[11] reference Thin layer chromatography studies are among the key identity tests in most pharmacopoeial monographs. Pharmacopoeial standards are typically used by industry as a basis for meeting QC requirements and current good manufacturing practices (cGMPs). An extension of TLC is high-performance thin layer chromatography (HPTLC) is robust, simplest, rapid, and efficient tool in quantitative analysis of compounds. HPTLC is an analytical technique based on TLC, but with enhancements intended to increase the resolution of the compounds to be separated and to allow quantitative analysis of the compounds. Some of the enhancements such as the use of higher quality TLC plates with finer particle sizes in the stationary phase which allow better resolution.

[12] The separation can be further improved by repeated development of the plate, using a multiple development device. As a consequence, HPTLC offers better resolution and lower Limit of Detection (LODs). Visual detection is suitable for qualitative analysis, but a more specific detection method is needed for quantitative analysis and for obtaining structural information on separated compounds. UV, diode-array and fluorescence spectroscopy, mass spectrometry (MS), Fourier-transform infrared (FTIR), and Raman spectroscopy have all been applied for the in situ detection of analyte zones on a TLC plate. Van Berkel and coworkers have recently described couplings of TLC to atmospheric pressure chemical ionization[13] and electrospray ionization.[14�C16] In both couplings, a special surface sampling probe is used for extracting the analyte on-line from the TLC plate to MS analysis.

The usage of HPTLC is well appreciated and accepted all over the world. Many methods are being established to standardize the assay methods. HPTLC remains one step ahead when compared with other tools of chromatography.[17] One of the available chromatographic techniques is HPTLC, which is used for the identification of constituents, identification and determination of impurities, and quantitative determination of active substances. The use of modern apparatus such as video scanners, densitometers, and new chromatographic chambers, and more effective elution techniques, high-resolution sorbents with selected particle size or chemically modified surface, the possibility of combining with other instrumental methods, and development of computer programs for method optimization all make HPTLC an important alternative method to HPLC or gas chromatography.

Specifically, HPTLC is one of the ideal TLC technique for the analytical purposes because of its increased accuracy, reproducibility, and ability to document the results, compared with standard TLC. Because of this, HPTLC technologies are also the most appropriate GSK-3 TLC technique for conformity with GMPs.

Figure 1 Phylogenetic tree highlighting the position of Alistipes senegalensis strain JC50T relative to other type strains within the Alistipes genus. GenBank accession numbers are indicated in parentheses. The tree was inferred from the comparison of 16S rRNA … Different growth temperatures (25, 30, 37, 45��C) were tested; no growth occurred at 25��C and especially 45��C, growth occurred between 30 and 37��C, and optimal growth was observed at 37��C. Colonies were 0.2 to 0.3 mm in diameter on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioM��rieux), and in the presence of air, with or without 5% CO2 and in aerobic conditions.

The optimal growth of the strain was obtained anaerobically, with weak growth being observed under microaerophilic conditions, and no growth observed under aerobic conditions. Gram staining showed Gram negative bacilli. A motility test was negative. Cells grown on agar are Gram-negative rod-shaped bacteria (Figure 2) and have a mean diameter of 0.56 ��m (Figure 3) by electron microscopy. Figure 2 Gram staining of A. senegalensis strain JC50T Figure 3 Transmission electron microscopy of A. senegalensis strain JC50T, using a Morgani 268D (Philips) at an operating voltage of 60kV.The scale bar represents 900 nm. Strain JC50T exhibited a catalase activity but no oxidase activity. Using API Rapid ID 32A, a positive reaction was obtained for mannose fermentation, proline arylimidase, leucyl glycine arylamidase, alanine arylamidase.

A weak reaction was obtained for indole production, ��-galactosidase, ��-galactosidase, ��-glucuronidase, arginine arlyamidase and glycine arylamidase. A. senegalensis is susceptible to penicillin G, imipeneme, amoxicillin + clavulanic acid and clindamycin but resistant to metronidazole and vancomycin. Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [20]. Briefly, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate, and spread as a thin film on an MTP 384 MALDI-TOF target plate (Bruker Daltonics, Germany). Four distinct deposits were done for strain JC50T from four isolated colonies.

Each smear was overlaid with 2��L of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50% acetonitrile, 2.5% tri-fluoracetic acid, and allowed to dry for five minutes. Measurements were performed with a Microflex spectrometer (Bruker). Spectra were recorded in the positive linear mode for the mass range of 2,000 to 20,000 Da (parameter settings: ion source 1 (ISI), 20kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 675 shots at a variable laser Dacomitinib power.

6% and HSP coverage of 64.4%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was “type”:”entrez-nucleotide”,”attrs”:”text”:”AF068800″,”term_id”:”7555793″,”term_text”:”AF068800″AF068800 selleck kinase inhibitor (‘hydrothermal vent clone VC2.1Bac24′), which showed an identity of 99.7% and an HSP coverage of 92.7%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were ‘hydrotherm’ (5.4%), ‘vent’ (4.9%), ‘microbi’ (3.6%), ‘water’ (2.9%) and ‘deep’ (2.0%) (167 hits in total). The most frequently occurring keyword within the labels of those environmental samples which yielded hits of a higher score than the highest scoring species was ‘hydrotherm, vent’ (50.

0%) (1 hit in total). Figure 1 shows the phylogenetic neighborhood of D. thermolithotrophum BSAT in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by two nucleotides from the previously published 16S rRNA sequence (“type”:”entrez-nucleotide”,”attrs”:”text”:”AJ001049″,”term_id”:”3063765″,”term_text”:”AJ001049″AJ001049). Figure 1 Phylogenetic tree highlighting the position of D. thermolithotrophum relative to the type strains of the other species within the order Aquificales. The tree was inferred from 1,422 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum … Table 1 Classification and general D. thermolithotrophum BSAT in accordance with the MIGS recommendations [18] and the NamesforLife database [19].

The cells of strain BSAT are small rods, about 1-2 ��m long and 0.4-0-5 ��m wide and occur singly or in pairs (Figure 2) [1]. Cells stain Gram-negative and are motile via three polar flagella; spores are not produced [1]. Strain BSAT grows between 40 and 75��C with an optimum around 70��C, while no growth is detected at 37 or 80��C after 48 h incubation [1]. Growth occurs between pH 4.4 and 8, with an optimum around AV-951 pH 6.25. No growth is detected at pH 3.7 or 8.5 after 48h incubation at 70��C [1]. Growth is observed in sea salts at concentrations ranging from 15 to 70g/l, with an optimum of approximately 35g/l (corresponding to 23 g NaCl/l [1]). No growth was observed in sea salts at concentrations of 10 and 80 g/l after 48 h incubation at 70��C [1]. Under optimal growth conditions (temperature, pH and NaCl), the doubling time of strain BSAT is around 135 min [1]. Strain BSAT is a strictly anaerobic chemolithotrophic organism that uses sulfur as an electron acceptor in the presence of H+ for growth [1]. It utilizes thiosulfate, sulfite and polysulfides as alternative electron acceptors with H2 as an electron donor.

algicola, with orthologs belonging to different COG categories. The LIM 21T genome contains 15 genes Rucaparib clinical coding for sulfatases, which are located in close proximity to glycoside hydrolase genes suggesting that sulfated polysaccharides may be used as substrates. ��-L-fucoidan could be a substrate, as three ��-L-fucosidases (Celly_0440, Celly_0442, Celly_0449) are located in close proximity to nine sulfatases (Celly_0432, Celly_0425, Celly_0426, Celly_0436, Celly_0431, Celly_0433, Celly_0435, Celly_0438, Celly_0444). Sakai and colleagues report the existence of intracellular ��-L-fucosidases and sulfatases, which enable ‘Fucophilus fucoidanolyticus’ to degrade fucoidan [42]. The above mentioned sulfatases and fucosidases containing region of C. lytica is similar to the recently described region of C.

algicola with five ��-L-fucosidases and three sulfatases [14]. This fucoidan degrading ability could be also shared by Coraliomargarita akajimensis, as the annotation of the genome sequence revealed the existence of 49 sulfatases and 12 ��-L-fucosidases [43]. Comparative genomics The genomes of the two recently sequenced Cellulophaga type strains differ significantly in their size, C. lytica having 3.8 Mb and C. algicola 4.9 Mb and their number of pseudogenes, 19 (0.6%) and 122 (2.8%), respectively. Liu et al., 2004 have shown that pseudogenes in prokaryotes are not uncommon; the analysis of 64 genomes, including archaea, pathogen and nonpathogen bacteria, revealed an occurrence of pseudogenes of at least 1-5% of all gene-like sequences, with some genomes containing considerably higher amounts [44].

To estimate the overall similarity between the genomes of C. lytica and C. algicola the GGDC-Genome-to-Genome Distance Calculator [45,46] was used. The system calculates the distances by comparing the genomes to obtain HSPs (high-scoring segment pairs) and interfering distances from the set of formulas (1, HSP length / total length; 2, identities / HSP length; 3, identities / total length). The comparison of the genomes of C. lytica with C. algicola revealed that 25% of the average of both genome lengths are covered with HSPs. The identity within these HSPs was 82%, whereas the identity over the whole genome was only 20%. These results demonstrate that according to the whole genomes of C. lytica and C. algicola the similarity is not very high, although the comparison of 16S rRNA gene sequences showed only 7.

7% differences. In order to compare the C. lytica and C. algicola genomes, correlation values (Pearson coefficient) according to the similarity on the level of COG category, pfam, enzymes and TIGRfam were calculated. The highest correlation value (0.98) was reached on the level of pfam data; the correlation values on the basis of COG, enzyme and TIGRfam GSK-3 data were 0.88, 0.92 and 0.93, respectively.

Curved and or articulated instruments have been used according to the surgeon’s preference [14], as they Ixazomib proteolytic may allow to work on the operative field without a straight approach from the access port. Using these instruments requires the instrument from the right hand to be on the left side of the screen and the left-hand instrument to be on the right side of the screen [6, 33]. One can choose an instrument with handles that are articulated so they are away from each other at the access port or use ports with a lower external or internal profile for a wider range of instrument motion. Also, instruments of variable lengths allow for external manipulation so that they are operated in different planes, thus avoiding collisions [25]. 3.

Patient Outcomes: SILC/LESS cholecystectomy versus Four-Port Cholecystectomy In spite of numerous reports regarding the safety and efficacy of the SILS/LESS cholecystectomy approach, laparoscopic cholecystectomy (LC) still remains the gold-standard for the surgical removal of the gallbladder [6]. Thus the comparison of patient outcomes between both procedures is of key importance. In this respect several prospective studies comparing LC and SILC/LESS Cholecystectomy have now been published [12�C20] (Table 2). Table 2 Comparison of clinical trials comparing SILC versus 4PLC��SILC/LESSC (single-incision laparoscopic cholecystectomy/laparoendoscopic single-site cholecystectomy), 4PLC (four port laparoscopic cholecystectomy). There are several blinded randomized trials comparing standard LC to SILC/LESS cholecystectomy with varied results regarding patient outcomes.

An outcome that has had a significant difference in several studies comparing SILC/LESS cholecystectomy versus LC is the cosmetic result. Patients are more satisfied with the hidden or infraumbilical single surgical scar than the four scars created by the LC [13, 17, 19]. In an attempt to try and reduce the bias associated with cosmetic evaluation, Marks et al. and Bucher et al. used body image scale, a scar scale photo series 10-point scoring questionnaire in order to compare results between SILC/LESS and LC patients. However regardless of the scale used, there is still an element of personal preference and opinion involved with the evaluation of cosmetic results. Aside from cosmetic perception, the only consistently reproducible and statistically significant result among series is a prolonged time of surgery for the SILC/LESS cholecystectomy groups versus standard LC groups [12�C14, 16�C20]. A study by Qiu et al. [34] focused specifically on the learning curve phenomenon associated with SILC/LESS cholecystectomy and saw an improvement in operative times as experience was gained [34] this Drug_discovery was similar to what was observed by others [18�C20].

Of the protein coding genes, 1,592 were assigned to a putative function, with the remaining being annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Tables 3 and and44. Table 3 Nucleotide content Src Bosutinib and gene count levels of the genome Table 4 Number of genes associated with the 25 general COG functional categories Comparisons with other fully sequenced genomes Comparison of the assembled draft genome sequence of strain BD11-00177 with publicly available F. tularensis genome sequences revealed that it clusters in the FTNF002-00 genomic group (B.Br.FTNF002-00 and BIV.FTNF002-00) defined by the FTNF002-00 genome sequence [28-30] within the B.IV clade. The presence of the 1.

59 kb RD23 deletion event [31] as well as the 464 bp size of the MLVA marker FtM24 [32], both typical for the FTNF002-00 genomic group, were confirmed in silico. Notably, isolates from this genomic group had previously been exclusively reported from Spain, France, Italy, Switzerland and Germany [28,31-35]. A BLAST Ring Image Generator (BRIG) analysis comparing the F. tularensis subsp. holarctica BD11-00177 genome against the F. tularensis subsp. holarctica genomes of F92, LVS, and FTNF002-00 revealed that the BD11-00177 draft genome shows considerable resemblance to FTNF002-00 (Figure 2). Figure 2 BRIG diagram of the F. tularensis subsp. holarctica BD11-00177, FTNF002-00 and SCHU S4 genomes using the F. tularensis subsp. holarctica FSC200 genome as a reference backbone. White regions represent absent genetic regions. Evolutionary history of F.

tularensis subspecies holarctica strain BD11-00177 was inferred using publicly available whole genome sequences. The trees in Figure 3 A and B are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the number of differences method and are in the units of the number of base differences per sequence. The overview of Francisella genus involved 52 public genome sequences using Piscirickettia salmonis as outgroup (Figure 3A). The detailed analysis involved 14 F. tularensis subsp. holarctica genome sequences using F. tularensis subsp. tularensis strain SCHU S4 as outgroup (Figure 3B) [17,30,33,36-41]. All positions containing gaps and missing data were eliminated.

There were a total of 1,599,589 positions in the final dataset. Figure 3 A) Overview of the Francisella genus phylogeny based on 52 public whole genome sequences. B) The phylogeny of F. tularensis subsp. holarctica strains based Cilengitide on whole genome sequences. The new isolate, BD11-00177 belongs to the FTNF002-00 genomic group … Conclusion Here we have presented the draft genome of the first member of FTNF002-00 genomic group of F. tularensis subspecies holarctica.

28,32 Despite these potential explanations, the exact mechanism of the anti-inflammatory action of L. sidoides is still unknown, and further www.selleckchem.com/products/Y-27632.html studies are required. The BI is a generally used dichotomous index for evaluation of gingivitis10,13,14, but it does not assess the severity of gingival inflammation. Studies evaluating the reduction of gingivitis by a grading index could be interesting to complement these results, as used in other works evaluating herbal agents.12,22,33 However, color change, used as a parameter in this grading index, is not necessarily an accurate indicator of gingivitis.33 Furthermore, laboratory tests such as enzyme immunoassays of gingival crevicular fluid would be required for a better understanding of the role of L. sidoides preparations as effective agents for gingivitis control.

Home-use studies are often influenced by a number of factors which can mask the superiority of a test agent over controls. Participants of clinical trials may experience some improvement not specifically associated to the therapeutic properties of the test agent, but rather related to a behavioral change; this is known as the Hawthorne effect.14,34 Subjects enrolled in oral hygiene studies usually improve their tooth brushing irrespective of the product they receive.14,34 Although the volunteers in the present study were not aware of which gel they were using, another main factor is the so-called novelty effect, which is the motivation to improve oral hygiene practices induced by the use of a new substance. In contrast, lack of compliance with correct use of the gel can occur as well.

14,34 In order to minimize this potential, participants were asked to bring the bottle at the end of the trial, so we could perform an indirect assessment of compliance. The reduction in gingivitis observed Drug_discovery in both test groups showed that participants used the product correctly, at least to some extent. Finally, the results showed that LS was an effective herbal antigingivitis agent, with performance similar to that of chlorhexidine digluconate, and could be advantageous in cases where patients spend little time on toothbrushing. Further clinical studies are required to evaluate the action of this herbal agent in other oral diseases, such as chronic periodontitis. CONCLUSION A gel formulation containing 10% L. sidoides essential oil was an efficient antiplaque and antigingivitis agent.
The conventional glass ionomer cement (GIC) has been advocated as a restorative material because of its ability to chemically bond to tooth structures1,2 and release fluoride.

Footnotes Competing Interests: The authors have declared that no competing interests exist. Funding: The authors were funded by the Institute for Pathology, University Hospital of Basel, Research pool account. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
autosomal recessive proximal http://www.selleckchem.com/products/azd9291.html renal tubular acidosis is characterized by severe proximal tubule bicarbonate wasting, hyperchloremic metabolic acidosis, and hypokalemia (11, 20, 21, 25�C27, 39, 50, 54). In addition to a renal phenotype, these patients have extrarenal manifestations involving the eye that includes glaucoma, cataracts, and band keratopathy. Mental retardation, short stature, teeth abnormalities, an elevated serum amylase, thyroid abnormalities, and basal ganglia calcification have also been reported (11, 20, 21, 25�C27, 39, 50, 54).

All patients characterized thus far have mutations in the electrogenic sodium bicarbonate cotransporter NBCe1 encoded by the SLC4A4 gene (1). Renal bicarbonate wasting results from the loss of normal NBCe1-A-mediated basolateral proximal tubule bicarbonate absorption. The extrarenal manifestations are due to the finding that NBCe1 also plays an important role in bicarbonate transport/pH regulation in various organs (46). Currently, 10 unique mutations throughout the cotransporter have been reported. The location of these mutations is depicted in Fig. 1 based on one of the putative current topological models of NBCe1.1 Fig. 1. Putative location of mutations in NBCe1-A in patients with autosomal recessive proximal renal tubular acidosis.

Of the known mutations in NBCe1, three mutations that cause premature truncation of the cotransporter have been described (21, 26, 27). In the NBCe1-A-Q29X nonsense mutation causing proximal renal tubular acidosis, a wild-type CAG codon encoding glutamine has been replaced by a UAG stop codon, resulting in premature truncation of the cotransporter (26). NBCe1 has three functional variants, NBCe1-A, NBCe1-B, and NBCe1-C; however, only NBCe1-A possesses a unique NH2 terminus containing Gln-29 and is therefore the only variant prematurely truncated in these patients as a result of the Q29X mutation.

Currently, hundreds of nonsense mutations causing human disease are known, including those causing Alport’s syndrome (28), diabetes insipidus (48), cystic fibrosis (63), Duchenne muscular dystrophy (23, 60), ataxia-telangiectasia (64), Hurler syndrome (32), hemophilia A (66), hemophilia B (34), and Cilengitide Tay-Sachs (2). Unfortunately, for many of those diseases there is presently no effective treatment. Although gene therapy seems like a potential possible solution for these genetic disorders, there are still many critical difficulties to be solved before this technique can be used in humans.

Fragments of the ERBB2 promote act differently in breast and in ovary or colon cancer cells we have analysed. Additional studies are needed to understand the mechanisms responsible http://www.selleckchem.com/products/carfilzomib-pr-171.html for the increased accumulation of the erbB-2 transcript and protein in non-breast cancer cells. Acknowledgments We thank Dr R Kiss for the pancreatic cell lines, Drs J Piette, M Grooteclaes and L Delacroix for critically reading the manuscript. This work was supported by a Grant from the Belgian ��Fonds National pour la Recherche Scientifique (F.N.R.S.)��, ��F��d��ration belge contre le Cancer��, ��Centre Anticanc��reux pr��s l’Universit�� de Li��ge�� and ��Fondation pour promouvoir la Recherche �� l’Ulg��. Rosita Winkler and Philippe Delvenne are Chercheur Qualifi�� du F.N.R.S. Douglas Vernimmen is a recipient of a T��l��vie Grant from the F.

N.R.S.
Cancer of the exocrine pancreas is the fourth leading cause of cancer-related deaths in both men and women (Fernandez et al, 1994). Even if advances in surgical techniques, radiation and chemotherapy have provided significant improvements in the overall survival and on the quality of life, fewer than 5% of pancreatic cancer (PC) patients survive 5 years after diagnosis (Parker et al, 1996). Therefore, it is not surprising that PC is a serious health problem. Thus, there is a great demand for new and more effective therapeutic strategies for the treatment of this disease. Recent studies on pancreatic tumour tissues and cell lines have shown that multiple subsets of genes undergo activation or inactivation during development and progression of disease (Hilgers and Kern, 1999).

Specific point mutations at codon 12 of the K-ras oncogene are detected in 75�C90% of PC specimens and constitute the most common genetic changes in PC (Almoguera et al, 1988). The mutated K-ras protein is not able to convert GTP to GDP, resulting in constitutively active GTP-bound species and constitutive activation of cell proliferative signals. It is therefore conceivable that the pancreatic carcinogenetic process and/or the maintenance of the transformed phenotype strongly rely on the dysregulated activity of the p21ras-mediated signalling. The inhibition of p21ras signalling has therefore been postulated as a possible target for anticancer therapy and might be specifically suitable for the treatment of PC.

Bisphosphonates (BPs), the most effective and widely used class of drugs in the treatment of metabolic bone disorders, including tumour-associated bone disease, are capable of inhibiting p21ras signalling (Luckman et al, 1998; GSK-3 Green, 2001). Therefore, the extensive evaluation of these compounds, and particularly the study of the recently available and more potent nitrogen-containing BPs, may suggest expanding applications of these drugs and warrant further investigation.

Women proved to have a more favorable overall outcome than men in the series. Sex has not been found to be of significance in population-based studies from Sweden and Iceland (Nilsson et al. 2005; Tryggvason et al. 2005), but in a study selleck by DeMatteo et al. (2000), male sex was a poor prognostic sign. The mean age at the time of diagnosis was almost equal between the sexes, and reasons other than age for longer survival among the women in our study must be considered. The location of the primary tumor has been found to be of importance by others (Emory et al. 1999; Nilsson et al. 2005) and is again confirmed in this study. Patients with gastric GISTs lived longer than patients with small bowel tumors. Nuclear atypia, either focal or diffuse, was found in this series to be an unfavorable morphologic variable, which has previously been reported by some (Miettinen et al.

2005) but not by others (Iesalnieks et al. 2005). In this study, we tested nuclear morphometric characteristics as potential independent variables for evaluating risk of malignancy in a large series of GISTs. In univariate models, some of these factors show a prognostic value for survival, but the significance is lost in multivariate models because of the strong correlation with mitoses and tumor size. Nuclei with less round features and with a lower SL ratio correspond to spindle cells, and those with rounder nuclei and higher SL ratios correspond to epithelioid cells. Investigation of predominant cell type, spindled or epithelioid, in GISTs has been evaluated in our study and also by others, without showing significance for overall survival (Carrillo et al.

1997; Iesalnieks et al. 2005; Miettinen et al. 2005). Nuclear roundness and nuclear ratio are interesting independent variables but should probably not be considered important as prognostic variables at this stage. Anatomical site of the primary tumor is now being accepted as a variable to be considered in risk stratification of primary GISTs (Hornick and Fletcher 2007). We suggest, however, that sex and nuclear atypia should also be considered when risk of malignancy in GISTs is evaluated. Further study of variables, which may provide information beyond that, gleaned from number of mitoses, tumor size, and site, is required in the prognostication of GISTs. Acknowledgments T.O.N. is a Scholar of the Michael Smith Foundation for Health Research.

The tissue microarray facility at the Genetic Pathology Evaluation Centre is supported in part by an unrestricted educational grant Dacomitinib from sanofi-aventis.
Objective To examine the risk of neurological and autoimmune disorders of special interest in people vaccinated against pandemic influenza A (H1N1) with Pandemrix (GlaxoSmithKline, Middlesex, UK) compared with unvaccinated people over 8-10 months.