bluuebunny/crossref_metadata_2025 huggingface dataset:
Concurrent evolution of anti-aging gene duplications and cellular phenotypes in long-lived turtles
Authors: Glaberman Scott , Bulls Stephanie E. , Vazquez Juan Manuel , Chiari Ylenia , Lynch Vincent J.
Published: July 2021

There are many costs associated with increased body size and longevity in animals, including the accumulation of genotoxic and cytotoxic damage that comes with having more cells and living longer. Yet, some species have overcome these barriers and have evolved remarkably large body sizes and long lifespans, sometimes within a narrow window of evolutionary time. Here, we demonstrate through phylogenetic comparative analysis that multiple turtle lineages, including Galapagos giant tortoises, concurrently evolved large bodies, long lifespans, and reduced cancer risk. We also show through comparative genomic analysis that Galapagos giant tortoises have gene duplications related to longevity and tumor suppression. To examine the molecular basis underlying increased body size and lifespan in turtles, we treated cell lines from multiple species, including Galapagos giant tortoises, with drugs that induce different types of cytotoxic stress. Our results indicate that turtle cells, in general, are resistant to oxidative stress related to aging, while Galapagos giant tortoise cells, specifically, are sensitive to endoplasmic reticulum stress, which may give this species an ability to mitigate the effects of cellular stress associated with increased body size and longevity.
Causes of variability in estimates of mutational variance from mutation accumulation experiments
Authors: Conradsen Cara , Blows Mark W. , McGuigan Katrina
Published: July 2021

Characteristics of the new phenotypic variation introduced via mutation have broad implications in evolutionary and medical genetics. Standardised estimates of this mutational variance,VM, span two orders of magnitude, but the causes of this remain poorly resolved. We investigated estimate heterogeneity using two approaches. First, meta-analyses of ~150 estimates from 37 mutation accumulation (MA) studies did not support a difference among taxa (which differ in mutation rate) in standardisedVM, but provided equivocal support for standardisedVMto vary with trait type (life history versus morphology, predicted to differ in mutation rate). Notably, several experimental factors were confounded with taxon and trait, and further empirical data are required to resolve their influences. Second, we analysed morphological data from an experiment inDrosophila serratato determine the potential for unintentional heterogeneity among environments in which phenotypes were measured (i.e., among laboratories or time points) or transient segregation of mutations within MA lines to affect standardisedVM. Approximating the size of an average MA experiment, variability among repeated estimates of (accumulated) mutational variance was comparable to variation among published estimates of standardisedVMfor morphological traits. This heterogeneity was (partially) attributable to unintended environmental variation or within line segregation of mutations only for wing size, not wing shape traits. We conclude that sampling error contributed substantial variation within this experiment, and infer that it will also contribute substantially to differences among published estimates. We suggest a logistically permissive approach to improve the precision of estimates, and consequently our understanding of the dynamics of mutational variance of quantitative traits.
Combinations of Peptides Synergistically Activate the Regenerative Capacity of Skin Cells In Vitro
Authors: Flagler Michael J. , Tamura Makio , Laughlin Tim , Hartman Scott , Ashe Julie , Adams Rachel , Kozak Kim , Cresswell Kellen , Mullins Lisa , Jarrold Bradley B. , Isfort Robert J. , Sherrill Joseph D.
Published: July 2021

OBJECTIVE

To explore synergistic effects related to skin regeneration, peptides with distinct biological mechanisms of action were evaluated in combination in different skin cell lines in the presence or absence of niacinamide (Nam). Furthermore, the synergistic responses of peptide combinations on global gene expression were compared to the changes that occur with fractional laser resurfacing treatment, a gold standard approach for skin rejuvenation, to further define optimal peptide combinations.

METHODS

Microarray profiling was used to characterize the biological responses of peptide combinations (+/− Nam) relative to the individual components in epidermal keratinocyte and dermal fibroblast cell lines. Cellular functional assays were utilized to confirm the synergistic effects of peptide combinations. Bioinformatics approaches were used to link the synergistic effects of peptide combinations on gene expression to the transcriptomics of the skin rejuvenation response from fractional laser treatment.

RESULTS

Microarray analysis of skin cells treated with peptide combinations revealed synergistic changes in gene expression compared to individual peptide controls. Bioinformatic analysis of synergy genes in keratinocytes revealed activation of NRF2-mediated oxidative stress responses by a combination of Ac-PPYL, Pal-KTTKS, and Nam. Additional analysis revealed direct downstream transcriptional targets of NRF2/ARE exhibiting synergistic regulation by this combination of materials, which was corroborated by a cellular reporter assay. NRF2-mediated oxidative stress response pathways were also found to be activated in the transcriptomics of the early skin rejuvenation response to fractional laser treatment, suggesting the importance of this biology in the early stages of tissue repair. Additionally, a second combination of peptides (pal-KT and Ac-PPYL) was found to synergistically restore cellular ATP levels that had been depleted due to the presence of ROS, indicating an additional mechanism whereby peptide synergies may accelerate skin repair.

CONCLUSION

Through combinatorial synergy studies, we have identified additional in vitro skin repair mechanisms beyond the previously described functions of individual peptides and correlated these to the transcriptomics of the skin rejuvenation response of fractional laser treatment. These findings suggest that specific peptides can act together, via complementary and synergistic mechanisms, to holistically enhance the regenerative capacity of in vitro skin cells.
The preeminent role of directional selection in generating extreme morphological change in Glyptodonts (Cingulata; Xenarthra)
Authors: Machado Fabio A. , Marroig Gabriel , Hubbe Alex
Published: July 2021

The prevalence of stasis on macroevolution has been classically taken as evidence of the strong role of stabilizing selection in constraining morphological evolution. Rates of evolution calculated over longer time scales tend to fall below the expected under genetic drift, suggesting that the signal for directional selection is erased at longer time scales. Here we investigated the rates of morphological evolution of the skull in a fossil lineage that underwent extreme morphological modification, the glyptodonts. Contrary to what was expected, we show here that directional selection was the main process during the evolution of glyptodonts. Furthermore, the reconstruction of selection patterns shows that traits selected to generate a glyptodont morphology are markedly different from those operating on extant armadillos. Changes in both direction and magnitude of selection are probably tied to glyptodonts’ invasion of a specialist-herbivore adaptive zone. These results suggest that directional selection might have played a more important role in the evolution of extreme morphologies than previously imagined.
Pharmacokinetic Effects of Combined Exposure to Nicotine and THC via E-Cigarettes in Pregnant Rats
Authors: Breit K. R. , Rodriguez C. G. , Hussain S. , Thomas K. , Zeigler M. , Gerasimidis I. , Thomas J. D.
Published: July 2021

Nicotine and cannabis are two of the most commonly consumed licit and illicit drugs during pregnancy, often consumed together via e-cigarettes. Vaping is assumed to be a safer alternative than traditional routes of consumption, yet the potential consequences of prenatal e-cigarette exposure are largely unknown, particularly when these two drugs are co-consumed. In a novel co-exposure model, pregnant Sprague-Dawley rats received nicotine (36 mg/mL), THC (100 mg/mL), the combination, or the vehicle via e-cigarettes daily from gestational days 5-20, mimicking the first and second human trimesters. Maternal blood samples were collected throughout pregnancy to measure drug and metabolite levels, and core body temperatures before and after exposure were also measured. Pregnant dams exposed to combined nicotine and THC had lower plasma nicotine and cotinine levels than those exposed to nicotine alone; similarly, the combined exposure group also had lower plasma THC and THC metabolite (THC-OH and THC-COOH) levels than those exposed to THC alone. Prenatal nicotine exposure gradually decreased basal core body temperatures each day, with chronic exposure, whereas exposure to THC alone decreased temperatures during the individual sessions. Despite these physiological effects, no changes were observed in food or water intake, weight gain, or basic litter outcomes. These data suggest that combined exposure to nicotine and THC elicits both separate and interactive physiological effects of nicotine and THC on pregnant dams. These data and use of this model can help improve education for pregnant mothers about prenatal e-cigarette use and has important implications for public policy.

HIGHLIGHTS

Repeated prenatal nicotine exposure via e-cigarettes gradually decreased temperatures

Prenatal THC exposure via e-cigarettes decreased temperatures during intoxication

Combined prenatal exposure via e-cigarettes altered plasma drug and metabolite levels

This co-exposure model elicits separate and interactive effects of nicotine and THC
The EGF-motif containing protein SPE-36 is a secreted protein required for sperm function at fertilization inC. elegans
Authors: Krauchunas Amber R. , Marcello Matthew R. , Looper A’Maya , Mei Xue , Putiri Emily , Singaravelu Gunasekaran , Ahmed Iqra I. , Singson Andrew
Published: July 2021

The growing number of genes specifically required for fertilization suggests that there is a significant amount of molecular complexity at the sperm-egg interface. Thus, we have adopted a model of a “fertilization synapse” where specialized zones of interaction and multi-protein complexes mediate gamete interaction and fusion. The fertilization synapse is likely to be composed of bothtransandcisprotein-protein interactions at the surface of each gamete. Mutations in theCaenorhabditis elegans spe-36gene result in a sperm-specific fertility defect. Surprisingly,spe-36encodes a secreted EGF-motif containing protein that functions cell autonomously. Despite the fact that morphology and migratory behavior ofspe-36sperm are indistinguishable from wild-type sperm,spe-36sperm make close contact with oocytes but fail to fertilize them. The genetic requirement for a secreted sperm-derived protein for fertilization is novel and represents a paradigm-shifting discovery in the molecular understanding of fertilization.
Identification of Acute Respiratory Distress Syndrome subphenotypes denovo using routine clinical data: a retrospective analysis of ARDS clinical trials
Authors: Duggal Abhijit , Kast Rachel , Van Ark Emily , Bulgarelli Lucas , Siuba Matthew T. , Osborn Jeff , Rey Diego , Zampieri Fernando G , Cavalcanti Alexandre B , Maia Israel S , Paisani Denise M , Laranjeira Ligia N , Neto Ary Serpa , Octávio Deliberato Rodrigo
Published: July 2021

Rationale

The acute respiratory distress syndrome (ARDS) is a heterogenous condition, and identification of subphenotypes may help in better risk stratification.

Objectives

Identify ARDS subphenotypes using new simpler methodology and readily available clinical variables.

Design

Retrospective Cohort Study of ARDS trials.

Setting

Data from the U.S. ARDSNet trials and from the international ART trial.

Participants

3763 patients from ARDSNet datasets and 1010 patients from the ART dataset.

Primary and secondary outcome measures

The primary outcome was 60-day or 28-day mortality, depending on what was reported in the original trial. K-means cluster analysis was performed to identify subgroups. For feature selection, sets. Sets of candidate variables were tested to assess their ability to produce different probabilities for mortality in each cluster. Clusters were compared to biomarker data, allowing identification of subphenotypes.

Results

Data from 4,773 patients was analyzed. Two subphenotypes (A and B) resulted in optimal separation in the final model, which included nine routinely collected clinical variables, namely: heart rate, mean arterial pressure, respiratory rate, bilirubin, bicarbonate, creatinine, PaO2, arterial pH, and FiO2. Participants in subphenotype B showed increased levels of pro-inflammatory markers, had consistently higher mortality, lower number of ventilator-free days at day 28, and longer duration of ventilation compared to patients in the subphenotype A.

Conclusions

Routinely available clinical data can successfully identify two distinct subphenotypes in adult ARDS patients. This work may facilitate implementation of precision therapy in ARDS clinical trials.

ARTICLE SUMMARY

Strengths and limitations of this study

Largest cohort of patients used to identify subphenotypes of ARDS patients.

Subphenotypes were validated in the population of a large international ARDS randomized controlled trial.

Subphenotypes were identified by using only routinely collected clinical data.

Our use of data exclusively from randomized controlled trials does not prove generalizability to unselected ARDS populations.

The clinical utility of the subphenotypes have to be validated in a prospective study.
Short-term diet intervention alters the small non-coding RNA (sncRNA) landscape of human sperm
Authors: Vaz Candida , Kermack Alexandra J , Burton Mark , Tan Pei Fang , Huan Jason , Yoo Tessa P X , Donnelly Kerry , Wellstead Susan J , Fisk Helena L , Houghton Franchesca D , Lewis Sheena , Chong Yap Seng , Gluckman Peter D , Cheong Ying , Macklon Nicholas S , Calder Philip C , Dutta Anindya , Godfrey Keith M , Kumar Pankaj , Lillycrop Karen A , Karnani Neerja
Published: July 2021

Offspring health outcomes are often linked with epigenetic alterations triggered by maternal nutrition and intrauterine environment. Strong experimental data also link paternal preconception nutrition with pathophysiology in the offspring, but the mechanism(s) routing the effects of paternal exposures remain elusive. Animal experimental models have highlighted small non-coding RNAs (sncRNAs) as potential regulators of paternal effects, though less is known about the existence of similar mechanisms in human sperm. Here, we first characterised the baseline sncRNA landscape of human sperm, and then studied the effects of a 6-week diet intervention on their expression profile. Baseline profiling identified 5’tRFs, miRNAs and piRNAs to be the most abundant sncRNA subtypes, primarily expressed from regulatory elements like UTRs, CpG-rich regions and promoters. Expression of a subset of these sncRNAs varied with age, BMI and sperm quality of the donor. Diet intervention enriched in vitamin D and omega-3 fatty acids showed a marked increase of these nutrients in circulation and altered the sperm sncRNA expression. These included 3 tRFs, 15 miRNAs and 112 piRNAs, with gene targets involved in fatty acid metabolism, vitamin D response (LXR/RXR activation, TGF-beta and Wnt signaling), and transposable elements. These findings provide evidence that human sperms are sensitive to alterations in exposures such as diet, and sncRNAs capture the epigenetic imprint of this change. Hence changes to paternal nutrition during preconception may improve sperm quality and offspring health outcomes. To benefit future research, we developed iDad_DB, an open access database of baseline and diet-altered sncRNA in human male germline.
Non-equilibrium conditions inside rock pores drive fission, maintenance and selection of coacervate protocells
Authors: Ianeselli Alan , Tetiker Damla , Stein Julian , Kühnlein Alexandra , Mast Christof , Braun Dieter , Dora Tang T-Y
Published: July 2021

Key requirements for the first cells on Earth include the ability to compartmentalize and evolve. Compartmentalization spatially localizes biomolecules from a dilute pool and an evolving cell which grows and divides permits mixing and propagation of information to daughter cells. Complex coacervate microdroplets are excellent candidates as primordial cells with the ability to partition and concentrate molecules into their core and support primitive and complex biochemical reactions. However, the evolution of coacervate protocells by fusion, growth and fission has not yet been demonstrated. In this work, a primordial environment initiated the evolution of coacervate-based protocells. Gas bubbles inside heated rock pores perturb the coacervate protocell distribution and drive the growth, fusion, division and selection of coacervate microdroplets. This setting provides a primordial non-equilibrium environment. Our findings describe how common gas bubbles within heated rock pores induce the early evolution processes of coacervate-based protocells, providing a compelling scenario for the evolution of membrane-free coacervate microdroplets on the early Earth.
Antibody and T cell memory immune response after two doses of the BNT162b2 mRNA vaccine in older adults with and without prior SARS-CoV-2 infection
Authors: Demaret Julie , Corroyer-Simovic Bénédicte , Alidjinou Enagnon Kazali , Goffard Anne , Trauet Jacques , Miczek Sophie , Vuotto Fanny , Dendooven Arnaud , Huvent-Grelle Dominique , Podvin Juliette , Dreuil Daniel , Faure Karine , Deplanque Dominique , Bocket Laurence , Duhamel Alain , Labreuche Julien , Sobaszek Annie , Puisieux Francois , Labalette Myriam , Lefèvre Guillaume
Published: July 2021

We quantified S1-specific IgG, neutralizing antibody titers, specific IFNγ secreting T cells and functionality of specific CD4+ and CD8+ T cells in 130 young adults (median age 44.0 years) and 106 older residents living in a long-term care facility (86.5 years) after 2 doses of BNT162b2. Three months after the first injection, humoral and cellular memory responses were dramatically impaired in the 54 COVID-19-naive older compared to the 121 COVID-19-naive younger adults. Notably, older participants’ neutralizing antibodies, detected in 76.5% (versus 100% in young adults,P< 0.0001), were ten times lower than the younger’s antibody titers (P< 0.0001). Antibody and T cell responses were greater among the 52 COVID-19-recovered than among the 54 COVID-19-naive older adults (P< 0.0001). Our study shows that 2 doses of BNT162b2 does not guarantee long-term protection against SARS-CoV-2 in the older. An additional dose should be considered to boost their specific memory response.
The geometry of domain-general performance monitoring in the human medial frontal cortex
Authors: Fu Zhongzheng , Beam Danielle , Chung Jeffrey M. , Reed Chrystal M. , Mamelak Adam N. , Adolphs Ralph , Rutishauser Ueli
Published: July 2021

Controlling behavior to flexibly achieve desired goals depends on the ability to monitor one’s own performance. It is unknown how performance monitoring can be both flexible to support different tasks and specialized to perform well on each. We recorded single neurons in the human medial frontal cortex while subjects performed two tasks that involve three types of cognitive conflict. Neurons encoding predicted conflict, conflict, and error in one or both tasks were intermixed, forming a representational geometry that simultaneously allowed task specialization and generalization. Neurons encoding conflict retrospectively served to update internal estimates of control demand. Population representations of conflict were compositional. These findings reveal how representations of evaluative signals can be both abstract and task-specific and suggest a neuronal mechanism for estimating control demand.
Growth temperature is the principal driver of chromatinization in archaea
Authors: Hocher Antoine , Borrel Guillaume , Fadhlaoui Khaled , Brugère Jean-François , Gribaldo Simonetta , Warnecke Tobias
Published: July 2021

Across the tree of life, DNA in living cells is associated with proteins that coat chromosomes, constrain their structure and influence DNA-templated processes such as transcription and replication. In bacteria and eukaryotes, HU and histones, respectively, are the principal constituents of chromatin, with few exceptions. Archaea, in contrast, have more diverse repertoires of nucleoid-associated proteins (NAPs). The evolutionary and ecological drivers behind this diversity are poorly understood. Here, we combine a systematic phylogenomic survey of known and predicted NAPs with quantitative protein abundance data to shed light on the forces governing the evolution of archaeal chromatin. Our survey highlights the Diaforarchaea as a hotbed of NAP innovation and turnover. Loss of histones and Alba in the ancestor of this clade was followed by multiple lineage-specific horizontal acquisitions of DNA-binding proteins from other prokaryotes. Intriguingly, we find that one family of Diaforarchaea, the Methanomethylophilaceae, lacks any known NAPs. Comparative analysis of quantitative proteomics data across a panel of 19 archaea revealed that investment in NAP production varies over two orders of magnitude, from <0.02% to >5% of total protein. Integrating genomic and ecological data, we demonstrate that growth temperature is an excellent predictor of relative NAP investment across archaea. Our results suggest that high levels of chromatinization have evolved as a mechanism to prevent uncontrolled helix opening and runaway denaturation – rather than, for example, to globally orchestrate gene expression – with implications for the origin of chromatin in both archaea and eukaryotes.
Ultrastructural analysis of nasopharyngeal epithelial cells from patients with SARS-CoV-2 infection
Authors: Saraiva Karina Lidianne Alcântara , Vasconcelos Luydson Richardson Silva , Bezerra Matheus Filgueira , Arcoverde Rodrigo Moraes Loyo , Brandão-Filho Sinval Pinto , Ayres Constância Flávia Junqueira , de Figueiredo Regina Célia Bressan Queiroz , Pereira-Neves Antonio
Published: July 2021

The nasal epithelium is an initial site for SARS-CoV-2 infection, responsible for the ongoing COVID-19 pandemic. However, the pathogenicity and morphological impact of SARS-CoV-2 on the nasopharynx cells from symptomatic patients with different viral loads remain poorly understood. Here, we investigated the ultrastructure of nasal cells obtained from individuals at distinct disease days and with high and low SARS-CoV-2 loads. Squamous and ciliated cells were the main cells observed in SARS-CoV-2 negative samples. We identified virus-like particles (VLPs) and replication organelles (RO)-like structures in the squamous cells from high viral load samples after 3- and 4-days of symptoms. Ultrastructural changes were found in those cells, such as the loss of microvilli and primary cilium, the increase of multivesicular bodies and autophagosomes, and signs of cell death. No ciliated cells were found in those samples. Squamous cells from low viral load sample after 5 days of symptoms showed few microvilli and no primary cilium. VLPs and RO-like structures were found in the ciliated cells only. No ultrastructural alterations were seen in the cells from low viral load individuals after 10- and 14-days of symptoms. Our results shed light on the ultrastructural effects of SARS-CoV-2 infection on the human nasopharyngeal cells.
Protein structural features predict responsiveness to pharmacological chaperone treatment for three lysosomal storage disorders
Authors: Woodard Jaie , Zheng Wei , Zhang Yang
Published: July 2021

Three-dimensional structures of proteins can provide important clues into the efficacy of personalized treatment. We perform a structural analysis of variants within three inherited lysosomal storage disorders, comparing variants responsive to pharmacological chaperone treatment to those unresponsive to such treatment. We find that predicted ΔΔG of mutation is higher on average for variants unresponsive to treatment, in the case of datasets for both Fabry disease and Pompe disease, in line with previous findings. Using both a single decision tree and an advanced machine learning approach based on the larger Fabry dataset, we correctly predict responsiveness of three Gaucher disease variants, and we provide predictions for untested variants. Many variants are predicted to be responsive to treatment, suggesting that drug-based treatments may be effective for a number of variants in Gaucher disease. In our analysis, we observe dependence on a topological feature reporting on contact arrangements which is likely connected to the order of folding of protein residues, and we provide a potential justification for this observation based on steady-state cellular kinetics.

Author summary

Pharmacological chaperones are small molecule drugs that bind to proteins to help stabilize the folded state. One set of diseases for which this treatment has been effective is the lysosomal storage disorders, which are caused by defective lysosomal enzymes. However, not all genotypes are equally responsive to treatment. For instance, missense mutants that are particularly destabilized relative to WT are less likely to respond. The availability of datasets containing responsiveness data for large numbers of mutants, along with crystal structures of the protein involved in each disease, make machine learning methods incorporating sequence-based and structural data feasible. We hypothesize that data from two diseases, Fabry and Pompe disease, may be useful for predicting responsiveness of variants in the related Gaucher disease. Results suggest that many rare variants in Gaucher disease could be amenable to existing drugs. Results suggest that drug responsiveness depends on protein topology is such a way that mutations in early-to-fold residues are more likely to be non-responsive to pharmacological chaperone treatment, which is consistent with a simple kinetic model of stability rescue. This study provides an example of how machine learning can be used to inform further studies towards personalized treatment in medicine.
Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development
Authors: Cardim-Pires Thyago R. , Sant’Anna Ricardo , Foguel Debora
Published: July 2021

Fungal infection is an important public health problem afflicting more than a billion people worldwide. Mycoses are especially important in Latin America, and in Brazil in particular. Paracoccidioides is the genus of fungi responsible for paracoccidioidomycosis comprising two species, P. brasiliensis and P. lutzii. The lungs are the primary infection site, but oral mucosa and airways can also be affected. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells and is the most studied protein of P. brasiliensis. Seminal work identified a specific stretch of 15 amino acids that spans the region 181-195 (called P10) as an important epitope of gp43, being recognized by T lymphocytes in peripheral blood mononuclear cells of mice and humans and is envisioned as a potential vaccine component. Here, we show by using thioflavin T (ThT), transmission electron microscopy and other methods that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. In silico, aggregation analysis reveals several aggregation-prone regions (APR) in the P10 sequence that are capable of forming amyloid cores with steric zipper architecture. Seeds of P10 obtained by fibril mechanical fragmentation were able to induce the aggregation of P4 but not P23, as evidenced by ThT binding and mass spectrometry. These two peptides, also derived from gp43, are potent modulators of local and systemic inflammation. In-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by the action of several proteases such as proteinase K, trypsin and pepsin, which suggests that P10 could be formed upon gp43 digestion in a physiological condition. Considering our data in the context of a potential vaccine development, we redesigned the sequence of the P10 peptide, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.
Heterogeneous substrate binding in a glutamate transporter homologue
Authors: Reddy Krishna D. , Ciftci Didar , Scopelliti Amanda , Boudker Olga
Published: July 2021

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family. Recent studies revealed transport rate heterogeneity in an archaeal homologue GltPh, inconsistent with simple kinetic models, but its structural and mechanistic determinants remain undefined. In a mutant GltPh, which exclusively populates the outward-facing state, we demonstrate the co-existence of at least two sub-states in slow equilibrium binding the substrate with different apparent affinities. Wild-type GltPh shows similar binding properties, and modulation of the sub-state equilibrium correlates with transport rates. Following binding, the low-affinity sub-state of the mutant is transient. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. In contrast, an equilibrated structure does not show such classes. The structure at 2.2 Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, while the extracellular labile half sets the affinity and dynamic properties.
Effects of Ionic Strength on Gating and Permeation of TREK-2 K2P channels
Authors: Conrad Linus J , Proks Peter , Tucker Stephen J
Published: July 2021

In addition to the classical voltage-dependent behavior mediated by voltage-sensing-domains (VSD), a growing number of voltage-dependent gating behaviors are being described in ion channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+channel TREK2 responds to membrane voltage through a gating process that is mediated by the interaction of K+with its selectivity filter. Recently, we have found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found that the channel is inhibited by high internal K+concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is more complex.
CARPOOL: A library-based platform to rapidly identify next generation chimeric antigen receptors
Authors: Kyung Taeyoon , Gordon Khloe S. , Perez Caleb R. , Holec Patrick V. , Ramos Azucena , Zhang Angela Q. , Liu Yunpeng , Koch Catherine , Starchenko Alina , Joughin Brian , Lauffenburger Douglas A. , Irvine Darrell J. , Hemann Michael T. , Birnbaum Michael E.
Published: July 2021

CD19-targeted CAR therapies have successfully treated B cell leukemias and lymphomas, but many responders later relapse or experience toxicities. CAR intracellular domains (ICDs) are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3ζ, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domain combinations that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in anin vitrotumor rechallenge condition, and comparable tumor controlin vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.
Adolescent Brain Cognitive Development (ABCD) Community MRI Collection and Utilities
Authors: Feczko Eric , Conan Greg , Marek Scott , Tervo-Clemmens Brenden , Cordova Michaela , Doyle Olivia , Earl Eric , Perrone Anders , Sturgeon Darrick , Klein Rachel , Harman Gareth , Kilamovich Dakota , Hermosillo Robert , Miranda-Dominguez Oscar , Adebimpe Azeez , Bertolero Maxwell , Cieslak Matthew , Covitz Sydney , Hendrickson Timothy , Juliano Anthony C. , Snider Kathy , Moore Lucille A. , Uriartel Johnny , Graham Alice M. , Calabro Finn , Rosenberg Monica D. , Rapuano Kristina M. , Casey BJ , Watts Richard , Hagler Donald , Thompson Wesley K. , Nichols Thomas E. , Hoffman Elizabeth , Luna Beatriz , Garavan Hugh , Satterthwaite Theodore D. , Ewing Sarah Feldstein , Nagel Bonnie , Dosenbach Nico U.F. , Fair Damien A.
Published: July 2021

The Adolescent Brain Cognitive Development Study (ABCD), a 10 year longitudinal neuroimaging study of the largest population based and demographically distributed cohort of 9-10 year olds (N=11,877), was designed to overcome reproducibility limitations of prior child mental health studies. Besides the fantastic wealth of research opportunities, the extremely large size of the ABCD data set also creates enormous data storage, processing, and analysis challenges for researchers. To ensure data privacy and safety, researchers are not currently able to share neuroimaging data derivatives through the central repository at the National Data Archive (NDA). However, sharing derived data amongst researchers laterally can powerfully accelerate scientific progress, to ensure the maximum public benefit is derived from the ABCD study. To simultaneously promote collaboration and data safety, we developed the ABCD-BIDS Community Collection (ABCC), which includes both curated processed data and software utilities for further analyses. The ABCC also enables researchers to upload their own custom-processed versions of ABCD data and derivatives for sharing with the research community. This NeuroResource is meant to serve as the companion guide for the ABCC. In section we describe the ABCC. Section II highlights ABCC utilities that help researchers access, share, and analyze ABCD data, while section III provides two exemplar reproducibility analyses using ABCC utilities. We hope that adoption of the ABCC’s data-safe, open-science framework will boost access and reproducibility, thus facilitating progress in child and adolescent mental health research.
Benchmarking long-read genome sequence alignment tools for human genomics applications
Authors: LoTempio Jonathan , Délot Emmanuèle , Vilain Eric
Published: July 2021

Background

The utility of long-read genome sequencing platforms has been shown in many fields including whole genome assembly, metagenomics, and amplicon sequencing. Less clear is the applicability of long reads to reference-guided human genomics, the foundation of genomic medicine. Here, we benchmark available platform-agnostic alignment tools on datasets from nanopore and single-molecule real-time platforms to understand their suitability in producing a genome representation.

Results

For this study, we leveraged publicly-available data from sample NA12878 generated on Oxford Nanopore and sample NA24385 on Pacific Biosciences platforms. Each tool that was benchmarked, including GraphMap2, LRA, Minimap2, NGMLR, and Winnowmap2 produced the same alignment file each time. However, the different tools widely disagreed on which reads to leave unaligned, affecting the end genome coverage and the number of discoverable breakpoints. Minimap2 and winnowmap2 were computationally lightweight enough for use at scale. No alignment from one tool independently resolved all large structural variants (10,000-100,000 basepairs) present in the Database of Genome Variants (DGV) for sample NA12878 or the truthset for NA24385.

Conclusions

It should be best practice to use an analysis pipeline that generates alignments with both minimap2 and winnowmap2 as both are lightweight and yield different views of the genome. If computational resources and time are not a factor for a given case or experiment, a third representation from NGMLR will provide another view, and another chance to resolve a case. LRA, while fast, did not work on the nanopore data for our cluster, but PacBio results were promising in that those computations completed faster than Mininmap2. Graphmap2 is not an ideal tool for exploration of a whole human genome generated on a long-read sequencing platform.
Allosteric effect of nanobody binding on ligand-specific active states of the β2-Adrenergic Receptor
Authors: Chen Yue , Fleetwood Oliver , Pérez-Conesa Sergio , Delemotte Lucie
Published: July 2021

Nanobody binding stabilizes the active state of G-protein-coupled receptor (GPCR) and modulates its affinity for bound ligands. However, the atomic level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like β2 adrenergic receptor (β2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes a highly active state of the β2AR with a ~14 Å outward movement of transmembrane helix 6 and close proximity of transmembrane (TM) helices 5 and 7. This is further supported by the residues located at hotspots located on TMs 5, 6 and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intercellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding can enhance the communication between the binding sites of Nb80 and of the ligand. We identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in presence of full agonist and G-protein biased partial agonist, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which could be useful for structure-based drug discovery.

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Amino acid availability is not essential for lifespan extension by dietary restriction in the fly
Authors: Gautrey Sarah L , Simons Mirre J P
Published: July 2021

Dietary restriction (DR) is one of the most potent ways to extend health- and lifespan. Key progress in understanding the mechanisms of DR, and ageing more generally, was made when dietary protein, and more specifically essential amino acids (EAA), were identified as the key dietary component to restrict to obtain DR’s health and lifespan benefits. This role of dietary amino acids has strongly influenced work on ageing mechanisms, especially in nutrient sensing, e.g. Tor and insulin(-like) signalling networks. Experimental biology in Drosophila melanogaster has been instrumental in generating and confirming the now dominant hypothesis that EAA availability is central to ageing. Here, we expand on previous work testing the involvement of EAA in DR through large scale (N=6,238) supplementation experiments across four diets and two genotypes in female flies. Surprisingly, we find that EAA are not essential to DR’s lifespan benefits. Importantly, we do identify the fecundity benefits of EAA supplementation suggesting the supplemented EAA were bioavailable. Furthermore, we find that the effects of amino acids on lifespan vary by diet and genetic line studied and that at our most restricted diet fecundity is constrained by other nutrients than EAA. We suggest that DR for optimal health is a concert of nutritional effects, orchestrated by genetic, diet and environmental interactions. Our results question the universal importance of amino acid availability in the biology of ageing and DR.
Ecology and transmission of a dengue virus serotype 4 identified in wild Aedes aegypti in Florida
Authors: Ayers Jasmine B. , Xie Xuping , Coatsworth Heather , Stephenson Caroline J. , Waits Christy M. , Shi Pei-Yong , Dinglasan Rhoel R.
Published: July 2021

1

Dengue virus is the most prevalent mosquito-borne virus, causing approximately 390 million infections and 25,000 deaths per year. Aedes aegypti, the primary mosquito vector of dengue virus, is well established throughout the state of Florida, USA. Autochthonous transmission of dengue virus to humans in Florida has been increasing since 2009, alongside consistent importation of dengue cases. However, most cases of first infection with dengue are asymptomatic and the virus can be maintained in mosquito populations, complicating surveillance and leading to an underestimation of disease risk. Metagenomic sequencing of Aedes aegypti mosquitoes in Manatee County, Florida revealed the presence of dengue virus serotype 4 (DENV-4) genomes in mosquitoes from multiple trapping sites over 2 years, in the absence of a human DENV-4 index case and even though a locally acquired case of DENV-4 has never been reported in Florida. This finding suggested that: i) DENV-4 may circulate amongst humans undetected, ii) the virus was being maintained in the mosquito population, or iii) the detected complete genome sequence may not represent a viable virus. This study demonstrates that an infectious clone generated from the Manatee County DENV-4 (DENV-4M) sequence is capable of infecting mammalian and insect tissue culture systems, as well as adult female Aedes aegypti mosquitoes when fed in a blood meal. However, the virus is subject to a dose dependent infection barrier in mosquitoes, and has a kinetic delay compared to a phylogenetically related wild-type (WT) control virus from a symptomatic child, DENV-4H (strain Homo sapiens/Haiti-0075/2015, GenBank accession MK514144.1). DENV-4M disseminates from the midgut to the ovary and saliva at 14 days post-infection. Viral RNA was also detectable in the adult female offspring of DENV-4M infected mosquitoes. These results demonstrate that the virus is capable of infecting vector mosquitoes, is transmissible by bite, and is vertically transmitted, indicating a mechanism for maintenance in the environment without human-mosquito transmission. These findings suggest undetected human-mosquito transmission and/or long-term maintenance of the virus in the mosquito population is occurring in Florida, and underscore the importance of proactive surveillance for viruses in mosquitoes.

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In order to better assess the public health risk posed by a detection of DENV-4 RNA in Manatee County, FL Aedes aegypti, we produced an infectious clone using the sequence from the wild-caught mosquitoes and characterized it via laboratory infections of mosquitoes and mosquito tissues.
Membrane activity of a DNA-based ion channel depends on the stability of its double-stranded structure
Authors: Morzy Diana , Joshi Himanshu , Sandler Sarah E. , Aksimentiev Aleksei , Keyser Ulrich F.
Published: July 2021

Structural DNA nanotechnology has emerged as a promising method for designing spontaneously-inserting and fully-controllable synthetic ion channels. However, both insertion efficiency and stability of existing DNA-based ion channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA-lipid interactions that hinder the formation of a stable transmembrane pore. Our all-atom MD simulations and experiments show that the insertion-driving cholesterol modifications, when introduced at an end of a DNA strand, are likely to cause fraying of the terminal base pairs as the DNA nanostructure adopts its energy-minimum configuration in the membrane. We also find that fraying of base pairs distorts nicked DNA constructs when embedded in a lipid bilayer. Here, we show that DNA nanostructures that do not have discontinuities (nicks) in their DNA backbones form considerably more stable DNA-induced conductive pores and insert into lipid membranes with a higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows to design and maintain membrane-spanning helices in a tilted orientation within lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.
Gluconobacter oxydans Knockout Collection Finds Improved Rare Earth Element Extraction
Authors: Schmitz Alexa M. , Pian Brooke , Medin Sean , Reid Matthew C. , Wu Mingming , Gazel Esteban , Barstow Buz
Published: July 2021

Rare earth elements (REE) are critical components of our technological society and essential for renewable energy technologies. Traditional thermochemical processes to extract REE from mineral ores or recycled materials are costly and environmentally harmful1, and thus more sustainable extraction methods require exploration. Bioleaching offers a promising alternative to conventional REE extraction2–4, and is already used to extract 5% of the world’s gold, and ≈ 15% of the world’s copper supply5,6. However, the performance of REE bioleaching lags far behind thermochemical processes2,7–9. Despite this, to the best of our knowledge no genetic engineering strategies have yet been used to enhance REE bioleaching, and little is known of the genetics that confer this capability. Here we build a whole genome knockout collection for Gluconobacter oxydans B58, one of the most promising organisms for REE bioleaching10, and use it to comprehensively characterize the genomics of REE bioleaching. In total, we find 304 genes that notably alter production of G. oxydans’ acidic biolixiviant, including 165 that hold up under statistical comparison with wild-type. The two most impactful groups of genes involved in REE bioleaching have opposing influences on acid production and REE bioleaching. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase all but eliminates bioleaching. In contrast, disruption of the phosphate-specific transport system accelerates acid production and enhances bioleaching. We identified 6 disruption mutants, that increase bioleaching by at least 11%. Most significantly, disruption of pstC, encoding part of the phosphate-specific transporter, pstSCAB, enhances bioleaching by 18%. Taken together, these results give a comprehensive roadmap for engineering multiple sites in the genome of G. oxydans to further increase its bioleaching efficiency.