Normalized neutral lipid quantitation by flow cytometry.

Interest in measuring tissue lipids has increased as the link between fat-laden tissues and metabolic disease has become obvious; however, linking disease to a specific cell type within a tissue has been hampered by methodological limitations. Flow cytometry (FC) has been used to assess relative lipid levels in cells. Unfortunately, its usefulness is limited because comparisons between samples generated over several hours is problematic. We show that: 1) in lipophilic fluorophore stained cells, fluorescence intensity measured by FC reflects lipid levels; 2) this technique can be used to assess lipid levels in a mixed cell population; 3) normalizing to a control condition can decrease experiment-to-experiment variation; and 4) fluorescence intensity increases linearly with lipid levels. This allows triacylglycerol (TG) mass to be estimated in mixed cell populations comparing cells with known fluorescence and TG levels. We exploited this strategy to estimate lipid levels in monocytes within a mixed population of cells isolated from human blood. Using this strategy, we also confirmed that perilipin (PLIN)1 increases TG accumulation by ectopically expressing fluorescently tagged PLIN1 in Huh7 cells. In both examples, biochemically assaying for TG in specific cell populations is problematic due to limited cell numbers and isolation challenges. Other advantages are discussed.

IL-38 binds to the IL-36 receptor and has biological effects on immune cells similar to IL-36 receptor antagonist.

The functional role of IL-1 family member 10, recently renamed IL-38, remains unknown. In the present study we aimed to elucidate the biological function of IL-38 and to identify its receptor. Heat-killed Candida albicans was used to stimulate memory T-lymphocyte cytokine production in freshly obtained human peripheral blood mononuclear cells from healthy subjects. The addition of recombinant IL-38 (152 amino acids) inhibited the production of T-cell cytokines IL-22 (37% decrease) and IL-17 (39% decrease). The reduction in IL-22 and IL-17 caused by IL-38 was similar to that caused by the naturally occurring IL-36 receptor antagonist (IL-36Ra) in the same peripheral blood mononuclear cells cultures. IL-8 production induced by IL-36γ was reduced by IL-38 (42% decrease) and also was reduced by IL-36Ra (73% decrease). When human blood monocyte-derived dendritic cells were used, IL-38 as well as IL-36Ra increased LPS-induced IL-6 by twofold. We screened immobilized extracellular domains of each member of the IL-1 receptor family, including the IL-36 receptor (also known as "IL-1 receptor-related protein 2") and observed that IL-38 bound only to the IL-36 receptor, as did IL-36Ra. The dose-response suppression of IL-38 as well as that of IL-36Ra of Candida-induced IL-22 and IL-17 was not that of the classic IL-1 receptor antagonist (anakinra), because low concentrations were optimal for inhibiting IL-22 production, whereas higher concentrations modestly increased IL-22. These data provide evidence that IL-38 binds to the IL-36R, as does IL-36Ra, and that IL-38 and IL-36Ra have similar biological effects on immune cells by engaging the IL-36 receptor.

Gossypium hirsutum gene of unknown function Gohir.A02G161000 encodes a potential transmembrane Root UVB Sensitive 4 Protein with a putative protein-protein interaction interface.

A gene of unknown function, Gohir.A02G161000.1, identified in Gossypium hirsutum was studied using computational sequence and structure bioinformatics tools. The associated protein GhRUS4-A0A1U8JPV7 (UniProt A0A1U8JPV7) is predicted to be a plastid-localized, transmembrane root UVB-sensitive 4 (RUS4) protein with a newly identified potential dimerization surface. Evidence from homology and sequence conservation suggest involvement in auxin transport and pollen maturation.

From classroom to clinic: Biochemistry lab for pre-health majors.

Undergraduate laboratory courses are vital for both enhancing student learning and preparing students for their future careers. Despite their importance, laboratory courses are often met with a lack of enthusiasm from students. For pre-health students specifically, laboratory courses are commonly seen as part of a check-list that needs to be completed in order to achieve future education or a career, instead of seeing the information that is taught in laboratories as essential preparation. A one-semester biochemistry laboratory module was designed to demonstrate the real-life applications of experimental techniques. This laboratory module introduced students to common clinical measurements and included learning metabolite analysis through hands-on experiments, connections to simulated patient visits, generation of a research question, and implementation of a student-designed independent experiment. Surveys indicate that this approach was helpful in creating a greater understanding of the applicability of undergraduate laboratory concepts appealing specifically to pre-health students. Additionally, students found this module to provide a variety of gained benefits, knowledge, and confidence in performing scientific techniques. The outcomes of this laboratory module indicate its success and potential to be used in curricula as an effective way to engage pre-health students in an undergraduate biochemistry laboratory.

Gossypium hirsutum gene of unknown function Gohir.A03G007700.1 encodes a potential VAN3-binding protein with a phosphoinositide-binding site.

A gene of unknown function, Gohir.A03G007700.1 (gene ID: Gohir.A03G007700_UTX-TM1_v2.1; transcript ID: Gohir.A03G007700.1_UTX-TM1_v2.1), identified in Gossypium hirsutum was studied using bioinformatic analyses of the sequence and structure of its encoded protein. Results from domain prediction, conserved residues and structure comparison indicate the encoded plant-specific protein (UniProt A0A1U8N485) is part of the VAN3-binding protein family with a conserved phosphoinositide-binding site. Homology comparison suggests functional similarity with Arabidopsis FORKED-like FL5 and 6, which localize to the Golgi apparatus and are linked to vein development and leaf size phenotypes.

Gossypium hirsutum gene of unknown function Gohir.A03G0737001 encodes a potential Chaperone-like Protein of protochlorophyllide oxidoreductase (CPP1).

A gene of unknown function identified in Gossypium hirsutum , Gohir.A03G0737001.1, was studied using sequence and bioinformatic tools. The encoded protein (referred to here as GhCPP1-A0A1U8HKT6) was predicted to function as a Chaperone-like protein of protochlorophyllide oxidoreductase (CPP1), which is involved with initiation of photochemical reactions of chlorophyll biosynthesis. Sequence analysis indicates it is embedded in the chloroplast envelope membrane through four transmembrane regions and contains a J-like domain that is structurally similar to the J domain of DnaJ/Hsp40 "holdase" chaperone proteins.

An Autocrine Role for CXCL1 in Progression of Hepatocellular Carcinoma.

BACKGROUND: One of the most prevalent causes of cancer fatalities is hepatocellular carcinoma (HCC), which has been linked to metabolic syndrome. Circulating levels of the saturated fatty acid palmitate are elevated in metabolic syndrome and lead to cellular stress. MATERIALS AND METHODS: Using enzyme-linked immunosorbent assay, flow cytometry, and migration assays, we characterized the response of rat hepatoma cells to palmitate treatment. RESULTS: We detected a 60% increase in secretion of C-X-C motif ligand 1 (CXCL1) which was dose-dependent and coincided with apoptosis. We measured expression of C-X-C motif chemokine receptor 2 (CXCR2) and observed a 4.5-fold increase on apoptotic hepatoma cells. Furthermore, we assayed migration of hepatoma cells and saw a 2-fold increase in the number of migrating cells towards CXCL1. CONCLUSION: These findings suggest that HCC cells secrete CXCL1 in response to metabolic syndrome signals and may promote the progression of cancer through apoptosis recovery or metastasis.

iCURE (iterative course-based undergraduate research experience): A case-study.

Several models suggest ways to expose undergraduates at minority serving institutions or institutions with limited research infrastructures to the iterative process of research. Apprentice-based research experiences allow students to work one-on-one with a research mentor in the hands-on discovery process, but with teaching being a priority for faculty at the aforementioned institutions, financial, spatial, and time limitations for research progress exist. Course-based undergraduate research experiences (CUREs) provide opportunities for a greater number of undergraduates to become familiar with the questions, techniques, and failure involved in research. However, designing projects that a group of students can complete in a semester can be challenging. Inclusive Research Education Communities are intended to promote retention in STEM courses for early college students but have limited benefit for upper-level courses. We sought to create an iterative CURE between fall semester BIOL3900 at the University of North Texas and spring semester CHE397 at Bethel University (Saint Paul, MN) to promote collaboration between unique learning communities. The research goal was to use a tobacco (Nicotiana benthamiana) transient expression system as a platform to test gene functions and to engineer valuable bioproducts in plant vegetative tissues. The outcomes of this 2-year integrative module included novel discoveries leading to publications in peer-reviewed journals, cost benefits due to shared resources, continual movement of the project, course-based training for future independent research projects, and improved student attitudes about research. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):565-572, 2019.

Decreasing Phosphatidylcholine on the Surface of the Lipid Droplet Correlates with Altered Protein Binding and Steatosis.

Alcoholic fatty liver disease (AFLD) is characterized by an abnormal accumulation of lipid droplets (LDs) in the liver. Here, we explore the composition of hepatic LDs in a rat model of AFLD. Five to seven weeks of alcohol consumption led to significant increases in hepatic triglyceride mass, along with increases in LD number and size. Additionally, hepatic LDs from rats with early alcoholic liver injury show a decreased ratio of surface phosphatidylcholine (PC) to phosphatidylethanolamine (PE). This occurred in parallel with an increase in the LD association of perilipin 2, a prominent LD protein. To determine if changes to the LD phospholipid composition contributed to differences in protein association with LDs, we constructed liposomes that modeled the LD PC:PE ratios in AFLD and control rats. Reducing the ratio of PC to PE increased the binding of perilipin 2 to liposomes in an in vitro experiment. Moreover, we decreased the ratio of LD PC:PE in NIH 3T3 and AML12 cells by culturing these cells in choline-deficient media. We again detected increased association of specific LD proteins, including perilipin 2. Taken together, our experiments suggest an important link between LD phospholipids, protein composition, and lipid accumulation.

A potential role for fructose-2,6-bisphosphate in the stimulation of hepatic glucokinase gene expression.

The effects of fructose-2,6-bisphosphate (F-2,6-P(2)) on hepatic glucokinase (GK) and glucose-6-phosphatase (G-6-Pase) gene expression were investigated in streptozotocin-treated mice, which exhibited undetectable levels of insulin. Hepatic F-2,6-P(2) levels were manipulated by adenovirus-mediated overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Streptozotocin treatment alone or with infusion of control adenovirus leads to a dramatic decrease in hepatic F-2,6-P(2) content compared with normal nondiabetic mice. This is accompanied by a 14-fold decrease in GK and a 3-fold increase in G-6-Pase protein levels, consistent with a diabetic phenotype. Streptozotocin-treated mice that were infused with adenovirus-overexpressing an engineered 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with high kinase activity and little bisphosphatase activity showed high levels of hepatic F-2,6-P(2). Surprisingly, these mice had a 13-fold increase in GK protein and a 2-fold decrease in G-6-Pase protein compared with diabetic controls. The restoration of GK is associated with increases in the phosphorylation of Akt upon increasing hepatic F-2,6-P(2) content. Moreover, the changes in levels of F-2,6-P(2) and Akt phosphorylation revealed a pattern similar to that of streptozotocin mice treated with insulin, indicating that increasing hepatic content of F-2,6-P(2) mimics the action of insulin. Because G-6-Pase gene expression was down-regulated only after the restoration of euglycemia, the effect of F-2,6-P(2) was indirect. Also, the lowering of blood glucose by high F-2,6-P(2) was associated with an increase in hepatic nuclear factor 1-alpha protein, a transcription factor involved in G-6-Pase gene expression. In conclusion, F-2,6-P(2) can stimulate hepatic GK gene expression in an insulin-independent manner and can secondarily affect G-6-Pase gene expression by lowering the level of plasma glucose.

The role of SREBP-1c in nutritional regulation of lipogenic enzyme gene expression.

A high carbohydrate diet up-regulates the transcription of enzymes of triglyceride biosynthesis (lipogenesis) in the mammalian liver. This treatment stimulates hepatic insulin signaling, leading to transcription of sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c has been implicated as a major factor that up-regulates lipogenic genes in response to carbohydrate feeding. However, we presented evidence for another factor, carbohydrate response factor, which is also involved in this response, and we proposed a model wherein SREBP-1c and carbohydrate response factor are independent transcription factors that act in response to insulin and glucose, respectively. In this study, we examined the contribution of SREBP-1c to the expression of lipogenic genes in glucose- and insulin-treated primary rat hepatocytes using an inducible adenovirus system. We found that SREBP-1c overexpression leads to a modest induction of fatty acid synthase, S(14), and acetyl-CoA carboxylase mRNAs to 20% (fatty acid synthase), 10% (S(14)), and 5% (acetyl-CoA carboxylase) of the induction seen by high glucose and insulin treatment. Restoring insulin to cells overexpressing SREBP-1c did not further increase these mRNA levels. In contrast, adenovirus-expressed SREBP-1c did not induce pyruvate kinase mRNA, suggesting that induction of this gene is SREBP-1c-independent. SREBP-1c does indeed play a role in the induction of lipogenic enzyme genes in response to insulin treatment, but it is not sufficient for the induction seen when hepatocytes are treated with insulin and high glucose.

Mlx is the functional heteromeric partner of the carbohydrate response element-binding protein in glucose regulation of lipogenic enzyme genes.

The expression of genes encoding enzymes involved in de novo triglyceride synthesis (lipogenesis) is transcriptionally induced in the liver in response to increased glucose metabolism. The carbohydrate response element-binding protein (ChREBP) is a newly identified basic helix-loop-helix/leucine zipper transcription factor proposed to regulate the expression of the glucose-responsive gene pyruvate kinase. This gene contains a carbohydrate response element (ChoRE) consisting of two E box motifs separated by 5 bp that is necessary and sufficient for glucose regulation. We demonstrate that overexpression of ChREBP in primary rat hepatocytes activates other ChoRE-containing promoters in a manner consistent with their ability to respond to glucose. In vitro binding of ChREBP to ChoRE sequences was not detected. Because E box-binding proteins function as obligate dimers, we performed a yeast two-hybrid screen of a mouse liver cDNA library to identify potential heteromeric partners. Mlx (Max-like protein X) was selected as the only basic helix-loop-helix/leucine zipper interaction partner in this screen. When a plasmid expressing either Mlx or ChREBP was cotransfected with a ChoRE-containing reporter plasmid into human embryonic kidney 293 cells, no increase in promoter activity was observed. However, the expression of both proteins dramatically enhanced promoter activity. This activation was observed with reporters containing ChoREs from several different lipogenic enzyme genes. In contrast, reporters containing non-glucose-responsive E box elements were not activated by ChREBP-Mlx expression. In vitro binding of ChREBP to ChoRE-containing oligonucleotides was observed only in the presence of Mlx. ChREBP-Mlx binding discriminated between E box sites that are glucose-responsive and those that are not. We conclude that Mlx is a functional heteromeric partner of ChREBP in regulating the expression of glucose-responsive genes.