In case of such an unlikely event, the production steps have sufficient capacity to remove prions. This research was supported in its entirety by Biotest AG, Dreieich, Germany and Biotest Pharmaceuticals, Boca Raton, FL, USA. We also thank NewLab; Cologne, Germany for excellently performing the studies

on HIV, WNV and BPV, and all technicans in the Pathogen Safety Lab at Biotest Dreieich. The technical support provided by John Hooper (BioCatalyst LLC, Liberty, Missouri, USA) is greatfully appreciated. “
“The CD2 family receptor (CD2f), part of the immunoglobulin (Ig) superfamily, includes 11 cell surface molecules in mammals. GS-7340 solubility dmso The CD2f are widely expressed on various types of leukocytes, such as natural killer (NK), T- and B-cells, macrophages, and dendritic (DC) cells, and they have different functions in relation to regulation or activation of immune responses. All CD2f consist of IgV and IgC2 domains, a transmembrane, and a cytoplasmic tail; however, some members only possess a short

cytoplasmic tail [3], [6], [7], [14] and [22]. The CD2 and CD58 genes are on the short arm of human chromosome 1 and the other members are clustered close together on the long arm of chromosome 1 [8]. This structural similarity and genomic localization suggest that the CD2f arose from an ancestral gene through successive gene duplications. The CD150 (signaling lymphocytic activating molecule: SLAM) subfamily mTOR target of the CD2f consists of CD150 along with NTB-A, CD319 (CS1), CD84, CD229, and CD244 (2B4). The most characteristic feature of the CD150 family is the presence of 2–4 immunoreceptor tyrosine-based switch motifs (ITSM) in their cytoplasmic tails. The ITSM motif has been shown to interact with small adaptor molecules through

Src homology 2 (SH2) domains (examples include SAP, EAT-2A, and EAT-2B (ERT) and their interaction triggers) to induce activating or inhibiting responses of Farnesyltransferase T- and B-cells or NK cells mediated by tyrosine phosphorylation signals [18], [22], [36] and [37]. Therefore, the presence of the ITSM motif is important for classification of the CD2f based on their functionality. Ig, TCR, MHC I, and MHC II as well as other proteins that function in immune recognition have been identified in teleosts in forms resembling their mammalian orthologs [2], [4], [9], [11], [23] and [30]. In addition, novel immune-type receptors (NITRs), which are encoded by clusters of multigene families, have been identified in a number of bony fish species [32] and [40]. This discovery of NITRs indicates that Ig superfamily (IgSF) receptors diverged extensively before the emergence of teleosts. These studies have shown that fish leukocytes express many types of IgSF receptors for contact with other immune cells. A recent report has also shown that at least seventy CD2-like genes are present in the genome and cDNA databases of Siurana tropicalis, suggesting that CD2f genes expanded and diverged in the lower vertebrates [10].

The number (quantity) of clinic staff members was positively associated with net income in 1996. However, in 2006, the number of staff members was no longer associated with net income. Instead, net income was associated with the types of services provided or the source of payment for those services. These results suggest that financial retrenchment has occurred in private dental clinics MK-2206 price in the recent years. Using data from a survey on the management of private dental clinics in October, 2008, 1240 private dental clinics were categorized into quintiles

according to net income. The higher quintiles (having higher net income) had younger owners (dentists) and larger median values of the number of patients per month. The number of full-time dental hygienists increased with the level of quintile (Fig. 5). Private dental clinics established after 1989 were more frequently categorized into the highest or second

highest quintile compared to those established before 1989. Moreover, the proportion of income from treatments not covered by health insurance in the total income gradually increased from the lowest quintile (4.1%) to the highest quintile (8.5%). These results suggest that private dental clinics opened after 1989 (new), and having dental hygienists (dental hygienists may conduct dental maintenance) had a large net income, suggesting that they may be better managed, they may have a different case-mix of

care, or they may have different sources enough of revenue. MG-132 Prevalence of dental caries has decreased, while that of periodontal disease has remained increasing [8]. Moreover, the number of private dental clinics has been increasing. Therefore, dental treatment needs may have shifted from dental caries to periodontal disease. As young dentists may have received new information, especially on periodontology, they employ dental hygienists, run a system of supportive periodontal treatment in their clinics, and consequently, have large net income. Analyses using data from the Central Social Insurance Medical Council also showed that dental clinics with more dental hygienists made higher profits [9]. A similar association between employing dental hygienists and income of dentists has also been reported in the United States [10] and [11]. Moreover, as young dentists have knowledge of dental implants, they treat patients using dental implants, which is not covered by public health insurance and brings in a large net income. Fig. 6 shows changes in the net income as well as total expenses of private dental clinics from 1996 to 2010, based on surveys conducted by the Japan Dental Association [5], [7] and [12]. Net income decreased from 1996 to 2004 with the lowest net income in 2004, after which point the value gradually increased until 2008.

It should be noted that using oxidants such as methylene blue in patients with G6PD deficiency may cause severe hemolytic crisis.5 and 6 There are two forms of methemoglobinemia, congenital and acquired forms. Diaphorase-I deficiency, hemoglobin variants (Hgb H, Hgb M) and G6PD deficiency are major causes of congenital form.1 Environmental exposure and toxins are acquired causes of the disorder. Nitrates, chlorates, aniline are agents which may lead to methemoglobinemia.7 In a retrospective study 138 cases of acquired methemoglobinemia were selleck examined and etiologic agents were found to be dapsone in 42%, benzocaine

in 4%, primaquine in 4% of the patients. A known side effect of dapsone therapy is methemoglobinemia during its usage in autoimmune diseases and prophylaxis against Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii. In another study performed on 242 cases of methemoglobinemia related with anesthesia and benzocaine found to be responsible in 60% of the cases.8 In our case there was no history of drug use or environmental exposure, and the most significant complaint was shortness of breath induced by exercise. Considering that the patient’s brother also died

with similar complaints at the age of fifteen, strengthening the idea that methemoglobinemia Autophagy inhibitor cost is congenital in this patient. The diagnosis and treatment of bronchial asthma by previous physicians, may be explained with insufficient investigation of the complaints, findings and history. After exposure to fenasetin and sulfanamid a rare disorder, sulfhemoglobinemia, may occur which can be clinically confused with methemoglobinemia. The distinction can be made by non-responsiveness of sulfhemoglobinemia to treatment with methylene blue. Additionally, the definitive diagnosis can be made by spectrometric examination.9 Administration of 1–2 mg/kg IV methylene blue and oral vitamin C have great help for the treatment of methemoglobinemia. N-acetylcysteine, cimetidine and ketokanazol all are experimental but promising treatments in methemoglobinemia yet. Exchange transfusion is

an alternative treatment in patients who are refractory to methylene blue.10 We met a similar methemoglobinemia case in the literature who was treated as asthma previously. Because cyanosis and shortness of breath complaints were constant despite the asthma therapy, the patient was detected and homozygous type 1b5r deficiency was found in the patient. The authors stated that the patient was freed from unnecessary treatment and the use of unnecessary drugs.11 Existence of dyspnea, hypoxemia and cyanosis in a patient firstly signs heart diseases (atrial septal defect, ventricular septal defect, etc.) and lung diseases (pulmonary embolism, etc.). Therefore evaluation of these diseases is a normal procedure.

However, to date, no studies have been published on the production and stability evaluation of bixin nanocapsules. Indications that bixin may be important to human health and bixin’s prevalence in the food industry as a colourant and antioxidant motivates the study of nanoencapsulation as a suitable technique for increasing the solubility of bixin in aqueous media. Therefore, the aim of this work was to prepare and characterise bixin nanocapsules and to evaluate their stability during storage. The polymer poly-ɛ-caprolactone (PCL) (Mw = 80,000) and sorbitan monostearate (Span 60) were

obtained from Sigma (St. Louis, MO, USA). The capric/caprylic triglycerides (CCT) and polysorbate 80 (Tween 80) were purchased from Delaware (Porto Alegre, Brazil). Annatto seeds were obtained from the local market Selleck ABT199 in Porto Alegre, Brazil. All Z-VAD-FMK mw other chemicals and solvents were of analytical or pharmaceutical

grade. A bixin standard was prepared in triplicate according to the method of Rios and Mercadante (2004). This method consisted in the production of a bixin standard extracted from annatto seeds. Annatto seeds (25 g) were twice washed with hexane (100 mL). The solvent was discarded and the seeds were washed twice with methanol (100 mL). Methanol was also discarded and bixin was extracted from the seeds with ethyl acetate (100 mL). Each wash or extraction was carried out under magnetic stirring during 15 min. The extract was TCL filtered and concentrated under reduced pressure in a rotary evaporator (Fisatom, model 801/802, São Paulo, SP, Brazil). After concentration, the recipient containing the extract was placed in a cold bath and dichloromethane (5 mL) was added slowly to this extract. After the addition of dichloromethane, ethanol

(99.7%) was added slowly (20 mL). This solution was held at −18 °C during 12 h for crystallisation. The crystals formed in the bottom of the recipient were filtered, washed with 50 mL of ethanol (99.7%) and dried under reduced pressure (T < 30 °C). The purity of the standard was evaluated by high performance liquid chromatography (HPLC). Bixin nanocapsules were prepared by the technique of interfacial deposition of preformed polymers according to the method of Venturini et al. (2011). The polymer (PCL) (250 mg), triglycerides (CCT) (400 μL), span 60 (95 mg) and bixin were dissolved in a mixture of acetone (60 mL) and ethanol (7.5 mL) under magnetic stirring at 40 °C. After the solubilisation of PCL, CCT and Span 60, the standard of bixin (98.7%) was added and remained under magnetic stirring for 10 min (40 °C). This organic phase was added into an aqueous phase (130 mL) containing Tween 80 (195 mg) and remained under stirring for 10 min. The dispersion was concentrated under reduced pressure until it reached a final volume of 25 mL. In this method, acetone and ethanol (a water-miscible solvent) were used to solubilise PCL and Span 60.

The quadratic regression coefficient of time (X1) and temperature (X2) significantly increased the total phenols while the concentration (X3) decreased. The interation between the time (X1) and temperature (X2) had a negative and significant effect, while interation of time (X1) and concentration (X3) had a positive and significant effect, as can be observed in Eq. (6): equation(6) Y=580.77+30.01X3+73.56X12+58.09X22-52.34X32-58.73X1X2+122.06X1X3.

Total flavonoids varied statistically (p   < 0.001) from 197.92 (assay number 12) to 333.76 mg/100 g (assay number 2). The highest values were found in the extraction with 65% acetone, for 20 min at 10 °C. The model of flavonoids was significant (p   < 0.001), did not present lack of fit (p   = 0.20), and it could explain 98.20% of variance in data (( Radj2 = 0.96). The temperature (X2) this website and acetone concentration (X3) significantly decreased the flavonoid levels and the quadratic regression coefficient of time (X1) was positive and significant, whereas concentration (X3) was

negative and significant Trametinib research buy Eq. (7): equation(7) Y=266.29+5.99X1-8.84X2-11.64X3+34.20X12-34.47X32-18.34X12X2+22.54X12X3-14.01X2X3. The DPPH varied significantly (p   < 0.001) from 1615.61 (assay number 3) to 3194.00 mg/100 g (central point). Extraction with 65% acetone for10 min at 40 °C had the lowest values, but higher antioxidant capacity. The RSM application on DPPH showed that the model was significant (p   < 0.001), did not present lack of fit (p   = 0.11), and could explain 77.55% of all variance in data (( Radj2 = 0.71). The acetone concentration (X3) significantly increased the DPPH levels. The quadratic regression coefficient of time (X1) and temperature (X2) was negative and significant, according to Eq. (8): equation(8) Y=2994.92+248.19X3-734.81X12-495.26X22 The FRAP

values ranged statistically (p   < 0.001) from 1009.62 (assay number 6) to 2021.15 μM/100 g (assay number 2). For obtaining compounds with high antioxidant capacity, extraction with 65% acetone at 10 °C for 20 min selleck compound should be performed. The RSM application of FRAP values showed that the model was significant (p   < 0.001), did not present lack of fit (p   = 0.06), and could explain 91.21% of all variance in data (( Radj2 = 0.85). The time (X1) and concentration (X3) significantly increased the FRAP levels. The quadratic regression coefficient of time (X1) and concentration (X3) was negative and significant, and the quadratic regression coeficient of temperature (X2) was positive and significant. The interation of time (X1) and concentration (X3) had a significant effect, as shown in Eq. (9): equation(9) Y=1880.04+135.05X1+105.41X3-327.96X12+216.34X22-227.16X32+278.60X1X3. The best yields in phenolic extraction were obtained with 65% acetone solution. This indicates that aqueous solutions are better in the phenolic extraction of apples.

The ethanol was removed using a rotary evaporator, before the resulting aqueous solution, containing catechins, was dissolved in acetate buffer (pH 6.0, 0.2 M) for identification of the compounds present. Fifty milliliter of distilled water and 250 mg of each sample of tea were combined in 125 ml Erlenmeyer flasks. The extraction of compounds from green tea and yerba mate was performed in a water bath Selleckchem GS 1101 at 100 °C for 30 min. After being filtered on filter paper, the extracts were freeze-dried. The

resulting powder was called dried tea extract and used for antioxidant assays (Cao et al., 1996). As an identified representative polyphenol from green tea, the commercial standard epigallocatechin gallate (EGCG, 95%) was used as a control sample, as was the chlorogenic acid (95%) from yerba mate tea. These samples were tested for antioxidant power (by DPPH and ORAC assays) and treated with tannase, using the same procedures that were employed on the tea extracts. The extracts obtained from the green tea, yerba mate and the commercial control samples were used as substrates for enzymatic hydrolysis by tannase isolated from Paecilomyces variotii ( Battestin, Macedo, & Freitas, 2008). The dried tea extract (5 mg) was dissolved in 1 ml of phosphate buffer (pH 7.4, 75 mM) and incubated with selleck chemicals 5 mg of tannase at 40 °C

for 30 min. The hydrolysis process was stopped by placing the reaction in an ice bath for 15 min. The biotransformed tea was used for the antioxidant assay after suitable dilution with the same phosphate buffer (pH 7.4, 75 mM) for ORAC and with a 70% methanol solution for DPPH. A Finnigan Surveyor-series liquid chromatograph, equipped with a 150 × 4.6 mm i.d., 5 μm LicroCART® (Merck,

Darmstadt, Germany), reversed-phase C18 column maintained at 25 °C by a thermostat, was used. Mass detection was carried out using Doxacurium chloride a Finnigan LCQ DECA XP MAX (Finnigan Corp., San José, CA, USA) mass detector with an API (atmospheric pressure ionisation) source of ionisation and an ESI (ElectroSpray ionisation) interface. The solvents used were formic acid in H2O (1%, v/v) and acetonitrile. The capillary voltage was 4 V and the capillary temperature was 275 °C. The spectra were recorded in the positive-ion mode between 120 and 1500 m/z. The mass spectrometer was programmed to carry out a series of three scans: a full mass, a zoom scan of the most intense ion in the first scan, and a MS–MS of the most intense ion using relative collision energy of 30 and 60. The ORAC method used, with fluorescein (FL) as the ‘‘fluorescent probe”, was described by Ou, Huang, Hampsch-Woodill, Flanagan, and Deemer (2002) and modified by Dávalos et al. (2004). The automated ORAC assay was carried out on a NovoStar Microplate reader (BMG LABTECH, Germany) with fluorescence filters for an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The measurements were made in a COSTAR 96 plate.

A catalogue of generic and specific exposure scenarios (ESs) has been developed for engineered nanomaterials (ENM), taking into account the release scenarios over the entire life-cycle of these materials (Brouwer et al., 2010 and Clark et al., 2012). For occupational exposure scenarios, published measurement data and contextual information were collected. These were reviewed to describe and characterize occupational exposure and the available tools and models to predict occupational exposure

to the ENMs. For the development of generic exposure scenario descriptions, a library for the collection of exposure scenarios according to REACH Guidance was developed. From the 57 occupational exposure scenarios PCI-32765 manufacturer (Brouwer et al., 2010), 14 are related to carbon-based nanomaterials, generating 35 contributing

exposure scenarios describing some facet of occupational exposure. Most of the ESs were from the production/synthesis of carbon-based nanomaterials or from handling materials (weighting, removing, sonication, etc.); two scenarios addressed tasks related to the machining of composites containing CNT. Based on the process of developing these ESs, several main conclusions could be drawn (Clark selleck chemical et al., 2012): Most studies reported had an explorative character and were focused on concentration/emission analysis. Therefore, the reports from these studies did not include most of the information necessary to build ESs, e.g. amount used and frequency of activities. Basic characterization of the products used was often not available and operational conditions were often not described. Most concentration/emission-related measurement results were task-based. An important observation was the

lack of harmonization of either the measurement strategy including distinction between manufactured nanoaerosols and ‘background’ aerosols, or the ifenprodil analysis and reporting of measurement data. ENM-release during synthesis is best described by an emission factor (EF), which is defined as number, surface area and/or mass (volume) per unit of time released to the environment (Fissan and Horn, 2013). The ENM-release per unit of mass of produced material is best described by a release factor (RF), defined as number, surface area and/or mass (volume) per unit of mass of nanostructured material (Fissan and Horn, 2013). This depends on nanostructured material properties and the amount and kind of energy input during the different kinds of treatments of the material. The ENM emission and release factors can be considered to be important process and material properties, since without emission and release there is no exposure and therefore no risk.

Khwaja and Roy [4] have given nutrient ranges in ginseng based on extensive sampling of growers’ fields. Minimum and maximum B concentrations in leaves of 2–4-yr-old plants were: 5 μg/g, deficient; 5–15 μg/g, low; 16–50 μg/g, sufficient; 51–100 μg/g, high; and >100 μg/g, excessive. Konsler and Shelton [5] and Konsler et al [6] described the effect of lime and phosphorus on the growth, nutrient status, and ginsenoside content of the ginseng root. Ginseng production in Ontario, Canada, the major center for American ginseng culture,

is on sandy and sandy-loam soil with low organic matter content, along the north shore of Lake Pictilisib cost Erie [7]. In general, these soils are low in B for production of many crops [8] and [9]. Previously, we reported that the rusty root of ginseng and associated internal browning of roots grown in the above-mentioned soils may be linked to B deficiency [10]. B is required by plants only in small amounts, therefore, overapplication click here to crops can occur easily.

Oliver [11] recommended that to maintain adequate soil levels of B for ginseng cultivation, 1–2 kg/ha should be applied when soil tests show ≤0.5 μg/mL. B is taken up through the plant roots as boric acid and transported with the transpiration flow. In most plants, B is highly immobile [12], being restricted to the transpiration stream. Accumulation of B can occur at the end of the transpiration stream in the leaves [13]. Manifestation of B toxicity shows as damage to tissues where it accumulates. Although B toxicity is crop-specific, Ceramide glucosyltransferase it generally leads to chlorosis and necrosis starting at the edges of mature leaves [12] and [13]. This development of necrotic areas can reduce leaf photosynthetic potential, cause a reduction of photosynthetic supply to the

developing root system, the economic part of the ginseng plant, and restrict activity in the meristematic tissues. It is unclear why B is toxic to plants, or why some plants can tolerate B and evade toxicity [13]. Reid et al [14] concluded that, at high B concentrations, many cellular processes are retarded and these are often made worse in light by photoxidative stress. Ginseng is a perennial plant requiring about 4 yr from seeding to root harvest, therefore, we examined the possibility of using radish as a time-saving model system in our B nutritional studies. Radish requires 3–6 wk from seeding to root harvest and B deficiency induces root splitting and brown heart disorder [15], similar to brown heart in ginseng [10]. Also, B toxicity in radish reduces root growth [16] and [17]. Lack of definitive data on B nutrition of American ginseng, the supposed deleterious effects on the leaves, roots, and meristematic regions, and an application of a high concentration of B to commercial ginseng plantings prompted this investigation.

, 2008). In addition, since geographically-proximate timber trees are (typically) more similar than those farther apart, even trees not individually fingerprinted before harvesting can be tracked based on reference samples, allowing discrimination between legal concessions and illegal harvest zones (see, e.g., GTTN, 2014). To respond to climate change, Alfaro et al. (2014) indicate the importance of new breeding approaches (e.g., El-Kassaby et al., 2012). This is because current methods are often too slow to respond adequately

due to long generation times in breeding cycles (Yanchuk and Allard, 2009). Such approaches are facilitated by advances in genomics, but the importance of participatory domestication, working with local communities, also has much find more to offer (Dawson et al., 2014 and Leakey et al., 2012). Another important issue to address is the role of epigenetic buffering in climate change responses (Aitken et al., 2008). The most well known example of epigenetic effects in trees is variation in the phenology of bud set in Norway spruce (Picea abies; Johnsen et al., 2009), but similar effects have been observed in other species (e.g., Greenwood and Hutchison, 1996 and Webber et al., 2005). There is, however, a general

lack of information on epigenetic VX 809 effects in angiosperm trees ( Rohde and Junttila, 2008). Finally, further studies on geographic patterns of molecular genetic variation in trees in combination with more advanced ensemble methods of past-, present- and predicted future-climate ecological niche modelling are required to understand http://www.selleck.co.jp/products/Verteporfin(Visudyne).html climate impacts on species and forests,

and prioritise geographic regions for conservation (Cavers and Dick, 2013, Lefèvre et al., 2013 and Thomas et al., 2012). Because data on tree species distributions are often deficient, the utility of vegetation maps as proxies for distributions is also an important area of research (VECEA, 2014). Bioversity International and ICRAF are part of the CGIAR Consortium Research Programme on Forests, Trees and Agroforestry (www.foreststreesagroforestry.org/). We thank colleagues within the Forest Genetic Resources Programme (Bioversity International), Science Domain 3: Tree Diversity, Domestication and Delivery (ICRAF) and Forestry Department (FAO) for their advice in writing this editorial. “
“The elemental role played by trees in the lives of rural people in the tropics appears obvious through the many uses made of tree products, in construction, fencing, furniture, foods, medicines, fibres, fuels and in livestock feed, and in their cultural value. Indeed, in a World Bank report published a few years ago, forests and trees-outside-forests were reported to contribute to the livelihoods of more than 1.6 billion people worldwide (World Bank, 2008).

Amplified samples and allelic ladder from the PowerPlex® ESI 17 Fast System were processed for electrophoresis on the ABI PRISM® 310 Genetic Analyzer with POP-6™ polymer Selleckchem ATM/ATR inhibitor as per instructions in the PowerPlex® ESI 17 Fast System Technical Manual [15]. POP-6™ polymer provided better resolution than POP-4™ polymer of larger alleles that are 1 base apart, such as is the case with D2S441, D12S391, and D1S1656 in the PowerPlex® ESI Fast Systems. One microliter of amplification product or allelic

ladder was combined with 23 μL Hi-Di™ formamide and 2 μL of CC5 ILS 500 Pro. Samples were heat denatured as described above. Injection was performed at 15 kV for 3 s. Data were analyzed using GeneMapper®ID 3.2.1 software (Life Technologies, Foster City, CA) and a 50 RFU detection threshold. To provide information on the effect of increased magnesium chloride or the effects of magnesium chelation on the results, titrations of increasing magnesium chloride (MgCl2) concentration (0.25 mM, 0.5 mM HDAC activation and 1 mM) or increasing EDTA concentration (0.1 mM, 0.25 mM, 0.5 mM, and 1.0 mM) were carried out with all four systems. To evaluate the effect of pipetting errors on performance of the PowerPlex® ESI Fast and ESX Fast Systems, amplification reactions were performed with final concentrations of either the Master Mix or Primer

Pair Mix of 0.8×, 0.9×, 1.0× (recommended), 1.1×, and 1.2×. Cycle number was examined with both purified DNA and all direct amplification samples. For purified DNA samples, amplification reactions were performed at 28, 30 (recommended), and 32 cycles of PCR. For direct amplification samples, amplification reactions were performed at 25, 26, and 27 cycles. The effect SDHB of annealing temperature was examined with both purified DNA and blood and buccal samples on 1.2 mm FTA® punches. Amplification reactions were performed at annealing temperatures of 56 °C, 58 °C, 60 °C (recommended), 62 °C and 64 °C. Purified DNA and direct amplification samples (blood on FTA® cards, blood on ProteinSaver™

903®, and buccal cells collected on OmniSwabs™) were amplified at full (25 μL) and half-volume (12.5 μL) reactions. For purified DNA samples, amplification reactions were performed with 500 pg and 50 pg 2800M Control DNA (constant mass) as well as no-template. Reactions were also performed with 20 pg/μL and 2 pg/μL 2800M Control DNA (constant concentration) in both reaction volumes. For direct amplification samples, 25 μL and 12.5 μL reactions were performed with 26, and 25 cycles, respectively (reduced cycle number required for 12.5 μL amplification reaction due to the two-fold increase in DNA concentration that results from a two-fold reduction in volume). A single 1.2 mm punch was used for both reaction volumes.