TP53/p53 Facilitates Stress-Induced Exosome and Protein Secretion by Adipocytes.

Besides the secretion of fatty acids, lipolytic stimulation of adipocytes results in the secretion of triglyceride-rich extracellular vesicles and some free proteins (e.g., fatty acid binding protein 4) that, in sum, affect adipose homeostasis as well as the development of metabolic disease. At the mechanistic level, lipolytic signals activate p53 in an adipose triglyceride lipase-dependent manner, and pharmacologic inhibition of p53 attenuates adipocyte-derived extracellular vesicle (AdEV) protein and FABP4 secretion. Mass spectrometry analyses of the lipolytic secretome identified proteins involved in glucose and fatty acid metabolism, translation, chaperone activities, and redox control. Consistent with a role for p53 in adipocyte protein secretion, activation of p53 by the MDM2 antagonist nutlin potentiated AdEV particles and non-AdEV protein secretion from cultured 3T3-L1 or OP9 adipocytes while the levels of FABP4 and AdEV proteins were significantly reduced in serum from p53-/- mice compared with wild-type controls. The genotoxin doxorubicin increased AdEV protein and FABP4 secretion in a p53-dependent manner and DNA repair-depleted ERCC1-/Δ-haploinsufficient mice expressed elevated p53 in adipose depots, along with significantly increased serum FABP4. In sum, these data suggest that lipolytic signals, and cellular stressors such as DNA damage, facilitate AdEV protein and FABP4 secretion by adipocytes in a p53-dependent manner.

Safety Net Program to Improve Statin Initiation Among Adults With High Low-Density Lipoprotein Cholesterol.

INTRODUCTION: Despite their effectiveness in reducing low-density lipoprotein cholesterol and cardiovascular disease risk, high-intensity statins are underutilized among adults with low-density lipoprotein cholesterol ≥190 mg/dL. This study determined whether a safety net program (SureNet) facilitating medication and laboratory test orders improved statin initiation and laboratory test completions after (SureNet period: April 2019-September 2021) and before implementation (pre-SureNet period: January 2016-September 2018). METHODS: Kaiser Permanente Southern California members aged 20-60 years with low-density lipoprotein cholesterol ≥190 mg/dL and no statin use in previous 2-6 months were included in this retrospective cohort study. Statin orders within 14 days and statin fills, laboratory test completions, and improved low-density lipoprotein cholesterol within 180 days of the high low-density lipoprotein cholesterol (pre-SureNet) or outreach (SureNet period) were compared. Analyses were conducted in 2022. RESULTS: Overall, 3,534 and 3,555 adults were eligible for statin initiation during the pre-SureNet and SureNet periods, respectively. Overall, 759 (21.5%) and 976 (27.5%) had a statin approved by their physician during pre-SureNet and SureNet periods, respectively (p<0.001). After multivariable adjustment for demographics and clinical characteristics, adults during the SureNet period had a higher likelihood of receiving a statin order (prevalence ratio=1.36, 95% CI=1.25, 1.48), filling their statin (prevalence ratio=1.32, 95% CI=1.26, 1.38), completing their laboratories (prevalence ratio=1.41, 95% CI=1.26, 1.58), and improving low-density lipoprotein cholesterol (prevalence ratio=1.21, 95% CI=1.07, 1.37) than in pre-Surenet period. CONCLUSIONS: The SureNet program was able to improve prescription orders, fills, laboratory test completions, and lower low-density lipoprotein cholesterol. Optimizing both physician adherence to treatment guidelines; and patient adherence to the program may improve low-density lipoprotein cholesterol lowering.

Comparison of Blood Pressure Measurements from Clinical Practice and a Research Study At Kaiser Permanente Southern California.

BACKGROUND: Accurate blood pressure (BP) measurement is essential to identify and manage hypertension. Prior studies have reported a difference between BP measured in routine patient care and in research studies. We aimed to investigate the agreement between BP measured in routine care and research-grade BP in Kaiser Permanente Southern California, a large, integrated healthcare system with initiatives to standardize BP measurements during routine patient care visits. METHODS: We included adults ≥65 years old with hypertension, taking antihypertensive medication, and participating in the Ambulatory Blood Pressure in Older Adults (AMBROSIA) study in 2019-2021. Clinic BP from routine care visits was extracted from the electronic health record. Research-grade BP was obtained by trained AMBROSIA study staff via an automatic oscillometric device. The mean difference between routine care and research-grade BP, limits of agreement, and correlation were assessed. RESULTS: We included 309 participants (mean age 75 years; 54% female; 49% non-Hispanic white). Compared with measurements from routine care, mean research-grade systolic BP (SBP) was 0.1 mm Hg higher (95% CI: -1.5 to 1.8) and diastolic BP (DBP) was 0.4 mm Hg lower (95% CI: -1.6 to 0.7). Limits of agreement were -29 to 30 mm Hg for SBP and -21 to 20 mm Hg for DBP. The intraclass correlation coefficient was 0.42 (95% CI: 0.33 to 0.51) for SBP and 0.43 (95% CI: 0.34 to 0.52) for DBP. CONCLUSIONS: High within-person variation and moderate correlation were present between BP measured in routine care and following a research protocol suggesting the importance of standardized measurements.

Demographic and cultural correlates of traditional eating among Alaska Native adults at risk for cardiovascular disease.

This cross-sectional study assessed how traditional eating relates to cultural and community factors. Alaska Native adults from the Norton Sound region were recruited and surveyed between 2015-2018 for a randomized clinical trial of multiple risk behavior change interventions for cardiovascular disease prevention. Participants (n = 291) were 49% female with a mean age of 47 years (SD = 14). A 34-item food frequency questionnaire assessed consumption of foods traditional and nontraditional to the regional Alaska Native diet. A novel measure, termed the "traditional foods index", was computed as weekly servings of culturally traditional food consumption divided by total foods reported. Overall, the sample's traditional foods index averaged 21%±16%, with higher values reported by participants assessed in summer (23%±17%) than winter (19%±15%, p<0.05); by women (22%±16%) than men (19%±16%, p < .05); and by residents of smaller communities (22%±17%) than the comparatively larger community of Nome (17%±14%, p<0.05). The traditional foods index was correlated with age (r = .26, p < .01), as well as the cultural variables of community connectedness (r = .19, p < .01), community standing (r = .15, p < .01), and traditional language comprehension (r = .19, p < .01). In a multivariate regression model, age, community connectedness, and community standing remained significantly associated with traditional diet. These findings may inform the design and evaluation of community-based, culturally-relevant dietary initiatives for heart health.

Best practices and tools for reporting reproducible fluorescence microscopy methods.

Although fluorescence microscopy is ubiquitous in biomedical research, microscopy methods reporting is inconsistent and perhaps undervalued. We emphasize the importance of appropriate microscopy methods reporting and seek to educate researchers about how microscopy metadata impact data interpretation. We provide comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities. We aim to improve microscopy reporting, thus improving the quality, rigor and reproducibility of image-based science.

Imaging methods are vastly underreported in biomedical research.

A variety of microscopy techniques are used by researchers in the life and biomedical sciences. As these techniques become more powerful and more complex, it is vital that scientific articles containing images obtained with advanced microscopes include full details about how each image was obtained. To explore the reporting of such details we examined 240 original research articles published in eight journals. We found that the quality of reporting was poor, with some articles containing no information about how images were obtained, and many articles lacking important basic details. Efforts by researchers, funding agencies, journals, equipment manufacturers and staff at shared imaging facilities are required to improve the reporting of experiments that rely on microscopy techniques.

A proof-of-concept study for developing integrated two-photon microscopic and magnetic resonance imaging modality at ultrahigh field of 16.4 tesla.

Functional magnetic resonance imaging (fMRI) based on the blood oxygen level dependent (BOLD) contrast has gained a prominent position in neuroscience for imaging neuronal activity and studying effective brain connectivity under working state and functional connectivity at resting state. However, the fundamental questions in regards to fMRI technology: how the BOLD signal inferences the underlying microscopic neuronal activity and physiological changes and what is the ultimate specificity of fMRI for functional mapping of microcircuits, remain unanswered. The capability of simultaneous fMRI measurement and functional microscopic imaging in a live brain thus holds the key to link the microscopic and mesoscopic neural dynamics to the macroscopic brain activity at the central nervous system level. Here we report the first demonstration to integrate high-resolution two-photon fluorescence microscopy (TPM) with a 16.4 tesla MRI system, which proves the concept and feasibility for performing simultaneous high-resolution fMRI and TPM imaging at ultrahigh magnetic field.

Plasmon resonance and the imaging of metal-impregnated neurons with the laser scanning confocal microscope.

The staining of neurons with silver began in the 1800s, but until now the great resolving power of the laser scanning confocal microscope has not been utilized to capture the in-focus and three-dimensional cytoarchitecture of metal-impregnated cells. Here, we demonstrate how spectral confocal microscopy, typically reserved for fluorescent imaging, can be used to visualize metal-labeled tissues. This imaging does not involve the reflectance of metal particles, but rather the excitation of silver (or gold) nanoparticles and their putative surface plasmon resonance. To induce such resonance, silver or gold particles were excited with visible-wavelength laser lines (561 or 640 nm), and the maximal emission signal was collected at a shorter wavelength (i.e., higher energy state). Because the surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, our novel protocol holds enormous promise of a rebirth and further development of silver- and gold-based cell labeling protocols.

Engineered protein nano-compartments for targeted enzyme localization.

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (ß-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.

Insulin degradation by acinar cell proteases creates a dysfunctional environment for human islets before/after transplantation: benefits of α-1 antitrypsin treatment.

BACKGROUND: Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of α-1 antitrypsin (A1AT). METHODS: Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations. The effect of proteases on ultrastructure of islets and ß-cell insulin granules were examined by transmission electron microscopy. The number of insulin granules and insulin-labeled immunogold particles were counted. The in vivo effect of proteases on islet function was studied by transplanting acinar cells adjacent to islet grafts in diabetic mice. The effects of A1AT culture supplementation on protease activity, insulin levels, and islet function were assessed in pure and impure islets. RESULTS: Islet loss after culture was significantly higher in impure relative to pure preparations (30% vs. 14%, P<0.04). Lower islet purity was associated with increased protease activity and decreased insulin levels in culture supernatants. Reduced ß-cell insulin granules and insulin degradation by proteases were confirmed by transmission electron microscopy. Transplantations in mice showed delayed islet graft function when acinar cells were transplanted adjacent to the islets under the kidney capsule. Supplementation of A1AT to impure islet cultures maintained islet cell mass, restored insulin levels, and preserved islet functional integrity. CONCLUSION: Culture of impure human islet fractions in the presence of A1AT prevents insulin degradation and improves islet recovery.

Identification and characterization of a small molecule inhibitor of Fatty Acid binding proteins.

Molecular disruption of the lipid carrier AFABP/aP2 in mice results in improved insulin sensitivity and protection from atherosclerosis. Because small molecule inhibitors may be efficacious in defining the mechanism(s) of AFABP/aP2 action, a chemical library was screened and identified 1 (HTS01037) as a pharmacologic ligand capable of displacing the fluorophore 1-anilinonaphthalene 8-sulfonic acid from the lipid binding cavity. The X-ray crystal structure of 1 bound to AFABP/aP2 revealed that the ligand binds at a structurally similar position to a long-chain fatty acid. Similar to AFABP/aP2 knockout mice, 1 inhibits lipolysis in 3T3-L1 adipocytes and reduces LPS-stimulated inflammation in cultured macrophages. 1 acts as an antagonist of the protein-protein interaction between AFABP/aP2 and hormone sensitive lipase but does not activate PPARgamma in macrophage or CV-1 cells. These results identify 1 as an inhibitor of fatty acid binding and a competitive antagonist of protein-protein interactions mediated by AFABP/aP2.

Advanced laboratory techniques for sample processing and immunolabeling using microwave radiation.

A better understanding of improved microwave technology has increased the benefits and versatility of the technique as it applies to all aspects of immunohistochemistry. The role of continuous magnetron power output (wattage) combined with precise control of sample heating demonstrated their significance to complex labeling protocols. Here, we present results for microwave-assisted formaldehyde fixation and its effect on GFP expression in transfected HeLa cells. Rat brain sections and cultured hippocampal cells were labeled with 11 different primary antibodies using a unified microwave protocol. Microwave-assisted immunohistochemistry made it possible to sequentially label tissues and cells with several primary antibodies in a very short period of time with excellent labeling characteristics.

Mapping of the hormone-sensitive lipase binding site on the adipocyte fatty acid-binding protein (AFABP). Identification of the charge quartet on the AFABP/aP2 helix-turn-helix domain.

The hormone-sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP/aP2) form a physical complex that affects basal and hormone-stimulated adipocyte fatty acid efflux. Previous work has established that AFABP/aP2-HSL complex formation requires that HSL be in its activated, phosphorylated form and AFABP/aP2 have a bound fatty acid. To identify the HSL binding site of AFABP/aP2 a combination of alanine-scanning mutagenesis and fluorescence resonance energy transfer was used. Mutation of Asp17, Asp18, Lys21, or Arg30 (but not other amino acids in the helix-turn-helix region) to alanine inhibited interaction with HSL without affecting fatty acid binding. The cluster of residues on the helical domain of AFABP/aP2 form two ion pairs (Asp17-Arg30 and Asp18-Lys21) and identifies the region we have termed the charge quartet as the HSL interaction site. To demonstrate direct association, the non-interacting AFABP/aP2-D18K mutant was rescued by complementary mutation of HSL (K196E). The charge quartet is conserved on other FABPs that interact with HSL such as the heart and epithelial FABPs but not on non-interacting proteins from the liver or intestine and may be a general protein interaction domain utilized by fatty acid-binding proteins in regulatory control of lipid metabolism.

Interaction of the adipocyte fatty acid-binding protein with the hormone-sensitive lipase: regulation by fatty acids and phosphorylation.

Adipocyte fatty acid-binding protein (AFABP/aP2) forms a physical complex with the hormone-sensitive lipase (HSL) and AFABP/aP2-null mice exhibit reduced basal and hormone-stimulated lipolysis. To identify the determinants affecting the interaction fluorescence resonance energy transfer (FRET) imaging was used in conjunction with a mutagenesis strategy to evaluate the roles AFABP/aP2 fatty acid binding and HSL phosphorylation have in complex formation as well as determine the HSL binding site on AFABP/aP2. The nonfatty acid binding mutant of AFABP/aP2 (R126Q) failed to form a FRET-competent complex with HSL either under basal or forskolin-stimulated conditions, indicating that lipid binding is required for association. Once bound to HSL and on the surface of the lipid droplet, YFP-AFABP/aP2 (but not YFP-HSL) exhibited energy transfer between the fusion protein and BODIPY-C12-labeled triacylglycerol. Serine to alanine mutations at the two PKA phosphorylation sites of HSL (659 and 660), or at the AMPK phosphorylation sites (565), blocked FRET between HSL and AFABP/aP2. Substitution of isoleucine for lysine at position 21 of AFABP/aP2 (K21I), but not 31 (K31I), resulted in a non-HSL-binding protein indicating that residues on helix alphaI of AFABP/aP2 define a component of the HSL binding site. These results indicate that the ligand-bound form of AFABP/aP2.interacts with the activated, phosphorylated HSL and that the association is likely to be regulatory; either delivering FA to inhibit HSL (facilitating feedback inhibition) or affecting multicomponent complex formation on the droplet surface.

William Cowper and his decorated copperplate initials.

William Cowper was an 18th-century London surgeon/anatomist who made important contributions to several medical disciplines. He achieved lasting fame for describing the bulbourethral glands and lasting infamy for pirating plates for his anatomical atlas, the Anatomy of Humane Bodies. Cowper wrote the books and papers that taught the forerunners of today's surgeons and anatomists. His most famous pupil, the great surgeon William Cheselden, was one of the teachers of John Hunter, the founder of modern surgery. Cowper's magnificent atlas of human myology, the Myotomia Reformata, is a showcase for exquisitely rendered illustrations of muscles and bones. Cowper populated his masterwork with dozens of delightfully decorated initial letters depicting anatomical scenes. This article introduces readers to the long-forgotten William Cowper and exhibits his stunning historiated letters in all their glory.

Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis.

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.

Asymmetric distribution of nuclear pore complexes and the cytoplasmic localization of beta2-tubulin mRNA in Chlamydomonas reinhardtii.

Although it is generally accepted that nuclear architecture is an important determinant of nuclear activity, it is not clear whether cytoplasmic events, such as transcript localization and cell polarity, are affected by this architecture. Characterization of the nuclear architecture of the single-cell alga Chlamydomonas reinhardtii revealed a polarized nucleus, with nuclear pore complexes preferentially concentrated at the posterior side of the nucleus. Nuclear asymmetry was greatly exaggerated during the upregulation of genes encoding flagellar proteins, when nuclear pore complexes (NPCs) were observed to hyperpolarize to the posterior side of the nucleus while heterochromatin polarized to the anterior side. Interestingly, prior to deflagellation, the beta2-tubulin gene was preferentially located in the posterior region of the nucleus, and following deflagellation, beta2-tubulin transcripts accumulated posteriorly in polysome-rich cytoplasmic regions adjacent to the highest concentration of NPCs, suggesting a connection between nuclear architecture and cytoplasmic transcript localization.