Use of Rep‐PCR for Genetic Diversity Analyses in Fusarium culmorum

Fusarium culmorum is a pathogen of economically important grain crops. In this work, Rep‐PCR was used to identify genetic diversity in F. culmorum isolates which have been collected from wheat fields in Turkey. Reproducible genomic fingerprints were amplified in each strain by PCRs of prokaryotic repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC) and BOX sequences. Totally 104 molecular markers were evaluated and similarity comparisons were shown as a dendrogram. The average genetic diversity was 52.3% ranging from 15.8% to 88.7% according to the Rep‐PCR data. Cluster analysis showed agreement with the distance of sampling locations. The highest genetic similarity (84.2%) was determined between two F. culmorum isolates (F1 and F2) originated from the same agro‐ecological region. Our results showed that Rep‐PCR is convenient and rapid for genetic diversity analyses and strain differentiation in F. culmorum.     

Hexokinase cellular trafficking in ischemia–reperfusion and ischemic preconditioning is altered in type I diabetic heart

Diabetes mellitus (DM) has been reported to alter the cardiac response to ischemia–reperfusion (IR). In addition, cardioprotection induced by ischemic preconditioning (IPC) is often impaired in diabetes. We have previously shown that the subcellular localisation of the glycolytic enzyme hexokinase (HK) is causally related to IR injury and IPC protective potential. Especially the binding of HK to mitochondria and prevention of HK solubilisation (HK detachment from mitochondria) during ischemia confers cardioprotection. It is unknown whether diabetes affects HK localisation during IR and IPC as compared to non-diabetes. In this study we hypothesize that DM alters cellular trafficking of hexokinase in response to IR and IPC, possibly explaining the altered response to IR and IPC in diabetic heart. Control (CON) and type I diabetic (DM) rat hearts (65 mg/kg streptozotocin, 4 weeks) were isolated and perfused in Langendorff-mode and subjected to 35 min I and 30 min R with or without IPC (3 times 5 min I). Cytosolic and mitochondrial fractions were obtained at (1) baseline, i.e. after IPC but before I, (2) 35 min I, (3) 5 min R and (4) 30 min R. DM improved rate-pressure product recovery (RPP; 71 ± 10 % baseline (DM) versus 9 ± 1 % baseline (CON) and decreased contracture (end-diastolic pressure: 24 ± 8 mmHg (DM) vs 77 ± 4 mmHg (CON)) after IR as compared to control, and was associated with prevention of HK solubilisation at 35 min I. IPC improved cardiac function in CON but not in DM hearts. IPC in CON prevented HK solubilisation at 35 min I and at 5 min R, with a trend for increased mitochondrial HK. In contrast, the non-effective IPC in DM was associated with solubilisation of HK and decreased mitochondrial HK at early reperfusion and a reciprocal behaviour at late reperfusion. We conclude that type I DM significantly altered cellular HK translocation patterns in the heart in response to IR and IPC, possibly explaining altered response to IR and IPC in diabetes.     

A two-stage pretreatment of seedlings improves adventitious shoot regeneration in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The effects of a two-stage pretreatment of seedlings on the subsequent shoot regeneration capacity were investigated. Pretreated seedlings were obtained by germinating seeds on three different germination media and then further culturing on six different growth media. Lamina and petiole explants of two sugar beet (Beta vulgaris L.) breeding lines were then excised from the pretreated seedlings and cultured on five different shoot regeneration media. In both breeding lines, petiole explants produced significantly more shoots than lamina explants with higher frequencies of organogenic capacities; petiole explants of the lines M1195 and ELK345 produced a mean of 2.1 and 2.7 shoots per explant while their lamina explants produced 1.5 and 2.2 shoots per explant, respectively. A genotypic variation was evident as the line ELK345 was more productive for shoot development from both types of explants. In overall comparisons of different germination, growth and regeneration media, germination medium was most effective when supplemented with 0.5 mg/l 6-benzyladenine (BA) while both growth and regeneration media were most productive when contained a combination of 0.25 mg/l BA and 0.10 mg/l indole-3-butyric acid (IBA). Of all the treatments tested, the highest mean number of shoots per explant (8.3 shoots) and frequency of organogenic explants (75.6%) were obtained on regeneration medium supplemented with 0.25 mg/l BA and 0.10 mg/l IBA when petiole explants of the line ELK345 were excised from the seedlings that had been germinated on medium containing 0.5 mg/l BA followed by further growth on medium containing 0.25 mg/l BA and 0.10 mg/l IBA.     

Regeneration and cardiotonic glycoside production in <Emphasis Type="Italic">Digitalis davisiana</Emphasis> Heywood (Alanya Foxglove)

A high-frequency in vitro regeneration of Digitalis davisiana Heywood (Alanya foxglove) and cardiotonic glycoside production from both in vitro produced materials (regenerated plantlets or germinated seedlings) and leaves of natural populations were obtained. Cardiac glycosides regulate heart rhythm and are effective in cancer chemotherapy, in particular for prostate and breast cancer treatments. Testing six different types of culture media revealed that Linsmaier and Skoog (LS) was the most effective for shoot production. Shoot regeneration efficiency was higher when flamingo-bill or hypocotyl explants were cultured on LS medium containing 0.5 mg/l thidiazuron (TDZ) and 0.25 mg/l indoleacetic acid (IAA). Rooting of all shoots that regenerated was readily achieved, even in the absence of plant growth regulators (PGRs). Production of cardenolides (lanatoside C and digoxin) in the materials grown in vitro seemed to correlate with several parameters, such as nutritional and hormonal compositions of the culture medium as well as the duration of culture on the initial regeneration and/or final growth medium. Higher amounts of digoxin accumulation were obtained when shoots were regenerated on LS or Gamborg’s B5 medium containing 0.5 mg/l TDZ and 0.25 mg/l IAA, producing 12.59 and 11.93 mg/kg dry weight (dw) digoxin, respectively. For natural populations, seasonal variations seemed to affect the production of digoxin in the leaves. The highest amount of digoxin (246.58 mg/kg dw) was in leaf samples collected in July, which coincides with the flowering stage of the plant in the region of collection.     

Functional polymorphisms of cyclooxygenase-2 gene and risk for hepatocellular carcinoma

Cyclooxygenase-2 (COX-2) influences carcinogenesis through immune response suppression, apoptosis inhibition, regulation of angiogenesis and tumor cell invasion, and metastasis. It is now well established that COX-2 is overexpressed in many premalignant, malignant, and metastatic cancers, including hepatocellular carcinoma (HCC). DNA sequence variations in the COX-2 gene may lead to altered COX-2 production and/or activity, and so they cause inter-individual differences in the susceptibility to HCC. Functional coding region polymorphisms -1195A>G (rs689466), -765G>C (rs20417), and +8473T>C (rs5275) in the COX-2 gene have recently been shown to be associated with several human cancers but their association with HCC has yet to be investigated. We used hospital-based case-control study to assess the hypothesis that the functional COX-2 variation may affect individual susceptibility to the HCC. COX-2 polymorphisms were investigated in 129 confirmed subjects with HCC and 129 cancer-free control subjects matched on age, gender, smoking, and alcohol consumption using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The distribution of the COX-2 -1195A>G and +8473T>C genotypes were not significantly different between HCC cases and control. However, proportion of the COX-2 -765CC genotype which leads to a 30% reduction of the COX-2 promoter activity was significantly lower in patients with HCC (3.1%) when compared to control subjects (11.6%) (P < 0.05). Logistic regression analyses revealed that the COX-2 -765G>C variant genotype (-765CC) was associated with a significantly decreased risk of HCC compared with the -765GG wild-type homozygotes [P < 0.05, odds ratio (OR) = 0.25, 95% confidence interval (CI) = 0.08-0.79]. Our results suggest for the first time that the -765CC genotype of COX-2 -765G>C polymorphism, causing lower COX-2 gen expression, is a genetic protective factor for HCC. However, because this is the first report concerning the COX-2 -1195A>G, -765G>C, and +8473T>C polymorphisms and the risk of HCC, independent studies are needed to validate our findings.     

Changing pattern of antibiotic susceptibility in intensive care units: ten years experience of a university hospital

The study was performed to assess microorganisms and antibiotic susceptibility patterns during ten years in intensive care units of a University Hospital. Infection Control Committee has active, prospective surveillance in ICUs for thirteen years. Ten years data of ICUs was evaluated retrospectively from surveillance forms. Microorganisms and their antibiotic resistance were recorded according to the years. During ten years, gram negative microorganisms were the most frequent isolated microorganisms from clinical specimens. Acinetobacter baumannii (21.8%), Pseudomonas aerigunosa (16%), Escherichia coli (10.4%) and Klebsiella pneumoniae (8%) were the most common gram negative microorganisms. However, Staphylococcus aureus was the most prevalent gram positive microorganism, the incidence decreased from 18.6% to 4.8% during ten years. Also antibiotic susceptibility of microorganisms changed during ten years. Carbapenem resistance increased from 44% to 92% in A. baumannii and ciprofloxacin resistance increased in E. coli from 28% to 60% and in K. pneumoniae from 21% to 55% during ten years. However, methicilin resistance decreased in S. aureus from 96% to 54%. In conclusion, antibiotic resistance is growing problem in ICUs. Rationale antibiotic policies and infection control measures will prevent the development of resistance.     

MDR1 gene polymorphisms may be associated with Behçet's disease and its colchicum treatment response

Behçet's disease (BD) is a chronic multisystem disorder. Infectious agents, immune system mechanisms, and genetic factors are implicated in the etiopathogenesis of BD, which remains to be explained. The human MDR1 (ABCB1) gene encoder P-glycoprotein (P-gp) plays a key role in drug disposition, serves as a protective mechanism against xenobiotics, and provides additional protection for the brain, testis, and fetus. We investigated the genotype and haplotype distributions of three MDR1 gene polymorphisms (C1236T, G2677T/A, and C3435T) in 104 BD patients and 130 control subjects. The genotyping analysis was performed by using PCR–RFLP methods.     

Lack of an association of programmed cell death-1 PD1.3 polymorphism with risk of hepatocellular carcinoma susceptibility in Turkish population: A case–control study

Aim

The programmed cell death-1 (PD-1) is a potent immunoregulatory molecule which is responsible for the negative regulation of T-cell activation and peripheral tolerance. Recently, overexpression of PD-1 has been reported to contribute to immune system evasion and poor survival of hepatocellular carcinoma (HCC). A common single nucleotide polymorphism in intron 4 of PD-1 gene called PD-1.3 has been reported to influence PD-1 expression, but its association with HCC has yet to be investigated. The aim of the present study was to investigate whether this polymorphism could be involved in the risk of HCC susceptibility.

Methods

The genotype frequency of PD-1.3 polymorphism was determined by using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method in 236 subjects with HCC and 236 cancer-free control subjects matched on age, gender, smoking and alcohol status.

Results

No statistically significant differences were found in the genotype distributions of the PD-1.3 polymorphism among HCC and cancer-free control subjects (P = 0.22).

Conclusion

Our results demonstrate for the first time that the PD-1.3 polymorphism has not been in any major role in genetic susceptibility to hepatocellular carcinogenesis, at least in the population studied here. Independent studies are needed to validate our findings in a larger series, as well as in patients of different ethnic origins.     

Neuroprotective effects of Caffeic acid phenethyl ester on experimental traumatic brain injury in rats

The aim of this study was to evaluate the therapeutic efficacy of caffeic acid phenethyl ester (CAPE) with an experimental traumatic brain injury (TBI) model in rats. Twenty-four adult male Sprague–Dawley rats were randomly divided into three groups of 8 rats each: control, TBI, and TBI + CAPE treatment. In TBI and TBI + CAPE treatment groups, a cranial impact was delivered to the skull from a height of 7 cm at a point just in front of the coronal suture and over the right hemisphere. Rats were sacrificed at 4 h after the onset of injury. Brain tissues were removed for biochemical and histopathological investigation. To date, no biochemical and histopathological changes of neurodegeneration in the frontal cortex after TBI in rats by CAPE treatment have been reported. The TBI significantly increased tissue malondialdehyde (MDA) levels, and significantly decreased tissue superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, but not tissue catalase (CAT) activity, when compared with controls. The administration of a single dose of CAPE (10 μmol/kg) 15 min after the trauma has shown protective effect via decreasing significantly the elevated MDA levels and also significantly increasing the reduced antioxidant enzyme (SOD and GPx) activities, except CAT activity. In the TBI group, severe degenerative changes, shrunken cytoplasma and extensively dark picnotic nuclei in neurons, as well as vacuolization indicating tissue edema formation. The morphology of neurons in the CAPE treatment group was well protected. The number of neurons in the trauma alone group was significantly less than that of both the control and TBI +CAPE treatment groups. The caspase 3 immunopositivity was increased in degenerating neurons of the traumatic brain tissue. Treatment of CAPE markedly reduced the immunoreactivity of degenerating neurons. TBI caused severe degenerative changes, shrunken cytoplasma, severely dilated cisternae of endoplasmic reticulum, markedly swollen mitochondria with degenerated cristae and nuclear membrane breakdown with chromatin disorganization in neurons of the frontal cortex. In conclusion, the CAPE treatment might be beneficial in preventing trauma-induced oxidative brain tissue damage, thus showing potential for clinical implications. We believe that further preclinical research into the utility of CAPE may indicate its usefulness as a potential treatment on neurodegeneration after TBI in rats.     

Tuning Optical Discs for Plasmonic Applications

We present simple physical and chemical procedures that allow tuning and modification of the topography of gratings present in optical storage discs into geometries optimal for grating coupled plasmon resonance excitation. After proper metal coating, the tuned surfaces exhibit sharp plasmon resonances that can be excited at wavelengths ranging from 260 nm to over 2.7 μm with relatively high quality factors. As an immediate exemplary application, use of such optimized gratings in aqueous medium for refractive index measurement is demonstrated.