Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.     

Formation of insulin-secreting, Sertoli-enriched tissue constructs by microgravity coculture of isolated pig islets and rat Sertoli cells

Pancreatic islets, isolated from neonatal pigs, and Sertoli cells, isolated from prepubertal rats, were cocultured in simulated microgravity utilizing the NASA-developed highly accelerating, rotating vessel (HARV) biochamber. Following 5 d of incubation, three-dimensional Sertoli-islet cell aggregates (SICA) retained the ability to secrete insulin when exposed to elevated glucose. SICA contained FasL-positive Sertoli cells and insulin-positive beta-cells randomly organized within the spherical construct. The addition of 1% Matrigel induced the reorganization of aggregates (SICAs formed in the presence of Matrigel [SICAmgs]) showing the peripherialization and epithelialization of Sertoli cells and the centralization of islets in association with lumen-like spaces. The Sertoli cells, but not Matrigel, aided in preserving the structural integrity of HARV-incubated islets. Neither Matrigel nor Sertoli cells appeared to interfere with the ability of SICA or SICA mg to secrete insulin and express FasL.     

Increased abundance of the non-indigenous zooplanktivore, <Emphasis Type="Italic">Bythotrephes longimanus</Emphasis>, is strongly correlated with greater spring prey availability in Canadian Shield lakes

The non-indigenous zooplanktivore, Bythotrephes longimanus, is a large Palaearctic cladoceran that is spreading rapidly in the Great Lakes watershed in North America. As a voracious predator, Bythotrephes can reduce herbivorous cladoceran abundance and diversity; however, the variables that affect its abundance are not well understood. To determine what bottom-up factors are associated with the abundance and seasonal dynamics of established Bythotrephes populations, two Bythotrephes datasets from lakes in south-central Ontario, Canada, were analysed using multiple regression and multivariate analyses: a multi-lake dataset of nine lakes sampled in 2003 and a multi-year dataset of one of these lakes, Harp Lake, sampled from 1994–1998 and 2001–2004. Bottom-up variables tested were Secchi disk depth, epilimnetic temperature, cladoceran (prey) density, total phosphorus, dissolved organic carbon and Chlorophyll a, as well as maximum depth for the multi-lake dataset. In both analyses and datasets, springtime abundance of herbivorous cladocerans was consistently found to be a significant factor associated with Bythotrephes (June–September) abundance; Bythotrephes annual abundance was significantly and positively associated with mean May and June prey abundance, along with mean Secchi disk depth for the multi-lake dataset, and groups of lakes or years with similar Bythotrephes seasonal abundance patterns were predicted by June prey abundance. Additionally, prey availability was the dominant contributor towards changes in weekly Bythotrephes birth rates calculated for two of the study lakes. Our study suggests that prey availability influences Bythotrephes abundance, which provides evidence that Bythotrephes establishment success is affected by the abundance of its prey.     

Imaging and artificial intelligence for progression of age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of severe vision loss. With our aging population, it may affect 288 million people globally by the year 2040. AMD progresses from an early and intermediate dry form to an advanced one, which manifests as choroidal neovascularization and geographic atrophy. Conversion to AMD-related exudation is known as progression to neovascular AMD, and presence of geographic atrophy is known as progression to advanced dry AMD. AMD progression predictions could enable timely monitoring, earlier detection and treatment, improving vision outcomes. Machine learning approaches, a subset of artificial intelligence applications, applied on imaging data are showing promising results in predicting progression. Extracted biomarkers, specifically from optical coherence tomography scans, are informative in predicting progression events. The purpose of this mini review is to provide an overview about current machine learning applications in artificial intelligence for predicting AMD progression, and describe the various methods, data-input types, and imaging modalities used to identify high-risk patients. With advances in computational capabilities, artificial intelligence applications are likely to transform patient care and management in AMD. External validation studies that improve generalizability to populations and devices, as well as evaluating systems in real-world clinical settings are needed to improve the clinical translations of artificial intelligence AMD applications.     

Engineered,highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side‐chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site‐specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100‐fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5‐cd]indolizine‐type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α‐helical, β‐sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q‐GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG‐reactive glutamines can be readily introduced into a protein domain for fluorescent labeling.     

Sequential rather than interactive effects of multiple stressors as drivers of phytoplankton community change in a large lake

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Assessing Anticalcification Treatments in Bioprosthetic Tissue by Using the New Zealand Rabbit Intramuscular Model

The objective of this work was to demonstrate that the New Zealand White (NZW) rabbit intramuscular model can be used for detecting calcification in bioprosthetic tissue and to compare the calcification in the rabbit to that of native human valves. The rabbit model was compared with the commonly used Sprague–Dawley rat subcutaneous model. Eighteen rabbits and 18 rats were used to assess calcification in bioprosthetic tissue over time (7, 14, 30, and 90 d). The explanted rabbit and rat tissue discs were measured for calcium by using atomic absorption and Raman spectroscopy. Calcium deposits on the human valve explants were assessed by using Raman spectroscopy. The results showed that the NZW rabbit model is robust for detecting calcification in a shorter duration (14 d), with less infection complications, more space to implant tissue groups (thereby reducing animal use numbers), and a more metabolically and mechanically dynamic environment than the rat subcutaneous model . The human explanted valves and rabbit explanted tissue both showed Raman peaks at 960 cm⁻¹ which is representative of hydroxyapatite. Hydroxyapatite is the final calcium and phosphate species in the calcification of bioprosthetic heart valves and rabbit intramuscular implants. The NZW rabbit intramuscular model is an effective model for assessing calcification in bioprosthetic tissue.Abbreviations: BHV, bioprosthetic heart valve; FET, formaldehyde–ethanol–Tween 80; NZW, New Zealand WhiteBecause of their outstanding durability, mechanical heart valves were 1 of the first replacement valves used in humans. However, drawbacks to mechanical valves include patient complaints regarding the noise the valve makes and the risk of lifelong anticoagulation therapy.^4,20 Currently bioprosthetic heart valves (BHV) are 1 of the most common medical devices used to replace damaged mitral and aortic heart valves in men and women 65 y and older.¹⁷A leading drawback to BHV is dystrophic calcification, which is typically the primary failure mode for these devices.¹⁵ Calcification in BHV is believed to be caused by multiple factors, with glutaraldehyde (used to crosslink the tissue), residual cellular debris, and mechanical stress as the major factors. Patient factors including diabetes, renal failure, atherosclerosis, and calcium metabolism disorders can be important contributors as well.¹⁴ BHV are designed by using bovine pericardium, porcine valve leaflets, and porcine pericardium or dura mater; the most common tissues used are bovine pericardium and porcine valve leaflets.³⁶ Preventing or delaying calcification of BHV is an ongoing research dilemma of biomedical device companies. A 1982 study assessed explanted human valves that had been treated with glutaraldehyde only and found that calcification was the main cause of valve explantation and valves typically lasted only 9 1/2 y on average.³ To increase the durability and delay the onset of calcification of glutaraldehyde-treated valves, researchers and heart valve manufacturers began developing anticalcification treatments. In early studies, chemical compounds like protamine were used to block charged chemical groups and thus reduce calcification in glutaraldehyde-treated BHV.¹³ Other treatments used ethanol, AlCl₃, FeCl₃, L-Hydro, and osteopontin.^11,23,25,35 Many of these treatments were either proven ineffective or were not able to be manufactured. Effective new anticalcification treatments currently available commercially include α-amino oleic acid (Medtronic, Minneapolis, MN), an ethanol-based treatment (Linx, St Jude Medical, St Paul, MN), and a process involving formaldehyde, ethanol, and Tween 80 (ThermaFix, Edwards Lifesciences, Irvine, CA).^10,12 However, young adolescents and patients with calcium metabolic disorders still receive mechanical valves due to rapid calcification of BHV implants in these patient groups.²⁶An animal model is necessary to assess the effects of anticalcification treatments on bioprosthetic tissue in a short and effective timeframe. When assessing anticalcification tissue treatments, the animal models used are typically rats and rabbits. Wistar and Sprague–Dawley rats and New Zealand White (NZW) rabbits are the strains of choice.^5,29 Small animals are chosen because they are inexpensive, the tissue calcifies in a short period of time, the surgical procedure requires minimal pain and discomfort, and the animals are easy to care for.⁹ Typically, the BHV leaflet tissue is cut into discs or is used as whole leaflets and implanted subcutaneously in either rats or rabbits. Typical study duration to assess anticalcification efficacy in the rat model varies from 90 d to 6 mo.^24,41 The Sprague–Dawley rat is one of the most common models used to assess BHV tissue calcification properties.²⁷ The NZW rabbit model is another small animal model available, but to date only the subcutaneous route has been used for tissue biocompatibility and toxicology in drug efficacy studies only.Large animal models used to assess the safety and efficacy of BHV have included pig, sheep, cows, and nonhuman primates, but the primary model currently used is the juvenile sheep.³⁸ The juvenile sheep is a robust model that performs similarly to humans with regards to hemodynamics, valve annulus sizing, and thrombogenicity.^1,10 A typical safety and efficacy study in juvenile sheep lasts 20 wk.²¹ The drawbacks to using sheep to assess anticalcification treatments are the large sample size needed to demonstrate statistically significant differences, high surgical costs, and high animal-care costs.The age of the animal is important when assessing the calcification potential of BHV tissue. Compared with their older counterparts, juvenile animals demonstrate higher calcium metabolism because of bone-building, organ function, and structural tissues.⁷ Ways to assess calcium metabolism in small animals include measuring skeletal length and bone growth relative to sexual maturation.³² In Wistar rats, bone growth increased from 276 µm/d in 21-d-old weanlings to 330 µm/d in 35-d-old rats.^19,42 The bone growth spurt in the rats began to slow, falling to 85 µm/d by day 80, with full maturity by 24 wk. Compared with rats, rabbits mature more slowly, reaching maturity by 34 wk of age. A study involving 17 male and 12 female NZW rabbits assessed growth of the tibia and femur, assessing the correlation of tibial and femoral lengths and sexual maturity in rabbits.³⁹ This knowledge helps researchers assess the progressive growth and maturity of rabbits as they change from juvenile to adults.²⁸ This type of assessment may be important for understanding how intramuscular implants calcify in juvenile (6- to 8-mo-old) rabbits.The NZW rabbit intramuscular model offers a particular advantage for assessing tissue calcification properties. Epinephrine in rabbits had a higher absorbance and diffusion rate when injected intramuscularly compared with subcutaneously.¹⁶ The intramuscular region in rabbits has a rich vascular supply due to the high density in the latissimus dorsi compared with the subcutaneous of either rats or rabbits. The vascular transport mechanisms of muscle allow it to respond to foreign material (such as BHV tissue) more efficiently than the response to subcutaneous implants.⁴² The intramuscular region also offers a more mechanically dynamic environment than the static subcutaneous region, better mimicking some of the stresses on a tissue that a BHV tissue might endure when implanted in humans.Many analytical methods are used to assess the type of calcification that occurs in arteries or bioprosthetic tissue; currently gaining popularity is near-infrared Fourier transform Raman spectroscopy.³⁴ This method measures light scattered inelastically from photons.⁴⁰ Elastically scattered photons have the same energy (frequency) and therefore wavelength as the incident beam. However, a small fraction of light (approximately 1 in 10⁷ photons) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. This process of inelastic scattering of photons is called Raman scattering, which can occur with a change in vibrational, or rotational, or electronic energy of the molecule being studied. The difference in energy between the incident photon and the Raman-scattered photon is equal to the energy of a vibration of the scattering molecule, such as calcium or phosphate. A plot of intensity of scattered light versus energy difference is called a Raman spectrum. When used to assess the type of calcification in BHV, Raman spectroscopy has been shown to be effective in analyzing the presence of calcium phosphate species and, when combined with a calcium assay, the relative calcium:phosphate ratios.⁶ The most common type of calcification is the mineral hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂]. Roughly 70% of all bone is composed of hydroxyapatite.¹⁸ By using Raman spectroscopy (830 nm), hydroxyapatite was found at wavelengths of 960 to 1200 cm⁻¹ in human explanted BHV.³³ Other calcium–phosphate combinations found on explanted human heart valves include carbonate apatites, octacalcium, dicalcium, and amorphous calcium phosphates.⁸ Nascent calcification in biologic tissues goes through phase transformations of unstable calcium–phosphate salts to more stable calcium phosphate salts, ultimately maturing into hydroxyapatite.²² Raman spectroscopy can be applied to a variety of different morphologies, giving it a unique advantage when analyzing biologic samples, which are generally mixtures of fluids, tissues, and mineral deposits.³⁰ Raman analysis requires minimal preparation of biologic samples and is nondestructive to the sample. In the current study, near-infrared Fourier transform Raman spectroscopy was used to detect the presence of calcification in bioprosthetic valves explanted from humans and in bioprosthetic valve tissue from intramuscular rabbit explants.The objective of this study was to validate the rabbit intramuscular model for assessing the calcification potential of bioprosthetic tissue. The outcome of this study was to assess the length of study (days) necessary to see significant differences in calcification among 3 test groups and 1 control group, compare the type of calcification seen in the rabbit model with that of human valve explants (Raman spectroscopy), and to correlate the rabbit model to human BHV calcification.     

Synthesis and SAR of novel histamine H3 receptor antagonists

The synthesis and biological evaluation of novel tetrahydroisoquinoline, tetrahydroquinoline, and tetrahydroazepine antagonists of the human and rat H(3) receptors are described. The substitution around these rings as well as the nature of the substituent on nitrogen is explored. Several compounds with high affinity and selectivity for the human and rat H(3) receptors are reported.