SDS-PAGE and Western blotting Electrophoresis was performed in 12% SDS polyacrylamide gels and
the recombinant proteins were detected by Western blotting using a monoclonal antibody (mAb) against the polyhistidine (His) tag in the C-terminal region of the fusion protein. Briefly, the transferred PVDF membrane was blocked with 2% (w/v) BSA in TBS for 1 h at 37°C, and washed thrice with TBS – 0.05% (v/v) Tween 20, then the membrane was incubated with a 1:5,000 dilution of anti-His tag (mouse mAb, CWBIO, Beijing, China) Tideglusib manufacturer in a 0.2% BSA-TBS – 0.05% Tween 20 solution for 1 h at 37°C, and washed thrice with TBS – 0.05% Tween 20. Protein bands were probed with 1:2,000 dilution of HRP-conjugated goat anti-mouse IgG (CWBIO, Beijing, China) and washed thrice as described above. Chemiluminescence was applied as instructed by the manufacturer (Li-COR Odyssey, USA). Electron microscopy The formation of HBcAg VLPs and chimeric VLPs (HBc-N149-VP4N20) was analyzed by negative staining electron microscopy according a previously described method . Briefly, proteins were adsorbed ABT-263 datasheet to 230 mesh carbon-coated copper grids and incubated for 1 min. The grids were then washed once with PBS and stained for 45 s with 2% phosphotungstic acid. Specimens were evaluated using an electron microscope (H-7650, HITACHI, Japan). Immunization of animals Pathogen-free female BALB/c mice were purchased from
Beijing HFK Bioscience Co. (Beijing, China).
All animals were housed at pathogen-free conditions. Animal experiments were performed in accordance with current guidelines for the Care and Use of Laboratory Animals of Experimental Animal Center of Military Medical Sciences and approved by the center. For mice experiments, five female BALB/c mice (6–8 weeks) per group were vaccinated intramuscularly (i.m.) with recombinant proteins Selleckchem SB431542 HBc-N149 (5 μg/mouse) or HBc-N149-VP4N20 (5 μg/mouse) at week FER 0. The second injection was performed at week 3. QuickAntibody™ from KBQ Biotechnology Co. (Beijing, China) was used as an adjuvant. Control group was immunized with PBS plus adjuvant. The immunized animals were bled at week 0, 2, 5, 8 for antibody detection. ELISA Direct ELISA was used for detection of antibodies in the sera of immunized animals. The peptide VP4N20 was synthesized by Scilight-Peptide (Beijing, China) and conjugated with Bull Serum Albumin (BSA-VP4N20). The peptides were purified using high-pressure liquid chromatography. ELISA plates (96-well) were coated with 250 ng/well of BSA-VP4N20 in coating buffer (50 mM Na2CO3–NaHCO3, pH 9.6) overnight at 4°C. After washing with PBS-0.05% (v/v) Tween 20 thrice, the plates were blocked with 2% (w/v) BSA in PBS for 2 h at 37°C. Sera were tested at 2-fold serial dilutions starting at 1:100. The plates were incubated at 37°C for 1 h and washed thrice with PBS-0.05% Tween 20.
Further, VX-765 clinical trial SpiC is involved in the AZD6244 chemical structure expression of
the fliC gene at the transcription level . These results suggest the possibility that SpiC participates in flagellar phase variation or the fliC gene expression directly. However, in addition to the FliC protein, we newly identified a FliD flagella protein that was decreased in the spiC mutant using proteomic analysis with liquid chromatography-tandem mass spectrometry (K. Uchiya, unpublished result). Taken together, these results suggest that SpiC contributes to the flagellar system by mechanisms other than phase variation or direct expression of the fliC gene in S. enterica serovar Typhimurium. Flagella expression in S. enterica serovar Typhimurium is controlled in a hierarchical manner. At the top of the hierarchy is the class 1 flhDC operon that is essential for transcription of all of the genes in the flagellar cascade. The class 2 operons contain the genes encoding the hook-basal body-associated proteins, a few regulatory proteins, and a component of the type III export pathway. The class 3 operons contain genes involved in filament formation, flagella rotation and chemotaxis [17, 18]. As described above, proteomic analysis showed that the spiC
mutant had lower expression levels of FliC and FliD proteins, suggesting that SpiC is involved in the expression of the class 3 flagellar genes. Therefore, we first investigated the effect of the spiC mutation on the expression of the class 3 genes. The total RNA was isolated from bacteria grown to an OD600 of 1.6 in LB to induce the expression of the spiC gene (Fig. 1B). CB-839 We analyzed the transcript levels of the fliD and motA genes that encode the flagella cap and motor torque proteins , respectively, using quantitative real-time PCR (RT-PCR). The transcript levels of the fliD and motA genes in the spiC mutant
were reduced by approximately 15-fold and 6-fold compared to the wild-type strain, respectively (Fig. 2). Complementation of the spiC mutant with a plasmid carrying the wild-type Cyclin-dependent kinase 3 spiC gene (pEG9127) restored the fliD and motA transcripts to about 80% of the level of the wild-type strain. Further, to confirm the contribution of SpiC in the regulation of class 3 flagellar gene transcription, we constructed newly a deletion mutant of the spiC gene using the lambda Red mutagenesis technique and examined the motA mRNA level. The deletion mutant showed the same phenotype as the spiC mutant (EG10128) used in this study (data not shown). These data indicate that SpiC has an influence on the flagellar system. Figure 2 Expression of the class 3 fliD and motA genes in the spiC mutant. Bacteria were cultured in LB to an OD600 of 1.6, and the total RNA was extracted from the wild-type Salmonella (WT), spiC mutant strain, or spiC mutant strain carrying the spiC gene-containing plasmid pEG9127 (spiC +). Quantitative RT-PCR was conducted using a TaqMan probe.
Guidry SP, Poole GV: The anatomy of appendicitis. Am Surg 1994, 60 (1) : 68–71.PubMed 15. Marbury WB: The retroperitoneal (retrocolic) appendix. Ann Surg 1938, 107 (5) : 819–28.PubMedCrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions HK, JD and RG participated in the care of the patient, including the operative part. HK, JD and RG envisioned the concept of the manuscript. HK wrote the first draft of the manuscript JD and RG critically reviewed
the manuscript. HK, JD and RG all read and approved the final manuscript.”
“Introduction Multiple diverticulosis of the jejunum constitutes an uncommon pathology of the small bowel. The disease GW-572016 concentration is often asymptomatic and must be taken into consideration in cases of unexplained malabsorption, anemia, selleckchem chronic abdominal pain and discomfort. Related complications such as diverticulitis, hemorrhage, obstruction and perforation present high mortality and morbidity
rates. We herein report a case of a 55 year-old man presented at the emergency department because of acute abdominal pain, vomiting and fever. Preoperative radiological examination followed by laparotomy revealed multiple and giant jejunal diverticula causing Selleckchem PCI-34051 intestinal obstruction. We also review the literature for this uncommon disease. Case Presentation A 55-year old man arrived at the emergency department complaining of 48-hour lasting intense abdominal pain and vomiting. The patient had a free medical history and was not receiving any drugs Montelukast Sodium at that time. He mentioned a two-year-lasting remittent abdominal pain, fullness and often abdominal distension. The
patient also mentioned a particular intolerance of pulse and vegetables. Physical examination revealed a distended abdomen with increased bowel peristalsis. Rectal examination was normal. Only his temperature was elevated (38.2°C) while other vital parameters were within normal limits. Abnormal laboratory findings included leukocytosis (13300/mm3), anemia (Hct:30%), hypokalemia (3.2 mmol/l) and hypoalbuminemia (2.80 mmol/l). C-reactive protein was also elevated (4.57 mg/dl). A plain abdominal X-ray showed multiple air-fluid levels and dilated intestinal loops suggesting intestinal obstruction but not signs of perforation (Figure 1). Abdominal ultrasonography revealed dilated and hyperactive intestinal loops but not free intraperitoneal fluid. Gallstones were also incidentally found. The abdominal computed tomography (CT) scan demonstrated multiple distended small bowel loops and jejunal diverticula. The patient had a nasogastric tube and received intravenously fluids, antibiotics (ciprofloxacin and metronidazole) and parenteral nutrition. Within next 72 hours, temperature and leukocytosis were decreased while the X-ray of the abdomen did not reveal gas-fluid levels.
For environments that lack cultured isolates or are relatively underexplored, researchers are often unable to find an appropriate training set to reveal the taxonomic identity of the extracted sequences [11–13]. However, if previous clone libraries have generated full length, high-quality 16S rRNA gene sequences, then these sequences can be utilized in a training set and taxonomy framework, potentially increasing the precision of the classification provided by the RDP-NBC. Our primary goal in this study was to test the effect of training set on the RDP-NBC-based classification of Apis mellifera (European honey bee) gut derived 16S rRNA gene sequences. Insect guts are Torin 2 relatively
underexplored and host novel bacterial groups for which there do not exist close, cultured relatives, making taxonomic assignments for 16S sequences and metatranscriptomic data difficult [14–16]. We also sought to improve the classification of sequences from the honey bee gut by the RDP-NBC ISRIB cost through the creation of training sets
that include full-length sequences identified as core honey bee microbiota as part of a phylogenetic framework first put forward by Cox-Foster et al., 2006 and extended by Martinson et al., 2010 [17, 18]. Below we compare the precision and reproducibility of classification of the honey bee gut microbiota using six different training sets: RDP, Greengenes, arb-silva, and custom, honey bee specific databases Mannose-binding protein-associated serine protease generated from each. Methods Generating a bee-specific seed alignment Sequences that corresponded to accession numbers published in analyses of bee-associated microbiota and that were near full
length (at least 1250 bp) were used to generate the seed alignment for our subsequent analyses (A total of 5,713 sequences were downloaded and 5,158 passed the length threshold) [18–22]. These sequences were clustered at 99% identity, reducing the dataset to 276 representatives. This set of sequences is OSI-744 solubility dmso referred to as the honey bee database (HBDB) throughout and were aligned using the SINA aligner (v 1.2.9, ) to the arb-silva SSU database (SSURef_108_SILVA_NR_99_11_10_11_opt_v2.arb) and visually inspected using ARB . We refer to this custom seed alignment as the arb-silva SSU + honey bee alignment (ASHB). To generate a phylogeny we used the ASHB as input to RAxML (GTR + γ with 1,000 bootstrap replicates) using a maximum likelihood framework (Stamatakis 2006). This phylogeny was used to inform the taxonomic designations (see below). In addition, we used the RAxML evolutionary placement algorithm to identify the placement of short reads within this framework (raxmlHPC-SSE3 –f v –m GTRGAMMA –n Placement). Alignment (ASHB) and phylogeny are available in TreeBase at http://purl.
Figure 5 Specificity of the aptamer by immunohistochemical staining. After incubating the MMP2 aptamer with MMP2 protein in PBS at room temperature for 2 h, the immnohistochemical staining in gastric cancer tissues was significantly reduced. Scale bar, 100 μm. Finally, we used the aptamer for ex vivo imaging. To do this, the aptamer was conjugated to fluorescent nanoprobe using EDC (Figure 6). To induce atherosclerosis in mice, ApoE knockout mice were fed a high cholesterol Depsipeptide manufacturer diet for 4 months. After injecting the
aptamer-conjugated fluorescent nanoprobe into a tail vein, fluorescent see more signals from atherosclerotic plaques were observed. The presence of atherosclerotic plaques was confirmed by oilred O staining. The MMP2 aptamer-conjugated nanoprobe produced significantly stronger signals in atherosclerotic plaques than the control aptamer-conjugated probe (Figure 7). Figure 6 Construction of the MMP2 aptamer-conjugated LY2606368 price fluorescent nanoprobe. The MMP2 aptamer was conjugated into magnetic fluorescent nanoprobe using EDC. Figure 7 Ex vivo imaging of atherosclerotic plaques using the MMP2 aptamer-conjugated fluorescent nanoprobe. Atherosclerotic plaques were induced by feeding ApoE knockout mice a high
cholesterol diet for 4 months and were confirmed by oilred O staining (middle panels). Ex vivo imaging was performed 2 h after intravenously injecting mice with the MMP2 aptamer-conjugated fluorescent nanoprobe. The MMP2 aptamer (right panels) showed much stronger signals in atherosclerotic plaques than the control aptamer
(left panels). Many studies have tried to visualize MMP molecules. Small molecular MMP inhibitors attached to radioisotopes, such as123I, 99mTC, and 18 F have been used for the imaging of atherosclerotic lesions and myocardial infarctions [12–15]. Notably, a peptide substrate, which fluoresces when cleaved by MMPs, was used to visualize MMP activity L-gulonolactone oxidase [16–18]. However, considerable time is required for in vivo imaging using this peptide substrate. We considered that aptamers could overcome this problem because aptamers bind directly to target proteins. In addition, due to its small size and easy chemical modification, it can be easily applied to construct new nanoparticles as presented in this study (, Figure 6). The specificity of the MMP2 aptamer produced during the present study was confirmed in vitro and ex vivo. Precipitation and immunohistochemistry studies demonstrated specific protein binding by MMP2 aptamer, and in particular, immunohistochemical staining of MMP2 aptamer was blocked by MMP2 protein. Furthermore, ex vivo imaging demonstrated that whereas MMP2 aptamer visualized atherosclerotic plaques, control aptamer did not. These results suggest that the devised MMP2 aptamer has clinical merit. Conclusions We developed an aptamer targeting MMP2 protein using a modified DNA SELEX technique.
e. by day 5 itself. Also the decrease in bacterial load was significantly greater than the monotherapy groups (group 2 and 3) on all days. Also
selleck peak phage titres were observed on day 2 and declined thereafter. In the co-therapy group, phage titres persisted till day 3 only and no plaque was seen on day 5. As phages are highly specific and thus replicate and increase in number at the expense of their respective host bacteria [53,54] hence no phage activity observed on different days, points towards complete eradication of their host bacteria (MRSA 43300) following treatment with phage. Complete eradication of bacteria was possible due to the combined administration of two agents after allowing successful colonisation of the bacteria in the nasal tissue of mice. The presence of S. aureus in the nose elicits a subclinical immune response, as reported in an earlier study where sero-conversion occurred after carriage was established . Also the host elicits a number of immune factors that constantly impose pressure to eliminate the foreign colonising population [34,56]. Neutrophils are the most PCI-34051 in vivo prominent cellular component of the innate immune system and act as an essential primary defence against S. aureus . In this study, neutrophil recruitment
was studied in terms of MPO GSK2118436 in vitro levels in all the groups. MPO levels were highest in the untreated colonised group on all post treatment days. The groups receiving phage and mupirocin alone showed peak MPO levels on day 2 and the activity declined to the basal value by day 7. This observation correlates well with the declining bacterial load seen on day 7 in both these groups. Combination therapy group exhibited maximum reduction in MPO levels on day 2 onwards. These results further confirm the efficacy of phages in eliminating the colonized S. aureus from the anterior nares of mice. PRKD3 The results of histopathological examination of control (untreated) and treated nasal tissue also substantiated these observations. In the
combined therapy group, minimal or no tissue infiltration was seen and the skin of nasal mucosa appeared normal. The present study indicates that the phage when given along with mupirocin was able to effectively eradicate the colonising population due to their combined action. The dual approach showed maximum nasal protection (better than use of either agent alone i.e. monotherapy) in terms of reduced nasal bacterial load, reduced catalase and MPO levels; with complete elimination of MRSA 43300 occurring by day 5. Coates et al.  advocated the need to develop potent bactericidal agent than mupirocin on the ground that the newer agents might reduce the relapse rate, clearing the patient of S. aureus for a longer period of time than mupirocin. The success obtained with this dual approach is based on the fact that mupirocin being a bacteriostatic antibiotic was able to significantly halt the multiplication and growth of S.
The OMVs were also studied with regard to lipooligosaccharide (LOS) patterns using SDS-PAGE and silver staining of preparations treated with Proteinase K. The LOS was detected in the OMV samples and the pattern was identical to that of the whole cell samples (data not shown). The relative intensity of the major bands indicated that the LOS in the OMVs represented ca 0.2-0.5% of the total LOS of whole bacterial cells. Figure 3 Immunoblot detection of intra- and extra-cellular CDT of C. jejuni. Immunoblot
analyses of samples from C. jejuni wild type strains 81-176 (lanes 1-4) and the cdtA::km mutant (lanes 5-8). Samples: 1&5; whole cells (WC), JNK-IN-8 chemical structure 2&6; supernatants 1 (S1), 3&7; supernatants 2(S2), 4&8; OMVs, (A) Immunoblot detection with anti-CdtA polyclonal antiserum, (B) immunodetection with anti-CdtB polyclonal antiserum. (C) immunoblot detection with anti-CdtC polyclonal antiserum. (D) immunoblot detection with anti-Omp50 polyclonal antiserum.
Pictilisib nmr Immunoelectron microscopic analysis of proteis in OMVs To more directly monitor the association of CDT proteins with OMVs, we performed immunoelectron microscopic analyses. By immunolocalization using anti-CdtA, anti-CdtB, and anti-CdtC antibodies in the immunogold labeling method we detected the deposition of gold Wortmannin order particles on the vesicles obtained from CDT-producing bacteria (Figure 4A-C), whereas there was no labeling of OMVs from the CDT-negative strain (Figure 4D-F). We observed that some CDT containing vesicles were ruptured when the OMVs samples were mixed with antiserum in the immunogold experiment. The gold particles were mainly
observed on the material of the ruptured vesicles. It appeared that due to the rupture of the OMVs some of the released CDT subunits were accessible to the antiserum. The results strongly support the suggestion that the CDT proteins were indeed associated with OMVs of C. jejuni strain 81-176 and it appeared that the proteins might be internal or integral to the vesicle membrane. Since the C. jejuni Hsp60 protein that was somehow associated with OMVs as detected by SDS-PAGE analysis after the ultrcentrifugation step we also performed the immnunogold labelling and electron microscopic examination Reverse transcriptase using an Hsp60 recognizing polyclonal antiserum raised against the E. coli GroEL protein (Sigma-Aldrish). As shown in Figure 5B the gold particles labelled with anti-Hsp60 antiserum were observed not in direct association with OMVs but gold particles were associated with some amorphous material outside the OMVs. A similar immunogold labelling and analysis of the OMVs preparation with anti-Omp50 antiserum was shown in Figure 5C. In this case the gold particles were found to be localized in direct association with the OMVs as expected for an outer membrane protein. The results from these analyses indicated that the Hsp60 protein of C.
e., (12) and are
the matrix elements of the Hamiltonians, (13) and (14) respectively. Here, V(r) stands for an external potential. The proposed calculation procedure employs linearly independent multiple correction vectors for updating the one-electron wave function. The pth one-electron wave function in the Ath SD is updated by (15) where C j (j = 1, 2,…, L + N c ) and N c are the expansion coefficient and the number of correction vectors, respectively. The components of the correction vectors G μ,m A determine N c linearly independent correction functions ξ μ (r) which are defined as linear combinations of Gaussian basis functions as (16) Since the linearly independent correction vectors can be given arbitrarily, randomly chosen values are employed in the present study. A larger number of correction vectors N c realize a larger volume search space; however, the number of the linearly independent AZD8931 manufacturer vectors N c is restricted to the dimension of the space defined by the basis set used. Thus, we have a linear combination of L + N c SDs as the new N-electron wave function (17) where (18) Figure 1 illustrates the flow of the present calculation procedure. Unrestricted
Hartree-Fock (UHF) solutions for a target system are used for initial one-electron wave functions. The coefficients of Equation 17 are given by solving the generalized eigenvalue equations AG 14699 obtained by employing the variational principle applied to the total energy, and we can have a new N-electron wave function as a linear combination of L SDs as shown in Equation 17. Iteration of the above updating process for all the one-electron wave functions of all SDs increasing the number of the SDs’ L leads to an essentially exact numerical solution of the ground state. Bindarit in vitro Figure 1 Flow of the present algorithm. Applications to few-electron molecular systems Convergence from performances for searching for the ground state of a C atom
with the 6-31G** basis set are shown in Figure 2. The UHF solutions are adopted as initial states, and the number of employed SDs is 30. The steepest descent direction and acceleration parameter are adopted for the calculation using one correction vector (N c =1), and seven randomly chosen linearly independent correction vectors are added to the steepest descent correction to create a calculation with eight correction vectors (N c =8). An indispensable advantage of the multi-direction search over the single steepest descent direction search is clearly demonstrated. Although the steepest descent vector gives the direction with the largest gradient, it does not necessarily point toward the global energy minimum state. On the contrary, a linear combination of multiple correction vectors can be used to obtain the minimum energy state within the given space by adopting the variation principle. Figure 2 Effectiveness of multi-direction search on total energy convergence.
realize the transient indentation in AFM, we introduced a novel experimental method. Viscoelastic nanoindentation theories were then developed based on the functional equation method . The adhesion between the AFM tip and the sample, which significantly affected the determination of the viscoelastic properties , was included in the indentation model . The viscoelastic responses of the sample with respect to different mechanical stimuli, including stress relaxation and strain creep, were further studied. The transition from transient properties to dynamic properties was also addressed. Methods The TMV/Ba2+ superlattice solution was obtained from the mixture of the TMV and BaCl2 solution (molar ratio of Ba2+/TMV = 9.2 × 104:1) as stated GSK872 cost in the reference . It was further diluted with deionized
water (volume ratio 1:1). A 10-μL drop of the diluted solution on a silicon wafer was spun at 800 rpm for 10 s to form a mono-layer dispersion of the sample. The sample was dried for 30 min under ambient conditions (40% R.H., 21°C) for AFM (Dimension 3100, Bruker, Santa Barbara, CA, USA) observation and subsequent indentation tests. The sample was observed with FESEM and AFM. The indentation 17DMAG ic50 D-malate dehydrogenase was performed using the AFM nanoindentation
mode (AFM probe type: Tap150-G, NanoAndMore USA, Lady’s Island, SC, USA). The geometry of the cantilever was precisely measured using FESEM (S-4700, Hitachi, Troy, MI, USA), with a learn more length of 125 μm, width of 25 μm, and thickness of 2.1 μm. To accurately measure the tip radius, the tip was scanned on the standard AFM tip characterizer (SOCS/W2, Bruker) and the scanned data was curve fitted using PSI-Plot (Poly Software International, Orangetown, NY, USA). The tip radius calculated to be 12 nm. For a typical indentation test, the tip was pressed onto the top surface of the sample until a predefined force of ~100 nN. The cantilever end remained unchanged in position during the controlled delay time. A series of indentations of the same predefined indentation force and different delay times were performed to track the viscoelastic responses. A 10-min time interval of the two consecutive indentations was set for the sample to fully recover prior to the next indentation. The sample drift was minimized by turning off the light bulb in the AFM controller during scanning to keep the AFM chamber temperature constant and by shrinking the scan area gradually down to 1 nm × 1 nm on the top surface of the sample to rid the scanner piezo of the hysteresis effect.
Dislocation cores are represented by thin tubes, in which Shockley partial dislocation with 1/6 <112 > Burgers vector and perfect dislocation with 1/2 <110 > Burgers vector are APR-246 concentration colored gray and red, respectively. It is seen from Figure 4b that the dislocation loop consists of four
partial dislocations and one perfect dislocation. In addition, there is one vacancy formed beneath the probe. Upon further penetration, the other Selleckchem IPI-549 three 111 slip planes are activated sequentially, and Figure 4c shows that the defect zone beneath the probe expands greatly. The glide of dislocations on adjacent slip planes leads to the formation of stair-rod dislocations with 1/6 <110 > Burgers vector highlighted by the arrows in Figure 4d. Figure 4e,f presents dislocation network after the completion of scratching and penetration, respectively. It is seen from Figure 4e that there is less dislocations but more
vacancies in the wake of the probe than that in the vicinity of the probe due to the plastic recovery. In addition to the stair-rod dislocations, there are glissile prismatic dislocation loops formed by dislocation reaction and cross-slip events. In particular, the prismatic dislocation half-loops in front of the probe glide parallels to the free surface to transport the materials displaced by the probe without the formation of surface steps . Although small part of the dislocations beneath the probe annihilates at the free surface during the retraction,
Figure 4f shows that the defect structures are stable. Figure MK-1775 4 Close inspections of defect structures in friction with a probe radius of 8 nm. The scratching depth is 0.82 nm. (a,c) Bottom views of defect structures at penetration depths of 0.72 and 0.82 nm, respectively. Atoms are colored according to their BAD values and FCC atoms are not shown. (b,d) Dislocation networks shown in (a) and (c), respectively. (e,f) Dislocation networks after the completion of scratching and retraction, respectively. Effect of probe radius on minimum wear depth To investigate the influence of probe radius on the minimum wear depth, friction simulations Reverse transcriptase with another three probe radiuses of 6, 10, and 12 nm are conducted, in addition to the probe radius of 8 nm. For each probe radius, the penetration stage stops at a penetration depth that is 0.1 nm deeper than the critical penetration depth at which the phenomenon of force drop occurs. Figure 5a,b plots the contact pressure-penetration depth curves and the friction coefficient-scratching length curves during the penetration and scratching stages with the four probe radiuses, respectively. The contact pressure is defined as the ratio of the penetration force to the contact area. A detailed description about the calculation of the contact area during spherical penetration can be found elsewhere .