A Novel Method for Separation of Somatic Embryos from Embryogenic Suspension Cultures by Cold Treatment

This study reports a novel method for embryo separation by cold treatment of heterogeneous suspension cultures which contain embryogenic single cells, cell clusters and embryos at various stages of development. The method was applied to embryo suspension cultures of pepper (Capsicum annuum) and sugarcane (Saccharum officinarum). In both plants, single cells lost their viability dramatically over a few days while the viability of embryos remained above 95% for 25-30 days when kept at 5 °C. The effect of duration of cold treatment on embryo germination was also tested. The optimal duration of cold treatment was found to be 10 days for sugarcane and 21 days for pepper. After the cold treatment, the germination percentages were 90% and 96% for sugarcane and pepper, respectively.     

Comprehensive Analysis of Deafness Genes in Families with Autosomal Recessive Nonsyndromic Hearing Loss

Comprehensive genetic testing has the potential to become the standard of care for individuals with hearing loss. In this study, we investigated the genetic etiology of autosomal recessive nonsyndromic hearing loss (ARNSHL) in a Turkish cohort including individuals with cochlear implant, who had a pedigree suggestive of an autosomal recessive inheritance. A workflow including prescreening of GJB2 and a targeted next generation sequencing panel (Illumına TruSight^TM Exome) covering 2761 genes that we briefly called as mendelian exome sequencing was used. This panel includes 102 deafness genes and a number of genes causing Mendelian disorders. Using this approach, we identified causative variants in 21 of 29 families. Three different GJB2 variants were present in seven families. Remaining 14 families had 15 different variants in other known NSHL genes (MYO7A, MYO15A, MARVELD2, TMIE, DFNB31, LOXHD1, GPSM2, TMC1, USH1G, CDH23). Of these variants, eight are novel. Mutation detection rate of our workflow is 72.4%, confirming the usefulness of targeted sequencing approach in NSHL.     

Synthesis,structural characterization and cell death-inducing effect of novel palladium(II) and platinum(II) saccharinate complexes with 2-(hydroxymethyl)pyridine and 2-(2-hydroxyethyl)pyridine on cancer cells in vitro

Four palladium(II) and platinum(II) saccharinate (sac) complexes with 2-(hydroxymethyl)pyridine (2-hmpy) and 2-(2-hydroxyethyl)pyridine (2-hepy), namely trans-[Pd(2-hmpy)₂(sac)₂]·H₂O (1), trans-[Pt(2-hmpy)₂(sac)₂]·3H₂O (2), trans-[Pd(2-hepy)₂(sac)₂] (3) and trans-[Pt(2-hepy)₂(sac)₂] (4), have been synthesized and characterized by elemental analysis, UV–vis, IR and NMR. Single crystal X-ray analysis reveals that the metal(II) ions in each complex are coordinated by two sac and two 2-hmpy or 2-hepy ligands with a trans arrangement. Anticancer effects of 1–4 were tested against four different cancer cell lines (A549 and PC3 for lung cancer, C6 for glioblastoma, and Hep3B for liver cancer). Cytotoxicity was first screened by the MTT assay and the results were further confirmed by the ATP assay. The mode of cell death was determined by both histological and biochemical methods. Among the metal complexes, complex 2 resulted in relatively stronger anti-growth effect in a dose-dependent manner (3.13–200 μM), compared to the others, by inducing apoptosis.     

Molecular karyotyping of an isolated partial trisomy 11q patient with additional findings

Isolated partial duplication of the long arm of chromosome 11 is very rare. The main features are dysmorphic facial features, pre/postnatal growth retardation, speech delay, mental retardation, hypotonia, microcephaly, and cardiac, vertebral, limb and genital anomalies. In this case, we report a patient with partial trisomy of 11q13.5 → qter due to a de novo rearrangement consisting of the whole X chromosome and part of chromosome 11; 46,X,der(X)(Xqter → Xp22.33::11q13.5 → 11qter). Additional findings were a separated clavicle, lacrimal duct stenosis and prenatally detected renal hypoplasia. SNP array results revealed a duplication between 11q13.5 and 11qter, measuring 58 Mb, from nucleotide 76,601,607 to 134,926,021. As a result, molecular karyotyping could be performed in such cases in order to establish a definite phenotype–genotype correlation using conventional or molecular cytogenetics techniques.     

<Emphasis Type="Italic">MEFV</Emphasis> mutations in patients with familial Mediterranean fever from the Aegean region of Turkey

Familial Mediterranean Fever (FMF) which is frequently present in Mediterranean populations is caused by mutations in the MEFV gene. According to recent data, MEFV mutations are not the only cause of FMF, but these are major genetic determinants which cause FMF. It has also been suggested that there may be a number of other genes causing FMF. The MEFV gene is located at 16p13.3 and encodes a protein, pyrin/marenostrin. More than 70 disease associated mutations and totally 186 mutations and polymorphisms have been defined in affected individuals. We have retrospectively evaluated the molecular test results of 1,201 patients identified as having FMF clinical symptoms referred to the Molecular Genetics Laboratory of the Department of Medical Genetics, Faculty of Medicine, Ege University, Izmir/Turkey over the last 4 years. Patients were tested for 12 common mutations in the MEFV gene using a strip assay method (Innogenetics, Belgium). Out of the 1,201 patients tested (2,402 chromosomes) in the Aegean region in Turkey, 654 (54.45%) did not carry any mutations, among the 547 (45.55%) patients with mutations 246 patients were either homozygous (101) or compound heterozygous (145), 296 carried only one detected mutation, and five patients had three mutations. Allelic frequencies for the four most common mutations in the mutation positive groups were 47.60% (M694V), 16.75% (E148Q), 12.95% (V726A), 11.94% (M680I G/C).The remaining alleles (10.76%) showed rare mutations which were R761H, P369S, A744S, K695R, F479L, M694I. When the frequencies of mutations detected in our group were compared to the frequencies reported in the other regions of Turkey, an increase in V726A mutation frequency was observed. No patient showed a I692del mutation which is sometimes evident in other Mediterranean populations.     

Unilateral nephrectomy leads to up-regulation of syndecan-2- and TGF-beta-mediated glomerulosclerosis in syndecan-4 deficient male mice.

Syndecan-4 is an ubiquitous, plasma membrane-spanning heparan sulfate proteoglycan involved in proliferation, differentiation, adhesion and migration of cells in vitro. Syndecan-4 knockout (KO) mice show no obvious defects but respond abnormally to experimental stress conditions. In the adult, syndecan-4 is the most abundant syndecan of renal tissue. We therefore investigated the consequences of syndecan-4 deficiency during progression of kidney disease using unilaterally nephrectomized mice, a model of glomerular hyperfiltration and renal hypertrophy. 60 days after unilateral nephrectomy (UNX), mesangial expansion, enhanced matrix production (collagens I and IV, fibronectin) and focal segmental glomerulosclerosis, resembling early stages of diabetic nephropathy, was apparent in male but not female syndecan-4 KO mice. No defect was detected in wild type UNX males. Syndecan-2 mRNA and protein were not detectable in renal glomeruli of wild type mice, but were induced specifically in the glomeruli of the syndecan-4 deficient kidneys after unilateral nephrectomy. Due to the structural similarities of syndecans-2 and -4 we hypothesize that de novo-production of syndecan-2 in kidneys after unilateral nephrectomy reflects a compensatory response. However, this response is counterproductive since syndecan-2 supports the pro-sclerotic activity of TGF-beta1 which is increased in parallel with syndecan-2 synthesis. By contrast, signaling through syndecan-4 negatively controls the production of pro-sclerotic TGF-beta1.     

Palladium(II) saccharinate complexes with bis(2-pyridylmethyl)amine induce cell death by apoptosis in human breast cancer cells in vitro

The outcomes of breast cancer patients are still poor although new compounds have recently been introduced into the clinic. Therefore, novel chemical approaches are required. In the present study, palladium(II) and corresponding platinum(II) complexes containing bis(2-pyridylmethyl)amine (bpma) and saccharine were synthesized and tested against human breast cancer cell lines, MCF-7 and MDA-MB-231, in vitro. Cytotoxicity was first screened by the MTT assay and the results were further confirmed by the ATP assay. The palladium complexes 1 and 3 yielded stronger cytotoxicity than the corresponding platinum complexes 2 and 4 at the same doses. The palladium complex 3 was found to be the most cytotoxic one. Therefore, a more comprehensive study was carried out with this complex only. The mode of cell death was determined morphologically under fluorescent microscope and biochemically with detection of active caspase-3 and PARP cleavage by Western blot. Changes in apoptosis-related gene expressions were measured with qPCR. It was demonstrated that complex 3 caused cell death by apoptosis determined by fluorescence imaging and Western blot. As a sign of apoptosis, PARP was cleaved in both of the cell lines. In addition, caspase-3 was cleaved in MDA-MB-231 cells while this cleavage was not observed in MCF-7. The results show that the complex 3 is a promising anti-cancer compound against breast cancer with an IC₅₀ value of 3.9 μM for MCF-7 and 4.2 μM for MDA-MB-231 cells, which warrants further animal experiments.     

Combined genetic mutations have remarkable effect on deep venous thrombosis and/or pulmonary embolism occurence

Purpose

Although deep vein thrombosis and thromboembolic diseases differ among various races, they are still important in our day. The difficulties in treatment and following-up of these diseases are caused by secret genetic mutations rather than predisposing factors.

Methods

Between January 2011 and May 2013, patients who were traced for deep vein thrombosis and/or pulmonary embolism were evaluated retrospectively. 84 patients (53.6% males and 46.4% females) were included in the study. Their family histories, predisposing factors and treatments were researched. Factor V Leiden (G 1691A), Factor II G20210A, Plasminogen Activator Inhibitor-Type 1 (4G/5G), and Methylene Tetrahydrofolate Reductase (C677T, A1298C) mutations were investigated from peripheral venous blood.

Results

Among the genetic mutations we searched, the incidence of single mutation rate was observed at 11.9%, double mutation collocation at 44%, triple mutation collocation at 29.8%, quadruple mutation collocation at 13.1%, and finally, quintuplet mutation collocation at 1.2%. Our approximate mutation number was found as 2.47 ± 0.91.

Conclusion

We observed that multiple mutations were high in number compared to single genetic mutations. The patients who have multiple mutations should be more in the front line considering their diagnosis, treatment and following up, and also in terms of decreasing mortality, morbidity and recurrence.     

Effectiveness of PET/CT with 18F-fluorothymidine in the staging of patients with squamous cell head and neck carcinomas before radiotherapy

Aim

The aim of our study was to compare the staging of the disease declared before anticancer treatment was begun with the staging that was found after the planning PET/CT scanning with ¹⁸F-FLT was performed.

Background

PET/CT in radiotherapy planning of head and neck cancers can facilitate the contouring of the primary tumour and the definition of metastatic lymph nodes.

Materials and methods

Between November 2010 and November 2013, 26 patients suffering from head and neck carcinomas underwent planning PET/CT examination with ¹⁸F-FLT. We compared the staging of the disease and the treatment strategy declared before and after ¹⁸F-FLT-PET/CT was performed.

Results

The findings from ¹⁸FLT-PET/CT led in 22 patients to a change of staging: in 19 patients it led to upstaging of the disease and in 3 patients it led to downstaging of the disease. In one patient, a secondary malignancy was found.

Conclusions

We have confirmed in this study that the use of ¹⁸F-FLT-PET/CT scanning in radiotherapy planning of squamous cell head and neck carcinomas has a great potential in the precise evaluation of disease staging and consequently in the precise determination of target volumes.