The staining intensity was also scored on a four-tiered scale (ne

The staining intensity was also scored on a four-tiered scale (negative scored 0, low intensity positive staining 1, moderate intensity positive staining 2, and strong intensity AZD7762 purchase positive staining 3). The staining intensity score plus the positive cell score is the overall score. 0 score was negative staining (−), more than 2 scores were positive staining (+), more than 6 scores was strong positive (++). Immunoreactive score was performed by two pathologists independently. Western

blotting The antibodies used in the Western blot, following manufacturer’s protocols, were anti-DLC1, anti-PAI-1 and anti-β-actin (Santa Cruz, USA). Tissue lysates containing equal amounts of total protein were separated by SDS-PAGE. To detect proteins of interest, enhanced chemiluminescence system was used according to the supplier’s protocol (Lumi-Light Western Blotting substrate; Roche). Relative levels of proteins were estimated densitometrically using β-actin as internal reference. Statistical analysis SPSS 17.0 software was used for the statistical analysis. Continuous variables were expressed as . Chi-square test, Logistic

regression analysis and Partial Correlate were performed to evaluate the association between DLC1 selleck compound and PAI-1 with clinicopathological characteristics. Overall survival was estimated by Kaplan-Meier curves and multivariate Cox analysis. The relationships between DLC1 and PAI-1 protein selleck screening library expression were analyzed by Pearson’s correlation coefficient. Results were considered statistically significant when P less than 0.05. Results Expression of DLC1 and PAI-1 in epithelial ovarian cancer tissues and normal ovarian tissues Positive staining for DLC1 observed in malignant and normal ovarian tissues were 33/75 (44.0%) and 25/25 (100.0%) respectively, Methamphetamine but were 51/75 (68.0%) and 9/25 (36%) for PAI-1 (Figures 1 and 2). The Western Blotting showed that the expression of DLC1 protein in normal and malignant ovarian tissues were (0.984 ± 0.010) and (0.497 ± 0.028),

but (0.341 ± 0.019) and (0.718 ± 0.017) for PAI-1 (Figures 3 and 4). The expression of DLC1 in ovarian carcinoma tissues was significantly lower than that in normal ovarian tissues (P < 0.05), whereas it was converse for PAI-1. Figure 1 Positive expression of DLC1 and PAI-1 in different ovarian tissues detected by immunohistochemistry staining. Normal ovary cells showed a higher staining of DLC1 (Up-left), but ovarian cancer cells showed lower density staining (Up-right); normal ovary cells showed a lower staining of PAI-1 (Down-left), but ovarian cancer cells showed higher density staining (Down-left). Immunoreactive Score method performed followed Remmele’s method, the number of positive-staining cells in 10 representative microscopic fields was counted, and the percentage of positive cells was calculated (DAB staining, ×400). Figure 2 The immunoreactive scores of DLC1 and PAI-1 in different ovarian tissues detected by immunohistochemistry staining.

5 mg; a half of the optimal dose) (LOS/HCTZ) is thus worth evalua

5 mg; a half of the optimal dose) (LOS/HCTZ) is thus worth evaluating in terms of BP lowering potency and avoiding side effects. In the present study, we made an attempt to evaluate the clinical

benefit of a single-tablet formulation of LOS/HCTZ, by conducting a multicenter observational trial, the Jikei Optimal Antihypertensive Treatment (JOINT) study in uncontrolled hypertensive patients. Methods Study subjects STA-9090 in vitro Eligible patients were men and women between 20 and 75 years of age with essential hypertension and those with CKD with hypertension. Ethnic extraction of all participants was Japanese with all four biological grandparents born in Japan and of Japanese descent. The inclusion criteria were outpatients whose BP was more than 130/80 mmHg despite the antihypertensive agents prescribed for more than 3 AZD1480 chemical structure months prior to study entry. The exclusion criteria were patients whose S63845 concentration serum creatinine (Cr) concentration exceeded 220 μmol/L (compatible with CKD stage 5), those with liver dysfunction (defined

as an elevation of aspartate aminotransferase/alanine aminotransferase 3 times higher than the upper normal limit), pregnant, expecting, or lactating women, CKD patients with massive proteinuria of nephrotic range (defined as a daily protein excretion of 3 g/day or more), and patients whose doctor in charge judged it inappropriate to enroll. Study protocol All institutions received prior ethics committee and or institutional review board approval, and the trial was conducted in accordance with the principles of Good Clinical Practice and the ethical principles of the concurrent Declaration of Helsinki which also protected the privacy of the patients. All patients gave written informed consent before study enrollment. The JOINT

was a multicenter observational self-controlled study to evaluate the antihypertensive effect of a fixed-dose combination formulation of LOS/HCTZ (Clinical trial Number by UMIN 000001950). The study was conducted at 28 centers and clinics for the JOINT study group (“Appendix”) in the vicinity of Tokyo, Japan. Patients were previously treated with either one or more antihypertensive agents on an outpatient basis. The protocol for the administration of LOS/HCTZ was the following. If the patient was being treated with either ARB or calcium channel blocker (CCB) alone or together, LOS/HCTZ was substituted for either Montelukast Sodium drug or the combination. If the patient was being treated with three drugs including RAS inhibitors, the RAS inhibitor was switched to LOS/HCTZ. In all of the protocol patterns, LOS/HCTZ was administered once a day in the morning. Advices on life-style modification plan were carried out throughout the study. Namely, from the run-in and the observation period, the patients were required to maintain a daily salt intake of 6 g or less. A protein restriction of 0.6–0.8 g/kg/day was also required when the patient’s CCr was below 30 mL/min/1.73 m2.

In a very similar vein, Student 8 based her rejection of cohabita

In a very similar vein, Student 8 based her rejection of cohabitation before marriage on religious grounds and said, “Where I live is independent of my religious views, the latter will always prevail.” In regards to housework, student 8 also said, “Housework should be done by women. Our religion suggests that this is the woman’s responsibility.” Theme 2: No Change Because of

Cultural and Social Values Some of the participants explained that the reason why they haven’t changed was mainly because they felt really strongly about the cultural and social values of their home country. For instance, while talking about gender expectations in dating, both Student 4 and Student 6 expressed strong feelings favoring traditional gender roles. More specifically, Student 4 mentioned, I expect MK-0457 supplier men to be chivalrous and gentlemanly, that’s what I grew up with and that is what

I believe to be true. Gestures like asking out, paying the bill, and picking up the girl from their home should always come from men. I could not change this culturally instilled GSK1120212 mouse value in me even if I wanted to. Similarly, when discussing economic responsibilities, Student 8 said that it is the responsibility of men to be the head of the selleck inhibitor household and to provide and that even though she plans on working, she sees this as a choice, whereas for men “it is a necessity.” In a similar vein, Student 2 reported, “Man needs to take care of the household; my money should go to my clothes and kids.” These traditional cultural norms were also prevalent in participants’ understanding of gender and housework. Student 7 said, “There is no need for the man, men are not skilled, and can’t really do any of the housework right anyways, so why bother?” The overpowering influence of cultural values was also evident in talking about

number of sex partners for some of the participants. For instance, Student 1 explained that in the Turkish culture, having Florfenicol more than one sex partner is indicative of lack of moral values and added that regardless of where she lives, she still carries this cultural piece with her. Similarly, on the topic of cheating, Student 7 said that she really is against the idea of cheating being so publicly discussed in the US. She then added, In my time in the US, I observed so many people cheating and sharing this with family and friends. To me, this is a topic that should not get discussed with outsiders. I simply can not understand people’s attitudes toward cheating here. Theme 3: Social Isolation Due to Language Barriers Another theme that emerged in this group of participants who reported ‘no change’ was that their romantic socialization with Americans was limited due to language barriers. To illustrate, Student 4 said, Living in the US has not really changed me because I do not have any American friends. I find it very tiring to communicate with others in English.

J Bacteriol 2006, 188:2027–2037 CrossRefPubMed 28 Perrin C, Bria

J Bacteriol 2006, 188:2027–2037.CrossRefPubMed 28. Perrin C, Briandet R, Jubelin G, Lejeune P, Mandrand Berthelot MA, Rodrigue A, Dorel C: Nickel promotes biofilm formation by Escherichia coli K-12 strains that produce curli. Appl Environ Microbiol 2009, 75:1723–1733.CrossRefPubMed 29. Gualdi L, Tagliabue L, Bertagnoli S, Ieranò T, De Castro C, Landini P: Cellulose modulates biofilm formation by counteracting

curli-mediated colonization of solid surfaces in Escherichia coli. Microbiology 2008, 154:2017–2024.CrossRefPubMed 30. Tariquidar price Zogaj X, Nimtz M, Rohde M, Bokranz W, Romling U: The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix. Mol Microbiol 2001, 39:1452–1463.CrossRefPubMed 31. Solano C, García B, Valle J, Berasain C, Ghigo JM, Gamazo C, Lasa I: Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose. Mol Microbiol 2002, 43:793–808.CrossRefPubMed 32. Spiers AJ, Bohannon J, Gehrig SM, Rainey PB: Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Mol Microbiol 2003, 50:15–27.CrossRefPubMed 33. Hoffman BIBW2992 molecular weight LR, D’Argenio DA, McCoss MJ, Zhang Z, Jones RA, Miller SI: Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 2005, 436:1171–1175.CrossRefPubMed 34. Lewis K: Multidrug tolerance of biofilms and persister cells. Curr

Top Microbiol Immunol 2008, 322:107–131.CrossRefPubMed 35. Chen X, Smith LM, Bradbury EM: Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification. Anal Chem 2000, 72:1134–1143.CrossRefPubMed 36. Anacetrapib Banin E, Vasil ML, Greenberg EP: Iron and Pseudomonas aeruginosa biofilm formation. Proc Natl Acad Sci USA 2005, 102:11076–11081.CrossRefPubMed 37. Johnson M, Cockayne A, Morrisey JA: Iron-regulated biofilm formation in Staphylococcus aureus Newman requires ica and the secreted protein Emp. Infect Immun 2008, 76:1756–1765.CrossRefPubMed 38. Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK: The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-γ-mediated macrophage killing. J Immunol 2005, 175:7512–7518.PubMed 39. Crosa JH, Walsh CT: Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 2002, 66:223–249.CrossRefPubMed 40. Vallenet D, Nordmann P, Barbe V, Poirel L, Mangenot S, Bataille E, Dossat C, Gas S, Kreimeyer A, Lenoble P, Oztas S, Poulain J, Segurens B, Robert C, Abergel C, Claverie JM, Raoult D, Médigue C, Weissenbach J, Cruveiller S: Comparative analysis of Acinetobacters : three genomes for three lifestyles. PLoS ONE 2008, 3:e1805.CrossRefPubMed 41.

E The colocalization of Francisella with TfR1, Rab5, or Rab7 is

E. The colocalization of Francisella with TfR1, Rab5, or Rab7 is described quantitatively for each time point by analyzing 100 infected cells from triplicate independent infection experiments. buy P505-15 Means +/- 1 standard error of mean (SEM) are shown. Early recycling endosomes are characterized by carrying TfR1, EEA1, and Rab5, while excluding Rab7 unless they are destined for further trafficking along the lysosomal degradation pathway [27]. Macrophages infected with Francisella were stained with antisera to Rab5 and Rab7. This

demonstrated that Francisella very early on at the membrane recruits Rab5 (Figure 2C and 2E; p = 0.09 for 15 and 30 minutes). Colocalization of Francisella and Rab5 decreases over time as Francisella escapes from the vacuole (Figure 2E; p = 0.03 for comparison of 30 and NVP-BSK805 45 minutes, p = 0.83 for 45 and 60 minutes, Student’s t-test). However, there is no co-localization with Rab7-containing vesicles (Figure 2D and 2E; p = 0.88 for comparison of 15 and 30 minutes, p = 0.91 for 30 and 45 minutes, p = 0.89 for 45 and 60 minutes, Student’s t-test). These findings suggest that Francisella enters through an early endosome, which is characterized by carrying TfR1 and Rab5. The Francisella-containing vacuole does not mature

further by acquiring Rab7 and does not retain TfR1. This is most likely due to exit from the vacuole [13] rather than to trafficking to a different vesicle environment with concomitant loss of TfR1. Infection of macrophages with Francisella upregulates transferrin receptor Expression of TfR1 remains unchanged during infection with MYO10 wild-type Salmonella [28]. However, when expression of the transferrin receptor in uninfected macrophages was compared by microscopy to the expression in cells infected with Francisella, it became evident that Francisella-infected macrophages have a higher level of transferrin receptor expression (Figure 3A). This

was confirmed by comparing the expression level of the transferrin receptor in Francisella-infected macrophages to the level found in uninfected cells by immunblotting at one hour and twenty-four hours after infection (Figure 3B). We also tested the expression level of transferrin receptor in cells, which had taken up formalin-fixed Francisella. This did not lead to a comparable upregulation of TfR1 (Figure 3B). Synthesis of the transferrin receptor is mainly regulated at the translational level as a response to the iron level or to other inputs. Indeed, after two hours of infection there was no increase in the mRNA level for Tfr1 as determined by real-time RT-PCR (Figure 3C; p = 0.29). However, after 24 h of infection, the mRNA level for TfR1 had more than doubled (Figure 3C; p = 0.002). Figure 3 Infection with Francisella increases expression of transferrin receptor. A. RAW264.7 macrophages were infected with Francisella that MEK162 constitutively expressed Gfp.

All groups can be seen to exhibit the same peaks, which match wel

All groups can be seen to exhibit the same peaks, which match well with the standard Fe3O4 XRD pattern (JCPDS 75–0030). The mean particle size (D) can be calculated by the check details full-width at half-maximum

(FWHM) and the area/height ratio (β) of the XRD peaks with instrumental correction, using the equation D = Kλ / β × cosθ, where K is the Scherrer constant, λ is the wavelength, β is the FWHM (in radians), and θ is the peak angular position [22, 23]. The XRD information gave crystallite sizes of 14.9, 13.2, 12.1, and 7.3 nm (Figure 3). As MNPs synthesized by coprecipitation may contain some iron oxide crystals, the particle size calculated from the TEM images was larger than that from the XRD data (Figure 3b). Selleckchem Captisol Figure 3 A stack plot of XRD patterns of MNPs and size calculation. The nanoparticles were well crystallized, H 89 clinical trial and the peaks are in accordance with the typical CoFe2O4 XRD spectrum in which the main peaks are (111), (220), (311), (400), (511), and (440) (a). The mean diameters of the crystal particles calibrated from signal width for the four groups from A to D

were 14.9, 13.2, 12.1, and 7.3 nm, respectively (b). The size-dependent MR contrast (T2 relaxivity) of the MNPs was measured on a 4.7-T MRI system. Figure 4a shows the dependence of the T2 relaxation rate (R 2, s−1) on the MNPs of the four groups. The T2 relaxation rate was increased with increased Co/Fe

concentration, and the T2 relaxivities (r 2) for the groups were measured from the slopes of the data. The r 2 values were found to be 302 ± 9, 268 ± 8, 179 ± 5, and 66 ± 4 mM−1s−1 for groups A, B, C, and D, respectively (Figure 4b). These values are comparable to those Rebamipide in the study of Joshi et al. [24], in which the T2 relaxivity of cobalt ferrite nanostructures synthesized by the thermal decomposition method was reported to be 110 to 301 mM−1s−1 depending on the particle size (6 to 15 nm). Figure 4c shows an MRI phantom image with the four groups depending on the Co/Fe concentration measured on the 4.7-T MRI system. The increase in MR T2 negative contrast was shown to depend on both the particle diameter and the Co/Fe concentration, indicating that a well-controlled contrast with each size-selected group of MNPs could be obtained. The particle size dependence of T2 relaxivity was in accordance with other reports [25, 26], in which T2 spin-spin relaxation is affected by mass magnetization depending on the magnetic particle size in the range lower than approximately 1 μm. This demonstrates that each group of MNPs could be used for specific applications depending on the particle diameter. One concern regarding these as-prepared MNPs is that they are not stable to variations in pH. This is a problem that needs to be overcome if they are to be successfully employed in vivo.

However, 5 5% of the cells showed

However, 5.5% of the cells showed double septa/mini cells (Figure 1B), which are never observed in wild type cells (Figure 1A). Additionally, 2.5% of mutant cells were larger than 5.5 μm (Figure 1C), while only 0.5% of wild type cells reach this size (250 cells measured for each strain). In contrast to e.g. a deletion of sftA, encoding for a DNA translocase that couples late states of chromosome segregation and cell division [25, 26], DNA was never observed to be trapped in a closed selleck screening library division septum in dynA mutant cells. Therefore, chromosome

segregation occurs normally in the mutant cells, but cell division is noticeably defective. Figure 1 Phenotypes of exponentially growing wild type (PY79) or mutant Bacillus subtilis cells. A) Wild type cells, B) dynA (ypbR) mutant cells, white triangles indicate double septa, C) dynA (ypbR) mutant cells, grey triangle

indicates highly elongated cell, D) ezrA mutant cells, E) ezrA/dynA double mutant cells, F) ezrA/dynA double mutant cells, white triangles indicate double septa, G) divIB mutant cells grown at 30°C, H) divIB/dynA double mutant cells grown at 30°C, I) divIB mutant cells grown at 42°C, J) divIB/dynA double mutant cells grown at 42°C. White or grey bars 2 μm. We wished to investigate the effect of a combination of the dynA deletion with that find more of a protein known to be important for an initial step in cell division. EzrA is a regulator of FtsZ, and therefore acts at a very early time point during cell division. The deletion of ezrA leads to the generation of elongated cells, to the formation of double septa and mini cells in rich medium [27]. In minimal medium used in this study, ezrA mutant cells were elongated, and formed mini cells (9%), but did not show any double septa (Figure 1D). Interestingly, ezrA dynA double mutant cells were more elongated than ezrA single mutant cells (Figure 1E), and contained more double septa than both single mutants (Figure 1F). Double mutant cells measured on average 5.16 ± 0.5 μm versus 4.07 ± 0.45 4-Aminobutyrate aminotransferase μm for ezrA

mutant cells, and contained double septa in 15% of the cells versus 5% in dynA single mutant cells (with 200 cells measured for each strain from 2 independent check details experiments). Occasionally, long ezrA dynA double mutant cells showed a single condensed or decondensed nucleoid indicating a segregation defect, but this referred only to a subpupulation of long cells (Figure 1E, white triangle). Thus, the increase in cell length is largely due to an effect on cell division. These data suggest that EzrA and DynA affect two distinct steps early in cell division, each of which contributes to efficient cell division, because all phenotypes are exacerbated by the loss of both proteins. We also tested if the dynA deletion is affected by the deletion of a gene involved in a later step of cell division. We used divIB mutant cells, which show a pronounced defect in cell division when they are shifted from 30 to 42°C.

5 ± 1 0, medium 7 0 ± 1 2) How this finding should be applied in

5 ± 1.0, medium 7.0 ± 1.2). How this finding should be applied into practical conservation is further discussed under the last section ‘Practical implication’. From our field observations of the study sites we noticed a distinguishable difference of the four largest sand pits from the smaller ones. Selleck Nepicastat The large sand pits could all be described as more homogenous in terms of topology and vegetation; with large

plane areas, steep edges and either even-aged young trees or almost no vegetation at all. We believe this difference between sites could explain why no more sand species were found in large sand pits compared to medium-sized ones. Of the two prominent hypotheses for SAR this observation would give more support to the ‘habitat heterogeneity hypothesis’ than the ‘area per se hypothesis’ (Báldi 2008). However, the strong interactions between the features that the two hypotheses are based upon make drawing clear conclusion difficult without further direct studies (Connor and McCoy 1979; Kallimanis et al. 2008). The rate of increase JPH203 in species number with area were illustrated by the log–log SA-curves of the power function (Fig. 2) and showed a more rapid increase in species number for carabids (z = 0.25) than for all beetles (z = 0.12). According

to Connor and McCoy (1979), z values regularly fall between 0.20 and 0.40, and according to a review by Drakare et al. (2006), the average z value obtained in investigations using independent sampling schemes (among 794 SAR studies considered) was 0.24. Whether the z value has any further biological significance has been debated, often with scepticism (Connor and McCoy 1979; He and Legender 1996; Martin 1981).

However, Drakare et al. (2006) detected apparent systematic correlations between z values and latitude (negative), organism size (negative; explained by the Metalloexopeptidase higher dispersal ability of small organisms) and habitat (lower in non-forested habitats). As this study examined relationships of small organisms dwelling in non-forested habitats at high latitude we should expect low z values, which was true for beetles (0.12), but not for carabids (for which the value was close to the average cited above; 0.25). YH25448 influence of the surrounding matrix In contrast to the sand species, no SAR was found when all species (irrespective of habitat-preferences) were included in the analysis. The same pattern has been observed for other terrestrial habitat islands, in which positive SARs have only been found for the habitat-specific species (Lövei et al. 2006; Magura et al. 2001; Vries de et al. 1996). This can be explained by an influence of the surrounding matrix where matrix species invade the habitat island resulting in an increase of species richness along the edges (Cook et al. 2002; Ewers and Didham 2006; Magura 2002; Niemelä 2001). This edge effect then counteracts the area effect because of the greater edge:area ratio in smaller patches (Lövei et al.

For each activity, the frequency, duration

in minutes, an

For each activity, the frequency, duration

in minutes, and MET score were multiplied and then divided by 14 days (i.e., (frequency × duration × MET)/14). The minutes spent per activity per day were summed to a total physical activity score (minutes/day × MET). For example, a participant who walks outside for 60 min four times per 2 weeks (4 × 60 × 3.5/14 = 60) and does light SBI-0206965 in vivo household work for 30 min per day (14 × 30 × 2.5/14 = 75) has a physical activity score of 135 min/day × MET. Potential effect modifiers Physical functioning was measured by physical performance and functional limitations. Physical performance was measured using the chair stands test (time needed to stand up from a chair and sit down for five times), the walk test (time needed to walk 3 m, turn 180°, and walk back), and the tandem stand (the participant stands unsupported with one foot behind the other (heel against toe) up to 10 s) [23, 29]. In order to calculate a total physical performance score, the time needed for the chair stands and walk test were categorized into quartiles (1 = slowest, 4 = fastest). For the tandem stand, 2

points were scored when able to hold for 3 to 9 s, and 4 points for 10 s. For each test, the score of 0 was assigned when the participant was unable to complete the test. The three scores were summed (range 0–12), a score of 12 representing optimal physical performance. Functional Ferrostatin-1 research buy limitations were assessed using a validated questionnaire about the degree of difficulty with climbing stairs, walking 5 min outdoors without resting, getting PF-01367338 concentration over up and sitting down

in a chair, dressing and undressing oneself, cutting one’s toenails, and using own or public transportation [30]. The scores on these six items were dichotomized (0 = no difficulty, 1 = at least some difficulty) and summed (range 0–6). A score of 6 indicates difficulties with all six activities. We dichotomized both measures, because, in case of a significant interaction with physical activity, further analyses would have to be stratified into low and high physical functioning, and stratification for more than two groups would have severely decreased the power to detect a significant association between physical activity and fall risk. Physical performance was dichotomized using the median score of 7 as the cut-off value (0–7 vs 8–12). Functional limitations were dichotomized using the median score of 1 as a cut-off value (0 vs ≥1 limitations). Confounders BMI (Body Mass Index) was calculated as weight (kilograms)/height (square meter). The number of chronic diseases was assessed using self-reports on chronic diseases, which included chronic nonspecific lung diseases, cardiac diseases, vascular diseases, stroke, diabetes mellitus, malignant neoplasms, and joint disorders (i.e., osteoarthritis and rheumatoid arthritis; range 0–7) [31]. Medication use was assessed by recording the names of the medications directly from the containers.

The contact pressure and contact diameter were evaluated using th

The contact pressure and contact diameter were evaluated using the Hertzian equation. At 1 and 6 μN, the contact pressures were 6.9 and 12.5 GPa, respectively.The selleck chemicals scanning density selleck products decreased with the scanning cycle number. The total contact sliding width can be evaluated from the product of the contact diameter

and scan number. Then, to evaluate the overlap ratio, the total contact width is divided by the scanning width. For example, at 6-μN load, the Hertzian contact diameter is nearly 30.3 nm; therefore, the total contact width for 128 scans was 30.3 × 128 nm and the overlap ratio was nearly 0.97, as shown in Figure  6b. In this case, the total contact width was smaller than the scanning width. The natural oxide layer formed on the Si surface was removed at low scan number conditions; overlap of the sliding contact area appeared to produce an etching-resistant layer. Figure 3 Etching profile for 128-scan pre-processing. (a) Surface profile. (b) Section profile (1 and 2 μN). (c) Section profile (4 and 6 μN). Figure 4 Etching profile for 256-scan pre-processing. (a) Surface profile. (b) Section profile (1 and 2 μN). (c) Section profile (4 and 6 μN). Figure 5 Etching profile for 512-scan pre-processing. (a) Surface Poziotinib mw profile. (b) Section profile (1 and 2 μN). (c) Section profile (4 and 6 μN). Figure 6 Dependence of etching depth

(a) and overlap ratio (b) on load and scanning number of pre-mechanical processing. Owing to the removal of the natural oxide layer, 512 scans at 1-μN load also increased the etching rate. Processing at higher loads of 4 and 6 μN increased the amount of mechanochemical oxidation owing to the high density of the scanning and thus decreased the etching depth. At 512 scans, the total contact width was larger than the scanning width, so the contact area overlapped. Pre-processing at low load and scanning density efficiently removed the natural oxide layer by mechanical action while also mechanochemically generating a thin oxide layer because of the sliding overlap.To clarify the etch properties of pre-processed areas at higher

load, the etching profiles obtained at 8-, 10-, 15-, and 20-μN load after 256 scans were evaluated as shown in Figure  7. In these cases, etching grooves could not be detected in any of the processed areas. The Bortezomib order heights of all of the processed areas were slightly greater than those of the unprocessed areas. Thus, the effect of any increases in etching rate resulting from the removal of the natural oxide layer could not be obtained. This is conceivable because mechanochemical oxidization increases at higher load, resulting in improved resistance towards etching with KOH solution.To compare the resistances of the natural oxide layer and the mechanochemically generated oxide layer to etching, we extended the etching time by 5 min. Figure  8 shows the etching profiles of pre-processed areas at 2-, 4-, 8-, and 15-μN loads.