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Abstract High volume hemofiltration (HVHF) could remove from plasma inflammatory mediators involved in sepsis-associated acute kidney injury (SA-AKI). The IVOIRE trial did not show improvements of outcome and organ dysfunction using HVHF. The aim of this study was to evaluate in vitro the biological effects of plasma of patients treated by HVHF or standard volume hemofiltration (SVHF). We evaluated leukocyte adhesion, apoptosis and functional alterations of endothelial cells (EC) and tubular epithelial cells (TEC). In vitro data were correlated with plasma levels of TNF-α, Fas-Ligand (FasL), CD40-Ligand (CD40L), von Willebrand Factor (vWF) and endothelial-derived microparticles. An experimental model of in vitro hemofiltration using LPS-activated blood was established to assess cytokine mass adsorption during HVHF or SVHF. Plasma concentrations of TNF-ɑ, FasL, CD40L and von Willebrand Factor (vWF) were elevated at the start (d1h0) of both HVHF and SVHF, significantly decreased after 6 h (d1h6), remained stable after 12 h (d1h12) and then newly increased at 48 h (d3h0). Plasma levels of all these molecules were similar between HVHF- and SVHF-treated patients at all time points considered. In addition, the levels of endothelial microparticles remained always elevated, suggesting the presence of a persistent microvascular injury. Plasma from septic patients induced leukocyte adhesion on EC and TEC through up-regulation of adhesion receptors. Moreover, on EC, septic plasma induced a cytotoxic and anti-angiogenic effect. On TEC, septic plasma exerted a direct pro-apoptotic effect via Fas up-regulation and caspase activation, loss of polarity, altered expression of megalin and tight junction molecules with an impaired ability to internalize albumin. The inhibition of plasma-induced cell injury was concomitant to the decrease of TNF-α, Fas-Ligand and CD40-Ligand levels. The protective effect of both HVHF and SVHF was time-limited, since a further increase of circulating mediators and plasma-induced cell injury was observed after 48 h (d3h0). No significant difference of EC/TEC damage were observed using HVHF- or SVHF-treated plasma. The in vitro hemofiltration model confirmed the absence of a significant modulation of cytokine adsorption between HVHF and SVHF. In comparison to SVHF, HVHF did not increase inflammatory cytokine clearance and did not reverse the detrimental effects of septic plasma-induced EC and TEC injury. Further studies using adsorptive membranes are needed to evaluate the potential role of high dose convective therapies in the limitation of the harmful activity of plasma soluble factors involved in SA-AKI. Trial registration IVOIRE randomized clinical trial; ClinicalTrials.gov (NCT00241228) (18/10/2005).

Abstract Canada thistle is a pervasive perennial weed, causing challenges to agricultural and natural ecosystems globally. Although research has focused on the phenology, genetics, and control of Canada thistle, little is known about the rhizosphere microbiome and the role plant–microbe interactions play in invasion success. This study investigated the rhizosphere microbiome of Canada thistle across diverse climates, soils, and crops in the U.S. northern Great Plains. Soil and rhizosphere samples were collected and bacterial 16S and fungal ITS2 sequencing were performed to characterize the core microbiome and identify potential factors contributing to invasion success. Amplicon sequencing revealed a stable core microbiome that was detected in the Canada thistle rhizosphere across all locations. The core microbiome was dominated by the bacterial phyla Actinobacteriota and Proteobacteria and fungal phyla Ascomycota and Basidiomycota. Differential abundance analysis showed rhizosphere fungal communities were enriched in pathogen-containing genera with a 1.7-fold greater abundance of Fusaria and a 2.6-fold greater abundance of Gibberella compared to bulk soil. Predictive functional profiling showed rhizosphere communities were enriched (p

Abstract Reactivation of latent herpes simplex type 1 results in virus returning to the cornea leading to recurrent herpetic stromal keratitis (rHSK). We compare two competing models to reactivate viruses from latency, UV-B irradiation and cyclophosphamide (CP). Results revealed that while both result in corneal recrudescence, only UV-B irradiation results in rHSK. To better understand the dynamics of reactivation, we analyzed corneas for both the presence of infectious viruses and the dynamics of exposure to multiple reactivations using UV-B. We noted that multiple reactivations result in progressively worse corneal disease. We also noted that expression of IFNα and STING, surragate markers for the presence of virus, are induced by the presence of reactivated virus. Studies to determine the importance of STING to the development of HSK revealed that in the absence of STING, mice do not develop significant HSK and the magnitude of the infiltrate of CD45+ cells in these corneas is significantly reduced. The resulting paucity of CD45+CD11b+GR-1+F4/80-neutrophils, and to a lesser extent CD45+CD11b+GR-1-F4/80+ macrophages in B6-STING KO mice following reactivation is likely the underlying cause for lack of rHSK as has been noted by ourselves and others. These results underscore the critical importance of STING’s role in developing rHSK.

Abstract Structural domains and domain walls, inherent in single crystalline perovskite oxides, can significantly influence the properties of the material and therefore must be considered as a vital part of the design of the epitaxial oxide thin films. We employ 4D-STEM combined with machine learning (ML) to comprehensively characterize domain structures at both high spatial resolution and over a significant spatial extent. Using orthorhombic LaFeO3 as a model system, we explore the application of unsupervised and supervised ML in domain mapping, which demonstrates robustness against experiment uncertainties. The results reveal the consequential formation of multiple domains due to the structural degeneracy when LaFeO3 film is grown on cubic SrTiO3. In situ annealing of the film shows the mechanism of domain coarsening that potentially links to phase transition of LaFeO3 at high temperatures. Moreover, synthesis of LaFeO3 on DyScO3 illustrates that a less symmetric orthorhombic substrate inhibits the formation of domain walls, thereby contributing to the mitigation of structural degeneracy. High fidelity of our approach also highlights the potential for the domain mapping of other complicated materials and thin films.

Abstract The cardiac responses to vagus nerve stimulation (VNS) are still not fully understood, partly due to uncontrollable confounders in the in-vivo experimental condition. Therefore, an ex-vivo Langendorff-perfused rabbit heart with intact vagal innervation is proposed to study VNS in absence of cofounding anesthetic or autonomic influences. The feasibility to evoke chronotropic responses through electrical stimulation ex-vivo was studied in innervated isolated rabbit hearts (n = 6). The general nerve excitability was assessed through the ability to evoke a heart rate (HR) reduction of at least 5 bpm (physiological threshold). The excitability was quantified as the charge needed for a 10-bpm HR reduction. The results were compared to a series of in-vivo experiments rabbits (n = 5). In the ex-vivo isolated heart, the baseline HR was about 20 bpm lower than in-vivo (158 ± 11 bpm vs 181 ± 19 bpm). Overall, the nerve remained excitable for about 5 h ex-vivo. The charges required to reduce HR by 5 bpm were 9 ± 6 µC and 549 ± 370 µC, ex-vivo and in-vivo, respectively. The charges needed for a 10-bpm HR reduction, normalized to the physiological threshold were 1.78 ± 0.8 and 1.22 ± 0.1, in-vivo and ex-vivo, respectively. Overall, the viability of this ex-vivo model to study the acute cardiac effects of VNS was demonstrated.

Abstract Understanding the interplay between nanoparticles (NPs) and cells is essential to designing more efficient nanomedicines. Previous research has shown the role of the cell cycle having impact on the efficiency of cellular uptake and accumulation of NPs. However, there is a limited investigation into the biological fate of NPs in cells that are permanently withdrawn from the cell cycle. Here we utilize senescent WI-38 fibroblasts, which do not divide and provide a definitive model for tracking the biological fate of silica nanoparticles (SiNPs) independent of cell cycle. We use several methods to measure the cellular uptake kinetics and intracellular retention of SiNPs, including confocal laser scanning microscopy (CLSM), flow cytometry, and transmission electron microscopy (TEM). We demonstrate that SiNPs readily enter into senescent cells. Once internalized, SiNPs do not exit and accumulate in the cytoplasm for long term. Our study provides a basis for future development of NP-based tools that can detect and target senescent cells for therapy.

Abstract Recurrent influenza epidemics and pandemic potential are significant risks to global health. Public health authorities use clinical surveillance to locate and monitor influenza and influenza-like cases and outbreaks to mitigate hospitalizations and deaths. Currently, global integration of clinical surveillance is the only reliable method for reporting influenza types and subtypes to warn of emergent pandemic strains. The utility of wastewater surveillance (WWS) during the COVID-19 pandemic as a less resource intensive replacement or complement for clinical surveillance has been predicated on analyzing viral fragments in wastewater. We show here that influenza virus targets are stable in wastewater and partitions favorably to the solids fraction. By quantifying, typing, and subtyping the virus in municipal wastewater and primary sludge during a community outbreak, we forecasted a citywide flu outbreak with a 17-day lead time and provided population-level viral subtyping in near real-time to show the feasibility of influenza virus WWS at the municipal and neighbourhood levels in near real time using minimal resources and infrastructure.

Abstract Variability in response to short-acting β2-agonists (e.g., albuterol) among patients with asthma from diverse racial/ethnic groups may contribute to asthma disparities. We sought to identify genetic variants associated with bronchodilator response (BDR) to identify potential mechanisms of drug response and risk factors for worse asthma outcomes. Genome-wide association studies of bronchodilator response (BDR) were performed using TOPMed Whole Genome Sequencing data of the Asthma Translational Genomic Collaboration (ATGC), which corresponded to 1136 Puerto Rican, 656 Mexican and 4337 African American patients with asthma. With the population-specific GWAS results, a trans-ethnic meta-analysis was performed to identify BDR-associated variants shared across the three populations. Replication analysis was carried out in three pediatric asthma cohorts, including CAMP (Childhood Asthma Management Program; n = 560), GACRS (Genetics of Asthma in Costa Rica Study; n = 967) and HPR (Hartford-Puerto Rico; n = 417). A genome-wide significant locus (rs35661809; P = 3.61 × 10–8) in LINC02220, a non-coding RNA gene, was identified in Puerto Ricans. While this region was devoid of protein-coding genes, capture Hi-C data showed a distal interaction with the promoter of the DNAH5 gene in lung tissue. In replication analysis, the GACRS cohort yielded a nominal association (1-tailed P

Abstract The human microbiome is comprised of a complex and diverse community of organisms that is subject to dynamic changes over time. As such, cross-sectional studies of the microbiome provide a multitude of information for a specific body site at a particular time, but they fail to account for temporal changes in microbial constituents resulting from various factors. To address this shortcoming, longitudinal research studies of the human microbiome investigate the influence of various factors on the microbiome of individuals within a group or community setting. These studies are vital to address the effects of host and/or environmental factors on microbiome composition as well as the potential contribution of microbiome members during the course of an infection. The relationship between microbial constituents and disease development has been previously explored for skin and soft tissue infections (SSTIs) within congregate military trainees. Accordingly, approximately 25% of the population carries Staphylococcus aureus within their nasal cavity, and these colonized individuals are known to be at increased risk for SSTIs. To examine the evolution of the nasal microbiota of U.S. Army Infantry trainees, individuals were sampled longitudinally from their arrival at Fort Benning, Georgia, until completion of their training 90 days later. These samples were then processed to determine S. aureus colonization status and to profile the nasal microbiota using 16S rRNA gene-based methods. Microbiota stability differed dramatically among the individual trainees; some subjects exhibited great stability, some subjects showed gradual temporal changes and some subjects displayed a dramatic shift in nasal microbiota composition. Further analysis utilizing the available trainee metadata suggests that the major drivers of nasal microbiota stability may be S. aureus colonization status and geographic origin of the trainees. Nasal microbiota evolution within the congregate setting imposed by military training is a complex process that appears to be affected by numerous factors. This finding may indicate that future campaigns to prevent S. aureus colonization and future SSTIs among high-risk military trainees may require a ‘personalized’ approach.

Abstract Single domain antibodies (sdAbs), also called nanobodies, have substantial biophysical advantages over conventional antibodies and are increasingly being employed as components of immunotherapeutic agents. One particularly favorable property is the ability to link different sdAbs into heteromultimers. This feature allows production of single molecules capable of simultaneously targeting more than one antigen. In addition, cooperative binding of multiple linked sdAbs to non-overlapping epitopes on the same target can produce synergistic improvements in target affinity, variant specificity, and in vivo potencies. Here we seek to test the option of increased component sdAbs in these heteromultimers by testing different sdAb heterohexamers in which each of the six camelid sdAb components (VHHs) can neutralize one of three different Botulinum neurotoxin (BoNT) serotypes, A, B or E. Each heterohexamer bound all three targeted BoNT serotypes and protected mice from at least 100 MIPLD50 of each serotype. To test the potential of mRNA therapeutics encoding long sdAb heteromultimers, one heterohexamer was encoded as replicating RNA (repRNA), formulated with a cationic nanocarrier, and delivered to mice via intramuscular injection. Heterohexamer antitoxin serum expression levels were easily detected by 8 h post-treatment, peaked at 5–10 nM around two days, and persisted for more than three days. Mice treated with the formulated repRNA one day post-treatment survived challenge with 100 MIPLD50 of each toxin serotype, demonstrating the function of all six component VHHs. Use of long sdAb multimers, administered as proteins or repRNA, offer the potential for substantially improved versatility in the development of antibody-based therapeutics.

Abstract Resveratrol is a polyphenol that has been well studied and has demonstrated anti-viral and anti-inflammatory properties that might mitigate the effects of COVID-19. Outpatients (N = 105) were recruited from central Ohio in late 2020. Participants were randomly assigned to receive placebo or resveratrol. Both groups received a single dose of Vitamin D3 which was used as an adjunct. The primary outcome measure was hospitalization within 21 days of symptom onset; secondary measures were ER visits, incidence of pneumonia, and incidence of pulmonary embolism. Five patients chose not to participate after randomization. Twenty-one-day outcome was determined of all one hundred participants (mean [SD] age 55.6 [8.8] years; 61% female). There were no clinically significant adverse events attributed to resveratrol. Outpatients in this phase 2 study treated with resveratrol had a lower incidence compared to placebo of: hospitalization (2% vs. 6%, RR 0.33, 95% CI 0.04–3.10), COVID-19 related ER visits (8% vs. 14%, RR 0.57, 95% CI 0.18–1.83), and pneumonia (8% vs. 16%, RR 0.5, 95% CI 0.16–1.55). One patient (2%) in each group developed pulmonary embolism (RR 1.00, 95% CI: 0.06–15.55). This underpowered study was limited by small sample size and low incidence of primary adverse events consequently the results are statistically similar between treatment arms. A larger trial could determine efficacy. Trial Registrations: ClinicalTrials.gov NCT04400890 26/05/2020; FDA IND #150033 05/05/2020.

Abstract The acute respiratory distress syndrome (ARDS) is a life-threatening clinical condition. The number of ARDS cases has risen dramatically recently but specific treatment options are limited. ARDS is associated with an overshooting inflammatory response and neutrophils play a central role in its pathogenesis. Neutrophils express the integrin Mac-1 on their surface which adopts a resting and activated conformation depending on leukocyte activation. The aim of this study was to investigate the anti-inflammatory effects of the unique activation-specific anti-Mac-1 DARPin ‘F7’ in a mouse model of ARDS. ARDS was induced by intratracheal lipopolysaccharide (LPS) instillation and the acute (day 1–4) and chronic phase (day 5–10) were studied. After expression and purification, F7, a control DARPin and PBS, were applied daily via the intraperitoneal route. Survival and weight loss were recorded. Histological analysis of lung sections, flow cytometric leukocyte analysis of blood and bronchioalveolar lavage (BALF) were performed. Moreover, protein concentration and cytokine levels were determined in the BALF. Treatment with F7 improved survival and reduced weight loss significantly compared to treatment with the control DARPin or PBS. Neutrophil count in the BALF and peripheral blood were significantly reduced in mice treated with F7. Histology revealed significantly reduced pulmonary inflammation in the F7 treated group. Treatment with DARPin F7 inhibited neutrophil accumulation, reduced signs of local and systemic inflammation and improved survival in a mouse model of ARDS. F7 may be a novel anti-inflammatory drug candidate for the treatment of severe ARDS.

Abstract Genetic variants that regulate lipid phosphate phosphatase 3 (LPP3) expression are risk factors for the development of atherosclerotic cardiovascular disease. LPP3 is dynamically upregulated in the context of vascular inflammation with particularly heightened expression in smooth muscle cells (SMC), however, the impact of LPP3 on vascular pathology is not fully understood. We investigated the role of LPP3 and lysophospholipid signaling in a well-defined model of pathologic aortic injury and observed Angiotensin II (Ang II) increases expression of PLPP3 in SMCs through nuclear factor kappa B (NF-κB) signaling Plpp3 global reduction (Plpp3 +/− ) or SMC-specific deletion (SM22-Δ) protects hyperlipidemic mice from AngII-mediated aneurysm formation. LPP3 expression regulates SMC differentiation state and lowering LPP3 levels promotes a fibroblast-like phenotype. Decreased inactivation of bioactive lysophosphatidic acid (LPA) in settings of LPP3 deficiency may underlie these phenotypes because deletion of LPA receptor 4 in mice promotes early aortic dilation and rupture in response to AngII. LPP3 expression and LPA signaling influence SMC and vessel wall responses that are important for aortic dissection and aneurysm formation. These findings could have important implications for therapeutics targeting LPA metabolism and signaling in ongoing clinical trials.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in a variety of clinical symptoms ranging from no or mild to severe disease. Currently, there are multiple postulated mechanisms that may push a moderate to severe disease into a critical state. Human serum contains abundant evidence of the immune status following infection. Cytokines, chemokines, and antibodies can be assayed to determine the extent to which a patient responded to a pathogen. We examined serum and plasma from a cohort of patients infected with SARS-CoV-2 early in the pandemic and compared them to negative-control sera. Cytokine and chemokine concentrations varied depending on the severity of infection, and antibody responses were significantly increased in severe cases compared to mild to moderate infections. Neutralization data revealed that patients with high titers against an early 2020 SARS-CoV-2 isolate had detectable but limited neutralizing antibodies against the emerging SARS-CoV-2 Alpha, Beta and Delta variants. This study highlights the potential of re-infection for recovered COVID-19 patients.

Abstract Widespread adoption of MON 87701 × MON 89788 soybean, expressing Cry1Ac Bt protein and glyphosate tolerance, has been observed in Brazil. A proactive program was implemented to phenotypically and genotypically monitor Cry1Ac resistance in Chrysodeixis includens (Walker). Recent cases of unexpected injury in MON 87701 × MON 89788 soybean were investigated and a large-scale sampling of larvae on commercial soybean fields was performed to assess the efficacy of this technology and the distribution of lepidopteran pests in Brazil. No significant shift in C. includens susceptibility to Cry1Ac was observed eight years after commercial introduction of this technology in Brazil. F2 screen results confirmed that the frequency of Cry1Ac resistance alleles remains low and stable in C. includens. Unexpected injury caused by Rachiplusia nu (Guenée) and Crocidosema aporema (Walsingham) in MON 87701 × MON 89788 soybean was detected during the 2020/21 season, and studies confirmed a genetically based alteration in their susceptibility to Cry1Ac. MON 87701 × MON 89788 soybean remains effective against Anticarsia gemmatalis (Hübner), C. includens, Chloridea virescents (Fabricius) and Helicoverpa armigera (Hübner) in Brazil. However, there is evidence of field-evolved resistance to MON 87701 × MON 89788 soybean by the secondary soybean pests R. nu and C. aporema.

Abstract Tooling is associated with complex cognitive abilities, occurring most regularly in large-brained mammals and birds. Among birds, self-care tooling is seemingly rare in the wild, despite several anecdotal reports of this behaviour in captive parrots. Here, we show that Bruce, a disabled parrot lacking his top mandible, deliberately uses pebbles to preen himself. Evidence for this behaviour comes from five lines of evidence: (i) in over 90% of instances where Bruce picked up a pebble, he then used it to preen; (ii) in 95% of instances where Bruce dropped a pebble, he retrieved this pebble, or replaced it, in order to resume preening; (iii) Bruce selected pebbles of a specific size for preening rather than randomly sampling available pebbles in his environment; (iv) no other kea in his environment used pebbles for preening; and (v) when other individuals did interact with stones, they used stones of different sizes to those Bruce preened with. Our study provides novel and empirical evidence for deliberate self-care tooling in a bird species where tooling is not a species-specific behaviour. It also supports claims that tooling can be innovated based on ecological necessity by species with sufficiently domain-general cognition.

Abstract Grain size is a key agronomic trait that contributes to grain yield in hexaploid wheat. Grain length and width were evaluated in an international collection of 157 wheat accessions. These accessions were genetically characterized using a genotyping-by-sequencing (GBS) protocol that produced 73,784 single nucleotide polymorphism (SNP) markers. GBS-derived genotype calls obtained on Chinese Spring proved extremely accurate when compared to the reference (> 99.9%) and showed > 95% agreement with calls made at SNP loci shared with the 90 K SNP array on a subset of 71 Canadian wheat accessions for which both types of data were available. This indicates that GBS can yield a large amount of highly accurate SNP data in hexaploid wheat. The genetic diversity analysis performed using this set of SNP markers revealed the presence of six distinct groups within this collection. A GWAS was conducted to uncover genomic regions controlling variation for grain length and width. In total, seven SNPs were found to be associated with one or both traits, identifying three quantitative trait loci (QTLs) located on chromosomes 1D, 2D and 4A. In the vicinity of the peak SNP on chromosome 2D, we found a promising candidate gene (TraesCS2D01G331100), whose rice ortholog (D11) had previously been reported to be involved in the regulation of grain size. These markers will be useful in breeding for enhanced wheat productivity.

Abstract With polygenic risk score (PRS) for autoimmune type 1 diabetes (T1D), this study identified T1D cases with low T1D PRS and searched for susceptibility loci in these cases. Our hypothesis is that genetic effects (likely mediated by relatively rare genetic variants) of non-mainstream (or non-autoimmune) T1D might have been diluted in the previous studies on T1D cases in general. Two cohorts for the PRS modeling and testing respectively were included. The first cohort consisted of 3302 T1D cases and 6181 controls, and the independent second cohort consisted of 3297 T1D cases and 6169 controls. Cases with low T1D PRS were identified using PRSice-2 and compared to controls with low T1D PRS by genome-wide association (GWA) test. Thirteen novel genetic loci with high imputation quality (Quality Score r2 > 0.91) were identified of SNPs/SNVs associated with low PRS T1D at genome-wide significance (P ≤ 5.0 × E−08), in addition to 4 established T1D loci, 3 reported loci by our previous study, as well as 9 potential novel loci represented by rare SNVs, but with relatively low imputation quality (Quality Score r2

Abstract P2Y12 blockade improves patient outcomes after myocardial infarction. As well as antithrombotic effects, anti-inflammatory effects may contribute to this beneficial clinical outcome. Here we aimed to identify potential anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages. Using flow cytometry, migration assays, flow chambers and RNA microarrays, we investigated the effects of adenosine diphosphate (ADP) and P2Y12 receptor blockers on blood monocytes, THP-1 monocytes and THP-1 monocytes after differentiation to macrophages. P2Y12 -expressing platelets can form aggregates with monocytes in circulating blood. Mediated by platelets, ADP results in activation of the integrin receptor Mac-1 on blood monocytes, as detected by the conformation-specific single-chain antibody MAN-1. Via the same association with platelets, THP-1 monocyte adhesion to the endothelial intercellular adhesion molecule 1 (ICAM-1) is induced by ADP. P2Y12 receptor blockers prevent these ADP effects on monocytes. Interestingly, in contrast to THP-1 monocytes, THP-1 monocytes, after differentiation to macrophages, directly expressed the P2Y12 receptor and consequently ADP was found to be a potent chemoattractant. Again, P2Y12 receptor blockers antagonised this effect. Accordingly, stimulation of THP-1 macrophages with ADP caused a substantial change in gene expression pattern and upregulation of several genes associated with inflammation and atherogenesis. These data establish novel anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages, which are expected to contribute to cardiovascular risk reduction.

Abstract The soybean technology MON 87701 × MON 89788, expressing Cry1Ac and conferring tolerance to glyphosate, has been widely adopted in Brazil since 2013. However, pest shifts or resistance evolution could reduce the benefits of this technology. To assess Cry1Ac soybean performance and understand the composition of lepidopteran pest species attacking soybeans, we implemented large-scale sampling of larvae on commercial soybean fields during the 2019 and 2020 crop seasons to compare with data collected prior to the introduction of Cry1Ac soybeans. Chrysodeixis includens was the main lepidopteran pest in non-Bt fields. More than 98% of larvae found in Cry1Ac soybean were Spodoptera spp., although the numbers of Spodoptera were similar between Cry1Ac soybean and non-Bt fields. Cry1Ac soybean provided a high level of protection against Anticarsia gemmatalis, C. includens, Chloridea virescens and Helicoverpa spp. Significant reductions in insecticide sprays for lepidopteran control in soybean were observed from 2012 to 2019. Our study showed that C. includens and A. gemmatalis continue to be primary lepidopteran pests of soybean in Brazil and that Cry1Ac soybean continues to effectively manage the target lepidopteran pests. However, there was an increase in the relative abundance of non-target Spodoptera spp. larvae in both non-Bt and Cry1Ac soybeans.

Abstract Naïve individuals of some bird species can rapidly solve vertical string-pulling tasks with virtually no errors. This has led to various hypotheses being proposed which suggest that birds mentally simulate the effects of their actions on strings. A competing embodied cognition hypothesis proposes that this behaviour is instead modulated by perceptual-motor feedback loops, where feedback of the reward moving closer acts as an internal motivator for functional behaviours, such as pull-stepping. To date, the kea parrot has produced some of the best performances of any bird species at string-pulling tasks. Here, we tested the predictions of the four leading hypotheses for the cognition underpinning bird string-pulling by presenting kea with a horizontal connectivity task where only one of two loose strings was connected to the reward, both before and after receiving perceptual-motor feedback experience. We find that kea fail the connectivity task both before and after perceptual-motor feedback experience, suggesting not only that kea do not mentally simulate their string-pulling actions, but also that perceptual-motor feedback alone is insufficient in eliciting successful performance in the horizontal connectivity task. This suggests a more complex interplay of cognitive factors underlies this iconic example of animal problem-solving.

Abstract Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.

Abstract Nicotinic acetylcholine receptors (nAChRs) play a critical role in the neuropharmacology of learning and memory. As such, naturally occurring alkaloids that regulate nAChR activity have gained interest for understanding and potentially improving memory function. In this study, we tested the acute effects of three known nicotinic alkaloids, nicotine, cotinine, and anatabine, in suppressing scopolamine-induced memory deficit in rodents by using two classic memory paradigms, Y-maze and novel object recognition (NOR) in mice and rats, respectively. We found that all compounds were able to suppress scopolamine-induced spatial memory deficit in the Y-maze spontaneous alternation paradigm. However, only nicotine was able to suppress the short-term object memory deficit in NOR, despite the higher doses of cotinine and anatabine used to account for their potential differences in nAChR activity. These results indicate that cotinine and anatabine can uniquely regulate short-term spatial memory, while nicotine seems to have more robust and general role in memory regulation in rodents. Thus, nAChR-activating alkaloids may possess distinct procognitive properties in rodents, depending on the memory types examined.

Abstract Intercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and non-excitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g., cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells (e.g., cardiomyocytes) that are light-insensitive. Cell–cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. Its potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modelling and proper calibration. Lastly, the sensitivity of OptoGap to intrinsic cell-scale excitability is robustly characterized via computational analysis.

Abstract An accurate model of the factors that contribute to individual differences in reading ability depends on data collection in large, diverse and representative samples of research participants. However, that is rarely feasible due to the constraints imposed by standardized measures of reading ability which require test administration by trained clinicians or researchers. Here we explore whether a simple, two-alternative forced choice, time limited lexical decision task (LDT), self-delivered through the web-browser, can serve as an accurate and reliable measure of reading ability. We found that performance on the LDT is highly correlated with scores on standardized measures of reading ability such as the Woodcock-Johnson Letter Word Identification test (r = 0.91, disattenuated r = 0.94). Importantly, the LDT reading ability measure is highly reliable (r = 0.97). After optimizing the list of words and pseudowords based on item response theory, we found that a short experiment with 76 trials (2–3 min) provides a reliable (r = 0.95) measure of reading ability. Thus, the self-administered, Rapid Online Assessment of Reading ability (ROAR) developed here overcomes the constraints of resource-intensive, in-person reading assessment, and provides an efficient and automated tool for effective online research into the mechanisms of reading (dis)ability.

Abstract Juvenile idiopathic arthritis (JIA) is one of the most common chronic diseases in children. While clinical outcomes for patients with juvenile JIA have improved, the underlying biology of the disease and mechanisms underlying therapeutic response/non-response are poorly understood. We have shown that active JIA is associated with distinct transcriptional abnormalities, and that the attainment of remission is associated with reorganization of transcriptional networks. In this study, we used a multi-omics approach to identify mechanisms driving the transcriptional abnormalities in peripheral blood CD4+ T cells of children with active JIA. We demonstrate that active JIA is associated with alterations in CD4+ T cell chromatin, as assessed by ATACseq studies. However, 3D chromatin architecture, assessed by HiChIP and simultaneous mapping of CTCF anchors of chromatin loops, reveals that normal 3D chromatin architecture is largely preserved. Overlapping CTCF binding, ATACseq, and RNAseq data with known JIA genetic risk loci demonstrated the presence of genetic influences on the observed transcriptional abnormalities and identified candidate target genes. These studies demonstrate the utility of multi-omics approaches for unraveling important questions regarding the pathobiology of autoimmune diseases.

Abstract The imidazolium compound YM155, first discovered as a potent inhibitor of Survivin, effectively kills many carcinomas in preclinical models. However, the upstream signaling mechanism triggered by YM155 remains unclear. Here we studied early signaling responses in vitro in prostate and renal cancer cell lines in a dose-dependent manner. We found that YM155 rapidly activates the retinoblastoma protein, correlating with the loss of expression of all three Cyclin Ds. Using Western blot, various selective chemical inhibitors and q-PCR, we show that YM155-mediated decrease in protein levels of Cyclin Ds, Survivin and Mcl-1 is independent of transcription or proteasomal control mechanisms. Moreover, we provide the first evidence that YM155 changes the phosphorylation status of known mTOR-target proteins involved in translational control, namely ribosomal protein S6 (rS6) and 4E-BP1. Our data support that YM155 achieves this by blocking mTORC1 via the phosphorylation of Raptor at S792 through activated AMPKα (T172). Furthermore, we also used a polysome profile, supporting that YM155 markedly suppresses cap-dependent translation of mRNAs which include Survivin, Cyclin D1 and Mcl-1. We provide the first evidence that YM155 functions as a potent activator of AMPKα, a robust suppressor of mTORC1 and an attenuator of global protein synthesis.

Abstract Males of hymenopteran insects, which include ants, bees and wasps, develop as haploids from unfertilized eggs. In order to accommodate their lack of homologous chromosome pairs, some hymenopterans such as the honeybee have been shown to produce haploid sperm through an abortive meiosis. We employed microscopic approaches to visualize landmark aspects of spermatogenesis in the jewel wasp Nasonia vitripennis, a model for hymenopteran reproduction and development. Our work demonstrates that N. vitripennis, like other examined hymenopterans, exhibits characteristics indicative of an abortive meiosis, including slight enlargement of spermatocytes preceding meiotic initiation. However, we saw no evidence of cytoplasmic buds containing centrioles that are produced from the first abortive meiotic division, which occurs in the honeybee. In contrast to other previously studied hymenopterans, N. vitripennis males produce sperm in bundles that vary widely from 16 to over 200, thus reflecting a range of cellular divisions. Our results highlight interesting variations in spermatogenesis among the hymenopteran insects, and together with previous studies, they suggest a pattern of progression from meiosis to a more mitotic state in producing sperm.

Abstract Suprachiasmatic nucleus circadian oscillatory protein (SCOP) (a.k.a. PHLPP1) regulates long-term memory consolidation in the brain. Using a mouse model of controlled cortical impact (CCI) we tested if (1) brain tissue levels of SCOP/PHLPP1 increase after a traumatic brain injury (TBI), and (2) if SCOP/PHLPP1 gene knockout (KO) mice have improved (or worse) neurologic outcomes. Blood chemistry (pH, pCO2, pO2, pSO2, base excess, sodium bicarbonate, and osmolarity) and arterial pressure (MAP) differed in isoflurane anesthetized WT vs. KOs at baseline and up to 1 h post-injury. CCI injury increased cortical/hippocampal SCOP/PHLPP1 levels in WTs 7d and 14d post-injury. Injured KOs had higher brain tissue levels of phosphorylated AKT (pAKT) in cortex (14d post-injury), and higher levels of phosphorylated MEK (pMEK) in hippocampus (7d and 14d post-injury) and in cortex (7d post-injury). Consistent with an important role of SCOP/PHLPP1 on memory function, injured-KOs had near normal performance on the probe trial of the Morris water maze, whereas injured-WTs were impaired. CA1/CA3 hippocampal survival was lower in KOs vs. WTs 24 h post-injury but equivalent by 7d. No difference in 21d cortical lesion volume was detected. SCOP/PHLPP1 overexpression in cultured rat cortical neurons had no effect on 24 h cell death after a mechanical stretch-injury.

Abstract Botulinum neurotoxins (BoNTs) are highly potent toxins that cleave neuronal SNARE proteins required for neurotransmission, causing flaccid paralysis and death by asphyxiation. Currently, there are no clinical treatments to delay or reverse BoNT-induced blockade of neuromuscular transmission. While aminopyridines have demonstrated varying efficacy in transiently reducing paralysis following BoNT poisoning, the precise mechanisms by which aminopyridines symptomatically treat botulism are not understood. Here we found that activity-dependent potentiation of presynaptic voltage-gated calcium channels (VGCCs) underlies 3,4-diaminopyridine (3,4-DAP)-mediated rescue of neurotransmission in central nervous system synapses and mouse diaphragm neuromuscular junctions fully intoxicated by BoNT serotype A. Combinatorial treatments with 3,4-DAP and VGCC agonists proved synergistic in restoring suprathreshold endplate potentials in mouse diaphragms fully intoxicated by BoNT/A. In contrast, synapses fully intoxicated by BoNT serotypes D or E were refractory to synaptic rescue by any treatment. We interpret these data to propose that increasing the duration or extent of VGCC activation prolongs the opportunity for low-efficiency fusion by fusogenic complexes incorporating BoNT/A-cleaved SNAP-25. The identification of VGCC agonists that rescue neurotransmission in BoNT/A-intoxicated synapses provides compelling evidence for potential therapeutic utility in some cases of human botulism.

Abstract Beyond classic “allergic”/atopic comorbidities, atopic dermatitis (AD) emerges as systemic disease with increased cardiovascular risk. To better define serum inflammatory and cardiovascular risk proteins, we used an OLINK high-throughput proteomic assay to analyze moderate-to-severe AD (n = 59) compared to psoriasis (n = 22) and healthy controls (n = 18). Compared to controls, 10 proteins were increased in serum of both diseases, including Th1 (IFN-γ, CXCL9, TNF-β) and Th17 (CCL20) markers. 48 proteins each were uniquely upregulated in AD and psoriasis. Consistent with skin expression, AD serum showed up-regulation of Th2 (IL-13, CCL17, eotaxin-1/CCL11, CCL13, CCL4, IL-10), Th1 (CXCL10, CXCL11) and Th1/Th17/Th22 (IL-12/IL-23p40) responses. Surprisingly, some markers of atherosclerosis (fractalkine/CX3CL1, CCL8, M-CSF, HGF), T-cell development/activation (CD40L, IL-7, CCL25, IL-2RB, IL-15RA, CD6) and angiogenesis (VEGF-A) were significantly increased only in AD. Multiple inflammatory pathways showed stronger enrichment in AD than psoriasis. Several atherosclerosis mediators in serum (e.g. E-selectin, PI3/elafin, CCL7, IL-16) correlated with SCORAD, but not BMI. Also, AD inflammatory mediators (e.g. MMP12, IL-12/IL-23p40, CXCL9, CCL22, PI3/Elafin) correlated between blood and lesional as well as non-lesional skin. Overall, the AD blood signature was largely different compared to psoriasis, with dysregulation of inflammatory and cardiovascular risk markers, strongly supporting its systemic nature beyond atopic/allergic association.

Abstract Mammals host diverse bacterial and archaeal symbiont communities (i.e. microbiomes) that play important roles in digestive and immune system functioning, yet cetacean microbiomes remain largely unexplored, in part due to sample collection difficulties. Here, fecal samples from stranded pygmy (Kogia breviceps) and dwarf (K. sima) sperm whales were used to characterize the gut microbiomes of two closely-related species with similar diets. 16S rRNA gene sequencing revealed diverse microbial communities in kogiid whales dominated by Firmicutes and Bacteroidetes. Core symbiont taxa were affiliated with phylogenetic lineages capable of fermentative metabolism and sulfate respiration, indicating potential symbiont contributions to energy acquisition during prey digestion. The diversity and phylum-level composition of kogiid microbiomes differed from those previously reported in toothed whales, which exhibited low diversity communities dominated by Proteobacteria and Actinobacteria. Community structure analyses revealed distinct gut microbiomes in K. breviceps and K. sima, driven by differential relative abundances of shared taxa, and unique microbiomes in kogiid hosts compared to other toothed and baleen whales, driven by differences in symbiont membership. These results provide insight into the diversity, composition and structure of kogiid gut microbiomes and indicate that host identity plays an important role in structuring cetacean microbiomes, even at fine-scale taxonomic levels.

Abstract Engineering apparatus that harness quantum theory promises to offer practical advantages over current technology. A fundamentally more powerful prospect is that such quantum technologies could out-perform any future iteration of their classical counterparts, no matter how well the attributes of those classical strategies can be improved. Here, for optical direct absorption measurement, we experimentally demonstrate such an instance of an absolute advantage per photon probe that is exposed to the absorbative sample. We use correlated intensity measurements of spontaneous parametric downconversion using a commercially available air-cooled CCD, a new estimator for data analysis and a high heralding efficiency photon-pair source. We show this enables improvement in the precision of measurement, per photon probe, beyond what is achievable with an ideal coherent state (a perfect laser) detected with 100% efficient and noiseless detection. We see this absolute improvement for up to 50% absorption, with a maximum observed factor of improvement of 1.46. This equates to around 32% reduction in the total number of photons traversing an optical sample, compared to any future direct optical absorption measurement using classical light.

Abstract It is well established that visual sensitivity to motion is correlated with reading skills. Yet, the causal relationship between motion sensitivity and reading skills has been debated for more than thirty years. One hypothesis posits that dyslexia is caused by deficits in the motion processing pathway. An alternative hypothesis explains the motion processing deficit observed in dyslexia as the consequence of a lack, or poor quality, of reading experience. Here we used an intensive reading intervention program to test the causal relationship between learning to read and motion processing in children. Our data show that, while the reading intervention enhanced reading abilities, learning to read did not affect motion sensitivity. Motion sensitivity remained stable over the course of the intervention. Furthermore, the motion sensitivity deficit did not negatively impact the learning process. Children with poor motion sensitivity showed the same improvement in reading skills as children with typical motion sensitivity. Our findings call into question the view that motion processing deficits are due to poor reading experience. We propose that the correlation between the two measures arises from other common mechanisms, or that motion processing deficits are among a collection of correlated risk factors for reading difficulties.

Abstract The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host–symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.

Variability in response to short-acting β2-agonists (e.g., albuterol) among patients with asthma from diverse racial/ethnic groups may contribute to asthma disparities. We sought to identify genetic variants associated with bronchodilator response (BDR) to identify potential mechanisms of drug response and risk factors for worse asthma outcomes. Genome-wide association studies of bronchodilator response (BDR) were performed using TOPMed Whole Genome Sequencing data of the Asthma Translational Genomic Collaboration (ATGC), which corresponded to 1136 Puerto Rican, 656 Mexican and 4337 African American patients with asthma. With the population-specific GWAS results, a trans-ethnic meta-analysis was performed to identify BDR-associated variants shared across the three populations. Replication analysis was carried out in three pediatric asthma cohorts, including CAMP (Childhood Asthma Management Program; n = 560), GACRS (Genetics of Asthma in Costa Rica Study; n = 967) and HPR (Hartford-Puerto Rico; n = 417). A genome-wide significant locus (rs35661809; P = 3.61 × 10-8) in LINC02220, a non-coding RNA gene, was identified in Puerto Ricans. While this region was devoid of protein-coding genes, capture Hi-C data showed a distal interaction with the promoter of the DNAH5 gene in lung tissue. In replication analysis, the GACRS cohort yielded a nominal association (1-tailed P

The salamander chytrid fungus (Batrachochytrium salamandrivorans [Bsal]) is causing massive mortality of salamanders in Europe. The potential for spread via international trade into North America and the high diversity of salamanders has catalyzed concern about Bsal in the U.S. Surveillance programs for invading pathogens must initially meet challenges that include low rates of occurrence on the landscape, low prevalence at a site, and imperfect detection of the diagnostic tests. We implemented a large-scale survey to determine if Bsal was present in North America designed to target taxa and localities where Bsal was determined highest risk to be present based on species susceptibility and geography. Our analysis included a Bayesian model to estimate the probability of occurrence of Bsal given our prior knowledge of the occurrence and prevalence of the pathogen. We failed to detect Bsal in any of 11,189 samples from 594 sites in 223 counties within 35 U.S. states and one site in Mexico. Our modeling indicates that Bsal is highly unlikely to occur within wild amphibians in the U.S. and suggests that the best proactive response is to continue mitigation efforts against the introduction and establishment of the disease and to develop plans to reduce impacts should Bsal establish.