Flavonols improve thermotolerance in tomato pollen during germination and tube elongation by maintaining ROS homeostasis.

Elevated temperatures impair pollen performance and reproductive success, resulting in lower crop yields. The Solanum lycopersicum anthocyanin reduced ( are ) mutant has a FLAVANONE 3 HYDROXYLASE ( F3H ) gene mutation resulting in impaired synthesis of flavonol antioxidants. The are mutant has reduced pollen performance and seed set relative to the VF36 parental line, which is accentuated at elevated temperatures. Transformation of are with the wild-type F3H gene, or chemical complementation with flavonols, prevented temperature-dependent ROS accumulation in pollen and reversed are's reduced viability, germination, and tube elongation to VF36 levels. VF36 transformed with an F3H overexpression construct prevented temperature driven ROS increases and impaired pollen performance, revealing thermotolerance results from elevated flavonol synthesis. Although stigmas of are had reduced flavonols and elevated ROS, the growth of are pollen tubes were similarly impaired in both are and VF36 pistils. RNA-Seq was performed at optimal and stress temperatures in are , VF36, and the VF36 F3H overexpression line at multiple timepoints across pollen tube elongation. Differentially expressed gene numbers increased with duration of elevated temperature in all genotypes, with the largest number in are . These findings suggest potential agricultural interventions to combat the negative effects of heat-induced ROS in pollen that leads to reproductive failure. One sentence summary: Flavonol antioxidants reduce the negative impacts of elevated temperatures on pollen performance by reducing levels of heat induced reactive oxygen species and modulation of heat-induced changes in the pollen transcriptome.

SARS-CoV-2 mRNA vaccines decouple anti-viral immunity from humoral autoimmunity.

mRNA-based vaccines dramatically reduce the occurrence and severity of COVID-19, but are associated with rare vaccine-related adverse effects. These toxicities, coupled with observations that SARS-CoV-2 infection is associated with autoantibody development, raise questions whether COVID-19 vaccines may also promote the development of autoantibodies, particularly in autoimmune patients. Here we used Rapid Extracellular Antigen Profiling to characterize self- and viral-directed humoral responses after SARS-CoV-2 mRNA vaccination in 145 healthy individuals, 38 patients with autoimmune diseases, and 8 patients with mRNA vaccine-associated myocarditis. We confirm that most individuals generated robust virus-specific antibody responses post vaccination, but that the quality of this response is impaired in autoimmune patients on certain modes of immunosuppression. Autoantibody dynamics are remarkably stable in all vaccinated patients compared to COVID-19 patients that exhibit an increased prevalence of new autoantibody reactivities. Patients with vaccine-associated myocarditis do not have increased autoantibody reactivities relative to controls. In summary, our findings indicate that mRNA vaccines decouple SARS-CoV-2 immunity from autoantibody responses observed during acute COVID-19.

Functional connectomics reveals general wiring rule in mouse visual cortex.

To understand how the brain computes, it is important to unravel the relationship between circuit connectivity and function. Previous research has shown that excitatory neurons in layer 2/3 of the primary visual cortex of mice with similar response properties are more likely to form connections. However, technical challenges of combining synaptic connectivity and functional measurements have limited these studies to few, highly local connections. Utilizing the millimeter scale and nanometer resolution of the MICrONS dataset, we studied the connectivity-function relationship in excitatory neurons of the mouse visual cortex across interlaminar and interarea projections, assessing connection selectivity at the coarse axon trajectory and fine synaptic formation levels. A digital twin model of this mouse, that accurately predicted responses to arbitrary video stimuli, enabled a comprehensive characterization of the function of neurons. We found that neurons with highly correlated responses to natural videos tended to be connected with each other, not only within the same cortical area but also across multiple layers and visual areas, including feedforward and feedback connections, whereas we did not find that orientation preference predicted connectivity. The digital twin model separated each neuron's tuning into a feature component (what the neuron responds to) and a spatial component (where the neuron's receptive field is located). We show that the feature, but not the spatial component, predicted which neurons were connected at the fine synaptic scale. Together, our results demonstrate the "like-to-like" connectivity rule generalizes to multiple connection types, and the rich MICrONS dataset is suitable to further refine a mechanistic understanding of circuit structure and function.

Towards a Foundation Model of the Mouse Visual Cortex.

Understanding the brain's perception algorithm is a highly intricate problem, as the inherent complexity of sensory inputs and the brain's nonlinear processing make characterizing sensory representations difficult. Recent studies have shown that functional models-capable of predicting large-scale neuronal activity in response to arbitrary sensory input-can be powerful tools for characterizing neuronal representations by enabling high-throughput in silico experiments. However, accurately modeling responses to dynamic and ecologically relevant inputs like videos remains challenging, particularly when generalizing to new stimulus domains outside the training distribution. Inspired by recent breakthroughs in artificial intelligence, where foundation models-trained on vast quantities of data-have demonstrated remarkable capabilities and generalization, we developed a "foundation model" of the mouse visual cortex: a deep neural network trained on large amounts of neuronal responses to ecological videos from multiple visual cortical areas and mice. The model accurately predicted neuronal responses not only to natural videos but also to various new stimulus domains, such as coherent moving dots and noise patterns, underscoring its generalization abilities. The foundation model could also be adapted to new mice with minimal natural movie training data. We applied the foundation model to the MICrONS dataset: a study of the brain that integrates structure with function at unprecedented scale, containing nanometer-scale morphology, connectivity with >500,000,000 synapses, and function of >70,000 neurons within a ~1mm3 volume spanning multiple areas of the mouse visual cortex. This accurate functional model of the MICrONS data opens the possibility for a systematic characterization of the relationship between circuit structure and function. By precisely capturing the response properties of the visual cortex and generalizing to new stimulus domains and mice, foundation models can pave the way for a deeper understanding of visual computation.

High-throughput identification of autoantibodies that target the human exoproteome.

Autoantibodies that recognize extracellular proteins (the exoproteome) exert potent biological effects but are challenging to detect. Here, we developed rapid extracellular antigen profiling (REAP), a high-throughput technique for the comprehensive discovery of exoproteome-targeting autoantibodies. Patient samples are applied to a genetically barcoded yeast surface display library containing 2,688 human extracellular proteins. Antibody-coated yeast are isolated, and sequencing of barcodes is used to identify displayed antigens. To benchmark REAP's performance, we screened 77 patients with autoimmune polyglandular syndrome type 1 (APS-1). REAP sensitively and specifically detected both known and previously unidentified autoantibodies in APS-1. We further screened 106 patients with systemic lupus erythematosus (SLE) and identified numerous autoantibodies, several of which were associated with disease severity or specific clinical manifestations and exerted functional effects on cell signaling ex vivo. These findings demonstrate the utility of REAP to atlas the expansive landscape of exoproteome-targeting autoantibodies and their impacts on patient health outcomes.

Diverse functional autoantibodies in patients with COVID-19.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-6. Although pathological innate immune activation is well-documented in severe disease1, the effect of autoantibodies on disease progression is less well-defined. Here we use a high-throughput autoantibody discovery technique known as rapid extracellular antigen profiling7 to screen a cohort of 194 individuals infected with SARS-CoV-2, comprising 172 patients with COVID-19 and 22 healthcare workers with mild disease or asymptomatic infection, for autoantibodies against 2,770 extracellular and secreted proteins (members of the exoproteome). We found that patients with COVID-19 exhibit marked increases in autoantibody reactivities as compared to uninfected individuals, and show a high prevalence of autoantibodies against immunomodulatory proteins (including cytokines, chemokines, complement components and cell-surface proteins). We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signalling and by altering peripheral immune cell composition, and found that mouse surrogates of these autoantibodies increase disease severity in a mouse model of SARS-CoV-2 infection. Our analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics. Our findings suggest a pathological role for exoproteome-directed autoantibodies in COVID-19, with diverse effects on immune functionality and associations with clinical outcomes.

Diverse Functional Autoantibodies in Patients with COVID-19.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-8. While pathological innate immune activation is well documented in severe disease1, the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the "exoproteome"). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with a high prevalence of autoantibodies against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.

Sex differences in immune responses that underlie COVID-19 disease outcomes.

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However, whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes, and whether such differences correlate with the sex difference in the disease course of COVID-19, is currently unknown. Here we examined sex differences in viral loads, SARS-CoV-2-specific antibody titres, plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast, female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably, we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients, but not in female patients. By contrast, higher levels of innate immune cytokines were associated with worse disease progression in female patients, but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19, and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19.

Sex differences in immune responses to SARS-CoV-2 that underlie disease outcomes.

A growing body of evidence indicates sex differences in the clinical outcomes of coronavirus disease 2019 (COVID-19)1-4. However, whether immune responses against SARS-CoV-2 differ between sexes, and whether such differences explain male susceptibility to COVID-19, is currently unknown. In this study, we examined sex differences in viral loads, SARS-CoV-2-specific antibody titers, plasma cytokines, as well as blood cell phenotyping in COVID-19 patients. By focusing our analysis on patients with mild to moderate disease who had not received immunomodulatory medications, our results revealed that male patients had higher plasma levels of innate immune cytokines and chemokines including IL-8, IL-18, and CCL5, along with more robust induction of non-classical monocytes. In contrast, female patients mounted significantly more robust T cell activation than male patients during SARS-CoV-2 infection, which was sustained in old age. Importantly, we found that a poor T cell response negatively correlated with patients' age and was predictive of worse disease outcome in male patients, but not in female patients. Conversely, higher innate immune cytokines in female patients associated with worse disease progression, but not in male patients. These findings reveal a possible explanation underlying observed sex biases in COVID-19, and provide important basis for the development of sex-based approach to the treatment and care of men and women with COVID-19.

Machine Learning and Primary Total Knee Arthroplasty: Patient Forecasting for a Patient-Specific Payment Model.

BACKGROUND: Value-based and patient-specific care represent 2 critical areas of focus that have yet to be fully reconciled by today's bundled care model. Using a predictive naïve Bayesian model, the objectives of this study were (1) to develop a machine-learning algorithm using preoperative big data to predict length of stay (LOS) and inpatient costs after primary total knee arthroplasty (TKA) and (2) to propose a tiered patient-specific payment model that reflects patient complexity for reimbursement. METHODS: Using 141,446 patients undergoing primary TKA from an administrative database from 2009 to 2016, a Bayesian model was created and trained to forecast LOS and cost. Algorithm performance was determined using the area under the receiver operating characteristic curve and the percent accuracy. A proposed risk-based patient-specific payment model was derived based on outputs. RESULTS: The machine-learning algorithm required age, race, gender, and comorbidity scores ("risk of illness" and "risk of morbidity") to demonstrate a high degree of validity with an area under the receiver operating characteristic curve of 0.7822 and 0.7382 for LOS and cost. As patient complexity increased, cost add-ons increased in tiers of 3%, 10%, and 15% for moderate, major, and extreme mortality risks, respectively. CONCLUSION: Our machine-learning algorithm derived from an administrative database demonstrated excellent validity in predicting LOS and costs before primary TKA and has broad value-based applications, including a risk-based patient-specific payment model.

Reduced Cortical Activity Impairs Development and Plasticity after Neonatal Hypoxia Ischemia.

Survivors of preterm birth are at high risk of pervasive cognitive and learning impairments, suggesting disrupted early brain development. The limits of viability for preterm birth encompass the third trimester of pregnancy, a "precritical period" of activity-dependent development characterized by the onset of spontaneous and evoked patterned electrical activity that drives neuronal maturation and formation of cortical circuits. Reduced background activity on electroencephalogram (EEG) is a sensitive marker of brain injury in human preterm infants that predicts poor neurodevelopmental outcome. We studied a rodent model of very early hypoxic-ischemic brain injury to investigate effects of injury on both general background and specific patterns of cortical activity measured with EEG. EEG background activity is depressed transiently after moderate hypoxia-ischemia with associated loss of spindle bursts. Depressed activity, in turn, is associated with delayed expression of glutamate receptor subunits and transporters. Cortical pyramidal neurons show reduced dendrite development and spine formation. Complementing previous observations in this model of impaired visual cortical plasticity, we find reduced somatosensory whisker barrel plasticity. Finally, EEG recordings from human premature newborns with brain injury demonstrate similar depressed background activity and loss of bursts in the spindle frequency band. Together, these findings suggest that abnormal development after early brain injury may result in part from disruption of specific forms of brain activity necessary for activity-dependent circuit development. SIGNIFICANCE STATEMENT: Preterm birth and term birth asphyxia result in brain injury from inadequate oxygen delivery and constitute a major and growing worldwide health problem. Poor outcomes are noted in a majority of very premature (<25 weeks gestation) newborns, resulting in death or life-long morbidity with motor, sensory, learning, behavioral, and language disabilities that limit academic achievement and well-being. Limited progress has been made to develop therapies that improve neurologic outcomes. The overall objective of this study is to understand the effect of early brain injury on activity-dependent brain development and cortical plasticity to develop new treatments that will optimize repair and recovery after brain injury.

Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity.

Semicarbazide-sensitive amine oxidase (SSAO, amine oxidase, copper-containing 3, and vascular adhesion protein-1) is a copper-containing enzyme that catalyzes the oxidative deamination of primary amines to an aldehyde, ammonia, and hydrogen peroxide. SSAO is also involved in leukocyte migration to sites of inflammation, and the enzymatic activity of SSAO is essential to this role. Thus, inhibition of SSAO enzyme activity represents a target for the development of small molecule anti-inflammatory compounds. Here, we have characterized the novel SSAO inhibitor, Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride (LJP 1586), and assessed its anti-inflammatory activity. LJP 1586 is a potent inhibitor of rodent and human SSAO activity, with IC(50) values between 4 and 43 nM. The selectivity of LJP 1586 was confirmed with a broad panel of receptors and enzymes that included the monoamine oxidases A and B. Oral administration of LJP 1586 resulted in complete inhibition of rat lung SSAO, with an ED(50) between 0.1 and 1 mg/kg, and a pharmacodynamic half-life of greater than 24 h. In a mouse model of inflammatory leukocyte trafficking oral dosing with LJP 1586 resulted in significant dose-dependent inhibition of neutrophil accumulation, with an effect comparable to that of anti-leukocyte function-associated antigen-1 antibody. In a rat model of LPS-induced lung inflammation, administration of 10 mg/kg LJP 1586 resulted in a 55% significant reduction in transmigrated cells recovered by bronchoalveolar lavage. The results demonstrate that a selective, orally active small molecule inhibitor of SSAO is an effective anti-inflammatory compound in vivo and provide further support for SSAO as a therapeutic anti-inflammatory target.

Design, synthesis, and biological evaluation of semicarbazide-sensitive amine oxidase (SSAO) inhibitors with anti-inflammatory activity.

In an attempt to examine the effect of inhibition of semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6, also known as VAP-1) as a novel anti-inflammatory target, the structure/mechanism based design and synthesis of a series of novel hydrazino-containing small molecules are described. The in vitro biological results show that compounds 4a,c are highly potent SSAO inhibitors with notable selectivity toward SSAO over monoamine oxidases A and B (MAO-A and MAO-B). SAR studies based on compound 4c were performed, and the results are discussed. The most potent and selective compound, 4a (IC(50) = 2 nM), is an orally active, competitive, and apparently irreversible inhibitor of SSAO that is effective at reducing disease incidence and severity in an in vivo animal disease model of multiple sclerosis.

Vascular adhesion protein-1 plays an important role in postischemic inflammation and neuropathology in diabetic, estrogen-treated ovariectomized female rats subjected to transient forebrain ischemia.

Endothelial vascular adhesion protein-1 (VAP-1) facilitates leukocyte adhesion and infiltration. This relates partly to the function of VAP-1 as a semicarbazide-sensitive amine oxidase (SSAO). We examined the effects of VAP-1/SSAO inhibition [via LJP-1207 (N'-(2-phenyl-allyl)-hydrazine hydrochloride)] on pial venular leukocyte adhesion and infiltration (at 2-10 h of reperfusion) and neuropathology (at 72 h of reperfusion) after transient forebrain ischemia (TFI). A model associated with increased postischemic inflammation was used-i.e., diabetic ovariectomized (OVX) female rats given chronic estrogen replacement therapy (ERT). We compared rats treated, either at the onset or at 6 h of reperfusion, with saline or LJP-1207. Additional rats, rendered neutropenic 24 h before TFI, were studied. In saline-treated controls, intravascular accumulation of adherent leukocytes gradually increased, reaching 15 to 20% of the venular area, at which point neutrophil infiltration commenced (at approximately 6 h). In the rats given LJP-1207 at the onset of reperfusion, limited neutrophil adhesion ( approximately 5% maximum) and no infiltration were observed. These results generally paralleled those in neutropenic rats. In rats treated at 6 h of reperfusion, the pattern of neutrophil adhesion was similar to that of the saline-treated group up to 6 h, but further infiltration was essentially prevented. Neurologic outcomes and histopathology were similar to one another in the LJP-1207-treated and neutropenic groups and significantly improved over those in saline-treated controls. Thus, VAP-1-mediated post-TFI leukocyte adhesion/infiltration in diabetic OVX females given chronic ERT contributes substantially to neuropathology. One implication is that specifically preventing leukocyte infiltration provides a substantial measure of neuroprotection. This could explain the finding of LJP-1207 having at least a 6-h therapeutic window in this model.