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.     

Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition comparison with other mammalian secreted phospholipases A2.

Human secreted group IIA phospholipase A2 (hGIIA) was reported to inhibit prothrombinase activity because of binding to factor Xa. This study further shows that hGIIA and its catalytically inactive H48Q mutant prolong the lag time of thrombin generation in human platelet-rich plasma with similar efficiency, indicating that hGIIA exerts an anticoagulant effect independently of phospholipid hydrolysis under ex vivo conditions. Charge reversal of basic residues on the interfacial binding surface (IBS) of hGIIA leads to decreased ability to inhibit prothrombinase activity, which correlates with a reduced affinity for factor Xa, as determined by surface plasmon resonance. Mutation of other surface-exposed basic residues, hydrophobic residues on the IBS, and His48, does not affect the ability of hGIIA to inhibit prothrombinase activity and bind to factor Xa. Other basic, but not neutral or acidic, mammalian secreted phospholipases A2 (sPLA2s) exert a phospholipid-independent inhibitory effect on prothrombinase activity, suggesting that these basic sPLA2s also bind to factor Xa. In conclusion, this study demonstrates that the anticoagulant effect of hGIIA is independent of phospholipid hydrolysis and is based on its interaction with factor Xa, leading to prothrombinase inhibition, even under ex vivo conditions. This study also shows that such an interaction involves basic residues located on the IBS of hGIIA, and suggests that other basic mammalian sPLA2s may also inhibit blood coagulation by a similar mechanism to that described for hGIIA.     

Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.     

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.     

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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