Total DNA Content Research Articles

An Analysis and Survey on Interleukin-10 Receptor Mutation in Inflammatory Bowel Disease in Iranian IBD Cohort

Background: Early-onset inflammatory bowel disease (IBD) is classified to Crohn’s disease, ulcerative colitis and unclassified disorders which have chronic, relapsing course and can result in substantial long-term morbidity. IBD is a multifactorial disorder with genetic susceptibility, immunological predisposition and environmental triggers. Objective: To generally determine prevalence of IL10R mutation in IBD patients in Iran-Isfahan, we performed sequencing of all exons in IL10RA and IL10RB in cohort of IBD patients and healthy control. Material and Method: Total DNA content of 76 patients and 50 healthy controls were extracted from whole blood and PCR amplification and sequencing was done. Result: Overall identified IL-10RA mutations were P.(I224V), P.(A153V), P.(A153A), P.(S159G), P.(R263Q), P.(R284C), P.(R351Q), P.(Q376Q), P.(T416I), P.(A493V), P.(A511A) and P.(S563S). In IL10RB gene the only detected mutation was P. (K47E). Of them, P.(A153V), P.(A153A), P.(R284C), P.(T416I), P.(A493V), P.(A511A), P.(S563S) were Not reported variant in IBD variants. Conclusion: Our results also confirmed that early-onset IBD could be attributed to a synergistic effect of several variant alleles of the genes encoding IL10 receptors. These variants, alone, could only give rise to a sub-clinical manifestation of the IBD.

The association between faecal host DNA or faecal calprotectin and feed efficiency in pigs fed yeast-enriched protein concentrate

Gut cell losses contribute to overall feed efficiency due to the energy requirement for cell replenishment. Intestinal epithelial cells are sloughed into the intestinal lumen as digesta passes through the gastrointestinal tract, where cells are degraded by endonucleases. This leads to fragmented DNA being present in faeces, which may be an indicator of gut cell loss. Therefore, measuring host faecal DNA content could have potential as a non-invasive marker of gut cell loss and result in a novel technique for the assessment of how different feed ingredients impact upon gut health. Faecal calprotectin (CALP) is a marker of intestinal inflammation. This was a pilot study designed to test a methodology for extracting and quantifying DNA from pig faeces, and to assess whether any differences in host faecal DNA and CALP could be detected. An additional aim was to determine whether any differences in the above measures were related to the pig performance response to dietary yeast-enriched protein concentrate (YPC). Newly weaned (∼26.5 days of age) Large White × Landrace × Pietrain piglets (8.37 kg ±1.10, n = 180) were assigned to one of four treatment groups (nine replicates of five pigs), differing in dietary YPC content: 0% (control), 2.5%, 5% and 7.5% (w/w). Pooled faecal samples were collected on days 14 and 28 of the 36-day trial. Deoxyribonucleic acid was extracted and quantitative PCR was used to assess DNA composition. Pig genomic DNA was detected using primers specific for the pig cytochrome b (CYTB) gene, and bacterial DNA was detected using universal 16S primers. A pig CALP ELISA was used to assess gut inflammation. Dietary YPC significantly reduced feed conversion ratio (FCR) from weaning to day 14 (P<0.001), but not from day 14 to day 28 (P = 0.220). Pig faecal CYTB DNA content was significantly (P = 0.008) reduced in YPC-treated pigs, with no effect of time, whereas total faecal bacterial DNA content was unaffected by diet or time (P>0.05). Faecal CALP levels were significantly higher at day 14 compared with day 28, but there was no effect of YPC inclusion and no relationship with FCR. In conclusion, YPC reduced faecal CYTB DNA content and this correlated positively with FCR, but was unrelated to gut inflammation, suggesting that it could be a non-invasive marker of gut cell loss. However, further validation experiments by an independent method are required to verify the origin of pig faecal CYTB DNA as being from sloughed intestinal epithelial cells.

Fragmented Nuclear DNA is the Predominant Genetic Material in Human Hair Shafts.

While shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, nuclear DNA short tandem repeat (STR) profiling is generally unsuccessful and DNA testing of shed hair is instead performed by targeting the mitochondrial DNA control region. Although the high copy number of mitochondrial DNA relative to nuclear DNA routinely permits the recovery of mitochondrial DNA (mtDNA) data in these cases, mtDNA profiles do not offer the discriminatory power of nuclear DNA profiles. In order to better understand the total content and degradation state of DNA in single shed hairs and assess the feasibility of recovering highly discriminatory nuclear DNA data from this common evidence type, high throughput shotgun sequencing was performed on both recently collected and aged (approximately 50-year-old) hair samples. The data reflect trends that have been demonstrated previously with other technologies, namely that mtDNA quantity and quality decrease along the length of the hair shaft. In addition, the shotgun data reveal that nuclear DNA is present in shed hair and surprisingly abundant relative to mitochondrial DNA, even in the most distal fragments. Nuclear DNA comprised, at minimum, 88% of the total human reads in any given sample, and generally more than 95%. Here, we characterize both the nuclear and mitochondrial DNA content of shed hairs and discuss the implications of these data for forensic investigations.

Hydrolytic enzymes mediated lipid-DNA catabolism and altered gene expression of antioxidants under combined application of lead and simulated acid rain in Fenugreek (Trigonella foenum graecum L.) seedlings.

Understanding ill effects of simultaneous existence of various abiotic stresses, commonly observed due to various anthropogenic activities and global climate change these days, over plants growth, metabolic activity and yield responses are important for continued agricultural productivity and food security. In the present study, seedlings of Fenugreek (Trigonella foenum graecum L.) were subjected to lead (Pb, 1200 ppm) and/or simulated acid rain (SAR, pH 3.5) for 30 days, and were then analysed. The results revealed reduced growth, and total lipid and DNA contents, while enhanced Pb accumulation, biological concentration factor, biological accumulation coefficient, translocation factor, lipase activity, and levels of free fatty acid, conjugated diene, lipid hydroperoxide, DNA oxidation and DNase activity under Pb and/or SAR exposure. Additionally, activities and gene expression levels of antioxidants (superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase) were enhanced in response to applied treatments. The results also suggested that inhibitions/ accelerations determined under joint addition of Pb and SAR were comparatively more profound than those measured under their single application. Additionally, root was more sensitive to Pb treatment, compared to both leaf and shoot. Hence, under simultaneous presence of two or more number of abiotic stresses, the strategy opted by plants for survival is chiefly governed by the interaction between prevailing stressors, which is then conceived by plants as a new state of stress.

Estimation of the burden of human placental micro- and nano-vesicles extruded into the maternal blood from 8 to 12 weeks of gestation

BackgroundThe human placenta extrudes a variety of extracellular vesicles (EVs) into the maternal blood daily. These vesicles may be crucial to the adaptation of the maternal cardiovascular and immune systems to pregnancy. Quantifying the EVs that are released in early gestation is important to our understanding of how placental EVs may contribute to the regulation of maternal physiology. MethodsEVs were isolated from first trimester placental explants and separated into micro- and nano-vesicles by differential centrifugation. The numbers of each type of EVs extruded from each milligram of placentae between gestational weeks 8 and 12 was determined by Nanoparticle Tracking Analysis. The total protein or DNA content of the vesicles was determined by BCA assay or Qubit® 2.0. ResultsNeither the number of micro- nor nano-EVs/mg explant (n = 49), nor the total protein (n = 19) and DNA content (n = 29) of these EVs changed significantly between 8 and 12 weeks of gestation. When the increasing placental weight with gestation was accounted for, the daily number of placental EVs extruded into the maternal blood increased by more than 100 fold between 8 and 12 weeks (micro-EVs 6.23 X 1014 and nano-EVs 1.84 X 1014 at 12 weeks, p = 0.0003). DiscussionConstant production of micro- and nano-EVs per-milligram placenta, regardless of gestational age, and the increased daily burden of EVs across gestational age indicate these EVs have the potential to regulate maternal physiology from early pregnancy. Since total EV protein content, like EV numbers was, constant, this is a potentially reliable surrogate for quantifying EVs.

Massive mitochondrial DNA content in diplonemid and kinetoplastid protists.

SummaryThe mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans‐splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper‐inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4′,6‐diamidino‐2‐phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein‐coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet‐like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology., 70(12):1267–1274, 2018

Enhanced corrosion resistance and cytocompatibility of biomimetic hyaluronic acid functionalised silane coating on AZ31 Mg alloy for orthopaedic applications.

This paper reports the corrosion resistant and cytocompatible properties of the hyaluronic acid-silane coating on AZ31 Mg alloy. In this study, the osteoinductive properties of high molecular weight hyaluronic acid (HA, 1-4 MDa) and the corrosion protection of silane coatings were incorporated as a composite coating on biodegradable AZ31 Mg alloy for orthopaedic applications. The multi-step fabrication of coatings first involved dip coating of a passivated AZ31 Mg alloy with a methyltriethoxysilane-tetraethoxysilane sol-gel to deposit a dense, cross-linked and corrosion resistant silane coating (AZ31-MT). The second step was to create an amine-functionalised surface by treating coated alloy with 3-aminopropyl-triethoxy silane (AZ31-MT-A) which facilitated the immobilisation of HA via EDC-NHS coupling reactions at two different concentrations i.e 1 mg.ml-1 (AZ31-MT-A-HA1) and 2 mg.ml-1 (AZ31-MT-A-HA2). These coatings were characterised by Fourier transform infrared spectroscopy, atomic force microscopy and static contact angle measurements which confirmed the successful assembly of the full coatings onto AZ31 Mg alloy. The influence of HA-silane coating on the corrosion of Mg alloy was investigated by electrical impedance spectroscopy and long-term immersion studies measurements in HEPES buffered DMEM. The results showed an enhanced corrosion resistance of HA functionalised silane coated AZ31 substrate over the uncoated equivalent alloy. Furthermore, the cytocompatibility of MC3T3-E1 osteoblasts was evaluated on HA-coated AZ31-MT-A substrates by live-dead staining, quantification of total cellular DNA content, scanning electron microscope and alkaline phosphatase activity. The results showed HA concentration-dependent improvement of osteoblast cellular response in terms of enhanced cell adhesion, proliferation and differentiation. These findings hold great promise in employing such biomimetic multifunctional coatings to improve the corrosion resistance and cytocompatibility of biodegradable Mg-based alloy for orthopaedic applications.

Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice

Aims/hypothesisIslet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.MethodsHuman islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1−/− mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining.ResultsCFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min).Conclusions/interpretationThe ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.

Preliminary analysis of the cause for the failure of non-invasive prenatal testing using cell-free fetal DNA derived from peripheral maternal blood

To explore the cause of failure of non-invasive prenatal testing (NIPT) using cell-free fetal DNA from peripheral maternal blood. A total of 31 832 cases of NIPT were retrospectively analyzed. The clinical data of pregnant women were analyzed and the outcome of pregnancy was followed up. Among the 31 832 cases, 200 patients have failed for the first NIPT test. Second test has succeeded in 171 (85.9%) of 199 cases, while 28 cases (14.1%) still yielded no effective results. This gave rise for a total failure rate of 0.088%. Of the 28 cases, 11 (39.2%) were due to high content of total free DNA and could not be sequenced, 17 (60.7%) were found to have the fetal DNA content of less than 4%. Among the 171 cases which have obtained a valid result, NIPT showed that there were 4 patients with high risk of trisomy 21, 18 cases with high risk of 18 trisomy and 1 case with high risk of 13 trisomy. Karyotyping analysis of the amniocytic chromosomes has identified 3 cases with 47,XN,+21, 1 case with 46,XN,rob(21;21), 1 case with 47,XN,+18, while the 13 trisomy case was found to be false positive. For the 28 cases with failed NIPT retest, 14 had normal delivery, with no anomaly noticed in the neonates. Nine patients had opted for artificial abortion during middle or late pregnancy due to maternal factors (4 cases) or fetal factors (5 cases). Four patients developed complications of pregnancy. One case was in good condition upon follow-up. Four cases were lost during follow-up. Of the 11 pregnant women who had failed the NIPT test due to high content of total free DNA, 6 (54.5%) had opted for artificial abortion during midterm pregnancy, which was significantly higher than that of pregnant women with low free DNA content (17.6%). Failure of NIPT testing should attract attention from researchers. Failure of single NIPT test should not be regarded as a high risk signal for fetal chromosomal aneuploidies. For those where the test has failed again, genetic counseling and strengthened perinatal care should be provided for the pregnant women.

Effects of secondary carbon supplement on biofilm-mediated biodegradation of naphthalene by mutated naphthalene 1, 2-dioxygenase encoded by Pseudomonas putida strain KD9

Polycyclic aromatic hydrocarbons (PAHs) belong to a diverse group of environmental pollutants distributed ubiquitously in the environment. The carcinogenic properties of PAHs are the main causes of harm to human health. The green technology, biodegradation have become convenient options to address the environmental pollution. In this study, we analyzed the biodegradation potential of naphthalene with secondary carbon supplements (SCSs) in carbon deficient media (CSM) by Pseudomonas putida strain KD9 isolated from oil refinerary waste. The rigid-flexible molecular docking method revealed that the mutated naphthalene 1,2-dioxygenase had lower affinity for naphthalene than that found in wild type strain. Moreover, analytical methods (HPLC, qRT-PCR) and soft agar chemotaxis suggest sucrose (0.5 wt%) to be the best chemo-attractant and it unequivocally caused enhanced biodegradation of naphthalene (500 mg L−1) in both biofilm-mediated and shake-flask biodegradation methods. In addition, the morphological analysis detected from microscopy clearly showed KD9 to change its size and shape (rod to pointed) during biodegradation of naphthalene in CSM as sole source of carbon and energy. The forward versus side light scatter plot of the singlet cells obtained from flow cytometry suggests smaller cell size in CSM and lower florescence intensity of the total DNA content of cells. This study concludes that sucrose may be used as potential bio-stimulation agent.

Nondestructive Assessment of Glycosaminoglycans in Engineered Cartilages Using Hexabrix-Enhanced Micro-Computed Tomography.

It is very useful to evaluate the content and 3D distribution of extracellular matrix non-destructively in tissue engineering. This study evaluated the feasibility of using micro-computed tomography (µCT) with Hexabrix to measure quantitatively sulfated glycosaminoglycans (GAGs) of engineered cartilage. Rabbit chondrocytes at passage 2 were used to produce artificial cartilages in polyglycolic acid scaffolds in vitro. Engineered cartilages were incubated with Hexabrix 320 for 20min and analyzed via µCT scanning. The number of voxels in the 2D and 3D scanning images were counted to estimate the amount of sulfated GAGs. The optimal threshold value for quantification was determined by regression analysis. The 2D µCT images of an engineered cartilage showed positive correlation with the histological image of Safranin-O staining. Quantitative data obtained with the 3D µCT images of 14 engineered cartilages showed strong correlation with sulfated GAGs contents obtained by biochemical analysis (R2 = 0.883, p < 0.001). Repeated exposure of engineered cartilages to Hexabrix 320 and µCT scanning did not significantly affect cell viability, total DNA content, or the total content of sulfated GAGs. We conclude that µCT imaging using Hexabrix 320 provides high spatial resolution and sensitivity to assess the content and 3D distribution of sulfated GAGs in engineered cartilages. It is expected to be a valuable tool to evaluate the quality of engineered cartilage for commercial development in the future.

Comparative Analysis of Repetitive DNA between the Main Vectors of Chagas Disease: Triatoma infestans and Rhodnius prolixus.

Chagas disease or American trypanosomiasis affects six to seven million people worldwide, mostly in Latin America. This disease is transmitted by hematophagous insects known as “kissing bugs” (Hemiptera, Triatominae), with Triatoma infestans and Rhodnius prolixus being the two most important vector species. Despite the fact that both species present the same diploid chromosome number (2n = 22), they have remarkable differences in their total DNA content, chromosome structure and genome organization. Variations in the DNA genome size are expected to be due to differences in the amount of repetitive DNA sequences. The T. infestans genome-wide analysis revealed the existence of 42 satellite DNA families. BLAST searches of these sequences against the R. prolixus genome assembly revealed that only four of these satellite DNA families are shared between both species, suggesting a great differentiation between the Triatoma and Rhodnius genomes. Fluorescence in situ hybridization (FISH) location of these repetitive DNAs in both species showed that they are dispersed on the euchromatic regions of all autosomes and the X chromosome. Regarding the Y chromosome, these common satellite DNAs are absent in T. infestans but they are present in the R. prolixus Y chromosome. These results support a different origin and/or evolution in the Y chromosome of both species.

Analysis of Gene Expression Changes in PHA-M Stimulated Lymphocytes – Unraveling PHA Activity as Prerequisite for Dicentric Chromosome Analysis

Dicentric chromosome analysis (DCA) is the gold standard for individual radiation dose assessment. However, DCA is limited by the time-consuming phytohemagglutinin (PHA)-mediated lymphocyte activation. In this study using human peripheral blood lymphocytes, we investigated PHA-associated whole genome gene expression changes to elucidate this process and sought to identify suitable gene targets as a means of meeting our long-term objective of accelerating cell cycle kinetics to reduce DCA culture time. Human peripheral whole blood from three healthy donors was separately cultured in RPMI/FCS/antibiotics with BrdU and PHA-M. Diluted whole blood samples were transferred into PAXgene tubes at 0, 12, 24 and 36 h culture time. RNA was isolated and aliquots were used for whole genome gene expression screening. Microarray results were validated using qRT-PCR and differentially expressed genes [significantly (FDR corrected) twofold different from the 0 h value reference] were analyzed using several bioinformatic tools. The cell cycle positions and DNA-synthetic activities of lymphocytes were determined by analyzing the correlated total DNA content and incorporated BrdU level with flow cytometry after continued BrdU incubation. From 42,545 transcripts of the whole genome microarray 47.6%, on average, appeared expressed. The number of differentially expressed genes increased linearly from 855 to 2,858 and 4,607 at 12, 24 and 36 h after PHA addition, respectively. Approximately 2-3 times more up- than downregulated genes were observed with several hundred genes differentially expressed at each time point. Earliest enrichment was observed for gene sets related to the nucleus (12 h) followed by genes assigned to intracellular structures such as organelles (24 h) and finally genes related to the membrane and the extracellular matrix were enriched (36 h). Early gene expression changes at 12 h, in particular, were associated with protein classes such as chemokines/cytokines (e.g., CXCL1, CXCL2) and chaperones. Genes coding for biological processes involved in cell cycle control (e.g., MYBL2, RBL1, CCNA, CCNE) and DNA replication (e.g., POLA, POLE, MCM) appeared enriched at 24 h and later, but many more biological processes (42 altogether) showed enrichment as well. Flow cytometry data fit together with gene expression and bioinformatic analyses as cell cycle transition into S phase was observed with interindividual differences from 12 h onward, whereas progression into G2 as well as into the second G1 occurred from 36 h onward after activation. Gene set enrichment analysis over time identifies, in particular, two molecular categories of PHA-responsive gene targets (cytokine and cell cycle control genes). Based on that analysis target genes for cell cycle acceleration in lymphocytes have been identified ( CDKN1A/B/C, RBL-1/RBL-2, E2F2, Deaf-1), and it remains undetermined whether the time expenditure for DCA can be reduced by influencing gene expression involved in the regulatory circuits controlling PHA-associated cell cycle entry and/or progression at a specific early cell cycle phase.

Influence of dynamic mechanical stimulation on tissue engineering annulus fibrosus

Objective To investigate the influence of dynamic mechanical stimulation on the annulus fibrosus (AF) cells seeded on silk scaffolds. Methods AF cells were isolated from rabbits and were seeded on the scaffold, then cultured for 3, 7, 14 days with different range of dynamic compression. Stereomicroscope and scanning electron microscope (SEM) was used to observe the surface morphology of tissue engineering annulus fibrosus cells (TE-AFs). After fixation, samples were harvested for histological staining. AF cells related extracellular matrix (ECM) was evaluated by the quantitative analysis of total DNA, proteoglycan and collagen I. The mechanical properties were compared within different groups. Results Stereomicroscope and SEM results showed that the colors of TE-AFs in all groups were deepening with time going. SEM showed cell adhesion on the scaffold and the secretion of extracellular matrix. Histological, immunohistochemical staining, biochemical quantitative analysis and total DNA content showed that the AF cells inside scaffolds could support AF cell attachment, proliferation and secretion. As a result, the compressive properties were enhanced with increasing culture time. Stereomicroscope showed that the colors of TE-AFs in all groups were deepening with time going after dynamic compression. HE staining, Safranin O staining and Type I collagen staining showed that cell proliferation and secretion, GAG secretion and collagen secretion were increased with time going within different groups. Quantitation of GAG achieved maximum in 15% strain group, and quantitation of collagen achieved maximum in 10% strain group. The total DNA content achieved maximum in 5% strain group, and compression elastic modulus achieved maximum in 15% strain group. The height of TE-AFs did not change after mechanical stimulation for 14 days. Conclusion Suitable mechanical stimulation is a positive factor for new AF tissue engineering that will tend to the nature tissue. Excessive compression can accelerate the progress of cell apoptosis. Key words: Tissue engineering; Silk; Tissue scaffolds; Biomechanics

Evaluation of genotoxicity by comet assay (single-cell gel electrophoresis) in tissues of the fish Cyprinus carpio during sub-lethal exposure to Karanjin

BackgroundAdverse effects caused by the synthetic pesticides has forced scientists to develop an alternative against the synthetic pesticides. Therefore, it has come a boon to mankind in the form of neem tree having secondary metabolites showing pesticidal properties which is in use since many years. Like neem tree, there has been research done on karanja plant, and its secondary metabolite karanjin has been found to possess good pesticidal properties. Comet assay or alkaline single-cell gel electrophoresis (SCG) was performed in fish Cyprinus carpio after exposure to sub-lethal concentration (0.28 ppm) of karanjin. The assay was carried out in the gill, liver, kidney, and blood. The total DNA content in each tissue was also estimated using Nano Drop.ResultsThe present study clearly shows that though karanjin is a derived secondary metabolite from plant, it is capable of producing genotoxicity in common carp. As the days of sub-lethal exposure increased, all the four tissues showed decrease in total DNA content which was directly proportional to the formation of DNA comet tails.ConclusionThe use of any kind of plant metabolites in the agriculture and aquaculture farms should be monitored well.

Cell Cycle Inhibition by Constricted Migration

As a cancer cell invades adjacent tissue, penetrates a basement membrane barrier, or squeezes into the smallest blood capillaries, its nucleus can be highly constricted. Such nuclear stress has recently been shown to enhance DNA damage and genome variation. However, little is known about the interaction between constriction-induced damage and cell cycle/DNA replication, even though the latter is increasingly being appreciated as a major source of cancerous mutations. Here, U2OS osteosarcoma cells, U251 glioblastoma cells, and A549 lung carcinoma cells were seeded at three different densities (low, medium, high) on 2D plastic and were also migrated through rigid 3 μm and 8 μm pores. In each case, cell cycle analysis was performed using total DNA content combined with EdU incorporation, as measured by fluorescence microscopy. For all three cell types, G2 was found to be suppressed most in 2D by high density, indicating possible contact inhibition. In 3D, G2 was suppressed most among cells that migrated through 3 μm pores, even though this was the lowest density condition. These findings suggest that constricted migration inhibits cell cycle progression, perhaps due to intra-S-phase checkpoint activation by constriction-induced DNA damage. Indeed, super-resolution microscopy of migrated cells reveals DNA damage throughout the nucleoplasm even though nuclear lamina disruption and chromatin blebs localize to the poles.

Evaluation of ITGB2 (CD18) and SELL (CD62L) genes expression and methylation of ITGB2 promoter region in patients with systemic sclerosis.

Systemic sclerosis (SSc), an autoimmune disease of connective tissue, is characterized by inflammation, fibrosis, and vessel endothelial damage. Products of Integrin subunit beta 2 (ITGB2) and selectin L (SELL) genes participate in several functional pathways of immune system. The aim of this investigation was to survey the transcript level of ITGB2 and SELL genes as well as methylation status of CpG sites in promoter region of differently expressed gene in PBMCs of SSc patients. PBMCs were isolated from whole blood of 50 SSc patients and 30 healthy controls. Total RNA and DNA contents of PBMCs were extracted. Gene expression was analyzed by real-time PCR using the SYBR Green PCR Master Mix. To investigate the methylation status of CpG sites, DNA samples were treated by bisulfite, amplified through nested PCR, and sequenced through Sanger difficult sequencing method. ITGB2 gene in PBMCs of SSc patients was overexpressed significantly in comparison to healthy controls. However, no altered SELL expression was observed. Three CpG sites of 12, 13 and 14 were significantly hypomethylated in patients group, despite overall methylation status of ITGB2 gene promoter revealed no significant difference between study groups. There was no statistically significant correlation between methylation status of ITGB2 promoter and the gene expression in patients. Regarding to lack of correlation of increased expression of ITGB2 with its promoter hypomethylation in SSc patients, our study suggests that upregulation of ITGB2 in PBMCs from SSc patients is probably due to another mechanism other than methylation alteration.

Cytological and genome size data analyzed in a phylogenetic frame: Evolutionary implications concerning Sisyrinchium taxa (Iridaceae: Iridoideae).

Sisyrinchium is the largest genus of Iridaceae in the Americas and has the greatest amount of cytological data available. This study aimed at investigating how genomes evolved in this genus. Chromosome number, genome size and altitude from species of sect. Viperella were analyzed in a phylogenetic context. Meiotic and pollen analyses were performed to assess reproductive success of natural populations, especially from those polyploid taxa. Character optimizations revealed that the common ancestor of sect. Viperella was probably diploid (2n = 2x =18) with two subsequent polyplodization events. Total DNA content (2C) varied considerably across the phylogeny with larger genomes detected mainly in polyploid species. Altitude also varied across the phylogeny, however no significant relationship was found between DNA content changes and altitude in our data set. All taxa presented regular meiosis and pollen viability (> 87%), except for S. sp. nov. aff. alatum (22.70%), suggesting a recent hybrid origin. Chromosome number is mostly constant within this section and polyploidy is the only source of modification. Although 2C varied considerably among the 20 taxa investigated, the diversity observed cannot be attributed only to polyploidy events because large variations of DNA content were also observed among diploids.

The human microbiome: Role in health and disease and its potential applications

The human microbiota comprises all microorganisms living within the human body and microbiome is their collective genome. They include eukaryotes, archaea, bacteria and viruses. Most microbes live in the gut, particularly in the large intestine. The number of genes contributed to by the microbes is 100 times the number of genes in the human genome. The sheer microbial abundance suggests that the human body is a collection of human and microbial cells and their genes resulting in a blend of human and microbial traits. The microbiome is essential for maintenance of human life. The microbes that live in and on us are mostly beneficial colonizers and have many important functions such as assisting in digestion, regulating immune system, producing essential vitamins and protecting against bacteria that cause diseases. Research has demonstrated that dysbioses in the human microbiome correlate with numerous disease states, including inflammatory bowel disease, malignancy and autoimmune diseases. The metagenome of the human microbiome, the total DNA content of microbes inhabiting our bodies shows a large degree of interpersonal diversity. With the advent of culture-independent techniques such as high throughput next generation sequencing of DNA and advances in computational biology, research on human microbiome has reached new heights. A breakthrough in research was the development of Human Microbiome Project (HMP), which was established in 2008, which is also known as the second human genome project, with the objective of generating a metagenomic reference database for “normal” individuals to serve as a resource for researchers. The introduction of new technologies has permitted the study of the functional component of the microbe–host interactions through metabolomics. The resultant biomarkers have been used to predict and diagnose diseases early. Future research should focus on understanding mechanisms responsible for pathology and the causality role of the microbiome to use them as therapeutic modalities .

Fatty Acid Profile and Anti-Inflammatory Activity of Fixed Plant Oils

Background: Many seed oils have been used as anti-inflammatory agents, administred by ingestion or topical application in traditional medicine. The objective of this research was to perform a chemical analysis of fatty profile and a pharmacological study through a topical experiment of TPA-induced ear edema test and an internal assay - acetic acid-induced vascular permeability in Swiss mice of some fixed oils popularly used for inflammatory problems, trying to confirm their action.Materials, Methods &amp; Results: Fixed lipids of Ouratea fieldingiana (batiputá), Caryocar coreaceum (pequi), Annacardium occidentale (cashew-nuts), Cocos nucifera (coco-da-bahia), Byrsonima crassifolia (murici) e Elaeis guineenses (palm) were selected for the identification of fatty acids profile by gas chromatography coupled to mass spectrometry (GC/MS) analysis and evaluation of anti-inflammatory activity by TPA-induced ear edema test and acetic acid-induced vascular permeability in Swiss male mice. The oils were purchased in local markets or extracted in Soxhlet apparatus with hexane. The oils of cashew nut, murici fruit, and pequi nut presented a high percentage of unsaturated fatty acids (81.80, 74.46 and 60.72 %, respectively). In the oils of batiputá and murici, linoleic acid was the main unsaturated fatty acid (45.06% and 74.66%, respectively) and oleic acid was main constituent in cashew nut, pequi and palm seed oils. Batiputá and palm oils exibit approximately equivalent content of saturated and unsaturated fatty acid and coconut oil more saturated fatty acids (80.72%) with predominance of lauric acid. The result of TPA-induced ear edema test revealed that all oils presented similar anti-inflammatory activity. In the acetic acid-induced vascular permeability model, the oil of O. fieldingiana was the only one who showed anti-inflammatory activity, while C. coreaceum and B. crassifolia oils showed pro-inflammatory activities. The presence of phenols and flavonoids was evaluated in the O. fieldingiana oil by spectrophometric methods.Discussion: All the oils showed anti-inflammatory action in the TPA-induced ear edema, probably the action of unsaturated fatty acids was more important in topical application, nevertheless in internal inflammation process the presence of antioxidant phenolic compounds could contribute to the higher activity of the oil from O. fieldingiana. The effect of linoleic and oleic acids was demonstrated on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in a prevoius study using sunflower oil. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. This pro-inflammatory effect of oleic and linoleic acids may contribute to the wound healing process. In this study with six plant oils, some of them have higher content in linoleic acid and others oleic acid is the major constituent so the antiinflamatory action on ear edema can be associated to these two unsaturaded fatty acids mechanism of action. In the internal model, probably other chemical constituents revealed in Ouratea fieldingiana as phenols, condensed tannins, flavones and flavanones, could contribute to the anti-inflammatory activity.