene-expression changes indicative of reduced B-cell activity. As such, killifish represent a powerful laboratory model to study adaptive immune aging.
Combining repetition suppression and pattern analysis provides new insights into the role of M1 and parietal areas in skilled sequential actions
Authors: Berlot Eva , Popp Nicola J. , Grafton Scott T. , Diedrichsen Jörn
Published: August 2020

How does the brain change during learning? In functional magnetic resonance imaging studies, both multivariate pattern analysis and repetition suppression (RS) have been used to detect changes in neuronal representations. In the context of motor sequence learning, the two techniques have provided discrepant findings: pattern analysis showed that only premotor and parietal regions, but not primary motor cortex (M1), develop a representation of trained sequences. In contrast, RS suggested trained sequence representations in all these regions. Here we applied both analysis techniques to a 5-week finger sequence training study, in which participants executed each sequence twice before switching to a different sequence. Both RS and pattern analysis indicated learning-related changes for parietal areas, but only RS showed a difference between trained and untrained sequences in M1. A more fine-grained analysis, however, revealed that the RS effect in M1 reflects a fundamentally different process than in parietal areas. On the first execution, M1 represents especially the first finger of each sequence, likely reflecting preparatory processes. This effect dramatically reduces during the second execution. In contrast, parietal areas represent the identity of a sequence, and this representation stays relatively stable on the second execution. These results suggest that the RS effect does not reflect a trained sequence representation in M1, but rather a preparatory signal for movement initiation. More generally, our study demonstrates that across regions RS can reflect different representational changes in the neuronal population code, emphasizing the importance of combining pattern analysis and RS techniques.

Significance statement

Previous studies using pattern analysis have suggested that primary motor cortex (M1) does not represent learnt sequential actions. However, a study using repetition suppression (RS) has reported M1 changes during motor sequence learning. Combining both techniques, we first replicate the discrepancy between them – with learning-related changes in M1 in RS, but not pattern dissimilarities. We further analysed the representational changes with repetition, and found that the RS effects differ across regions. M1’s activity represents the starting finger of the sequence, an effect that vanishes with repetition. In contrast, activity patterns in parietal areas exhibit sequence dependency, which persists with repetition. These results demonstrate the importance of combining RS and pattern analysis to understand the function of brain regions.
The receptor binding domain of SARS-CoV-2 spike is the key target of neutralizing antibody in human polyclonal sera
Authors: Steffen Tara L. , Stone E. Taylor , Hassert Mariah , Geerling Elizabeth , Grimberg Brian T. , Espino Ana M. , Pantoja Petraleigh , Climent Consuelo , Hoft Daniel F. , George Sarah L. , Sariol Carlos A. , Pinto Amelia K. , Brien James D.
Published: August 2020

Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.
Regulation of extracellular matrix assembly and structure by hybrid M1/M2 macrophages
Authors: Witherel Claire E. , Sao Kimheak , Brisson Becky K. , Han Biao , Volk Susan W. , Petrie Ryan J. , Han Lin , Spiller Kara L.
Published: August 2020

Aberrant extracellular matrix (ECM) assembly surrounding implanted biomaterials is the hallmark of the foreign body response, in which implants become encapsulated in thick fibrous tissue that prevents their proper function. While macrophages are known regulators of fibroblast behavior, how their phenotype influences ECM assembly and the progression of the foreign body response is poorly understood. In this study, we usedin vitromodels with physiologically relevant macrophage phenotypes, as well as controlled release of macrophage-modulating cytokines from gelatin hydrogels implanted subcutaneouslyin vivoto investigate the role of macrophages in ECM assembly. Primary human macrophages were polarized to four distinct phenotypes, which have each been associated with fibrosis, including pro-inflammatory M1, pro-healing M2, and a hybrid M1/M2, generated by exposing macrophages to M1- and M2-promoting stimuli simultaneously. Additionally, macrophages were first polarized to M1 and then to M2 (M1→M2) to generate a phenotype typically observed during normal wound healing. Human dermal fibroblasts that were cultured in macrophage-conditioned media upregulated numerous genes involved in regulation of ECM assembly, especially in M2-conditioned media. Hybrid M1/M2 macrophage-conditioned media caused fibroblasts to produce a matrix with thicker and less aligned fibers, while M2 macrophage-conditioned media caused the formation of a more aligned matrix with thinner fibers. Gelatin methacrylate hydrogels containing interleukin-4 (IL4) and IL13-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were designed to promote the M2 phenotype in a murine subcutaneousin vivomodel. NanoString multiplex gene expression analysis of hydrogel explants showed that hydrogels with and without drug caused markers of both M1 and M2 phenotypes to be highly expressed, but the release of IL4+IL13 promoted upregulation of M2 markers and genes associated with regulation of ECM assembly, such asCol5a1andCol6a1. Biochemical analysis and second harmonic generation microscopy showed that the release of IL4+IL13 increased total sulfated glycosaminoglycan content and decreased fibril alignment, which is typically associated with less fibrotic tissue. Together, these results show that hybrid M1/M2 macrophages regulate ECM assembly, and that shifting the balance towards M2 may promote architectural and compositional changes in ECM with enhanced potential for downstream remodeling.
Cyclophilin A is a mitochondrial factor that forms antiapoptotic complexes with p23
Authors: Daneri-Becerra Cristina , Valeiras Brenda , Lagadari Mariana , Galigniana Mario D.
Published: August 2020

Cyclophilin A (CyPA) is an abundant and ubiquitously expressed protein belonging to the immunophilin family that has intrinsic peptidyl-prolyl-(cis/trans)-isomerase enzymatic activity. In addition to mediating the immunosuppressive effects of the drug cyclosporine A, CyPA is involved in multiple cellular processes such as protein folding, intracellular trafficking, signal transduction, and transcriptional regulation. Because CyPA is also a molecular chaperone, its expression is induced by several stressor agents and is a highly abundant protein in cancer cells. In this study, it is demonstrated that in several cell types and at least in murine liver, a significant pool of this immunophilin is primarily an intramitochondrial factor that migrates to the nucleus upon the onset of stress. It is also shown that CyPA has antiapoptotic action. Importantly, the capability of CyPA to form complexes with the small acidic cochaperone p23 is proven, this interaction being independent of the usual association of p23 with the heat-shock protein of 90-kDa, Hsp90. Furthermore, it is demonstrated that the CyPA•p23 complex enhances the antiapoptotic response of the cell, suggesting that both proteins form a functional unit whose high level of expression plays a significant role in cell survival.
Analysis of the oxidative stress regulon identifiessoxSas a genetic target for resistance reversal in multi-drug resistantKlebsiella pneumoniae
Authors: Anes João , Dever Katherine , Eshwar Athmanya , Nguyen Scott , Cao Yu , Sivasankaran Sathesh K , Sakalauskaitė Sandra , Lehner Angelika , Devineau Stéphanie , Daugelavičius Rimantas , Fanning Séamus , Srikumar Shabarinath
Published: August 2020

In bacteria, the defense system deployd to counter oxidative stress is orchestrated by three transcriptional factors – SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data is not available forKlebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced inK. pneumoniaeMGH 78578 using paraquat and the corresponding oxidative stress regulon recorded using RNA-seq. ThesoxSgene was significantly induced during oxidative stress and a knock-out mutant was constructed, to explore its functionality. The wild-type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate thesoxSregulon inK. pneumoniaeMGH78578. Genes that are commonly regulated both in the oxidative stress regulon andsoxSregulon were identified and denoted as the ‘oxidative SoxS regulon’ – these included a stringent group of genes specifically regulated by SoxS. Efflux pump encoding genes such asacrAB-tolC, acrE, and global regulators such asmarRABwere identified as part of this regulon. Consequently, the isogenicsoxSmutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. ThesoxSmutant was also susceptible to tetracyclinein vivo, in a zebrafish embryo model. We conclude that thesoxSgene could be considered as a genetic target against which an inhibitor could be developed in the future and used in combinatorial therapy with tetracycline to combat infections associated with multi-drug resistantK. pneumoniae.
Solution structures of theShewanella woodyiH-NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP-051
Authors: Chen Cheng-Yu , Lee Woonghee , Montfort William R.
Published: August 2020

Heme-nitric oxide/oxygen binding (H-NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacteriumShewanella woodyi(Sw), H-NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H-NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H-NOX and coiled-coil domains. Here, we report the full NMR structure for CO-ligatedSwH-NOX in the presence and absence of stimulator compound IWP-051, and its backbone dynamics. Non-planar heme geometry was retained using a semi-empirical quantum potential energy approach. Although IWP-051 binding is weak, a single binding conformation was found at the interface of the two H-NOX subdomains. Binding lead to rotation of the subdomains and closure of the binding pocket. Backbone dynamics for the protein are similar across both domains except for two helix-connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure-based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H-NOX proteins. The largest dynamical loop lies at the interface betweenSwH-NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.
Recombinant SARS-CoV-2 RBD molecule with a T helper epitope as a built in adjuvant induces strong neutralization antibody response
Authors: Su Qiudong , Zou Yening , Yi Yao , Shen Liping , Ye Xiangzhong , Zhang Yang , Wang Hui , Ke Hong , Song Jingdong , Hu Keping , Cheng Bolin , Qiu Feng , Yu Pengcheng , Zhou Wenting , Cao Lei , Bi Shengli , Wu Guizhen , Gao George Fu , Zheng Jerry
Published: August 2020

Without approved vaccines and specific treatment, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading around the world with above 20 million COVID-19 cases and approximately 700 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 – Gly548 of Spike protein of SARS-CoV-2 linked with Gln830 – Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5), and prokaryotic expression, chromatography purification and the rational renaturation of the protein were performed. The antigenicity and immunogenicity of S1-4 protein was evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency of it was measured by virus neutralization test in Vero E6 cells with SARS-CoV-2. S1-4 protein was prepared to high homogeneity and purity by prokaryotic expression and chromatography purification. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity compared with S1-5 protein. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralization antibody GMT 256. The candidate subunit vaccine molecule could stimulate strong humoral and Th1 and Th2-type cellular immune response in mice, giving us solid evidence that S1-4 protein could be a promising subunit vaccine candidate.
Mutually Beneficial Symbiosis Between Human and Gut-DominantBacteroidesSpecies Through Bacterial Assimilation of Host Mucosubstances
Authors: Sato Masahiro , Kajikawa Kanta , Kumon Tomoya , Watanabe Daisuke , Takase Ryuichi , Hashimoto Wataru
Published: August 2020

The composition of gut microbiota is influenced by the quantity and type of nutrients in host. Even with someBacteroidesspecies being categorized as pathogens,Bacteroidesis one of the most dominant gut bacteria. Here we indicate the physiological determinants of the species ofBacteroidesfor being dominant in human gut microbiota. Each of the host extracellular mucosubstances including glycosaminoglycans (GAGs) and mucin has grown human gut microbiota. In spite of the differences among initial microbiota profiles,Bacteroidesspecies dominated the community when GAG (e.g., chondroitin sulfate or hyaluronan) was used as a sole carbon source. In fact, GAGs and theBacteroidesgenes which are vital for the degradation of GAGs were commonly detected in human feces. Mucin has encouraged the growth ofBacteroidesand several other genera. A comprehensive analysis on the degradation and assimilation of mucosubstances by the genusBacteroidesusing around 30 species has shown that most species degrade and assimilate GAGs and mucin, showing thatBacteroidesspecies can survive even in the undernutrition condition including the fasting state. In the assimilation of GAG or mucin,Bacteroidesspecies significantly secreted essential amino acids, γ-amino butyrate (GABA), and/or short-chain fatty acids which are needed for human health. This is the first report as regards mutually beneficial interaction between human andBacteroidesspecies via bacterial assimilation of host mucosubstances and secretion of metabolites for host health promotion.

Significance

The genusBacteroidesis one of the most dominant gut bacteria, although its beneficial effects on human health have not been well understood. Here, we show modes of action in human-Bacteroidesinterrelationship. Mucosubstances including GAGs and mucin secreted by human host are abundant in gut for microbiota to grow well.Bacteroidesspecies are dominant in the community in the presence of GAGs, and provide human host with a considerable amount of essential amino acids, γ-amino butyrate, and short-chain fatty acids produced from mucosubstances. These results postulate mutually beneficial symbiosis system between human andBacteroidesthrough bacterial assimilation of host mucosubstances and secretion of metabolites for human body and mental health promotion even in the undernutrition condition including the fasting state.
Molecular basis for substrate recruitment to the PRMT5 methylosome
Authors: Mulvaney Kathleen M. , Blomquist Christa , Acharya Nischal , Li Ruitong , O’Keefe Meghan , Ranaghan Matthew , Stokes Matthew , Nelson Alissa J , Jain Sidharth S. , Columbus Josie , Bozal Fazli K. , Skepner Adam , Raymond Donald , McKinney David C. , Freyzon Yelena , Baidi Yossef , Porter Dale , Ianari Alessandra , McMillan Brian , Sellers William R.
Published: August 2020

PRMT5 is an arginine methyltransferase and a therapeutic target inMTAPnull cancers. PRMT5 utilizes adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (pICln/CLNS1A, RIOK1 and COPR5) and show it is necessary and sufficient for interaction with PRMT5. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosome assembly complexes. Genetic disruption of the PRMT5-substrate adaptor interface leads to a hypomorphic decrease in growth ofMTAPnull tumor cells and is thus a novel site for development of therapeutic inhibitors of PRMT5.
Active Perception during Angiogenesis: Filopodia speed up Notch selection of tip cellsin silicoandin vivo
Authors: Zakirov Bahti , Charalambous Georgios , Aspalter Irene M. , Van-Vuuren Kelvin , Mead Thomas , Harrington Kyle , Thuret Raphael , Regan Erzsébet Ravasz , Herbert Shane Paul , Bentley Katie
Published: August 2020

How do cells make efficient collective decisions during tissue morphogenesis? Humans and other organisms utilize feedback between movement and sensing known as ‘sensorimotor coordination’ or ‘active perception’ to inform behaviour, but active perception has not before been investigated at a cellular level within organs. Here we provide the first proof of conceptin silico/in vivostudy demonstrating that filopodia (actin-rich, dynamic, finger like cell-membrane protrusions) play an unexpected role in speeding up collective endothelial decisions during the time-constrained process of ‘tip cell’ selection during blood vessel formation (angiogenesis).

We first validate simulation predictionsin vivowith live imaging of zebrafish intersegmental vessel growth. Further simulation studies then indicate the effect is due to the coupled positive feedback between movement and sensing on filopodia conferring a bistable switch-like property to Notch lateral inhibition, ensuring tip selection is a rapid and robust process. We then employ measures from computational neuroscience to assess whether filopodia function as a primitive (‘basal’) form of active perception and find evidence in support. By viewing cell behaviour in tissues through the ‘basal cognitive lens’ we acquire a fresh perspective on not only the well-studied tip cell selection process, revealing a hidden, yet vital, time-keeping role for filopodia, but on how to interpret and understand cell behaviour in general, opening up a myriad of new and exciting research directions.
Live imaging of avian embryos revealing a new head precursor map and the role for the anterior mesendoderm in brain development
Authors: Yoshihi Koya , Kato Kagayaki , Iida Hideaki , Teramoto Machiko , Kawamura Akihito , Watanabe Yusaku , Nunome Mitsuo , Nakano Mikiharu , Matsuda Yoichi , Sato Yuki , Mizuno Hidenobu , Iwasato Takuji , Ishii Yasuo , Kondoh Hisato
Published: August 2020

We investigated the initial stages of head development using a new method to randomly label chicken epiblast cells with enhanced green fluorescent protein, and tracking the labeled cells. This analysis was combined with grafting mCherry-expressing quail nodes, or node-derived anterior mesendoderm (AME). These live imagings provided a new conception of the cellular mechanisms regulating brain and head ectoderm development. Virtually all anterior epiblast cells are bipotent for the development into the brain or head ectoderm. Their fate depends on the positioning after converging to the AME. When two AME tissues exist following the ectopic node graft, the epiblast cells converge to the two AME positions and develop into two brain tissues. The anterior epiblast cells bear gross regionalities that already correspond to the forebrain, midbrain, and hindbrain axial levels shortly after the node is formed. Therefore, brain portions that develop with the graft-derived AME are dependent on graft positioning.
Age effects aggressive behavior: RNA-seq analysis in cattle with implications for studying neoteny under domestication
Authors: Eusebi Paulina G. , Sevane Natalia , O’Rourke Thomas , Pizarro Manuel , Boeckx Cedric , Dunner Susana
Published: August 2020

Aggressiveness is one of the most basic behaviors, characterized by targeted intentional actions oriented to cause harm. The reactive type of aggression is regulated mostly by the brain’s prefrontal cortex; however, the molecular changes underlying aggressiveness in adults have not been fully characterized. Here we used an RNA-seq approach to investigate differential gene expression in the prefrontal cortex of bovines from the aggressive Lidia breed at different age stages: young three-year old and adult four-year-old bulls. A total of 50 up and 193 down-regulated genes in the adult group were identified. Furthermore, a cross-species comparative analysis retrieved 29 genes in common with previous studies on aggressive behaviors, representing an above-chance overlap with the differentially expressed genes in adult bulls.

Particularly, we detected changes in the regulation of networks such as synaptogenesis, involved in maintenance and refinement of synapses, and the glutamate receptor pathway, which acts as excitatory driver in aggressive responses. Our results provide insights into candidate genes and networks involved in the molecular mechanisms leading to the maturation of the brain. The reduced reactive aggression typical of domestication has been proposed to form part of a retention of juvenile traits as adults (neoteny). The significant age-associated differential expression of genes implicated in aggressive behaviors and concomitant increase in Lidia cattle aggression validates this species as a novel model comparator to explore the impact of behavioral neoteny under domestication.
Functional validation of a finding from a mouse genome-wide association study demonstrates that a mutant allele ofAzi2alters sensitivity to methamphetamine
Authors: Zhou Xinzhu , Barkley-Levenson Amanda , Montilla-Perez Patricia , Telese Francesca , Palmer Abraham A.
Published: August 2020

Methamphetamine is a widely abused psychostimulant. In a previous genome-wide association study (GWAS), we identified a locus that influenced the stimulant response to methamphetamine. That locus was also an eQTL for the geneAzi2. Based on those findings, we hypothesized that heritable differences in the expression ofAzi2were causally related to the differential response to methamphetamine. In this study, we created a mutantAzi2allele that caused lowerAzi2expression and enhanced the locomotor response to methamphetamine; however, based on the GWAS findings, we had expected lowerAzi2to decrease rather than increase the stimulant response to methamphetamine. We then sought to explore the mechanism by whichAzi2influenced methamphetamine sensitivity. A recent publication had reported that the 3’UTR ofAzi2mRNA downregulates the expression ofSlc6a3, which encodes the dopamine transporter (DAT), which is a key target of methamphetamine. We evaluated the relationship betweenAzi2/Azi23’UTR andSlc6a3expression in the VTA in the mutantAzi2mice and in a new cohort of CFW mice. We did not observe any correlation betweenAzi2andSlc6a3in the VTA in either cohort. However, RNA sequencing confirmed that theAzi2mutation alteredAzi2expression and also revealed a number of potentially important genes and pathways that were regulated byAzi2, including the metabotropic glutamate receptor group III pathway and nicotinic acetylcholine receptor signaling pathway. Our results support a role forAzi2in methamphetamine sensitivity; however, the exact mechanism does not appear to involve regulation ofSlc6a3and thus remains unknown.
Leading edge maintenance in migrating cells is an emergent property of branched actin network growth
Authors: Garner Rikki M. , Theriot Julie A.
Published: August 2020

Animal cell migration is predominantly driven by the coordinated, yet stochastic, polymerization of thousands of nanometer-scale actin filaments across micron-scale cell leading edges. It remains unclear how such inherently noisy processes generate robust cellular behavior. We employed high-speed imaging of migrating neutrophil-like HL-60 cells to explore the fine-scale shape fluctuations that emerge and relax throughout the process of leading edge maintenance. We then developed a minimal stochastic model of the leading edge that reproduces this stable relaxation behavior. Remarkably, we find lamellipodial stabilitynaturally emergesfrom the interplay between branched actin network growth and leading edge shape – with no additional feedback required – based on a synergy between membrane-proximal branching and lateral spreading of filaments. These results thus demonstrate a novel biological noise-suppression mechanism based entirely on system geometry. Furthermore, our model suggests that the Arp2/3-mediated ~70-80° branching angle optimally smooths lamellipodial shape, addressing its long-mysterious conservation from protists to mammals.

Significance Statement

All cellular functions are driven by the stochastic dynamics of macromolecules, and thus are subject to biological noise. Here, as a model system for noise-suppression in the context of cell migration, we investigate lamellipodial maintenance – where thousands of stochastically polymerizing filaments self-organize into a highly-stable, micron-scale leading edge. Combining experiment and computational modeling, we (1) establish lamellipodial stability is anemergent propertyof dendritically-branched actin network growth, (2) outline a noise-suppression mechanism based on thegeometryof lamellipodial actin, and (3) determine the evolutionarily-conserved Arp2/3-mediated ~70-80° branching angle optimally suppresses stochastic fluctuations. Our results not only explain the essential role of Arp2/3-mediated branching in lamellipodial formation, but also address the decades-old question ofwhythis specific geometry is so well-conserved.