Increase In DNA Concentration Research Articles

Assessing the dynamics and macromolecular interactions of the intrinsically disordered protein YY1.

YY1 is a ubiquitously expressed, intrinsically disordered transcription factor involved in neural development. The oligomeric state of YY1 varies depending on the environment. These structural changes may alter its DNA binding ability and hence its transcriptional activity. Just as YY1's oligomeric state can impact its role in transcription, so does its interaction with other proteins such as FOXP2. The aim of this work is to study the structure and dynamics of YY1 so as to determine the influence of oligomerisation and associations with FOXP2 on its DNA binding mechanism. The results confirm that YY1 is primarily a disordered protein, but it does consist of certain specific structured regions. We observed that YY1 quaternary structure is a heterogenous mixture of oligomers, the overall size of which is dependent on ionic strength. Both YY1 oligomerisation and its dynamic behaviour are further subject to changes upon DNA binding, whereby increases in DNA concentration result in a decrease in the size of YY1 oligomers. YY1 and the FOXP2 forkhead domain were found to interact with each other both in isolation and in the presence of YY1-specific DNA. The heterogeneous, dynamic multimerisation of YY1 identified in this work is, therefore likely to be important for its ability to make heterologous associations with other proteins such as FOXP2. The interactions that YY1 makes with itself, FOXP2 and DNA form part of an intricate mechanism of transcriptional regulation by YY1, which is vital for appropriate neural development.

A Sensitive Method for Detecting Beauveria bassiana, an Insecticidal Biocontrol Agent, Population Dynamics, and Stability in Different Substrates.

Beauveria bassiana is a well-known insecticidal biocontrol agent. Despite its broad field applications, its survival, colonization, and stability under field conditions remained unclear, mainly due to the lack of a quick and reliable detection method. In this study, we developed a quantitative real-time PCR technology to monitor the stability and population dynamics of B. bassiana in different substrates (water, soil, and on the cotton leaves surface), different spores of B. bassiana applied on Chinese cabbage leaves surface, and the lethality of Pieris rapae spraying with different spores of B. bassiana. Our results showed a decreased concentration of B. bassiana DNA in all three substrates from the 1st day till 9th day of post inoculation (dpi) period, possibly due to the death of B. bassiana. After this decrease, a quick and significant rebound of B. bassiana DNA concentration was observed, starting from the 11th dpi in all three substrates. The B. bassiana DNA concentration reached the plateau at about 13th dpi in water and 17th dpi in the soil. On cotton leaves surface, the B. bassiana DNA concentration reached the highest level at the 17th dpi followed by a small decline and then stabilized. This increase of DNA concentration suggested recovery of B. bassiana growth in all three substrates. We found that the most suitable killing effectiveness of P. rapae was the 1.0 × 107 spores/mL of B. bassiana. In summary, we have established a detection technology that allows a fast and reliable monitoring for the concentration and stability of B. bassiana under different conditions. This technology can benefit and help us in the development of proper management strategies for the application of this biocontrol agent in the field.

Small Molecule Interactions with Biomacromolecules: DNA Binding Interactions of a Manganese(III) Schiff Base Complex with Potential Anticancer Activities.

A manganese(III) complex, [MnIII(L)(SCN)(enH)](NO3)·H2O (1•H2O) (H2L = 2-((E)-(2-((E)-2-hydroxy-3-methoxybenzylidene-amino)-ethyl-imino)methyl)-6-methoxyphenol), has been synthesized and characterized by single-crystal X-ray diffraction analysis. The interaction of 1•H2O with DNA was studied by monitoring the decrease in absorbance of the complex at λ = 324 nm with the increase in DNA concentration, providing an opportunity to determine the binding constant of the 1•H2O-ct-DNA complex as 5.63 × 103 M-1. Similarly, fluorescence titration was carried out by adding ct-DNA gradually and monitoring the increase in emission intensity at 453 nm on excitation at λex = 324 nm. A linear form of the Benesi-Hildebrand equation yields a binding constant of 4.40 × 103 M-1 at 25 °C, establishing the self-consistency of our results obtained from absorption and fluorescence titrations. The competitive displacement reactions of dyes like ethidium bromide, Hoechst, and DAPI (4',6-diamidine-2'-phenylindole dihydrochloride) from dye-ct-DNA conjugates by 1•H2O were analyzed, and the corresponding KSV values are 1.05 × 104, 1.25 × 104, and 1.35 × 104 M-1 and the Kapp values are 2.16 × 103, 8.34 × 103, and 9.0 × 103 M-1, from which it is difficult to infer the preference of groove binding over intercalation by these DNA trackers. However, the molecular docking experiments and viscosity measurement clearly indicate the preference for minor groove binding over intercalation, involving a change in Gibbs free energy of -8.56 kcal/mol. The 1•H2O complex was then evaluated for its anticancer potential in breast cancer MCF-7 cells, which severely abrogates the growth of the cells in both 2D and 3D mammospheres, indicating its promising application as an anticancer drug through a minor groove binding interaction with ct-DNA.

Electrochemical and Optical Detection of Plant DNA for Sex Determination in a Lab-on-Chip Prototype

INTRODUCTION The fruit industry employs millions of hectares for production; of these, a significant percentage uses only female or hermaphrodite plants. The selection is carried out by morphological analysis until blooming, which represents an inefficient use of resources. Typically, well-equipped laboratories and trained personnel are required to perform DNA analysis and give valid results1,2. Devices capable of generating this information within a short timespan could contribute to a fast and effective selection of productive plants. Nonetheless, it is necessary to have high sensitivity to differentiate between samples. The objective of this work is the development of an electrochemical and an optical detection system, with high sensitivity and repeatability. With the aim to incorporate the best option in a fully integrated device that ensures an easy-to-use product, safeguarding the correct use by non-trained operators and reducing the time to obtain a result. MATERIALS AND METHODS To evaluate the detection systems a random sequence of DNA (5’-TAGTCGTCAATCCTCCGCTT) was used (50 – 540 ng/µL). The electrochemical detection design is composed by three electrodes (WE-Au, AE-Au, RE-Ag|AgCl) and 0.5 M Ru(NH3)6Cl3 as a DNA intercalating agent3 in 40 mM TAE buffer. Three electrochemical techniques (SWV, DPV and CV) were compared (Bio Logic potentiostat). The optical detection system is composed of a phototransistor (Max. Sens. @ 550 nm), a filter (520 nm) and a LED (Max. WL peak @ 455 nm) to detect the fluorescent DNA intercalator SYBRgreen. Different concentrations of fluorescein (0.005 – 10 µM) were used to evaluate the system capabilities. The system was evaluated with the DNA sequence and SYBRgreen, then compared with a fluorescence microscope (Nikon Eclipse Ti-U). Finally, both systems were tested with plant DNA samples. Genomic material was extracted (CTAB protocol4) form 6 plants (3 female/3 hermaphrodite), amplified by PCR (Thermal cycler miniPCR Amplyus) and analyzed by electrophoresis. RESULTS With the electrochemical detection system, we observed a proportional decrease in the Ru(NH3)6Cl3 signal as the DNA concentration increases, as expected. The data analysis demonstrated that SWV was the most sensitive technique with a R2=0.980, LOD 30.968 ng/µL and LOQ 103.226 ng/µL. Results showed that female plants present an average current of 1.774 μA while hermaphrodite plants had 1.694 μA, allowing us to differentiate hermaphrodite plants accurately with a 90 % confidence interval. On the other hand, in the optical detection system with a R2=0.9568, LOD 2.12 ng/µL and LOQ 7.06 ng/µL, we observed an increase in the signal as de DNA concentration increases. When the optical analysis was compared with the fluorescence microscope results, we observed a similar trend in the signals with a R2=0.995, that validate the functionality of our system. However, the difference between plant samples was detected with only the 70 % confidence interval, female samples showed an average signal of 381 mV while hermaphrodite plants had 352.56 mV. CONCLUSIONS Plant DNA detection was evaluated through two different systems. The optical detection system showed more sensitivity with synthetic samples. However, in the evaluation of plant samples, the electrochemical system is less susceptible to interferences. Therefore, we can differentiate between female and hermaphrodite plants with a 90 % confidence interval. In this way, it represents the best option to evaluate a lab-on-a-chip device. REFERENCES Barrantes-Santamaría W, Loría-Quirós C, Gómez-Alpízar L. Evaluation of two-sex determining systems in papaya plants (Carica papaya) Pococí hybrid. Agron Mesoamerican. 2019;30(2):437-446. doi:10.15517/am.v30i1.34916Cornelis S. FORENSIC LAB-ON-A-CHIP DNA ANALYSIS. 2019.Li LY, Jia HN, Yu HJ, et al. Synthesis, characterization, and DNA-binding studies of ruthenium complexes [Ru(tpy)(ptn)] 2 + and Ru(dmtpy)(ptn)] 2 +. J Inorg Biochem. 2012;113:31-39. doi:10.1016/j.jinorgbio.2012.03.008Biosciences. CTAB extraction solution for genomic DNA extraction. Biosciences. 2016:3-7. Figure 1

Fragmentomics of urinary cell-free DNA in nuclease knockout mouse models.

Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We explored the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in the fragmentation of ucfDNA using mouse models. The deletion of Dnase1 in mice (Dnase1-/-) caused aberrations in ucfDNA fragmentation, including a 24-fold increase in DNA concentration, and a 3-fold enrichment of long DNA molecules, with a relative decrease of fragments with thymine ends and reduction of jaggedness (i.e., the presence of single-stranded protruding ends). In contrast, such changes were not observed in mice with Dnase1l3 deletion (Dnase1l3-/-). These results suggested that DNASE1 was an important nuclease contributing to the ucfDNA fragmentation. Western blot analysis revealed that the concentration of DNASE1 protein was higher in urine than DNASE1L3. The native-polyacrylamide gel electrophoresis zymogram showed that DNASE1 activity in urine was higher than that in plasma. Furthermore, the proportion of ucfDNA fragment ends within DNase I hypersensitive sites (DHSs) was significantly increased in Dnase1-deficient mice. In humans, patients with bladder cancer had lower proportions of ucfDNA fragment ends within the DHSs when compared with participants without bladder cancer. The area under the curve (AUC) for differentiating patients with and without bladder cancer was 0.83, suggesting the analysis of ucfDNA fragmentation in the DHSs may have potential for bladder cancer detection. This work revealed the intrinsic links between the nucleases in urine and ucfDNA fragmentomics.

Herpesviruses in patients after renal transplantation

To estimate graft function after kidney transplantation during active herpesviruses or superinfection Materials and methods. The study included 32 patients (men 21, women 11) with end-stage chronic kidney disease. The median age was 43 years. Cytomegalovirus (CMV), EpsteinBarr virus (EBV) and human herpes virus 6 (HHV-6) DNAs were screened by RT-PCR in the donor's transplant biopsy, and recipients peripheral blood and urine after kidney transplantation (KT) on 0, 1, 2, 4, 6, 12 months. Antiviral antibodies (IgM and IgG) were also screened by Enzyme-linked immunoassay analysis (ELISA) along with PCR. The 500 or less copies of viral DNA per 105 nuclear cells or 1 ml of urine was considered as low, more than 1000 copies high. On the first month after KT CMV DNA was detected in 50% of pts., EBV DNA in 40% and HHV-6 DNA in 33%. During first year after KT two or three viruses simultaneously were found in 12 recipients: CMV, EBV, and HHV-6 were detected in 5 recipients; CMV and EBV in 4 patients; CMV and HHV-6 in 2 pts; EBV and HHV-6 in 1 pt. Graft dysfunction was observed in 9 patients with a high concentration of viral DNA of one, two or three viruses simultaneously. An upraise of the concentration of virus DNA (CMV, EBV and HHV 6) was detected primarily in the urine, while in the blood its concentration was less than 500 cop or undetectable. Renal dysfunction was not observed on the background of low concentrations of viral DNA in urine and blood. However, with an increase of DNA concentration, an impaired graft function in 8 of 12 patients appeared. Low viral DNA level proved to be a background for another virus activation or bacterial/fungal superinfection. Graft dysfunction occurs at high viral DNA levels detection during mono-or superinfection. Low viral load can serve as a background for another virus activation and/or bacterial/fungal superinfection.

Achieving enhanced biological phosphorus removal utilizing waste activated sludge as sole carbon source and simultaneous sludge reduction in sequencing batch reactor

Achieving enhanced biological phosphorus removal dominated by Tetrasphaera utilizing waste activated sludge (WAS) as carbon source could solve the problems of insufficient carbon source and excessive discharge of WAS in biological phosphorus removal. Up to now, the sludge reduction ability of Tetrasphaera remained largely unknown. Furthermore, the difference between traditional sludge fermentation and sludge fermentation dominated by Tetrasphaera was still unclear. In this study, two different sequencing batch reactors (SBRs) were operated. WAS from SBR-parent was utilized as sole carbon source to enrich Tetrasphaera with the relative abundance of 91.9% in SBR-Tetrasphaera. PO43−-P removal and sludge reduction could simultaneously be achieved. The effluent concentration of PO43−-P was 0, and the sludge reduction efficiency reached about 44.14% without pretreatment of sludge. Cell integrity detected by flow cytometry, the increase of DNA concentration in the sludge supernatant and decrease of particle size of activated sludge indicated that cell death and lysis occurred in sludge reduction dominated by Tetrasphaera. Stable structure of activated sludge was also damaged in this process, which led to the sludge reduction. By analyzing the excitation-emission matrix spectra of extracellular polymeric substances and the changes of carbohydrate and protein concentration, this study proved that slowly biodegradable organics (e.g., soluble microbial byproduct, tyrosine and tryptophan aromatic protein) could be better hydrolyzed and acidized to volatile fatty acids (VFAs) in sludge fermentation dominated by Tetrasphaera than traditional sludge fermentation, which provided carbon source for biological nutrients removal and saved operation cost in wastewater treatment.

Forensic human identification: Investigation into tooth morphotype and DNA extraction methods from teeth

When a body is decomposed, hard tissues such as teeth may provide the only DNA source for human identification. There is currently no consensus as to the best DNA extraction method, and there is a lack of empirical data regarding tooth morphotype and condition that may impact DNA recovery. Therefore, this study sought to investigate which variables significantly improved DNA concentration, integrity and profiling success. A total of 52 human teeth were assessed, representing all tooth morphotypes from three deceased individuals. DNA was extracted using both the QIAamp® DNA Investigator Kit and the phenol-chloroform method. DNA concentration and degradation index were assessed using real time PCR, prior to conventional DNA profiling. Contrary to international guidelines promoting the use of molars, DNA profiling from molars was the least successful, with premolars, followed by canines, performing the best. The presence of fillings reduced the DNA quantity and quality obtained and may explain the poor performance of molars. DNA from the maxillae were significantly less degraded when the QIAamp® was used, although this did not influence DNA profiling success. A significant increase in DNA concentration, integrity and profiling success was observed in diseased teeth (periodontitis) compared to those without disease. This may be due to increased white blood cell presence at the site. There was no significant difference in DNA profiling success between the two DNA extraction methods. However, different teeth yielded failed DNA profiles for each extraction method, suggesting that repeated attempts, using alternative DNA extraction methods, is recommended. The recovery of additional DNA profiling information from degraded samples may help to ultimately reduce the burden of unidentified human remains.

Helical aggregates of bis(styryl) dyes formed by DNA templating

In order to further investigate mechanism of the styryl dye – DNA interaction, three different bis(styryl)pyridinium dyes possessing OMe or/and NMe2 substituents in phenyl ring were selected and interaction of these compounds with the calf thymus DNA (ct-DNA) was monitored by using of UV–vis and fluorescence spectroscopic techniques, circular dichroism (CD), Hoechst 33258 displacement experiments and quantum-chemical calculations. The experimental results indicated a higher fluorescence enhancement for dyes containing NMe2 group upon binding with DNA. The results proved the interaction of the molecules with ct-DNA occurs through the formation of aggregates in minor groove at high dye concentration. Bis(styryl) dye with OMe substituent forms the helical dye aggregates of right-handed chirality, whereas, dye containing NMe2 group demonstrates formation of left-handed chiral aggregates in minor groove of DNA. As the DNA concentration increases, the dyes begin to spread out along the minor groove. This research provided a better understanding of mechanism of styryl dye - DNA interaction and effect of dye structure on interacting mode with DNA.

Evaluation of the Roche MagNA Pure 96 nucleic acid extraction platform for the Seegene Anyplex II HPV28 detection assay.

Validate the Roche, MagNAPure96 (MP96) nucleic acid extraction platform for Seegene Anyplex II HPV28 (Anyplex28) detection of Human Papillomavirus. Comparisons were made for Anyplex28 genotyping from 115 cervical samples extracted on the Hamilton, STARlet and the MP96. Two DNA concentrations were used for the MP96, one matched for sample input to the STARlet and another 5× concentration (laboratory standard). Agreement of HPV detection was 89·8% (κ=0·798; P=0·007), with HPV detected in 10 more samples for the MP96. There was a high concordance of detection for any oncogenic HPV genotype (κ=0·77; P=0·007) and for any low-risk HPV genotype (κ=0·85; P=0·008). DNA extracted at laboratory standard had a lower overall agreement 85·2% (κ=0·708; P<0·001), with 17/115 discordant positive samples that tested negative after STARlet extraction. Of the discordant genotypes, 72·7% were detected in the lowest signal range for Anyplex28 ('+'). MP96 performed with high concordance to STARlet, although produced DNA with a higher analytical sensitivity on the Anyplex28. This analysis supports the use of samples extracted on the MP96 for HPV genotyping using the Anyplex28. Furthermore, an increase in DNA concentration increased analytical sensitivity of the Anyplex28, particularly appropriate for prevalence studies.

In vivo Tracking of DNA for Precise Determination of the Stratum Corneum Thickness and Superficial Microbiome Using Confocal Raman Microscopy

The skin barrier function is mostly provided by the stratum corneum (SC), the uppermost layer of the epidermis. To noninvasively analyze the physiological properties of the skin barrier functionin vivo, it is important to determine the SC thickness. Confocal Raman microscopy (CRM) is widely used for this task. In the present in vivo study, a new method based on the determination of the DNA concentration profile using CRM is introduced for determining the SC thickness. The obtained SC thickness values are compared with those obtained using other CRM-based methods determining the water and lipid depth profiles. The obtained results show almost no significant differences in SC thickness for the utilized methods. Therefore, the results indicate that it is possible to calculate the SC thickness by using the DNA profile in the fingerprint region, which is comparable with the SC thickness calculated by the water depth profiles (ANOVA test p = 0.77) and the lipid depth profile (ANOVA test p = 0.74). This provides the possibility to measure the SC thickness by using the DNA profile, in case the water or lipid profile analyses are influenced by a topically applied formulation. The increase in DNA concentration in the superficial SC (0–2 µm) is related to the DNA presence in the microbiome of the skin, which was not present in the SC depth below 4 µm.

Mononuclear copper(II) complexes with 2-thiophene carboxylate and N-N donors; DNA interaction, antioxidant/anti-inflammatory and antitumor activity

Redox-active compounds such as copper-phenanthroline are known as artificial/chemical nucleases with a great impact and potential for their applications as metallotherapeutics. In that vein, the mononuclear copper(II) complexes [Cu(L)2(bipy)] (1), [Cu(L)2(bipy)(H2O)] (2) and [Cu(L)2(phen)(H2O)] (3), where L = 2-thiophene carboxylate, bipy = 2,2΄-bipyridine and phen = 1,10-phenanthroline, have been prepared and pharmacochemically studied, while the crystal structure of 1 is also reported. All the tested complexes preferably bind to CT-DNA via minor groove as resulted from UV spectroscopy studies, luminescent titration, EB competition assays and viscosity measurements. Complexes 2 and 3 in aqua behave like a “light switch” for DNA. The intensity enhancement, with the increase of DNA concentration, reached about 3–fold for 2 and 10-fold for 3. In vitro antioxidant activity of compounds 1–3, was evaluated using two different antioxidant assays: a) interaction with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical and b) inhibition of lipid peroxidation. Moreover, their inhibitory activity on soybean lipoxygenase (LOX) was evaluated for their anti-inflammatory potency. The tested complexes showed good activity on both lipid peroxidation and soybean LOX inhibition while complex 2 exhibited the best antioxidant/anti-inflammatory activity. A computational analysis over the LOX protein structure 1JNQ was performed, in an effort to support their possible mode of action. The cytotoxicity of the complexes was determined and their efficacy against several human cancer cell lines (ovarian, OAW-42; lung, A549; colon, HT29; breast, MDA-MB-231; kidney, Caki-2; and cervical, Hela) and human non-tumor cell lines (lung, MRC-5; and breast, MTSV1-7) were evaluated. The best cytotoxic activity was appeared for complex 3. In silico, computational methods support antiestrogen activity of the administered complexes on normal breast cells.

A comparative study of different laboratory storage conditions for enhanced DNA analysis of crime scene soil-blood mixed sample

Crime scene investigation is an important step in the entire criminal investigation process because this is where evidence is gathered. Blood from the perpetrator or victim of a crime can be left at crime scenes or transferred to other materials such as clothing, knives and guns. Most often, this body fluid is contaminated with soil at outdoor crime scenes but this might be the only or the most important evidence in solving a crime. This work aimed at identifying the most appropriate method of storing crime scene soil-blood mixed sample prior to analysis at the laboratory. Human blood was mixed with soil and stored at three different storage conditions (i.e., Room temperature/25°C, 4°C and −20°C). Samples stored at room temperature saw significant reduction in DNA concentration as storage time increased (P=0.001). Samples stored at 4°C saw a drastic decrease in DNA concentration just after two weeks of storage. By the eighth week of storage at 4°C, there was no detectable DNA (P=0.000). Samples stored at −20°C recorded no specific pattern in decrease or increase in DNA concentration for the entire 12week storage (P=0.324). There were full STR Profiles generated for room temperature stored samples and −20°C stored samples throughout the study. There were full, partial and null Profiles generated for 4°C stored samples depending on the sample storage duration. In conclusion, −20°C was identified as the best storage condition for soil-blood mixed sample followed by room temperature and 4°C, respectively.

Alu and Poet Ankyrine Detection and Quantization in Cell Free Dna of Cancer Patients

In this study, specimens of plasma were collected from 96 from patient diagnosed with cancer from Al Karama teaching hospital. Also plasma collected from 25 healthy individual as control. Cell-free DNA (cfDNA) was extracted from 1ml plasma using high pure viral Nucleic Acid Kit as an alternative to high expensive cfDNA kit. cfDNA extracted according to the manufacturer’s instructions, but without the use of carrier RNA. This represent the first recorded method of using viral nucleic acid extraction kit for collection of cell free DNA. Results indicate that all cancer samples show significant increase of cfDNA in compare with control (p 0.01). and this dramatic increase in DNA concentration provide good indicator about health condition. DNA integrity have been checked depending on amplification of Arthrobacter luteus (Alu sequence). Both small (115) and large (247) large Alu stretches have amplified using RT PCR using SYBR green dye. Results shows that integrity of DNA extracted from patients and control are suitable for any further molecular investigation, and short Alu repeats are much less abundant in health individual in compare with cancer patient of all cancer types under study. Depending on these results one can conclude that almost all DNA obtained from normal patients is finely fragmented due to apoptosis action and that’s why give very little positive. results in compare with corresponding cancer patient which release large DNA fragments resulted from necrosis and NK/Tc cells activity, which is amplifiable more efficiently. In this work another molecular study conducted to investigate the cancer specific sequences. Since we deals with different cancers type in this study, two types of prostate, ovary, testis expressed protein (POTE Ankyrine) were tested 2α and 2β. Results shows that almost all healthy group were negative for specific POTE test while cancer patient samples are positive for POTE specific amplification. This results are promising since it could be developed to be a building block of cfDNA cancer tests.

Impact of pre-incubation time of silk fibroin scaffolds in culture medium on cell proliferation and attachment.

Cell behaviours such as proliferation and attachment can be affected by the length of pre-incubation period of the scaffolds in the culture medium for long term. The aim of this study was to investigate the long term pre-incubation of 3D silk fibroin scaffolds in complete culture medium on cell attachment and proliferation. After the preparation of silk fibroin scaffolds by the technique of freeze drying, the scaffolds were pre-incubated in complete culture medium for 2 d, 6 d or 10 d before apical papilla stem cells (SCAP) seeding. Modifications of the scaffold surface and wettability were examined by FE-SEM and water contact angle, respectively. Results showed a decrease both in roughness and water contact angle as pre-incubation time increases. DNA measurement after 18h and 10 d cell seeding showed a significant increase of DNA concentration which represents better attachment and proliferation with pre-incubation time increase. Qualitative examination, live&dead assay or H&E staining method after 30h and 10 d cell seeding respectively, indicated that pre-incubation of scaffolds has time dependent effect on cell proliferation and attachment. This suggests that improvement of cell attachment and proliferation may be mediated by differences in the amount of wettability (decreased water contact angle) after exposure of scaffold to culture medium for long term which, in turn, causes more protein adsorption in the surface of silk fibroin scaffold (decreased roughness).

161 EFFECTS OF LIPOSOMES ON SPERM MOTILITY AND DNA-BINDING EFFICIENCY

In mice, microinjection is the most common gene transfer method used. Unfortunately, this strategy does not translate as well to livestock. Another potential method is sperm-mediated gene transfer, which takes advantage of sperm’s natural ability to bind to naked DNA. Gene transfer using sperm-mediated gene transfer has been shown in pigs (Gandolfi et al. 1989 J. Reprod. Fert. Abstr. Ser. 4) and cattle (Perez et al. 1991 Biotecnol. Apl. 8, 90–94). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analysed methods to select thawed bovine sperm for DNA binding and evaluated the binding of exogenous DNA to those sperm. To determine the optimal sperm-selection method, the sperm were analysed using a computer-assisted sperm analyzer (CASA), the parameters selected were: total motility, rapid motility, and progressive motility. To measure the binding of DNA we used an indirect analysis using NanoDrop technology (Thermo Scientific, Wilmington, DE, USA) to compare the different DNA concentrations among groups. Liposome preparation was done using a cationic lipid, 3-(trimethyl ammonium iodide) 1,2 dimystryl-propanediate and a neutral lipid, l-a Dioleoyl phosphatidyl-ethanolamine prepared according to the protocol of Russell (1997). Percoll or swim-up methods were used to select sperm after thawing (Rubessa et al. 2016), followed by incubation (3 h) with the liposome-DNA complexes according to liposome preparation protocol (Russell, 1997). We used enhanced green fluorescent protein in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analysed: 1) immediately after processing (Control), 2) After 3 h of incubation with no liposomes, 3) incubation with liposomes and no DNA, 4) incubation with 1 ng of DNA, and 5) incubation with 10 ng of DNA. This was repeated five times. The CASA results for total motility and rapid motility showed a greater amount of significant differences (P &lt; 0.01) between the control and the other treatments in the Percol group as opposed to swim-up. These results confirm that the sperm selected with swim-up is more stable. Following CASA analysis, sperm was washed with PBS twice and collected in tubes. The DNA from all samples was extracted to determine the quantity of attaching varying amounts of DNA to sperm. The results showed a general increase in DNA concentrations with the increase of DNA added for both methods, but the statistical variation was too large to draw any definite conclusion. In future studies, real-time PCR will be used to determine the quantity of enhanced green fluorescent protein bound to the sperm. Table 1. Results of the computer-assisted sperm analyzer (CASA)

The effects of 405 nm light on bacterial membrane integrity determined by salt and bile tolerance assays, leakage of UV-absorbing material and SYTOX green labelling.

Bacterial inactivation by 405 nm light is accredited to the photoexcitation of intracellular porphyrin molecules resulting in energy transfer and the generation of reactive oxygen species that impart cellular oxidative damage. The specific mechanism of cellular damage, however, is not fully understood. Previous work has suggested that destruction of nucleic acids may be responsible for inactivation; however, microscopic imaging has suggested membrane damage as a major constituent of cellular inactivation. This study investigates the membrane integrity of Escherichia coli and Staphylococcus aureus exposed to 405 nm light. Results indicated membrane damage to both species, with loss of salt and bile tolerance by S. aureus and E. coli, respectively, consistent with reduced membrane integrity. Increased nucleic acid release was also demonstrated in 405 nm light-exposed cells, with up to 50 % increase in DNA concentration into the extracellular media in the case of both organisms. SYTOX green fluorometric analysis, however, demonstrated contradictory results between the two test species. With E. coli, increasing permeation of SYTOX green was observed following increased exposure, with >500 % increase in fluorescence, whereas no increase was observed with S. aureus. Overall, this study has provided good evidence that 405 nm light exposure causes loss of bacterial membrane integrity in E. coli, but the results with S. aureus are more difficult to explain. Further work is required to gain greater understanding of the inactivation mechanism in different bacterial species, as there are likely to be other targets within the cell that are also impaired by the oxidative damage from photo-generated reactive oxygen species.

Enhancing the speed of morpholino-DNA biosensor by electrokinetic concentration of DNA in a microfluidic chip.

PSS) as a cation-selective membrane material. Using the microfluidic concentrator, we could increase the concentration of DNA by three orders of magnitude in less than 5 min at an electric field of 75 Vcm(-1). The 1000-fold increase in concentration of DNA led to an increase in the speed of MO-DNA hybridization by two orders of magnitude and enabled a detection sensitivity of ~1 nM within 15 min of concentration. Using the proposed microfluidic concentrator, we also demonstrated a rapid hybridization with a binary DNA mixture, containing a fully complementary and a non-complementary sequence to mimic molecular backgrounds present in real DNA samples.

Relaxation dynamics and structural changes in DNA soft gels

Abstract Herein, we report relaxation dynamics and structural changes occurring in DNA during transition of its solution to soft gel phase using data obtained from rheology, dynamic light scattering (DLS) and small angle neutron scattering (SANS) experiments. The viscoelastic response showed this transition taking place above a crossover concentration (CDNA = 2% (w/v)). It was observed that at low concentration CDNA = 1% (w/v) solution possessed multiple relaxation dynamics owing to the presence of internal modes at all temperatures in the range, T = 30–80 °C. At higher concentration (CDNA = 3% (w/v)) the system showed single relaxation dynamics because of the systematic arrest of many of these modes. However, at crossover concentration (CDNA = 2% (w/v)), samples showed multiple relaxation modes at high temperature (T = 80 °C) and single mode relaxation at lower temperature (T = 50 °C) with sub diffusive behaviour showing transition from ergodic to arrested phase owing to ceased Brownian dynamics. Normal diffusion in CDNA = 1% (w/v) and sub-diffusive behaviour for CDNA = 2% and 3% (w/v) samples was observed. SANS studies revealed interparticle distance decreased and mesh like structures began to appear with increase in DNA concentration, 2% ≤ CDNA ≤ 5% (w/v). Thus, the observed relaxation dynamics was hierarchical in DNA concentration and the same was temperature dependent. The results augment our general understanding of relaxation dynamics of DNA in aqueous dispersions.

Investigation on the toxic interaction of typical plasticizers with calf thymus DNA

The interactions of typical plasticizers dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) with calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopic techniques and molecular modeling. Experimental results indicated that the characteristic fluorescence intensity of phthalic acid rose with the increase of DNA concentration; while the characteristic fluorescence intensities of plasticizers decreased with the increase of DNA concentration. Experiments on native and denatured DNA determined that plasticizers interacted with DNA both in groove and electrostatic binding mode. The molecular modeling results further illustrated that there is groove binding between them; hydrogen bonding and Van der Waals interactions were the main forces. With the extension of branched-chains, the binding effects between plasticizers and DNA were weakened, which could be related to the increased steric hindrance.