A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.

Analyzing global steroid metabolism in humans can shed light on the etiologies of steroid-related diseases. However, existing methods require large amounts of serum and lack the evaluation of accuracy. Here, we developed an LC/MS/MS method for the simultaneous quantification of 12 steroid hormones: testosterone, pregnenolone, progesterone, androstenedione, corticosterone, 11-deoxycortisol, cortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, estriol, and estradiol. Steroids and spiked internal standards in 100 μl serum were extracted by protein precipitation and liquid-liquid extraction. The organic phase was dried by evaporation, and isonicotinoyl chloride was added for steroid derivatization, followed by evaporation under nitrogen and redissolution in 50% methanol. Chromatographic separation was performed on a reverse-phase PFP column, and analytes were detected on a triple quadrupole mass spectrometer with ESI. The lower limits of quantification ranged from 0.005 ng/ml for estradiol to 1 ng/ml for cortisol. Apparent recoveries of steroids at high, medium, and low concentrations in quality control samples were between 86.4% and 115.0%. There were limited biases (–10.7% to 10.5%) between the measured values and the authentic values, indicating that the method has excellent reliability. An analysis of the steroid metabolome in pregnant women highlighted the applicability of the method in clinical serum samples. We conclude that the LC/MS/MS method reported here enables steroid metabolome analysis with high accuracy and reduced serum consumption, indicating that it may be a useful tool in both clinical and scientific laboratory research.

This article focuses on the establishment of an accurate and sensitive quantitation method for the analysis of furan fatty acids. In particular, the sensitivity of GC/MS and UPLC/ESI/MS/MS was compared for the identification and quantification of furan fatty acids. Different methylation methods were tested with respect to GC/MS analysis. Special attention needs to be paid to the methylation of furan fatty acids, as acidic catalysts might lead to the degradation of the furan ring. GC/MS analysis in full-scan mode demonstrated that the limit of quantitation was 10 μM. UPLC/ESI/MS/MS in multiple reaction monitoring mode displayed a higher detection sensitivity than GC/MS. Moreover, the identification of furan fatty acids with charge-reversal derivatization was tested in the positive mode with two widely used pyridinium salts. Significant oxidation was unexpectedly observed using N-(4-aminomethylphenyl) pyridinium as a derivatization agent. The formed 3-acyl-oxymethyl-1-methylpyridinium iodide derivatized by 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide improved the sensitivity more than 2,000-fold compared with nonderivatization in the negative mode by UPLC/ESI/MS/MS. This charge-reversal derivatization enabled the targeted quantitation of furan fatty acids in human plasma. Thus, it is anticipated that this protocol could greatly contribute to the clarification of pathological mechanisms related to furan fatty acids and their metabolites.

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

Mandeep Bajaj
Nov 1, 2005; 54:3148-3153
Metabolism

Mariam Alatrach
Apr 1, 2020; 69:681-688
Pathophysiology

VOLUME 295 (2020) PAGES 4079–4092There was an error in the abstract. “The pyridinium cation hampers uptake of OPs into the central nervous system (CNS)” should read as “The pyridinium cation hampers uptake into the central nervous system (CNS).”

AS ARTISTES and musicians try to manage the situation brought on by COVID-19’s stranglehold on the world and its economy, in the interim, many have resorted to hosting live-stream events. But that only succeeds to a point. Performers retain their...

There has been an outpouring of grief following the death of legendary Jamaican singer Millicent Dolly May Small, popularly known as Millie Small. She died in the United Kingdom today at the age of 73 after suffering a stroke. The voice...

BEFORE MILLIE Small became the ‘lollipop girl’, she was a ‘sugar plum’. Not only is it a term of endearment, it is also the title of her first song – a duet as it was called back in 1960 – with singer Owen Gray. This collab was born out of the time...

Active maintenance of β-cell identity through fine-tuned regulation of key transcription factors ensures β-cell function. Tacrolimus, a widely used immunosuppressant, accelerates onset of diabetes after organ transplantation, but underlying molecular mechanisms are unclear. Here we show that tacrolimus induces loss of human β-cell maturity and β-cell failure through activation of the BMP/SMAD signaling pathway when administered under mild metabolic stress conditions. Tacrolimus-induced phosphorylated SMAD1/5 acts in synergy with metabolic stress–activated FOXO1 through formation of a complex. This interaction is associated with reduced expression of the key β-cell transcription factor MAFA and abolished insulin secretion, both in vitro in primary human islets and in vivo in human islets transplanted into high-fat diet–fed mice. Pharmacological inhibition of BMP signaling protects human β-cells from tacrolimus-induced β-cell dysfunction in vitro. Furthermore, we confirm that BMP/SMAD signaling is activated in protocol pancreas allograft biopsies from recipients on tacrolimus. To conclude, we propose a novel mechanism underlying the diabetogenicity of tacrolimus in primary human β-cells. This insight could lead to new treatment strategies for new-onset diabetes and may have implications for other forms of diabetes.

Those hoping to watch Ronald Acuna Jr. extend last season's incredible post-All-Star break production should remember that his pace would have equated to 45 homers and 33 stolen bases over 162 games.

Joseph G. Yu
Oct 1, 2002; 51:2968-2974
Obesity Studies

David E. Cummings
Aug 1, 2001; 50:1714-1719
Rapid Publications

Norikazu Maeda
Sep 1, 2001; 50:2094-2099
Pathophysiology

THE EDITOR, Madam: TO THE Jamaican Government: there are no trade laws that say you have to buy poison – foreign chicken, beef, produce and other agricultural staples are poisonous. They are chock-full of hormones, antibiotics, fillers, excess...

PET using radiolabeled prostate-specific membrane antigen (PSMA) is now being more widely adopted as a valuable tool to evaluate patients with prostate cancer (PC). Recently, 3 different criteria for interpretation of PSMA PET were published: the European Association of Nuclear Medicine (EANM) criteria, the Prostate Cancer Molecular Imaging Standardized Evaluation criteria, and the PSMA Reporting and Data System. We compared these 3 criteria in terms of interreader, intrareader, and intercriteria agreement. Methods: Data from 104 patients prospectively enrolled in research protocols at our institution were retrospectively reviewed. The cohort consisted of 2 groups: 47 patients (mean age, 64.2 y old) who underwent Glu-NH-CO-NH-Lys-(Ahx)-[⁶⁸Ga(HBED-CC)] (⁶⁸Ga-PSMA11) PET/MRI for initial staging of biopsy-proven intermediate- or high-risk PC, and 57 patients (mean age, 70.5 y old) who underwent ⁶⁸Ga-PSMA11 PET/CT because of biochemically recurrent PC. Three nuclear medicine physicians independently evaluated all ⁶⁸Ga-PSMA11 PET/MRI and PET/CT studies according to the 3 interpretation criteria. Two of them reevaluated all studies 6 mo later in the same manner and masked to the initial reading. The Gwet agreement coefficient was calculated to evaluate interreader, intrareader, and intercriteria agreement based on the following sites: local lesion (primary tumor or prostate bed after radical prostatectomy), lymph node metastases, and other metastases. Results: In the PET/MRI group, interreader, intrareader, and intercriteria agreement ranged from substantial to almost perfect for any site according to all 3 criteria. In the PET/CT group, interreader agreement ranged from substantial to almost perfect except for judgment of distant metastases based on the PSMA Reporting and Data System (Gwet agreement coefficient, 0.57; moderate agreement), in which the most frequent cause of disagreement was lung nodules. Intrareader agreement ranged from substantial to almost perfect for any site according to all 3 criteria. Intercriteria agreement for each site was also substantial to almost perfect. Conclusion: Although the 3 published criteria have good interreader and intrareader reproducibility in evaluating ⁶⁸Ga-PSMA11 PET, there are some factors causing interreader disagreement. Further work is needed to address this issue.

¹⁸F-PSMA-1007 is a novel prostate-specific membrane antigen (PSMA)–based radiopharmaceutical for imaging prostate cancer (PCa). The aim of this study was to compare the diagnostic accuracy of ¹⁸F-PSMA-1007 with ⁶⁸Ga-PSMA-11 PET/CT in the same patients presenting with newly diagnosed intermediate- or high-risk PCa. Methods: Sixteen patients with intermediate- or high-risk PCa underwent ¹⁸F-PSMA-1007 and ⁶⁸Ga-PSMA-11 PET/CT within 15 d. PET findings were compared between the 2 radiotracers and with reference-standard pathologic specimens obtained from radical prostatectomy. The Cohen -coefficient was used to assess the concordance between ¹⁸F-PSMA-1007 and ⁶⁸Ga-PSMA-11 for detection of intraprostatic lesions. The McNemar test was used to assess agreement between intraprostatic PET/CT findings and histopathologic findings. Sensitivity, specificity, positive predictive value, and negative predictive value were reported for each radiotracer. SUV_max was measured for all lesions, and tumor-to-background activity was calculated. Areas under receiver-operating-characteristic curves were calculated for discriminating diseased from nondiseased prostate segments, and optimal SUV cutoffs were calculated using the Youden index for each radiotracer. Results: PSMA-avid lesions in the prostate were identified in all 16 patients with an almost perfect concordance between the 2 tracers ( ranged from 0.871 to 1). Aside from the dominant intraprostatic lesion, similarly detected by both radiotracers, a second less intense positive focus was detected in 4 patients only with ¹⁸F-PSMA-1007. Three of these secondary foci were confirmed as Gleason grade 3 lesions, whereas the fourth was shown on pathologic examination to represent chronic prostatitis. Conclusion: This pilot study showed that both ¹⁸F-PSMA-1007 and ⁶⁸Ga-PSMA-11 identify all dominant prostatic lesions in patients with intermediate- or high-risk PCa at staging. ¹⁸F-PSMA-1007, however, may detect additional low-grade lesions of limited clinical relevance.

OP-ED CONTRIBUTION: COVID-19 AND THE ECONOMY FOR MOST countries in the Caribbean, the current testing for COVID-19 has not reached levels suitable for ascertaining an accurate picture of the state of outbreak and spread of the infection. This...

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

What President Trump calls a border crisis is in fact a crisis in the asylum system—one worsened at every turn by his administration’s harsh policies and rhetoric. Rather than spend $5.7 billion on a wall, it would be far more effective to use the money to retool an overwhelmed asylum system, adapt outmatched border enforcement infrastructure to respond to the changing composition of arrivals, and work cooperatively with Mexico to tackle the factors propelling Central Americans to flee.

The House is set to vote on two bills that would largely dismantle the U.S. asylum system at the southern border by significantly narrowing grounds to apply for asylum, eliminating protections for the vast majority of unaccompanied minors, and unilaterally declaring Mexico a safe third country. The result would be a sharp reduction in the number of people permitted to seek humanitarian protection, as this commentary explains.

PGA Tour star Brooks Koepka said he'll no longer smack talk Michael Jordan on the golf course after he lost a round to the basketball legend.

Joseph Tran
Oct 1, 2012; 30:173-178
Practical Pointers

OBJECTIVE

Type 2 diabetes mellitus (T2DM) is associated with dyslipidemia, but the detailed alterations in lipid species preceding the disease are largely unknown. We aimed to identify plasma lipids associated with development of T2DM and investigate their associations with lifestyle.

OBJECTIVE

To study plasma and dietary linoleic acid (LA) in relation to type 2 diabetes risk in post–myocardial infarction (MI) patients.

Kalevi Pyörälä
Mar 1, 1979; 2:131-141
Proceedings of the Kroc Foundation International Conference on Epidemiology of Diabetes and its Macrovascular Complications

A Google-related company has pulled the plug on its ambitious high-tech waterfront city project in Toronto, citing financial strains from the coronavirus, but the project had also run into local opposition.

Israel's Ministry of Defense ordered small drones for its ground forces working in urban areas, maker Rafael Advanced Defense Systems Ltd. said Monday.

Apr 1, 1994; 17:360-361
Syst[egrave]me International (SI) Units

Nov 1, 1995; 18:1524-1525
Syst[egrave]me International (SI) Units

Aug 1, 1995; 18:1223-1224
Syst[egrave]me International (SI) Units

The ADA, along with a large group of other dental organizations, told the U.S. Department of Labor that they are concerned about provisions in HR 6201, the Families First Coronavirus Response Act as related to family and medical leave and paid sick leave.

As Congress works on a third legislation package in response to the coronavirus pandemic, the ADA is asking lawmakers to include provisions on how to assist dental practices and other small businesses facing economic burdens.

The Association continues to wait for clear guidance from the U.S. Department of Treasury and Small Business Administration on the best way to help dentists considering applying for Paycheck Protection Program 7(a) loans and Economic Injury Disaster Loans.

Dentists can apply for both Economic Injury Disaster Loans and Paycheck Protection Program 7(a) loans, the Small Business Administration told the American Dental Association on April 6.

The ADA webinar, Small Business Administration Loans: Understanding the Options for Dentist Owners, is available online.

The ADA is urging Congress to continue supporting small businesses during the COVID-19 pandemic by increasing funding and streamlining the application process for Small Business Administration loans.

The Senate passed new coronavirus relief legislation April 21 calling for much-needed funding for depleted federal loan programs that could help businesses nationwide, including dentists and dental practices, recover from the economic fallout of the pandemic.

Johns Hopkins University will start two clinical trials of convalescent blood plasma for treatment of COVID-19, the disease caused by the new coronavirus.

OBJECTIVE

To investigate the association of the cardiovascular risk factor lipoprotein (Lp)(a) and vascular complications in patients with type 1 diabetes.

OBJECTIVE

Acrylamide exposure from daily-consumed food has raised global concern. We aimed to assess the exposure-response relationships of internal acrylamide exposure with oxidative DNA damage, lipid peroxidation, and fasting plasma glucose (FPG) alteration and investigate the mediating role of oxidative DNA damage and lipid peroxidation in the association of internal acrylamide exposure with FPG.

OBJECTIVE

To examine the association between manganese intake and the risk of type 2 diabetes in postmenopausal women and determine whether this association is mediated by circulating markers of inflammation.

Engineers in China have developed a fossil fuel-free jet propulsion prototype design that uses microwave air plasmas.

Artificial intelligence requires a lot of energy. Simply solving a puzzle can require the equivalent of the energy produced by three nuclear plants in a single hour.

"Real Housewives of New York" star Tinsley Mortimer gave an update on her wedding plans amid the coronavirus pandemic.

BRUSSELS — Even as the number of refugees in need of protection has reached an all-time high, the resettlement spots offered by countries in 2018 were less than half the level in 2016—and future commitments may shrink further. With refugee needs high and generosity dimming, there is increasing urgency for humanitarian actors to find new ways to bring refugees to safety as well as to rebuild public interest and consensus around the importance of protection.

Salmon and fennel at its best.

OBJECTIVE

Plasma protein N-glycan profiling integrates information on enzymatic protein glycosylation, which is a highly controlled ubiquitous posttranslational modification. Here we investigate the ability of the plasma N-glycome to predict incidence of type 2 diabetes and cardiovascular diseases (CVDs; i.e., myocardial infarction and stroke).