The estimated multi-locus outcrossing rate ™ for M huberi was

The estimated multi-locus outcrossing rate ™ for M. huberi was high (0.98 ± 0.111) suggesting that the species is predominantly allogamous. M. huberi showed high levels of genetic diversity, however this species also presents a high rate of endogamy, i.e. a deviation from Hardy–Weinberg equilibrium, most likely caused by crossing among related individuals

(tm − ts = 0.277) and a high spatial genetic structure up to 450 m. Pollen flow was the most restricted among the studied species. These results suggest that in situ conservation management programs for the species should include large areas, avoiding fragmentation to minimize isolation by distance effects. Azevedo et al. (2007) recommended reduced impact selective logging, and that removal of trees should be randomized to avoid fragmentation buy ABT-263 and sub-population losses. The significant spatial genetic structure observed in the population (as a whole) at a radius of 450 m was not detected after exploitation, with the genetic structure observed

in commercially exploited trees lost. There appears to be a significant difference in the pattern of genetic diversity and endogamy in the new generation. The fixation index of 0.26 in seedlings before logging was decreased to 0.06 after logging (unpublished data). AZD9291 cost Dipteryx odorata pollen from inside the plot originated from relatively few pollen donors per mother tree (2.6 trees pre-logging, 1.7 post-logging) relative to the total number of potential pollen donors (pre-logging 66, post-logging 39). Strong asynchrony in flowering is likely to be limiting reproduction, and this aspect has serious consequences for species being managed by selective logging due to the possibility of a mother tree having no breeding partners if the area being managed is a 500 ha (or smaller) fragment with no possibility of pollen flow from other fragments ( Vinson, 2009). Hymenea courbaril showed high pollen flow movement with low biparental inbreeding selleck chemical (tm − ts = 0.096), however, a high spatial genetic structure was observed (Fij = 0.227 up to 100 m and Fij = 0.139 up to 300 m), possibly as a consequence of gravity

seed dissemination ( Lacerda et al., 2008). The results suggest that logging produced an increase in the number of pollen donors and further pollen dispersal. Logging may also result in a significant reduction in the genetic diversity within the progeny of the species and an increase in self-fertilization ( Carneiro et al., 2011). Symphonia globulifera showed a distinct spatial genetic structure (θxy = 0.119 up to 50 m; comparable to that of half sibs with θxy = 0.125) possibly as a consequence of gravity seed dissemination ( Carneiro et al., 2007). Although S. globulifera has a low number of pollen donors (Nep = 2.4–4.0), low selfing and biparental inbreeding rates (ts = 0.0–0.11 and tm − ts = 0.063–0.093, respectively) were detected.

Excluding the primate species, no peaks were detectable above 50 

Excluding the primate species, no peaks were detectable above 50 RFU in either PowerPlex® ESI 17 Fast or ESX 17 Fast for any of the domestic animal or microbial samples except

for A. lwoffi which showed a CH5424802 research buy peak at 292–293 bases with a height of 94–108 RFU in the green dye channel (D10S1248) of PowerPlex® ESI 17 Fast and also at 89–90 bases with a height of 116–129 RFU in the green dye channel (D10S1248) of PowerPlex® ESX 17 Fast. In both cases the peaks migrated on-ladder as an 11 allele. A. lwoffi is part of the normal oropharynx and skin flora of about 25% of individuals [27] with a genome size of 3.48 Mb. At 10 ng DNA in an amplification reaction, this equates to 2.6 million genome copies which suggests a low level cross hybridization to give a peak of the height seen. Primates show the most amplification peaks with fewest being seen with macaque, followed

by gorilla and orangutan with a comparable number of peaks and the greatest number with chimpanzee (Supplemental Fig. 18). The results from the 20 mock crime stain samples amplified with the PowerPlex® ESI Fast Systems were either the same or better than the equivalent AmpFlSTR® SGM Plus® results in terms of number of alleles recovered (Supplemental Table 6). Full profiles were concordant PCI-32765 in vitro with the AmpFlSTR® SGM Plus® profile of the major donor. In general the 44 mock crime stain samples amplified with the PowerPlex® ESX Fast Systems generated allele calls that were concordant with those obtained with the Investigator®

ESSplex Plus Kit with recovery of similar numbers of alleles (Supplemental Table 7). However, a few samples had apparent discordant allele calls. In the case of samples 13-031518R-01-1, about 13-031880R-01-1, and 13-031881R-01-1, the PowerPlex® ESX Fast Systems called a 16.3 allele at D1S1656. This was labelled off-ladder with the Investigator® ESSplex Plus Kit due to the allele migrating just to the right edge of the 16.3 allele bin position. In addition, in sample 13-031881R-01-1 there was an apparent discordance at D1S1656 with the Investigator® ESSplex Plus Kit calling this as an OL, 17.3 whereas PowerPlex® ESX 17 Fast genotyped this sample as 16.3, 18.3. This sample was a low level mixture (PowerPlex® ESX 17 Fast profile showed three alleles at the SE33 locus) and gave a partial profile for the major contributor with Investigator® ESSplex Plus and a full profile for the major contributor with PowerPlex® ESX 17 Fast. However, amplification of the major contributor to this mixture with PowerPlex® ESI 17 Fast and ESX 17 Fast (primer pairs for D1S1656 being different between these two multiplexes) gave a genotype of 16.3, 18.3 with both kits as well as with the Investigator® ESSplex Plus Kit (Supplemental Fig. 19). Thus, the discordance seen with this casework sample appears to be due to this being a low level mixture with the 17.3 allele possibly being due to the second minor contributor in this mixed casework sample.

Symptoms of cerebral malaria evaluated through modified SHIRPA pr

Symptoms of cerebral malaria evaluated through modified SHIRPA protocol, such as: paralysis, Pictilisib order piloerection, and locomotor activity were only observed up to 5 days post-infection (data not shown). Furthermore, at day 5, an increase in parasitemia (19%) as well as in Evans blue accumulation in brain tissue and W/D lung ratio during P. berghei infection was observed ( Fig. 1C–D). P. berghei-infected mice demonstrated a greater number of areas with alveolar collapse ( Fig. 2A and D), neutrophil infiltration ( Fig. 2B and E) and interstitial oedema at days 1 and 5 compared to SAL mice ( Fig. 2C and F). However, the value of each of these parameters for infected

mice was higher at day 5 compared to day 1. Neutrophil infiltration was also observed when lung tissue was submitted to a Percoll gradient (neutrophil count in lung tissue SAL vs P. berghei-infected, at Neratinib in vivo day 1: 0.49 ± 0.11 × 106/lung tissue vs 0.73 ± 0.05 × 106/lung tissue, p < 0.05 and at day 5: 0.30 ± 0.07 × 106/lung tissue vs 0.67 ± 0.06 × 106/lung tissue,

p < 0.05). At day 1, there were more areas with interstitial oedema than observed at day 5 ( Fig. 1C). Since a heightened inflammatory response was observed in the lung tissue 1 day post-infection, cytokine production was also evaluated at this time point. IFN-γ production in the lung tissues of infected mice was lower at day 1 and higher than SAL mice at day 5 (Fig. 3A). TNF-α production was greater by day 5, but not by day 1, in these mice (Fig. 3B). Conversely, CXCL1 production was greater on both days 1 and 5 post-infection, greater at day 5 compared to day 1 (Fig. 3C). Levels of these cytokines were also measured in distal organs, but no significant differences were observed between P. berghei-infected mice and controls at days 1 and 5 (data not shown). At day 1, static lung elastance (Est,L) (Fig.

4A), resistive pressure (ΔP1,L) (Fig. 4B), and viscoelastic/inhomogeneous (ΔP2,L) pressure (Fig. 4C) were significantly greater in P. berghei-infected mice (+36%, 75% and 33%, respectively) compared to SAL mice, and these parameters remained elevated until day 5. These mechanical parameters were lower at day 5 post-infection than at day 1 in infected mice (Est, 27%; ΔP1, 60%; ΔP2, 20%). To evaluate CYTH4 the occurrence of pathological events in distal organs during P. berghei infection, photomicrographs of brain, heart, liver and kidney specimens from mice in the control and severe malaria groups were obtained at days 1 and 5 ( Fig. 5). The brains of P. berghei-injected mice exhibited cortical oedema, glial cell swelling, and congested capillaries, with erythrocytes adhered to the endothelium, causing occlusion, at days 1 and 5. However, an increase in the number of microglial cells was only observed 5 days post-infection ( Fig. 5, Table 1). The hearts of P. berghei-infected mice demonstrated interstitial oedema of the myocardium, which was more marked at day 5 than day 1.

Active bipolar superficial electrodes consisting of two parallel

Active bipolar superficial electrodes consisting of two parallel rectangular Ag/AgCl bars

(1 cm in length, 0.78 cm2 of contact area) were used with an internal amplifier to reduce the effects of electromagnetic interference and other noise. For SMM, the electrodes were fastened to the lower third of the muscle belly, which was identified by palpation during manually resisted flexion of the neck (Falla et al., 2002). For ABD, the electrodes were placed 2 cm away from the umbilicus on the rectus abdominal muscle (Duiverman et al., 2004). The ground electrode was fixed on the ulnar styloid process. All of the electrodes were fixed on the right side. The EMG signal collection and analysis were carried out as recommend by the International Society for Electrophysiology and Kinesiology (Merletti, 1999). The activity of the respiratory muscles was analyzed by the root mean square (RMS) method. The participants were asked to quantify their sensation of dyspnea at rest and immediately after ILB on a scale of Epacadostat 0–10 using the modified Borg scale. The sample size was based on being able to detect at least a difference of 300 ml in the chest wall tidal volume (Romagnoli

et al., 2011). Considering our data of pilot study with six subjects (mean and standard deviation), a two-sided alpha of 0.05 and a statistical power of 0.80, the target sample size was set at 13 individuals. Thus, 15 patients were selected to account for the possibility of dropouts. The chest wall volumes measured during the six minutes at rest and two minutes of ILB (90–210 s) were analyzed using specific software. The mean rest values were compared to the ILB values with Student’s t-test or Wilcoxon’s test, depending on the data distribution. The EMG signals were processed according to the time-domain. One minute of

the signal (30–90 s) from the second set of two minutes at rest and one minute of the signal from the ILB (120–180 s) were analyzed. We evaluated the change Thiamine-diphosphate kinase from rest to ILB period expressed as percentage (relative change) analyzing by the Mann–Whitney test. All of the statistical procedures were carried out using the Statistical Package for Social Science (SPSS, 15.0, Chicago, IL, USA). The level of significance was set at p < 0.05. Fifteen patients with COPD were initially evaluated. Two of the patients were excluded because they were not able to complete five minutes of ILB. Therefore, 13 participants were included in the analysis. However, the chest wall volume and muscular activity correlation was calculated from 12 participants, as artifacts in the EMG signal analyses precluded the use of the data from another participant.

The segment between the Garrison and Oahe dams was divided into f

The segment between the Garrison and Oahe dams was divided into five geomorphic reaches termed: Dam Proximal, Dam-Attenuating, River-Dominated Interaction, Reservoir-Dominated Interaction, and Reservoir. The divisions are based on changes in cross-sectional area,

channel planform, and morphology, which are often gradational. The Dam Proximal reach of the river is located immediately downstream of the dam and extends 50 km downstream. The cross-sectional data and aerial images suggest that the Dam Proximal reach of the river is eroding the bed, banks, and islands (Fig. 5). The ERK inhibitor in vivo standard spatial deviation of cross sectional area for all cross sections on the river in 1946 was 269 m2. All 22 sites examined in the Dam-Proximal

reach (Appendix A) experienced an increase in cross-sectional area that is greater than this natural variability. As an example, Fig. 3A is a typical cross-section in the Dam Proximal reach and has lost 1364 m2 of cross-sectional area between BMS-777607 purchase 1954 and 2007 (Fig. 3A, Eq. (2)). The thalweg elevation at the transect decreased by as much as 1.5 m between 1954 and 2007, evidence that much of the material scoured from the channel in this location came from the bed (Fig. 3A). Laterally, the banks scoured as much as 45 m in other areas. The aerial images shown in Fig. 5A also indicate that most of the islands in the area have eroded away (red areas). The historical aerial photo analysis indicates that the island surface area lost is approximately 35,000 m2. The areal extent of islands in 1999 was 43% of what is was in 1950. The Dam-Attenuating reach

extends from 50 to 100 km C-X-C chemokine receptor type 7 (CXCR-7) downstream of the dam. The islands in this reach are essentially metastable (adjusting spatially but with no net increase or decrease in areal extent). The reach itself has experienced net erosion with respect to the bed and banks, but to a lesser extent than the Dam Proximal reach. Twelve of the 14 cross sections in the Dam-Attenuating reach show an increase in cross-sectional area greater than the 1946 natural variability (269 m2). Fig. 3B is representative of the reach and has had an increase in cross-sectional area of 346 m2. The reach gained a net of 3300 m2 in island area from 1950 to 1999 which represents a 16% increase. All major islands present in 1950 were still present in 1999 with similar geometries and distribution (Fig. 5B). The River-Dominated Interaction reach extends from 100 to 140 km downstream of the dam. This reach is characterized by an increase in islands and sand bars and minimal change in channel cross-sectional area. 4 of the 11 sites have erosion greater than the natural variability (269 m2) and 5 of the 11 sites are depositional. The cross-section in Fig. 3C is typical of this reach and has a relatively small decrease in the cross-sectional area between 1958 and 2007 (25 m2), less than the natural variability. However, the banks widened more than 518 m (Fig. 3C).

Wooly mammoths survived on Wrangel Island off northeast Siberia <

Wooly mammoths survived on Wrangel Island off northeast Siberia until about 3700 years ago (Stuart et al., 2004 and Vartanyan et al., 2008) and on Alaska’s Pribilof Islands until ∼5000 years ago (Yesner et al., 2005). These animals survived the dramatic climate and vegetation changes of the Pleistocene–Holocene transition, in some cases on relatively small islands that saw dramatic environmental change. Climate change proponents suggest, however, that these cases represent refugia populations in favorable habitats in the far north. Ultimately, additional data on vegetation shifts (studies from pollen and macrofloral evidence) across the Pleistocene–Holocene boundary, including investigation of

seasonality patterns and climate fluctuations at decadal to century scales, will be important for continued evaluation of climate change models. The human overhunting Adriamycin model implicates humans as the primary driver of megafaunal extinctions in the late Quaternary. Hunting, however,

does not have to be the principal cause of megafauna deaths and humans do not necessarily have to be specialized, big game hunters. Rather, human hunting and anthropogenic ecological changes add a critical number of megafauna deaths, where death rates begin to exceed birth rates. Extinction, then, can be rapid or slow depending on the forcing of human hunting (Koch and Barnosky, 2006:231). The human overhunting model was popularized by Martin, 1966, Martin, 1967, Martin, 1973 and Martin, 2005 with his blitzkrieg model for extinction in the Americas. Martin however argued that initial human colonization of the New World by Clovis peoples, big game hunting specialists who swept across the Bering Land Bridge and down the Ice Free Corridor 13,500 years ago, resulted in megafaunal extinctions

within 500–1000 years as humans spread like a deadly wave from north to south. Similarly, the initial human colonization of Australia instigated a wave of extinctions from human hunting some 50,000 years ago. According to Martin (1973), this blitzkrieg was rapid and effective in the Americas and Australia because these large terrestrial animals were ecologically naïve and lacked the behavioral and evolutionary adaptations to avoid intelligent and technologically sophisticated human predators (Martin, 1973). Extinctions in Africa and Eurasia were much less pronounced because megafauna and human hunting had co-evolved (Martin, 1966). Elsewhere, Martin (1973) reasoned that since the interaction between humans and megafauna was relatively brief, very few archeological kill sites recording these events were created or preserved. Much of the supporting evidence for the overkill model is predicated on computer simulation, mathematical, and foraging models (e.g., Alroy, 2001, Brook and Bowman, 2004 and Mosimann and Martin, 1975). These suggest a rapid, selective extinction of megafauna was possible in the Americas and Australia at first human colonization.

3, 4, 5 and 6 It must be emphasized, however, that there is a nee

3, 4, 5 and 6 It must be emphasized, however, that there is a need for methods used in body composition determination that are practical, fast, MK-2206 concentration and easy to perform, with the possibility of being applicable to several working conditions, including in population-based studies in the field. Among these methods, bioelectrical impedance analysis (BIA) is highlighted, as it has all these characteristics at a relatively low cost, in addition to its portability and noninvasiveness.4, 5, 7, 8 and 9 The use of previous preparations (protocols) for

standardization of variables that affect body hydration is a recommendation to perform BIA.9, 10 and 11 However, its use may be restricted by lack of adherence or difficulty to follow these requirements by the adolescent. Given the importance of accurately determining body composition and the broad use of BIA, this study aimed to determine the predictive capacity of four different devices in the evaluation of adolescents with and without a protocol. This was an epidemiological, cross-sectional study, with a population of 215 adolescents of both genders, aged between 10 years to 14 years, 11 months, selected by simple random sampling from all public and private schools in the age range of interest, located NVP-BGJ398 mw in urban and rural areas of the city of Viçosa, state of Minas Gerais, Brazil. The following inclusion criteria were used: interest in participating in the study; absence of

prosthetics and/or pacemakers; absence of chronic Ureohydrolase diseases or use of continuous medication that could interfere with body hydration; and adherence to the recommended protocol to undergo BIA. Sample selection was based on the total number of adolescents in the city at the age of interest in 2010.12 The sample was calculated using

EpiInfo software, release 6.04 for cross-sectional studies, considering a total population of 5,754 individuals, the expected frequency of excess body fat of 17.5%, 13 and variability of 5%, totaling 214 individuals, with a confidence level of 95%. The sample draw was conducted among all who met the inclusion criteria and returned the signed informed consent, respecting the proportionality of the number of students that each school had in each age group. When the adolescent did not want to participate or abandoned the study, a new draw was made to replace him/her. The project was approved by the Ethics Committee on Human Research of Universidade Federal de Viçosa (protocol. N. 0140/2010); adolescents and their parents signed the consent form, prepared in accordance with standards established by 196/96 Resolution of the National Health Council. Weight was measured on a digital scale with a maximum capacity of 150 kg and a sensitivity of 50 g, whereas height was measured using a portable stadiometer with an extension of 2.13 m and 0.1 cm resolution. Measurements were made in duplicate, allowing the use of the mean values between the two measurements. In cases where the difference exceeded 0.

Another important associated change during breastfeeding relates

Another important associated change during breastfeeding relates to the regulation of sleep and wake patterns for both the mother and the child, helping the mother to feel less tired, which could also prevent symptoms of depression. Parents of infants who were exclusively breastfed slept an average of 40-45 minutes more and self-reported less sleep disturbance than parents of infants given

formula.14 Women with postpartum depression experienced poorer sleep than women without postpartum depression, and sleep quality worsened with increasing postpartum depression symptom severity.61, 70 and 71 Maternal sleep patterns are enhanced by breastfeeding,13 while this deregulation may cause postpartum depression.61, 70 and 71 Research also shows that breastfeeding improves some psychological conditions and processes that can protect mothers from emerging postpartum depression. Maternal selleck chemical self-efficacy, a condition inversely associated with postpartum depression,72 is improved in mothers who breastfeed.45 and 73 Regardless of maternal depression, mothers who breastfed rather than bottle-fed their infants had higher confidence levels and rated their infants as less alert and less Luminespib purchase irritable during feedings.45 However, breastfeeding self-efficacy

appears to play an important role on postpartum depression; mothers who show higher levels of breastfeeding self-efficacy present lower levels of postpartum depression symptoms.74 Maternal emotional involvement with the infant is also improved by breastfeeding75 and is negatively correlated with postpartum depression.65 and 76 In fact, feeding patterns appear to influence mother-child bonding, with non-breastfeeding mothers presenting more difficulties to establish an emotional involvement with the infant than breastfeeding mothers.77 Regarding the relationship with the partner, studies relate breastfeeding initiation Florfenicol with stronger parental bonds.78 Temperamental

difficulties and sleep problems are reduced when the child is breastfed,79 while the presence of those problems has been associated with postpartum depression.72 and 80 Depressed breastfeeding mothers were less likely to have infants with highly reactive temperaments.45 and 79 Infant competencies are enhanced by breastfeeding,4 and 81 and are adversely affected in the presence of postpartum depression.80 and 82 Breastfeeding also facilitates mother-infant interaction,45 and 83 which is poorer when the mother is depressed.78 Breastfeeding is associated with better mother-infant interactions, with breastfed infants showing more physical contact, vocalizations, and positive play, and mothers exhibiting more proximity towards the infant.68, 79, 83 and 84 Data also specifically suggests that depressed mothers and their infants, not unlike non-depressed mothers and their infants, may benefit from breastfeeding: depressed mothers and infants are more relaxed during breastfeeding versus bottle-feeding interactions.

This study was approved by the Ethics Committee of the Institutio

This study was approved by the Ethics Committee of the Institution (Ethics Committee for Research Project Analysis of the Clinical Board of Hospital das Clínicas and Faculdade de Medicina da Universidade de São Paulo) through research protocol 1383/09 and later adopted by the other institutions. Categorical data are shown with frequency distribution, and continuous data using mean and standard Metformin in vitro deviation, as indicated. The univariate analysis

of categorical variables used the chi-squared or Fisher’s exact test, when indicated, whereas Student’s t-test was used for continuous variables. To calculate the risk, after determining the odds ratio (95% CI), logistic binary regression and backward stepwise multiple regression were performed using MedCalc (Medical Calculator) software, version The statistical significance level was set at 5% (p < 0.05). A total of 1,097 newborns with BW = 400 to 999 g and GA < 33 weeks were admitted in 16 NICUs of the BNRN during the study period. Of these, 220 were excluded due to death or transfer during the first three days AZD6244 in vivo of life, malformations, and congenital infections; 494 newborns met the inclusion criteria with echocardiographic diagnosis of PDA and no information regarding the presence of symptoms. The infants were subdivided according

to the therapeutic approach, into: G1 – 187 (37.8%), G2 – 205 (41.5%), and G3 – 102 (20.6%) (Fig. 1). The characteristics of the population according to each study group is shown in Table 1;it can be observed that there were differences between the groups in relation to the GA, mean SNAPPE II score, frequency and time of mechanical ventilation, and occurrence of LONS. Regarding the analyzed outcomes, it was observed that mortality was higher in G1 (51.3%), while it was lower in G3 (14.7%). The highest incidence of BPD36wks (70.6%) and ROPsur (23.5%) was observed in G3, while the combined outcome death/BPD36wks was less frequent in G2 (58.0%). It was not possible to analyze the effects of the therapy used on the occurrence of NECsur, due to the very small number of cases, although a Vitamin B12 statistically

significant difference was observed between G1 and G2 (Table 2). The multivariate regression analysis showed no influence of the type of therapeutic approach on the probability of death or the occurrence of BPD36wks alone, rather only for the combined outcome death/BPD36wks. However, the following were identified as risk factors for the outcome of death: NECsur (OR 5.64, 95% CI: 1.03 to 30.7) and IVH-III/IV (OR 3.62, 95% CI: 1.30 to 10.11). For the male gender (OR 2.59, 95% CI: 1.33 to 5.02), LONS (OR 1.88, 95% CI: 1.00 to 3.54), GA (OR 1.49, 95% CI: 1.22 to 1.81), and time of MV (OR 1.04, 95% CI: 1.02 to 1.07) were factors related to the presence of BPD36wks. BW alone was a protective factor against the outcomes death and BPD36wks (OR 0.99, 95% CI: 0.99 to 0.99) (Table 3).

The siRNA sequences of the Cont siRNA-Chol were as follows : sens

The siRNA sequences of the Cont siRNA-Chol were as follows : sense strand: 5′-GAACUGUGUGUGAGAGGUCCU*Chol-3′, and antisense strand: 5′-AGGACCUCUCACACACAGUUc*g*C-3′. The lower-case letters represent 2′-O-methyl-modified nucleotides; asterisks represent phosphorothioate linkages. Cationic liposome was prepared from DOTAP/Chol at a molar ratio of 1/1 by a thin-film hydration method, as previously reported [9,10]. For preparation of rhodamine-labeled cationic liposome, Lissamine™ rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rhodamine-DHPE,

Invitrogen, Carlsbad, CA, USA) was incorporated at 1 mol% into the total lipids. The particle size and ζ-potential of cationic liposomes were measured by dynamic Selisistat purchase light-scattering and electrophoresis light-scattering methods, respectively (ELS-Z2, Otsuka Electronics Co., Ltd., Osaka, Japan). The size of the cationic liposomes was adjusted to approximately 80 nm. To prepare cationic liposome/siRNA complex (cationic lipoplex), cationic liposome suspension was mixed with siRNA by vortex-mixing for 10 s at a charge ratio (−/+) of 1/4, and left for 15 min at room temperature. The theoretical charge ratio (−/+) of siRNA

to cationic liposome was calculated as the molar ratio of siRNA phosphate to DOTAP PCI-32765 solubility dmso nitrogen. To prepare ternary complexes with anionic polymers, cationic lipoplex was mixed with CS, PGA and PAA solutions (CS-, PGA- and PAA-coated lipoplexes, respectively) at the indicated charge ratios. The theoretical charge ratios (−/+) of CS, PGA and PAA to DOTAP were calculated as the molar ratios of sulfate and carboxylic

acid of CS (two negative charges per disaccharide unit), carboxylic acid of PGA (one negative charge per glutamic acid) and carboxylic acid of PAA (one negative charge per aspartic acid) to nitrogen of DOTAP, respectively. After preparation of the cationic lipoplexes, CS-, PGA- and PAA-coated lipoplexes of 1 µg of siRNA or siRNA-Chol at the indicated charge ratios (−/+) of anionic polymer and siRNA to DOTAP, they were analyzed on an 18% acrylamide gel for siRNA in Tris–borate–EDTA (pH 8.0) buffer and were visualized by ethidium bromide staining, as previously reported [11]. siRNA condensation Megestrol Acetate by anionic polymer-coated lipoplexes was analyzed by exclusion assay using an SYBR® Green I Nucleic Acid Gel Stain (Takara Bio Inc., Shiga, Japan), as previously reported [11]. The anionic polymer-coated lipoplexes of 1 µg of siRNA at various charge ratios (−/+) in a volume of 100 µL of Tris–HCl buffer (pH 8.0) were mixed with 100 µL of 2500-fold diluted SYBR® Green I Nucleic Acid Gel Stain solution with Tris–HCl buffer, and then incubated for 30 min. The fluorescence was measured at an emission wavelength of 521 nm with an excitation wavelength of 494 nm using a fluorescent spectrophotometer, F-2700 (Hitachi Co., Ltd., Tokyo, Japan). As a control, the value of fluorescence obtained upon addition of 5 µg/ml free siRNA solution was set as 100%.