Derivation of genetically modified human pluripotent stem cells with integrated transgenes at unique mapped genomic sites

Many applications in human pluripotent stem cell (PSC) research require the genetic modification of PSCs to express a transgene in a stable and dependable manner. Random transgene integration commonly results in unpredictable and heterogeneous expression. We describe a protocol for the derivation of clonal populations of human embryonic stem cells or induced pluripotent stem cells (iPSCs) expressing a transgene from a single copy of an integrated lentiviral vector that is mapped to the genome. Using optimized transduction conditions, followed by single-cell subcloning and a round of antibiotic selection, we find that approximately half of the colonies retrieved contain a single vector copy. After expansion, the majority of these are confirmed to be clonal. The vector/genomic DNA junction is sequenced and the unique integration site is mapped to the genome. This protocol enables the efficient derivation of genetically modified PSCs containing an integrated transgene at a known genomic site in ～7 weeks.     

The cytosolic chaperone Hsc70 promotes traffic to the cell surface of intracellular retained melanocortin-4 receptor mutants

Inherited modifications in protein structure frequently cause a loss-of-function by interfering with protein synthesis, transport, or stability. For the obesity-linked melanocortin-4 receptor (MC4R) and other G protein-coupled receptors, many mutants are intracellular retained. The biogenesis and trafficking of G protein-coupled receptors are regulated by multiple factors, including molecular chaperone networks. Here, we have investigated the ability of the cytosolic cognate 70-kDa heat-shock protein (Hsc70) chaperone system to modulate cell surface expression of MC4R. Clinically occurring MC4R mutants S58C, P78L, and D90N were demonstrated to have reduced trafficking to the plasma membrane and to be retained at the endoplasmic reticulum (ER). Analyses by fluorescence recovery after photobleaching revealed that the mobility of MC4R mutant protein at the ER was reduced, implying protein misfolding. In cells expressing MC4R, overexpression of Hsc70 resulted in increased levels of wild-type and mutant receptors at the cell surface. MC4R and Hsc70 coimmunoprecipitated, and fluorescence recovery after photobleaching analyses showed that increasing cellular levels of Hsc70 promoted the mobility of ER retained MC4R. Moreover, expression of HSJ1b, a cochaperone that enhances degradation of Hsc70 clients, reduced cellular levels of MC4R. Hsp70 and Hsp90 chaperone systems collaborate in the cellular processing of clients. For MC4R, inhibition of endogenous Hsp90 by geldanamycin reduced receptor levels. By contrast, expression of the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase) increased cellular levels of MC4R. Finally, we demonstrate that signaling of intracellular retained MC4R mutants is increased in cells overexpressing Hsc70. These data indicate that cytosolic chaperone systems can facilitate rescue of intracellular retained MC4R by improving folding. They also support proteostasis networks as a potential target for MC4R-linked obesity.     

S100B protein stimulates microglia migration via RAGE-dependent up-regulation of chemokine expression and release

The Ca(2+)-binding protein of the EF-hand type, S100B, is abundantly expressed in and secreted by astrocytes, and release of S100B from damaged astrocytes occurs during the course of acute and chronic brain disorders. Thus, the concept has emerged that S100B might act an unconventional cytokine or a damage-associated molecular pattern protein playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases. S100B proinflammatory effects require relatively high concentrations of the protein, whereas at physiological concentrations S100B exerts trophic effects on neurons. Most if not all of the extracellular (trophic and toxic) effects of S100B in the brain are mediated by the engagement of RAGE (receptor for advanced glycation end products). We show here that high S100B stimulates murine microglia migration in Boyden chambers via RAGE-dependent activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Rac1/JNK/AP-1, Rac1/NF-κB, and, to a lesser extent, p38 MAPK. Recruitment of the adaptor protein, diaphanous-1, a member of the formin protein family, is also required for S100B/RAGE-induced migration of microglia. The S100B/RAGE-dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 results in the up-regulation of expression of the chemokines, CCL3, CCL5, and CXCL12, whose release and activity are required for S100B to stimulate microglia migration. Lastly, RAGE engagement by S100B in microglia results in up-regulation of the chemokine receptors, CCR1 and CCR5. These results suggests that S100B might participate in the pathophysiology of brain inflammatory disorders via RAGE-dependent regulation of several inflammation-related events including activation and migration of microglia.     

Focus: Antimicrobial Resistance: Gut Microbiota Modulation and Prevention of Dysbiosis as an Alternative Approach to Antimicrobial Resistance: A Narrative Review

Background: The importance of gut microbiota in human health is being increasingly studied. Imbalances in gut microbiota have been associated with infection, inflammation, and obesity. Antibiotic use is the most common and significant cause of major alterations in the composition and function of the gut microbiota and can result in colonization with multidrug-resistant bacteria. Methods: The purpose of this review is to present existing evidence on how microbiota modulation and prevention of gut dysbiosis can serve as tools to combat antimicrobial resistance. Results: While the spread of antibiotic-resistant pathogens requires antibiotics with novel mechanisms of action, the number of newly discovered antimicrobial classes remains very low. For this reason, the application of alternative modalities to combat antimicrobial resistance is necessary. Diet, probiotics/prebiotics, selective oropharyngeal or digestive decontamination, and especially fecal microbiota transplantation (FMT) are under investigation with FMT being the most studied. But, as prevention is better than cure, the implementation of antimicrobial stewardship programs and strict infection control measures along with newly developed chelating agents could also play a crucial role in decreasing colonization with multidrug resistant organisms. Conclusion: New alternative tools to fight antimicrobial resistance via gut microbiota modulation, seem to be effective and should remain the focus of further research and development.     

In Vitro Digestion of caseinate and Tween 20 Emulsions

Food Biophysics - In vitro digestion experiments are set-up with emulsions stabilized either with sodium caseinate and/or Tween 20, in the presence of mucin in the simulated GI fluids. The progress...     

Corticotrophin-Releasing Factor (CRF) and the Urocortins Are Potent Regulators of the Inflammatory Phenotype of Human and Mouse White Adipocytes and the Differentiation of Mouse 3T3L1 Pre-Adipocytes

Chronic activation of innate immunity takes place in obesity and initiated by the hypertrophic adipocytes which obtain a pro-inflammatory phenotype. The corticotrophin-releasing factor (CRF) family of neuropeptides and their receptors (CRF₁ and CRF₂) affect stress response and innate immunity. Adipose tissue expresses a complete CRF system. The aim of this study was to examine the role of CRF neuropeptides in the immune phenotype of adipocytes assessed by their expression of the toll-like receptor-4 (TLR4), the production of inflammatory cytokines IL-6, TNF-α and IL-1β, chemokines IL-8, monocyte attractant protein-1 (MCP-1) and of the adipokines adiponectin, resistin and leptin. Our data are as follows: (a) CRF, UCN2 and UCN3 are expressed in human white adipocytes as well as CRFR_1a, CRFR_2a and CRFR_2b but not CRFR_2c. 3T3L1 pre-adipocytes and differentiated adipocytes expressed both CRF₁ and CRF₂ receptors and UCN3, while UCN2 was detected only in differentiated adipocytes. CRF₂ was up-regulated in mouse mature adipocytes. (b) CRF₁ agonists suppressed media- and LPS-induced pre-adipocyte differentiation while CRF₂ receptor agonists had no effect. (c) In mouse pre-adipocytes, CRF₂ agonists suppressed TLR4 expression and the production of IL-6, CXCL1 and adiponectin while CRF₁ agonists had no effect. (d) In mature mouse adipocytes LPS induced IL-6 and CXCL1 production and suppressed leptin. (e) In human visceral adipocytes LPS induced IL-6, TNF-α, IL-8, MCP-1 and leptin production and suppressed adiponectin and resistin. (f) In mouse mature adipocytes CRF₁ and CRF₂ agonists suppressed basal and LPS-induced production of inflammatory cytokines, TLR4 expression and adiponectin production, while in human visceral adipocytes CRF and UCN1 suppressed basal and LPS-induced IL-6, TNF-α, IL-8 and MCP-1 production. In conclusion, the effects of the activation of CRF₁ and CRF₂ may be significant in ameliorating the pro-inflammatory activity of adipocytes in obesity.     

Array-based pharmacogenomics of molecular-targeted therapies in oncology

The advent of microarrays over the past decade has transformed the way genome-wide studies are designed and conducted, leading to an unprecedented speed of acquisition and amount of new knowledge. Microarray data have led to the identification of molecular subclasses of solid tumors characterized by distinct oncogenic pathways, as well as the development of multigene prognostic or predictive models equivalent or superior to those of established clinical parameters. In the field of molecular-targeted therapy for cancer, in particular, the application of array-based methodologies has enabled the identification of molecular targets with 'key' roles in neoplastic transformation or tumor progression and the subsequent development of targeted agents, which are most likely to be active in the specific molecular setting. Herein, we present a summary of the main applications of whole-genome expression microarrays in the field of molecular-targeted therapies for solid tumors and we discuss their potential in the clinical setting. An emphasis is given on deciphering the molecular mechanisms of drug action, identifying novel therapeutic targets and suitable agents to target them with, and discovering molecular markers/signatures that predict response to therapy or optimal drug dose for each patient.