Endoplasmic reticulum (ER) stress as well as oxidative stress have been shown to play important roles in metabolic and cardiovascular disease, and drugs that counteract the effects of ER and ...oxidative stresses may be clinically useful. To identify novel compounds that ameliorate ER and oxidative stresses, we screened two drug libraries purchased from Evotec, San Francisco, CA; the NIH clinical collection 1 (446 compounds) and the NIH clinical collection 2 (281 compounds). Human coronary artery endothelial cells (HCAEC) were tested for ER and oxidative stress. ER stress was measured with an ER stress-sensitive secreted alkaline phosphatase (SAP) assay. The cells were transfected with the plasmid pSAP2.Control, expressing a heat-resistant form of SAP, and treated with the ER stress inducer tunicamycin in the presence or absence of each of the various compounds for 24-h, at which time SAP activity was measured. Compounds exhibiting significant increases in SAP activity (41 compounds out of a total of 727 tested; 5.6%) were then assayed for their ability to suppress superoxide (SO) anion generation in cells treated with 27.5 mM dextrose. SO generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo1,2-Apyrazin-3-one hydrochloride chemiluminescence. Of the 41 compounds identified as ER stress reducers, only 33 (80.5%) suppressed dextrose-induced SO anion generation. Interestingly, 51% of the compounds found to be dual-stress modifiers consisted of cardioprotective drugs, including statins, angiotensin receptor blockers, angiotensin-converting enzyme inhibitors as well as β-blockers. Future studies to validate the clinical effectiveness of these agents remain to be performed in pre-clinical and clinical trials.
Black seed extract stimulates apolipoprotein A-I (apo A-I) gene expression in hepatocytes and intestinal cells in part by elevating peroxisome proliferator-activated receptor α (PPARα) and retinoid X ...receptor α (RXRα) levels. To explore potential ramifications of these observations, we examined the effects of black seed extract on hepatocyte lipid content and expression of key transcriptional regulators of fatty acid β-oxidation and lipogenesis in HepG2 cells. PPARα, peroxisome proliferator-activated receptor γ (PPARγ), RXRα, thyroid hormone receptor β (TRβ), sterol-responsive element binding protein 1 (SREBP1), and sterol-responsive element binding protein 2 (SREBP2) levels were measured in black seed extract treated liver-derived HepG2 cells. Black seed extract treatment increased PPARα and RXRα expression and decreased intracellular neutral lipid content. Black seed extract treatment increased TRβ expression and activity, and PPARα activity. In contrast, PPARγ, SREBP1 and SREBP2 levels were decreased in black seed extract treated cells. Black seed extract treatment also increased acyl-CoA synthetase long chain family member 5 (ACSL5), peroxisomal acyl-CoA oxidase 1 (ACOX1), and carnitine palmitoyl transferase 1A (CPT-1A) expression, three PPARα-dependent rate-limiting genes that facilitate fatty acid oxidation, similar to fenofibrate. PPARα knockdown reversed the effects of fenofibrate and blackseed on ACSL5, ACOX1, and CPT-1A expression. In conclusion, black seed extract-mediated lipid lowering in HepG2 cells is associated with increased expression of fatty acid oxidation enzymes and PPARα and reduced lipogenic signaling. Thus black seed extract may be potentially beneficial in metabolic diseases such as diabetes, cardiovascular disease, and metabolic syndrome.
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•Black seed extract decreases lipid content in HepG2 cells.•Black seed extract increased PPARα, RXRα, and TRβ expression.•Black seed extract suppressed SREBP1, SREBP2, and PPARγ expression.•Black seed extract may be a novel means to suppress lipid storage in hepactocytes.
Oxidative and endoplasmic reticulum (ER) stresses are implicated in premature cardiovascular disease in people with diabetes. The aim of the present study was to characterize the nature of the ...interplay between the oxidative and ER stresses to facilitate the development of therapeutic agents that can ameliorate these stresses.
Human coronary artery endothelial cells were treated with varying concentrations of dextrose in the presence or absence of three antioxidants (alpha tocopherol, ascorbate and ebselen) and two ER stress modifiers (ERSMs) (4-phenylbutyrate and taurodeoxycholic acid). ER stress was measured using the placental alkaline phosphatase assay and superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo1,2-Apyrazin-3-one hydrochloride chemiluminescence.
The SO generation was increased with increasing concentrations of dextrose. The ER stress was increased with both low (0 and 2.75mM) and high (13.75 and 27.5mM) concentrations of dextrose. The antioxidants inhibited the dextrose induced SO production while in high concentrations they aggravated ER stress. The ERSM reduced ER stress and potentiated the efficacy of the three antioxidants. Tunicamycin-induced ER stress was not associated with increased SO generation. Time course experiments with a high concentration of dextrose or by overexpressing glucose transporter one in endothelial cells revealed that dextrose induced SO generation undergoes adaptive down regulation within 2h while the ER stress is sustained throughout 72h of observation.
The nature of the cross talk between oxidative stress and ER stress induced by dextrose may explain the failure of antioxidant therapy in reducing diabetes complications.
Water impoundment imposes fundamental changes on natural landscapes by transforming rivers into reservoirs. The dramatic shift in physical conditions accompanying the loss of flow creates novel ...ecological and evolutionary challenges for native species. In this study, we compared the body shape of Cyprinella venusta collected from eight pairs of river and reservoir sites across the Mobile River Basin (USA). Geometric morphometric analysis of the body shape showed that river populations differ from reservoir populations. Individuals inhabiting reservoirs are deep-bodied and have a smaller head, a more anterior dorsal fin, a shorter dorsal fin base and a more ventral position of the eye than C. venusta in streams. The direction of shape divergence within reservoir–river pairs was consistent among pairs of sites, and the shape of C. venusta in reservoirs is strongly correlated with reservoir size. These findings show that physical characteristics of reservoirs drive changes in the morphological attributes of native fish populations, indicating that water impoundment may be an important, yet largely unrecognized, evolutionary driver acting on aquatic biodiversity.
During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. ...Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.
HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.
Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould ...pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary and systemic model of aspergillosis.
Endoplasmic reticulum (ER) stress plays a critical role in progression of diabetes and development of complications, notably cardiovascular disease. Some of the contemporary anti-hyperglycemic drugs ...have been shown to inhibit ER stress. To extend these observations, the effects of various anti-hyperglycemic agents were screened for their effects on ER stress. Seven classes of anti-hyperglycemic drugs were screened including sulfonylureas, meglitinides, metformin, α glucosidase inhibitors, thiazolidinedione, glucagon like peptide 1 (GLP-1) receptor agonists and sodium-glucose cotransporter 2 (SGLT-2) inhibitors. ER stress was measured in human coronary artery endothelial cells (HCAEC) either treated with tunicamycin (TM) or cultured in hyperglycemic conditions (27.5 mM dextrose). The ER stress was measured with the secreted alkaline phosphatase (ES-TRAP) assay. Mediators of the unfolded protein response, including activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), phospho-inositol-requiring enzyme 1α (pIRE1α), IRE1α, phospho-protein kinase R (PKR)-like endoplasmic reticulum kinase (pPERK), and PERK were measured by Western blot. Metformin, GLP-1 receptor agonists (GLP-1, exendin 4, liraglutide, albiglutide, and lixisenatide) and SGLT-2 inhibitors (canagliflozin, dapagliflozin, and empagliflozin) were the only anti-hyperglycemic drugs screened that reduced ER stress caused by pharmacological (tunicamycin) or hyperglycemic conditions. High-dextrose and TM increased IRE1α and PERK phosphorylation and ATF6 and GRP78 expression, while treatment with metformin, liraglutide (a GLP-1 receptor agonist) and dapagliflozin (a SGLT-2 inhibitor), suppressed IRE1α and PERK phosphorylation as well as ATF6 and GRP78 expression. Thus, the cardioprotective effects of metformin, some of the GLP-1 receptor agonists and SGLT2 inhibitors may be partly related to their ability to reduce ER stress.
A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million ...people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption.sup.1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
Food-based strategies to reduce nutritional iron deficiency have not been universally successful. Biofortification has the potential to become a sustainable, inexpensive, and effective solution.
This ...randomized controlled trial was conducted to determine the efficacy of iron-biofortified beans (Fe-Beans) to improve iron status in Rwandan women.
A total of 195 women (aged 18-27 y) with serum ferritin <20 μg/L were randomly assigned to receive either Fe-Beans, with 86 mg Fe/kg, or standard unfortified beans (Control-Beans), with 50 mg Fe/kg, 2 times/d for 128 d in Huye, Rwanda. Iron status was assessed by hemoglobin, serum ferritin, soluble transferrin receptor (sTfR), and body iron (BI); inflammation was assessed by serum C-reactive protein (CRP) and serum α1-acid glycoprotein (AGP). Anthropometric measurements were performed at baseline and at end line. Random weekly serial sampling was used to collect blood during the middle 8 wk of the feeding trial. Mixed-effects regression analysis with repeated measurements was used to evaluate the effect of Fe-Beans compared with Control-Beans on iron biomarkers throughout the course of the study.
At baseline, 86% of subjects were iron-deficient (serum ferritin <15 μg/L) and 37% were anemic (hemoglobin <120 g/L). Both groups consumed an average of 336 g wet beans/d. The Fe-Beans group consumed 14.5 ± 1.6 mg Fe/d from biofortified beans, whereas the Control-Beans group consumed 8.6 ± 0.8 mg Fe/d from standard beans (P < 0.05). Repeated-measures analyses showed significant time-by-treatment interactions for hemoglobin, log serum ferritin, and BI (P < 0.05). The Fe-Beans group had significantly greater increases in hemoglobin (3.8 g/L), log serum ferritin (0.1 log μg/L), and BI (0.5 mg/kg) than did controls after 128 d. For every 1 g Fe consumed from beans over the 128 study days, there was a significant 4.2-g/L increase in hemoglobin (P < 0.05).
The consumption of iron-biofortified beans significantly improved iron status in Rwandan women. This trial was registered at clinicaltrials.gov as NCT01594359.
The in situ esterification/transesterification of algal biomass lipids to produce FAME for potential use as biodiesel was investigated. Commercial algal biomass was employed, containing 20.9 wt% ...hexane extractable oil. This consisted of 35.1 wt% free fatty acids (FFA), 18.2 wt% TAG, and 8.8 wt% MAG, accounting for 62.1% of the extractable material. Other constituents of the hexane extractable material, accounting for 37.9% of the extracts, were not further characterized. The predominant fatty acids in the oil were palmitic (42.4 wt%), oleic (30.6 wt%), linoleic (22.8 wt%), and linolenic (16.1 wt%). Small amounts of 10‐keto 16:0 and 10‐OH 16:0 fatty acids were also present. Statistical experimental design was employed to coordinately examine the effects of the amounts of methanol, sulfuric acid, and reaction temperature (23–65°C) on the yield of FAME in 2 h reactions. Three methods of feedstock preparation were examined – as received, oven dried, and water‐washed/dried. For all feedstocks conditions could be identified which were predicted to yield greater than 90% maximum theoretical FAME production. Oven drying the feedstock reduced the amount of methanol required, with 83% of maximum yield obtained at a methanol/fatty acid molar ratio of 220:1 (4 mL methanol/g substrate). Water washing the biomass did not reduce the methanol required for high level transesterification.
Practical applications: In biodiesel production by the conventional method of alkali‐catalyzed transesterification of a refined vegetable oil/the combination of feedstock and process costs threatens the economic viability of biofuel production. In situ esterification/transesterification is a method for producing biodiesel wherein a lipid‐bearing material is directly treated with reagents that catalyze fatty acid alkyl ester production from FFA and acylglycerols. By eliminating the need to isolate and refine the feedstock lipid, this approach to biodiesel production eliminates some of the processing steps required by contemporary methods. This could provide a welcome reduction in the cost of biodiesel production. There is presently considerable interest in the possibility of biofuel production from algal biomass. This study describes the application and optimization of in situ transesterification to a lipid‐bearing algal biomass. Application of this technology could facilitate the economical production of biodiesel from algal biomass.
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