The Effect of Breast Milk from Different Lactation Stages on in Vitro Wound Healing.

Objective: Wound healing is a complex and dynamic process essential for restoring tissue integrity and homeostasis. It is thought that breast milk contributes positively to the wound healing process, thanks to the components it contains. The aim of this study is to compare the effects of breast milk on the wound healing process at different lactation stages and to evaluate the underlying mechanism(s). Materials and Methods: The effects of breast milk from different lactation stages (colostrum, transitional, and mature milk) on wound healing were determined by in vitro scratch assay in L929 fibroblast cells. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), total oxidant, and antioxidant capacity were used to confirm antioxidant effects. The effect of breast milk on netrin-1 levels in L929 cells was elucidated by ELISA. Results: Breast milk at different lactation stages promoted wound healing. While the wound closure percentage was determined as 48.7% in the control group, this rate was determined to be the highest at 81.6% in the mature milk group (p:0.0002). The free radical scavenging capacity of colostrum, transitional, and mature milk with DPPH was determined as 49.69%, 60.64%, and 80.85%, respectively, depending on the lactation stages. Netrin-1 levels detected by ELISA were determined as 490.1 ± 6.5 pg/mL in the control group, while the lowest level was determined as 376.6 ± 4.5 pg/mL in mature milk (p:0.0003). Conclusions: Breast milk, especially mature milk, promoted wound healing on L929 cells by suppressing netrin-1 levels and scavenging free radicals.

Determining Stable Structure and in Vitro Antiproliferative Properties of a Novel 3-(2-((4-Trifluoromethyl)phenyl)amino)thiazol-4-yl)-2H-chromen-2-one Molecule.

Since coumarin and thiazole derivatives are known to have antioxidant properties, a novel derivative was synthesized in this article. 3-(2-((4-(trifluoromethyl)phenyl)amino)thiazol-4-yl)-2H-chromen-2-one (ATC) was synthesized as a novel compound with high yield and characterized by Raman, FT-IR, 13 C-NMR, and 1 H-NMR spectroscopic procedures and DFT calculations. In this study, the potential in vitro antiproliferative properties of the ATC compound were evaluated on colorectal cancer (HT29) and melanoma (SK-MEL-30) cell lines. According to the results, the compound was found to be significantly active, approximately 2.6-fold, against melanoma cells compared to healthy fibroblast (L929) cells. Unlike melanoma cells, the compound did not have any adverse effects on colorectal cancer cells. Due to these findings, the compound can be harnessed as a promising antiproliferative drug candidate for preclinical studies against melanoma.

Green and eco-friendly biosynthesis of zinc oxide nanoparticles using Calendula officinalis flower extract: Wound healing potential and antioxidant activity.

This study aimed to produce zinc oxide nanoparticles with Calendula officinalis flower extract (Co-ZnO NPs) using the green synthesis method. In addition, the antioxidant and wound healing potential of synthesized ZnO NPs were evaluated. The absorbance band at 355 nm, which is typical for ZnO NPs, was determined from the UV-Vis absorbance spectrum. The energy-dispersive X-ray spectroscopy (EDS) measurements revealed a high zinc content of 42.90%. The x-ray diffractometer data showed Co-ZnO NPs with an average crystallite size of 17.66 nm. The Co-ZnO NPs did not have apparent cytotoxicity up to 10 µg/mL (IC50 25.96 µg/mL). C. officinalis ZnO NPs showed partial cell migration and percent wound closure (69.1%) compared with control (64.8%). In addition, antioxidant activities of Co-ZnO NPs with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2 diphenyl-1 picrylhydrazil (DPPH) were evaluated and radical scavenging activity of 33.49% and 46.63%, respectively, was determined. These results suggest that C. officinalis extract is an effective reducing agent for the green synthesis of ZnO NPs with significant antioxidant and wound healing potential.

Anticancer Effects of Vitis vinifera L. Mediated Biosynthesized Silver Nanoparticles and Cotreatment with 5 Fluorouracil on HT-29 Cell Line.

The aim of this study was to evaluate the anticancer effects of biosynthesized silver nanoparticles (Vv-AgNPs) from grape (Vitis vinifera L.) seed aqueous extract, alone or in combination with 5-Fluorouracil (5-FU) on HT-29 cell line. Vv-AgNPs were characterized by techniques such as UV-vis spectrophotometer (surface plasmon peak 454 nm), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). HT-29 cells were treated with different concentrations (0-80 µg/mL for MTT) and (0-20 µg/mL for BrdU) of Vv-AgNPs alone and combined with (200 µg/mL) 5-FU for 72 h. The cytotoxic effects were analyzed by [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay (IC50 values 13.74 and 5.35 µg/mL, respectively). Antiproliferative effects were examined 5-bromo-2'-deoxyuridine (BrdU) assay (IC50 values 9.65 and 5.00 µg/mL, respectively). Activation of caspase-3 and protein expression levels of p53 were determined by Western blotting analysis. It was observed that Vv-AgNPs significantly increased the cleavage of the proapoptotic proteins caspase 3 and obviously enhanced the expression of p53 in a dose-dependent manner. The increased amount of total oxidant status (TOS) in the 10 µg/mL Vv-AgNPs + 5-FU treatment group, despite the increasing amount of total antioxidant status (TAS), caused an increase in Oxidative Stress Index (OSI) compared to the control. In this study, it has been shown in vitro that the use of successfully biosynthesized Vv-AgNPs in combination with 5-FU exhibits synergistic cytotoxic, antiproliferative, apoptotic, and oxidative effects against HT-29 cell line.

The Role of Zinc Status on Spatial Memory, Hippocampal Synaptic Plasticity, and Insulin Signaling in icv-STZ-Induced Sporadic Alzheimer's-Like Disease in Rats.

Alzheimer's disease (AD), especially its sporadic form (sAD), is of multifactorial nature. Brain insulin resistance and disrupted zinc homeostasis are two key aspects of AD that remain to be elucidated. Here, we investigated the effects of dietary zinc deficiency and supplementation on memory, hippocampal synaptic plasticity, and insulin signaling in intracerebroventricular streptozotocin (icv-STZ)-induced sAD in rats. The memory performance was evaluated by Morris water maze. The expression of hippocampal protein and mRNA levels of targets related to synaptic plasticity and insulin pathway was assessed by Western blot and real-time quantitative PCR. We found memory deficits in icv-STZ rats, which were fully recovered by zinc supplementation. Western blot analysis revealed that icv-STZ treatment significantly reduced hippocampal PSD95 and p-GSK3ß, and zinc supplementation restored the normal protein levels. mRNA levels of BDNF, PSD95, SIRT1, GLUT4, insulin receptor, and ZnT3 were found to be reduced by icv-STZ and reestablished by zinc supplementation. Our data suggest that zinc supplementation improves cognitive deficits and rescues the decline in key molecular targets of synaptic plasticity and insulin signaling in hippocampus caused by icv-STZ induced sAD in rats.

Epithelial mesenchymal transition regulator TWIST1 transcription factor stimulates glucose uptake through upregulation of GLUT1, GLUT3, and GLUT12 in vitro.

TWIST1 is a major regulator of epithelial mesenchymal transition process, essential in cancer metastasis. Cancer cells increase glucose uptake capabilities to meet their high energy requirements. In this study, we explored the potential role of TWIST1 on glucose transport into the 293T cells in an insulin-dependent and insulin-independent manner. For this purpose, the ectopic expression of TWIST1 was successfully performed by electroporation. The altered mRNA expressions of GLUT-1, -3, -4, and -12, insulin receptor (InsR), and insulin receptor substrate (IRS)-1 and -2 were assessed in control and TWIST1-overexpressing cells. Glucose uptake rates of the cells were evaluated by fluorometric glucose uptake assay. Our findings showed that the transcriptional expression levels of GLUT-1, -3, and -12 genes were significantly upregulated by TWIST1. However, TWIST1 did not alter the mRNA and protein expressions of the InsR, its substrates (IRS-1 and -2), and GLUT-4 genes in 293T cells which are main factors for insulin-stimulated glucose uptake pathway. Also, the glucose transport activities were significantly increased in TWIST1-overexpressing cells compared to controls due to fetal bovine serum (FBS) stimulation, but there was a slight non-significant difference in insulin stimulation. Thus, our data suggest that TWIST1 could promote glucose uptake independently of insulin and is possible to be evaluated as a metabolic marker in cancer. Further investigations are needed to clarify the precise molecular mechanisms underlying the cells' glucose uptake and consumption during tumorigenesis.

Anti-proliferative and apoptotic effects of green synthesized silver nanoparticles using Lavandula angustifolia on human glioblastoma cells.

In this study, we aimed at the green synthesis of silver nanoparticles (AgNPs) using Lavandula angustifolia extract and the investigation of the anti-proliferative and apoptotic inducing effects of these nanoparticles in the U87MG glioblastoma cancer cell line. Green synthesized silver nanoparticles were characterized by various analytical techniques such as UV-Visible Spectrophotometer (UV-Vis), scanning electron microscopy (SEM) and Energy Dispersive X-ray (EDX). UV-Vis spectroscopy displayed a specific silver plasmon peak at 430 nm. U87MG cells were treated at increased concentrations with Lavandula angustifolia-AgNPs (La-AgNPs) (0-20 µg/mL) for 72 h and the anti-proliferative effects of green synthesized silver nanoparticles on U87MG cells were evaluated by MTT assay. The La- AgNPs induced a statistically significant dose-dependent decrease in proliferation and increased cytotoxicity in U87MG cells. The IC50 value is 7.536 µg/mL. Furthermore, the expression of apoptosis proteins caspase-3, caspase-8 and caspase-9 was analyzed using ELISA and caspase-3 and p53 using western blotting. The results suggest that La-AgNPs induce cell death in U87MG cells through the p53 mediated intrinsic apoptotic pathway. Together, the present findings suggest that La-AgNPs could be considered as a potential option for the treatment of glioblastoma.

AKT-mediated phosphorylation of TWIST1 is essential for breast cancer cell metastasis.

Previously, it was shown that human TWIST1 (basic helix-loop-helix (b-HLH) is phosphorylated by Akt kinase at S42, T121, and S123. To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. We hypothesized that alanine mutants would inhibit the metastatic ability of 4T1 cells while glutamic acid mutants would convert nonmetastatic 67NR cells into metastatic phenotype. To confirm this hypothesis, we created metastatic 4T1 and nonmetastatic 67NR cells expressing alanine mutants and glutamic acid mutants mouse TWIST1, respectively. Then, we injected 1 × 106 67NR and 1 × 105 4T1 cells overexpressing mutants of TWIST1 into the breast tissue of BALB/c mice. At the end of the 4th week, we sacrificed the animals, determined the numbers of tumors at lungs and liver. Although 67NR cells overexpressing wild-type TWIST1 did not show any metastasis, cells overexpressing S42E and T125E mutants showed 15-30 macroscopic metastasis to liver and lungs. Parallel to this, 4T1 cells expressing S42A and T125A mutants of TWIST1 showed no macroscopic metastasis. Our results indicate that phosphorylation of S42 and T125 by AKT is essential for TWIST1-mediated tumor growth and metastasis.

Identification of novel mutations of Insulin Receptor Substrate 1 (IRS1) in tumor samples of non-small cell lung cancer (NSCLC): Implications for aberrant insulin signaling in development of cancer.

Lung cancer is the leading cause of cancer-related death, and NSCLC constitutes nearly 85%-90% of all cases. The IRS proteins function as adaptors and transmit signals from multiple receptors. Upon binding of insulin to the insulin receptor (IR), IRS1 is phosphorylated at several YXXM motifs creating docking sites for the binding of PI3Kp85, which activates AKT kinase. Therefore, we thought that gain of function mutantions of IRS1 could be related to development of lung cancer. In line with this, we wanted determine whether the IRS1 gene was mutated in the coding regions surrounding YXXM motifs. We sequenced the coding regions surrounding YXXM motifs of IRS1 using tumor samples of 42 NSCLC patients and 40 matching controls and found heterozygote p.S668T mutation in nine of 42 samples and four of nine also had the p.D674H mutation. We generated IRS1 expression vectors harboring p.S668T, p.D674H and double mutants. Expression of the mutants differentially affected insulin-induced phosphorylation of IRS1, AKT, ERK, and STAT3. Also, our mutants induced proliferation, glucose uptake, inhibited the migration of 293T cells and affected the responsiveness of the cells to cisplatin and radiation. Our results suggest that these novel mutations play a role in the phenotype of lung cancer.

Differential expression pattern of Twist1 in mouse preimplantation embryos suggests its multiple roles during early development.

PURPOSE: The purpose of the present study is to understand Twist-related protein 1 (Twist1) spatiotemporal expression patterns and functions during early embryo development. METHODS: We performed whole-mount double immunofluorescence staining and reverse transcription (RT)-PCR analysis of the Twist1 protein and gene throughout the preimplantation development in mice. RESULTS: We determined that after compaction, the expression of Twist1 becomes developmentally differentiated and targeted in the inner cells of embryos. In blastocysts at E4.5, uniform staining of the inner cell mass was apparent, and it had been gradually translocated to the nucleus of hatched embryonic cells at E4.75. Furthermore, the effect of potential regulators of Twist on its expression level during blastocyst development was also sought. Accordingly, Twist1 expression appeared to be upregulated in both mRNA and protein level following culture of embryos in the presence of high glucose. CONCLUSIONS: Our study revealed the dynamic Twist localization within the early stage of embryo. The results are discussed in terms of potential roles of Twist1 in the processes of lineage segregation, hatching, and implantation in post-compaction embryos and in blastocysts.

Clinicogenetic study of Turkish patients with syndromic craniosynostosis and literature review.

BACKGROUND: Fibroblast growth factor receptor 2 mutations have been associated with the craniosynostotic conditions of Apert, Crouzon, Pfeiffer, Saethre-Chotzen, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes in various ethnic groups. METHODS: Thirty-three unrelated Turkish patients (12 with Apert syndrome, 14 with Crouzon syndrome, six with Pfeiffer syndrome, and one with Saethre-Chotzen syndrome) and 67 nonsyndromic craniosynostosis patients were screened for mutations in exons IIIa and IIIc of the FGFR2 gene by denaturing high-performance liquid chromatography and confirmed by direct sequencing. RESULTS: We detected several pathogenic mutations in 11/33 (33%) patients with Apert syndrome (four with p.Pro253Arg; seven with p.Ser252Trp) and 8/33 (24%) patients with Crouzon syndrome (three with p.Trp290Arg, one with p.Cys342Tyr, p.Cys278Phe, p.Gln289Pro, and a novel p.Tyr340Asn mutation) and five (15%) with Pfeiffer syndrome (p.Cys342Arg, p.Pro253Arg, p.Trp290Arg, and p.Ser351Cys). No FGFR2 gene mutation was detected in any of the patients with Saethre-Chotzen syndrome and nonsyndromic craniosynostosis. CONCLUSIONS: Our results indicate that the majority of Turkish patients with syndromic craniosynostosis have detectable genetic changes with an overall frequency of 72.7%. Because this is the first molecular genetic report from a Turkish cohort, the identified spectrum profile of FGFR2 mutations of the syndromic craniosynostotic patients would be very helpful for understanding the genotype-phenotype relationship and has a great value for diagnosis, prognosis, and genetic counseling.

Differential expression and activation of epidermal growth factor receptor 1 (EGFR1), ERK, AKT, STAT3, and TWIST1 in nonsmall cell lung cancer (NSCLC).

Lung cancer is the leading cause of death of both men and women across the world. Overexpression and activating mutations of the epidermal growth factor receptor-1 (EGFR1) are frequently observed and associated with poor prognosis. To inhibit the function of EGFR1, multiple antibodies and small-molecule tyrosine kinase inhibitors (TKI) that target EGFR1 have been developed. Even though some patients respond to these TKI, subsequent studies reveal that this is not the case for all nonsmall cell lung cancer (NSCLC) patients. In this study, we determine whether activation and expression levels of EGFR1, ERK, AKT, STAT3, and TWIST1 are dependent on the activating mutations of EGFR1. Protein lysates and DNA have been isolated from tumor and corresponding normal tissues of 16 NSCLC patients. Genomic-DNA is used to sequence the exons 18, 19, and 21 of EGFR1, and exon 2 of k-RAS. Protein lysates were used to determine the expression or phosphorylation levels of EGFR, STAT3, ERK, AKT, and TWIST1. Our results revealed that 16 tumor samples of NSCLC patients showed no mutation in any of the indicated exons of EGFR1 and k-RAS albeit significant levels of activation or expression of the above-mentined oncogenes. In NSCLC patients, the tumor micro-environment can be as important as the activating mutations of EGFR1. TK therapy may also be considered for patients who show high levels of activation of EGFR1 even in the absence of activating mutations.

Analysis of TPO gene in Turkish children with iodide organification defect: identification of a novel mutation.

The objective was to determine molecular genetic analysis of the TPO gene in Turkish children with iodide organification defect (IOD). Patients with a diagnosis of primary hypothyroidism were evaluated. Subjects having a definite diagnosis of autoimmune thyroiditis, thyroid gland dysplasia and, or iodine deficiency were excluded. A total of 10 patients from nine unrelated Turkish families, with an unknown etiology of hypothyroidism, and with a presumptive diagnosis of IOD were included in the study. A perchlorate discharge test (PDT) was performed to all subjects, and sequence analysis of TPO gene was applied in patients with a positive PDT. Five out of 10 patients have a total IOD, while the five remaining patients have a partial IOD according to PDT results. In two sisters, one has a partial and the other one has a total IOD a novel homozygous nonsense p.Q315X mutation was found in exon 8. Additionally, a previously known homozygous missense p.R314W mutation was detected in the same exon in another patient with a total IOD. No TPO gene mutation was detected in any of the seven remaining patients. Two different TPO gene mutations were found to be responsible for IOD in two unrelated Turkish families from the same ethnic background. More subjects should be screened for detecting the prevalence and spectrum profile of TPO mutations in our population that might be helpful for understanding the pathophysiology of congenital hypothyroidism.

Final diagnosis in children with subclinical hypothyroidism and mutation analysis of the thyroid peroxidase gene (TPO).

AIM: To determine the final diagnosis of patients with subclinical hypothyroidism (SCH), and to perform mutation screening of the thyroid peroxidase gene (TPO). METHODS: Infants with SCH without an identified etiology were included in the study. Patients with thyroid dysgenesis were excluded. Children > or = 2 years of age, and still on L-thyroxine (LT4) treatment underwent a diagnostic algorithm. After LT4 was discontinued for 4 weeks, thyroid function tests (TFT) were obtained. A perchlorate discharge test (PDT) was performed in patients with normal thyroid ultrasound but abnormal TFT. Sequence analysis of TPO was studied in all children who underwent a PDT. RESULTS: Forty-eight patients (23 males and 25 females) completed the trial. Among these children, 19 (39.5%) had transient SCH, and 29 (60.5%) had permanent SCH. Among patients with permanent SCH, 19 had thyroid hypoplasia, six had partial iodide organification defect with positive PDT, and four had other dyshormonogenesis with negative PDT. Mean LT4 dose before the medication ceased was 1.2 +/- 0.5 microg/kg/day in transient cases, and 1.7 +/- 0.4 in those with permanent SCH (p < 0.05). No TPO mutation was detected. However, in five patients, seven different previously known TPO polymorphisms were detected: c.102C > G, L4L; > A, A576A; c.2088C > T, D666D; c.2263A > C, T725P; c.2630 T >C, V847A. CONCLUSIONS: LT4 treatment should be stopped after the age of 2 years in infants with SCH without a definite pathology of the thyroid gland to exclude cases with transient hypothyroidism. Additionally, we should consider particularly thyroid gland hypoplasia, and also partial defects in iodide organification in infants with SCH.