We thank Associate Editor Veerle Vanacker and two anonymous reviewers for providing thoughtful comments and suggestions that helped us to improve the paper. “
“Large rivers deliver substantial amounts of terrestrial sediment, freshwater,

and nutrient to the sea, serving as the major EPZ-6438 molecular weight linkage between the continent and the ocean. Inputs of freshwater and terrestrial sediments have multiple morphological, physical and bio-geochemical implications for the coastal environment (Chu et al., 2006, Raymond et al., 2008, Blum and Roberts, 2009, Wang et al., 2010 and Cui and Li, 2011). Riverine material in a large system is a complex function of hydrologic variables influenced by a combination of natural and anthropogenic processes over the watershed (Milliman and Syvitski, 1992), and is thus considered a valuable indicator of global change. The past several decades have witnessed varying levels of changes in water and sediment discharges for large rivers, e.g. the Yangtze in China, the Nile in Egypt, the Chao Phraya river in Thailand,

the Red River in Vietnam, the Mississippi River and the Columbia River in the United States, in addition to the Huanghe (Yellow River) in China (Yang et al., 1998, Peterson et al., 2002, Yang et al., 2006, Wang et al., 2006, Wang et al., 2007, Meade and Moody, 2010 and Naik and Jay, 2011). The Pexidartinib ic50 five largest rivers in East and Southeast Asia (Huanghe, Changjiang (Yangtze River), Peal, Red and Mekong) now annually deliver only 600 × 109 kg of sediment to the ocean, representing a 60% N-acetylglucosamine-1-phosphate transferase decrease from levels in the year 1000 BP (Wang et al., 2011), whereas in the Arctic Ocean, an increase of freshwater delivered by rivers has been observed (Peterson et al., 2002 and Giles et al., 2012). Many studies have attempted to link these changes

to climatic and anthropogenic drivers (Vörösmarty et al., 2000, Syvitski et al., 2005, Wang et al., 2006, Wang et al., 2007, Walling, 2006, Milliman et al., 2008, Rossi et al., 2009, Dang et al., 2010 and Meade and Moody, 2010), with possibilities as diverse as changes in basin precipitation, North Atlantic Oscillation (NAO), EI Niño-Southern Oscillation (ENSO), land cover changes, large reservoir impoundment, and water consumption (Peterson et al., 2002, Wang et al., 2006, Wang et al., 2007 and Milliman et al., 2008). Anthropogenic processes play a significant role in changing the movement of riverine material to the sea (Vörösmarty et al., 2003 and Syvitski et al., 2005). This is particularly true for some mid-latitude rivers (Milliman et al., 2008), where water and sediment discharges to the sea have altered by an order of magnitude. Most of the world’s large rivers are dammed to generate power and regulate flow, in response to growing populations that have increased the demand for water (Dynesius and Nilsson, 1994, Milliman, 1997, Vörösmarty et al.

The DDF curves were created according

to the official and mandatory procedure described by the Adige-Euganeo Land Reclamation Consortium (2011), find more the local authority in charge of the drainage network management. The mandatory approach is based on the Gumbel (1958) distribution. In this method, the precipitation depth P  T (in mm) for any rainfall duration in hour, with specified return period T  r (in years) is computed using the following relation: equation(2) PT=P¯+KTSwhere P¯ the average and S is the standard deviation of annual precipitation data, and KT is the Gumbel frequency factor given by equation(3) KT=−6π0.5772+lnlnTrTr−1 The steps below briefly describe the process of creating DDF curves: (i) Obtain annual maximum series of precipitation depth for a given duration (1, 3, 6, 12 and 24 h); We considered rainfall data coming from an official database provided by the Italian National Research Council (CNR, 2013) (Table 1) for the rainfall station

of Este. For Talazoparib cost this station, the available information goes from the year 1955 to the year 1995, but we updated it to 2001 based on data provided by the local authorities. Given the DDF curves (Fig. 7), we considered all the return periods (from 3 up to 200 year), and we defined a design rainfall with a duration of 5 h. The choice of the rainfall duration is an operational choice, to create a storm producing, for the shortest return

time, a volume of water about 10 times larger than the total volume that can be stored in the 1954 network. This way, we have events that can completely saturate the network, and we can compare the differences in the NSI: by choosing a shorter rainfall duration, giving the DDF curves of the study area, for some return times we would not be able to reach the complete saturation to compute the NSI; by choosing longer durations, we would increase the computation time without obtaining any Cytoskeletal Signaling inhibitor result improvement. We want to underline that the choice of the rainfall duration has no effect on the results, as long as the incoming volume (total accumulated rainfall for the designed duration) is higher than the storage capacity of the area, enough to allow the network to be completely saturated with some anticipation respect the end of the storm. The considered rainfall amounts are 37.5 mm, 53.6 mm, 64.2 mm, 88.3 mm, 87.6 mm, 97.6 mm and 107.4 mm for a return time of 3, 5, 10, 30, 50, 100 and 200 year respectively. For these amounts, we simulated 20 different random hyetographs (Fig. 8), to reproduce different distributions of the rainfall during the time.

It is likely that this channel was one of the Brenta river mouths cited Docetaxel by Comel (1968) and by Bondesan and Meneghel (2004) closed by the Venetians in 1191 in order to slow down the filling process of the lagoon. Before this diversion the Brenta river flowed to the city of Venice through the ancient “Canal de Botenigo” into the Giudecca Channel (Fig. 3) through the island of Tronchetto. This

hypothesis is confirmed by the presence of a similar channel deposition in the transect B–B′ between Santa Marta and the Canal Grande shown on page 20 in Zezza (2008). This palaeochannel is further described in Zezza (2010), where it is observed that in the city area “the lithostratigraphic model of the subsoil reveals that alluvial processes lasted until the verge of the Holocene Period and, furthermore, that the Flandrian transgression determined first all the widening and successively the partial selleck chemicals llc filling of the alluvial channel, incised into the caranto and evolved into a tide channel during the Holocene”. Finally in the southern part of profile 4 (Fig. 2d) one can see the chaotic and structureless filling of a recent superficial palaeochannel (CL3). This kind of acoustic signal probably corresponds to a sandy filling of the channel. The absence

of stratified reflectors implies a highly energetic environment and a fast channel filling. The palaeochannel CL3 corresponds to the “Coa de Botenigo” (Fig. 4b). The map of the areal extension of all palaeochannels reconstructed in the study area is shown in Fig. 4 for five different times: Fig. 4a represents the palaeochannels that were dated between 2000 BC and 0 AD, active during the Bronze, Iron Age and Roman Times reconstructed using as a basis the acoustic survey and the geological data. This corresponds

to a natural environment immediately before the first stable human settlements. Instead, the map of 1691, which is one of the first detailed cartographic representation of the area, refers to a time when some of the main river and channel paths were already modified by the Venetians. Fig. 4b–d depicts not only the reconstructed palaeochannels but also channel paths (and when available the land extension), digitized from the historical maps of Methocarbamol 1691, 1810, 1901, respectively. The present situation is shown in Fig. 4e. Many palaeochannels were reconstructed in the area, adding more information to the historical maps. In general they flow almost parallel in the west-east direction, with a slightly sinuous path. This orientation can be explained by the fact that this hydrographic system probably belonged to the Brenta megafan (Bondesan and Meneghel, 2004 and Fontana et al., 2008). A few palaeochannels have a north–south direction. This orientation may be related to the natural development of tidal networks. We show the patterns of the palaeochannels that existed before or that formed immediately after the lagoon expansion in the area (Fig. 4a).

Interventions

that featured individuals with a chronic disease and a structured peer support intervention led or co-led by a peer were included. Studies needed to feature qualitative methods (see Appendix A for selection criteria). Original searches (October 2008–January 2009), were updated in March 2010 and April 2011. All abstracts were reviewed independently by two individuals for inclusion, with discrepancies between reviewers discussed, and agreement sought by consensus. A pair of reviewers independently evaluated each selected article using a quality assessment AZD6244 order tool [20] coding eligible papers into a data extraction form. A third researcher reviewed disputed papers. This process followed well established procedures; and those conducting meta-ethnographies

have not usually published inter rater reliability coefficients for example [19]. Concepts (ideas or metaphors with explanatory rather than descriptive potential) were identified within each included paper [18] and [19]. Selleckchem Venetoclax First order concepts refer to respondents’ terms (direct quotations) expressing key ideas; second order concepts are authors’ interpretations of participants’ key ideas (for example, themes identified by authors). Third order concepts are reviewers’ re-interpretation of these concepts, interpretations that must be congruent with interpretations of individual studies, while extending beyond with potentially richer explanatory potential [19]. During concept identification, reviewers extracted data on intervention format, disease, and type Thiamine-diphosphate kinase of participant (see Table 1), setting, mentors’ roles, training, and socio-demographic characteristics, to contextualize

results. To identify concepts across included articles, each article was independently reviewed by three to four individuals. This enabled a rich interpretation of each article from multiple perspectives, thereby encouraging identification of a broad range of concepts. First and second order concepts in each article were identified and defined. Definitions allowed reviewers to establish whether a particular concept meant the same thing across papers and whether new descriptors were needed. Thirty-six concepts were first identified. Similar or related concepts were grouped together to produce 13 key concepts. Next, a key concept grid was produced, with data extracted on how each article containing the concept defined or related to it from the perspectives of study participants (first order), and study authors (second order). A record was kept of whose first order perspective was represented – mentors, mentees, or both. Finally, the research team produced third order definitions for each key concept through the process of translation [18]. The final synthesis was achieved by analysing and representing the relationships between the third order translations of the 13 key concepts.

The deeper nearshore sampling points were located at depths of 7 m and 10 m (Figure 2). The paper includes the results of the grain-size analysis of 263 samples by dry sieving in an Eko-Lab analyser with 0.5 φ mesh sieves (from

4 to 0.004 mm). The lithodynamic indices – mean (MG), sorting (σG), skewness (SkG) and kurtosis (KG) – were calculated using the method of Folk & Ward (1957), which is the most accurate for sandy deposits in the marine coastal zone ( Racinowski et al. 2001) ( Tables 1 and 2). Grain-size values were calculated with the Gradistat program ( Blott & Pye 2001). The lithodynamic interpretation of all grain-size indices was done on the basis of the confidence interval calculated for the standard deviation of the mean (MG), sorting (σG), skewness (SkG) and kurtosis (KG), BGB324 in vitro with the confidence level of 90%. Passega C/M (1964) and Hjulström (1935) diagrams were constructed. The comparison was carried out on the mean (MG) and sorting (σG) of the samples collected from the shore by two different methods ( Figure 2). Lithological data were interpolated by kriging in Golden Software Surfer Selleckchem NU7441 8.0. The shore zone of the Vistula Spit consists of one or two (profiles 16p, 5mv, 3mv, 3a, 8a, 9a, 10a) foredunes developed to various degrees (Figure 3). In the north-eastern part of the Vistula Spit, on the 400 m long shore adjacent to the Strait

of Baltiysk, there are no foredunes owing to intensive erosion. In the south-western part of the Spit, the shore is represented by older, afforested dunes, with a relative height of 5.1–14 m STK38 (profiles 6a–10a, Figure 3). In the remaining area, between profiles 5p and 6a, the relative height of the foredune ranges between 4 and 9 m (Figure 3). At the base of the foredune, the 1–3.5 m high initial dunes are formed locally (profiles 16p, 5mv, 3mv, 3a, 8a–10a). The slope of the foredune is 3° near the Strait

of Baltiysk (profile 3p), 9.5°–13° on profiles 6mv, 5mv and 5a, and 24–30° on profiles 10p and 7a. The beach along the Vistula Spit is from 10 m (profile 3p) to 43–45 m (profiles 1mv, 6a) wide and from 1 m (profile 3p) to 3 m (profiles 5p–13p, 1mv, 10a) high. The slope of the beach is from 2.7°–2.9° (profiles 3mv, 4mv, 6a) to 6.4°–6.7° (profiles 13p, 9a). The system of one (profiles 1a–2a and 7a–10a) or two longshore bars is located in the nearshore (Figure 3). One longshore bar with a height from 0.3 m (profile 10p) to 2.6 m (profiles 13p and 1a) is separated from the shore by a trough located 80–100 m from the shoreline, at depths of 3.5–4.8 m (Figure 3). In the nearshore with two 0.5–1.9 m high bars, the trough separating the first bar from the shoreline is located closer to the shore (10–70 m), at depths of 2.2–3.4 m (profiles 3a–6a, Figure 3). The 3.6–5.7 m deep trough that separates the first and the second longshore bar is located 173–280 m from the shoreline (Figure 3).

However, the colorectal carcinoma cell lines HCT-15, DLD-1, LS 174 T, and LoVo cells that express Mdr-1 are growth inhibited by 17-AAG. We used Colo

320 cells as a positive control for Mdr-1 expression. MRP1 expression could be barely detected Selleck Galunisertib only in DLD-1 cells, which respond to 17-AAG. T98G cells were used as a positive control. On the contrary, BCRP1 expression was detected mainly in the sensitive Hs 766 T pancreatic carcinoma cells and to a lesser extent in several colorectal carcinoma cell lines: DLD-1, SW480, LS 174 T, SW620, HCT-15, and HGUE-C-1 sensitive to 17-AAG and in Caco-2 cells resistant to 17-AAG. The 17-AAG–resistant PANC-1 and CFPAC-1 cells do not express any of the ABC transporters used in our study. We wanted to confirm whether NQO1 was involved in the intrinsic resistance to 17-AAG found by others in pancreatic cancer cell lines [39] and to determine its role in our panel of pancreatic and colorectal carcinoma cell lines and primary tumor cell cultures. The protein NQO1 levels and enzymatic activity were undetectable in the 17-AAG–resistant CFPAC-1 and PANC-1 pancreatic

carcinoma cells and in Caco-2 colorectal cells, which are 17-AAG–resistant (Figure 8, A and B). In fact, there was a negative correlation between the IC50 for 17-AAG after 72 hours of drug exposure and NQO1 activity in the pancreatic and colorectal carcinoma cells studied ( Figure 8C). In addition, the primary cell cultures derived from colorectal tumors express different levels of NQO1 and Hsp70 ( Figure 8A). Interestingly, NQO1 protein levels were relatively high in the less sensitive primary culture to both 17-AAG and NVP-AUY922, HCUVA-CC-34. As expected, there was no GDC-0068 cell line correlation between the IC50 for NVP-AUY922 and NQO1 enzymatic activity in the pancreatic and colorectal carcinoma cell lines studied ( Figure 8C). To determine the role of NQO1 in sensitivity to 17-AAG, we performed cell proliferation assays in 17-AAG–sensitive cell lines in the presence of the NQO1-specific inhibitor ES936 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione], which

was added 30 minutes before exposure to 17-AAG and sustained throughout 17-AAG treatment for 72 hours. In spite of significantly reducing NQO1 activity (Figure 8B), this inhibitor was unable to confer 17-AAG resistance to sensitive cells ( Figure 9A). As expected, no effect was observed in cell lines devoid of NQO1 Cytidine deaminase protein or enzymatic activity, such as CFPAC-1, PANC-1, or Caco-2 cells (data not shown). Then, we wanted to determine the effects of NQO1 ablation in long-term clonogenic assays. First, we determined that after 4 fours of treatment with ES936, NQO1 activity was still inhibited ( Figure 9D). Then, we performed clonogenic experiments after incubating HT-29 cells for 4 hours with 17-AAG or a combination of the specific inhibitor ES936 and 17-AAG and found that clonogenic survival of cells was only slightly recovered after the combination treatment ( Figure 9, B and C).

Such associations between the color of the grains and levels of phenolic compounds may suffer variations as already noted by other authors (Barampama & Simard, 1993). When comparing the preparation methods within the same genotype (Table 1) it was found that the raw grains (R) had the highest content of total phenolics. This result can be explained by the high solubility of these compounds in water, as in soaking water as in broth after the cooking process. Which agrees with Jiratanan and Liu (2004) who analyzed peas, the cooking provided a significant decrease in the phenolic content in

this grain (p < 0.05). Another study ( Ranilla et al., 2009) also corroborates with

these results concluding that different cooking methods do not differ among themselves (p < 0.05) Ku-0059436 molecular weight as to the loss of phenolic compounds, independently of the used genotype. The high values Venetoclax manufacturer of the phenolic compounds obtained between genotypes in different preparation methods (2.0–5.0) may be explained by the form of preparation of the samples, because in this case the seed coat was not separated from its cotyledon, in which the whole seed was used ( Ranilla et al., 2009). Tannins were detected only on raw grain samples (R) due to its high solubility in water (Stanley, 1992) after the soaking or cooking process. Even though there were no significant differences between genotypes, there was a tendency of higher values in genotypes with black color of the seed (Uirapuru and BAF 55) (Table 1). This facilitated loss of phenolic compounds may be associated with higher antioxidant capacity of dark samples cooked with and without soaking water. The genotypes did not differ regarding to the phytate content (Table 1), specially within each bean preparation methods. But when the genotype was compared with the four distinct

BCKDHA preparation forms the IAPAR-81 and Uirapuru showed losses of up to 34.1% and 39.5% of phytate, respectively, in cooked beans without soaking water (COSW) compared to raw beans (R). The results agree with Nergiz and Gökgöz (2007), who found phytate reductions up to 58.4% when bean samples were soaked and cooked. Another research noted a 28% decrease in phytate of the black soaking beans (Kataria, Chauhan, & Gandhi, 1988), Barampama and Simard (1994) also detected a decrease of 47.2% of phytate in soaked and cooked beans compared to raw beans. The decrease of the phytate content occurs because during the soaking there are changes in the membrane permeability of the grains increasing the water absorption, therefore the intrinsic phosphatase is activated causing hydrolysis and the increase of phytate release to the environment (Khokhar & Chauhan, 1986).

In the context of the human relevance framework [6], the similarity of multi-organ carcinogenicity data and body weight gain profiles between Ticagrelor and other dopaminergic compounds is sufficient weight of evidence to establish inhibition

of dopamine reuptake and potentiation of endogenous dopamine agonist activity at the level of the anterior pituitary by Ticagrelor as its MOA for the findings in the rat carcinogenicity bioassay. In addition, since Ticagrelor is peripherally restricted it is likely that this inhibition of dopamine transport and potentiation of endogenous dopamine occurs at the level of the lactotrophs in the pituitary, thus peripheral and not central dopamine levels are most likely responsible for the rat carcinogenesis findings. Daporinad chemical structure The human relevance framework helps classify the human patient safety risk from high confidence in the rodent Selleck Forskolin carcinogenicity data translating into patient safety risk, to the mechanism of action studies determining the rat carcinogenicity data has a MOA not plausible in human and thereby no patient safety risk. Three characterized

examples of the application of the human relevance framework are: 1) High confidence in the human relevance of the ethylene oxide rat carcinogenicity data because it was found to be genotoxic in in vitro and in vivo studies, a mechanism which is not specific to a single

species [32], Based on the human relevance framework, the next step in evaluating patient safety risk was to determine if the Ticagrelor rat carcinogenicity MOA was plausible in humans. In order to determine this, there was a need to understand both the differences between Thiamet G DAT inhibition in the rat versus human as well as how hypoprolactinemia can lead to uterine tumors and if the mechanism is similar in humans. In normal reproductive cycling rats, the estrus cycle consists of 4 days (proestrus, estrus, diestrus-1 and diestrus-2). Prolactin levels are low throughout the estrus cycle except during the afternoon of proestrus, which is driven by the rising estrogen levels in the morning of proestrus [4]. The prolactin released during proestrus is luteotropic in that it promotes rescue of the corpus luteum from degradation, but prolactin is also essential for progesterone production after ovulation, which antagonizes the estradiol-stimulated uterine growth [16]. With aging in rats, there is a progressive loss of hypothalamic dopaminergic neurons, which decreases the level of dopamine at the pituitary and resulting in higher prolactin release [37] and [40].

gondii seropositivity nor serointensity was associated with depression. Our study design was cross-sectional and we are therefore limited in our ability to assess causality. While a convergence of evidence suggests that T. gondii exposure may contribute to anxiety, it is possible that the altered behavior of individuals with GAD increases the risk

of exposure to T. gondii. To our knowledge, however, no data exist to suggest that GAD increases exposure to undercooked meat or cat ownership, Roxadustat nmr two main routes of T. gondii infection. In addition, it is also possible that GAD-related stressors could suppress host immunity, permit T. gondii reactivation, and result in elevated T. gondii antibody levels. However, the specificity of the observed relationship between high T. gondii antibody level category and GAD but not PTSD or depression argues against non-specific

immunosuppression resulting from poor mental health. Another limitation is our measurement of T. gondii exposure, as we were unable to assess parasite strain, route, or timing learn more of infection. Although it is difficult to measure some of these parameters in a population-based study, future research should strive to include this information in assessment of T. gondii exposure in the community setting. Last, reporting of comorbid conditions were only available for 74% of our participants (360/484). Using this subset, we conducted sensitivity analyses to examine whether comorbidity was a potential confounder of the associations of interest in this study. First, we created a modified Charlson comorbidity index using data from the subset of participants who had complete data on 10 available health conditions included in the original Charlson index ( Charlson et al., 1994 and Charlson selleck kinase inhibitor et al., 1987). The modified Charlson comorbidity index was not significantly associated with either T. gondii serostatus or any of the mental health

outcomes. Therefore, the comorbidity index did not meet the criteria for considering a confounder in our data ( Rothman et al., 2012). Nonetheless, we conducted a sensitivity analysis by adding in the comorbidity index in the fully adjusted models for each of our outcomes. We observed that the odds of having GAD among seropositive individuals decreased slightly from 2.25 (95% CI, 1.11–4.53) to 2.16 (95% CI, 0.92–5.08). Among those in the highest antibody level category, the odds of having GAD increased from 3.35 (95% CI, 1.41–7.97) to 3.92 (95% CI, 1.41–10.87), suggesting that the association between high antibody levels to T. gondii and GAD are robust to control for comorbid conditions. Our novel findings suggest that T. gondii exposure, particularly among the highest antibody level category, is associated with GAD but not PTSD or depression even after adjusting for important covariates. Given the tremendous personal and societal burden of GAD in the United States ( Kessler et al.

1, Supplemental Table 1) of dead eggs/embryos, and the daily replacement of 80% of the seawater volume. Dead eggs and embryos were identified as being negatively buoyant and opaque. Total embryonic mortality was assessed at 1 day post-fertilization (1 dpf), 3 dpf, and 7 dpf. These time points correspond to blastula, gastrula, and segmentation periods, respectively, based on the embryonic development of Atlantic cod held at temperatures similar to those used in the current study (Hall

et al., 2004 and Rise et al., 2012). Our use of total mortality at 7 dpf and percent hatch as indices of egg quality is similar to approaches used by other groups studying the influence of fish maternal transcript expression on egg quality (Aegerter et al., 2004, Aegerter et AZD8055 al., 2005, Bonnet et al., 2007 and Mommens

et al., 2010). Each pool of 25 unfertilized eggs per female was homogenized in 400 μL of TRIzol Reagent (Invitrogen/Life Technologies) Epacadostat ic50 using a motorized Kontes RNase-Free Pellet Pestle Grinder (Kimble Chase, Vineland, NJ). An additional 400 μL of TRIzol Reagent was added, and each sample was then passed through a QIAshredder (QIAGEN, Mississauga, ON) following the manufacturer’s instructions. Two hundred μL of TRIzol was then added to each sample to make a total homogenate volume of approximately 1 mL, and the TRIzol total RNA extractions were completed following the manufacturer’s instructions. For the 7 hpf samples, a 0.25 mL volume of flash-frozen fertilized eggs from each female was homogenized in 2.5 mL of TRIzol using a Bio-Gen PRO200 tissue homogenizer (PRO Scientific Inc., Oxford, CT). This homogenizer was equipped with a 5 mm × 150 mm generator tip, and a speed setting of 2–3 was used until no solids were visible (approx. 30 sec). The generator tip was cleaned between samples by running it in a 500 mL beaker of RNase-free water to remove any retained solids, sequentially rinsing it with 0.1% SDS,

0.01% SDS, 0.001% SDS and Milli-Q water, and then running the generator tip 3 times (in separate 50 mL conical tubes) in RNase-free water to ensure that all SDS was removed. The homogenate samples were then passed through QIAshredder columns (QIAGEN) following the manufacturer’s instructions, and Tryptophan synthase centrifuged at 4 °C (12,000 ×g for 10 min) to pellet insoluble material. The TRIzol total RNA extractions were then completed following the manufacturer’s protocol. Individual total RNA samples were treated with 6.8 Kunitz units of DNaseI (RNase-Free DNase Set, QIAGEN) with the manufacturer’s buffer (1 × final concentration) at room temperature for 10 min to degrade any residual genomic DNA. The DNase-treated RNA samples were then column-purified using the RNeasy MinElute Cleanup Kit (QIAGEN) following the manufacturer’s methods.