Ral Overactivation in Malignant Peripheral Nerve Sheath Tumors

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.The Ras family of guanine-nucleotide bound proteins exerts a fundamental role in cell biology and constitutes an important area of cancer research due to its significant involvement in the development and progression of malignancies (8, 10, 17, 18, 32). Ras-like (Ral) proteins are crucial members of this family and have been shown to play a pivotal role in human tumors (7, 28, 41, 66, 70). Because Ral guanine nucleotide exchange factors (Ral-GEFs) are direct effectors of Ras, the Ral signaling pathway has been traditionally considered a Ras-effector pathway. Activation of Ras (in resemblance to Ral) is regulated by two classes of proteins: Ras-GEFs (e.g., SOS) and Ras- GTPase activating proteins (Ras-GAPs such as neurofibromin). The latter induces hydrolysis of Ras from the active (GTP) form to the inactive (GDP) form (13). Ral-GEFs include two main groups: the proteins that are stimulated by Ras because of their carboxy-terminal Ras binding domain (RalGDS, RGL1, and RGL2) and the proteins that are activated by substrates of PI3K through a pleckstrin homology domain on their C-terminal (RALGPS1 and RALGPS2) (19). Although highly similar to Ras, Ral proteins (RalA and RalB) involve a series of distinctly different effectors that influence gene expression and translation through interaction with ZO-1-associated nucleic acid binding protein (ZONAB) and RalA binding protein 1 (RalBP1) (11, 23, 33). RalB directly interacts with the SEC5 subunit of exocyst to facilitate the host defense response (48, 58).In addition to overactivation of GEFs, inactivation of GAPs is another mechanism for overactivation of GTP-bound proteins. The lack of neurofibromin (encoded by NF1 on human chromosome 17q11.2), a Ras-GAP protein, is the main molecular event in neurofibromatosis type 1 (NF-1), an autosomal-dominant human genetic disease occurring in approximately 1 in 2,500 to 3,500 births (22, 27, 42). One of the main tumor-causing effects of inactivating mutations in the tumor suppressor NF1 gene is postulated to be the subsequent activation of Ras (3, 29, 53, 57, 69). With two main functional domains, SEC14 and Ras-GAP, neurofibromin is best known for its Ras-GAP function. Although the yeast SEC14p is shown to be involved in regulating intracellular proteins and lipid trafficking, the function of its homologous domain in neurofibromin is unknown (49, 62). Although neurofibromas are the most common tumors in NF-1, 10% of patients with plexiform develop malignant peripheral nerve sheath tumors (MPNSTs), which are typically high grade and often fatal (21, 34, 65).The molecular events involved in the malignant transformation of benign neurofibromas to MPNST are poorly defined. Usually arising in the third through sixth decades of life, these tumors are composed of tightly packed hyperchromatic spindle-shaped cells with frequent mitotic figures. Inactivation of both copies of the NF1 gene has been demonstrated in benign human neurofibromas and shown to cause tumors in murine models (56). Loss of heterozygosity of NF1 and p53 has frequently been observed in human MPNST (35, 47, 54). Recombinant mouse strains (NP mice), which harbor inactivated Nf1 and p53 alleles (cis-Nf1^+/−:p53^+/−), demonstrate the cumulative effects of loss of both Nf1 and p53 genes in the etiology of MPNST (14, 68).In the present study, we show that while both Ras activation and activation of a series of its downstream effector pathways are observed in a fraction of MPNST cells, RalA is activated globally in all studied mouse and human MPNST cells and tumor samples. Our results also explain the involvement of this signaling molecule in a series of key biological functions of MPNST cells, as shown in a variety of in vitro assays and an in vivo model of MPNST. Such information may play a role in designing novel therapies for treatment of MPNST or other tumors with overactivation of the Ral pathway.     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.     

The early effects of uninephrectomy on rat kidney metabolic state using optical imaging

Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney, although it is uncertain if this alters the mitochondrial redox state and how soon such changes may occur. A custom‐designed fluorescence cryo‐imaging technique was used to quantitatively assess the effect of UNX by measuring the levels of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the remaining kidney. Kidneys were snap‐frozen 3 days following UNX, and the intrinsic fluorescence of NADH and FAD were optically acquired. The 3D images were created to characterize the NADH/FAD redox ratios (RR) of the right kidneys, which underwent UNX and the remaining kidneys 3 days following UNX. Both the NADPH‐oxidases (Nox2 and Nox4) and the mitochondria are the main sources of reactive oxygen species (ROS) production in tubular epithelial cells. Responses to the UNX were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt‐sensitive (SS) rats and SS rats in which NADPH‐oxidase isoform 4 (Nox4) was knocked out (SS^Nox4?/?). The results found that each of the strains exhibited similar increase in kidney weights averaging 17% after 3 days of UNX. SD and SS^Nox4?/?rats both exhibited global reductions of the RR (P < .05) with a similar tendency observed in SS rats (P < .08), indicating increased ROS production. The unexpected reduction of the RR in the remnant kidneys of SS^Nox4?/? rats indicates that mechanisms independent of H₂O₂ produced from Nox4 may be responsible for this global increase of ROS. We propose that the reduced RR was largely a consequence of enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney. The data indicate that mitochondria become the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.     

LAMP3 (CD208) Expression in Squamous Cell Carcinoma and Epithelial Dysplasia of the Oral Cavity and Clinicopathological Characteristics of Unfavorable Prognosis

Background:This study aimed to evaluate LAMP3 (CD208) gene expression in oral squamous cell carcinoma (OSCC) and dysplastic oral epithelium by quantitative real-time polymerase chain reaction (qPCR) and compare LAMP3 expression in different disease grades and stages.Methods:In this study, 60 OSCC and dysplastic oral epithelium samples were obtained from the Mashhad University of Medical Sciences together with their demographic and clinicopathological documents. LAMP3 expression was measured by qPCR.Results:LAMP3 expression was significantly greater in OSCC than in dysplasia samples (P=0.001), in grade III OSCC than in grades I and II, and also greater in advanced than in early OSCC disease stage (P=0.001).Conclusion:The significantly greater LAMP3 expression in OSCC than in dysplastic epithelium indicates a role for LAMP3 in carcinogenesis in oral mucosa. Our results suggest LAMP3 may be useful as an anticancer target and/or to predict disease pathogenesis in OSCC patient’s cells.Key Words: Clinicopathological, Grade, Epithelial dysplasia, LAMP3, Stage, Squamous cell carcinoma.     

A bioinspired 3D shape olibanum-collagen-gelatin scaffolds with tunable porous microstructure for efficient neural tissue regeneration

There are a number of procedures for regeneration of injured nerves; however, tissue engineering scaffolds seems to be a promising approach for recovery of the functionality of the injured nerves. Consequently, in this study, olibanum-collagen-gelatin scaffolds were fabricated by freeze-cast technology. For this purpose, the olibanum and collagen were extracted from natural sources. The effect of solidification gradient on microstructure and properties of scaffolds was investigated. Scanning electron microscopy micrographs showed the formation of lamellar-type microstructure in which the average pore size reduced with an increase in freezing rate. According to the results, the prepared scaffolds at lower freezing rate showed a slight reduction in mechanical strength while the swelling and biodegradation ratio were increased due to the presence of larger pores and unidirectional channels. The composition of scaffolds and oriented microstructure improved cellular interaction. In addition, scaffolds with lower freezing rate exhibited promising results in terms of adhesion, spreading, and proliferation. In brief, the synthesized scaffolds at lower solidification rate have the potential for more in vitro and in vivo analyses to regeneration of neural defects.     

Hydrogels Composed of Cyclodextrin Inclusion Complexes with PLGA-PEG-PLGA Triblock Copolymers as Drug Delivery Systems

Although conventional pharmaceuticals have many drug dosage forms on the market, the development of new therapeutic molecules and the low efficacy of instant release formulations for the treatment of some chronic diseases and specific conditions encourage scientists to invent different delivery systems. To this purpose, a supramolecular hydrogel consisting of the tri-block copolymer PLGA-PEG-PLGA and α-cyclodextrin was fabricated for the first time and characterised in terms of rheological, morphological, and structural properties. Naltrexone hydrochloride and vitamin B12 were loaded, and their release profiles were determined.     

Subcircuits of Deep and Superficial CA1 Place Cells Support Efficient Spatial Coding across Heterogeneous Environments

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