The photogalvanic current is measured in the unbiased structures at room temperature via a preamplifier and then is recorded by a lock-in amplifier in phase with the PEM. Besides, in order to normalize the data thus enabling a better comparison between BIA and SIA, a common photocurrent j 0 under selleckchem direct current (dc) bias is also measured by a chopper and a lock-in amplifier. Thus, we can use the common photocurrent j 0 as the denominator for normalizing the CPGE current to eliminate the influences of the anisotropic carrier mobility

and carrier density in different directions [26]. For QWs of zinc blende structures grown along the [001] direction, which belongs to C 2v point group symmetry, the Rashba term of the spin-orbital Hamiltonian can be written selleck compound as [2] (1) while the Dresselhaus term is (2) Here, σ is the Pauli spin matrix, k is the in-plane wave vector, α (or β) is the Rashba (or Dresselhaus) spin-orbital parameter, and the coordinate system is x∥ [100] and y∥ [010]. These two Hamiltonians will interfere with each other and result in anisotropic spin splitting in k-space. We can separate the spin splitting induced by Rashba and Dresselhaus terms according to the method suggested in [4, 7], since the Rashba and Dresselhaus terms contribute differently

for particular crystallographic directions. Thus, we can use the geometries shown in Figure 1, i.e., named as geometry CPGE-I shown in Figure 1b and geometry CPGE-II shown in Figure 1c,d, to separate the CPGE current induced by Rashba and Dresselhaus SOC, respectively. In the figures, denotes Resveratrol the direction of light propagation, and j R and j D indicate the CPGE current induced by Rashba and Dresselhaus spin splitting, respectively [4, 7, 26]. Thus, we can obtain j R and j D directly from geometry CPGE-I and obtain the sum and difference of j R and j D from geometry CPGE-II. Therefore, the j R and j D can be obtained separately by the

geometry CPGE-I and CPGE-II, respectively, and then be compared to each other to see whether they are self-consistent [26]. Figure 1 The schematic diagram of the experimental geometries and the spectra of the normalized CPGE current. The schematic diagram for geometries CPGE-I (a) and CPGE-II (b and c). The spectra of the normalized CPGE current obtained by geometry CPGE-II at different angles of incidence (d). The thin lines indicate the sum of j R and j D by the geometry shown in (b), and the thick lines indicate the difference of j R and j D obtained by the geometry shown in (c). All of the spectra are shifted vertically for clarity. In order to get the knowledge of the symmetry of the QW system, we perform reflectance-difference spectrum (RDS) measurement. RDS is an interface-sensitive and nondestructive technique [27, 28], and it can precisely measure the in-plane optical anisotropy (IPOA) between the [110] and directions.

Capitalizing on opportunities emerging in response to climate change Selleck INCB018424 Opportunities for conservation planning that may emerge as climate changes will range from ecological to social. Climate change may improve conditions for some species, ecosystems, and processes of conservation concern, allowing conservation resources currently directed at these elements to be redirected elsewhere. Societal responses

to climate change can provide novel opportunities to increase both the success and cost effectiveness of conservation. For example, strategies for REDD—Reduced Emissions from Deforestation and Forest Degradation (Angelsen 2008) use payments from developed countries to developing countries to reduce greenhouse gas emissions from deforestation and forest degradation. This approach provides a potentially powerful and well-funded mechanism to maintain ecologically intact forests that are also likely to have substantial biodiversity benefits, such as conserving greater numbers of species (Venter

et al. 2009; Busch et al. 2010). In addition to these biodiversity benefits, increasing the LY2157299 price representation and extent of ecosystem types under conservation management have been identified as two key principles for climate adaptation (Kareiva et al. 2008). While REDD itself is a climate change mitigation activity, using REDD to help conserve biodiversity at a regional scale is an adaptation strategy taking advantage of an emerging opportunity. In addition to REDD, opportunities might also emerge from carbon/biodiversity off-sets (Kiesecker et al. 2010), renewable energy developments (Wiens et al. 2011), human responses to climate change (Hale and Meliane 2009), and perhaps other ecosystem service opportunities (Tallis et al. 2008). These opportunities could influence the priorities for conservation areas that emerge from

why systematic conservation planning processes, and plans may need to explicitly consider how such opportunities might best intersect with conservation priorities. For example, initial efforts to incorporate ecosystem services into systematic conservation planning are promising (Chan et al. 2006; Egoh et al. 2010) but may involve trade-offs with biodiversity conservation. The climate change policy arena presents a special opportunity to focus on conservation actions that promote the ability of ecosystems, and the societies that depend on them, to deal with climate-induced changes. This approach is referred to as Ecosystem-Based Adaptation (EBA), a term favored by the International Union for the Conservation of Nature (IUCN; www.​iucn.​org/​) and the Climate Action Network (www.​climatenetwork.​org/​).

meliloti loci. Since homologs to EryA, EryB and EryD were ubiquitous through the data set, it was decided to construct phylogenies based on Maximum Likelihood and Bayesian

analysis using the EryA, EryB and EryD data sets. The topology of the phylogenetic tree using EryA is presented in Figure  2. A tree including branch lengths is included as Additional file 1: Figure S1. V. eiseniae Palbociclib price was also the most distant member with respect to the EryA phylogeny and again used as an outgroup. The phylogenetic trees of EryB and EryD are not shown but were generally consistent with the EryA phylogeny. The species tree, based on RpoD, was included as a mirror tree with the EryA tree to demonstrate possible horizontal gene transfer events (Figure  2). The data show that there is a high degree of correlation between the loci configuration and the EryA phylogenetic tree (Figure  1, 2). We note the similarity of the loci of A. radiobacter and R. leguminosarum to Brucella species and O. anthropi but not to the more closely related Sinorhizobium species. This suggests that a horizontal gene transfer may have occurred between these organisms. This is in agreement with what has been previously Volasertib supplier reported [20]. It also seems likely that a horizontal gene transfer event may have

occurred between the Brucella and E. fergusonii. This may explain the unique occurrence of the loci’s presence in a member of the gamma-proteobacteria.

Finally, our mirror tree suggests that a horizontal gene transfer of the more complex erythritol locus may have occurred between M. loti and an ancestral species the Sinorhizobium species (Figure  2). Modes of evolution for the Rutecarpine polyol utilization loci Comparison of the phylogenetic trees of EryA, EryB and EryD to the arrangement and content of the loci led us to more thoroughly investigate the phylogenies of a number of proteins that stood out as unique within the data set. These phylogenies have led us to postulate modes of evolution that may have occurred in these loci. BLASTP analysis showed a clear distinction between the type of transporter encoded by each of the loci and the remaining genetic content. In general, loci that contained adonitol/L-arabitol type genes contained a transporter homologous to the S. meliloti MptABCDE (Table  2, Figure  1). Loci that contained only erythritol genes contained a transporter homologous to the EryEFG of R. leguminosarum. One exception to this correlation was M. ciceri bv. biserrulae which contained a homologous transporter to EryEFG rather than MptABCDE. This is interesting because M. ciceri groups with the other Mesorhizobia in the EryABD trees. In order to analyze the evolution of these transporters more clearly, phylogenetic trees were constructed of homologs to EryG and homologs to MptA (Figure  3).

To our knowledge, this is the first study to examine the impact of implementing an ACS service on wait-times for elective surgeries. Miller et al.[27] and Barnes et al.[15] observed a 23% and 44% increase in operative productivity in terms of elective caseloads, respectively, but an overall decline in general surgery operative EPZ-6438 cell line volumes because of a reduction in emergent cases [15]. However, neither study considered wait-times for elective cases. While many studies examining the impact of ACS services originate from the United States, American ACS services often

differ significantly from Canadian models. In Canada, general surgeons participating in ACS services often also perform cancer operations as part of their elective practices, whereas many American acute care surgeons are trauma specialists who do not routinely perform oncological operations. One of the limitations of this study is that the effect of ACCESS on wait-times

for non-cancer elective operations, such as elective bowel resections for non-malignant pathology or hernia repair, was not explored. Because of the lack of organized databases to measure wait-times for elective non-cancer operations, it was difficult to ascertain the impact find more of ACCESS on wait-times for these cases. However, surgeons are given the discretion to book elective cases during ACCESS OR time if there are no emergency cases on the board. Most have reported excellent patient satisfaction with the development of “standby lists”, whereby patients who are booked for elective non-cancer surgeries are called into the hospital on the day of their operation. Additionally, as discussed earlier, the recent integration of elective and emergency operating databases, which also include non-cancer operations, may allow for future prospective studies to address this important issue. In conclusion, the reallocation

of operating room resources from elective surgical practice towards an ACS service did not appear to affect the timeliness of care provided to patients waiting for elective cancer surgeries, and thus such concerns should not serve as a barrier for centres considering implementing an ACS service. nearly References 1. Ball CG: Acute care surgery: a new strategy for the general surgery patients left behind. Can J Surg 2010, 53:84–85.PubMedCentralPubMed 2. Davis KA: Acute care surgery in evolution. Crit Care Med 2010, 38:S405-S410.PubMedCrossRef 3. Hameed SM, Brenneman FD, Ball CG, Pagliarello J, Razek T, Parry N, Widder S, Minor S, Buczkowski A, Macpherson C, Johner A, Jenkin D, Wood L, McLoughlin K, Anderson I, Davey D, Zabolotny B, Saadia R, Bracken J, Nathens A, Ahmed N, Panton O, Warnock GL: General surgery 2.0: the emergence of acute care surgery in Canada. Can J Surg 2010, 53:79–83.

GuaA, involved in guanine nucleotide metabolism, indirectly governs intracellular GTP level responsible for translation efficiency [35], while ribosomal protein S30EA limits protein synthesis by reducing translation initiation [40]. Both proteins were down-regulated in the sensitive strain following bile exposure, which is consistent with previous studies [14, 38]. All in all, 7 out of the 13 proteins directly involved in bile tolerance of the three-selected L. plantarum strains were not dedicated to one of the damaging effects of bile, but covered a wide range of environmental stresses instead. In contrast, other factors contribute in a specific way to bile tolerance.

This is the case of GshR1 and GshR4 selleck screening library which help protect the cell against oxidative injury [41]. This coincides with the

lower global levels of glutathione reductases in the sensitive strain in both standard and stimulating conditions found in our study. Another protein, the Cfa2, catalyzes the cyclopropane ring formation in phospholipid biosynthesis, which may help maintain integrity of the cell check details envelope. In Escherichia coli, the cytoplasmic membrane of a cfa-mutant displayed increased overall permeability to protons compared to the native strain [42]. This could for instance explain the higher acid sensitivity of a cfa-mutant of L. acidophilus NCFM [43]. In our study, a Cfa2 isoform was absent in the sensitive strain, while another isoform was not detected in the resistant one, suggesting different functional properties of the isoforms with regard to bile tolerance. Another specific mechanism of bile adaptation is the active removal of bile-related stress factors. Such is the case of the F0F1-ATP synthases which facilitate the extrusion of protons from the cytoplasm by proton motive force [28]. Previous findings reported that a bile-adapted B. animalis strain was able to tolerate bile by inducing proton pumping by a F0F1-ATP synthase, therefore tightly regulating the internal pH [44]. In our study, a representative F0F1-ATP synthase, AtpH, was absent in the weak strain and was

up-regulated in the intermediate strain, which is consistent with the up-regulation of the corresponding gene reported for L. plantarum WCFS1 when exposed to porcine bile Astemizole [45]. ABC transporters are also a major part of the efflux systems involved in the transport of harmful-compounds and cell detoxification [46]. A representative ABC transporter, OpuA, was more abundant in the resistant strain, less abundant in the intermediate one, and not detected in the sensitive one. This protein is known to be implied in the L. plantarum response to osmotic stress, one of the numerous deleterious effects of bile [47]. In addition, deletion of an opuA gene in Listeria monocytogenes was shown to significantly increase bacterial sensitivity to physiological concentrations of human bile [48].

1D). Although the amount of vimentin may vary throughout different HBCEC cultures, cytokeratin levels were always detected

at 95% or higher. Moreover, while the expression of intermediate filaments (Fig. 1C and 1D) was obtained from primary tumor cells after 34d, longer term culture remained stable displaying a similar pattern of intermediate filaments (data not shown). Together, these data suggested an almost exclusively epithelial-like cell population of HBCEC. To evaluate cell surface markers during Natural Product Library clinical trial long term culture of the breast tumors, an HBCEC population after 176 days was analyzed for CD24, CD44 and CD227, respectively, and compared to a tumor culture of the same patient after 462 days (Fig. 2A). Thus, CD24 was expressed in 89% of 176d HBCEC and in 86% of 462d HBCEC. Moreover, CD44 appearance was detectable in 94% of 176d HBCEC and in 99% of 462d HBCEC, suggesting little if any changes of both, CD24 and CD44 during long term tumor culture (Fig. 2A). In contrast, expression of the CD227 (MUC1) surface protein significantly R428 increased from 52% in 176d HBCEC to 88% in 462d HBCEC (Fig. 2A). Figure 2 Surface marker expression, SA-β-gal staining and telomerase activity in HBCEC. A. Determination of the percentage of cell surface marker expression in HBCEC at different ages. Expression of the surface marker proteins CD24, CD44, CD227 was maintained

during long term culture of HBCEC. Whereas CD24 and CD44 were similarly expressed after 176d and 462d, CD227 increased from 52% to 88% in HBCEC 462d. The flow cytometry measurements varied by about 8%. B. SA-β-gal staining of primary HBCEC and HMEC cultures. Staining for SA-β-gal of a HBCEC population after 722d in culture revealed little if any positive cell. Normal HMEC in passage 16, however, displayed already predominantly enlarged

senescent cells after 32d, demonstrated by the dark-green stain (bar = 200 μm). C. Telomerase (TRAP-)assay of this website primary cultures from breast cancer biopsies. Telomerase activity was analyzed according to the Telomeric Repeat Amplification Protocol (TRAP). HBCEC populations demonstrated telomerase activity independent of the age of the culture and the harvest method. The human embryonic kidney (HEK) 293T cell line was used as a positive control and 1× CHAPS buffer served as a negative control. Quantification was performed using densitometric analysis. Further characterization of the HBCEC cultures was performed to determine aging cells in a senescence-associated β-galatosidase (SA-β-gal) assay as compared to normal post-selection human mammary epithelia cells (HMEC) (Fig. 2B). Thus, SA-β-gal staining of primary cultures from breast cancer biopsies after 722d demonstrated majorly small young cells and only occasional positively-stained senescent cells in contrast to normal post-selection HMEC (P16) after 32d with almost exclusively large SA-β-gal positive senescent cells (Fig. 2B).

Renal pathology of ANCA-related vasculitis: proposal for standardization of pathological diagnosis in Japan. Clin Exp Nephrol. 2008;12:277–91.PubMedCrossRef 2. Bajema IM, Hagen EC, Hansen BE, et al. The renal histopathology in systemic vasculitis: an international survey study find more of inter- and intra-observer agreement. Nephrol Dial Transplant. 1996;11:1989–95.PubMedCrossRef 3. Lind

De, van Wijngaarden RA, Hauer HA, Wolterbeek R, et al. Clinical and histologic determinants of renal outcome in ANCA-associated vasculitis: a prospective analysis of 100 patients with severe renal involvement. J Am Soc Nephrol. 2006;17:2264–74.CrossRef 4. Yamagata K, Usui J, Saito C, et al. ANCA-associated systemic vasculitis in Japan: clinical features and prognostic changes. Clin Exp Nephrol. 2012;16:580–8.PubMedCrossRef 5. Berden AE, Ferrario F, Hagen EC, et al. Histopathologic classification of ANCA-associated glomerulonephritis. J Am Soc Nephrol. 2010;21:1628–36.PubMedCrossRef 6. Fujimoto S, Uezono S, Hisanaga S, et al. Incidence of ANCA-associated primary renal vasculitis in the Miyazaki Prefecture: the Omipalisib cell line first population-based, retrospective, epidemiologic survey in Japan. Clin J Am Soc Nephrol.

2006;1(5):1016–22.PubMedCrossRef 7. Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides: proposal of an international consensus committee. Arthritis Rheum. 1994;37:187–92.PubMedCrossRef 8. Chang DY, Wu LH, Liu G, et al. Re-evaluation of the histopathologic classification of ANCA-associated glomerulonephritis: a study of 121 patients in a single center. Nephrol Dial Transplant. 2012;27:2343–9.PubMedCrossRef 9. Watts RA, Scott DG, Jayne DR, et al. Renal vasculitis in Japan and the UK—are there differences in epidemiology

and clinical phenotype? Nephrol Dial Transplant. Bumetanide 2008;23:3928–31.PubMedCrossRef 10. Watts RA, Lane SE, Scott DG, et al. Epidemiology of vasculitis in Europe. Ann Rheum Dis. 2001;60:1156–7.PubMedCrossRef”
“Introduction Chronic kidney disease (CKD) is the leading risk factor for cardiovascular disease (CVD), a great threat to health and an economic burden [1]. In Japan, the prevalence of end-stage kidney disease (ESKD) requiring renal replacement therapy has been increasing over the last three decades. There were 38,893 new cases in 2010, bringing the total number of cases in Japan to 304,592 [2]. Since the number of patients requiring dialysis has continued to increase [3], there appear to be an enormous number of latent cases of CKD in the Japanese population. In a recent study, Imai et al. reported the prevalence of CKD by calculating the estimated glomerular filtration rate (eGFR) using an equation that estimates GFR based on data from the Japanese annual health check program in 2005 [4]. They predicted that 13 % of the Japanese adult population (approximately 13.3 million people) would have CKD in 2005.

In this study, we focused on the ability of FLP/FRT recombination to excise a long region of chromosomal DNA [29] and considered it to be suitable for introducing an unmarked mutation into a large gene. Here, we developed a new system for targeted gene disruption by FLP/FRT CH5424802 clinical trial recombination in non-competent Gram-negative bacteria, and then constructed an unmarked ataA mutant from Acinetobacter sp. Tol 5 in order to demonstrate the feasibility of our methodology. Results and discussion A new unmarked plasmid-based mutation for non-competent bacteria To apply the FLP/FRT recombination system to unmarked mutagenesis, a target gene has to be sandwiched between two identical

FRT sites on the chromosome. For non-competent bacteria that cannot uptake linear DNA, we developed a new plasmid-based method for unmarked mutagenesis in which the FLP/FRT recombination system can be employed. We constructed two new mobile plasmids (Figure 1): pJQFRT, which harbors the sacB counter-selection marker and the gentamicin resistance selection marker, and pKFRT/FLP, which harbors the kanamycin resistance selection marker and flp recombinase gene under the control of the tetR regulator. Both plasmids also harbor a single FRT site

adjacent to a multiple cloning site for the insertion of a homologous region upstream or downstream of a target gene. Since these plasmids contain oriT, which is the origin of conjugative Lenvatinib mouse transfer, they can be readily introduced into a non-competent bacterium from a donor strain that possesses tra genes by bacterial conjugation [4]. The scheme for the unmarked deletion of a target gene using these constructed plasmids is shown in Figure 2. ColE1 and p15A replicons do not work in many Gram-negative bacteria, except tuclazepam for Escherichia coli and a limited species of Enterobacteriaceae. Since the introduced plasmids cannot be replicated

in a non-enterobacterial cell, they are integrated into the chromosome by a single crossover event at the homologous site. When pJQFRT and pKFRT/FLP are integrated into the upstream and downstream regions of a target gene, respectively, in the resultant mutant, the original target gene is sandwiched between the sequences derived from the integrated vectors containing antibiotic resistance markers, the sacB marker, and flp recombinase under the control of the tetR regulator, all of which are bracketed by identical FRT sites in the same direction. In the absence of an inducer for the tet promoter, TetR tightly regulates the expression of flp recombinase, and the plasmid-integrated mutant is stable. When the expression of flp recombinase is induced, FLP recombinase excises the FRT bracketing sequences containing the target gene on the chromosome, resulting in the introduction of an unmarked mutation.

85. Estimates of pairwise linkage disequilibrium and departures from the Hardy–Weinberg equilibrium for each pair of loci in each population were calculated using

GenePop on the Web version 4.0.10 (Raymond and Rousset 1995); Bonferroni’s correction was applied to multiple comparisons. Evaluations of the PD98059 in vivo presence of null alleles were performed using MicroChecker version 2.2.3 (Van Oosterhout et al. 2004). Loci that consistently departed from equilibrium, showed linkage equilibrium or evidence of null alleles were removed from further analyses. The genetic variability of each locus within each feral population and also in ranch mink was estimated as the mean allele number (A), mean number of private alleles (A private), number of effective alleles (N e), heterozygosity (H O) and expected heterozygosity (H E) using FSTAT (Goudet 1995) and GenAlex version 6 (Peakall and Smouse 2006). The mean number of alleles per locus is expected

to be sensitive to sample size, therefore estimates of the expected allele number per locus and mink origin were corrected for unequal sample size (Ar). The inbreeding coefficient (F IS) and potential deviation from the Hardy–Weinberg equilibrium and linkage equilibrium for each locus and site were tested using the randomisation test in GENEPOP 3.4 (Raymond and Rousset 1995). We used a range of different analytical approaches for identifying genetic differentiation across samples of feral and

ranch American mink. Y-27632 price Population genetic structure was detected by determination of F ST (Fixation Index) levels among predefined populations using FSTAT 2.9.3 software (Goudet 1995) as well as the recently developed, alternative measure of genetic differentiation D est (Jost 2008), using the software SMOGD 1.2.5 (Crawford 2010). Cryptic genetic structure of American mink was assessed using STRUCTURE 2.2 software (Pritchard et al. 2000). The greatest rate of change of the likelihood Ceramide glucosyltransferase function with respect to K (ΔK) was used to find the most likely K (Evanno et al. 2005). In the first round of STRUCTURE analyses, we searched for the number of genetically different populations using the entire data set, including feral and ranch mink. This method usually detects only the uppermost level of genetic structure (Evanno et al. 2005). For each round of STRUCTURE analysis, we used the model which assumed no prior information about the population and the admixture model with correlated allele frequency parameters (λ = 1), and a burn-in phase of 500,000 interactions followed by a run phase of 500,000 interactions. Posterior probability values for the number of populations (K), ranging from 1 to 7, were calculated from 10 independent runs, to establish consistency. To assess the number of ranch mink in the feral population we estimated the proportion of individuals with membership q ≥0.8 in the first level of structure analysis.

bNo transconjugants were detected under the detection level (<10-10). cNumber of transconjugants analyzed. dNumber of transconjugants positive for the repA/C SCH772984 PCR marker. eNumber of transconjugants positive for the oriX1 PCR marker. We calculated that the transposition and co-integration events occurred within YU39 at frequencies between

10-6 and 10-9, based on the difference between the conjugation frequency of pA/C + pX1 and pX1::CMY transconjugants (10-7 and 10-10; Table 2 and Table 4) compared with that of pX1ydgA::Tn5 (10-1; Table 5). It is worth noting that these conjugation experiments involving a DH5α donor carrying pA/C and pX1 produced the same results observed as when the YU39 wild-type strain was used as donor, indicating that the interaction between these plasmids did not require additional elements from the YU39 genome. pColE1-like was preferentially

trans-mobilized along with pA/C To determine the genetic identity of the 5 kb plasmid the band was purified, digested and cloned. The sequences from the cloned fragments showed homology to the replication and mob genes of ColE1 plasmids, indicating that the 5 kb was a ColE1-like plasmid (pColE1-like). PCR screening using specific primers to amplify the pColE1-like mobA region (Additional file 3: Table S1) showed that YU39 and all the transconjugants displaying the 5 kb band were positive. The mobA PCR product was employed as a probe to hybridize YU39 and transconjugants selleck plasmid profiles. These hybridizations confirmed

the identity of the 5 kb band and, in addition, showed that the pColE1-like was not involved in the formation of pA/C + X1 co-integrates or Thiamet G pX1::CMY. The pColE1-like was mobilized in trans with all the DH5α pA/C + X1, with most of the SO1 pA/C transconjugants and with a few pX1::CMY transconjugants (Table 2), indicating stable co-existence with pA/C and pX1, and with pSTV when present. The YU39 pX1 is closely related to other E. coli and Salmonella pX1 The nucleotide sequences for the six regions selected for the pX1 PCR screening showed that the YU39 pX1 was highly similar to other pX1 plasmids. In a recent study, Johnson et al. proposed the use of the taxC sequence as a genetic marker to compare IncX plasmids [19]. The phylogenetic inference obtained by the comparison of the taxC partial sequence of the YU39 pX1 with those of IncX plasmids showed that it was closely related to other E. coli and Salmonella IncX1 plasmids (Figure 6). Similar phylogenetic reconstructions were observed for the other five YU39 pX1 sequences (data not shown). Figure 6 Genetic relationships of YU39 pX1 and other IncX plasmids. The dendrogram was constructed using the Maximum Likelihood method based on the HKY + G model with 500 bootstrap replicates.