Long-Term Weight-Loss in Gastric Bypass Patients Carrying Melanocortin 4 Receptor Variants

Background

The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in MC4R are predominantly found in obese individuals. Though some rare variants in MC4R discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects.

Subjects/Methods

In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of MC4R. We matched rare variant carriers to patients with the MC4R reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. In vitro, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy.

Results

While carrying a rare MC4R allele is associated with obesity, carriers of rare variants exhibited comparable weight-loss after RYGB to non-carriers. However, subjects carrying three of these variants, V95I, I137T or L250Q, lost less weight after surgery. In vitro, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between in vitro signaling and pre- or post-surgery clinical phenotype.

Conclusions

These data suggest that subtle differences in receptor signaling conferred by rare MC4R variants combined with additional factors predispose carriers to obesity. In the absence of complete MC4R deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention.     

Quirks of dye nomenclature. 2. Congo red

The history, origin, identity, chemistry and uses of Congo red are described. Originally patented in 1884, Congo red soon found applications in dyeing cotton, as a pH indicator for chemists and as a biological stain. Unlike the majority of the 19th century synthetic dyes, it still is available commercially.     

Differences in outcomes of mandatory motorcycle helmet legislation by country income level: A systematic review and meta-analysis

BackgroundThe recent Lancet Commission on Legal Determinants of Global Health argues that governance can provide the framework for achieving sustainable development goals. Even though over 90% of fatal road traffic injuries occur in low- and middle-income countries (LMICs) primarily affecting motorcyclists, the utility of helmet laws outside of high-income settings has not been well characterized. We sought to evaluate the differences in outcomes of mandatory motorcycle helmet legislation and determine whether these varied across country income levels.Methods and findingsA systematic review and meta-analysis were completed using the PRISMA checklist. A search for relevant articles was conducted using the PubMed, Embase, and Web of Science databases from January 1, 1990 to August 8, 2021. Studies were included if they evaluated helmet usage, mortality from motorcycle crash, or traumatic brain injury (TBI) incidence, with and without enactment of a mandatory helmet law as the intervention. The Newcastle–Ottawa Scale (NOS) was used to rate study quality and funnel plots, and Begg’s and Egger’s tests were used to assess for small study bias. Pooled odds ratios (ORs) and their 95% confidence intervals (CIs) were stratified by high-income countries (HICs) versus LMICs using the random-effects model. Twenty-five articles were included in the final analysis encompassing a total study population of 31,949,418 people. There were 17 retrospective cohort studies, 2 prospective cohort studies, 1 case–control study, and 5 pre–post design studies. There were 16 studies from HICs and 9 from LMICs. The median NOS score was 6 with a range of 4 to 9. All studies demonstrated higher odds of helmet usage after implementation of helmet law; however, the results were statistically significantly greater in HICs (OR: 53.5; 95% CI: 28.4; 100.7) than in LMICs (OR: 4.82; 95% CI: 3.58; 6.49), p-value comparing both strata < 0.0001. There were significantly lower odds of motorcycle fatalities after enactment of helmet legislation (OR: 0.71; 95% CI: 0.61; 0.83) with no significant difference by income classification, p-value: 0.27. Odds of TBI were statistically significantly lower in HICs (OR: 0.61, 95% CI 0.54 to 0.69) than in LMICs (0.79, 95% CI 0.72 to 0.86) after enactment of law (p-value: 0.0001). Limitations of this study include variability in the methodologies and data sources in the studies included in the meta-analysis as well as the lack of available literature from the lowest income countries or from the African WHO region, in which helmet laws are least commonly present.ConclusionsIn this study, we observed that mandatory helmet laws had substantial public health benefits in all income contexts, but some outcomes were diminished in LMIC settings where additional measures such as public education and law enforcement might play critical roles.

In a systematic review and meta-analysis, Jacob Lepard and colleagues evaluate the differences in outcomes of mandatory motorcycle helmet legislation by country-income level.     

Adverse effects of fluorescent dust marking on the behavior of western black widow spiderlings

Fluorescent dust marking is commonly employed to identify and track small arthropods for studies of ecology, demography, and behavior. Despite its widespread use, no study to date has empirically tested the suitability of dust marking for studies of spider behavior. Here, we test the effects of fluorescent dust marking on proximity of cohabitation, sibling cannibalism, and non‐cannibalistic mortality of western black widow spiderlings, Latrodectus hesperus Chamberlin & Ivie (Araneae: Theridiidae). Results indicate that dust‐marked spiderlings cohabitated at closer proximities and died sooner than undusted spiderlings due to a greater incidence of cannibalism in the dust‐marked group. Thus, we conclude that fluorescent dust marking significantly affected the cohabitation and cannibalistic behavior of L. hesperus spiderlings. Although few studies have reported adverse effects of dust marking on arthropods, our results should serve as a warning to future studies that normal behavior may be disrupted by the use of these fluorescent dust markers. Therefore, preliminary testing should be routine when determining the suitability of any marking technique for not only new species, but also new life stages and behaviors.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Quirks of dye nomenclature. 9. Fluorescein

Adolf Baeyer announced the discovery of fluorescein in 1871 and named it after its most striking property, i.e., fluorescence. I describe here the synthesis of fluorescein. There are seven molecular species in both the solid state or in solution. I also summarize some of the diverse applications of the dye, both medical and nonmedical, which depend mostly on the facile detection of fluorescein at low concentration. Both animal and human toxicity are examined.     

Quirks of dye nomenclature. 1. Evans blue

The history, origin, identity, chemistry and use of Evans blue dye are described along with the first application to staining by Herbert McLean Evans in 1914. In the 1930s, the dye was marketed under the name, Evans blue dye, which was profoundly more acceptable than the ponderous chemical name.     

A Single-Seed Assay for Endo-[beta]-Mannanase Activity from Tomato Endosperm and Radicle Tissues

Completion of germination (radicle emergence) is an all-or-none developmental event for an individual seed. Variation in germination timing among seeds in a population therefore reflects variation among seeds in the rates or extents of physiological or biochemical processes prior to radicle emergence. For tomato (Lycopersicon esculentum Mill.) seeds, correlative evidence suggests that endo-[beta]-mannanase activity weakens the endosperm cap tissue opposite the radicle tip to permit radicle emergence. To test whether endo-[beta]-mannanase activity is causally related to germination rates, we have developed a sensitive assay suitable for use with individual radicle tips or endosperm caps. We show that endo-[beta]-mannanase activity varies at least 100-fold and often more than 1000-fold among individual inbred tomato seeds prior to radicle emergence. Other sources of variation (tissue size and experimental error) were evaluated and cannot account for this range of activity. Endo-[beta]-mannanase activity was generally 10-fold greater in leachates from endosperm caps than from radicle tips. Release of reducing sugars from individual endosperm caps also varied over a considerable (9-fold) range. These extreme biochemical differences among individual tomato seeds prior to radicle emergence indicate that results obtained from bulk samples could be misleading if it is assumed that all seeds exhibit the "average" behavior.     

ALS/FTD‐associated FUS activates GSK‐3β to disrupt the VAPB–PTPIP51 interaction and ER–mitochondria associations

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca²⁺ uptake by mitochondria following its release from ER stores, which is a physiological read‐out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS‐expressing cells; mitochondrial ATP production is linked to Ca²⁺ levels. Finally, we demonstrate that the FUS‐induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase‐3β (GSK‐3β), a kinase already strongly associated with ALS/FTD.     

The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 mumol inorganic phosphate/g muscle in 20 min at 20 degrees C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 degrees C for 18 h caused ectoenzyme inactivation which plateaued at 35-40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 micrograms ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by alpha-methyl D-mannopyranoside. As the temperature increased from 10 to 40 degrees C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 degrees C, with a "break point" and a clear change in slope at 35 degrees C. When treated with ConA the activity increased linearly from 10 to 40 degrees C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 degrees C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.