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ATP synthase expression is localized exclusively in the mitochondria where it generates most cellular ATP. However, ATP synthase components have recently been identified as cell-surface receptors for apparently unrelated ligands in the course of studies carried out on angiogenesis [24–26], lipoprotein metabolism [27], innate

immunity [28–32], etc. by immunofluorescence, biochemistry, and proteomics analyses. Its molecular mechanism, function, and significance have not been clarified well. Dr. Jian Ni’s group prepared specific monoclonal antibody against the α-subunit of ATP synthase, named as HAI-178 antibody, and provided this to my group. Our primary studies showed that Selleck SHP099 the α-subunit of ATP synthase

also exhibited over-expression in gastric cancer cells and clinical gastric cancer tissues, with no or very low expression in normal gastric mucous tissues. Especially as one kind of self antibody which existed in human sera from patients with gastric cancer, this should be a potential biomarker with diagnosis value. In our previous work, we prepared fluorescent magnetic nanoparticles (FMNPs) composed of silicon-wrapped magnetic nanoparticles and CdTe quantum dots and used FMNPs-labeled MSC cells to realize the targeted imaging and hyperthermia therapy of in vivo gastric cancer [33]. We also confirmed that the prepared fluorescent magnetic nanoparticles show good biocompatibility [34]. In the present study, we fully used the advantages of FMNPs and potential gastric cancer biomarker Ro-3306 nmr α-subunit of ATP synthase, prepared HAI-178 monoclonal antibody-conjugated

FMNPs, and investigated the feasibility of prepared nanoprobes to target in vitro and in vivo gastric cancer cells. Our results show that Tucidinostat order as-prepared nanoprobes can be used for in vivo dual-model imaging and therapy of in vivo cancer, and have great potential in applications such as dual-model imaging and simultaneous therapy of early gastric cancer Tangeritin in the near future. Methods All animal experiments (no. SYXK2007-0025) were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. Expression of α-subunit of ATP synthase in gastric cancer tissues HAI-178 monoclonal antibody was presented as a gift by Dr. Jian Ni. HAI-178 monoclonal antibody was used as first antibody to stain 172 specimens of gastric cancer and control gastric mucous tissues with immunohistochemistry method [35], which were collected from Xi’an Central Hospital, Xianya Hospital, Changzheng Hospital, and the First People’s Hospital in Shanghai, and identified by pathological examination. Preparation and Surface functionalization of FMNPs FMNPs were prepared according to our previous report [36–38]. Before coupling the FMNPs with the HAI-178 antibody, we first functionalized the surface functional group of FMNPs as carboxyl group.

The trend of beta (the deteriorative degree of Emricasan in vitro dielectric relaxation) rises from 12.1 nm, peaks at 22.5 nm with the beta value of 0.03, and then declines within the range of 22.5 to 25 nm. The trend of tau decreases from 12.1 to 25 nm accordingly, similar to the CeO2 samples. It is well known that the optical and electrical properties of CeO2 are highly dependent on the surface and interface structure, morphology, and chemistry [10], which in turn is controlled by the fabrication technique and growth conditions [11]. The ability to tailor the properties so as to optimize performance requires a detailed understanding of the relationship

between electronic and geometric structures, particularly at nanoscale dimensions, of CeO2. CeO2 readily crystallizes in the fluorite form, but control

over the grain size formed is important due to the effect of grain boundary density on properties selleck chemicals llc like ionic conductivity and dielectric response [12]. Moreover, the intrinsic frequency dispersion (dielectric relaxation) studies [13, 14] have also been found to be relevant to grain size of the samples, especially those dealing with nanostructured materials. In this Selleckchem Androgen Receptor Antagonist paper, CeO2 is prepared by ALD under different deposition temperatures. The grain size of the samples is determined respectively by the fabrication technique and growth conditions. The focus of the present work is, therefore, on elucidating grain size effects on the electrical properties of CeO2. An interesting correlation between grain size and dielectric relaxation, which provides a reference to tailor the properties and performance of CeO2 as a high-k thin film, has been presented and discussed in the paper. Methods The CeO2 thin films were deposited by liquid injection ALD via a modified Aixtron AIX 200FE AVD reactor (Herzogenrath, Germany) fitted with a liquid injector system. The precursor was a 0.05-M solution

of [Ce(mmp)4] (SAFC Hitech Ltd, Dorset, England, UK) in toluene [9], and the source of oxygen was deionized water. ALD procedures were run at substrate temperatures of 150°C, 200°C, 250°C, 300°C, and 350°C, respectively. The evaporator temperature was 100°C, and the reactor pressure was 1 mbar. The CeO2 thin films were grown on n-Si(100) wafers. Argon carrier gas flow was performed with Bupivacaine 100 cm3/min. The flow of [Ce(mmp)4]/purge/H2O/purge was 2:2:0.5:3.5 s, and the number of growth cycles was 300. For physical characterization, X-ray diffraction (XRD) was achieved using a Rigaku miniflex diffractometer (Shibuya-ku, Japan) with CuKα radiation (0.154051 nm, 40 kV, 50 mA), spanning a 2θ range of 20° to 50° at a scan rate of 0.01°/min. Raman spectra were obtained with a Jobin-Yvon LabRam HR consisting of a confocal microscope coupled to a single grating spectrometer equipped with a notch filter and a charge-coupled device camera detector.

For all strains > 80% viability persisted until 8 h, where upon viability decreased to approximately 30% at 12 h and 1–2% at 24 h. Values represent the means ± SD of at least two experiments. Purified LXH254 gingipains can induce detachment and apoptosis in HGECs Our previous experiments with live bacteria Alisertib nmr and bacterial culture supernatant suggest that either Arg- or Lys-gingipains are necessary for apoptosis in HGECs. In order to determine if specific purified gingipains are also sufficient to induce apoptosis, HGECs were challenged with purified HRgpA, RgpB and Kgp for 2, 4, 8, 15 and 24 hours and DNA fragmentation was assessed by TUNEL (Fig. 6). All three gingipains were able to induce cell detachment

and apoptosis, although at different time points. For HRgpA, signs of apoptosis were already evident at 2 hours post-challenge, while for RgpB and Kgp, TUNEL positive cells appeared at 4 and 8 hours respectively. For all three gingipains, the percentage of apoptotic and detached HGECs increased progressively over time. By 24 hours, HGECs challenged with HRgpA and Kgp had completely detached from the plates, while some clumped cells still

remained on the plates challenged with www.selleckchem.com/products/sb273005.html RgpB (Fig. 6). Different WT P. gingivalis strains induce apoptosis with similar kinetics HGECs were challenged with live P. gingivalis 33277 or W50 at an MOI:100 for 4, 8, 12 and 24 hours and phosphatidylserine (PS) externalization was measured by Annexin-V staining. Untreated cells were used as a negative control. A slow gradual increase in both Annexin-V single and Annexin-V/7-AAD double positive cells was noted for HGECs challenged with both strains compared to the unchallenged control over 12 hours (Fig. 8). The percentage of apoptotic cells was 4–5 fold higher than the unchallenged control 24 hours after challenge with either WT

strain. The results of this kinetic study confirm our previous observations that apoptosis occurs late upon P. gingivalis challenge. Furthermore, the similarity in the kinetics of the response between the two strains suggests that the observed apoptosis is a characteristic of P. gingivalis and not an attribute of a single strain. Figure 8 Flow cytometry for Annexin-V staining to detect PS externalization, an early apoptotic event. HGECs were challenged with live WT P. gingivalis Urease 33277 and W50 at MOI:100 for 4, 8, 12, and 24 hours. The percent of apoptotic cells (7AAD+/AnnexinV+ and 7AAD-/AnnexinV+) is shown for unchallenged HGECs (control), and HGECs challenged with each of the WT strains (+33277, +W50). Values represent the means ± SD of at least two experiments. Statistical comparisons are between challenged and control cells at the same time points ** P < 0.01, *** P < 0.001. P. gingivalis challenge of HGECs results in upregulation of genes related to apoptosis HGECs were challenged with live or heat-killed P. gingivalis 33277 at an MOI:100 for 4 and 24 hours and qPCR was performed on a focused panel of 86 apoptosis-related genes (Fig. 9).

As reported in other studies [5, 9, 30], associated see more premorbid illness was documented in 7.1% of cases. Associated premorbid illnesses have been reported to influence the outcome of patients with perforated

peptic ulcers [5]. In the present study, associated premorbid illness predicted the outcome of patients with perforated peptic ulcers. CB-839 in vitro The prevalence of HIV infection among patients with perforated PUD in the present study was 9.5% that is higher than 6.5% [31] in the general population in Tanzania. This difference was statistically significant (P < 0.001). The high prevalence of HIV infection in our patients may be attributed to high percentage of the risk factors for HIV infection reported in the present study population. The overall HIV seroprevalence in our study may actually be an underestimate and the magnitude of the problem may not be apparent because many cases (8 patients) were excluded from the study due to failure to meet the inclusion criteria. We could not find any literature regarding the effect of HIV infection on the perforation rate and outcome in patient with perforated PUD. This calls for a need to research PF-562271 clinical trial on this observation. In this study, HIV infection was found to be associated with high perforation rate and poor

postoperative outcome. This observation calls for routine HIV screening in patients suspected to have perforated PUD. In agreement with other studies [3, 4, 21, 22, 32], the diagnosis of perforated PUD in this study was made from history and identification of free air under the diaphragm in plain abdominal and chest radiographs, and the diagnosis was confirmed at laparotomy. The value of the radiological investigation has been compared with other writers and with current radiological

techniques; 80-90% of cases are correctly diagnosed [4, 33]. In case of perforated TCL PUD ulcer, free intraperitoneal gas is less likely to be seen if the time interval between the perforation and radiological examination in short [4]. Recently, Computerized tomography (CT) scans with oral contrast are now considered the reliable method of detecting small pneumoperitonium before surgery and the gold standard for the diagnosis of a perforation [34, 35]. Abdominal ultrasonography has also been found to be superior to plan radiographs in the diagnosis of free intra-peritoneal air [35]. None of these imaging studies were used in the diagnosis of free intra-peritoneal air in our study. We relied on plain radiographs of the abdominal/chest to establish the diagnosis of free intra-peritoneal air which was demonstrated in 65.8% of cases. We could not establish, in our study, the reason for the low detection rate of free air under the diaphragm.

The color change was measured at 492 nm using a Synergy HT plate reader (Bio-Tek). Determination of % cell viability was performed using the appropriate control values, as described by the manufacturer. Lipid raft labeling HeLa cells were seeded into 8-well chamber slides (Lab-Tek) at 1 × 104 cells/well and were incubated overnight to achieve

70% confluence. The cells were washed with PBS prior to incubation with dilutions of HIS-PLD (0-50 ng) for 10 min at 37°C and 5% CO2. Dilutions of imidazole-containing elution buffer were used as a control. Lipid rafts were labeled using the Vybrant® Lipid Raft Labeling Kit (Molecular Probes). The Quisinostat cost slides were mounted in 2% 1,4-diazabicyclo [2.2.2]

octane (DABCO; Sigma) in 50% glycerol and visualized with a Nikon epifluorescence microscope fitted with a rhodamine filter. To assess the inhibitory effect of specific antibody, 1/1000 dilutions of anti-PLD or pre-immune serum were incubated with 50 ng HIS-PLD for 1 h at 37°C prior to addition of the mixture to the HeLa cell monolayer. To assess the effect of cholesterol sequestration, 5 mM MβCD was added to HeLa cells for 40 min at 37°C and 5% CO2 prior to stimulation of the cells with 50 ng HIS-PLD. PLD enzymatic activity was not inhibited by the presence of 5 mM MβCD (data not shown). Transmission electron microscopy (TEM) HeLa cell monolayers were inoculated and incubated as for the invasion assay described above. The cells were harvested by scraping and were fixed with 4% formaldehyde-1% glutaraldehyde in PBS, embedded in Epon-Araldite, ACY-738 molecular weight postfixed with 1% osmium tetroxide and stained with 5% uranyl acetate. Thin sections (50 nm) were examined using a Philips CM-12 electron microscope at an accelerating GPX6 voltage of 60 kV. Apoptosis assays HeLa cells were seeded into 96-well plates at 2 × 104 cells/well

and the cells were incubated overnight to achieve 80% confluence. Triplicate wells were inoculated with A. haemolyticum strains, as described above for the epithelial cell cytotoxicity assay. Apoptosis was measured using the 4SC-202 Caspase-Glo 3/7, 8 or 9 Assay Systems (Promega). HeLa cells were incubated with 1 μM staurosporine (Sigma) to induce apoptosis, as a positive control. Statistical analysis Statistical significance was determined at the p < 0.05 level with single factor ANOVA, calculated using Microsoft Excel. Nucleotide sequence accession number The pld gene region sequence data were submitted to the GenBank/EMBL/DDBJ databases under accession number FJ766092. Acknowledgements The authors acknowledge Maricela V. Pier, Stephanie E. Hastings and Ryan G. Miller, University of Arizona for excellent technical assistance and Deborah A. Schaefer for advice with fluorescence microscopy.

The clusters common and unique to the groups mentioned above are presented in additional file 3. In the BBH performed to all strains studied, 77 common genes were obtained, of which 17 (FixA, FixB, FixI, FixG, FixH, FixK, FixN, FixO, FixP, NifA, NifS, NodD, NodM, “”VirB234″”, VirG, TraG and TrbB) are related to biological nitrogen fixation and pathogenesis processes (Figure 2C).

Phylogenetic reconstructions were then performed to the proteins identified in the BBHs with more representativeness among the genomes analyzed. The PDGFR inhibitor topologies SHP099 molecular weight of Fix, Nif, Nod, Vir and Trb proteins (Figures 3 to 5, and additional file 4), have shown some incongruences when compared with the phylogeny model (Figure 1). The reconstruction obtained for FixNOP (Figure 3A) has a similar topology to the model with one exception. In the model reconstruction, Mesorhizobium BNC1 is close to the symbiont and pathogens branch, being grouped with M. loti, while in the FixNOP tree, Mesorhizobium BNC1 is distant from M. loti, in a highly reliable branch, suggesting that these genes in Mesorhizobium BNC1 could have originated from horizontal transfer. Figure 3 FixNOP, FixABC, TrbCFGIJ and FixS phylogenies. Phylogenies of selected

nitrogen fixation and conjugation proteins obtained by BBH, reconstructed with the Neighbor-Joining method of the Phylip 3.67 program, with 1,000 replicates for bootstrap support. (A) concatenated phylogeny for FixNOP proteins; (B) concatenated Ro-3306 cost phylogeny for FixABC proteins; (C) phylogeny for FixS protein; (D) concatenated phylogeny for TrbCFGIJ proteins. Figure 4 NodN and NodD phylogenies. Flavopiridol (Alvocidib) Phylogenies of selected nodulation proteins obtained by BBH, reconstructed with the Neighbor-Joining method of

the Phylip 3.67 program, with 1,000 replicates for bootstrap support. (A) phylogeny for NodN protein; (B) NodD protein. Figure 5 VirB 8 and VirB9 phylogenies. Phylogenies of selected proteins of type IV secretion system obtained by BBH reconstructed with the Neighbor-Joining method of the Phylip 3.67 program, with 1,000 replicates for bootstrap support. (A) phylogeny for VirB8 protein; (B) VirB9 protein. The phylogenetic tree obtained with FixABC (Figure 3B) was the most distinct from the phylogeny model. In the group of photosynthetic, methylotrophic and bioremediation bacteria, Azorhizobium caulinodans is close to Bradyrhizobium and distant from X. autotrophicus. In the pathogen and symbiont group, Rhizobium etli is grouped with M. loti and not with Rhizobium leguminosarum, which in turn is grouped with Ensifer (= Sinorhizobium)meliloti, while in the phylogeny model this bacterium is more related to M. loti. Interestingly, the same patterns of FixABC were obtained in NifAB, with the grouping of R. etli – M. loti and R. leguminosarum – E. meliloti (additional file 4). Furthermore, the grouping between R. etli and M. loti and the proximity between R. leguminosarum and E.

The amount of spores that needs to be added to yield this Cq should be determined for each new batch as it will vary with each new spore stock, and the DNA extraction protocol used. The observed inhibition highlights that multiplex qPCR can be problematic if it is used for the detection of mixed pathogens present in different quantities as amplification of targets from a dominant organism could inhibit the detection of an uncommon pathogen. Assays for the detection of single targets from multiple pathogens simultaneously, such as that described for B. anthracis, F. tularensis and Y. pestis detection [23], should therefore be carefully evaluated for this inhibition effect.

Environmental testing Application of the multiplex qPCR assays directly on human specimens or environmental samples could save time and prevent loss of DNA during extraction. However, we use the assays only after a OSI-906 order DNA extraction protocol, in order to prevent unanticipated inhibition by diverse matrices.

Our laboratory has compared several commercially available DNA extraction kits for use in a BSL3 facility, and selected one that combined efficient DNA extraction with ease-of-use and applicability in the restricted BSL3 environment. We FK228 solubility dmso have been using the developed qPCRs for the analysis of samples suspected for the presence of these pathogens with B. thuringiensis spores added before DNA extraction under BSL3 biosafety conditions. Hundreds of samples containing all sorts of solid materials and liquids have been analyzed without yielding false positive readings. Conclusion The multiplex qPCR assays that were developed for B. anthracis, F. tularensis and Y. pestis allow the rapid detection of 3 pathogen-specific targets simultaneously without compromising sensitivity.

Together with the application of an internal control for both DNA extraction and DNA amplification, this assures highly reliable detection, while template consumption and laboratory effort is kept at a minimum. These considerations see more are particularly advantageous in the context of biothreat samples which may be used for additional tests and for surge Selleck CP673451 capacity during an outbreak. The detection of multiple targets decreases the chance of false-positive and false-negative results and provides additional information about virulence. Methods Selection signature sequences An initial selection of potential signature sequences for specific detection of B. anthracis, F. tularensis and Y. pestis was based on previous reports and on the availability of sequences through public databases (NCBI/EMBL). The selection was based on functional and on technical criteria. Since 4 reporter dyes can be reliably differentiated by using qPCR instruments, and 1 channel was reserved for the internal control, we selected 3 signature sequences per organism.

pertussis 18323, and survival of challenge mice was monitored. For recombinant proteins immunized MGCD0103 groups, A, B, and C indicated 100 μg, 20 μg, and 4 μg dose of immunization. The reference vaccine is used as national reference standard in the intracerebral challenge assay in China and this standard have an assigned activity of 14 IU/ampoule. A, B and C indicated 0.5 IU, 0.1 IU and 0.02 IU dose of immunization. All mice of control group were immunized adjuvant alone. An asterisk symbol (*) indicates a significant difference (P < 0.05) between immunized

and control group. Discussion Because of its advantages in cost, yield and purity, vaccine based on recombinant components has been considered to be a valuable alternative for the vaccine production [22], in particular for the developing countries. In the present study, we showed that the recombinant Prn, Fim2 and Fim3 proteins can be readily expressed and purified in large quantities from E. coli, and each recombinant protein solution is stable for up to twelve months when stored at below -20°C. They

were prepared in a large quantity and freeze-dried. It was confirmed that the activity of freeze-dried preparation had no difference significantly compared with liquid preparation by ELISA method and in some animal experiments (data not shown). The three recombinant proteins LY2109761 can elicit both humoral and cellular immune responses when they were investigated in mice. Furthermore, this recombinant technology makes it possible to avoid contaminations from the B. pertussis components that may cause side effects in vaccine preparations [19]. Availability of the purified Fim2 and Fim3 also provided an opportunity to assess their individual roles in the immunogeniCity and protective efficacy. As a virulence factor of B. pertussis, the ability of Prn to function as adhesin has been investigated both in vitro and in vivo [10, 23]. It is reported that the Prn-mediated protection may be afforded by

blocking Prn-mediated attachment of B. pertussis to the host cells [24, 25]. Studies on the immunized children have also suggested that high level of circulating antibodies against Prn are associated with protection [26, 27]. Furthermore, evidence suggests that anti-Prn antibodies may promote Branched chain aminotransferase extracellular killing with complement or as opsonins, and mediate killing bacteria by phagocytes [25]. However, although antibodies specific to B. pertussis antigens confer protection, many studies have indicated that humoral immunity alone is not sufficient to provide long-term protection against B. pertussis infection and that the protection against B. pertussis requires both T cell- and B cell-mediated immunity [28, 29]. Our results showed that the antibody response increased significantly in mice immunized with rPrn. selleck compound immunization of rPrn also induced a Th1 response that is characterized by the enhanced production of IL-2- and TNF-α.

The films were grown at a deposition temperature of 300°C using pulsed laser deposition (PLD). We successfully demonstrated the temperature-dependent thermal conductivities of epitaxial Fe3O4 thin films via four-point probe 3-ω method in the temperature range of 20 to 300 K. The measured out-of-plane thermal conductivities Selleck Ion Channel Ligand Library of the Fe3O4 thin films (0.52 to 3.51 W/m · K) at 300 K are considerably reduced compared to those of

the bulk materials (approximately 6 W/m · K) [17] because of strongly enhanced phonon-boundary scattering, as expected in the Callaway model [18]. Furthermore, we clearly realized that the thermal conductivity increased with an increase in film thickness and grain size, which agreed well with the theoretical predictions of the Callaway model. Methods The epitaxial magnetite thin films were synthesized on SiO2/Si (100) substrates at a temperature of 300°C using PLD. The detailed growth processes can be found in our previous publication [19]. In brief, a krypton fluoride (KrF, 248 nm in wavelength) excimer laser whose energy density was approximately 2.1 J/cm2 at repetition rate of 4 Hz at selleck products a pressure of 10-3 Pa was used along with a ceramic target (pure, homogeneous, and highly dense α-Fe2O3 ceramic).

Our previous results confirmed that the surface roughness of the films increased with increasing temperature. Consequently, the deposition C-X-C chemokine receptor type 7 (CXCR-7) temperature was maintained at 300°C to obtain a uniform quality in the grown films. The deposition rate of the films was maintained at approximately 1.2 nm/min. To measure the thermal conductivity, we prepared three Fe3O4 thin films with thicknesses of 100, 300, and 400 nm using PLD. X-ray diffraction confirmed that the films were grown with a (111) Alisertib research buy preferred orientation with high-quality epitaxial growth, as detected from the in-plane phi-scans of the films [19]. Figure 1a,b,c

shows the cross-sectional scanning electron microscope (SEM) images of the as-grown Fe3O4 thin films, confirming that the thicknesses of the films were in the range of 100 to 400 nm. Atomic force microscope (AFM) images (insets of Figure 1ab,c) showed that the grown films exhibit smooth grain morphologies with a root-mean-square (rms) roughness of 1.4 to 6.0 nm, as summarized in Figure 1d. We also found that the grain size of the films increased from approximately 13.2 ± 5.2 nm to approximately 230 ± 23.10 nm when the film thickness was increased from 100 to 400 nm, indicating that thicker films have much rougher surface morphology and larger grain size. Figure 1 SEM cross-sectional images of Fe 3 O 4 thin films grown on a SiO 2 /Si substrate at 300°C using PLD. (a) 100 nm, (b) 300 nm, and (c) 400 nm. The insets show the AFM images of each thin film. (d) A summary of the prepared Fe3O4 thin film, including rms roughness, film thickness, deposition time, and grain size information.