bluuebunny/crossref_metadata_2025 huggingface dataset:
The Lipin Protein Family, Cellular Lipid Storage, and Disease
Authors: Reue Karen
Published: June 2021

Several years ago we identified the Lipin gene family in a mutant inbred mouse strain with lipodystrophy and insulin resistance. The lipin proteins are phosphatidate phosphatase enzymes, which dephosphorylate phosphatidic acid to diacylglycerol, the penultimate step in triacylglycerol biosynthesis. Lipins are unique among the glycerolipid biosynthetic enzymes in their ability to associate with multiple cellular compartments that include the cytosol, endoplasmic reticulum membrane, and nucleus. Lipin function has been evolutionarily conserved from a single ortholog in yeast to the mammalian family of three lipin proteins—lipin‐1, lipin‐2, and lipin‐3. In mice and humans, the levels of lipin activity are a determinant of triacylglycerol storage in several tissues, and humans with deficiency in lipin‐1 or lipin‐2 have severe metabolic diseases. Recent work has highlighted the complex physiological interactions between members of the lipin protein family, which exhibit both overlapping and unique functions in specific tissues. The analysis of “lipinopathies” in mouse models and in humans has revealed that lipin activity regulates lipid intermediates that influence fundamental cellular processes including adipocyte and chondrocyte differentiation, cerebellar function, intestinal fat absorption, and autophagy. The elucidation of lipin molecular and physiological functions could lead to novel approaches to modulate cellular lipid storage and metabolic disease.
Chemical Approaches to the Identification and Characterization of Protein‐Membrane Binding Interactions Using Synthetic Lipid Probes
Authors: Best Michael , Rowland Meng , Ricks Tanei , Alam Shahrina , Eni Eni Sammy , Mattern‐Schain Sam
Published: June 2021

Signaling lipids present in cellular membranes act as important regulators of biological processes and have been implicated in the onset of numerous diseases. A common role of these lipids is the recruitment of proteins to the cell membrane surface through binding events that generally regulate protein function and localization. While understanding these interactions at the molecular level is of great interest, such efforts are hindered by the complexity of the membrane environment in which binding occurs. Towards this end, chemical strategies will be presented by which protein–membrane recognition events can be efficiently characterized. Firstly, the development and application of lipid activity probes for the proteomic identification of lipid‐binding proteins will be described. In addition, microarray platforms for the characterization of protein‐membrane binding interactions will be presented. Finally, strategies for the labeling and detection of lipids in native environments will also be discussed.
Sphingolipids of Commensals Modulate Host Immunity through Regulation of iNKT Cells
Authors: Kasper Dennis
Published: June 2021

The microbiota modulate invariant natural killer T (iNKT) cell numbers in the colon. SInce iNKT cells recognize glycosphingolipids presented by CD1d and the Bacteroidetes are among the few microbial phyla with sphingolipids contributing to membrane architecture, we sought to find a molecule within the Bacteroidetes involved in modulating iNKT cell homeostasis.

In Bacteroides fragilis, we generated a mutant deleted of serine palmitoyl transferase (del SPT) that does not produce any sphingolipids in the microbial membranes. After monocolonization of germ‐free (GF) mice with either the wild‐type or the del SPT mutant, we found that the wild‐type B. fragilis colonized mice had significantly lower colonic iNKT cell numbers than the del SPT mutant mono‐colonized mice. Interestingly colonic iNKT cell numbers were very low in the wild type colonized mice as we had previously reported in conventionally colonized mice. In contrast, the del SPT mutant colonized mice had high iNKT cell numbers as we reported in GF mice. We purified and chemically identified a microbial sphingolipid molecule that when administered to the del SPT colonized mice, re‐capitulates the colonic iNKT phenotype seen in the WT mice. Sphingolipids have been reported previously to stimulate iNKT cell numbers, but the sphingolipid we have isolated from Bacteroides fragilis actually inhibits iNKT cell numbers in the colon, simulating the observation reported with the entire microbiota.

Our study revealed a previously unknown pathway by which the microbiota modulate the host immune system. It also points to a new direction for therapeutic intervention of diseases mediated by proinflammatory iNKT cells.
Testing Competencies with the New MCAT® Exam
Authors: Kroopnick Marc
Published: June 2021

The new Medical College Admission Test® (MCAT®) will be introduced in April of 2015. Periodic updates to the MCAT exam are important to ensure it keeps pace with medical school curricula, and provides admissions committees with valuable information about their applicants' readiness. These updates help ensure that the exam is keeping pace with changes in the study and practice of medicine, such as new and innovative treatments, health care system reforms, and the challenges that come with serving an increasingly diverse population. Highlights of the changes to the exam include the organization around competencies, use of interdisciplinary life sciences passage sets, the inclusion of biochemistry content, and the introduction of a test section that focuses on the behavioral and social sciences. The competencies that include biochemistry content can be found in the Biological and Biochemical Foundations of Living Systems section as well as the Chemical and Physical Foundations of Biological Systems section. Each section will feature interdisciplinary life sciences passages and questions that will ask students to combine their knowledge of the content domain with scientific inquiry and reasoning skills to answer the questions. This presentation will describe the new exam with a special focus on the competencies that include biochemistry as well as the resources available to aid faculty in preparing students for the exam.
Inter‐relationship Between the In vivo Metabolism of Apolipoprotein B100‐Containing Lipoproteins and LDL Particle Size and LDL Particle Number
Authors: Leclerc Myriam , Ooi Esther , Couture Patrick , Richard Caroline , Desroches Sophie , Marin Johanne , Tremblay André , Lamarche Benoît
Published: June 2021

Studies have shown that small dense LDL particles confer an increased risk of coronary heart disease (CHD) compared with large LDL. LDL particle number is also an important risk factor for CHD.

The objective was to investigate the inter‐relationship between the in vivo kinetics of apolipoprotein (apo) B100‐containing lipoproteins and LDL particle size (LDLsi) and LDL particle number (LDL‐apoB).

This analysis is based on data from 154 male and female subjects among whom in vivo lipoprotein kinetics were investigated using a bolus/infusion of D3‐leucine. LDLsi was assessed by non‐denaturing polyacrylamide gradient gel electrophoresis. Participants' mean age (±SD) was 44.7±12.6 yrs. Mean body mass index (BMI) and triglyceride levels were 28.6±4.7 kg/m2 and 1.97±1.4 mmol/l respectively.

LDLsi correlated positively with plasma adiponectin levels (age and BMI‐adjusted Spearman r=0.41, P<0.001) and the fractional catabolic rate (FCR) of VLDL‐apoB (r=0.43, P<0.001) and negatively with plasma TG (r=‐0.42 P<0.001) and the pool size of VLDL‐apoB (r=‐0.38, P<0.001). Plasma LDL‐apoB levels correlated positively with the production rate of VLDL‐apoB (r=0.27, P=0.006) and negatively LDL‐apoB FCR (r=‐0.59, P<0.001). LDL‐apoB showed no correlation with plasma TG (r=0.07, P=0.47), LDLsi (r=0.18, P=0.08) and adiponectin (r=‐0.02, P=0.82).

These data suggest that LDL size and LDL particle number are determined by distinct metabolic pathways.

Funding provided by the Canadian Institutes of Health Research and the Chair of Nutrition, Université Laval
Role of Acyl‐Coenzyme A:Cholesterol Acyltransferase 1 in Lipogenesis in Adipocytes
Authors: Zhu Yuyan , Chen Chih‐Yu , Kim Kee‐Hong
Published: June 2021

Dysregulated lipogenesis in adipose tissue contributes to obesity and its metabolic disorders. However, the molecular mechanisms regulating lipogenesis in adipocytes are not fully understood. Acyl‐coenzyme A: cholesterol acyltransferase (ACAT), an enzyme that catalyzes the formation of cholesteryl ester from free cholesterol (FC), has been identified as a therapeutic target to treat atherosclerosis. However, the role of ACAT in adipogenesis and lipogenesis in adipocytes is unknown. Here, we employed both pharmaceutical inhibitors and a lentivirus‐mediated shRNA‐based gene knockdown technique to investigate the role of ACAT in adipogenesis using murine 3T3‐L1 preadipocytes as an in vitro model of adipogenesis. Avasimibe, an ACAT inhibitor, was added at non‐toxic concentrations and replaced every other day during the course of adipogenesis. Avasimibe treatment inhibited lipid accumulation in a dose‐dependent manner, as judged by Oil red O staining and thin layer chromatography. Consistently, ACAT1 knockdown in 3T3‐L1 cells reduced lipid droplet accumulation by 40%. Real‐time PCR revealed that ACAT inhibition down‐regulated expressions of sterol regulatory element‐binding protein‐1 and genes involved in lipogenesis, with little effect on adipogenic transcription factors. These results suggest that ACAT is required for lipid droplet biogenesis in adipocytes. In addition, ACAT inhibition reduced 37% of intracellular FC and impaired cholesterol uptake in adipocytes, indicating a role of ACAT1 in the biochemical link between lipogenesis and cholesterol homeostasis. Taken together, our results reveal a novel function of ACAT1 in lipogenesis and highlight an important role of ACAT1 in integrating cholesterol metabolism and neutral lipid synthesis in adipocytes.
Vitamin D Inhibits ROS, ICAM‐1, and Monocyte Adhesion in High Glucose and Acetoacetate Treated Endothelial Cells by Upregulating GSH
Authors: Kanikarla Marie Preeti , Jain Sushil
Published: June 2021

The incidence of developing microvascular dysfunction is significantly higher in type 1 diabetic (T1D) patients. Monocyte adhesion to the endothelium plays a central role in the progression of endothelial dysfunction. This study examines whether vitamin D (VD) supplementation is beneficial in preventing endothelial dysfunction in diabetes. Human umbilical vein endothelial cells (HUVEC) were treated with 1, 25‐(OH)2‐D3 (0‐25 nM, 24 h) and later exposed to the ketone body acetoacetate (AA, 4 mM) or high glucose (HG, 25 mM) for 24 h. Treatment with AA or HG causes an increase in ROS, ICAM‐1 expression, and monocyte adhesion, along with a decrease in GSH levels in HUVEC. VD supplementation (25 nM) significantly lowered ROS (41%, p<0.05), ICAM‐1 (73%, p=0.05) and monocyte adhesion (37.5%, p=0.05) in AA treated HUVEC. VD had a similar beneficial effect on HG treated cells. Interestingly, we also saw an upregulation in GSH levels following treatment with VD. We hypothesize that the beneficial effect of VD is mediated by the upregulation of GSH. Glutamyl cysteine ligase (GCLC) catalyzes GSH biosynthesis. Knocking down GCLC using siRNA reduced GSH levels as well as abolished the beneficial effect of VD on ketone‐ or HG‐induced ROS, ICAM‐1, and monocyte adhesion. This suggests that VD mediates its beneficial effects via the upregulation of GCLC. This study provides the biochemical mechanism through which VD can lower ROS, ICAM‐1 expression, monocyte–endothelial adhesion, and thereby lower the risk of endothelial dysfunction in diabetes.

Supported by the NIH, the Malcolm Feist chair in Diabetes, and a fellowship from the Malcolm Feist Cardiovascular Research Endowment, LSUHSC‐Shreveport.
Micronutrient Status of Pre‐Bariatric Surgery Patients: the Prevalence of Rare and Poly‐micronutrient Deficiencies and Relationships with Pre‐Surgery BMI?
Authors: Crowley Nina , Petitpain Debbie , DellaValle Diane
Published: June 2021

Adequate micronutrient (MN) status plays a direct role in post‐bariatric surgery (BS) outcomes. The goal of this cross‐sectional analysis was to examine the prevalence of MN deficiencies in a cohort of patients preparing for BS (n=119; 87%F; 41.9±11.4y; 54.1±13.1 kg/m2). Prior to BS, in addition to routine lab work (Fe, B12, vitamin D, folate), serum levels of Zn, Cu, B1, and B6 were assessed. Sub‐optimal levels of vitamin B6 were observed with the most frequency (70.6% of the cohort), followed by vitamin B1 (35.3%), Zn (15.1%), and Cu (2.5%). Incidence of pre‐surgical B6 deficiency was negatively correlated with low Zn (r=‐0.24, p=0.008) and low B1 status (r=‐0.37, p<0.001). Seventy nine percent of the cohort exhibited a deficiency in one of the routine MNs (e.g. Fe, B12, vitamin D, folate) in addition to one of those pilot‐tested (Zn, Cu, B1, B6). Fifty‐two percent of the cohort had their BS delayed in order to address MN deficiencies. Patients with BMIs >51.9 kg/m2 were significantly more likely to have multiple MN deficiencies, as well as B1 deficiency compared to those with BMIs<51.9 (p=0.01 and p=0.04, respectively). Current practice guidelines for assessing nutrition status in BS patients do not recommend routine assessment of Zn, Cu, B1, and B6. This study clearly indicates a significant prevalence rate of sub‐optimal MN status in the BS candidate, and further research is needed to explore how this may impact post‐surgical outcomes.
Natural Resources and Food Security: Fish‐for‐Sex Relationships Around Lake Victoria, Kenya
Authors: Fiorella Kathryn , Camlin Carol , Salmen Charles , Omondi Ruth , Hickey Matthew , Omollo Dan , Milner Erin , Brashares Justin
Published: June 2021

For rural populations, natural resources provide for food and livelihoods. An estimated 10‐12% of people work in fisheries; while the majority of fishers are men, 90% of traders are women. To understand how natural resources affect food security, we study how changing fish catch in Lake Victoria, Kenya affects food security and a transactional sexual economy. Where 65% of households are severely food insecure and HIV prevalence is 27%, we analyze fish declines' influence on fish‐for‐sex participation and power dynamics. Our mixed methods approach includes a cross‐sectional household survey (n=303) and in‐depth interviews (n=30). Sexual exchanges are reported by 34% of men and 10% of women. Among those who exchanged sex, men were more likely to fish for low‐value fish (Pearson chi‐square: 4.06, p=0.04), and women were more likely to be unmarried (6.40, p=0.01), food insecure (Fisher's exact test, p=0.04), and have a fisher as a partner (6.15, p=0.01). Qualitative data show that relationships are critical to women's fish access. Further, fish scarcity increases dependence on fish‐for‐sex relationships to smooth fluctuations in fish availability. Fish declines motivate entry into fish‐for‐sex and affect power dynamics, especially negotiation of condoms. Our results have implications for understanding the Lake Victoria fish‐for‐sex economy, and how resource declines may affect the food security and well‐being of populations dependent on increasingly scarce resources.
Critical role of protein kinase D in VEGF‐induced vascular permeability and angiogenesis
Authors: Jin Zheng Gen , Zhao Jinjing , Yin Meimei , Kwon Il‐Sun , Mastrangelo Michael
Published: June 2021

Vascular endothelial growth factor (VEGF) is not only a powerful angiogenic factor but also a potent vascular permeability factor, which disrupts vascular barrier function in diseased tissues. In inflammatory and ischemia tissues, through impairing endothelial cell‐cell junctions VEGF causes vascular permeability and edema, resulting in extensive injury to ischemic tissues after stroke or myocardial infarction. In cancer, VEGF‐mediated disruption of the vascular barrier may potentiate tumour cell extravasation, resulting in widespread metastatic disease. All of these pathological conditions are also associated with increased angiogenesis. The OBJECTIVE of this study is to elucidate the signaling pathways whereby VEGF induces vascular permeability and angiogenesis. Emerging evidence from my group and others supports a crucial role of protein kinase D (PKD) in VEGF signaling and angiogenesis. PKD1 is a member of a novel family of serine/threonine protein kinases,which are potential druggable targets for therapeutics. My laboratory was the first to report that VEGF via its receptor 2 (VEGFR2) stimulated PKD1 activation in vascular ECs, which modulates EC proliferation. Subsequently, we found that PKD1, via phosphorylation of histone deacetylases, regulates EC migration and tube formation. Nascent observations from my group now reveal that PKD1, through its effects on cytoskeleton and adherens junction, regulates vascular permeability. Using Transwell EC permeability assay, we found that the inhibition of PKD1 by pharmacological inhibitors, PKD1 siRNA or adenoviral PKD1 dominant‐negative mutant significantly attenuated VEGF‐stimulated endothelial monolayer permeability. Using in vivo vascular permeability assays in mouse lung and ear microvascular beds, we found that EC‐specific knockout of PKD1 in mice greatly decreased VEGF‐induced vascular permeability. The inhibition of PKD1 by the pharmacological inhibitors also markedly diminished vascular permeability in mice. Mechanistically, we found that PKD1 mediated VEGF‐induced F‐actin stress fiber formation and the dismantlement of endothelial adherens junctions in ECs. In CONCLUSION, our results demonstrate a critical role of PKD1 in VEGF regulation of vascular permeability and angiogenesis. These findings would facilitate development of new therapeutic approaches to prevent or treat inflammation‐ and angiogenesis‐associated diseases.
Regulation of Tight Junction and Adherent Junction Disassembly by Connexin43 Hemichannels
Authors: Yao Jian , Chi Yuan , Gao Kun , Zhang Xiling , Yoda Ryuichi
Published: June 2021

Connexin (Cx) hemichannels regulate many cellular processes with little information available regarding their mechanisms. Given that many pathological factors that activate hemichannels also disrupt the integrity of cellular junctions, we speculated a potential participation of hemichannels in the regulation of the dissemble of tight/adherent junctions (TJs/AJs). Here we tested this hypothesis. 1) Exposure of renal tubular epithelial cells to Ca2+‐free medium led to disassembly of TJs and AJs, as indicated by the reduced ZO‐1 and E‐cadherin, disorganized F‐actin, as well as the reduced transepithelial resistance. 2) Ca2+ deprivation activated hemichannels, as revealed by extracellular release of ATP and intracellular influx of ethidium bromide. Inhibition of hemichannels with chemical inhibitors or Cx43 siRNA significantly prevented the disassembly of TJs. 3) Fetal fibroblasts derived from Cx43 wide‐type (Cx43+/+) and heterozygous (Cx43+/‐) mouse were more sensitive to Ca2+ deprivation‐induced disassembly of AJs, as compared with cells from Cx43 knockout (Cx43‐/‐) mouse. 4) Further analysis revealed that Cx43+/+ cells had severer protein oxidation, more dramatic changes in protein phosphorylation, and much stronger activation of P38. 5) Hemichannel opening was associated with extracellular loss of the major antioxidant GSH. Supplement of cells with exogenous GSH largely prevented the above mentioned changes in protein modifications and disassembly of TJs/AJs. 6) In a pathologic model of hemichannel opening induced by cadmium, hemichannels also contributed to cadmium‐induced disassembly of TJs. Taken together, our study thus characterizes Cx hemichannels as a presently unrecognized factor implicated in the regulation of TJ/AJ disassembly.
Isolation and Characterization of Human Breast Epithelial Cells
Authors: Hoynowski Steven , Ludlow John , Buehrer Benjamin
Published: June 2021

Breast cancer is one of the most common cancers among women in the United States and it is the second leading cause of cancer deaths in this population. Roughly 232,000 women in the U.S. were diagnosed with malignant breast cancer in 2013 and approximately 40,000 of these patients will die. The majority of breast cancers originate in the lobular or ductal cells of the milk‐producing glands. In these structures are two main cell types: luminal cells, which are surrounded by basal myoepithelium. These cells are structurally distinct and are the precursors to variable forms of breast cancer. As such, it is important to study them not only separately but also together. Current methods for culturing human mammary epithelial cells select for those of a basal phenotype. Isolation of pure basal and luminal cell populations was accomplished by using antibody‐linked magnetic beads to isolate essentially pure populations of both basal (CALLA/CD10) and luminal mammary (MUC‐1/CD227) epithelial cells from organoid explant cultures. Organoids were isolated and plated on collagen I coated flasks in either DFCI (basal) or MBCM (luminal) media and cultured at 37°C in 5% CO2 to induce cell migration. The media was replenished every 2 – 3 days until the cells reached confluence. Cultured cells were harvested and immunopurified using a CALLA/CD10 or MUC‐1/CD227 antibody and magnetic beads. Positively selected cells were seeded in tissue culture treated flasks and propagated with medium exchange every 2 – 3 days. A second round of immunopurification resulted in an isolated cell population that was greater than 95% MUC1 positive, suggesting a pure luminal epithelial cell population. These isolated cell types can now be tested individually for drug sensitivity.
Selection Gradients and Ecogeographic Variance in the Human Post‐Crania
Authors: Savell Kristen , Roseman Charles , Auerbach Benjamin
Published: June 2021

Recent work by Roseman and Auerbach (in press) indicates that the ecogeographic distribution of human body proportions is driven by a combination of neutral evolutionary forces and natural selection. This contrasts with assumptions that natural selection produces morphological variance along ecogeographic clines (i.e., Bergmann's and Allen's “rules”). Roseman and Auerbach's approach, however, does not distinguish between direct and indirect responses to the natural selection acting on these morphologies. This study uses retrospectively estimated selection gradients for limb lengths, femoral head size, and body breadth to assess the nature of selective forces on ecogeographic variance in human morphology.

We estimated the vectors of selection gradients required to evolve one group into another for pairs of populations across major climate regions, and used parametric bootstraps to determine 95% confidence intervals. Equatorial African populations are assumed to have been drifting atop a plateau on the adaptive landscape, reflecting a tropically‐adapted human state. Results indicate that strong selection on distal limb lengths, femoral head size, and body breadth effected an evolutionary transition to morphologies found in arctic populations. The patterns of selection necessary to effect a transition to proportions found in more temperate regions (i.e. North Africa and Europe) were qualitatively similar but much less strong. These results in part support the findings of Roseman and Auerbach, but suggest that the action of natural selection may be entangled with random genetic drift, gene flow, and neutral mutation.
Imaging Cellular Protein Fatty Acylation
Authors: Hannoush Rami
Published: June 2021

Wnts are secreted palmitoylated glycoproteins that are important in embryonic development and human cancers. Due to a lack of tools, studying the behavior of Wnts and other post‐translationally palmitoylated proteins has proven challenging. Over the past few years, there have been considerable advances in developing biochemical techniques for detecting palmitoylated cellular proteins with good sensitivity. Yet, methods that enable tracking the palmitoylated form of an individual protein in single cells have been lacking. Such approaches will be invaluable as they provide an extra layer of spatial information about the palmitoylated state of a protein and its intracellular dynamics. Here we describe a strategy which integrates click chemistry, proximity ligation and fluorescence microscopy to visualize the palmitoylated form of proteins with subcellular resolution. We applied the system to image Wnt proteins, thereby revealing new biological insights about Wnt fatty acylation, trafficking and its biochemical regulation. The system is also adaptable to a high throughput format, making it of potential use in drug discovery to screen for or validate modulators of Wnt palmitoylation that may interfere with Wnt‐driven cancers. Because the method developed here is modular in nature, it can be applied to imaging the palmitoylation of cellular proteins as well as other protein post‐translational modifications that are detectable by clickable chemical reporters.
A Cholesterol Story Never Told before
Authors: Cho Wonhwa , Sheng Ren , Liu Shu‐Lin
Published: June 2021

Cholesterol is known to interact with various cellular proteins but its direct involvement in cell regulation has not been fully demonstrated. To systematically investigate the regulatory function of cholesterol, we have taken a two‐pronged approach of identifying cellular cholesterol binding proteins and developing a quantitative in situ imaging analysis of cholesterol. Through genome‐wide screening of domains and proteins we have identified many cholesterol‐binding cellular proteins and elucidated how cholesterol specifically regulates key cell signaling pathways via these proteins. Our new quantitative imaging analysis has also revealed strikingly unique spatiotemporal distribution and dynamics of cholesterol in mammalian cells, which is crucial for its specific regulation of cell signaling pathways.
Expanding the Language of Bacterial Quorum Sensing Using Synthetic Lligands
Authors: Blackwell Helen
Published: June 2021

We are developing chemical tools that attenuate cell‐cell communication pathways in bacteria. Many bacteria communicate using small organic molecules and peptides to monitor their population densities in a process called “quorum sensing.” At high cell densities, bacteria use this signaling network to switch from an isolated, nomadic existence to that of a multicellular community. This lifestyle switch is significant; only in groups will pathogenic bacteria turn on virulence pathways and grow into drug‐impervious communities called biofilms that are the basis of myriad chronic infections. In turn, certain symbiotic bacteria will only colonize their hosts and initiate beneficial behaviors at high population densities. Our research is broadly focused on the design, synthesis, and characterization of non‐native ligands that can intercept quorum sensing and provide new insights into its role in host/microbe interactions. These molecules provide a novel approach to study quorum sensing with both spatial and temporal control in a range of settings. We have developed a series of efficient synthetic methods that provide us with straightforward access to these ligands. In addition, we have applied our quorum sensing antagonists and agonists in vitro and in vivo to investigate quorum sensing as an anti‐infective target. This talk will introduce our research approach and highlight recent results.
Signals that Distinguish Noncoding RNA Scaffolds from mRNAs
Authors: Moazed Danesh , Iglesias Nahid , Sevilimedu Aarti , Yu Ruby , Jih Gloria , Kruger Ryan , Gygi Steven
Published: June 2021

At the fission yeast pericentromeric DNA repeats, nascent noncoding transcripts act as scaffolds for the siRNA‐programmed RITS complex, which then recruits histone H3 lysine 9 (H3K9) methylation and promotes heterochromatin formation. However, siRNAs cannot promote heterochromatin formation at most complementary euchromatic regions, suggesting that mRNAs are poor scaffold siRNA‐mediated H3K9 methylation. We recently showed that cleavage and polyadenylation signals within the 3′ untranslated region of an mRNA antagonize siRNA‐mediated heterochromatin formation. Consistent with these results, we find that the recruitment of a conserved nuclear deadenylation complex to target RNAs is critical for RNAi‐mediated heterochromatin establishment. These results suggest that transcription termination signals and alternative polyadenylation pathways determine whether a nascent transcript can act as a scaffold for the recruitment of chromatin‐modifying complexes.
Mechanism of Ion‐Coupled Transport in Glutamate Transporters
Authors: Boudker Olga
Published: June 2021

Glutamate transporters clear the neurotransmitter from the synaptic cleft, thus permitting multiple rounds of signaling and preventing glutamate‐mediated excitotoxicity. To generate up to a million fold trans‐membrane concentration gradients of glutamate, the transporters couple uptake of the amino acid to movements of ions, primarily sodium and potassium, down their electrochemical gradients. Using crystallography and a range of biophysical techniques, we have defined a molecular mechanism for this family of proteins in the context of an archaeal homologue, aspartate transporter GltPh. Our studies explain how the solutes are carried across the membrane and how coupling between the uptake of the amino acid and movements of ions is achieved.
Visualizing Replication and Recombination on Single Molecules of DNA
Authors: Kowalczykowski Steven
Published: June 2021

It is now possible to image individual proteins acting on single molecules of DNA. Such imaging affords unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, a single DNA molecule is attached to a polystyrene bead, which is captured in an optical trap. The DNA is extended either by the force of solution flow in a micro‐flowcell, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Proteins and DNA are visualized via fluorescent reporters. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Molecules are introduced and supramolecular complexes are built, one component at a time, using microfluidic flowcells. Using these approaches, we have watched proteins functioning in the repair, replication, and manipulation of DNA. We have imaged unwinding of DNA by helicases, translocation along DNA by motor proteins, self‐assembly of protein filaments on DNA as well as regulation of nucleation and growth, the search for DNA sequence homology protein‐DNA filaments, real‐time replication of DNA, and nucleosome structure and its remodeling. I will summarize how these experiments were done, and what we've learned from a selection of these experiments.
Assessing Intrinsically Disordered Protein Structure and Function through Carbon Detected NMR
Authors: Showalter Scott , Sahu Debashish , Bastidas Monique , Lawrence Chad
Published: June 2021

Intrinsically Disordered Proteins (IDPs) partially or completely lack a co‐operatively folded structure under native conditions, preventing their equilibrium state from being adequately described by a single structural model. Our view is that IDPs do possess native structure that is responsible for imparting their specific functions; describing these structures simply requires a broadening of the traditionally narrow structure‐function paradigm, beyond the current models developed for cooperatively folding systems. We have shown that 13C direct‐detection NMR methods are well suited to generating quantitative and comprehensive structural constraints for IDP ensembles. Generally effective strategies for applying 13C‐detected NMR to highly flexible biomolecules will be presented. The structural ensembles we have generated will be discussed in the context of coupled folding and binding interactions, which have arisen as a prevalent mechanism for molecular recognition when IDPs interact with other proteins, or nucleic acids. Many investigators have proposed that a significant loss of chain entropy must oppose the coupled folding and binding process, thus explaining the generally weak (micromolar) binding affinities experimentally measured for IDP‐mediated interactions. Synergy between our NMR structural methodology and equilibrium binding measurements will be discussed, suggesting that this view is not generally valid. In contrast, we propose that coupled folding and binding has the characteristics of a bi‐molecular protein folding process, driven by a favorable change in solvent entropy.
Added Sugar Levels in Kids' Meals From Chain Restaurants
Authors: Scourboutakos Mary , Semnani‐Azad Zhila , L'Abbe Mary
Published: June 2021

The objective was to analyze the added sugar levels in kids' meals from chain restaurants in relation to the World Health Organization's proposed and current sugar recommendation (no more than 5%‐10% of total daily calories should come from added sugars). Total sugar levels were retrieved from the websites of 17 chain restaurants in 2010. The added sugar levels in 3178 kids' meals (including entrées, side dishes, beverages and desserts) from Canadian restaurants were calculated by subtracting all naturally occurring sugars from the total sugar level. Overall, added sugar levels in kids' meals ranged from 0g to 114g. The average amount of added sugar in a kids' meal (25±0.36 g) was equivalent to the WHO's proposed daily sugar recommendation (calculated using an average child's energy requirement). 50% of meals exceeded the WHO's proposed daily sugar recommendation (<5% of total calories), and 19% exceeded the WHO's current daily sugar recommendation (<10% of total calories). Beverages on average contained 16±20 g of added sugars, desserts contained 12±7 g, entrées contained 3±6 g and side dishes contained 0.6±2 g. Beverages with the highest sugar content were typically soft drinks, fruit juices with added sugars and chocolate milk. Entrées with the highest sugar content were chicken nugget meals containing honey mustard dipping sauces, as well as rib meals and sandwiches with sweet sauces. In conclusion, there is a wide range of sugar levels in kids' meals from restaurants, and many contain more than a day's worth of sugar.
A Novel Nutrient Profiling Score To Drive Food Reformulation
Authors: Masset Gabriel , Vlassopoulos Antonis , Potter Monika , Leroy Fabienne
Published: June 2021

Background

Nutrient profiling (NP) has been mainly used for regulation or food labeling purposes; few systems have been proposed to track food reformulation across a wide range of manufactured foods

Objective

To present a category‐specific NP model, the NF score, which aim is to facilitate the tracking of nutritional characteristics of foods; and to show examples of NF score implementation

Methods

The NF score is calculated as an unweighted average of common (energy, added sugars, fructose, sodium, and total, saturated, and trans fats) and category‐specific (e.g. calcium for dairy products, fiber for breads) nutrient ratios (R) calculated per serving: NF score=Average R. For nutrients to limit, R=content/criteria; for nutrients to encourage R=criteria/content. The criteria are category‐specific and linked to public health recommendations, target population (children or adults), and to the role of the food category in the diet. If the food content for one nutrient does not reach the criteria (i.e. R>1), then all Rs蠄1 are set to 1, to ensure that a nutritional weakness cannot be compensated for. Any score above 2 is capped at 2. The final NF score, ranging from 0 to 2, is reversed for a more intuitive reading; any score below 1 indicates that the food has at least one nutritional weakness.

Results

The NF score was used to assess the evolution of nutritional characteristics of reformulated foods. For all the examples but one, there was an improvement of the NF score (Figure). Improvements for the Cereal bar, Sugar candies, and Pepperoni pizza were also linked to a reduced serving size.

image

Conclusions

The NF score allows for a sensitive tracking of food reformulation. Testing the NF score in standard food composition databases should highlight how effective the NF score could be in improving the food environment.
Sustainability of Food and Nutrition Security Policy during Presidential Transitions
Authors: Escobar Alegria Jessica , Frongillo Edward , Blake Christine , Walker Lee , McDermott Gerald
Published: June 2021

Developing countries with democratic governments periodically experience presidential transitions that affect political and institutional performance. Sustainability is particularly important for food and nutrition security policies (FNSP) that require uninterrupted implementation, commitment, funding, collaboration, and capacity development. We aimed to understand what explains the sustainability of FNSP in a country that experiences presidential transitions every 4 years. A constructivist grounded‐theory approach with a retrospective and sequential explanatory design was used for a case study. Snowball sampling and interview requests were used to recruit 52 policy actors from all relevant sectors and levels, elected or appointed. Semi‐structured interviews data were triangulated using news, speeches, and policy documents. Data were analyzed using constant comparative method with open and axial coding. We identified 24 forces that simultaneously shape a dynamic process determining sustainability during transitions. These forces are molded by conditions for sustaining and for not sustaining actions. The forces favor or impede the sustainability of an action or part of it, and operate at all levels and sectors but affect actions differently in these. The forces do not compete, but combinations of them bring about expected outcomes. The potential of forces to shape sustainability depends on interacting actors responding to emerging events within specific contexts. Informed actors who strategically seek to sustain through transitions actions that work are more likely to reach long‐term goals of FSNP.
Whole‐Wheat Supplemented Diet Changed Fecal Microbial Ecology of Obese Diabetic Mice
Authors: Noratto Giuliana , García‐Pérez Enrique , Ivanov Ivan , Mills David
Published: June 2021

Diet affects bacterial diversity and abundance of the gastrointestinal microbiota. Our objective was to study the effect of whole‐wheat supplemented diet in fecal microbiota composition of obese diabetic (db/db) mice. The db/db mice were fed standard or whole‐wheat isocaloric diets for eight weeks. Fecal DNA was extracted and the V4 domain of the 16S rRNA was amplified using the universal primers F515 and R806; subsequently, amplicons were sequenced on an Illumina MiSeq instrument. Raw Illumina fastq files were demultiplexed, quality filtered, and analyzed using QIIME v1.8.0. Operational taxonomic units (OTUs) were assigned using QIIME's uclust‐based close‐reference OTU‐picking workflow, with a threshold of 97% pairwise identity against the Greengenes bacterial 16S rRNA database (13_5 release). Our results showed that the bacteroidetes/firmicutes ratio was three times higher in the whole‐wheat group compared to the db/db standard diet group. In addition, whole‐wheat diet decreased abundances of the families Enterococcaceae, Turicibacteraceae, and Ruminococcaceae. However, the alpha diversity, quantified by Shannon‐Wiener index was not significantly different between groups (p=0.0652). In conclusion, whole‐wheat supplemented diet changed the bacterial diversity and abundance of fecal microbiota of obese mice. Further studies are being conducted to analyze the relationship between microbiota changes and host metabolic responses.
Epigenetic Regulation of the FXR by High Fat Diet and APC in Colon Cells
Authors: Lyon Adam , Selmin Ornella , Fang Changming , Smith Jeffrey , Doetschman Tom , Thompson Patricia , Martinez Jesse , Lance Peter , Romagnolo Donato
Published: June 2021

The farnesoid‐X‐receptor (FXR) controls hepatic and intestinal bile acid (BA) homeostasis. BAs not reabsorbed via the enterohepatic circulation are converted in the colon to deoxycholic acid (DCA) and other cancer‐promoting secondary BAs. Loss of FXR expression is observed in adenomatous polyposis coli (APC) gene‐deficient colonocytes from mice and the majority of colon cancers.

OBJECTIVE

To investigate whether Western high‐fat diet (HFD) and APC status influence colonic mucosal epigenetic regulation of the Fxr.

METHODS

Weaned C57BL/6J male mice were fed a HFD (22% safflower oil=SO) or low fat diet (LFD; 5% SO) for 6 wks. Effects on CpG methylation of Fxr, and expression of FXR, peroxisome‐proliferator activated receptor (PPAR)‐γ, and cyclooxygenase‐2 (COX‐2) were examined. The influence of APC status on CpG methylation of the Fxr gene, and expression of FXR, ileal bile acid‐binding protein (IBABP), small heterodimer partner (SHP), and COX‐2 in normal colonic mucosa and colon tumors from APCMin/+ mice was investigated.

RESULTS

A 60% reduction in Fxr promoter methylation and 2 to 3‐fold increases in FXR, COX‐2, and PPARγ levels were observed with the HFD. APC‐deficiency was associated with constitutive hypermethylation of the Fxr gene, elevated COX‐2 levels, and 60‐90% reductions in baseline FXR, IBABP, and SHP expression. In human HCT‐116 colon cells, siRNA knock‐down of APC caused a 50% reduction in basal FXR expression, which was lowered by an additional 30% on exposure to DCA.

CONCLUSIONS

APC deficiency leads to constitutive epigenetic silencing of Fxr that combined with a HFD enhances BA‐induced inflammation and colon cancer.
Heterogeneity in the Proliferative Capacity of Smooth Muscle Cells (SMCs)
Authors: Sandison Mairi , McCarron John
Published: June 2021

In cardiovascular disease, artery walls remodel through an increase in SMC numbers. The predominant hypothesis for this is that SMCs in the tunica media undergo phenotypic modulation into a proliferative cell type. However, direct evidence for SMC phenotypic modulation is scant. We therefore exploited time‐lapse microscopy methods to track the fate of freshly isolated, contractile SMCs. As SMCs isolated from different smooth muscle (SM) tissues are heterogeneous in nature, we have used time lapse microscopy in combination with immunocytochemistry to investigate the proliferative capacity of SMCs from portal vein (PV), carotid artery (CA) and distal colon.

The adventia/endothelium and mucosa/serosa were mechanically removed before isolating cells by enzymatic digestion and trituration. Highly elongated, contractile SMCs that stained for both SM α‐actin (SMA) and SM myosin heavy chain (SM‐MHC) were obtained from all tissues. Significantly, both colon and PV tissues also contained large numbers of spherical cells that did not stain for SMA or SM‐MHC (nonSMCs). In standard culture conditions, the SMCs showed limited proliferative capacity: with the exception of one SMC that divided once (out of 15 tracked cells), colon SMCs did not proliferate. Of 11 PV SMCs tracked, 7 SMCs did divide, though none progressed beyond the 3rd generation (daughter of daughter) by confluency. Conversely, the nonSMCs proliferated rapidly (reaching the 5th generation in 5 days) and dominated the resulting cultures. In contrast, all CA cells stained for both SMA and SM‐MHC i.e. no nonSMCs were present. Whilst most CA SMCs underwent apoptosis early in culture (27 of 31 cells), those that survived went on to proliferate at varying rates (up to the 6th generation in 5 days). These results illustrate the complexities involved in creating models of SMC proliferation.
Tailoring Biomass for Biochemical, Chemical or Thermochemical Catalytic Conversion
Authors: McCann Maureen , Abu‐Omar Mahdi , Agrawal Rakesh , Bozell Joseph , Carpita Nicholas , Chapple Clint , Crowley Michael , Delgass Nicholas , Donohoe Bryon , Himmel Michael , Kenttamaa Hilkka , Makowski Lee , Meilan Richard , Mosier Nathan , Murphy Angus , Peer Wendy , Ribeiro Fabio , Tucker Melvin
Published: June 2021

Biomass has one‐third the energy density of crude oil and lacks petroleum's versatility as a feedstock for fuels and chemicals. Chemical catalysis and fast pyrolysis can overcome these limitations by transforming the main components of biomass (cellulose, xylan, and lignin) from grasses and trees directly to liquid hydrocarbons and aromatic co‐products. Our data show that, regardless of conversion process, biomass structural complexity at molecular, nanoscale, and mesoscale levels impacts the yields and selectivities of desired reaction products from catalytic and pyrolytic transformations. Therefore, we have modified the composition and architecture of cell wall components to optimize post‐conversion product yields without compromising pre‐conversion biomass yields. Our strategies to deliver metal catalysts throughout the cell wall structure and create functionalized sites ready for catalytic transformations may dramatically increase the effective surface area for catalysis of cellulose and xylan. Genetic redesign of the lignin network simplifies its architecture to enable facile catalytic disassembly and conversion of aromatics. Prior disassembly of about half of the lignin from intact woody biomass using a catalytic system dramatically improves subsequent saccharification yield and allows a re‐visioning of the cellulosic biorefinery concept.
GPCR‐TargetÍng Nanobodíes as Attractive Research Tools, Diagnostics and Therapeutics
Authors: Smit Martine
Published: June 2021

G protein‐coupled receptors (GPCRs) represent a major therapeutic target class as they play a key role in cellular communication. GPCRs have been successfully targeted with small molecules. With new and improved immunization‐related technologies and advances in GPCR purification and expression techniques, antibody‐based targeting of GPCRs has gained attention. Nanobodies, llama‐derived antibodies, are a relatively novel class of antibodies, which combine the advantages of both small molecules (e.g., molecular cavity binding, ease of production) and monoclonal antibodies (e.g. high affinity and specificity). We have identified the first inhibitory nanobodies targeting chemokine receptors, which play a role in leukocyte trafficking and are implicated in several pathologies, such as inflammatory diseases, cancer and HIV. We have identified nanobodies targeting the chemokine receptors CXCR4 and CXCR7. These nanobodies appeared to effectively modulate CXCR4 (stem cell mobilization, HIV co‐receptor activity) and CXCR7 (tumor progression) function in vitro and/or in vivo. In addition, we identified nanobodies targeting chemokines, acting as neutralizing antibodies. Hence nanobodies targeting the chemokine receptor system serve as interesting research tools, diagnostics and potentially therapeutics which may contribute to advancements in chemokine receptor research.