Differential localization of PD-L1 and Akt-1 involvement in radioresistant and radiosensitive cell lines of head and neck squamous cell carcinoma.

Immunotherapy by blockade of the PD-1/PD-L1 checkpoint demonstrated amazing tumor response in advanced cancer patients including head and neck squamous cell carcinoma (HNSCC). However, the majority of HNSCC patients still show little improvement or even hyperprogression. Irradiation is currently investigated as synergistic treatment modality to immunotherapy as it increases the number of T-cells thereby enhancing efficacy of immunotherapy. Apart from this immunogenic context a growing amount of data indicates that PD-L1 also plays an intrinsic role in cancer cells by regulating different cellular functions like cell proliferation or migration. Here, we demonstrate opposing membrane localization of PD-L1 in vital and apoptotic cell populations of radioresistant (RR) and radiosensitive (RS) HNSCC cell lines up to 72 h after irradiation using flow cytometry. Moreover, strong PD-L1 expression was found in nuclear and cytoplasmic cell fractions of RR. After irradiation PD-L1 decreased in nuclear fractions and increased in cytoplasmic fractions of RR cells. In contrast, RS cell lines did not express PD-L1, neither in the nucleus nor in cytoplasmic fractions. Additionally, overexpression of PD-L1 in RS cells led to a proportional increase of vital PD-L1 positive cells after irradiation. Moreover, co-immunoprecipitation experiments revealed an interaction between Akt-1 and PD-L1, mostly in irradiated RR cells compared to RS cells suggesting a differential influence of PD-L1 on cell signaling. In summary, our data imply the need for different therapeutic strategies dependent on the molecular context in which PD-L1 is embedded.

Different surgical techniques and L-carnitine supplementation in an experimental varicocele model.

We aimed to investigate the impact of various varicocelectomy techniques and/or L-carnitine as an adjunct treatment, following the emergence of oxidative stress, on the expression levels of SCF/c-kit signalling pathways in spermatogenesis. Forty-two rats were divided into seven groups: group 1 (G1) control; group 2 (G2) sham; group 3 (G3) varicocele; group 4 (G4) varicocele + varicocelectomy with testicular nonartery sparing; group 5 (G5) same as G4 but with artery sparing; group 6 (G6) same as G4 but with L-carnitine and group 7 (G7) same as G5 with L-carnitine. mRNA expression levels of SCF and c-kit were measured quantitatively using real-time polymerase chain reaction. CASP-3 activity at protein level was determined, and histological evaluation was performed. mRNA expression level of SCF increased in G6 as compared to control group (3.52-folds change; P = 0.035), whereas mRNA expression level of c-kit gene remained the same. We found that in the left testis of G6 group, mRNA expression level of SCF increased 2.2-folds in comparison with the right testis (P < 0.05). There were no statistically significant differences in the CASP-3 protein expression levels between the control and other groups. When Cosentino Score analyses of immunostaining were conducted, we observed no significant differences among groups. Spermatogenic failure could be primarily due to a sertoli cell dysfunction. Although surgical treatment has been the best option for management of varicocele, auxiliary agents like L-carnitine may be considered as supportive treatment regimes in addition to conventional surgical treatments.

C-banding visualized by atomic force microscopy.

C-banding is a method used for studying chromosome rearrangements near centromeres and for investigating polymorphisms. In human chromosomes, the C-bands are located at the centromere of all the chromosomes and the distal long arm of the Y chromosome. In this study, we aimed to detect the structural changes in chromosomes during the stages of C-banding by atomic force microscopy. We observed crater-like structures in the chromosomes after 2xSSC (saline sodium citrate) treatment and measured the relative difference between the heights of chromatid and centromere of the chromosomes. Results showed that the relative difference was 3 nm in chromosomes 1, 9, 16, and Y, whereas in the other chromosomes this value was 11.6 nm. After Giemsa staining, the relative difference increased by a factor of 16 in chromosomes 1, 9, 16, and Y. The other chromosomes showed no such increase, which is in accordance with our suggestion that nonhiston proteins associated with DNA in constitutive heterochromatin can make the constitutive heterochromatin resistant to C-banding.

The mechanism of G-banding detected by atomic force microscopy.

The morphologic changes occurring in human chromosomes during G-banding by trypsin treatment on the same metaphase were followed with the aid of an atomic force microscope (AFM). It was found that trypsin treatment alone caused a pattern of collapse in the chromosomes that was clearly dependent on the duration of trypsinization. The progressive pattern of collapse first indicated the loss of internal differentiation between chromatids, then bands, and finally all internal structures, except for edges running around the chromosomes' perimeter. When stained with Giemsa, the collapsed chromosomes partly regained their original form, and transverse ridges appeared that correspond to G-positive band regions. However, the treatment of fixed chromosomes with trypsin for 42 s diminished the chromosomal edges, and the z-dimensions could not be measured even with the subsequent application of Giemsa.

Data on the PCR Turkish population based loci: LDLR, GYPA, HBGG, D7S8, and Gc.

Allele and genotype frequencies for the five PCR-based loci were analyzed in 157 unrelated Turkish individuals. The five PCR-based loci included LDLR, GYPA, HBGG, D7S8, and Gc. The results of the chi-square and exact tests showed that the genotype distribution at the LDLR, GYPA, D7S8, and Gc loci did not significantly differ from the Hardy-Weinberg Expectation (HWE). However, the genotype distribution at the HBGG locus did not conform to HWE. Moreover, the genotype frequencies calculated in this study were compared with the published genotype frequencies of US African American and US Caucasian populations. The Turkish population was significantly different at the HBGG locus from the US Caucasian population. However, there were highly significant differences at the LDLR, HBGG, and Gc loci between the Turkish and African American populations.

Numerical chromosomal abnormalities detected by atomic force microscopy.

The numerical abnormalities of human metaphase chromosomes, fixed according to standard procedures for optical microscopy but not treated for banding, were detected by atomic force microscopy (AFM). High-resolution AFM imaging of chromosomes in trisomy 13, 21, and Klinefelter syndrome can be compared directly with the traditional optical image. The unbanded metaphase chromosomes, including the extra ones in trisomic patients showed a structural pattern very similar to G-banding. Comparison of AFM images with light microscopic data allows the identification of specific chromosomes, and images of chromosomes showing numerical and structural abnormalities can then be analysed.

Interactions of DNA with fluorescent dyes: by scanning tunneling microscopy.

Genomic DNA was obtained from peripheral blood samples of healthy volunteers and interacted with two fluorescent dyes (i.e. Hoechst 33,258 and ethidium bromide) in aqueous media. These media containing DNA-dye complexes deposited on the gold coated mica surfaces. Then, STM images were obtained in which the STM was operated in air at atmospheric pressure with a tip-to-substrate bias voltage of 250-1000 mV (sample positive) and the tunneling currents in the range of 10-20 pA by using etched tips of Pt/Ir, in constant current mode. Both dyes from molecular clusters on DNA. While, the Hoechst molecules were observed on the DNA chains at regular distances, the ethidium bromide molecular clusters did not.

Detection and typing of human papillomavirus DNAs by restriction endonuclease mapping of the PCR products.

Human papillomavirus (HPV) has been implicated strongly with human cervical, anal and penile cancers. The polymerase chain reaction (PCR) was used to detect HPV in cervical specimens of 88 women working at the public whorehouse. Using consensus primers which encode the L1 region of the HPV genome, the presence of HPV DNA was demonstrated in 2 specimens. Restriction endonuclease digestion of the amplified products was carried out for accurate typing. Samples which were positive by L1 PCR were digested with Hae III, BstN I and Dde I restriction enzymes. The patterns produced by digestion were identified as HPV types 6b and 16.

Serum beta-carotene, vitamin E, vitamin C and malondialdehyde levels in several types of cancer.

We measured the levels of serum carotenoids (beta-carotene), total tocopherol (vitamin E), ascorbic acid and malondialdehyde (MDA) in newly diagnosed cancer cases. Levels of the antioxidants and MDA in serum samples from 208 subjects with cancer affecting different sites (59 breast, 38 head and neck, 46 genitourinary, 12 lung, 20 gastrointestinal and 33 other sites) were compared with levels in 156 controls. Cases and controls were compared with respect to a number of potentially confounding factors: age, sex, smoking status, Quetelet index (kg/m2), diet and alcohol intake. Mean (+/- SD) levels of beta-carotene, vitamin E and vitamin C were significantly lower among the cases than the controls (49.35 +/- 36.55 micrograms/l, 0.60 +/- 0.14 mg/dl, 0.40 +/- 0.27 mg/dl and 75.31 +/- 28.59 mg/dl, 0.98 +/- 0.13 mg/dl, 0.88 +/- 0.47 mg/dl, respectively) (P < 0.05). On the other hand, mean levels of MDA were significantly higher among the cases than the controls (6.79 +/- 1.22 nmol/ml and 3.52 +/- 0.97 nmol/ml, respectively) (P < 0.05). The results obtained suggest that measurement of serum antioxidants and MDA levels may provide further useful information when evaluating cancer patients.

Sister-chromatid exchange inducing effect of smokeless tobacco using on T-lymphocyte chromosomes.

A kind of a smokeless tobacco (Maras powder) is widely used instead of cigarettes in the South Eastern region of Turkey. In this study we investigated the sister-chromatid exchange (SCE) inducing effect of this powder on the chromosomes of its users compared with smokers and nonsmokers using standard cell culture methods and SCE staining techniques. Average SCE per metaphase and total SCEs increased significantly among both smokeless tobacco users and smokers compared to nonsmokers (p < 0.01). However, the effect is significantly lower in smokeless tobacco users than in smokers (p < 0.05).

A chemical modification approach to the olfactory code: vapor phase labeling using photoaffinity odorants.

A photoaffinity labeling technique was used to study the receptors involved in the discrimination of odorants. Aromatic azides, 1-azidonaphthalene (AzN) and 1-azido-4-nitronaphthalene (AsNN), were found to be pleasant-smelling compounds and produced good responses, giving standard EOG's (electro-olfactogram) of the kind observed for normal odorants. Following irradiation of the frog olfactory mucosa with light during constant stimulation with one of the azides vapor, there was a specific partial inhibition of the receptors for that odorant. The extent of reduction in amplitude of the EOG responses to AzN and AzNN varied between 40 to 60% of the original amplitude.

[Salmonella tester strain TA104 for the detection of mutagenic and carcinogenic chemicals in our environment]. / Cevremizdeki mutajenik ve karsinojenik maddelerin Salmonella typhimurium TA104 susu ile saptanmasi.

We used the Salmonella mutagenicity test for detecting chemical carcinogens as mutagens in the Salmonella typhimurium tester strain TA104 . The mutagenicity of several compounds was assessed by induction of histidine revertants in the TA104 . In each experiment we routinely included positive mutagenesis controls using three different concentrations of known mutagens. The mutagenic chemicals such as sodium azide, hydrogen peroxide and hydroxylamine were found to be mutagenic to TA104 at very low concentration (10(-4) mg/ml). Their mutagenic activity decreased while their concentrations were increased. The effect of acridine orange, 2, 4, 6-trinitrobenzene sulphonic acid, 2- phenylnaphthalene and 20- methylcholanthrene were also found to be mutagenic to TA104 at the concentration of 10(-2) mg/ml. The mutagenicity of other materials such as hair dyes, meat- broth preparations+ and cigarette smoke condensates were also tested, and all of them were found to be mutagenic to TA104 . The highest mutagenic activities were observed at the concentration of 10 mg/ml for two different hair dyes and of 1 mg/ml for cigarette smoke condensates.

A chemical-modification approach to the olfactory code. Studies with a thiol-specific reagent.

The effects of thiol-specific reagents on the amplitude of the electro-olfactogram (E.O.G.) responses elicited from frog olfactory mucosa by pulses of odorant vapours was studied. The impermeant thiol-specific reagent mersalyl [(3-{[2-(carboxymethoxy)-benzoyl]amino}-2-methoxypropyl)hydroxymercury monosodium salt] brings about a rapid decrease in the E.O.G. signal obtained with the odorant pentyl acetate. The extent of the decrease is proportional to the concentration of the mersalyl applied and the effect of the reagent is partially but incompletely reversed by treatment of the labelled mucosa with dithiothreitol. The sites labelled by mersalyl can be protected by pretreating the mucosa with a dilute solution of the odorant pentyl acetate and leaving the solution in contact with the tissue after the addition of mersalyl. When the protecting odorant is washed out of the tissue, the original E.O.G. amplitude is regained. Pentyl acetate applied to the mucosa protected the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: n-butyric acid, n-butyl acetate, phenylacetaldehyde and cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane). The pentyl acetate applied to the mucosa failed to protect the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: butan-1-ol, benzyl acetate, nitrobenzene, beta-ionone and linalyl acetate. The significance of the differential protection effects for the odour-quality-coding mechanism in the olfactory primary neurons is discussed. It is suggested that the olfactory code at this level of the olfactory system may be elucidated by chemical-modification methods.