STAT3 Is the Master Regulator for the Forming of 3D Spheroids of 3T3-L1 Preadipocytes

To elucidate the currently unknown mechanisms responsible for the diverse biological aspects between two-dimensional (2D) and three-dimensional (3D) cultured 3T3-L1 preadipocytes, RNA-sequencing analyses were performed. During a 7-day culture period, 2D- and 3D-cultured 3T3-L1 cells were subjected to lipid staining by BODIPY, qPCR for adipogenesis related genes, including peroxisome proliferator-activated receptor γ (Pparγ), CCAAT/enhancer-binding protein alpha (Cebpa), Ap2 (fatty acid-binding protein 4; Fabp4), leptin, and AdipoQ (adiponectin), and RNA-sequencing analysis. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by a quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Bioinformatic analyses were performed on DEGs using a Gene Ontology (GO) enrichment analysis and an Ingenuity Pathway Analysis (IPA). Significant spontaneous adipogenesis was observed in 3D 3T3-L1 spheroids, but not in 2D-cultured cells. The mRNA expression of Pparγ, Cebpa, and Ap2 among the five genes tested were significantly higher in 3D spheroids than in 2D-cultured cells, thus providing support for this conclusion. RNA analysis demonstrated that a total of 826 upregulated and 725 downregulated genes were identified as DEGs. GO enrichment analysis and IPA found 50 possible upstream regulators, and among these, 6 regulators—transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL6), angiotensinogen (AGT), FOS, and MYC—were, in fact, significantly upregulated. Further analyses of these regulators by causal networks of the top 14 predicted diseases and functions networks (IPA network score indicated more than 30), suggesting that STAT3 was the most critical upstream regulator. The findings presented herein suggest that STAT3 has a critical role in regulating the unique biological properties of 3D spheroids that are produced from 3T3-L1 preadipocytes.

Clinical Utility of a Biomarker to Detect Contrast-Induced Acute Kidney Injury during Percutaneous Cardiovascular Procedures

<b><i>Introduction:</i></b> Contrast-induced acute kidney injury (CI-AKI) is a major clinical complication of percutaneous cardiovascular procedures requiring iodinated contrast. Despite its relative frequency, practicing physicians are unlikely to identify or treat this condition. <b><i>Methods:</i></b> In a 2-round clinical trial of simulated patients, we examined the clinical utility of a urine-based assay that measures liver-type fatty acid-binding protein (L-FABP), a novel marker of CI-AKI. We sought to determine if interventional cardiologists’ ability to diagnose and treat potential CI-AKI improved using the biomarker assay for 3 different patient types: pre-procedure, peri-procedure, and post-procedure patients. <b><i>Results:</i></b> 154 participating cardiologists were randomly divided into either control or intervention. At baseline, we found no difference in the demographics or how they identified and treated potential complications of AKI, with both groups providing less than half the necessary care to their patients (46.4% for control vs. 47.6% for intervention, <i>p</i> = 0.250). The introduction of L-FABP into patient care resulted in a statistically significant improvement of 4.6% (<i>p</i> = 0.001). Compared to controls, physicians receiving L-FABP results were 2.9 times more likely to correctly identify their patients’ risk for AKI (95% CI 2.1–4.0) and were more than twice as likely to treat for AKI by providing volume expansion and withholding nephrotoxic medications. We found the greatest clinical utility in the pre-procedure and peri-procedure settings but limited value in the post-procedure setting. <b><i>Conclusion:</i></b> This study suggests L-FABP as a clinical marker for assessing the risk of potential CI-AKI, has clinical utility, and can lead to more accurate diagnosis and treatment.

Effect of cogon grass root ethanol extract on fatty acid binding protein 4 and oxidative stress markers in a sepsis mouse model

Background: Sepsis causes several immunological and metabolic alterations that induce oxidative stress. The modulation of fatty acid-binding protein 4 (FABP4) has been shown to worsen this condition. Extract of cogon grass root (ECGR) contains flavonoids and isoeugenol compounds that exhibit anti-inflammatory and antioxidant properties. This study aimed to assess the effects of ECGR on FABP4 and oxidative stress–related factors in a sepsis mouse model. Methods: Twenty-nine male mice (Mus musculus) of the Deutsche Denken Yoken strain were divided into four groups: group 1, control; group 2, mice treated with 10 μL/kg body weight (BW) lipopolysaccharide (LPS); and groups 3 and 4, mice pre-treated with 90 and 115 mg/kg BW, respectively, and then treated with 10 μL/kg BW LPS for 14 d. Blood, liver, lymph, and cardiac tissue samples were collected and subjected to histological and complete blood examinations. Antioxidant (Glutathione peroxidase 3 (GPx3) and superoxide dismutase), FABP4 levels, and immune system-associated biomarker levels (TNF-α, IL-6 and IL-1β ) were measured. Results: Significant increases in platelet levels (p = 0.03), cardiomyocyte counts (p =0.004), and hepatocyte counts (p = 0.0004) were observed in group 4 compared with those in group 2. Conversely, compared with those in group 2, there were significant decreases in TNF-α expression in group 3 (p = 0.004), white pulp length and width in group 4 (p = 0.001), FABP4 levels in groups 3 and 4 (p = 0.015 and p = 0.012, respectively), lymphocyte counts in group 4 (p = 0.009), and monocyte counts (p = 0.000) and polymorphonuclear cell counts in the livers (p = 0.000) and hearts (p = 0.000) of groups 3 and 4. GPx3 activity was significantly higher in group 3 than in group 1 (p = 0.04). Conclusions: ECGR reduces FABP4 level and modulating oxidative stress markers in sepsis mouse model.

Metabolic and proteomic signatures of type 2 diabetes subtypes in an Arab population

Background. Type 2 diabetes (T2D) has a heterogeneous etiology which is increasingly recognized to influence the risk of complications and choice of treatment. A data driven cluster analysis in four separate European populations of patients with type 2 diabetes identified four subtypes of severe insulin dependent (SIDD), severe insulin resistant (SIRD), mild obesity-related (MOD), and mild age-related (MARD) (Ahlqvist et al., Lancet Diabetes Endocrinology, 2018). Our aim was to extend this classification to the Arab population of Qatar and characterize the biological processes that differentiate these subtypes in relation to metabolomic and proteomic signatures. Methods. The Ahlqvist et al. subtype clustering approach was applied to 631 individuals with T2D from the Qatar Biobank (QBB) and validated in an independent set of 420 participants from the same population. The association between blood metabolites (n=1,159) and protein levels (n=1,305) with each cluster were established. Findings. The four subtypes of T2D were reproduced and validated in the population of Qatar. Cluster-specific metabolomic and proteomic associations revealed subtype-specific molecular processes. Activation of the complement system with many features of autoimmune diabetes and reduced 1,5-anhydroglucitol (1,5-AG) characterized SIDD, with evidence of impaired insulin signaling in SIRD, elevated leptin and fatty acid binding protein in MOD, whilst MARD appeared to be the healthiest subgroup. Interpretation. We have replicated the four T2D clusters in an Arab population and identified distinct metabolic and proteomic signatures, providing insights into underlying etiology with the potential to deploy subtype-specific treatment options.

Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

Background: FabB (3-oxoacyl-[acyl-carrier-protein] synthase 1) is part of the fatty acid synthesis II pathway found in bacteria and a potential target for antibiotics. The enzyme catalyses the Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP via an acyl-enzyme intermediate. Here, we report the crystal structure of the intermediate-mimicking Pseudomonas aeruginosa FabB (PaFabB) C161A variant. Methods: His-tagged PaFabB C161A was expressed in E. coli Rosetta DE3 pLysS cells, cleaved by TEV protease and purified using affinity and size exclusion chromatography. Commercial screens were used to identify suitable crystallization conditions which were subsequently improved to obtain well diffracting crystals. Results: We developed a robust and efficient system for recombinant expression of PaFabB C161A. Conditions to obtain well diffracting crystals were established. The crystal structure of PaFabB C161A was solved by molecular replacement at 1.3 Å resolution. Binding site comparison between PaFabB and PaFabF revealed a conserved malonyl binding site but differences in the fatty acid binding channel. Conclusions: The PaFabB C161A crystal structure can be used as a template to facilitate the design of FabB inhibitors.

Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.