Determination Of DNA Content Research Articles

ALTMAN: A Novel Method for Cell Cycle Analysis.

Accurate analysis of S-phase fraction is crucial for the assessment of cell proliferation levels, tumor malignancy and prognostic effects of treatment. Most of the currently developed methods for S-phase cell analysis rely on flow cytometric analysis of DNA content determination. However, the lack of standardized procedures for sample analysis and interpretation of cell cycle fitting graphs poses a significant limitation in clinical practice for utilizing flow cytometry to measure the cell cycle based on DNA content. Herein, we developed an approach for analyzing S-phase cells based on telomerase activity determination. Briefly, this approach distinguishes S-phase cells in cell populations via direct fluorescence tracking of telomerase activity within individual cells. The dynamic analysis of telomerase activity in different cell cycles was made possible by the ALTMAN strategy developed in our previous studies, which has been successfully employed to distinguish S-phase cells in cultured cells. This method offers a novel avenue for the assessment of cell cycle status and the evaluation of the proliferation status of tumor cells and the prognosis effect of tumor patients via analyzing the differences in telomerase activity during different cell cycle processes.

Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Modulation of early osteoarthritis by tibiofemoral re-alignment in sheep

ObjectiveTo investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA. MethodNormal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone. ResultsMultivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface–to–volume ratio (all P ≤ 0.0359 vs. normal). ConclusionThe data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

Decellularization of Pig Lung to Yield Three-Dimensional Scaffold for Lung Tissue Engineering.

Lately, the need for three-dimensional (3D) cell culture has been recognized in order to closely mimic the organization of native tissues. Thus, 3D scaffolds started to be employed to facilitate the 3D cell organization and enable the artificial tissue formation for the emerging tissue engineering applications. 3D scaffolds can be prepared by various techniques, each with certain advantages and disadvantages. Decellularization is an easy method based on removal of cells from native tissue sample, yielding extracellular matrix (ECM) scaffold with preserved architecture and bioactivity. This chapter provides a detailed protocol for decellularization of pig lung and also some basic assays for evaluation of its effectivity, such as determination of DNA content and histological verification of the selected ECM components. Such decellularized scaffold can subsequently be used for various tissue engineering applications, for example, for recellularization with cells of interest, for natural ECM hydrogel preparation, or as a bioink for 3D bioprinting.

Cytotoxicity of poly-guanidine in medulloblastoma cell lines.

Medulloblastoma (MB) is the most common pediatric brain tumor. The therapy frequently causes serious side effects, and new selective therapies are needed. MB expresses hyper sialylation, a possible target for selective therapy.The cytotoxic efficacy of a poly guanidine conjugate (GuaDex) incubated with medulloblastoma cell cultures (DAOY and MB-LU-181) was investigated. The cells were incubated with 0.05-8µM GuaDex from 15min to 72h. A fluorometric cytotoxicity assay (FMCA) measured the cytotoxicity. Labeled GuaDex was used to study tumor cell interaction. FITC-label Sambucus nigra confirmed high expression of sialic acid (Sia). Immunofluorescence microscopy was used to visualize the cell F-actin and microtubules. The cell interactions were studied by confocal and fluorescence microscopy. Annexin-V assay was used to detect apoptosis. Cell cycle analysis was done by DNA content determination. A wound-healing migration assay determined the effects on the migratory ability of DAOY cells after GuaDex treatment.IC50 for GuaDex was 223.4 -281.1nM. FMCA showed potent growth inhibition on DAOY and MB-LU-181 cells at 5 uM GuaDex after 4h of incubation. GuaDex treatment induced G2/M phase cell cycle arrest.S. nigraFITC-label lectin confirmed high expression of Sia on DAOY medulloblastoma cells. The GuaDex treatment polymerized the cytoskeleton (actin filaments and microtubules) and bound to DNA, inducing condensation. The Annexin V assay results were negative. Cell migration was inhibited at 0.5µM GuaDex concentration after 24h of incubation.GuaDex showed potent cytotoxicity and invasion-inhibitory effects on medulloblastoma cells at low micromolar concentrations. GuaDex efficacy was significant and warrants further studies.

265-LB: Human Skeletal Muscle Mitochondrial Dynamics across the Insulin-Sensitivity Spectrum

Introduction: There is immense dispute as to whether perturbations in skeletal muscle mitochondrial function contribute to the onset and progression of type 2 diabetes (T2D). The purpose of this study was to examine differences in mitochondrial dynamics, structure, and energetic function in humans across the spectrum of insulin sensitivity. Methods: 58 sedentary adults (37±12 years) were enrolled into one of three groups based upon the following criteria: (1) healthy weight without T2D (HW; BMI: 22.6±1.8 kg/m2 and HbA1c: 5.0±1.1 %); (2) overweight/obesity without T2D (Ov/Ob; BMI: 32.6±3.3 kg/m2 and HbA1c: 5.6±0.4 %); or (3) overweight/obesity with T2D (T2D; BMI: 36.1±6.5 kg/m2 and HbA1c: 6.9±0.9 %). Participants underwent a 3-day inpatient stay consisting of body composition (DXA), aerobic capacity (VO2MAX), and insulin sensitivity (hyperinsulinemic euglycemic clamp). Prior to insulin sensitivity testing, a skeletal muscle biopsy was obtained for determination of mitochondrial dynamics (Western blot), DNA content (qPCR), ultrastructure (electron microscopy), and respiratory capacity (respirometry). Comparisons were made using a one-way ANOVA with contrasts. Results: Insulin sensitivity and aerobic capacity were lower in Ov/Ob and T2D compared to HW. Markers of mitochondrial fission were higher in T2D (Drp1Ser616, MiD49), and fusion lower in T2D and Ov/Ob (Mfn2). The mitophagy marker Parkin was higher in T2D only while Pink1 was lower in T2D relative to Ov/Ob but not HW. The autophagy marker LC3II was higher in Ov/Ob. Mitochondrial content was lower in Ov/Ob and T2D relative to HW. Mitochondrial respiratory capacity was similar between groups. Conclusions: T2D is associated with heightened expression of proteins required for mitochondrial quality control and reduced mitochondrial volume despite intact respiratory function. These data support the notion that mitochondria adapt to progressive insulin resistance by increasing fragmentation to maintain bioenergetic capacity. Disclosure C. L. Axelrod: None. C. Fealy: Employee; Mission Therapeutics. C. L. Hoppel: None. J. P. Kirwan: None. W. S. Dantas: None. E. R. M. Zunica: None. K. Belmont: None. E. C. Heintz: None. G. Davuluri: None. J. T. Mey: None. M. Erickson: None. H. Fujioka: None. Funding National Institutes of Health (DK108089, GM104940)

Effect of Hydrogen Sulfide on Essential Functions of Polymorphonuclear Leukocytes.

Impaired polymorphonuclear leukocyte (PMNL) functions contribute to increased infections and cardiovascular diseases in chronic kidney disease (CKD). Uremic toxins reduce hydrogen sulfide (H2S) levels and the anti-oxidant and anti-inflammatory effects of H2S. Its biosynthesis occurs as a side process of transsulfuration and in the disposal of adenosylhomocysteine, a transmethylation inhibitor and proposed uremic toxin. PMNL chemotaxis was measured by the under-agarose method, phagocytosis, and oxidative burst by flow cytometry in whole blood and apoptosis by determining DNA content by flow cytometry and morphological features by fluorescence microscopy. Sodium hydrogen sulfide (NaHS), diallyl trisulphide (DATS) and diallyl disulphide (DADS), cysteine, and GYY4137 were used as H2S-producing substances. Increased H2S concentrations did not affect chemotaxis and phagocytosis. NaHS primed PMNL oxidative burst activated by phorbol 12-myristate 13-acetate (PMA) or E. coli. Both DATS and cysteine significantly decreased E. coli-activated oxidative burst but had no effect on PMA stimulation. While NaHS, DADS, and cysteine attenuated PMNL apoptosis, GYY4137 decreased their viability. Experiments with signal transduction inhibitors suggest that the intrinsic apoptosis pathway is mainly involved in GYY4137-induced PMNL apoptosis and that GYY4137 and cysteine target signaling downstream of phosphoinositide 3-kinase.

Gender Preselection in the Buffalo Species (Bubalus Bubalis): Preliminary Data on Different Strategies and Pregnancy Outcome

In this study, a new comparison was tested in Mediterranean Italian buffaloes (Bubalus bubalis), between two different strategies for obtaining sperm cells of the desired sex. In addition, a control group of animals was included in the study, where only ordinary semen straws, containing unselected sper cells were used. Sexed and control straws were then used for Artificial Insemination (AI) in order to confront pregnancy and gender rates. The first of the two strategies to preselect sperm cells relies on the physical determination of DNA content in single spermatozoa though cell sorting machines. The second strategy instead, relies on the coincubation of either fresh or frozen/thawed semen with a specific solution specifically designed for the species of interest. The trial involved a total of 206 heifers and 211 pluriparous buffaloes belonging to a buffalo farm in the south of the Lazio Region in Italy. In this study, no significant differences between treated and control groups were detected in terms of pregnancy rates both on heifers (P=0.6) and pluriparous buffaloes (P=7). With regard to gender rates, pregnancy rates were significantly different among the three groups of heifers (P=0.001) and the three groups of pluriparous buffaloes (P=0.002). Sperm cells preselected for gender determination through Cell Sorting (CST) resulted effective in shifting the gender ratio towards the female, when compared to both the Coinbubation Technique (COiT) and control group of animals.

Weeds in Cereal Crop Rotations May Host Fusarium Species That Cause Fusarium Head Blight and Grain Weight Losses in Wheat

Fusarium Head Blight (FHB) is one of the most common worldwide wheat and other small grain diseases. The infection is caused by Fusarium graminearum and other related species, which significantly reduce grain yield and contaminate grains with mycotoxins which are harmful for humans and animals. Fusarium pathogen survives the winter well in plant debris left on the field. Weeds around and within crops are alternative hosts of Fusarium fungi when an economically important host plant is not present. This article focuses on the determination of DNA content of Fusarium species (F. graminearum and F. avenaceum) in artificially inoculated wheat plants with isolates from weeds, as well as its influence on the severity of FHB and spring wheat 1000-grain weight under field conditions. Fungal DNA content in grains was evaluated by quantitative real-time PCR. The results showed that the DNA concentration of F. graminearum was significantly higher in the grain than F. avenaceum. The severity of FHB when wheat heads were inoculated with F. graminearum was significantly higher than with F. avenaceum. All F. graminearum strains statistically significantly reduced the weight of spring wheat grains, while F. avenaceum did not affect the weight of 1000 grain. This investigation has shown that weeds in crop rotations are a potential source of FHB infection. However, the severity of the disease is more affected by the Fusarium species than the host plant. This experiment is, to our knowledge, the first report on the estimation of Fusarium DNA content in artificially inoculated wheat plants with isolates from weeds, as well as its comparison with pathogenicity to wheat and its effect on 1000-grain weight.

Study of DNA ploidy in newly diagnosed multiple myeloma by flow cytometry and its correlation with disease prognosis and outcome

Objectives: Multiparameter flow cytometry is increasingly being used in determining DNA content in several hematological malignancies where abnormal DNA ploidy is a useful prognostic marker. This study was done to analyze the DNA ploidy of plasma cells in multiple myeloma (MM) by flow cytometry and explore its role as a prognostic factor. Material and Methods: Propidium-iodide staining technique and gating based on light scatter properties were used. DNA index (DI) of the tumor sample was calculated as the ratio of the DNA content of the G0-G1 population of the myeloma cells with the normal control cells (lymphocytes) present in the same sample. Based on DI, ploidy was categorized as diploidy, hypodiploidy, and hyperdiploidy and the results were correlated with the clinical outcome. Results: Among 32 patients, none had hypodiploid DNA content, 8 (25%) patients had hyperdiploid DNA, and 24 (75%) patients had diploid DNA. There was no significant association between DI and international staging system staging (P = 0.68), Eastern Cooperative Oncology Group Performance Status (P = 0.59), and post-induction remission status (P = 0.10). The median overall survival (OS) in the study patients was 20 (CI 11.4–28.8) months and the median progression-free survival was not reached. There was no difference in the OS among patients with diploid MM and hyperdiploid MM (P = 0.84). Conclusion: Although hyperdiploid MM has been reported to have a better prognosis than diploid MM, we did not find any significant difference possibly due to the small sample size. Nevertheless, flow cytometry is a useful tool in DNA ploidy analysis and its role as a prognostic factor in various hematologic malignancies including MM can be further explored.

Hydrostatic pressure shock induced diploid/tetraploid mosaic in mandarin fish (Siniperca chuatsi), with the observation of embryo development and change in body spots

The mandarin fish, Siniperca chuatsi, is an economically fish with delicious flesh and rich nutrients. Artificially induced polyploid is an effective methods of breeding superior species of fish. The increasing frequency of mosaicism in the research of fish polyploidy has attracted the attention of many scholars. In this study, hydrostatic pressure shock caused an abnormal cleavage of fertilized, larvae treated with pressure shock displayed reduced fertilization, hatch and survival rates. Mosaics that both have diploid and tetraploid cells were induced in mandarin fish. The effective condition was applying 500 kg/cm2 pressure at 45 min after fertilization for 2 min at 25 °C, the occurrence of 44.5% diploid/tetraploid mosaic fish was confirmed by flow cytometry determination of DNA content. Mosaic mandarin fish has the ability to incubate and grow, and the body weight and body length analysis showed no significant difference between mosaic and diploid individuals. However, after the hydrostatic pressure shock, delayed embryonic development and body deformities were observed, and the body spot and stripe have largely changed in experimental mandarin fish.

Decellularization alters the unfavorable regenerative adverse microenvironment of the injured spinal cord to support neurite outgrowth.

BackgroundAcellular tissue has been transplanted into the injury site as an external microenvironment to intervene with imbalance microenvironment that occurs after spinal cord injury (SCI) and stimulating axonal regeneration, although the mechanism is unclear. Given decellularization is the key means to obtain acellular tissues, we speculated changes in the internal components of tissue caused by decellularization may be the key reason why acellular tissues affect remodeling of the microenvironment.MethodsComplete spinal cord crush in a mouse model was established, and the dynamic of extracellular matrix (ECM) expression and distribution during SCI was studied with immunohistochemistry (IHC). Normal spinal cord (NSC) and 14-day injury spinal cord (ISC) were obtained to prepare the decellularized NSC (DNSC) and decellularized ISC (DISC) through a well-designed decellularization method, and the decellularization effects were evaluated by residual DNA content determination, hematoxylin and eosin staining (H&E), and IHC. Rat dorsal root ganglia (DRG) were co-cultured with NSC, ISC, DNSC, and DISC to evaluate their effect on neurite outgrowth. Furthermore, the mechanisms by which decellularized tissue promotes axonal growth were explored with proteomics analysis of the protein components and function of 14-day ISC and DISC.ResultsWe found the expression of the four main ECM components (collagen type I and IV, fibronectin, and laminin) gradually increased with the progression of SCI compared to NSC, peaking at 14 days of injury then slightly decreasing at 21 days, and the distribution of the four ECM proteins in the ISC also changed dynamically. H&E staining, residual DNA content determination, and IHC showed decellularization removed cellular components and preserved an intact ECM. The results of co-cultured DRG with NSCs, ISCs, DNSCs, and DISCs showed DNSCs and DISCs had a stronger ability in supporting neurite outgrowth than NSC and ISC. We found through proteomics that decellularization could remove proteins associated with inflammatory responses, scarring, and other pathological factors, while completely retaining the ECM proteins.ConclusionsTaken together, our findings demonstrate decellularization can optimize the imbalanced microenvironment after SCI by removing components that inhibit spinal cord regeneration, providing a theoretical basis for clinical application of acellular tissue transplantation to repair SCI.

Chromosome and ploidy analysis of winter hardy Hibiscus species by FISH and flow cytometry

Determination of nuclear DNA content, genome size, and ploidy level and, information on cytogenetic characteristics are all prerequisite of modern plant breeding. However, identification of individual chromosomes of Hibiscus species is extremely difficult due to high number, small size and similar shape of mitotic chromosomes. The goal of the study was to ascertain the chromosome number, karyomorphology, distribution of 5S and 18S rDNA signals, chromosome length, and centromere positions as well as the ploidy level, genome sizes, 2C - DNA content of winter-hardy Hibiscus (H. syriacus ‘Saejamyung’, H. sinosyriacus ‘Seobong’, H. moscheutos ‘Luna Red’ and, H. paramutabilis). 5S rDNA and 18S rDNA signals were detected by fluorescence in situ hybridization (FISH). According to the FISH results, there are two 5S rDNA signals (green) in H. syriacus, H. sinosyriacus, and H. moscheutos, and four 5S rDNA signals in H. paramutabilis. The range in length of somatic chromosomes in H. syriacus, H. sinosyriacus, H. moscheutos, and H. paramutabilis is 2.66–7.06, 3.18–7.31, 2.91–5.23, and 4.75–7.60, respectively. The 2C - DNA content of H. syriacus, H. sinosyriacus, and H. paramutabilis are very similar, the amount was 4.06, 4.11, and 4.18 pg, respectively whereas, H. moscheutos has nearly half and that amount was 2.06 pg. These findings will contribute to the detailed cytogenetic assessment of Hibiscus and thus benefit plant breeding in this genus.

6-Methyl-1-(3-(trifluoromethyl)phenyl)-1H-pyrazolo[4,3- c]pyridin-4(5H)-one (FMPPP) exhibits anti-proliferative effect on prostate cancer via autophagy induction and mTOR/p70S6K inhibition


 
 
 
 Purpose: To investigate 6-methyl-1-(3-(trifluoromethyl)phenyl)-1H-pyrazolo[4,3-c]pyridin-4(5H)-one (FMPPP) as anti-proliferative agent against prostate cancers.
 Methods: The FMPPP-mediated changes in cell proliferation were measured using cell counting kit-8 (CCK-8). Flow cytometry and propidium iodide staining were used for cellular DNA content determination. Proteins expression in cells was probed by western blotting assay.
 Results: A significant (p < 0.05) dose-dependent suppression of DU145 and PC 3 cell proliferation was observed following FMPPP treatment. FMPPP treatment at 20 μM raised DU145 cell fraction to 75.08 ± 4.87 % in G1 phase when compared to 48.32 ± 3.44 % for control. The population of PC 3 cells in G1- phase reached to 72.78 ± 5.21 % on treatment with 20 μM FMPPP compared to 49.65 ± 4.62 % in control. The FMPPP treatment of DU145 and PC-3 cells elevated LC3-II expression and suppressed SQSTM1/p62 expression. In FMPPP-treated DU145 and PC-3 cells, p-ERK1/2 level was promoted whereas mTOR and p70S6K phosphorylation significantly decreased. Exposure to U0126 (ERK pathway inhibitor) reduced FMPPP-induced increase of LC3-II expression and promotion of p-ERK1/2 level in DU145 and PC-3 cells.
 Conclusion: FMPPP exhibits anti-proliferative effect by increasing autophagy in prostate cancer cells. The cytotoxicity of FMPPP involves elevation of ERK1/2 phosphorylation and targeting mTOR pathway in DU145 and PC-3 cells. Therefore, FMPPP may be beneficial for the treatment of prostate cancer in patients.
 
 
 

Reference standards for flow cytometric estimation of absolute nuclear DNA content in plants.

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1/G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well‐established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

Comparison of nuclear DNA yield and STR typing success in Second World War petrous bones and metacarpals III

DNA yield and STR typing success differ among skeletal element types and within individual bones. Consequently, it is necessary to identify the skeletal elements and their intra-skeletal parts that will most likely yield utilizable and informative endogenous DNA for human identification of skeletal remains. The petrous portion of the temporal bone has been shown to be the most suitable skeletal part for sampling archaeological skeletons, and it has also been used successfully in some forensic cases. When all representative bone types were analyzed for three complete Second World War skeletons, metatarsals and metacarpals yielded more DNA than petrous bones (which generated full profiles even if the DNA yield was lower) and, among almost 200 Second World War metatarsals and metacarpals analyzed, metacarpals III were found to be the highest-yielding bones. To further improve the sampling strategy in DNA analysis of aged skeletal remains, a comparison between 26 petrous bones and 30 metacarpals III from Second World War skeletons was made considering intra-bone DNA yield variability. In metacarpals III only epiphyses were sampled, and in petrous bones only the dense part within the otic capsule was used. To exclude the influence of taphonomic issues as much as possible, petrous bones and metacarpals III from a single Second World War mass grave were examined. The difference between petrous bones and metacarpals III was explored by measuring nuclear DNA yield and success of STR typing. After cleaning the samples, full demineralization extraction was used to decalcify 0.5 g of powdered bone. PowerQuant (Promega) was used to determine DNA content and DNA degradation rates, and STR typing was performed using the PowerPlex ESI 17 Fast System (Promega). Metacarpals III produced the same DNA yields and STR typing success as petrous bones with no intra-individual difference observed in concentration of DNA, degradation rate, percentage of successfully amplified alleles, and intensity of electrophoretic signals. Sampling and processing of metacarpal III epiphyses is consequently recommended for genetic identification of highly degraded skeletal remains in routine forensic casework and in buried non-commingled aged skeletal remains whenever metacarpals III are preserved. Metacarpals III are easy to sample and less prone to contamination with modern DNA because no saw is needed for sampling in comparison to the petrous portion of the temporal bone. The data obtained in this study further improve the sampling strategy for genetic identification of Second World War skeletal remains in Slovenia.

Determination of Core DNA Contents and Use in Species Identification by Ploidy Analysis of Dactylis glomerata ssp., and Festuca arundinaceae ssp. of Trifolium repens ssp. Collected from the Central Black Sea Region

Bu çalışma, Orta Karadeniz Bölgesi doğal florasından toplanan domuz ayrığı (Dactylis glomerata ssp.), kamışsı yumak (Festuca arundinaceae ssp.) ve ak üçgül (Trifolium repens ssp.) popülasyonlarının çekirdek DNA içeriklerinin flow sitometri yöntemiyle belirlenmesi ve ploidi analizi ile tür teşhisinde kullanılması amacıyla yürütülmüştür. Proje kapsamında ak üçgülden mera tipi 80, domuz ayrığından ot ve mera tipi 80 ve kamışsı yumaktan ot, mera ve çim tipi 130 adet genotip kullanılmıştır. Analizlerde tarlada yetişmekte olan bitkilerden alınan taze yaprak dokuları kullanılmıştır. Floresan boya olarak ise propidium iodide kullanılmıştır. İnternal standard, Dactylis glomerata ve Trifolium repens genotiplerinde Vicia sativa, Festuca arundinecea genotiplerinde ise Hordeum vulgare kullanılmıştır. Elde edilen sonuçlara göre araştırmada kullanılan Dactylis glomerata genotiplerinin ortalama 2C çekirdek DNA içeriği 9.12 pg (8.32 pg-9.66 pg); Trifolium repens genotiplerinin ortalama 2C çekirdek DNA içeriği 2.33 pg (2.22 pg-2.44 pg); Festuca arundinecea genotiplerinin ortalama 2C çekirdek DNA içeriği 17.47 pg (17.02 pg-17.98 pg) arasında değiştiği belirlenmiştir. Çekirdek DNA içerikleri ile genotiplerin ploidi düzeylerini ilişkilendirmek amacıyla yapılan kromozom sayımlarında domuz ayrığı genotiplerinin 2n=4x=28, ak üçgül genotiplerinin 2n=4x=32 ve kamışsı yumak genotiplerinin 2n= 6x=42 kromozoma sahip oldukları belirlenmiştir. Elde edilen bu sonuçlara göre çalışmada kullanılan Dactylis ve Trifolium genotiplerinin tetraploid, Festuca genotiplerinin ise hekzaploid olduğu saptanmıştır. Ayrıca çalışmada kullanılan tüm genotipler çekirdek DNA içeriklerinin de yardımıyla taksonomik olarak teşhis edilmiş ve Trifolium repens ssp.’nin Trifolium repens var. repens, Dactylis glomerata ssp.’nin Dactylis glomerata subsp. glomerata ve Festuca arundineceae ssp.’nin de Festuca arundinacea subsp. arundinacea olduğu ve genotiplerin içerisinde başka türlere ait hiç bir genotipin bulunmadığı belirlenmiştir.

QS9: Decellularization Of Vascularized Composite Allografts In The Rat

Purpose:The decellularization of Vascularized Composite Allotransplantation (VCA) represents a first step in circumventing the need for immunosuppression by depleting donor cells from VCAs. Here we present a model of decellularization of VCAs in rats.Methods:Rat hind limbs based upon the common femoral artery were harvested and perfused through the vascular pedicle at 1ml/min through with 1% sodium dodecyl sulfate (SDS) (group 1) or 0.2% SDS (group 2) or were immersed in SDS 1% (group 3), followed by perfusion (group 1 and 2) or immersion (group 3) in 1% Triton X-100. The degree of decellularization was assessed quantitatively by determining DNA content and qualitatively by histology in all groups at the same locations. The group yielding best decellularization outcome was selected for further assessment for the preservation of ECM content and vasculature and was transplanted orthotopically to assess the ability to reperfuse these decellularized limbs in vivo. RESULTS - Gross examination and DNA quantification of less than 50 ng/mg tissue demonstrated successful decellularization only in group 2 while preserving tissue matrix architecture, including vessels. The arterial network was found to be perfusable and intact during in vivo orthotopic transplantation of the decellularized limb. No outflow was observed, however, after 115 minutes of arterial reperfusion.Conclusions:We describe a successful decellularization protocol for rat hindlimb VCAs by perfusion ex-vivo of 1% SDS. During its in vivo transplantation, decellularization alone allows for a perfusable arterial network and with further optimization, we aim to achieve venous perfusion as well.

First report on genome size and ploidy determination of five indigenous coffee species using flow cytometry and stomatal analysis

The nuclear 2C DNA content and ploidy level determination of five indigenous coffee species from India was carried out using flow cytometry and by assessing the stomatal characteristics. The nuclear DNA content (2C/pg) analyzed varied from 1.29 pg in Coffea wightiana Wall. ex Wight & Arn. to 1.48 pg in Coffea jenkinsii Hook.f. Based on the genome size, all five species were divided into two groups. The first group comprises Coffea travancorensis Wight & Arn and C. wightiana with lower genome size (2C DNA 1.29–1.30 pg), and the second group consists of Coffea bengalensis Roxb.ex Schult, Coffea khasiana (Korth.) Hook.f and C. jenkinsii with larger genome size (2C DNA 1.45–1.48 pg). The species included in the first group were inhabited in dry environmental conditions in contrast to the species included in the second group which was predominantly from mesic habitat. Among the stomatal characteristics, no significant variation in stomatal density was observed among the species, although stomatal guard cell length and stomatal chloroplast number recorded significant variation among some species. Based on flow cytometry and stomatal characteristics, all five endangered coffee species analyzed in the study were identified as diploids. The study revealed that stomatal guard cell length and stomatal chloroplast number could be fast, inexpensive, and reliable methods for determining the ploidy status of indigenous coffee species.