Anatomy, palynology and nutlet micromorphology of Turkish endemic Teucrium sandrasicum (Lamiaceae)

In this study, the anatomical features of the leaf and stem, besides the pollen and nutlet characteristics of Teucrium sandrasicum are investigated. T. sandrasicum, belonging to sect. Teucrium, is an endemic perennial herb growing on serpentine around Muğla province. The anatomical studies on T. sandrasicum revealed that the stem shares the general characteristics of the Labiatae family. The leaves clearly exhibit xeromorphy due to features such as the distribution of stomata on the lower surface (hipostomatic), the occurrence of guard cells below the epidermis (xeromorphic type), inrolled margins, thick cuticle layer, thick outer epidermal cell wall, a high density of trichomes and thick palisade layer of the mesophyll. The anatomical studies showed that the upper epidermal cells of the leaf include many spherocrystals. The pollen grains are prolate, medium in size, 3-colpate with verrucate ornamentation. The nutlets are ellipsoid with a reticulate-verrucate surface. The results have proven that T. sandrasicum is different from the other species of the sect. Teucrium because of the branched trichomes on the stem and the lack of eglandular trichomes on the nutlets.     

Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer

The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new opportunities for noninvasive prenatal diagnosis (NIPD). However, the extremely low levels of cfDNA within a high background of the maternal DNA in maternal circulation necessitate highly sensitive molecular techniques for its reliable use in NIPD. In this proof of principle study, we evaluated the earliest possible detection of cfDNA in the maternal plasma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, amplification, magnetics). Blood samples were collected from in vitro fertilization (IVF) patients at 2 to 6 weeks following embryo transfer (i.e., 4 to 8 week pregnancies) and plasma DNA was extracted. The genomic regions of both X and Y chromosome-specific sequences (AMELX and AMELY) were concurrently amplified in two sequential PCRs; first by conventional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene sequences were counted by a flow cytometer. Our results showed that the pregnancies yielding boys had significantly higher plasma AMELY gene fractions (0.512 ± 0.221) than the ones yielding girls (0.028 ± 0.003) or non-pregnant women (0.020 ± 0.005, P= 0.0059). Here, we clearly demonstrated that the BEAMing technique is capable of reliably detecting cfDNA in the blood circulation of 4-week-pregnant women, which is only two weeks after the embryo transfer. BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.     

The Role of Epigenetic Regulation and Pluripotency‐Related MicroRNAs in Differentiation of Pancreatic Stem Cells to Beta Cells

In this study, we aimed to research the effects of class‐I HDACs and glucose on differentiation of pancreatic islet derived mesenchymal stem cells (PI‐MSCs) to beta cells. Beta cell differentiation determined by flow cytometric analysis and gene expression levels of PDX1, PAX4, PAX6, NKX6.1, NGN3, INS2, and GLUT2. As a result the valproic acid, is an inhibitor of class‐I HDACs, caused the highest beta cell differentiation in PI‐MSCs. However, the cells in this group were at early stages of differentiation. Glucose co‐administration to this group carried the differentiation to higher levels, but these newly formed beta cells were not functional. Moreover, reduction in the levels of pluripotency factors that Oct3/4, c‐Myc, and Nanog were parallel to beta cell differentiation. Also, the levels of HDAC1 and acetylated H3/H4 were increased and methylated H3 was decreased by VPA treatment. In addition, we have detected over expression in genes of miR‐18a‐5p, miR‐19b‐5p, miR‐30d‐3p, miR‐124, miR‐146a‐5p, miR‐184, miR‐335, and miR‐433‐5p in parallel to beta cell differentiation. As the conclusion, this study is important for understanding the epigenetic mechanism that controls the beta cell differentation and it suggests new molecules that can be used for diagnosis, and treatment of diabetes. J. Cell. Biochem. 119: 455–467, 2018. © 2017 Wiley Periodicals, Inc.     

Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish

Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82 % similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.     

Phototherapy increases DNA damage in lymphocytes of hyperbilirubinemic neonates

Phototherapy is commonly used in the treatment of hyperbilirubinemia in newborns. No serious side effects related to phototherapy have been observed, but concerns regarding its potential to damage DNA have been expressed, based on animal or cell-culture studies. The aim of this study was to investigate, in neonates with hyperbilirubinemia, the possible relation between phototherapy and DNA damage. The study included 33 full-term newborns with non-physiological jaundice and 14 healthy newborns with physiological jaundice as controls. Phototherapy was performed with an array of six fluorescent lamps producing radiation with wavelengths of 480–520 nm at 12 μW/cm²/nm. DNA damage in lymphocytes was determined by use of the alkaline comet assay. The DNA damage increased significantly with the duration of phototherapy, as shown by measurements at 24, 48, and 72 h (P < 0.001). These findings indicate that phototherapy, widely used in neonatology units, increases DNA damage in newborns. It remains to be seen whether the genotoxic effect observed in the present study can cause any long-term health effect in phototherapy-treated infants in later life.     

Pregnenolone protects the PC-12 cell line against amyloid beta peptide toxicity but its sulfate ester does not

Pregnenolone (P), the main precursor of the steroids, and its sulfate ester, pregnenolone sulfate (PS), are the major neurosteroids produced in the neural tissue. Many neuroendocrinological studies stressed the neuroprotective role of neurosteroids although it has been suggested that the inhibition of P and PS synthesis can delay neuronal cell death. The potential roles of P and PS in vital neuronal functions and in amyloid beta peptide (Aβ) toxicity are not clearly identified. This work aims to investigate the effects of P and PS on cell viability and Aβ peptide toxicity in a concentration and exposure time-dependent manner in rat PC-12 cells. The cells were treated with 20 μM Aβ peptide 25-35 and variable concentrations of P and PS ranging from 0.5 μM to 100 μM. To examine the effects of steroid treatment on Aβ peptide toxicity, 0.5 μM (low) and 50 μM (high) neurosteroids were used. The cell viability and lactate dehydrogenase release of cells were evaluated after 24, 48 and 72 h. Morphological changes of cells were also examined. The treatment with higher than 1 μM concentrations of P and PS significantly decreased the cell viability comparing to untreated cells. At lower concentrations, P and PS had no toxic actions until 72 h. The Aβ treatment resulted in a significant decrease in cell viability comparing to untreated cells. P showed a dose-dependent protective effect against Aβ peptide in PC-12 cells. But its sulfate ester did not have the same effect on Aβ peptide toxicity, even it significantly decreased cell viability in Aβ-treated cells. Consequently, the discrepant effects of P and PS on Aβ peptide toxicity may provide insight on the pathogenesis of Alzheimer’s disease.     

The effect of pretreatment or combined treatment of quercetin on menadione toxicity in rat primary mixed glial cells in vitro

Neurons and glia are highly susceptible to reactive oxygen species that play a key role in various neurodegenerative diseases. Menadione, a synthetic derivative of vitamin K, induces reactive oxygen generation. Quercetin one of the most ubiquitous bioflavonoids in food of plant origin, has strong antioxidant activities on different cell types, however recent studies demonstrated that it has also prooxidant and cytotoxic potentials. We examined the action of pre- and co-treatment of quercetin on menadione induced glial toxicity. The primary mixed glial cells obtained from 1 to 3 day old rat brain were pretreated with 10, 25, 100 or 250 μM quercetin for 1 h, washed out and 10, 25, 50, 75 or 100 μM menadione was added for 6 h. The other group of cells was treated with respective doses of quercetin combined simultaneously with the same doses of menadione for 6 h. The cells were washed and incubated for additional 24 h for recovery period and the viability was measured by using MTT assay. Menadione was dose-dependently toxic to glia cells and pretreatment with respective quercetin doses for 1 h could not eliminate this toxicity. Although 10 and 25 μM quercetin combined with 10 and 25 μM menadione could not change, 100 and 250 μM quercetin together with 10 or 25 μM menadione for 6 h increased further the menadione induced toxicity. We conclude that when combined with menadione, quercetin at high doses could be toxic to primary rat glia cells in culture.     

Genetic Variation on Chromosome 6 Influences F Cell Levels in Healthy Individuals of African Descent and HbF Levels in Sickle Cell Patients

Fetal haemoglobin (HbF) is a major ameliorating factor in sickle cell disease. We investigated if a quantitative trait locus on chromosome 6q23 was significantly associated with HbF and F cell levels in individuals of African descent. Single nucleotide polymorphisms (SNPs) in a 24-kb intergenic region, 33-kb upstream of the HBS1L gene and 80-kb upstream of the MYB gene, were typed in 177 healthy Afro-Caribbean subjects (AC) of approximately 7% European admixture, 631 healthy Afro-Germans (AG, a group of African and German descendents located in rural Jamaica with about 20% European admixture), 87 West African and Afro-Caribbean individuals with sickle cell anaemia (HbSS), as well as 75 Northern Europeans, which served as a contrasting population. Association with a tag SNP for the locus was detected in all four groups (AC, P = 0.005, AG, P = 0.002, HbSS patients, P = 0.019, Europeans, P = 1.5×10⁻⁷). The association signal varied across the interval in the African-descended groups, while it is more uniform in Europeans. The 6q QTL for HbF traits is present in populations of African origin and is also acting in sickle cell anaemia patients. We have started to distinguish effects originating from European and African ancestral populations in our admixed study populations.