One way analysis of variance (ANOVA) statistical test was used to compare the groups, and post hoc tests were used where there is significant click here difference to compare between and within groups. BYL719 results and discussion Animal grouping Rats were arranged into four treatment and one control group at the commencement of the study as shown in Table 1. Morbidity and mortality Morbidity, mortality and gross pathology results of sub-acute toxicity study in rats after repeated oral doses were presented

in this study. Weight changes during the study The animals treated with zinc-aluminium layered hydroxide nanocomposite intercalated and unintercalated with levodopa over 28 days showed no mortality. The food and water intake in both control and treatment groups were unaffected during the study period. No signs of toxicity, such as vomiting, diarrhoea, paralysis, convulsion, restless, irritation, bleeding and breathing difficulties were observed in any of the groups (Table 2). During the course of experiment, rats treated with high and low doses of nanocomposite showed a sustained weight gain similar to their counterpart in the vehicle control group. The weight gain was shown to be continuous over the study period; statistically, the difference in weight gain between day 0 and all other days in all the groups is significant (p < 0.05) (Figure 1).

However, body weight changes between weeks were found to MM-102 be statistically significant (p < 0.05), meaning the weight gain in all group from day zero (0) is statistically significant compared to weight in

the subsequent weeks. The coefficient of the brain, liver, spleen, heart and kidney was presented in Table 3. It is the ratio of these organs to the whole body taken on the 28th day. There were no significant differences observed in the coefficients of these organs. Thus, 28 days of repeated doses of ZAL and ZA at 5 and 500 mg/kg, via oral route did not show any effect on these organs’ weight in relation to the whole body weight. This implies that orally administered ZAL Thiamet G and ZA at 5 or 500 mg/kg respectively do not induce any obvious clinical toxicity or do they resulted in any animal demise. Table 2 Morbidity, mortality and gross pathology results of sub-acute toxicity study in rats after repeated oral doses Group Dose (mg/kg) body weight Toxicity sign t/n Mortality d/a Gross pathology l/nl ZALH 500 0/8 0/8 0/8 ZALL 5 0/8 0/8 0/8 ZAH 500 0/8 0/8 0/8 ZAL 5 0/8 0/8 0/8 VC 0 (vehicle) 0/8 0/8 0/8 Based on the doses used, no rats showed any clinical toxicity sign, no death was recorded, and no obvious gross pathology seen on the organs observed. Data are expressed as means ± SD, n = 8. t/n (toxic/normal), d/a (dead/alive), l/nl (lesion/no lesion). ZALH, zinc aluminium levodopa nanocomposite high dose; ZALL, zinc aluminium levodopa nanocomposite low dose; ZAH, zinc aluminium nanocomposite high dose; ZAL, zinc aluminium nanocomposite low dose; VC, vehicle control.

Additionally, the experiments CH5183284 clinical trial indicated

that the toxin is the most active, or best activated, when first exposed to a short 10 min pulse at 47°C and then continuously incubated at 42°C for 120 hrs. The detection of the 2281 m/z (NT) and 1762 m/z (CT) product ions in each experiment confirmed that the lots of commercial toxin used were active. Relative quantification of type G toxin and NAPs was determined by use of MSE Label-free relative protein quantification was obtained for each component of the type G toxin complex (Table 2). When calculated by weight, the BoNT/G complex contained 30% of toxin, 38% of NTNH, 28% of HA70, and 4% of HA17. These percentages and nanogram amounts indicate that the overall weight ratio of BoNT:NAPs present within the complex is 1:3. The percentages of each molecule present in the complex are as follows: 17.2% of toxin, 23.1% of NTNH, 42.0% HA70, and 17.8% HA17. These percentages and femtomole

amounts indicate a 1:1:2:1 BoNT:NTNH:HA70:HA17 ratio, or a 1:4 BoNT:NAPs ratio, of molecules within the complex. Table 2 Relative quantification of Type G toxin and NAPs. Protein Description Accession # Avg Mass (kDa) Amount OnColumn % in the Complex       femtomoles nanograms molecules weight BoNT/G CAA52275 149034 110.0 16.4 17.2 30.4 NTNH type G CAA61228 139083 147.6 20.5 23.1 38.1 HA-70 (III) type G CAA61225 55791 268.5 BMS-907351 price 14.9 42.0 27.8 HA-17 (II) type G CAA61226 17372 113.8 1.9 17.8 3.7 The proteins identified in the/G complex, NCBI accession numbers, and average masses are shown, in addition to the calculated amounts on column, femtomoles and nanograms, and the percent Nintedanib (BIBF 1120) of each

protein, by weight and molarity, within the BoNT complex. Discussion BoNT/G is the least-studied and the most recently reported of the seven serotypes produced by C. botulinum. Although BoNT/G is associated with a distinct species and metabolic group, the toxin shares multiple characteristics with the other six progenitor toxins. The seven serotypes have similar biochemical and molecular mechanisms of cell entry and https://www.selleckchem.com/p38-MAPK.html membrane translocation. They cause disease by inhibiting synaptic transmission as a result of the enzymatic cleavage of the SNARE protein complex. In the present work, we detail the in silico comparison of BoNT/G progenitor toxin proteins to the other six serotypes of C. botulinum, as well as methods for the digestion, detection, and relative quantification of BoNT/G and its NAPs. The comparison of the BoNT/G progenitor toxin with the other six serotypes was completed to determine/G’s phenotypic relationship with the other BoNTs. In general, past analyses [7, 10, 23] have included a comparison at the gene level; this study focuses solely on protein level.

In fact, many clinical and other types of studies of CCTA have reported the administration of β-blockers to lower heart rate for CCTA [3, 4]. One recent study reported high diagnostic capability with the assistance of the latest devices that shorten the imaging time and improve time resolution, without the use of β-blockers [5]. However, those results were obtained using only a specific model such as dual-source CT in an Fludarabine manufacturer updated facility,

and thus CT equipment commonly used in clinical practice still require the use of β-blockers to lower heart rate during CCTA. Furthermore, it is essential to lower the heart rate to reduce GDC-0994 cost exposure volume [6, 7] as many techniques to reduce the volume of exposure to radiation are applicable only at low heart rates. Injectable or oral β-blockers, which not only take more than 1 h to become effective but also have long half-lives [2.3 h for injection (propranolol), and 2.8 (metoprolol) to 3.9 h (propranolol) for tablets], thus constraining patients for a longer time, were widely used in previous studies. Therefore, Adriamycin chemical structure short-acting β-blockers have been demanded in order to achieve safer and more efficient inspection. The pharmacokinetic profile of landiolol hydrochloride shows high β1-selectivity as well as a very short half-life

(3.97 min) [8]. Landiolol hydrochloride has been a useful agent for improving the image quality of CCTA by 64- and 320-slice multi-detector CT (MDCT) as it was confirmed to reduce heart rate significantly and rapidly after intravenous injection [9–11]. Although

there are some studies in which the efficacy, safety, or usefulness of β-blockers has been explored [11, 12], no study has examined the usefulness and safety of short-acting β-blockers at an approved dosage and with approved administration in CCTA by 16-slice MDCT. Nowadays, 64-slice CT or newer CT equipment with more slices have the most advanced functions. However, due to the cost of 64-slice CT, most small- and medium-sized hospitals still have 16-slice CT. Sixteen-row CT is less expensive than the newer CTs and is still widely used in Japan. In ADAM7 addition, new low-dose algorithms for the reduction of radiation exposure are also available in CCTA with 16-slice CT, and the X-ray exposure dose of 16-slice MDCT is less than that of the 64-slice MDCT [13, 14]. It is possible to obtain an appropriate coronary image by 16-slice MDCT [15–22] if the patient’s heart rate during CCTA is properly controlled. In the present study, the usefulness and safety of the short-acting β1-receptor blocker landiolol hydrochloride (ONO-1101) 0.125 mg/kg for CCTA were assessed using 16-slice CT. 2 Methods 2.

Higher taxonomic ranks (phylum, class, order and family) have approximately the low specificity percentages, while for genera and especially species there is a clear increase in the amount of specific taxa. Nevertheless, the percentage of specific species does not even reach 20% for the most favourable PCI-32765 price case of environment supertypes (using 90% for the specificity criterion, Figure 1). Some of these species belong to well-known examples of specificity, such as

the marine bacteria Prochlorococcus marinus and Pelagibacter ubique. These taxa are thought to be amongst the most abundant microorganisms in the Earth [22], but at the same time they are specific from the pelagic marine environment: they are typical examples of specialists living on a widely extended habitat on the Earth. For these taxa, however, genetic differences that can be associated to niche differentiation have been reported, showing that specificity could be found on subspecific (ecotype) level [23]. The gastrointestinal tract of animals is, once more, the environment where more specific bacteria can be found. Figure 1 Quantification of specific and cosmopolitan taxa. Left side: percentage of specific taxa for the three levels of environmental classification. A particular taxa is defined as specific when a given percentage of its observations

belong to a single environment. That percentage is shown in the abscissa axis. Right CH5183284 solubility dmso side: percentage of cosmopolitan taxa for the three levels of environmental classification, in relation to the number of environments in which the taxa is present. It must also be remarked that for environmental subtypes, the most selleck products detailed level

of the environmental classification, specificity is GF120918 mouse almost inexistent at any taxonomic depth (Figure 1). The relatively low numbers of specific bacteria, even at the species level, indicate that, using this environmental classification, environment-specific clades of bacteria are not abundant and therefore clear-cut specialization is not a widely used strategy in prokaryotes. We can define a cosmopolitan taxa as having five or more observations in 90% of the environments (5 of 5 for supertypes, 18 of 20 for types and 41 of 46 for subtypes). While the upper taxonomic ranks can be considered as eurioic (tolerant to highly diverse conditions), that behaviour does not necessarily hold for their constituents. This trend can indeed be appreciated in Figure 1, where cosmopolitanism decreases greatly for the genus level and disappears almost completely for species. Again, the upper taxonomic levels (phylum, class and order) show a uniform behaviour, with high levels of cosmopolitanism (around 70% of the taxa for environmental supertypes, and 30% for subtypes).

However, no significant change was shown in the abundance of genes involved in recalcitrant C (e.g., lignin) degradation. Therefore, our results indicate that eCO2 significantly affected metabolic potentials for C fixation and degradation. However, it appears that such changes have little effect on soil C storage [25], probably due to accelerated degradation of increased C inputs, which is consistent with increased soil CO2 flux over the course of the experiment. Another important question is Selleckchem Inhibitor Library whether eCO2 affects soil N cycling processes and/or

soil N dynamics. Our previous study has Belnacasan supplier showed that soil N supply is probably an important constraint on global terrestrial productivity in response to eCO2[32]. When N is limiting, decomposers may respond to increased C inputs by decomposing soil organic matter to gain access to N and constrain the plant biomass accumulation at eCO2[42, 43]. In this study, our GeoChip analysis showed that the abundance of nifH genes significantly increased at eCO2. Presumably, an increase

in N2 fixation under eCO2 may lead to enhanced CO2 fertilization of plant biomass production by alleviating some of the N constraints on plant response to eCO2. In the plots examined in the present Selumetinib mw study, no N fertilizer was supplemented, but significant increases were observed in the total plant biomass and aboveground plant biomass, especially the biomass of legume plant species Lupinus perennis, which may be associated with significant increases of N2 fixers in soil under eCO2 measured by the abundance of nifH genes in this study. At eCO2, if the increased nifH abundance is interpreted as potential increase of soil microbial N2 fixation, such increase could supplement N nutrients for the plant growth to eliminate the N limitation constraint. In addition, the abundance of Rucaparib nirS genes significantly increased at eCO2 while all others genes involved in denitrification

remained unaffected. The results suggest that eCO2 could significantly impact microbial N2 fixation and denitrification, and probably enhance the production of the greenhouse gas N2O. However, it appears that no significant changes were observed in soil N dynamics under eCO2, which may be largely due to the large N pool size in soil. It is largely unknown whether or how eCO2 and eCO2-induced effects, such as increased C inputs into soil and changes in soil properties, shape soil microbial community structure. The direct effects of elevated atmospheric CO2 concentration on soil microbial communities were expected to be negligible compared to potential indirect effects such as increased plant C inputs to soil, since the CO2 concentrations in the pore space of soil generally is much higher than those in the atmosphere even under ambient CO2 concentrations [5]. However, this has not been well studied.

Together, these data imply that the ability of cells to persist in the face of antibiotic treatment depends on the specific mechanism by which the persister phenotype is generated, and the precise manner in which the antibiotic acts: cells that persist in one antibiotic may not persist in a second antibiotic, even if that

antibiotic has a very similar mode of action. These C59 wnt solubility dmso data contrast strongly with data from experimental studies on lab strains of E. coli, which have generally shown that when mutants exhibit higher levels of persistence in one antibiotic, they also exhibit higher levels of persistence in other antibiotics (multidrug tolerance) [6, 7, 11, 13, 19–22]. However, there do appear to be a subset of cells that persist after treatment with multiple antibiotics, as evidenced by using combination treatments. Finally, the data here suggest that the parameter that has the largest influence on the fraction of Selleckchem BIBF 1120 persisters exhibited by any strain is the rate of switching from a normal cellular phenotype to a persister state; in contrast, the rate of switching from

persister to normal cell has a much smaller influence. Results Consistent quantification of persister fractions A critical issue when studying bacterial persistence is the precise definition of the persister fraction. Previous studies have defined persister cells as the VX-680 chemical structure surviving fraction after antibiotic exposure for an arbitrary amount of time, ranging from hours [4, 8, 10, 11, 19, 23–25] up to several days [15]. In addition, these fractions have been assessed at different growth states: mid-exponential [8, 10, 11, 19, 25], late exponential [24] and in rare cases, stationary phase [4, 24, 25]. Most often, these studies are performed in liquid cultures of rich media. However, some studies have assayed persisters on agar [6, 12, 13], by plating samples of logarithmically growing cultures on LB agar with ampicillin, incubating overnight, spraying the plates with penicillinase, and again incubating for 24 hours to count the number of surviving cells. These

different methods tremendously complicate comparisons across studies. To quantify the fraction of persisters in a consistent manner, we use a model motivated by triclocarban observations of persister cell dynamics first reported by Balaban et al. [6], who observed two types of persister cells, which they proposed arose through two different mechanisms. Type I persisters occurred through unspecified events that occur during stationary phase, and remained fully dormant until switching to a normal growth state. These have been associated with a specific genotype, the hipA7 allele. Type II persisters arise through an infrequent stochastic switch to a slow-growth state, and remain so until switching to a normal growth state. These were associated with a mutation at a second locus, hipQ. A similar model of persister formation has been proposed by Wiuff et al. [23].

O-glycosylation occurs at Ser and Thr residues respectively. Although glycosylations of the tryptic or AspN-digested N-terminal peptides of LprF and LppX were identified, the exact glycosylation

site within the peptide could not be determined. No glycosylations were found for N-terminal fragments of LpqH and LpqL. This possibly is due to the use of proteases which have cleavage sites close to the N-terminus and therefore the peptide fragment may be too short to include O-glycosylation sites. The information about the exact molecular nature and function of the glycosylation is scarce, but its influence on subcellular lipoprotein localization and its protection from proteolytic degradation are proposed [45, 62]. In B. subtilis lipoprotein CAL101 glycosylation is discussed to control a lipoprotein “shaving” mechanism and thus their release into the culture medium [63]. In our study, glycosylations were found also in lipoproteins from the Δlnt mutant, demonstrating that N-acylation is not a prerequisite for glycosylation. Lnt independent glycosylation was also I BET 762 demonstrated in C. glutamicum[16]. In C. glutamicum Cg-Ppm1 is responsible for glycosylation. Cg-ppm1 (Ppm buy AMN-107 synthase) and Cg-ppm2 (Lnt) are similar organized as MSMEG_3859 (Ppm synthase) and MSMEG_3860 (Lnt) in

M. smegmatis (Figure 2). Deletion of the Lnt domain of BCG_2070c obviously did not abolish Ppm activity encoded in the same ORF. Of note, Lnt is dispensable while Ppm is 4-Aminobutyrate aminotransferase essential in M. tuberculosis[64]. In Gram-negative bacteria, the efficient lipoprotein transport to the outer membrane depends on the localization of lipoproteins (Lol) transport system and there is good evidence that N-acylation by Lnt facilitates lipoprotein translocation in E. coli[6, 65]. Lnt is essential in E. coli, however deletion of lnt was possible upon overexpression of proteins from the Lol system, indicating an important role

of N-acylation in targeting lipoproteins to the outer membrane [9]. Mycobacteria have an outer membrane mycolic acid bilayer [66–68] and are known to localize lipoproteins to the cell surface [66]. Nevertheless, no mechanisms for translocation or transport systems are identified and whether N-acylation and glycosylation, alone or in combination are involved in the translocation of specific lipoproteins to the mycolate layer is not known so far. In the present study we show that lipoproteins from M. bovis BCG, the live vaccine for tuberculosis are triacylated and we identified the lipid modifications at the molecular level. BCG_2070c is a functional homologue of E. coli Lnt, but differs in substrate specificity. The identification of N-linked tuberculostearic acid shows for the first time, to our knowledge, that mycobacteria-specific fatty acids are used by mycobacterial Lnts.

5. Note that the thresholds for categories of risk differ from those used in men and those used in women (which also differ from each other—see Table 1). With this SB202190 cost proviso, the general pattern remained similar. Discordances in classification were relatively few. In the consolidated map, two countries coded low risk had been previously coded at intermediate risk (men in India and China). At the other extreme, one country coded as high risk had been previously coded at intermediate risk (men and women in Argentina). As might

be expected, there were more discordances in the moderate risk category. Six countries coded at moderate risk had MEK phosphorylation been previously coded at low risk (men in Portugal, Thailand and Spain; women in Croatia, Jordan and Romania). Twelve countries coded at moderate risk had been previously coded at high risk (women in Hong Kong, Turkey, Italy, Lebanon and the

UK; men in Kuwait, Japan, Russia, South Korea and Finland; men and women from Greece and Singapore). FRAX A total of 45 country and/or ethnic models were available for inclusion into the distribution of fracture probability. The FRAX models used are summarised in Table 7 of the Appendix. There was a marked heterogeneity https://www.selleckchem.com/products/icg-001.html in the 10-year probability of a major fracture between countries. In men (Fig. 6), the lowest probabilities were found in Tunisia (1.9%), Ecuador (2.5%), Philippines (4.8%) and China (5.4%). The highest rates were observed in Denmark (23%), Sweden (21%), Norway (19%) and Switzerland (18%). Numerical data for other countries is given in Table 7 of the

Appendix. Thus, there was a greater than 10-fold range in fracture probability. Fig. 6 Ten-year probability of a major fracture (in percent) in men and women aged 65 years with a prior fragility fracture (and no other clinical risk factors) at Non-specific serine/threonine protein kinase the threshold of osteoporosis as judged by BMD at the femoral neck (i.e. a T-score of −2.5 SD). The body mass index was set at 24 kg/m2 Fracture probabilities were consistently higher in women than in men but the difference was relatively modest. On average, probabilities were 23% higher in women than in men. This contrasts, therefore, with hip fracture incidence which was twofold higher in women than in men. As expected, there was a close correlation between probabilities in men and those in women (r = 0.88; p < 0.001). The geographic distribution by fracture risk is shown in men and women in Figs. 7 and 8, respectively. High-risk regions for men were Taiwan, Austria, USA (Caucasian), Switzerland, Norway, Sweden and Denmark. Those at low risk included Africa (Tunisia), Oceania, the Latin American countries of Ecuador and Colombia and several European countries (Spain, Poland, Romania, France and Turkey). Other countries at low risk were China, Lebanon, Philippines and the US Black population. Fig.

The LD spectrum shows a large negative band just above 810 nm, which is due to several overlapping sub-bands. This means that the corresponding transition dipole moments are preferentially oriented along the symmetry axis. The opposite is true for the bands at 805 and Ferrostatin-1 order 825 nm, which exhibit positive LD. On combining these results with the results of polarized fluorescence spectroscopy, an absolute calibration is possible (PF01367338 Wendling et al. 2002). The size of

the LD appears to be in agreement with the orientations of the BChls a in the crystal structure, provided that the Q y transition dipole moment is parallel to the Y-axis in the BChls a. This finding shows that the red-most transition dipole moment of BChl a indeed closely coincides with the Y-axis of the molecule, this is implicitly assumed in many theoretical simulations of the spectroscopic properties of BChl a containing proteins. The absolute calibration of the LD spectrum allowed Wendling et al. (2002) to quantitatively relate the crystal structure to

the LD spectrum, including the precise transition energies (site energies) of all the 7 BChl a pigments (which are influenced by the direct protein environment). Fig. 2 LD spectrum of the FMO (Fenna Matthews Olson) complex from Prosthecochloris aestuarii obtained with a squeezed gel. The spectrum is represented upside down, and the peak at 815 nm indicates that the corresponding transition dipole moments are MK-1775 more or less perpendicular to the C3-symmetry axis of the complex (Vulto et al. 1998a) The FMO complex of Chlorobium tepidum was analyzed N-acetylglucosamine-1-phosphate transferase in a similar way. The spectra are grosso modo quite similar to those of Prosthecochloris aestuarii, and the spectral simulations based on the crystal structure agree even better with the experimental results (Vulto et al. 1998a). The linear-dichroism measurements were not sufficient for the

complete assignment of the site energies and interaction strengths, but they turned out to be crucial. Additional information was obtained from other (polarized) spectroscopic techniques, including CD. Moreover, the pathways of excitation energy transfer and relaxation were studied with transient absorption experiments and could satisfactorily be extracted from the data, using the results of the steady-state (polarized) experiments (Vulto et al. 1998b, 1999). Graham Fleming and coworkers (Brixner et al. 2005), at the University of California at Berkeley, have been able to visualize the flow of excitation energy in the FMO complex using 2D ultrafast spectroscopy. The results were in rather good agreement with those of Thijs Aartsma and coworkers (Vulto et al. 1998b, 1999). It is important to point out, however, that the assignment of the pigment site energies based, amongst others, on the LD experiments, was also essential for the interpretation of the 2D experiments.

The PCR product was cut with BamHI and HindIII and cloned into the plasmid pSP72 (Promega, Madison, WI) which had been cut with the same enzymes, transformed into DH5α, and selected for bright blue

colonies on LB-amp plates containing 40 μg/ml GSK3326595 in vivo X-gal. The plasmid was subsequently transformed to the restriction minus methylation plus strain YS501 before transforming other Salmonella strains. β-gal assays were performed according to the instructions for the Galacto-Star™ chemiluminescent reporter gene assay system (Applied Biosystems, Bedford, Massachusetts). Briefly, 1 ml of bacterial culture expressing β-gal from pSP72lacZ was pelleted at 13,000 × g for 5 min. Supernatants were filtered through a 0.2 μm syringe filter and then assayed immediately or frozen at -80°C until assayed with no further processing. Cell pellets were quickly freeze-thawed and suspended in 50 μl or 200 μl B-PER™ bacterial cell lysis reagent (Pierce click here Chemical) containing Selleck XL184 10 mg/ml lysozyme (Sigma). Bacteria were allowed to lyse for 10–20 min. at room temperature and were then placed on ice. All reagents and samples were allowed to adjust to room temperature before use. Filtered supernatants and bacterial lysates were diluted as needed in Galacto-Star™ Lysis Solution or assayed directly.

β-gal standard curves were made by preparing recombinant β-gal (Sigma, 600 units/mg) to 4.3 mg/ml stock concentration in 1× PBS. The stock was diluted in Lysis Solution to

prepare a standard curve of 100 ng/ml- 0.05 ng/ml in doubling dilutions. 20 μl of standard or sample was added to each well of a 96-well tissue culture plate. 100 μl of Galacto-Star™ Subtrate, diluted 1:50 in Reaction Buffer Diluent, was added to each well and the plate rotated gently to mix. The plate was incubated for 90 minutes at 25°C in the dark and then read for 1 second/well in an L-max™ plate luminometer (Molecular Devices). Sample light units/ml were compared to the standard curve and values converted to units β-gal/ml. Percent release of β-gal was determined by dividing units/ml supernatant by total units/ml (units/ml supernatant + units/ml pellet). All samples were assayed in triplicate. Acknowledgements We wish to thank the reviewers Sulfite dehydrogenase for helpful suggestions, and Diana Downs and Eugenio I. Vivas (University of Wisconsin, Madison) for expeditiously providing gnd mutants. This work was supported by Vion Pharmaceuticals, New Haven, CT. SRM was supported by NIH Grant 1SC2 GM084860-01. DB thanks Caroline Clairmont for informing him of the plating results at the NCI. References 1. Nikaido H: Outer membrane. Escherichia coli and Salmonella: Cellular and molecular biology (Edited by: Neidhardt F, Curtiss R, Ingraham J, Lin ECC, Low KB, Magasanik B, Reznikoff M, Riley M, Schaechter M, Umbager HE). Washington D.C.: ASM Press 1996, 29–47. 2.