Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted

The Marine Stratus/Stratocumulus Experiment (MASE): Aerosol-cloud relationships in marine stratocumulus

Journal of Geophysical Research, Vol. 112, D10209The article of record as published may be located at http://dx.doi.org/10.1029/2006JD007985.The Marine Stratus/Stratocumulus Experiment (MASE) field campaign was undertaken in July 2005 off the coast of Monterey, California to evaluate aerosol-cloud relationships in the climatically important regime of eastern Pacific marine stratocumulus. Aerosol and cloud properties were measured onboard the Center for Interdisciplinary Remotely-Piloted Aircraft Studies (CIRPAS) Twin Otter aircraft. One cloud that was clearly impacted by ship emissions as well as the ensemble of clouds observed over the entire mission are analyzed in detail. Results at both the individual and ensemble scales clearly confirm the Twomey effect (first indirect effect of aerosols) and demonstrate drizzle suppression at elevated aerosol number concentration. For the ship track impacted cloud, suppressed drizzle in the track led to a larger cloud liquid water path (LWP) at the same cloud thickness, in accord with the so-called second indirect effect. Ensemble averages over all clouds sampled over the entire 13-flight mission show the opposite effect of aerosol number concentration on LWP, presumably the result of other dynamic influences (e.g., updraft velocity and ambient sounding profile). Individual polluted clouds were found to exhibit a narrower cloud drop spectral width in accord with theoretical prediction (M.-L. Lu and J. H. Seinfeld, Effect of aerosol number concentration on cloud droplet dispersion: A large-eddy simulation study and implications for aerosol indirect forcing, Journal of Geophysical Research, 2006). This field experiment demonstrates both the indirect aerosol effect on ship track perturbed clouds, as well as the subtleties involved in extracting these effects over an ensemble of clouds sampled over a 1-month period

The Marine Stratus/Stratocumulus Experiment (MASE): Aerosol-cloud relationships in marine stratocumulus

The Marine Stratus/Stratocumulus Experiment (MASE) field campaign was undertaken in July 2005 off the coast of Monterey, California to evaluate aerosol-cloud relationships in the climatically important regime of eastern Pacific marine stratocumulus. Aerosol and cloud properties were measured onboard the Center for Interdisciplinary Remotely-Piloted Aircraft Studies (CIRPAS) Twin Otter aircraft. One cloud that was clearly impacted by ship emissions as well as the ensemble of clouds observed over the entire mission are analyzed in detail. Results at both the individual and ensemble scales clearly confirm the Twomey effect (first indirect effect of aerosols) and demonstrate drizzle suppression at elevated aerosol number concentration. For the ship track impacted cloud, suppressed drizzle in the track led to a larger cloud liquid water path (LWP) at the same cloud thickness, in accord with the so-called second indirect effect. Ensemble averages over all clouds sampled over the entire 13-flight mission show the opposite effect of aerosol number concentration on LWP, presumably the result of other dynamic influences (e.g., updraft velocity and ambient sounding profile). Individual polluted clouds were found to exhibit a narrower cloud drop spectral width in accord with theoretical prediction (M.-L. Lu and J. H. Seinfeld, Effect of aerosol number concentration on cloud droplet dispersion: A large-eddy simulation study and implications for aerosol indirect forcing, Journal of Geophysical Research, 2006). This field experiment demonstrates both the indirect aerosol effect on ship track perturbed clouds, as well as the subtleties involved in extracting these effects over an ensemble of clouds sampled over a 1-month period

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies

A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus

Background: Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial. Results: We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 μM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9. Conclusions: A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity

Mechanisms underlying reduced weight gain in intestinal fatty acid-binding protein (IFABP) null mice

Intestinal- fatty acid binding protein (IFABP; FABP2) is a 15-kDa intracellular protein abundantly present in the cytosol of the small intestinal (SI) enterocyte. High-fat (HF) feeding of IFABP-/- mice resulted in reduced weight gain and fat mass relative to wild-type (WT) mice. Here, we examined intestinal properties that may underlie the observed lean phenotype of high fat-fed IFABP-/- mice. No alterations in fecal lipid content were found, suggesting that the IFABP-/- mice are not malabsorbing dietary fat. However, the total excreted fecal mass, normalized to food intake, was increased for the IFABP-/- mice relative to WT mice. Moreover, intestinal transit time was more rapid in the IFABP-/- mice. IFABP-/- mice displayed a shortened average villus length, a thinner muscularis layer, reduced goblet cell density, and reduced Paneth cell abundance. The number of proliferating cells in the crypts of IFABP-/- mice did not differ from that of WT mice, suggesting that the blunt villi phenotype is not due to alterations in proliferation. IFABP-/- mice were observed to have altered expression of genes and proteins related to intestinal structure, while immunohistochemical analyses revealed increased staining for markers of inflammation. Taken together, these studies indicate that the ablation of IFABP, coupled with high-fat feeding, leads to changes in gut motility and morphology, which likely contribute to the relatively leaner phenotype occurring at the whole-body level. Thus, IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, intestinal structure, and nutrient absorption, thereby impacting systemic energy metabolism.Fil: Lackey, Atreju I.. Rutgers University; Estados Unidos. Rutgers Center for Lipid Research; Estados UnidosFil: Chen, Tina. Rutgers University; Estados UnidosFil: Zhou, Yin X.. Rutgers University; Estados UnidosFil: Bottasso Arias, Natalia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Doran, Justine M.. Rutgers University; Estados UnidosFil: Zacharisen, Sophia M.. Rutgers University; Estados UnidosFil: Gajda, Angela M.. Rutgers University; Estados Unidos. Rutgers Center for Lipid Research; Estados UnidosFil: Jonsson, William O.. Rutgers University; Estados Unidos. Rutgers Center for Lipid Research; Estados UnidosFil: Córsico, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Anthony, Tracy G.. Rutgers University; Estados Unidos. Rutgers Center for Lipid Research; Estados UnidosFil: Joseph, Laurie B.. Rutgers Center for Lipid Research; Estados Unidos. Rutgers University; Estados UnidosFil: Storch, Judith. Rutgers University; Estados Unidos. Rutgers Center for Lipid Research; Estados Unido

Discovery and Mechanistic Characterization of Novel SARS Coronavirus Inhibitors that Block Viral Entry [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionSevere acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS-associated coronavirus (SARS-CoV). Viral entry is a key target step for therapies because it can prevent the propagation of virus at early stages of the disease. We used a cell-based assay to identify inhibitors of SARS-CoV entry. We prepared a pseudotyped virus in which the core is from HIV and envelop is from the SARS-CoV (HIV-luc/SARS env). This pseudotyped virus was used to infect, 293T cells expressing the receptor for SARS-CoV, Aangiotensin-converting enzyme-2 (ACE2). Using this assay we screened a chemical library of more than 2000 compounds and identified three compounds that specifically inhibit entry of the HIV-luc/SARS env. These compounds did not inhibit another pseudotyped virus which had same core from HIV but envelop was from Vesicular Stomatitis Virus. The compounds had strong potencies (EC50s were 2.9, 4.8 and 5.8 µM) and low cytotoxicities (high CC50s) resulting in promising Selectivity Indices (CC50/EC50 were >175, >65, and >86, respectively). Importantly, the compounds were found to have excellent antiviral activities, blocking SARS-CoV replication at low nM concentrations. Only one of the compounds was a moderate inhibitor of cathepsin L, a cellular protease whose activity is required to process the SARS-CoV env glycoprotein (Spike) and allow viral entry. Moreover, none of the compounds affects the cleavage activity of furin, another host protease, which may also be involved in SARS-CoV entry. Using a flow cytometry binding assay, we found that all three compounds decrease binding of the SARS-CoV Spike receptor binding domain to ACE2 receptor expressed on the surface of 293Tcells. Hence, we have discovered three promising compounds as the first small molecule inhibitors that can block receptor-dependent entry of SARS-CoV

Lower Rates of Heart Failure and All-Cause Hospitalizations During Pulmonary Artery Pressure-Guided Therapy for Ambulatory Heart Failure: One-Year Outcomes From the CardioMEMS Post-Approval Study.

BACKGROUND: Ambulatory hemodynamic monitoring with an implantable pulmonary artery (PA) sensor is approved for patients with New York Heart Association Class III heart failure (HF) and a prior HF hospitalization (HFH) within 12 months. The objective of this study was to assess the efficacy and safety of PA pressure-guided therapy in routine clinical practice with special focus on subgroups defined by sex, race, and ejection fraction. METHODS: This multi-center, prospective, open-label, observational, single-arm trial of 1200 patients across 104 centers within the United States with New York Heart Association class III HF and a prior HFH within 12 months evaluated patients undergoing PA pressure sensor implantation between September 1, 2014, and October 11, 2017. The primary efficacy outcome was the difference between rates of adjudicated HFH 1 year after compared with the 1 year before sensor implantation. Safety end points were freedom from device- or system-related complications at 2 years and freedom from pressure sensor failure at 2 years. RESULTS: Mean age for the population was 69 years, 37.7% were women, 17.2% were non-White, and 46.8% had preserved ejection fraction. During the year after sensor implantation, the mean rate of daily pressure transmission was 76±24% and PA pressures declined significantly. The rate of HFH was significantly lower at 1 year compared with the year before implantation (0.54 versus 1.25 events/patient-years, hazard ratio 0.43 [95% CI, 0.39-0.47], CONCLUSIONS: In routine clinical practice as in clinical trials, PA pressure-guided therapy for HF was associated with lower PA pressures, lower rates of HFH and all-cause hospitalization, and low rates of adverse events across a broad range of patients with symptomatic HF and prior HFH. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02279888