Expression of V-nitrogenase and Fe-nitrogenase in Methanosarcina acetivorans is controlled by molybdenum, fixed nitrogen, and the expression of Mo-nitrogenase.

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N2) to ammonia, with some also containing vanadium (V) and iron-only (Fe) nitrogenases that lack Mo. Among diazotrophs, the regulation and usage of the alternative V-nitrogenase and Fe-nitrogenase in methanogens are largely unknown. Methanosarcina acetivorans contains nif, vnf, and anf gene clusters encoding putative Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. This study investigated nitrogenase expression and growth by M. acetivorans in response to fixed nitrogen, Mo/V availability, and CRISPRi repression of the nif, vnf, and/or anf gene clusters. The availability of Mo and V significantly affected growth of M. acetivorans with N2 but not with NH4Cl. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N2 in medium containing Mo, and the slowest growth in medium lacking Mo and V. qPCR analysis revealed the transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo, whereas vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being key. Immunoblot analysis revealed Mo-nitrogenase is detected when fixed nitrogen is depleted regardless of Mo availability, while V-nitrogenase and Fe-nitrogenase are detected only in the absence of fixed nitrogen and Mo. CRISPRi repression studies revealed that V-nitrogenase and/or Fe-nitrogenase are required for Mo-independent diazotrophy, and unexpectedly that the expression of Mo-nitrogenase is also required. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and dependent on Mo-nitrogenase expression. IMPORTANCE Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens.

Systems immunology-based drug repurposing framework to target inflammation in atherosclerosis.

The development of new immunotherapies to treat the inflammatory mechanisms that sustain atherosclerotic cardiovascular disease (ASCVD) is urgently needed. Herein, we present a path to drug repurposing to identify immunotherapies for ASCVD. The integration of time-of-flight mass cytometry and RNA sequencing identified unique inflammatory signatures in peripheral blood mononuclear cells stimulated with ASCVD plasma. By comparing these inflammatory signatures to large-scale gene expression data from the LINCS L1000 dataset, we identified drugs that could reverse this inflammatory response. Ex vivo screens, using human samples, showed that saracatinib-a phase 2a-ready SRC and ABL inhibitor-reversed the inflammatory responses induced by ASCVD plasma. In Apoe-/- mice, saracatinib reduced atherosclerosis progression by reprogramming reparative macrophages. In a rabbit model of advanced atherosclerosis, saracatinib reduced plaque inflammation measured by [18F] fluorodeoxyglucose positron emission tomography-magnetic resonance imaging. Here we show a systems immunology-driven drug repurposing with a preclinical validation strategy to aid the development of cardiovascular immunotherapies.

Erratum: Publisher Correction: Systems immunology-based drug repurposing framework to target inflammation in atherosclerosis.

[This corrects the article DOI: 10.1038/s44161-023-00278-y.].

Subclinical Atherosclerosis in Young, Socioeconomically Vulnerable Hispanic and Non-Hispanic Black Adults.

BACKGROUND: Non-Hispanic Black persons are at greater risk of cardiovascular (CV) events than other racial/ethnic groups; however, their differential vulnerability to early subclinical atherosclerosis is poorly understood. OBJECTIVES: This work aims to study the impact of race/ethnicity on early subclinical atherosclerosis in young socioeconomically disadvantaged adults. METHODS: Bilateral carotid and femoral 3-dimensional vascular ultrasound examinations were performed on 436 adults (parents/caregivers and staff) with a mean age of 38.0 ± 11.1 years, 82.3% female, 66% self-reported as Hispanic, 34% self-reported as non-Hispanic Black, and no history of CV disease recruited in the FAMILIA (Family-Based Approach in a Minority Community Integrating Systems-Biology for Promotion of Health) trial from 15 Head Start preschools in Harlem (neighborhood in New York, New York, USA). The 10-year Framingham CV risk score was calculated, and the relationship between race/ethnicity and the presence and extent of subclinical atherosclerosis was analyzed with multivariable logistic and linear regression models. RESULTS: The mean 10-year Framingham CV risk was 4.0%, with no differences by racial/ethnic category. The overall prevalence of subclinical atherosclerosis was significantly higher in the non-Hispanic Black (12.9%) than in the Hispanic subpopulation (6.6%). After adjusting for 10-year Framingham CV risk score, body mass index, fruit and vegetable consumption, physical activity, and employment status, non-Hispanic Black individuals were more likely than Hispanic individuals to have subclinical atherosclerosis (OR: 3.45; 95% CI: 1.44-8.29; P = 0.006) and multiterritorial disease (P = 0.026). CONCLUSIONS: After adjustment for classic CV risk, lifestyle, and socioeconomic factors, non-Hispanic Black younger adults seem more vulnerable to early subclinical atherosclerosis than their Hispanic peers, suggesting that the existence of emerging or undiscovered CV factors underlying the residual excess risk (Family-Based Approach in a Minority Community Integrating Systems-Biology for Promotion of Health [FAMILIA (Project 2)]; NCT02481401).

The challenge of sustainability: Long-term results from the Fifty-Fifty peer group-based intervention in cardiovascular risk factors.

BACKGROUND: The Fifty-Fifty trial demonstrated that a peer-group-based intervention was able to improve healthy behaviors in individuals with cardiovascular (CV) risk factors immediately post-intervention. OBJECTIVES: To determine the long-term sustainability of a one-year peer-group-based intervention focused on CV health and behavior. METHODS: A total of 543 adults aged 25 to 50 years with at least 1 CV risk factor were screened and recruited, received initial training through workshops, and were then randomized 1:1 to a peer-group-based intervention group (IG) or a self-management control group (CG) for 12 months. At a median of 52 months from baseline, 321 participants were re-assessed (~60% retention). The primary outcome was the mean change in a composite health score related to blood pressure, exercise, weight, alimentation, and tobacco use (Fuster-BEWAT score [FBS], range 0-15). Intervention effects were assessed using linear-mixed effects models. RESULTS: The mean age of retained participants was 48.0 years (SD: 5.4), and 73% were female. Consistent with previous results, the change of overall FBS was significantly greater in the IG than in the CG at 12-month follow-up (between-group difference, 0.60 points; 95% CI, 0.08-1.12; P = .025). Assessment of long-term sustainability (52-month follow-up) showed that there were no between-group differences in the mean overall FBS (IG mean score, 8.52; 95% CI, 7.97-9.07 vs CG mean score, 8.51; 95% CI, 7.93-9.10; P = .972) or in the change of overall FBS from screening (IG mean change, 0.64; 95% CI, 0.00-1.28; CG mean change, 0.46; 95% CI, -0.20-1.12; P = .497). CONCLUSIONS: A one-year peer-group-based intervention showed favorable results at immediate post-intervention but did not demonstrate significant differences between the IG and CG at 52 months. Combination of an initial training period (workshops) with the maintenance of peer-support groups or other re-intervention strategies may be required to achieve sustained effects on healthy behaviors. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02367963. Registered (https://clinicaltrials.gov/show/NCT02367963).

Single cell analyses to understand the immune continuum in atherosclerosis.

Atherosclerosis is initiated by the accumulation of lipids in the arterial wall that trigger a complex and poorly understood network of inflammatory processes. At the same time, recent clinical findings reveal that targeting specific immune alterations in patients with cardiovascular disease (CVD) represents a promising approach to preventing recurrent cardiovascular events. In order to achieve these tailored therapies, it is critical to resolve the heterogenous environment of the atherosclerotic lesion and decipher the complex structural and functional changes which immune cells undergo throughout disease progression. Recently, single-cell approaches including single cell mass cytometry by time of flight (CyTOF), single cell RNA sequencing (scRNA-seq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) have emerged as valuable tools in resolving cellular plasticity within atherosclerotic lesions. In this review, we will discuss the most important insights that have been gleaned from the application of these single-cell approaches to validated experimental models of atherosclerosis. Additionally, as clinical progress in treatment of the disease depends on the translation of discoveries to human tissues, we will also examine the challenges associated with the application of single-cell approaches to human vascular tissue and the discoveries made by the initial efforts in this direction. Finally, we will analyze the advantages and limitations of dissociative single-cell approaches and how novel in-situ technologies could advance the field by allowing for the investigation of individual cells while preserving the heterogenous architecture of the atherosclerotic lesion.

Different Lifestyle Interventions in Adults From Underserved Communities: The FAMILIA Trial.

BACKGROUND: The current trends of unhealthy lifestyle behaviors in underserved communities are disturbing. Thus, effective health promotion strategies constitute an unmet need. OBJECTIVES: The purpose of this study was to assess the impact of 2 different lifestyle interventions on parents/caregivers of children attending preschools in a socioeconomically disadvantaged community. METHODS: The FAMILIA (Family-Based Approach in a Minority Community Integrating Systems-Biology for Promotion of Health) study is a cluster-randomized trial involving 15 Head Start preschools in Harlem, New York. Schools, and their children's parents/caregivers, were randomized to receive either an "individual-focused" or "peer-to-peer-based" lifestyle intervention program for 12 months or control. The primary outcome was the change from baseline to 12 months in a composite health score related to blood pressure, exercise, weight, alimentation, and tobacco (Fuster-BEWAT Score [FBS]), ranging from 0 to 15 (ideal health = 15). To assess the sustainability of the intervention, this study evaluated the change of FBS at 24 months. Main pre-specified secondary outcomes included changes in FBS subcomponents and the effect of the knowledge of presence of atherosclerosis as assessed by bilateral carotid/femoral vascular ultrasound. Mixed-effects models were used to test for intervention effects. RESULTS: A total of 635 parents/caregivers were enrolled: mean age 38 ± 11 years, 83% women, 57% Hispanic/Latino, 31% African American, and a baseline FBS of 9.3 ± 2.4 points. The mean within-group change in FBS from baseline to 12 months was ∼0.20 points in all groups, with no overall between-group differences. However, high-adherence participants to the intervention exhibited a greater change in FBS than their low-adherence counterparts: 0.30 points (95% confidence interval: 0.03 to 0.57; p = 0.027) versus 0.00 points (95% confidence interval: -0.43 to 0.43; p = 1.0), respectively. Furthermore, the knowledge by the participant of the presence of atherosclerosis significantly boosted the intervention effects. Similar results were sustained at 24 months. CONCLUSIONS: Although overall significant differences were not observed between intervention and control groups, the FAMILIA trial highlights that high adherence rates to lifestyle interventions may improve health outcomes. It also suggests a potential contributory role of the presentation of atherosclerosis pictures, providing helpful information to improve future lifestyle interventions in adults.

Single-cell immune landscape of human atherosclerotic plaques.

Atherosclerosis is driven by multifaceted contributions of the immune system within the circulation and at vascular focal sites. However, specific characteristics of dysregulated immune cells within atherosclerotic lesions that lead to clinical events such as ischemic stroke or myocardial infarction are poorly understood. Here, using single-cell proteomic and transcriptomic analyses, we uncovered distinct features of both T cells and macrophages in carotid artery plaques of patients with clinically symptomatic disease (recent stroke or transient ischemic attack) compared to asymptomatic disease (no recent stroke). Plaques from symptomatic patients were characterized by a distinct subset of CD4+ T cells and by T cells that were activated and differentiated. Moreover, some T cell subsets in these plaques presented markers of T cell exhaustion. Additionally, macrophages from these plaques contained alternatively activated phenotypes, including subsets associated with plaque vulnerability. In plaques from asymptomatic patients, T cells and macrophages were activated and displayed evidence of interleukin-1ß signaling. The identification of specific features of innate and adaptive immune cells in plaques that are associated with cerebrovascular events may enable the design of more precisely tailored cardiovascular immunotherapies.

A causal account of the brain network computations underlying strategic social behavior.

During competitive interactions, humans have to estimate the impact of their own actions on their opponent's strategy. Here we provide evidence that neural computations in the right temporoparietal junction (rTPJ) and interconnected structures are causally involved in this process. By combining inhibitory continuous theta-burst transcranial magnetic stimulation with model-based functional MRI, we show that disrupting neural excitability in the rTPJ reduces behavioral and neural indices of mentalizing-related computations, as well as functional connectivity of the rTPJ with ventral and dorsal parts of the medial prefrontal cortex. These results provide a causal demonstration that neural computations instantiated in the rTPJ are neurobiological prerequisites for the ability to integrate opponent beliefs into strategic choice, through system-level interaction within the valuation and mentalizing networks.

Fiber-based 1.5 microm lidar vibrometer in pulsed and continuous modes.

A fiber-based 1.5 mum heterodyne lidar that is easily switched between pulse-pair and cw modes is described. In laboratory experiments using well-controlled vibrating targets, and in computer simulations, the performance of the two modes is compared given the same average laser power. The accuracy of Doppler frequency (target velocity) estimates, and the signal-to-noise ratio in spectrally resolved plots of vibrational features, are evaluated. When the target-induced frequency modulation is wideband, pulse-pair often has clearly higher carrier-to-noise. But its advantage in signal-to-noise is smaller because combining the more numerous cw measurements improves the estimates of vibration frequencies and amplitudes. They are combined here through autocorrelation-based demodulation, one of several methods that can outperform phase-differencing.

Lidar frequency modulation vibrometry in the presence of speckle.

We report laboratory target vibration measurements that use an easily aligned and adjusted fiber-based 1.5-microm heterodyne lidar. The targets are simple spherically curved retroreflectors with well-controlled vibration frequencies and amplitudes. A rotating ground-glass screen creates Gaussian speckle. We wish to understand the modulated and fast-fading lidar returns seen from real target. We frequency demodulated the recorded laboratory data by phase differencing to provide estimates of dphi/dt, where phi is the phase of the received carrier-plus-noise phasor. Experimental results for signal strength and signal-to-noise ratio, for specific target modulation parameters, agree well with our recently developed dphi/dt correlation-function theory.