SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells

Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.     

Ways of coping as predictors of satisfaction with curriculum and academic success in medical school

The purpose of the present study was to determine the coping strategies of medical students and to investigate the effects of coping strategies on student satisfaction and academic achievement with different instruction methods. A total of 152 medical students was followed throughout the first 2 yr of medical education between 2008 and 2010. Students completed a sociodemographic questionnaire and revised form of the Ways of Coping questionnaire both at the beginning of the first year and at the end of the second year. These forms provided data about the characteristics and main coping strategies (problem focused or emotion focused) of the students and revealed the change over time. At the end of the second year, participants also completed a satisfaction questionnaire asking their satisfaction with lectures, problem-based learning, and practicals. The authors used block, problem-based learning, and practical exam scores of the students attained in the past 2 yr as academic achievement indicators. No sociodemographic variable was related to coping strategy. The majority of students (80.9%) adopted problem-focused coping. A shift occurred in the main coping strategies of some students in both sides. Problem-focused coping scores decreased over time. Problem-focused coping positively correlated with satisfaction with practicals and practical exam scores, whereas emotion-focused coping showed the same correlation negatively. The main coping strategy also predicted satisfaction and exam success in practicals. In conclusion, a main coping strategy may be helpful to predict student satisfaction and academic achievement with some student-centered instruction methods. Determining undesired coping strategies may provide an opportunity for intervention to prevent relevant dissatisfaction and failure.     

Ubiquitously expressed hematological and neurological expressed 1 downregulates Akt-mediated GSK3β signaling, and its knockdown results in deregulated G2/M transition in prostate cells

As the molecular mechanism of β-catenin deregulation is not well understood, and stabilized β-catenin is known to translocate into the nucleus and activate genes for proliferation, a novel regulatory factor, hematological and neurological expressed 1 (HN1), for Akt-GSK3β-β-catenin axis is reported here. In our studies, HN1 gene structure was characterized. HN1 expression was found to be epidermal growth factor-responsive in PC-3 cells, and protein expression was also upregulated in PC-3 and LNCaP but not in DU145 cells. Additionally, HN1 was found to be downregulated by the specific AKT inhibitor wortmannin but not with PI3K or MAPK inhibitors, LY294002 and PD98059, respectively, in PC-3 and MCF-7 cells. Further, siRNA-mediated knockdown of HN1 resulted in considerable increase in Akt((S473)) and GSK3β((S9),(Y216)) phosphorylations; moreover, subsequent accumulation of β-catenin, increase in c-myc expression, and nuclear accumulation of cyclin D1 were observed in PC-3 cells. Knockdown of HN1 also resulted in prolongation of G(1) phase in cell cycle, increasing tetraploidy, presumably because of cells escaping from abnormal mitosis in PC-3 cells. Consistently, overexpression of HN1 reversed the cell-cycle-specific observations, resulted in accumulation of cells in G(2)/M, and reduced the proliferation rate, which were investigated using flow cytometry and methylthiazol tetrazolium assays. As activating mutations of β-catenin have been demonstrated in late-stage tumors, and β-catenin stabilization was correlated with poor prognosis in previous reports, epidermal growth factor-upregulated HN1 expression might have a role in deregulating the AKT-GSK3β((S9))-mediated signaling as a novel compensating mechanism.     

Electrochemical cysteine determination in serum samples by Hg thin film sensor

Cysteine is a nonessential aminoacid, meaning that cysteine can be made in the human body. It is one of the few amino acids that contain sulfur. This allows cysteine to bond in a special way and maintain the structures of proteins in the body. Cysteine strengthens the protective lining of the stomach and intestines, which may help prevent damage caused by aspirin and similar drugs. In addition, cysteine may play an important role in the communication between immune system cells. In this study, glassy carbon electrodes modified with mercury (Hg) were used as working electrode. Mercury thin film on glassy carbon electrode was deposited by holding the electrode potential at -0.7 V; the measurement period for the coating process was 2 minutes. pH and temperature effects on the electrode response were carried out by working at different pHs and temperatures. The calibration graph for cysteine was drawn in the range of 5-120 μM cysteine. Repeatability and interferences studies were investigated. GSH had an interference effect of about 13% of cysteine response. Finally, the sensor was applied to real samples for cysteine determination and the method was validated by Ellman's reagent.     

Investigating paraoxonase-1 gene Q192R and L55M polymorphism in patients with renal cell cancer

Increased oxidative stress can help promote carcinogenesis, including development of renal cell carcinoma. The enzyme protects low-density lipoproteins from oxidation and can be a factor in this process. PON1 Q192R and L55M paraoxonase gene polymorphisms were assessed in 60 renal cell carcinoma patients and 60 healthy controls. Genotypes were examined by PCR; the restriction enzyme AlwI was used to examine the Q192R polymorphism and Hsp92II for the L55M polymorphism. Significant differences in the PON1 Q192R polymorphism were found between patients and controls. The Q allele was more frequent in the patient group than in controls, while the R allele was more frequent in the control group. No significant differences were found in the L55M polymorphism. Additionally, there were no significant differences in L and M allele frequencies. We conclude that the R allele may protect against renal cell carcinoma.     

A karyotypic study on Silene sect. Sclerocalycinae in Turkey

The karyology of fifteen taxa of Silene sect. Sclerocalycinae from Turkey has been investigated. Karyotype analyses has been carried out and the chromosome number of Silene caramanica, S. peduncularis, S. armena, S. laxa, S. swertiifolia, S. caesarea, S. sclerophylla, S. haradjianii and S. lycaonica have been determined for the first time. For all the taxa studied, the diploid chromosome number and the basic chromosome number were found to be 2n=24 and x=12, respectively. Except for S. chlorifolia and S. doganii, the karyomorphology of the taxa studied is here described for the first time. S. laxa was found to have the smallest chromosomes whereas the largest ones were observed in S. haradjianii. Silene chlorifolia had the highest A₁ index and S. bupleuroides subsp. bupleuroides had the highest interchromosomal asymmetry coefficient (A₂).     

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs.