Vikas Kumar

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Meckel's diverticulum is the most common congenital malformation of gastrointestinal tract. It can cause complications in the form of ulceration, haemorrhage, intussusception, intestinal obstruction, perforation and, very rarely, vesicodiverticular fistulae and tumours. These complications, especially bleeding, are more common in the paediatric age group than in adults; however it is not uncommon to miss the diagnosis of Meckel's diverticulum in adults. Here, we reviewed the literature regarding the complications of this forgotten clinical entity in adults with potential diagnostic difficulties and management strategies.

Gastric outlet obstruction (GOO) is characterized by epigastric pain and postprandial vomiting secondary to mechanical obstruction. Management of GOO is usually focused on alleviating the symptoms of obstruction and can be achieved by surgical gastrojejunostomy or enteral stenting. Recent studies have shown success with EUS-guided gastroenterostomy (EUS-GE) in the management of GOO but data is limited. We, therefore, conducted a meta-analysis to evaluate the safety and efficacy of EUS-GE in the management of GOO. A comprehensive literature review was conducted by searching the Embase and PubMed databases from inception to January 2019 to identify all studies that evaluate the efficacy and safety of EUS-GE in GOO. Our primary outcome was to evaluate technical success and clinical success. Secondary outcomes were to evaluate the need for reintervention and adverse events of the procedure. Twelve studies including 285 patients were included in the meta-analysis. Technical success was achieved in 266 patients with a pooled technical success of 92% (95% confidence interval [CI]: 88%–95%). Clinical success was achieved in 90% of the patients (95% CI: 85%–94%). Recurrence of symptoms or unplanned reintervention was needed in 9% of the patients (95% CI: 6%–13%) and adverse events were reported in 12% of the patients (95% CI: 8%–16%). The heterogeneity tests among studies were nonsignificant with I2 = 0. EUS-GE is a safe and efficacious treatment modality for the management of benign and malignant GOO. Larger prospective studies are needed to further evaluate its utility in GOO.

G-protein-coupled receptors are thought to transmit extracellular signals to the cytoplasm from their position on the cell surface. Some receptors, including the metabotropic glutamate receptor 5 (mGluR5), are also highly expressed on intracellular membranes where they serve unknown functions. Here, we show that activation of cell surface versus intracellular mGluR5 results in unique Ca(2+) signatures leading to unique cellular responses. Specifically, activation of either cell surface or intracellular mGluR5 leads to JNK, Ca(2+)/calmodulin-dependent protein kinase (CaMK), and cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas activation of only intracellular mGluR5 leads to ERK1/2 and Elk-1 phosphorylation. Using pharmacological and genetic approaches, the present findings support a role for CaMK kinase in mediating mGluR5-dependent cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas CaMKII is upstream of intracellular mGluR5-mediated Elk-1 phosphorylation. Consistent with models showing Elk-1 regulating cascades of gene expression, the known Elk-1 targets c-fos and egr1 were up-regulated following intracellular mGluR5 activation, whereas a representative non-Elk-1 target, c-jun, was not. These findings emphasize that glutamate not only serves as a neurotransmitter for cell surface receptors but, when transported into the cell, can also activate intracellular receptors such as mGluR5. Glutamate activation of intracellular mGluR5 serves an important role in the regulation of nuclear Ca(2+), transcriptional activation, and gene expression necessary for physiological processes such as synaptic plasticity.