Targeting Glutamatergic Signaling and the PI3 Kinase Pathway to Halt Melanoma Progression

Our group has previously reported that the majority of human melanomas (> 60%) express the metabotropic glutamate receptor 1 (GRM1) and that the glutamate release inhibitor riluzole, a drug currently used to treat amyotrophic lateral sclerosis, can induce apoptosis in GRM1-expressing melanoma cells. Our group previously reported that in vitro riluzole treatment reduces cell growth in three-dimensional (3D) soft agar colony assays by 80% in cells with wildtype phosphoinositide 3-kinase (PI3K) pathway activation. However, melanoma cell lines harboring constitutive activating mutations of the PI3K pathway (PTEN and NRAS mutations) showed only a 35% to 40% decrease in colony formation in soft agar in the presence of riluzole. In this study, we have continued our preclinical studies of riluzole and its effect on melanoma cells alone and in combination with inhibitors of the PI3 kinase pathway: the AKT inhibitor, API-2, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. We modeled these combinatorial therapies on various melanoma cell lines in 3D and 2D systems and in vivo. Riluzole combined with mTOR inhibition is more effective at halting melanoma anchorage-independent growth and xenograft tumor progression than either agent alone. PI3K signaling changes associated with this combinatorial treatment shows that 3D (nanoculture) modeling of cell signaling more closely resembles in vivo signaling than monolayer models. Riluzole combined with mTOR inhibition is effective at halting tumor cell progression independent of BRAF mutational status. This makes this combinatorial therapy a potentially viable alternative for metastatic melanoma patients who are BRAF WT and are therefore ineligible for vemurafenib therapy.     

In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2 restricted CD8 T cell response

Background

As a potent CD8⁺ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population.

Methods and Findings

Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8⁺ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2⁺ individuals recovered from L. major. HLA-A2⁻ individuals recovered from L. major and HLA-A2⁺ healthy donors were included as control groups. Individual response of HLA-A2⁺ recovered volunteers as percent of CD8⁺/IFN-γ⁺ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2⁻ recovered individuals. Based on cutoff scores calculated from the response of HLA-A2⁻ recovered individuals, 31.6% and 13.3% of HLA-A2⁺ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2⁻ recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2⁺ recovered individuals.

Conclusion

Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis.     

Quantification of protein deposits on silicone hydrogel materials using stable-isotopic labeling and multiple reaction monitoring

This study was designed to use multiple reaction monitoring (MRM) for accurate quantification of contact lens protein deposits. Worn lenses used with a multipurpose disinfecting solution were collected after wear. Individual contact lenses were extracted and then digested with trypsin. MRM in conjunction with stable-isotope-labeled peptide standards was used for protein quantification. The results show that lysozyme was the major protein detected from both lens types. The amount of protein extracted from contact lenses was affected by the lens material. Except for keratin-1 (0.83 ± 0.61 vs 0.77 ± 0.20, p = 0.81) or proline rich protein-4 (0.11 ± 0.04 vs 0.15 ± 0.12, p = 0.97), the amounts of lysozyme, lactoferrin, or lipocalin-1 extracted from balafilcon A lenses (12.9 ± 9.01, 0.84 ± 0.50 or 2.06 ± 1.6, respectively) were significantly higher than that extracted from senofilcon A lenses (0.88 ± 0.13, 0.50 ± 0.10 or 0.27 ± 0.23, respectively) (p < 0.05). The amount of protein extracted from contact lenses was dependent on both the individual wearer and the contact lens material. This may have implications for the development of clinical responses during lens wear for different people and with different types of contact lenses. The use of MRM-MS is a powerful analytical tool for the quantification of specific proteins from single contact lenses after wear.     

Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies

Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host–parasite interactions at the cellular level.     

Investigate of AQP gene expression in the liver of mice after ischemia–reperfusion

Ischemia–reperfusion (IR) injury usually occurs during liver transplantation. Aquaporins (AQPs) are transmembrane channels that facilitate water permeability through cell membranes and are essential for the regulation of water homeostasis. Changes in the AQPs expression have been correlated with several inflammatory diseases. Less is known about AQPs expression in hepatic ischemia reperfusion injury. To clarify the roles of AQPs in IR injury, in this current study we examined the gene expression patterns of AQP1, 8 and 9 in the liver after IR injury. Male balb/c mice were exposed to partial (70%) hepatic ischemia for 65 min and then randomized into five groups of reperfusion [0 h (A), 8 h (B), 1 day (C), 3 days (D), and 7 days (E)]. A surgical group was also selected as the sham group. Serum and liver tissue samples were collected for evaluation of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver histopathology. Real time PCR was performed to evaluate the AQPs expression. I/R injury resulted in a significant increase in ALT and AST (p?<?0.05) compared to sham mice in each group. The gene expression of AQPs was significantly increased in the IR group compared with the sham group (p?<?0.05). AQP8 and AQP1 after 8 h (group B) showed the highest gene expression in comparison with other groups, but the highest level of AQP9 gene expression was observed after 1 day (group C). Pathologic changes in the liver after reperfusion were confirmed the IR. In the IR group cytoplasmic vacuolization, inflammatory cell infiltration and focal necrosis were detected. In conclusion, our findings indicated that the damage caused by ischemia–reperfusion in the liver can change the expression of AQP genes, which can interfere with hepatocellular homeostasis and their function. Upregulation of AQP1, 8 and 9 could contribute to the development of hepatocellular swelling after hepatic IR injury.     

Occurrence and Characterization of Candida nivariensis from a Culture Collection of Candida glabrata Clinical Isolates in Malaysia

Background

Candida nivariensis and C. bracarensis have been recently identified as emerging yeast pathogens which are phenotypically indistinguishable from C. glabrata. However, there is little data on the prevalence and antifungal susceptibilities of these species.

Objective

This study investigated the occurrence of C. nivariensis and C. bracarensis in a culture collection of 185 C. glabrata isolates at a Malaysian teaching hospital.

Methods

C. nivariensis was discriminated from C. glabrata using a PCR assay as described by Enache-Angoulvant et al. (J Clin Microbiol 49:3375–9, 2011). The identity of the isolates was confirmed by sequence analysis of the D1D2 domain and internal transcribed spacer region of the yeasts. The isolates were cultured on Chromogenic CHROMagar Candida ^® agar (Difco, USA), and their biochemical and enzymic profiles were determined. Antifungal susceptibilities of the isolates against amphotericin B, fluconazole, voriconazole and caspofungin were determined using E tests. Clotrimazole MICs were determined using a microbroth dilution method.

Results

There was a low prevalence (1.1 %) of C. nivariensis in our culture collection of C. glabrata. C. nivariensis was isolated from a blood culture and vaginal swab of two patients. C. nivariensis grew as white colonies on Chromogenic agar and demonstrated few positive reactions using biochemical tests. Enzymatic profiles of the C. nivariensis isolates were similar to that of C. glabrata. The isolates were susceptible to amphotericin B, fluconazole, voriconazole and caspofungin. Clotrimazole resistance is suspected in one isolate.

Conclusion

This study reports for the first time the emergence of C. nivariensis in our clinical setting.     

The frequency and distribution of thiopurine S-methyltransferase alleles in south Iranian population

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Variability in TPMT activity is mainly due to genetic polymorphism. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in an Iranian population from south of Iran (n = 500), using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. Four hundred seventy four persons (94.8%) were homozygous for the wild type allele (TPMT*1/*1) and twenty five people were TPMT*1/*3C (5%). One patient was found to be heterozygous in terms TPMT*1 and *2 alleles with genotype of TPMT*1/*2 (0.2%). None of the participants had both defective alleles. The TPMT*3C and *2 were the only variant alleles observed in this population. The total frequency of variant alleles was 2.6% and the wild type allele frequency was 97.4%. The TPMT*3B and *3A alleles were not detected. Distributions of TPMT genotype and allele frequency in Iranian populations are different from the genetic profile found among Caucasian or Asian populations. Our findings also revealed inter-ethnic differences in TPMT frequencies between different parts of Iran. This view may help clinicians to choose an appropriate strategy for thiopurine drugs and reduce adverse drug reactions such as bone marrow suppression.     

The Effect of Leptin on Sex Hormones and Antioxidant Enzyme Levels in Obese and Normal Male Rats

International Journal of Peptide Research and Therapeutics - The correlation between leptin, nutritional status and reproductive performance is known but its effect on male obese is not fully...     

Molecular 14C evidence for contrasting turnover and temperature sensitivity of soil organic matter components

Climate projection requires an accurate understanding for soil organic carbon (SOC) decomposition and its response to warming. An emergent view considers that environmental constraints rather than chemical structure alone control SOC turnover and its temperature sensitivity (i.e., Q₁₀), but direct long-term evidence is lacking. Here, using compound-specific radiocarbon analysis of soil profiles along a 3300-km grassland transect, we provide direct evidence for the rapid turnover of lignin-derived phenols compared with slower-cycling molecular components of SOC (i.e., long-chain lipids and black carbon). Furthermore, in contrast to the slow-cycling components whose turnover is strongly modulated by mineral association and exhibits low Q₁₀, lignin turnover is mainly regulated by temperature and has a high Q₁₀. Such contrasts resemble those between fast-cycling (i.e., light) and mineral-associated slow-cycling fractions from globally distributed soils. Collectively, our results suggest that warming may greatly accelerate the decomposition of lignin, especially in soils with relatively weak mineral associations.