Generating Mouse Models Using CRISPR-Cas9-Mediated Genome Editing.

The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including mice. The CRISPR-Cas9 reagents can be delivered directly into the mouse zygote to derive a mutant animal carrying targeted genetic modifications. The major components of the system include the guide RNA, which provides target specificity, the Cas9 nuclease that creates the DNA double-strand break, and the donor oligonucleotide or plasmid carrying the intended mutation flanked by sequences homologous to the target site. Here we describe the general considerations and experimental protocols for creating genetically modified mice using the CRISPR-Cas9 system.

Pneumocystis cell wall beta-glucan stimulates calcium-dependent signaling of IL-8 secretion by human airway epithelial cells.

BACKGROUND: Respiratory failure secondary to alveolar inflammation during Pneumocystis pneumonia is a major cause of death in immunocompromised patients. Neutrophil infiltration in the lung of patients with Pneumocystis infection predicts severity of the infection and death. Several previous studies indicate that airway epithelial cells release the neutrophil chemoattractant proteins, MIP-2 (rodents) and IL-8 (humans), in response to Pneumocystis and purified Pneumocystis cell wall beta-glucans (PCBG) through the NF-kappaB-dependent pathway. However, little is known about the molecular mechanisms that are involved in the activation of airway epithelium cells by PCBG resulting in the secretion of IL-8. METHOD: To address this, we have studied the activation of different calcium-dependent mitogen-activated protein kinases (MAPKs) in 1HAEo- cells, a human airway epithelial cell line. RESULTS: Our data provide evidence that PCBG induces phosphorylation of the MAPKs, ERK, and p38, the activation of NF-kappaB and the subsequently secretion of IL-8 in a calcium-dependent manner. Further, we evaluated the role of glycosphingolipids as possible receptors for beta-glucans in human airway epithelial cells. Preincubation of the cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) a potent inhibitor of the glycosphingolipids synthesis, prior to PCBG stimulation, significantly decreased IL-8 production. CONCLUSION: These data indicate that PCBG activates calcium dependent MAPK signaling resulting in the release of IL-8 in a process that requires glycosphingolipid for optimal signaling.

The Pneumocystis meiotic PCRan1p kinase exhibits unique temperature-regulated activity.

Pneumocystis organisms are opportunistic fungal pathogens that cause significant pneumonia in immune-compromised hosts. Recent evidence has suggested that Pneumocystis carinii exists as separate mating types, and expresses and regulates proteins that govern meiosis and progression of the life cycle. This study was undertaken to investigate the activity of three life cycle-regulatory proteins in Pneumocystis, including two proteins essential in mating signaling, and a putative meiotic regulator, to determine the conditions under which they are most active. This study used V5/HIS-tagged PCRan1p, PCSte20p, and PCCbk1, purified from Saccharomyces cerevisiae strain, INVSC, as well as an in vitro Escherichia coli protein expression system to determine the optimal expression conditions of each protein in the presence of varying pH, temperature, and metal ions. These studies demonstrate an atypical enzymatic activity in PCRan1p, whereby the kinase was most active in the environmental conditions between 10 and 25 degrees C, compared with a dramatic reduction in activity above 30 degrees C, temperatures typically found within mammalian hosts. Circular dichroism and fluorescence spectroscopy suggest that PCRan1p becomes partially unfolded at 25 degrees C, leading to its most active conformation, whereas continued unfolding as temperature increases results in strongly suppressed activity. These studies suggest that, in vivo, while under conditions within the mammalian lung (typically 37 degrees C), PCRan1p kinase activity is largely suppressed, allowing better conditions for the activation of meiosis, whereas in ex vivo environments, PCRan1p kinase activity increases to arrest progression of the life cycle until conditions become more favorable.

Disruption of parallel and converging signaling pathways contributes to the synergistic antitumor effects of simultaneous mTOR and EGFR inhibition in GBM cells.

Elevated epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) signaling are known to contribute to the malignant properties of glioblastoma multiforme (GBM), which include uncontrolled cell proliferation and evasion of apoptosis. Small molecule inhibitors that target these protein kinases have been evaluated in multiple clinical trials for cancer patients, including those with GBM. Here we have examined the cellular and molecular effects of a combined kinase inhibition of mTOR (rapamycin) and EGFR (EKI-785) in U87 and U251 GBM cells. Simultaneous treatment with rapamycin and EKI-785 results in synergistic antiproliferative as well as proapoptotic effects. At a molecular level, rapamycin alone significantly decreases S6 phosphorylation, whereas EKI-785 alone promotes substantially reduced signal transducer and activator of transcription (STAT3) phosphorylation. Treatment with rapamycin alone also increases Akt phosphorylation on Ser-473, but this effect is blocked by a simultaneous administration of EKI-785. Individually, EKI-785 diminishes while rapamycin promotes the binding of the translation inhibitor eukaryotic initiation factor 4E binding protein (4EBP1) to the eukaryotic translation initiation factor 4E (eIF4E). In spite of these opposing effects, the highest level of 4EBP1-eIF4E binding occurs with the combination of the two inhibitors. These results indicate that the inhibition of EGFR and mTOR has distinct as well as common signaling consequences and provides a molecular rationale for the synergistic antitumor effects of EKI-785 and rapamycin administration.