bluuebunny/crossref_metadata_2025 huggingface dataset:
nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase
Authors: Bennett Sophie P. , Torres Maria J. , Soriano-Laguna Manuel J. , Richardson David J. , Gates Andrew J. , Le Brun Nick E.
Published: September 2020

Nitrous oxide (N2O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N2OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N2OR contains two important copper cofactors (CuA and CuZ centres) that are essential for activity, and in copper-limited environments, N2OR fails to function, contributing to rising levels of atmospheric N2O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A

P. denitrificans

ΔnosX deletion mutant failed to reduce N2O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N2OR activity. N2OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the CuA and CuZ centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N2OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from

Pseudomonas stutzeri

, which functions in the flavinylation of another N2OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N2OR and cellular N2O reduction.
Clinical Performance Evaluation of a SARS-CoV-2 Rapid Antibody Test for Determining Past Exposure to SARS-CoV-2
Authors: Findeisen Peter , Stiegler Hugo , Lopez-Calle Eloisa , Schneider Tanja , Urlaub Eva , Hayer Johannes , Zemmrich Claudia
Published: September 2020

The true prevalence and population seropositivity of SARS-CoV-2 infection remains unknown, due to the number of asymptomatic infections and limited access to high-performance antibody tests. To control the COVID-19 pandemic it is crucial to understand the true seroprevalence, but not every region has access to extensive centralized PCR and serology testing. Currently available rapid antibody tests lack the accuracy needed for recommendation by health authorities. To fill this gap, we analyzed and validated the clinical performance of a new point-of-care SARS-CoV-2 Rapid Antibody Assay, a chromatographic immunoassay for qualitative detection of IgM/IgG antibodies for use in near-patient settings. Analysis was performed using 42 Anti-SARS-Cov-2 positive (CoV+) and 92 Anti-SARS-Covid-2 negative (CoV-) leftover samples from before December 2019, using the Elecsys® Anti-SARS-CoV-2 as the reference assay. Analytical specificity was tested using leftover samples from individuals with symptoms of common cold collected before December 2019. The SARS-CoV-2 Rapid Antibody Test was 100.0% (95% CI 91.59–100.00) sensitive and 96.74% (95% CI 90.77–99.32) specific with an assay failure rate of 0.00%. No cross-reactivity was observed against the common cold panel. Method comparison was additionally conducted by two external laboratories, using 100 CoV+/275 CoV-samples, also comparing whole blood versus plasma matrix. The comparison demonstrated for plasma 96.00% positive/96.36% negative percent agreement with the Elecsys Anti-SARS-CoV-2 and overall 99.20% percent agreement between whole blood and EDTA plasma. The SARS-CoV-2 Rapid Antibody Test demonstrated similar clinical performance to the manufacturer's data and to a centralized automated immunoassay, with no cross-reactivity to common cold panels.
Historical trends of the ecotoxicological pesticide risk from the main grain crops in Rolling Pampa (Argentina)
Authors: Ferraro D.O. , Ghersa F , de Paula R. , Duarte Vera A.C. , Pessah S.
Published: September 2020

We showed the results of the first long-term analysis (1987-2019) of pesticide impact in the main agricultural area of Argentina. Using a clear and meaningful tool, based not only on acute toxicity but also on scaling up the results to total sown area, we identified time trends for both total pesticide impact and the ecoefficiency of modal pesticide profiles. By the end of the time series, soybean showed a pesticide impact four times greater than maize crop in the studied area. However, the time trend in the last years showed a sustainable pattern of pesticide use, with an improvement in the ecoefficiency. Oppositely, maize showed a relatively constant ecoefficiency value during most of the time series, suggesting a possible path towards an unsustainable cropping system. Findings from this study suggest that some efforts have to be made to improve the pest management decisions towards a more efficient pesticide profiles in maize crop and to keep improving the ecotoxicity pesticide profile in soybean crops because of its large sown area in the studied area.
Locus coeruleus integrity and the effect of atomoxetine on response inhibition in Parkinson’s disease
Authors: O’Callaghan Claire , Hezemans Frank H. , Ye Rong , Rua Catarina , Jones P. Simon , Murley Alexander G. , Holland Negin , Regenthal Ralf , Tsvetanov Kamen A. , Wolpe Noham , Barker Roger A. , Williams-Gray Caroline H. , Robbins Trevor W. , Passamonti Luca , Rowe James B.
Published: September 2020

Cognitive decline is a common feature of Parkinson’s disease, and many of these cognitive deficits fail to respond to dopaminergic therapy. Therefore, targeting other neuromodulatory systems represents an important therapeutic strategy. Among these, the locus coeruleus-noradrenaline system has been extensively implicated in response inhibition deficits. Restoring noradrenaline levels using the noradrenergic reuptake inhibitor atomoxetine can improve response inhibition in some patients with Parkinson’s disease, but there is considerable heterogeneity in treatment response. Accurately predicting the patients who would benefit from therapies targeting this neurotransmitter system remains a critical goal, in order to design the necessary clinical trials with stratified patient selection to establish the therapeutic potential of atomoxetine. Here, we test the hypothesis that integrity of the noradrenergic locus coeruleus explains the variation in improvement of response inhibition following atomoxetine. In a double-blind placebo-controlled randomised crossover design, 19 people with Parkinson’s disease completed an acute psychopharmacological challenge with 40 mg of oral atomoxetine or placebo. A stop-signal task was used to measure response inhibition, with stop-signal reaction times obtained through hierarchical Bayesian estimation of an ex-Gaussian race model. Twenty-six control subjects completed the same task without undergoing the drug manipulation. In a separate session, patients and controls underwent ultra-high field 7T imaging of the locus coeruleus using a neuromelanin-sensitive magnetisation transfer sequence. The principal result was that atomoxetine improved stop-signal reaction times in those patients with lower locus coeruleus integrity. This was in the context of a general impairment in response inhibition, as patients on placebo had longer stop-signal reaction times compared to controls. We also found that the caudal portion of the locus coeruleus showed the largest neuromelanin signal decrease in the patients compared to controls. Our results highlight a link between the integrity of the noradrenergic locus coeruleus and response inhibition in Parkinson’s disease patients. Furthermore, they demonstrate the importance of baseline noradrenergic state in determining the response to atomoxetine. We suggest that locus coeruleus neuromelanin imaging offers a marker of noradrenergic capacity that could be used to stratify patients in trials of noradrenergic therapy and to ultimately inform personalised treatment approaches.
Cellular and molecular phenotypes ofC9orf72ALS/FTD patient derived iPSC-microglia mono-cultures
Authors: Lorenzini Ileana , Alsop Eric , Levy Jennifer , Gittings Lauren M , Lall Deepti , Rabichow Benjamin E , Moore Stephen , Pevey Ryan , Bustos Lynette , Burciu Camelia , Bhatia Divya , Singer Mo , Saul Justin , McQuade Amanda , Tzioras Makis , Mota Thomas A , Logemann Amber , Rose Jamie , Almeida Sandra , Gao Fen-Biao , Marks Michael , Donnelly Christopher J , Hutchins Elizabeth , Hung Shu-Ting , Ichida Justin , Bowser Robert , Spires-Jones Tara , Blurton-Jones Mathew , Gendron Tania F , Baloh Robert H , Van Keuren-Jensen Kendall , Sattler Rita
Published: September 2020

While motor and cortical neurons are affected inC9orf72ALS/FTD, it remains still largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We generatedC9orf72ALS/FTD patient-derived induced pluripotent stem cells differentiated into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons,C9orf72ALS/FTD iPSC-MG mono-cultures form G4C2repeat RNA foci, exhibit reduced C9orf72 protein levels and generate dipeptide repeat proteins. Healthy control andC9orf72iPSC-MG equivalently express microglial specific genes and display microglial functions including inflammatory cytokine release and phagocytosis of extracellular toxic cargos such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. SelectC9orf72iPSC-MG patient lines show inability to efficiently remove phagocytosed contents, suggesting dysfunction of the endosomal-lysosomal pathways. Finally, RNA sequencing revealed overall transcriptional changes in diseased microglia yet no significant differentially expressed microglial-enriched genes. These minimal differences in cellular, molecular and functional characteristics of microglial mono-cultures suggest that a diseased microenvironment is associated with microglial activation and subsequent regulation of neuronal dysfunction.
Single-cell Characterization of Acute Myeloid Leukemia and its Microenvironment Following PD-1 Blockade Based Therapy
Authors: Abbas Hussein A. , Hao Dapeng , Tomczak Katarzyna , Barrodia Praveen , Im Jin Seon , Reville Patrick K. , Alaniz Zoe , Wang Wei , Wang Ruiping , Wang Feng , Al-Atrash Gheath , Takahashi Koichi , Ning Jing , Ding Maomao , Mathews Jairo T. , Little Latasha , Zhang Jianhua , Basu Sreyashi , Konopleva Marina , Garcia-Manero Guillermo , Green Michael R. , Sharma Padmanee , Allison James P. , Kornblau Steven M. , Rai Kunal , Wang Linghua , Daver Naval , Futreal Andrew
Published: September 2020

Acute myeloid leukemia (AML) and effector cells of immune checkpoint blockade (ICB) therapy co-reside in a complex bone marrow (BM) milieu. The interplay of tumor intrinsic and microenvironment (TME) mechanisms that influences the response to ICB-based therapies in AML have not been elucidated. Here we report our analyses of single cell RNA profiling of more than 127,000 BM cells from healthy donors and relapsed/refractory (R/R) AML patients at pre/post treatment with azacitidine/nivolumab, paired with single cell T cell receptor (TCR) repertoire profiles, to uncover factors impacting response and resistance. Loss of chromosome 7/7q conferred an immunosuppressive TME and was associated with resistance to ICB-based therapy in R/R AML. Our trajectory analysis revealed a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B (GZMB) and GZMK. GZMK expression defined a BM residing memory CD8+ T cell subset with stem-like properties likely an intermediary between naïve and cytotoxic lymphocytes. Responses to ICB-based therapy were primarily driven by novel and expanded T cell clonotypes. Our findings support an adaptable T cell plasticity in response to PD-1 blockade in AML. Disentangling AML cells from their complex, immune-rich microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities.

Significance

Determining the cellular and molecular underpinnings of response and resistance to PD-1 blockade based therapy in AML can guide immune-based therapeutic strategies. Our results reveal AML intrinsic characteristics (chromosome 7/7q status and oxidative stressors) and tumor microenvironment to modulate responses to checkpoint blockers. CD8 cells exist in the bone marrow in a continuum with GZMK expression defining a memory, stem-like T cell population that could play a role in response to therapy.
Bacterial Peptidoglycan Fragments Differentially Regulate Innate Immune Signaling
Authors: Bersch Klare L. , DeMeester Kristen E. , Zagani Rachid , Wodzanowski Kimberly A. , Reinecker Hans-Christian , Grimes Catherine L.
Published: September 2020

The human innate immune system responds to both pathogen and commensal bacteria at the molecular level using bacterial peptidoglycan (PG) recognition elements. Traditionally, synthetic and commercially accessible PG monosaccharide units known as muramyl dipeptide (MDP) andN-glycolyl MDP (ng-MDP) have been used to probe the mechanism of innate immune activation of pattern recognition receptors (PRRs) such as NOD-like receptors (NLRs). However, bacterial PG is a dynamic and complex structure, with various chemical modifications and trimming mechanisms that result in the production of disaccharide containing elements. These molecules pose as attractive targets for immunostimulatory screening; however, studies are limited due to their synthetic accessibility. Inspired by disaccharide containing compounds produced from the gut microbe,Lactobacillus acidophilus, a robust and scalable chemical synthesis of PG-based disaccharide ligands was implemented. Together with a monosaccharide PG library, compounds were screened for their ability to stimulate proinflammatory genes in bone marrow derived macrophages (BMDMs). The data reveal a diverse gene induction pattern between monosaccharide and disaccharide PG units, suggesting that PG innate immune signaling is more complex than a one-activator-one pathway program, as biologically relevant fragments induce distinct transcriptional programs. These disaccharide molecules will serve as critical immunostimulatory tools to more precisely define specialized innate immune regulatory mechanisms that distinguish between commensal and pathogenic bacteria residing in the microbiome.
A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity In Mice
Authors: de Alwis Ruklanthi , Gan Esther S , Chen Shiwei , Leong Yan Shan , Tan Hwee Cheng , Zhang Summer L , Yau Clement , Matsuda Daiki , Allen Elizabeth , Hartman Paula , Park Jenny , Alayyoubi Maher , Bhaskaran Hari , Dukanovic Adrian , Bao Belle , Clemente Brenda , Vega Jerel , Roberts Scott , Gonzalez Jose A. , Sablad Marciano , Yelin Rodrigo , Taylor Wendy , Tachikawa Kiyoshi , Parker Suezanne , Karmali Priya , Davis Jared , Sullivan Sean M. , Hughes Steve G. , Chivukula Pad , Ooi Eng Eong
Published: September 2020

A self-transcribing and replicating RNA (STARR™) based vaccine (LUNAR®-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolong SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell mediated immunity produced a strong viral antigen specific CD8+T lymphocyte response. Assaying for intracellular cytokine staining for IFN-γ and IL-4 positive CD4+T helper lymphocytes as well as anti-spike glycoprotein IgG2a/IgG1 ratios supported a strong Th1 dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of Lunar-COV19 as a single dose vaccine.
Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering
Authors: Jarrell Dillon K. , Vanderslice Ethan J. , Lennon Mallory L. , Lyons Anne C. , VeDepo Mitchell C. , Jacot Jeffrey G.
Published: September 2020

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implementin vivoand introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that the simple inclusion of bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) in the fibrinogen solvent slows the degradation rate of fibrin by providing additional crosslinking. In this study, we aimed to further improve the longevity of fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. In this study, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels bothin vitro(when treated with proteases and when seeded with amniotic fluid stem cells) andin vivo(when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.
A fully defined pluripotent stem cell derived multi-liver-cell model for steatohepatitis and fibrosis
Authors: Kumar Manoj , Toprakhisar Burak , Van Haele Matthias , Antoranz Asier , Boon Ruben , Chesnais Francois , De Smedt Jonathan , Idoype Teresa Izuel , Canella Marco , Tilliole Pierre , De Boeck Jolan , Tricot Tine , Bajaj Manmohan , Ranga Adrian , Bosisio Francesca Maria , Roskams Tania , van Grunsven Leo A , Verfaillie Catherine M
Published: September 2020

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC)-derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFβ-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.
Exploring and explaining properties of motion processing in biological brains using a neural network
Authors: Rideaux Reuben , Welchman Andrew E
Published: September 2020

Visual motion perception underpins behaviours ranging from navigation to depth perception and grasping. Our limited access to biological systems constrain our understanding of how motion is processed within the brain. Here we explore properties of motion perception in biological systems by training a neural network (‘MotionNetxy’) to estimate the velocity image sequences. The network recapitulates key characteristics of motion processing in biological brains, and we use our complete access to its structure explore and understand motion (mis)perception at the computational-, neural-, and perceptual-levels. First, we find that the network recapitulates the biological response to reverse-phi motion in terms of direction. We further find that it overestimates the speed of slow reverse-phi motion while underestimating the speed of fast reverse-phi motion because of the correlation between reverse-phi motion and the spatiotemporal receptive fields tuned to motion in opposite directions. Second, we find that the distribution of spatiotemporal tuning properties in the V1 and MT layers of the network are similar to those observed in biological systems. We then show that compared to MT units tuned to fast speeds, those tuned to slow speeds primarily receive input from V1 units tuned to high spatial frequency and low temporal frequency. Third, we find that there is a positive correlation between the pattern-motion and speed selectivity of MT units. Finally, we show that the network captures human underestimation of low coherence motion stimuli, and that this is due to pooling of noise and signal motion. These findings provide biologically plausible explanations for well-known phenomena, and produce concrete predictions for future psychophysical and neurophysiological experiments.
Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS
Authors: Gregorova Pavlina , Sipari Nina H. , Sarin L. Peter
Published: September 2020

Post-transcriptional RNA modifications play an important role in cellular metabolism with homeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukemia and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9 min sample-to-sample run. To validate the performance and versatility of our method, we analyzed tRNA modification patterns of representative microorganisms from each kingdom of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae) and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homeostasis studies of RNA modification in complex biological samples.
Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides
Authors: Holgersen Erle M. , Gandhi Shreshth , Zhou Yongchao , Kim Jinkuk , Vaz Brandon , Bogojeski Jovanka , Bugno Magdalena , Shalev Zvi , Cheung-Ong Kahlin , Gonçalves João , O’Hara Matthew , Sun Mark , Kakaradov Boyko , Delong Andrew , Merico Daniele , Deshwar Amit G.
Published: September 2020

Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to mRNA and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization-driven, while differential expression events are more common and driven by other mechanisms. We further evaluated the performance of in silico screens for off-target events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR. Despite these large improvements in performance, the approach does not detect the majority of events at a false discovery rate below 99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs.
Oxidized Lipids and CD36-Mediated Lipid Peroxidation in CD8 T Cells Suppress Anti-Tumor Immune Responses
Authors: Xu Shihao , Chaudhary Omkar , Rodríguez-Morales Patricia , Sun Xiaoli , Zappasodi Roberta , Xu Ziyan , Pinto Antonio F. M. , Williams April , Chen Dan , Low Jun Siong , Farsakoglu Yagmur , Tang Wenxi , Wang Haiping , Varanasi Siva Karthik , McDonald Bryan , Tripple Victoria , Downes Michael , Evans Ronald M. , Abumrad Nada A. , Merghoub Taha , Wolchok Jedd D. , Shokhirev Maxim N. , Ho Ping-Chih , Witztum Joseph L. , Emu Brinda , Cui Guoliang , Kaech Susan M.
Published: September 2020

T cell metabolic fitness plays a pivotal role in anti-tumor immunity and metabolic deregulation causes T cell dysfunction (i.e., ‘exhaustion’) in cancer. We identify that the scavenger receptor CD36 limits anti-tumor CD8+T cell effector functions through lipid peroxidation. In murine tumors, oxidized phospholipids (OxPLs) were highly abundant and CD8+TILs increased uptake and accumulation of lipids and lipid peroxidation. Functionally ‘exhausted’ CD8+TILs substantially increased CD36 expression and CD36-deficient CD8+TILs had more robust anti-tumor activity and cytokine production than wild-type cells. We further show that CD36 promotes uptake of oxidized low-density lipoproteins (OxLDL) and induces lipid peroxidation in CD8+TILs, and OxLDL inhibits CD8+T cell functions in a CD36-dependent manner. Moreover, glutathione peroxidase 4 (GPX4) over-expression lowers lipid peroxidation and restores functionalities in CD8+TILs. These results define a key role for an oxidized lipid-CD36 axis in promoting intratumoral CD8+T cell dysfunction.
A vapor exposure method for delivering heroin alters nociception, body temperature and spontaneous activity in female and male rats
Authors: Gutierrez Arnold , Creehan Kevin M. , Taffe Michael A.
Published: September 2020

Background

The ongoing crisis related to non-medical use of opioids makes it of continued importance to understand the risk factors for opioid addiction, the behavioral and neurobiological consequences of opioid exposure and to seek potential avenues for therapy. Pre-clinical rodent models have been critical to advancing understanding of opioid consequences for decades, but have been mostly limited to drug delivery by injection or by oral dosing. Inhalation, a significant route for many human users, has not been as well-established.

Method

We adapted an e-cigarette based exposure system, previously shown efficacious for delivery of other drugs to rats, to deliver heroin vapor. Effectsin vivowere assessed in male and female Sprague-Dawley rats using a warm-water assay for anti-nociception and an implanted radiotelemetry system for evaluating changes in body temperature and spontaneous activity rate.

Results

Inhalation of vapor created by heroin 100 mg/mL in the propylene glycol (PG) vehicle significantly slowed tail-withdrawal from a 52°C water bath, bi-phasically altered activity, and increased temperature in male and female rats. Inhalation of heroin 50 mg/mL for 15 minutes produced significant effects, as the lower bound on efficacy, whereas inhalation of heroin 100 mg/mL for 30 minutes produced robust effects across all endpoints and groups.

Conclusions

This work shows that e-cigarette devices deliver psychoactive doses of heroin to rats, using concentrations of ∼50-100 mg/mL and inhalation durations of 15-30 minutes. This technique may be useful to assess the health consequences of inhaled heroin and other opioid drugs.
Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications
Authors: Drepper Friedel , Biernat Jacek , Kaniyappan Senthillvelrajan , Meyer Helmut E. , Mandelkow Eva Maria , Warscheid Bettina , Mandelkow Eckhard
Published: September 2020

Abnormal changes in the neuronal microtubule-associated protein Tau, such as hyperphosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Hyperphosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to ∼20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pmshowed overall occupancies centered at 8 Pi(± 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Phhad 14 Pi(± 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination). None of the Tau fractions self-assemble readily, arguing that Tau aggregation is not promoted by phosphorylation per se but requires additional factors.
Subcutaneous nanotherapy repurposes the immunosuppressive mechanism of rapamycin to enhance allogeneic islet graft viability
Authors: Burke Jacqueline A. , Zhang Xiaomin , Reddy Bobbala Sharan Kumar , Frey Molly A. , Fuentes Carolina Bohorquez , Haddad Helena Freire , Allen Sean D. , Richardson Reese A.K. , Ameer Guillermo A. , Scott Evan A.
Published: September 2020

Oral rapamycin administration rapamycin is plagued by poor bioavailability and wide biodistribution. Thus, this pleotropic mTOR inhibitor has a narrow therapeutic window, numerous side effects and provides inadequate transplantation protection. Parental formulation was not possible due to rapamycin’s hydrophobicity (log P 4.3). Here, we demonstrate that subcutaneous rapamycin delivery via poly(ethylene glycol)-b-poly(propylene sulfide)(PEG-b-PPS) polymersome (PS) nanocarriers modulates cellular biodistribution of rapamycin to change its immunosuppressive mechanism for enhanced efficacy while minimizing side effects. While oral rapamycin inhibits naïve T cell proliferation directly, subcutaneously administered rapamycin-loaded polymersomes (rPS) instead modulated Ly-6Clowmonocytes and tolerogenic semi-mature dendritic cells, with immunosuppression mediated by CD8+ Tregs and rare CD4+ CD8+ double-positive T cells. As PEG-b-PPS PS are uniquely non-inflammatory, background immunostimulation from the vehicle was avoided, allowing immunomodulation to be primarily attributed to rapamycin’s cellular biodistribution. Repurposing mTOR inhibition significantly improved maintenance of normoglycemia in a clinically relevant, MHC-mismatched, allogeneic, intraportal (liver) islet transplantation model. These results demonstrate the ability of engineered nanocarriers to repurpose drugs for alternate routes of administration by rationally controlling cellular biodistribution.
NHR-49/PPAR-α and HLH-30/TFEB promoteC. eleganshost defense via a flavin-containing monooxygenase
Authors: Wani Khursheed A. , Goswamy Debanjan , Taubert Stefan , Ratnappan Ramesh , Ghazi Arjumand , Irazoqui Javier E.
Published: September 2020

During bacterial infection, the host is confronted with multiple overlapping signals that are integrated at the organismal level to produce defensive host responses. How multiple infection signals are sensed by the host and how they elicit the transcription of host defense genes is much less understood at the whole-animal level than at the cellular level. The model organismCaenorhabditis elegansis known to mount transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, but the regulation of such responses is not well understood. Direct comparison ofC. elegansthat were starved or infected withStaphylococcus aureusrevealed a large infection-specific transcriptional signature. This signature was almost completely abrogated by deletion of transcription factorhlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene,fmo-2/FMO5. Deletion offmo-2/FMO5severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Moreover, the mechanism offmo-2/FMO5induction required the nuclear hormone receptor, NHR-49/PPAR-α, which inducedfmo-2/FMO5and host defense cell non-autonomously. These findings for the first time reveal an infection-specific host response toS. aureus, identify HLH-30/TFEB as its main regulator, reveal that FMOs are important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPAR-α inC. elegans, with important implications for innate host defense in higher organisms.
Correlation imputation in single cell RNA-seq using auxiliary information and ensemble learning
Authors: Gan Luqin , Vinci Giuseppe , Allen Genevera I.
Published: September 2020

Abstract

Single cell RNA sequencing is a powerful technique that measures the gene expression of individual cells in a high throughput fashion. However, due to sequencing inefficiency, the data is unreliable due to dropout events, or technical artifacts where genes erroneously appear to have zero expression. Many data imputation methods have been proposed to alleviate this issue. Yet, effective imputation can be difficult and biased because the data is sparse and high-dimensional, resulting in major distortions in downstream analyses. In this paper, we propose a completely novel approach that imputes the gene-by-gene correlations rather than the data itself. We call this method SCENA: Single cell RNA-seq Correlation completion by ENsemble learning and Auxiliary information. The SCENA gene-by-gene correlation matrix estimate is obtained by model stacking of multiple imputed correlation matrices based on known auxiliary information about gene connections. In an extensive simulation study based on real scRNA-seq data, we demonstrate that SCENA not only accurately imputes gene correlations but also outperforms existing imputation approaches in downstream analyses such as dimension reduction, cell clustering, graphical model estimation.
SS-Detect: Development and Validation of a New Strategy for Source-Based Morphometry in Multi-Scanner Studies
Authors: Ge Ruiyang , Ding Shiqing , Keeling Tyler , Honer William G. , Frangou Sophia , Vila-Rodriguez Fidel
Published: September 2020

Background and Purpose

Source-based morphometry (SBM) has been used in multi-centre studies pooling magnetic resonance imaging (MRI) data across different scanners to advance the reproducibility of neuroscience research. In the present study, we developed an analysis strategy forScanner-SpecificDetection (SS-Detect) of SBPs in multi-scanner studies, and evaluated its performance relative to a conventional strategy.

Methods

In the first experiment, the SimTB toolbox was used to generate simulated datasets mimicking twenty different scanners with common and scanner-specific SBPs. In the second experiment, we generated one simulated SBP from empirical gray matter volume (GMV) datasets from two different scanners. Moreover, we applied two strategies to compare SBPs between schizophrenia patients’ and healthy controls’ GMV from two different scanners.

Results

The outputs of the conventional strategy were limited to whole-sample-level results across all scanners; the outputs of SS-Detect included whole-sample-level and scanner-specific results. In the first simulation experiment, SS-Detect successfully estimated all simulated SBPs, including the common and scanner-specific SBPs whereas the conventional strategy detected only some of the whole-sample SBPs. The second simulation experiment showed that both strategies could detect the simulated SBP. Quantitative evaluations of both experiments demonstrated greater accuracy of the SS-Detect in estimating spatial SBPs and subject-specific loading parameters. In the third experiment, SS-Detect detected more significant between-group SBPs, and these SBPs corresponded with the results from voxel-based morphometry analysis, suggesting that SS-Detect has higher sensitivity in detecting between-group differences.

Conclusions

SS-Detect outperformed the conventional strategy and can be considered advantageous when SBM is applied to a multi-scanner study.
Inferring number of populations and changes in connectivity under the n-island model
Authors: Arredondo Armando , Mourato Beatriz , Nguyen Khoa , Boitard Simon , Rodríguez Willy , Noûs Camille , Mazet Olivier , Chikhi Lounès
Published: September 2020

Inferring the demographic history of species is one of the greatest challenges in populations genetics. This history is often represented as a history of size changes, thus ignoring population structure. Alternatively, structure is defined a priori as a population tree and not inferred. Here we propose a framework based on the IICR (Inverse Instantaneous Coalescence Rate), which can be estimated using the PSMC method of Li and Durbin (2011) for a single diploid individual. For an isolated population, the IICR matches the population size history, which is how the PSMC outputs are generally interpreted. However, it is increasingly acknowledged that the IICR is a function of the demographic model and sampling scheme. Our automated method fits observed IICR curves of diploid individuals with IICR curves obtained under piecewise-stationary symmetrical island models, in which we assume a fixed number of time periods during which gene flow is constant. We infer the number of islands, their sizes, the periods at which connectivity changes and the corresponding rates of connectivity. Validation with simulated data showed that the method can accurately recover most of the scenario parameters. Our application to a set of five human PSMCs yielded demographic histories that are in agreement with previous studies using similar methods and with recent research suggesting ancient human structure. They are in contrast with the widely accepted view of human evolution consisting of one ancestral population branching into three large continental and panmictic populations with varying degrees of connectivity and no population structure within each continent.
Mammalian ANP32A and ANP32B proteins drive alternative avian influenza virus polymerase adaptations
Authors: Peacock Thomas. P. , Sheppard Carol M. , Staller Ecco , Frise Rebecca , Swann Olivia C. , Goldhill Daniel H. , Long Jason S. , Barclay Wendy S.
Published: September 2020

ANP32 proteins, which act as influenza polymerase co-factors, vary between birds and mammals. The well-known mammalian adaptation, PB2-E627K, enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbour this adaptation. Here, we show that alternative PB2 adaptations, Q591R and D701N also allow influenza polymerase to use mammalian ANP32 proteins. PB2-E627K strongly favours use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs and horses where ANP32A proteins are more strongly pro-viral. In an experimental evolution approach, passage of avian viruses in human cells drives acquisition of PB2-E627K, but not when ANP32B is ablated. The strong pro-viral support of ANP32B for PB2-E627K maps to the LCAR region of ANP32B.
Inferring metabolic rewiring in embryonic neural development using single cell data
Authors: Jatav Shashank , Malhotra Saksham , Miller Freda D , Jha Abhishek , Goyal Sidhartha
Published: September 2020

Metabolism is intricately linked with cell fate changes. Much of this understanding comes from detailed metabolomics studies averaged across a population of cells which may be composed of multiple cell types. Currently, there are no quantitative techniques sensitive enough to assess metabolomics broadly at the single cell level. Here we presentscMetNet, a technique that interrogates metabolic rewiring at the single cell resolution and we apply it to murine embryonic development. Our method first confirms the key metabolic pathways, categorized into bioenergetic, epigenetic and biosynthetic, that change as embryonic neural stem cells differentiate and age. It then goes beyond to identify specific sub-networks, such as the cholesterol and mevalonate biosynthesis pathway, that drive the global metabolic changes during neural cortical development. Having such contextual information about metabolic rewiring provides putative mechanisms driving stem cell differentiation and identifies potential targets for regulating neural stem cell and neuronal biology.
Thermodynamic constraints are sufficient for the emergence of flux sensors in metabolism
Authors: Euler Christian Karl , Mahadevan Radhakrishnan
Published: September 2020

Metabolism is a precisely coordinated phenomenon, the apparent goal of which is to balance fluxes to maintain robust growth. However, coordinating fluxes requires information aboutrates, which is not obviously reconcilable with known regulatory mechanisms in whichconcentrationsare sensed through metabolite binding. While flux sensor examples have been characterized, the fundamental principles underlying the phenomenon in general are not well understood. Specifically, the questions of which fluxes can be sensed, and the mechanism by which they are remain open. We address this by showing that the concentrations of substrates of thermodynamically constrained reactions reflect upstream flux and therefore carry information about rates which can be propagated through regulatory interactions to control other fluxes in the network. Using fluxomic, metabolomic, and thermodynamic data inE coli, we show that the concentrations of a few metabolites in central carbon metabolism reflect their producing fluxes and demonstrate that they can transmit information about these rates because of their positions in the network and their roles as effectors.
SILAC-Based Quantitative Proteomics Identifies Multifactorial Mechanism of Oxaliplatin Resistance in Pancreatic Cancer Cells
Authors: Kim Young Eun , Kim Eun-Kyung , Song Min-Jeong , Kim Tae-Young , Jang Ho Hee , Kang Dukjin
Published: September 2020

Oxaliplatin is a commonly used chemotherapeutic drug for the treatment of pancreatic cancer. Understanding the cellular mechanisms of oxaliplatin resistance is important for developing new strategies to overcome drug resistance in pancreatic cancer. In this study, we performed a stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative proteomic analysis of oxaliplatin-resistant and sensitive pancreatic cancer PANC-1 cells. We identified 107 proteins whose expression levels changed between oxaliplatin-resistant and sensitive cells, which were involved in multiple biological processes, including DNA repair, drug response, apoptotic signalling, and the type 1 interferon signalling pathway. Notably, myristoylated alanine-rich C-kinase substrate (MARCKS) and wntless homolog protein (WLS) were upregulated in oxaliplatin-resistant cells compared to sensitive cells, as confirmed by qRT-PCR and Western blot analysis. We further demonstrated the activation of AKT and β-catenin signalling (downstream targets of MARCKS and WLS, respectively) in oxaliplatin-resistant PANC-1 cells. Additionally, we show that the siRNA-mediated suppression of both MARCKS and WLS enhanced oxaliplatin sensitivity in oxaliplatin-resistant PANC-1 cells. Taken together, our results provide insights into multiple mechanisms of oxaliplatin resistance in pancreatic cancer cells and reveal that MARCKS and WLS might be involved in the chemotherapeutic resistance in pancreatic cancer.
Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing
Authors: Aganezov Sergey , Goodwin Sara , Sherman Rachel M. , Sedlazeck Fritz J. , Arun Gayatri , Bhatia Sonam , Lee Isac , Kirsche Melanie , Wappel Robert , Kramer Melissa , Kostroff Karen , Spector David L. , Timp Winston , McCombie W. Richard , Schatz Michael C.
Published: September 2020

Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of the disease and its progression. We performed whole-genome sequencing of the SKBR3 breast cancer cell line and patient-derived tumor and normal organoids from two breast cancer patients using Illumina/10x Genomics, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT) sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings show that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long reads even at relatively low coverage (25×–30×). Furthermore, we integrated SV and CNV data into a unifying karyotype-graph structure to present a more accurate representation of the mutated cancer genomes. We find hundreds of variants within known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.