Wednesday, February 29, 2012

Chromium DNA, and Soil Microbial Communities. DNA Working and RNA

Chromium DNA, and Soil Microbial Communities. DNA Working and RNA

The experiments were conducted on a previously uncontaminated soil amended with various levels of hexavalent Cr. Only 2-3% of the Cr was bio available 10 days after addition of the metal. Even though Cr bioavailability decreased rapidly, the bacterial community richness, as measured using denaturing gradient gel electrophoresis (DGGE), was reduced for both the 200 and 1000 mg kg-1 Cr treatments. One year after contamination, bacterial community richness had not returned to pre-disturbance levels. However, the fungal community had increased richness for the Cr-contaminated soils that was maintained for the duration of experiment. Changes in bacterial community richness were significantly related to both the total amount of Cr added and Cr bioavailability. From this research, there appears to be an antagonistic relationship between the fungal and bacterial communities stressed by Cr, which is significantly related to the total metal in the system. Due to technical problems with the amplification of DNA isolated from these chromium containing soils, I hypothesized that Cr was extracted along with the DNA isolated directly from soil. Using size exclusion chromatography with inductively coupled plasma mass spectrometry (SEC-ICP-MS), I determined the DNA extracts contained Cr. However, Cr was not free in solution indicating that inhibition of PCR was related to the environmental DNA samples containing low levels of Cr (0.5-0.7 ng g-1). To further understand the impact of Cr bound to DNA, DNA from an eight member model bacterial community was extracted in the presence of varying concentrations of Cr and community structure was measured using DGGE. The DGGE profiles of the DNA extracted in the presence of Cr had decreases in the number of bands corresponding to the model community, as well as decreases in the intensity of individual bands, as Cr concentrations increased. These results indicate that PCR-based molecular analyses of metal containing DNA may indicate altered community profiles that do not reflect actual changes in the community.

hydralazine-DNA pyrimidine interaction

hydralazine-DNA pyrimidine interaction

Hydralazine is a potent arteriolar vasodilator by producing relaxation of vascular smooth muscle.
The vasodilation is most marked in the splanchnic, coronary, cerebral and renal arterial beds. Some of the symptoms may be caused by vasodilation and histaminic effects. Iron chelation may lead to anemia. (Alarcon-Segovia et al, 1967) A hydralazine-DNA pyrimidine interaction resulting in immune responses to hydralazine and nuclear antigens in which antibodies to native DNA occur can explain the hydralazine-induced lupus erythematosus

STR Amplification and Detection

STR amplification systems can accommodate a range of template DNA concentrations. Most of the Promega PowerPlex® STR systems provide optimal sister allele balance and locus-to-locus balance with 0.5–1.0ng of DNA template, and studies performed at Promega show that full profiles can be observed with less than 100pg (Ensenberger and Fulmer, 2009; McLaren, 2007; Krenke et al. 2005; Krenke et al. 2002). However, amplification and detection instrumentation can vary. You may need to optimize protocols, including cycle number and detection conditions (e.g., injection time or loading volume), for each laboratory instrument. Most of the PowerPlex® systems use a thermal cycling program with 30 or 32 cycles for 0.5–1ng of purified DNA template. For larger amounts of input DNA (i.e., FTA® paper) or to decrease sensitivity, fewer cycles should be evaluated. In-house validation should be performed to determine the optimal amplification and detection conditions.

The sensitive nature of PCR works in a lab's favor, but it can cause problems if great care is not taken to avoid contaminating the reaction with exogenous DNA. Three main categories of exogenous DNA have the biggest impact on DNA-typing laboratories: 1) DNA from the analyst, 2) DNA from other samples in the lab and 3) allelic ladder fragments. DNA from nonhuman sources, such as bacteria and fungi, will not be amplified and detected because STR systems are species-specific. Extreme care must be taken to avoid cross-contamination when preparing sample DNA, handling primer pairs, assembling amplification reactions and analyzing amplification products. Reagents and materials used prior to amplification should be stored separately from those used following amplification. Amplification reactions should be assembled in a room dedicated for reaction setup, using equipment and supplies reserved for amplification setup. We highly recommend the use of gloves and aerosol-resistant pipette tips. To detect reagent contamination, assemble a negative control reaction (i.e., no template) for every set of reactions, and scrutinize the reactions for the presence of unexpected peaks. After setting up reactions, wash all surfaces with a dilute bleach solution.

high-molecular-weight DNA Human Identification

high-molecular-weight DNA Human Identification

In the early days of DNA-based identification, the hypervariable regions of interest were variable number tandem repeat (VNTR) loci, which had a high level of heterozygosity and were relatively large in size (300–10,000bp) (Nakamura et al., 1987; Budowle et al., 1991). VNTRs were analyzed using restriction fragment length polymorphism (RFLP), where high-molecular-weight target DNA is digested with a restriction enzyme that has recognition sites at both ends of the hypervariable region. The size of the DNA fragment resulting from the restriction enzyme digestion is dictated by the number of repeat elements. These fragments are separated by size using agarose or polyacrylamide gel electrophoresis and detected using a labeled VNTR probe. Analysis of multiple VNTR loci results in a unique pattern of DNA fragments on the gel. The patterns generated from a DNA sample of unknown origin and DNA of known origin are compared. Matching patterns indicate that the sources of the unknown and known DNA samples are likely the same. RFLP analysis of VNTR loci works well to resolve immigration and paternity disputes and for other applications where large amounts of intact DNA can be collected. However, RFLP is not ideally suited to forensic investigations because microgram amounts of high-molecular-weight DNA are required.Thus, VNTR analysis is limited to investigations where large amounts of DNA are recovered.

Promega’s DNA isolation systems

The majority of Promega’s DNA isolation systems for genomic, plasmid and PCR product purification are based on purification by silica. Regardless of the method used to create a cleared lysate, the DNA of interest can be isolated by virtue of its ability to bind silica in the presence of high concentrations of chaotropic salts (Chen and Thomas, 1980; Marko et al. 1982; Boom et al. 1990). These salts are then removed with an alcohol-based wash and the DNA eluted in a low-ionic-strength solution such as TE buffer or water. The binding of DNA to silica seems to be driven by dehydration and hydrogen bond formation, which competes against weak electrostatic repulsion (Melzak et al. 1996). Hence, a high concentration of salt will help drive DNA adsorption onto silica, and a low concentration will release the DNA.

Promega has sold and supported silica-based DNA purification systems for nearly two decades. The first technology available was silica resin, exemplified by the Wizard® Plus Minipreps DNA Purification System. The protocol for purification by silica resin involves combining the cleared lysate with a resin slurry and using vacuum filtration to wash the bound DNA, followed by centrifugation to elute the purified DNA.

Monday, February 6, 2012

Ofloxacin Evaluation

Ofloxacin Evaluation

Most ocular treatments like eye drops and suspensions call for the topical administration of ophthalmically active drugs to the tissues around the ocular cavity. These dosage forms are easy to instill but suffer from the inherent drawback that the majority of the medication they contain is immediately diluted in the tear film as soon as the eye drop solution is instilled into the culde-sac and is rapidly drained away from the precorneal cavity by constant tear flow and lacrimo-nasal drainage.
The eye as a portal for drug delivery is generally used for local therapy against systemic therapy in order to avoid the risk of eye damage from high blood concentrations of the drug, which is not intended. The unique anatomy, physiology and biochemistry of the eye render this organ impervious to foreign substances, thus presenting a constant challenge to the formulator to circumvent the protective barriers of the eye without causing permanent tissue damage [‎ 1].
Therefore, only a very small fraction of the instilled dose is absorbed by the target tissue for this reason, concentrated solutions and frequent dosing are required
for the instillation to achieve an adequate level of therapeutic effect. One of the new classes of drug delivery systems, ocular inserts, which are gaining worldwide praise, release drugs at a pre-programmed rate for a longer period by increasing the precorneal residence time [‎ 2-‎ 4].
Ofloxacin is a pale yellow or bright yellow, crystalline powder. Ofloxacin is antibacterial and is the most commonly used fluroquinolone. It inhibits the enzyme bacterial DNA gyrase, which nicks double stranded DNA, introduces negative supercoils and then reseals the nicked ends. This is necessary to prevent excessive positive supercoiling of the strands when they separate to permit replication or transcription. The bactericidal action probably results from digestion of DNA by exonucleases whose production is signaled by the damaged DNA

Tramadol | analgesic | pain | cyclooxygenase

Tramadol | analgesic | pain | cyclooxygenase

pain is defined by the International Association for the Study of Pain (IASP) as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage".Pain has now been equated to a “fifth vital sign” highlighting the significance of pain management in patient care. Tramadol is a centrally acting analgesic, structurally related to codeine and morphine .It is effectively used to treat moderate-to-severe acute and chronic pain in diverse conditions. Tramadol is placed on the second step of WHO analgesic ladder and in contrast to traditional opioids, exerts its analgesic activity, a dual mechanism of action inhibiting transmission as well as perception of pain. Tramadol is more suitable than NSAIDs and coxibs for patient with GI, renal and cardiovascular problems. Combined with low dependence/abuse potential, it has proven to be of significant advantage over other agents, especially in the elderly.

Spherical crystallization | Flowability | Compactability | Bioavailability

Spherical crystallization | Flowability | Computability | Bioavailability

Spherical crystallization is a particle design technique, by which crystallization and agglomeration can be carried out simultaneously in one step and which has been successfully utilized for improvement of flowability, compactability and bioavailability of crystalline drugs. General methods of spherical crystallization are spherical agglomeration, emulsion solvent diffusion and ammonia diffusion method. The principle steps involved in the process of spherical crystallization are flocculation zone, zero growth zone, fast growth zone and constant size zone. Spherical crystallization is having wide applications in pharmaceuticals like improvement of flowability and compressibility of poorly compressible drugs, masking bitter taste of drugs and improving the solubility and dissolution rate of poorly soluble drug.


Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.