[18] LC-MS LC-MS or HPLC-MS refers to the coupling of an LC with

[18] LC-MS LC-MS or HPLC-MS refers to the coupling of an LC with a mass spectrometer (MS) [Figure 3]. The separated sample emerging from the column can be identified on the basis of its mass spectral data. A switching valve can help make a working combination of the two techniques. kinase inhibitor KPT-330 A typical automated LC-MS system consists of double three-way diverter in-line with an autosampler, an LC system, and the mass spectrometer. The diverter generally operates as an automatic switching valve to divert undesired portions of the eluate from the LC system to waste before the sample enters the MS. Figure 3 Schematic of an LC-MS (electrospray ionization interface) system An LC-MS combines the chemical separating power of LC with the ability of an MS to selectively detect and confirm molecular identity.

MS is one of the most sensitive and highly selective methods of molecular analysis, and provides information on the molecular weight as well as the fragmentation pattern of the analyte molecule. The information obtained from MS is invaluable for confirming the identities of the analyte molecules. This qualitative analysis makes it possible to reconstruct an unknown compound from MS data. The ionization techniques used in LC-MS are generally soft ionization techniques that mainly display the molecular ion species with only a few fragment ions. Hence, the information obtained from a single LC-MS run, on the structure of the compound, is rather poor. However, this problem has now been tackled by the introduction of tandem mass spectrometry (MS-MS), which provides fragments through collision-induced dissociation of the molecular ions produced.

[19] The use of LC-MS-MS is increasing rapidly. Hyphenated techniques such as HPLC coupled to UV and mass spectrometry (LC-UV-MS) have proved to be extremely useful in combination with biological screening for a rapid survey of natural products. Nowadays, various types of LC-MS systems incorporating different types of interfaces are available commercially. The interfaces are designed in such a way that they offer adequate nebulization and vaporization of the liquid, ionization of the sample, removal of the excess solvent vapor, and extraction of the ions into the mass analyzer. The two most widely used interfaces, especially in relation to natural product analysis, are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

The latter is considered as ��the chromatographer’s LC-MS interface�� Brefeldin_A because of its high solvent flow rate capability, sensitivity, response linearity, and fields of applicability. With these interfaces, various types of analyzers, e.g., quadrupole, ion trap, or TOF, can be used. Each of these analyzers, however, offers varying degree of mass accuracy and resolution. In the LC-UV-MS mode, thermospray (LC-TSP-MS) and continuous-flow FAB (LC-CF-FAB) interfaces can also be applied.

) The five most frequent keywords within the labels of environmen

) The five most frequent keywords within the labels of environmental samples which yielded hits were ‘microbi’ (9.4%), ‘hypersalin’ (9.1%), http://www.selleckchem.com/products/pazopanib.html ‘mat’ (8.6%), ‘len, miniprim, new, view, world’ (8.5%) and ‘food’ (3.4%). The single most frequent keyword within the labels of environmental samples which yielded hits of a higher score than the highest scoring species was ‘hypersalin, len, mat, microbi, miniprim, new, view, world’ (12.5%). These key words are in line with the ecology and the niche from where strains of H. praevalens have been isolated. Figure 1 shows the phylogenetic neighborhood of H. praevalens GSLT in a 16S rRNA gene based tree.

The sequences of the four 16S rRNA gene copies in the genome differ from each other by up to five nucleotides, and differ by up to five nucleotides from the previously published 16S rRNA gene sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”AB022034″,”term_id”:”4127263″,”term_text”:”AB022034″AB022034). Figure 1 Phylogenetic tree highlighting the position of H. praevalens GSLT relative to the other type strains within the family Halanaerobiaceae. The tree was inferred from 1,460 aligned characters [15,16] of the 16S rRNA gene sequence under the maximum likelihood … The cells of strain GSLT are straight and rod-shaped (1 �� 2.4 ��m) (Figure 2) when grown to the mid-log phase at 37oC on CS medium containing 12.5% NaCl and 0.5% glucose [2]. When grown at higher NaCl concentrations (> 20%) the cells appear granulated and shorter in length [2]. Single colonies were reported as white to translucent in color, 0.5-2.

0 mm in diameter, and glistened, when grown on agar plates containing CS medium, 12.5% NaCl, and 0.5% glucose [2]. H. praevalens cells stain Gram-negative [2] and electron microscopy in thin section revealed architectural features typical of Gram-negative bacteria [2]. However, the positive D-BAPA-ase reaction [11] confirms its phylogenetic affiliation to the endospore-forming firmicutes (Table 1). In this respect, H. praevalens is able to hydrolyze only the D- but not the L- isomer of N��-benzoyl-arginine-p-nitroanilide (BAPA) [11]. The activity of D-BAPA-ase was highest at low NaCl concentration (100 mM) and completely inhibited at NaCl concentration equal or higher than 1.0 M (~12%) [11]. Strain GSLT is described to be non-motile, although many flagellar genes have been identified in the genome (see below).

Other isolates Brefeldin_A of H. praevalens were described as motile [5,6], as were other members of the genus [7,9,10], suggesting strain GSLT is atypical with regard to motility. The organism is a strictly anaerobic chemoorganotroph [2]. It grows at NaCl concentrations between 2% and 30%, with optimal growth at approximately 13% [2]. The doubling time is 4 h at 12.5% NaCl and 7 h at 25% NaCl in complex CS medium [2]. The temperature range for growth ranges from 5oC to 60oC, with an optimum at 37oC [2]. The pH range for growth is between 6.0 and 9.0, with an optimum at pH between 7.0 and 7.4 [2].

Unfortunately, these assessment methods cannot reliably diagnose

Unfortunately, these assessment methods cannot reliably diagnose early selleck chemicals cartilage injury and degeneration prior to loss of articular cartilage surface integrity, following which the pathological changes may be irreversible [4]. Experimental evidence shows that chondrocyte metabolic deficits occurring prior to breakdown of the articular surface may be reversible [5]. As such, clinically useful methods to detect subsurface cartilage injury and degeneration are important for development and testing of chondroprotective and chondrorestorative therapies. Optical Coherence Tomography (OCT) is a novel, nondestructive imaging technology capable of near-real-time cross-sectional imaging of articular cartilage at high resolutions comparable to low power histology [6�C11].

The following describes the advent of OCT for arthroscopic imaging of articular cartilage and the potential use of OCT as a new clinical tool for enhanced clinical diagnosis and staging of early cartilage injury and degeneration. 2. Current Clinical Imaging Modalities Minimally invasive arthroscopic imaging of the articular cartilage is considered the clinical standard for detection of early cartilage injury and degeneration. During arthroscopy, the cartilage is graded from 0 to 4 using the Outerbridge scoring system (0 = firm cartilage, 1 = softening, 2 = fissuring of <50% of cartilage thickness, 3 = fissuring >50% of cartilage thickness, and 4 = exposed bone) [12]. Arthroscopy is primarily a surface imaging technology combined with subjective tactile probing.

As such, arthroscopy falls short of the laboratory assessment standards of histopathology, metabolic study and biomechanical testing. Experimentally, biopsy and histology can detect matrix degradation and structural breakdown in cartilage that exhibits no gross surface abnormalities when observed by arthroscopy [7]. However, this is not a practical means for routine clinical detection of early arthritis since histology requires removal and destruction of the tissue being examined. Historically, radiographs were used to diagnose osteoarthritis. However only end-stage bone-related changes are reliably detectable by radiographic exam which does not adequately show soft tissues or directly image articular cartilage. MRI, while being a noninvasive cross-sectional imaging technology, suffers from low resolution and the inability of standard MRI to discern matrix changes leading to cartilage ��softening�� [13].

As such, arthroscopy remains the current clinical standard for diagnosis and staging of early articular cartilage injury and degeneration. 3. Optical Coherence Tomography Optical Coherence Tomography (OCT) is a novel imaging modality that allows for a nondestructive, Cilengitide cross-sectional ��optical biopsy�� of tissue [10, 14].

All 14 patients had successful stabilization with cementoplasty a

All 14 patients had successful stabilization with cementoplasty and symptomatic pain relief was achieved within 24 hours after procedure in 13 of the 14 patients. Moreover, mobility after procedure was improved in 13 of the 14 cases by 1 week [52]. Many other studies have evaluated the success of cementoplasty alone selleck chemicals Imatinib or in combination with other interventional procedures (Table 5). Table 5 Summary of cementoplasty studies in metastatic disease. Hoffmann et al. reviewed 22 patients with 28 metastatic lesions in the spine, pelvis, and lower extremities treated with RA followed by cementoplasty. Pain relief occurred in all patients within 24 hours and after 3 months of the performed procedure. Moreover, the amount of pain medications used was also reduced in 15 of the 22 patients.

The complication rates were also low [54]. 4. Conclusion Surgery and chemotherapy have long been the mainstay of treatment in metastatic disease. However, due to medical comorbidities, intolerance of systemic drug therapy, patient preference, and progression of disease, minimally invasive methods may be utilized in these scenarios. These techniques are becoming more applicable for the treatment of patients with metastatic disease and give the option of less invasive surgical approaches for palliation and local control. With the advancement of research and technology, new and innovative minimally invasive procedures are continually being developed and will benefit increasing numbers of patients with metastatic disease. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.

Years after surgical procedures are performed, operative reports are often the only source of information another surgeon possesses when attempting to understand the history and internal anatomy of a patient. Evidence shows that a structured format for documenting findings improves overall accuracy of reporting and, by extension, is likely to improve patient outcomes [1, 2]. An appropriately detailed report may greatly improve treatment strategy and general preparedness for a case, theoretically leading to better patient safety and care. While efforts have been made in the general surgical field to improve and standardize operative reports, these efforts are still lacking in gynecology surgery. Pelvic anatomy is unique in that various pathologies can be missed if not intentionally sought out for identification. These anatomical characteristics could influence Anacetrapib the detailed description of pelvic findings during surgery in general and, more specifically, during laparoscopy. Classically the pelvis is divided into a true and false pelvis.

The right-skewed distribution of the bRPD values (see Figure 2) m

The right-skewed distribution of the bRPD values (see Figure 2) manifests itself in the fact that only about 2,000 strains … Using bRPD as primary http://www.selleckchem.com/products/Paclitaxel(Taxol).html selection criterion and matching the GOLD database in the current version during the GEBA phase I planning period (December 2010) resulted in the following numbers. Among the total of 8,029 strains, 453 had a ��completed�� genome-sequencing project, 38 a project ��in progress��, and 766 a ��targeted�� project. Among the remaining strains lacking a genome-sequencing project registered in GOLD at the time being, 7 were Cyanobacteria, 970 were not contained in the holdings of the DSMZ, 36 had to be rejected for technical reasons, 685 were set aside as replacement strains in case any of the 1,000 targeted ones turned out to pose difficulties in sequencing.

Finally, 4,074 strains with low scores or expected technical difficulties remained that were postponed and not considered for this phase of the project. Some of the strains not available at DSMZ were selected using the same procedure for potential targeting by the ATCC, Manassas, VA. Table 3 shows the results for the 20 highest-scoring strains according to the bRPD. Apparently, strains from a considerable diversity of phyla are included in the list, and mainly from sparsely sampled phyla with accordingly high inter-species differences [32,36]. Only comparatively few strains had to be postponed or rejected because of their current unavailability or for technical reasons related to cultivation and gDNA extraction. Most of the strains that were not selected were known as targets of other genome projects (or the GEBA pilot project).

Stability of the scoring The comparison of the LTP release ��LTPs106�� with release ��LTPs102�� revealed that 7,991 of the INSDC 16S RNA accessions used in LTPs102 were still in use in the more recent dataset. The Kendall correlation between the bRPD values from both releases after restricting the dataset to the common accessions was 0.925; for uRPD, it was 0.917. Among the 1,000 accessions of the LTPs102 release with the highest bRPD score, 76 were not among the highest-scoring ones from LTPs106; if uRPD was used, this number amounted to 83. This result indicates an additional advantage of bRPD over uRPD. Suitable targets for genome sequencing within the Roseobacter clade The phylogenetic tree used for target selection within the Roseobacter clade is shown in Figure 4, whereas Supplementary Table 1 includes the scores for the species.

As expected, the scoring preferred species situated in isolated positions (e.g., Methylarcula terricola) and/or at long branches (e.g., Rubellimicrobium spp.). Eight species were selected for genome sequencing (see Supplementary Table 1 for details), among them was Roseibacterium elongatum, the one with the Brefeldin_A overall highest score.

Figure 2 Laparoscopic findings and subsequent management of patie

Figure 2 Laparoscopic findings and subsequent management of patients with undescended testes. DSD: disorders of sexual differentiation. A ��high�� position of EPZ-5676 buy the testis was defined as being above the external iliac vessels; orchiopexy for these patients consisted of a two-stage Fowler-Stephens procedure. A ��low�� intra-abdominal testis was usually managed by one-stage laparoscopic orchiopexy. Orchidectomy was performed on an atrophic testis accompanied by a contralateral normal testis. All patients were routinely followedup at our outpatient clinic. A ��successful�� procedure was defined as a testis palpable in the scrotum and of similar or increased size. Factors evaluated included patient age at operation, side of the impalpable testis, clinical and laparoscopic findings, operative intervention, and outcomes.

3. Results We identified 91 patients, 9 with bilateral and 82 with unilateral impalpable testes, between January 2006 and December 2010, for a total of 100 testes. There was a trend of increasingly performed cases over that period (Figure 1). Mean patient age at the time of surgical intervention was 19 + (interquartile range [IQR], 12�C36) months. The sides of impalpable testes are shown in (Figure 3). Figure 3 Distribution of impalpable testes according to the side affected. Thirty-seven patients received no further treatment after laparoscopy due to absent testes. In contrast, we found that 11 intra-abdominal testes were high, above the iliac vessels, and 5 were low. The 11 high intra-abdominal testes were managed using the two-stage Fowler-Stephens procedure.

This procedure was successful for 3 testes, after a mean followup period of 3.5 months, but unsuccessful in 4; of the latter, 3 underwent orchidectomy for atrophic testes. The remaining 3 were lost to followup. Seven ��low�� intra-abdominal testes were managed using the one-stage Fowler-Stephens procedure, which was successful in 1 and unsuccessful in 1; the remaining 5 were lost to followup. In 42 testes, the vas and vessels entered the internal inguinal ring; open-standard inguinal orchiopexy was successful in 20 patients and unsuccessful in 10; the latter underwent a second orchiopexy. The remaining 12 were lost to followup Table 1. Table 1 Outcomes relative to intraoperative laparoscopic categorization of impalpable testes.

None of these patients experienced any immediate or postoperative complications from laparoscopy. No port site hernia was detected at followup. 4. Discussion Testicular descent, although not yet fully understood, takes place Cilengitide during two different gestational stages, occurring during intrauterine weeks 8 to 15 and 25 to 35. Failure of the first phase of descent is rarer than of the second phase and results in an intra-abdominal undescended testis [8].

Figure 2 Mean rank values

Figure 2 Mean rank values www.selleckchem.com/products/tofacitinib-cp-690550.html of ��E associated with the two bleaching methods. (Note: ��E1 = Bleaching effectiveness; ��E2 = Rebound effect; ��E3 = Color difference between post-treatment and unbleached teeth) Table 1 Mean (SD) values of ��E associated with the two bleaching methods The difference between the bleaching methods immediately after bleaching, then at 2 weeks, 1 month, 3 month and 6 month follow-up periods was compared using the Mann-Whitney test. Between groups, the bleaching effectiveness (��E1) and rebound effect (��E2) and ��E3 were compared using the Mann-Whitney test because of non-matching data comparison between the two groups. The Friedman test was used to identify differences among ��E1, ��E2 and ��E3 for each bleaching technique that was indicated for matching comparisons among three or more groups.

The Wilcoxon test was done as the Friedman test showed some difference between ��E1, ��E2 and ��E3 for each bleaching technique. The Wilcoxon is indicated for matching comparisons between only two groups. The distribution of tooth sensitivity for comparing both methods and jaws was evaluated using the Chi-square test (�� =0.05), that was indicated for qualitative data, for a non-matching comparison between two groups. RESULTS Eight participants from the power bleaching phase and six from the at-home bleaching phase complained about tooth sensitivity after treatment. Two of them experienced sensitivity after both treatment methods. Three participants were dissatisfied with the taste of the at-home bleaching agent. Nonetheless, all were able to complete the bleaching intervals successfully.

It should be noted that there was no significant difference between both bleaching methods immediately after bleaching, or at the 2 week, 1 month and 3 month follow-up periods. (P > 0.05 Mann-Whitney). The only statistic significant difference between the two bleaching methods was found in ��E2 (P = 0.001 < 0.05, Mann-Whitney) and at the 6 months follow-up period suggesting that the Dacomitinib rebound effect in power bleaching method was significantly faster than that of at-home bleaching after 6 months. In both groups, there was no significant difference between ��E1 and ��E3 (home bleach = 0.59 > 0.05 and office bleach = 0.069 > 0.05 Wilcoxon). The Chi-square test showed that post-treatment sensitivity was identical (P > 0.05) for both methods [Figure 3]. Figure 3 Prevalence of sensitivity with each bleaching method DISCUSSION The aim of this clinical trial was to address practitioner concerns regarding the efficacy, longevity and degree of hypersensitivity of bleached teeth after in-office vital bleaching compared with at-home bleaching techniques.

Stabilization of HIF-1�� protein led to

Stabilization of HIF-1�� protein led to sellectchem the transcriptional increase of HIF-1 target genes BNIP3, CA9 as well as EPO, a target gene shared with HIF-2 (Figure 1B). To further verify that SIRT1 protein is not altered by hypoxia, HCC cells were exposed to 1% O2 for up to 48 hours. There was no change in SIRT1 protein or mRNA in cells exposed for 12, 24 or 48 hours to 1% O2 (Figure 1C & D). In addition, there was no significant change in SIRT1 mRNA in Hep3B cells exposed to more severe hypoxia of 0.1% O2 for 24 hours (Figure 1E). These data show that both SIRT1 and HIF-1�� are present in cells under hypoxic conditions and HIF-1 is transcriptionally active. Moreover, unlike HIF-1��, SIRT1 expression is not tightly regulated by hypoxia in HCC cells. Figure 1 SIRT1 and HIF-1�� are simultaneously expressed in hypoxic cells.

Inhibition of SIRT1 decreases HIF transcriptional activity and suppresses HIF-1�� protein accumulation As demonstrated above, we observed a simultaneous expression of both SIRT1 and HIF-1�� protein under hypoxic conditions. A recent report by Lim et al. has suggested that SIRT1 negatively regulates HIF-1 activity by suppressing the transcriptional regulation of its target genes [27]. Following the model proposed by their data, inhibition of SIRT1 should provoke an exaggerated HIF-mediated response. Therefore, we tested the effect of inhibiting SIRT1 on HIF-1 activity using sirtinol, a cell permeable specific inhibitor of SIRT deacetylase activity [41]. The IC50 value of sirtinol is 70 ��M for SIRT1 activity; hence concentrations of 25, 50 and 100 ��M were used in this study.

First, we confirmed the functionality of sirtinol on SIRT1 deacetylase activity in HepG2 cells. HepG2 cells have abundant SIRT1 protein, as well as an intact p53 response to DNA damage. The acetylation of lysine 382 of p53 is modulated by SIRT1 [21]. We demonstrated that sirtinol had no detectable effect on the lysine 382 acetylation in the absence of DNA damage, whereas, sirtinol produced a time-dependent increase of acetylated p53 in cells treated with the DNA damaging agent, doxorubicin (Figure S1). These data confirm that sirtinol is an efficient inhibitor of SIRT1 activity in vitro. We next investigated the effect of inhibiting SIRT1 activity on HIF-mediated transcriptional activation.

Cells were transfected with a HRE reporter construct that contains multiple HRE sites and is specifically induced by HIF proteins and thus represents a direct measurement Carfilzomib of HIF activation [42]. Twenty-four hours after transfection, cells were treated with increasing doses of sirtinol and incubated 24 hours at 1% O2. Hypoxia increased reporter activity 38-fold and there was no significant change in cells treated with DMSO at a concentration equal to the amount needed to treat cells with 100 ��M sirtinol.


Other selleck compound studies have noted similar results with lower fetal biometry at 20�C24 weeks gestation (Hanke, Sobala, and Kalinka, 2004), higher risk for fetal death, preterm delivery, and low birth weight (Gray et al., 2010; Jaddoe et al., 2008). SHS exposure during pregnancy has also been associated with poorer respiratory health among infants over the first 6 months of life (Jedrychowski et al., 2007), and among children with asthma, higher exposure to SHS is associated with higher externalizing behavior problems in the early school years (Yolton et al., 2008). Thus, accurate identification of SHS exposure among pregnant women is an essential first step to preventing negative consequences for maternal and infant health. The study is guided by social�Cecological theory (e.g., Corbett, 2001; Ennett et al.

, 2010; Green, Richard, and Potvin, 1996; Stokols, 1996) suggesting that multiple domains of influence such as household/family, peers, and workplace may provide a context for maintenance of smoking through pregnancy. For example, contexts characterized by the absence of antismoking rules and policies and a high number of individuals who smoke and continue to smoke around the pregnant woman are likely to support the maintenance of smoking during pregnancy. Alternatively, these sources of influence may provide motivation for quitting if social network pressures inhibit access or opportunity and provide social pressures to quit smoking during pregnancy. Finally, this theory would suggest that it is important to consider multiple domains of influence to understand the social context of smoking.

Thus, identification of multiple sources of SHS exposure across several contexts is important for understanding the social ecology of pregnancy smoking and of quitting and maintaining quit status. There is no gold standard for self-report measurement of SHS exposure during pregnancy. Most studies of pregnant women measuring SHS exposure (SHS) use one single item measure of SHS exposure. The most common methods of measuring SHS exposure among pregnant women have been a single question about partner smoking status or the number of cigarettes smoked by the partner (see Windham, Eaton, and Hopkins, 1999, review). When studies of pregnant smokers have included other questions, they consist of either a question about number of smokers in the household or hours of exposure to SHS (Leonardi-Bee et al.

, 2008; Newman et al., 2010; Pogodina, Brunner Huber, Racine, and Platonova, 2009; Windham et al.). Few prospective studies of maternal cigarette smoking during pregnancy have examined ongoing SHS exposure using more than single indicator measures of SHS exposure, although studies of postnatal SHS exposure Cilengitide or lifetime SHS exposure have more comprehensive measures (e.g., Edwards, 2009; Rise and Lund, 2005).

A secondary aim was to identify the potential role of tobacco dep

A secondary aim was to identify the potential role of tobacco dependence as a mediator underlying significant relations. METHODS Participants selleck chem inhibitor and Procedures Data were collected as part of a longitudinal study examining changes in risk perceptions over time among community smokers attempting to quit smoking. Inclusion criteria were as follows: English-speaking current smokers aged 18�C65 who endorse smoking ��5 cigarettes/day for ��12 months, willingness to quit smoking within the next week, functioning telephone number and a permanent home address, and sixth grade literacy level. Exclusion criteria were as follows: regular use of tobacco products other than cigarettes, the use of pharmacological smoking cessation treatments at enrollment, reported medical contraindications to the nicotine patch, pregnancy or lactation, other household members enrolled in the study, participation in a smoking cessation program in the last 3 months, or active substance use or dependence (other than tobacco).

Participants in the parent study were 200 smokers from Houston, TX, who were enrolled in 2006�C2007; however, due to the inadequate representation of some racial and ethnic groups in the sample, the current study was limited to the non-Hispanic White and non-Hispanic Black participants in the parent project (n = 183). Data were collected during a phone screening for eligibility and five in-person clinic visits. These visits occurred 1 week prior to the participants�� quit date (baseline), on the quit date (week 0), and weeks 1, 2, and 3 following the quit date (week 1, week 2, and week 3).

All participants in the parent protocol had access to four brief cessation counseling sessions and 4 weeks of nicotine replacement therapy (the patch), and were compensated for their time and effort with a $25 department store gift card at each in-person clinic visit they attended. The University of Texas MD Anderson Cancer Center Institutional Review Board approved this study. Written informed consent was obtained before data collection. Measures Sociodemographics Sociodemographics collected during the phone screen included age, race, gender, total annual household income, educational level, employment status, and partner status. Menthol Use Status Menthol use was assessed during the phone screen by a single item assessing if participant��s usual brand of cigarettes was menthol or nonmenthol.

Results yielded a binary predictor variable (nonmenthol vs. menthol; reference group = nonmenthol). Continuous Short-Term Smoking Abstinence Smoking status was assessed at postquit weeks 1, 2, and 3. Continuous abstinence from smoking was defined as a self-report of no cigarettes smoked since the quit date (not even a puff) and an expired Brefeldin_A carbon monoxide level of <10 ppm at each assessment.