A zoom-in of the photo clearly shows the strong luminescence of our ZnO homojunction device. Figure 4 PL measurements of Sb-doped ZnO microrod array (red) and intrinsic ZnO microrod array (black). The inset shows a photo taken on the Sb-doped PD0332991 solubility dmso ZnO microrod array and a zoom-in showing violet luminescence. Figure 5 shows the temperature-dependent PL spectra of the Sb-doped ZnO microrod array from T = 30 K to T = 300 K. The red shift of the PL peak along with increasing temperature can be described by the Varshni equation [19]: (1) where E(0) is the transition energy of the free exciton or the free electron-to-acceptor level (FA) transition at zero temperature, and α and β are constants. The result of the fitting

curve is shown in the inset

of Figure 5 with α = 7.8 × 10-4 eV/K, β = 510 K, and E(0) = 3.322 eV. Moreover, the peak of the photoluminescence can be attributed to the free electron-to-acceptor level transition [16, 20]. The acceptor binding energy is given by (2) where Eg, E_D, E_A, ϵ_0, ϵ, and r are the bandgap energy, Mitomycin C the donor binding energy, the acceptor binding energy, the permittivity of ZnO in vacuum, the dielectric constant of ZnO, and the distance of the electron-hole pair, respectively. The donor energy ED is reported to be about 60 meV, the value of is 30 to 60 meV, and the bandgap of ZnO is 3.437 eV; therefore, the estimated EA is 161 ± 15 meV [21]. Strong violet luminescence at room temperature was revealed in this work. This particular phenomenon was induced by replacement of the Teicoplanin Zn sites, instead of the O ones, with Sb atoms (Sb_Zn) to form a complex with two V_Zn, which is the Sb_Zn-2V_Zn complex. This Sb_Zn-2V_Zn complex has a lower formation energy and acts as a

shallow acceptor; therefore, strong violet luminescence was induced as shown in Figure 5. From the room-temperature PL spectra shown in Figure 4, an estimation of the activation energy of 140 meV for Sb-doped ZnO was obtained. This value is in good agreement with the theoretical ionization energy of the Sb_Zn-2V_Zn complex acceptors [21]. The particular phenomenon has a potential application in violet light emission. Figure 5 Temperature-dependent PL spectra of the Sb-doped ZnO microrod array. From top to bottom: T = 30, 45, 60, 75, 90, 105, 120, 135, 150, 180, 210, 240, 270, and 300 K, respectively. The peaks centered at around 2.8 eV are laser background signals. The inset shows the PL peak positions in energy as a function of temperature of the Sb-doped ZnO microrod array. The squares are experimental data of the FA emission, and the red line is the fitting curve to the Varshni equation. The I-V measurement of the ZnO homojunction device is shown in Figure 6. Ohmic contacts for each device were assured by the linear I-V relations shown in the inset of Figure 6. Therefore, the observed non-linear I-V characteristics as shown in Figure 6 must be due to the device rather than non-ideal electrical contacts.

Sorption capacity and potentiometric measurements Ion exchange

capacity of the membranes has been determined by their treatment with a HCl solution (100 mol m−3), washing with deionized water followed by treatment with a NaOH solution (100 mol m−3) and analysis of the eluate using an I-160 M potentiometer and Cl−-selective electrode. The solution was neutralised Selleck Enzalutamide with HNO3 before the measurements. Membrane potential (E m) was measured at 298 K using a two-compartment cell [16, 17]. HCl solutions (10 and 15 to 100 mol m−3) filled their chambers, where Ag/AgCl electrodes were placed. Transport numbers of counter ions (t m) through the membrane were calculated as [16] (3) where a 1 and a 2 are the activities of counter ions in less and more concentrated solutions, respectively; indexes ‘+’ and ‘−’ correspond to cations and anions, respectively; R is the gas constant; F is the Faraday constant; T is the temperature; NVP-LDE225 nmr and a ± is the activity of ions in a solution of varied concentration. The equation is valid

for a 1:1 electrolyte like HCl. The transport numbers of counter ions (Cl−) were found from a derivative of the function, which describes a deviation of the membrane potential from theoretical value : (4) The difference of was found, and then its dependence on a ± (i.e. on activity of more concentrated solution, a 2) was plotted. At last, the transport number was calculated from a slope of the curve. Electrodialysis isometheptene The experimental setup involved a four-compartment cell, three independent liquid lines, power supplier and measurement instrumentation described earlier

[7] (Figure 1). A scheme of the membrane system was as follows: cathode compartment, polymer cation-exchange membrane (Nafion 117, Dupont, Wilmington, DE, USA), desalination compartment filled with glass spacers (6 × 10−4 m of a diameter), inorganic membrane, concentration compartment, polymer cation-exchange membrane and anode compartment. The distance from each membrane to the other (and from cation-exchange membrane to the opposite electrode) was 1 cm, the cross-sectional area of each compartment was 4 cm, and the effective area of each membrane was 16 cm (4 cm × 4 cm). Figure 1 Scheme of the electrodialysis setup. A solution containing NaCl (10 mol m−3), the volume of which was 50 cm3, circulated from the desalination compartment with a flow velocity of 1 cm3 s−1 (first liquid line). The second line provided circulation of the solution, which contained initially K2SO4 (1,000 mol m−3), through the cathode and anode compartments (second line). At last, a H2SO4 solution (100 mol m−3) circulated through the concentration compartment. The content of Cl− and Na+ species in the solution being purified was controlled by means of ion-selective electrodes. The removal degree of NaCl from the solution was calculated as , where C is the concentration at time τ and C i is the initial concentration.

The clinical and pathological data collected included gender, age, hepatitis B surface antigen (HBsAg) status, serum

alpha-fetoprotein (AFP) level, tumor number, tumor size, degree of tumor differentiation, Child-Pugh class, Barcelona Clinic Liver Cancer (BCLC) stage, presence of cirrhosis, ascites, tumor thrombus, and extrahepatic metastasis. The PFS and OS were defined as the time from initiation of sorafenib therapy to the time of disease progression detected by computed tomography or magnetic resonance imaging, or death, respectively. Immunohistochemical staining Expression of VEGFR-2, PDGFR-β, and c-Met were determined by two-step PV-6000 click here immunohistochemistry staining. Specimen slices were dewaxed, rinsed in phosphate-buffered saline (PBS). Antigen retrieval was performed by placing the slides in a high pressure cooker in 0.01 mmol/L citrate buffer, pH 6.0, for 3 minutes at 100°C, followed by cooling for 20 min at room temperature,

rinsing in PBS, treating with 3% hydrogen peroxide in deionized water for 10 min to block endogenous peroxidase, and rinsing again in PBS. Specimens were then incubated at 37°C for 1 hour with primary antibody against VEGFR-2 (dilution ratio 1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA), PDGFR-β (dilution ratio 1:40; Santa Cruz Biotechnology Inc., CA), and c-Met (rabbit anti-human c-Met monoclonal antibody working solution; Epitomics, California, US), followed by rinsing three times in PBS for 2 min each time. Specimens were incubated at 37°C for 20 min with universal IgG antibody-HRP polymer (Zhongshan Jinqiao Co., Beijing, China), and rinsed three times in PBS Small molecule library cost for 2 min each time. Specimens

were placed in DAB solution for color development, rinsed with distilled water, stained again, dehydrated, and sealed with transparent strips. Primary antibodies were replaced with PBS to produce a negative control, and a known positive tissue slice was used as a positive control. Analysis of immunohistochemistry results Two pathologists who were blind to diagnosis independently inspected the slices. The rate of agreement between the two pathologists was 95%. The scores from both pathologists were averaged to provide Clostridium perfringens alpha toxin the final score for each case. A combination of positive cell count and staining intensity was used for scoring. Positive cell count was scored based on the average percentage of positive cells per 100 cells in 10 high-power fields, as follows: 0–10%, score 0; 11–25%, score 1; 26–50%, score 2; 51–75%, score 3; and >75%, score 4. Staining intensity was scored as follows: negative, score 0; faint yellow, score 1; yellow or deep yellow, score 2; brown or dark brown, score 3. The final score was obtained by multiplying the cell count and staining intensity scores. For VEGFR-2 and c-Met, a score of ≥ 5 was defined as high expression and a score of < 5 was defined low expression.

J Alloys Compd 2007, 438:258–262. 10.1016/j.jallcom.2006.08.030CrossRef 25. Li Y, Li Y, Zhu M, Yang T, Huang J, Jin H, Hu Y: Structure and magnetic properties of Cr-doped ZnO nanoparticles prepared under APO866 supplier high magnetic field. Solid State Commun 2010, 150:751–754. 10.1016/j.ssc.2010.01.027CrossRef 26. Wesselinowa J, Apostolov A: A possibility to obtain room temperature ferromagnetism by transition metal doping of ZnO nanoparticles. J Appl Phys 2010, 107:053917–053917–053915.CrossRef 27. Yousefi R, Zak AK, Jamali-Sheini F: The effect of group-I elements on the structural and optical properties of ZnO nanoparticles. Ceram Int 2013,

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electroluminescence of ZnO nanorods/MEH-PPV heterostructure by using a ZnS buffer layer. Org Electron 2011, 12:92–97. 10.1016/j.orgel.2010.09.018CrossRef 31. Khorsand Zak A, Razali R, Abd Majid WH, Darroudi M: Synthesis and characterization of a narrow size distribution of zinc oxide nanoparticles. Int J Nanomedicine 2011, 6:1399–1403.CrossRef 32. Zak AK, Majid WHA: Effect of solvent on structure and optical properties of PZT nanoparticles prepared by sol–gel method, in infrared region. Ceram Int 2011, 37:753–758. 10.1016/j.ceramint.2010.10.020CrossRef 33. Deng X, Sun J, Yu S, Xi J, Zhu W, Qiu X: Steam reforming of ethanol for hydrogen production

over NiO/ZnO/ZrO 2 catalysts. Int J Hydrog Energy 2008, 33:1008–1013. Competing interests The authors declare that they do not have competing interests. Authors’ contributions AKZ carried out the sample preparation, XRD, and UV section. MD carried out the TEM imaging and Auger spectroscopy MycoClean Mycoplasma Removal Kit part. AMH was the project leader and contributed in analyzing the data. All authors read and approved the final manuscript.”
“Background The layered transitional quasi-two-dimensional (Q2D) semiconductor oxides MO3 (M = Mo, W), have recently attracted significant interest because they demonstrate quantum confinement effects at the few-layer limit [1, 2]. Among them, tungsten trioxide (WO3) is an n-type semiconductor in an indirect bandgap of 2.6 to 2.9 eV [3] with excellent electrochromic and gasochromic properties [4]. It has electron Hall mobility of ~12 cm2V-1 s-1 at room temperature and responsive to the blue end of the visible spectrum (λ < 470 nm) [5].

Cancer 1995, 76 (5) : 853–9.CrossRefPubMed 16. Arai Y, Tsukuda M, Ito K, Enomoto H, Furukawa M, Kubota A, Yanoma S, Okamoto N: Analysis of DNA ploidy using fresh frozen tissues of head and neck squamous cell carcinomas. Auris Nasus Larynx 1997, 24 (2) : 193–8.CrossRefPubMed 17. Rubio Bueno P, Naval Gias L, Garcia Delgado R, Domingo Cebollada J, Diaz Gonzalez FJ: Tumor DNA content as a prognostic indicator in squamous cell carcinoma of the oral cavity and tongue base. Head & Neck 1998, 20 (3) : 232–39.CrossRef 18. Goldsmith MM, Cresson DH, Arnold LA, Postma DS, Askin FB, Pillsbury HC: DNA flow cytometry as a prognostic indicator in head and neck cancer. Otolaryngol Head Neck

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in predicting survival of patients with pancreatic carcinoma. J Gastrointest Surg 2003, 7 (8) : 953–9. discussion 59–60.CrossRefPubMed 21. Halfpenny W, Hain SF, Biassoni L, Maisey MN, Sherman JA, McGurk M: FDG-PET. A possible prognostic factor in head and neck cancer. Br J Cancer 2002, 86 (4) : 512–6.CrossRefPubMed 22. Kunkel M, Reichert TE, Benz P, Lehr HA, Jeong JH, Wieand S, Bartenstein P, Wagner selleck Farnesyltransferase W, Whiteside TL: Overexpression of Glut-1 and increased glucose metabolism in tumors are associated with a poor prognosis in patients with oral squamous cell carcinoma. Cancer 2003, 97 (4) : 1015–24.CrossRefPubMed 23. Brun E, Kjellen

E, Tennvall J, Ohlsson T, Sandell A, Perfekt R, Wennerberg J, Strand SE: FDG PET studies during treatment: prediction of therapy outcome in head and neck squamous cell carcinoma. Head Neck 2002, 24 (2) : 127–35.CrossRefPubMed 24. Schoder H, Yeung HW: Positron emission imaging of head and neck cancer, including thyroid carcinoma. Semin Nucl Med 2004, 34 (3) : 180–97.CrossRefPubMed 25. Smith TA, Titley JC, McCready VR: Proliferation is associated with 2-deoxy-D-[1–3H]glucose uptake by T47D breast tumour and SW480 and SW620 colonic tumour cells. Nucl Med Biol 1998, 25 (5) : 481–5.CrossRefPubMed 26. Minn H, Joensuu H, Ahonen A, Klemi P: Fluorodeoxyglucose imaging: a method to assess the proliferative activity of human cancer in vivo. Comparison with DNA flow cytometry in head and neck tumors. Cancer 1988, 61 (9) : 1776–81.CrossRefPubMed 27. Smith TA, Titley J: Deoxyglucose uptake by a head and neck squamous carcinoma: influence of changes in proliferative fraction. Int J Radiat Oncol Biol Phys 2000, 47 (1) : 219–23.CrossRefPubMed 28.

Our research showed that the amounts of EPS produced by P. aeruginosa strains were also significantly inhibited by 0.5 and 1 mg/ml of NAC. Taking into account the results given above, NAC may be a potent agent for treating P. aeruginosa biofilms associated infections, and can be used

in combination with ciprofloxacin. Stafanger [21] studied the effect of peroral NAC in patients with cystic fibrosis and chronic pulmonary P. aeruginosa infection, a significant improvement of the spirometric values was proved after NAC treatment in the patients with peak expiratory flow rate below or equal to this website 70% of predicted normal values. Stey [22] reviewed the publications on the effect of oral NAC in chronic bronchitis, eleven randomized controlled NAC trials were analysed (a total of 2,011 patients), concluded that oral NAC reduced the risk of exacerbation and improved symptoms in patients with chronic bronchitis compared with palcebo. But the benefit it achieved still remains unclear. We are not sure whether it took into account the other elements such as anti-bacterial activities and detach biofilms or not? It needs further study. NAC can be administered by nebulization or direct instillation, orally or intravenously. The concentrations tested in our study are much higher than those reach in serum when administer by an intravenous or oral route. Nevertheless, it may be possible that using local respiratory application (10% solution may be used undiluted

for inhalation) obtains Ibrutinib nmr useful concentrations to disrupt biofilms and control biofilm-associated infections of P. aeruginosa. Conclusions In conclusion, our results suggest that NAC has anti-bacterial properties against P. aeruginosa and may detach P. aeruginosa biofilms. It may

be a new strategy for the treatment of biofilm-associated chronic respiratory infections, although it would be appropriate to conduct in vivo animal models and clinical studies to confirm this. Methods Bacterial strains P. aeruginosa Glycogen branching enzyme PAO1 expressing a green fluorescent protein (GFP) plasmid (pMRP9-1) was kindly donated by Dr. E. P. Greenberg (University of Washington, Seattle). An additional 20 strains of P. aeruginosa isolated from respiratory samples were studied. Determination of minimum inhibitory concentrations (MIC) and drug-drug interactions Crystalline NAC (Sigma-Aldrich, USA) was dissolved in distilled water to make a 100 mg/ml solution; the pH of solution was adjusted to 7.2 before use. Stock solution of ciprofloxacin (National Institute for the Control of Pharmaceutical and Biological Products, China) was prepared at concentrations of 4096 μg/ml in the distilled water. MICs of NAC and ciprofloxacin were determined using a broth micro-dilution assay according to Clinical Laboratory Standards Institute (CLSI) guidelines [23]. Each well of a 96-well microtiter plate containing 100 μl from a series of diluted NAC with Mueller-Hintor broth was inoculated with 100 μl of P.

aureus and S. epidermidis generated biofilms. AKBA is reported to be

active against a large number of inflammatory diseases, cancer, arthritis, chronic colitis, ulcerative colitis, Crohn’s disease, and bronchial asthma [21, 26, 20, 27, 28]. The anticancer activity of AKBA is attributed to the inhibitory effect on the lipoxygenases leading to the inhibition PLX3397 chemical structure of cell proliferation and induction of apoptosis in tumor cells [29]. There are numerous reports available on the antibacterial activity of oleo-gum resin extracts and oleo-gum resin essential oils from Boswellia spp. (Burseraceae) [30–32]. Weckessera et al. [33] reported the antibacterial activity of Boswellia dry extract and keto-ß-boswellic acid. Their findings revealed that the extract was highly effective against selected aerobic and anaerobic bacteria such as Streptococcus, Corynebacteria, C. perfringens and P. acnes; whereas KBA was not effective against these pathogens, suggesting that the effective components are other boswellic acids or essential oils contained in the extract. In this study, we extensively evaluated the boswellic acids for the antibacterial activity and further for the first time established that AKBA is the single most potent antibacterial compound

present in the gum exudates of Boswellia serrata. We further investigated the effect of AKBA on the bacterial cell membrane integrity through propidium iodide uptake assay. Propidium iodide is fluorescent nucleic acid stain that

binds to DNA by intercalating between the bases with little or no sequence preference. It is membrane impermeant and generally excluded from viable cells. The increased uptake of propidium AZD2281 mouse iodide in the AKBA treated cells of S aureus in our study indicated that AKBA altered the cell membrane structure, resulting in the disruption of the permeability barrier of microbial membrane structures. Leakage of cytosolic constituents (260 and 280 nm absorbing materials) from S. aureus cells in the presence 64 μg/ml AKBA over a period of two h was significantly higher than background levels (P < CYTH4 0.05). These observations indicate that the antimicrobial activity of AKBA results from its ability to disrupt the permeability barrier of microbial membrane structures. The lack of antibacterial activity of AKBA against Gram-negative bacteria may be attributed due to the presence of lipophilic outer membrane. This outer layer of the Gram-negative outer membrane is composed primarily of lipopolysaccharide molecules and forms a hydrophilic permeability barrier providing protection against the effects of highly hydrophobic compounds [34, 35]. This may be the probable explanation of the resistance of Gram-negative bacteria to lipophilic AKBA. Similar observations have been made in other studies also, where lipohilic terpenes such as carvacrol, thymol, eugenol, geraniol, linalyl acetate, (-) menthol and bakuchiol have reported low sensitivities against Gram-negative bacteria [36–38].

Arch Intern Med 2002, 162:2113–2123.PubMedCrossRef 26. Usha PR, Naidu MU: Randomised, Double-Blind, Parallel, Placebo-Controlled Liproxstatin-1 solubility dmso Study of Oral Glucosamine, Methylsulfonylmethane and their Combination in Osteoarthritis. Clin Drug Investig 2004, 24:353–363.PubMedCrossRef 27. Petersen SG, Saxne T, Heinegard D, Hansen M, Holm L, Koskinen S, Stordal C, Christensen H, Aagaard P, Kjaer M: Glucosamine but not ibuprofen alters cartilage turnover in osteoarthritis patients in response to physical training. Osteoarthritis Cartilage 2010, 18:34–40.PubMedCrossRef

28. Ostojic SM, Arsic M, Prodanovic S, Vukovic J, Zlatanovic M: Glucosamine administration in athletes: effects on recovery of acute knee injury. Res Sports Med 2007, 15:113–124.PubMedCrossRef 29. Hespel P, Maughan RJ, Greenhaff PL: Dietary supplements for football. J Sports Sci 2006, 24:749–761.PubMedCrossRef 30. Heavin G: Permanent Results Without Permanent Dieting: The

Curves for Women Wight Loss Method. Waco, TX: Curves Interational Inc; 1999. 31. Almada A, Kreider R: Comparison of the reliability of repeated whole body DEXA scans to repeated spine and hip scans. J Bone Miner Res 1999, 14:S369. 32. Kaminsky LA, Bryant CX, Mahler DA, Durstine JL, Humphrey RH: ACSM’s Guidelines for Exercise Testing and Prescription. 8th edition. Baltimore, MD: Lippincott, Williams & Wilkins; 2009. 33. Wessel J: Isometric strength measurements of knee extensors in women with osteoarthritis of the knee. J Rheumatol 1996, 23:328–331.PubMed 34. Carter ND, Khan KM, Petit

PLX4720 MA, Heinonen A, Waterman C, Donaldson MG, Janssen PA, Mallinson A, Riddell L, Kruse K, Prior JC, Flicker L, Oxaprozin McKay HA: Results of a 10 week community based strength and balance training programme to reduce fall risk factors: a randomised controlled trial in 65–75 year old women with osteoporosis. Br J Sports Med 2001, 35:348–351.PubMedCrossRef 35. Cuka S, Dvornik S, Drazenovic K, Mihic J: Evaluation of the Dade Behring Dimension RxL clinical chemistry analyzer. Clin Lab 2001, 47:35–40.PubMed 36. McAuley KA, Williams SM, Mann JI, Walker RJ, Lewis-Barned NJ, Temple LA, Duncan AW: Diagnosing insulin resistance in the general population. Diabetes Care 2001, 24:460–464.PubMedCrossRef 37. Ware JE, Kosinski M, Bayliss MS, McHorney CA, Rogers WH, Raczek A: Comparison of methods for the scoring and statistical analysis of SF-36 health profile and summary measures: summary of results from the Medical Outcomes Study. Med Care 1995, 33:AS264–279.PubMedCrossRef 38. Denegar CR, Perrin DH: Effect of transcutaneous electrical nerve stimulation, cold, and a combination treatment on pain, decreased range of motion, and strength loss associated with delayed onset muscle soreness. J Athl Train 1992, 27:200–206.PubMed 39.

Marketing services Agricultural products are frequently subjected to market analyses by the USDA such as economic and census reports. As the commercialization of algae progresses, market analyses will be advantageous to assess the strengths and weaknesses of the industry, the interplay between the agricultural and energy aspects of algae, and the outlook of the industry. The USDA also provides marketing

assistance to farmers through financial assistance, research and promotion (AMS 2013). To successfully break Selleckchem 5-Fluoracil into the agricultural market, algae would benefit from the marketing services available from the USDA. State programs Defining the commercial cultivation of algae as agriculture provides opportunities at the state level as well. Many states offer additional loan and financing programs, especially for first-time farmers, such as “Aggie Bonds” that encourage private lenders to loan to beginning farmers (CDFA 2005). Beyond financial assistance, states can control laws associated with agricultural property and zoning. For example, the Ohio state legislatures recently defined algaculture as agriculture to allow use value Opaganib assessments of algae cultivation land for tax purposes, thus lowering property taxes for land used for commercial algaculture (OH-H.R. 2012). The

law additionally limits the authority of zoning laws to restrict algae cultivation on lands. Although decisions on specific investments in algae development are made at the regional and local levels, a federal initiative is still imperative to establish and influence direction and focus for the industry, as well as to develop guidance for new algae programs. Application of agricultural programs to algae Opportunities currently exist for algae cultivation to expand commercialization DCLK1 within agriculture if it were defined as such.

The most notable is the potential to fill a large void in agriculture of the use of non-arable land to produce renewable hydrocarbons and high value protein. Unlike terrestrial crops, algae do not require fertile soil or arable land for growth, thus expanding the areas of the country in which algae can be cultivated. Algae do require other inputs such as salt or freshwater, nutrients, and consistent year-round sunlight. Taking all of these factors into account, a recent study by the Pacific Northwest National Laboratory (PNNL) identified ~90,000 sites in the U.S. that would be suitable for algaculture, comprising ~5.5 % of the contiguous U.S. land mass and consisting predominantly of shrub/scrub landscape. These sites exclude any cropland, urban land, protected lands, wetlands, wilderness, or significantly sloping landscapes (Wigmosta et al. 2011). To compare, agricultural land currently utilizes over 40 % of the total U.S. land mass. The USDA currently asserts jurisdiction of algae as an agricultural crop, and can potentially offer agricultural safety net programs to algal biomass companies.

To confirm that the inocula contained or lacked the kan cassette and that

the kan cassette was not lost Sorafenib datasheet by the mutant during the course of infection, individual colonies from the inocula, surface cultures and biopsy specimens were picked, suspended in freezing medium and frozen in 96-well plates. If available, thirty colonies from an individual specimen were scored for susceptibility to kanamycin on kanamycin-containing chocolate agar plates as described [31]. Recombinant fusion protein construction and expression The ompP4 ORF, without the signal peptide sequence, was amplified from 35000HP genomic DNA using synthetic primers (5’-TGTACTTATCATCATAATCATAAGCAT-3’ and 5’-TGAATAACGAGTTAATCCTAACAAAA-3’) and then cloned into the pCR-XL-TOPO vector using the TOPO XL Cloning Kit (Invitrogen Corp, San Diego, Calif). The fragment was excised using EcoRI and then cloned into pRSETB (Invitrogen). Transformation of recombinant plasmid into BL21(DE3)pLysS cells allowed for fusion protein expression. Recombinant OmpP4 was expressed in inclusion bodies and was purified under conditions using urea following find more the QIAexpressionist System (Qiagen, Inc, Valencia, Calif). Stepwise dialysis with decreasing

urea concentrations was used to remove urea from the recombinant proteins and then concentrated with a Centricon-10 microconcentrator (Amicon Corp., Beverly, Mass). Purified recombinant OmpP4 was used to inoculate BALB/c mice to produce polyclonal antibodies (Harlan Bioproducts for Science) that were used in bactericidal and phagocytosis assays. Immune serum bactericidal assays 35000HP was grown for 16–18 h from a freezer stock on chocolate agar plates at 33°C with 5% CO2 and harvested in phosphate-buffered saline. After vortexing for 30 sec, cells were suspended

in GC medium and diluted to a final concentration of approximately 103 to 104 CFU/ml. Bactericidal assays were performed in 96-well plates. Each well received 50 μl 35000HP and 10 μl (or 10%) of heat-inactivated NMS or HMS-P4 and brought to 65 μl with GC broth. Plates were incubated for 30 min at 33°C with Edoxaban 5% CO2. Then, 25 μl of either active or heat-inactivated normal human serum, which was used as the complement source, was added and the plates were incubated for an additional 60 min at 33°C with 5% CO2. Bacteria were quantified by plating 100 μl from each well onto chocolate agar and incubating for 48 h at 33°C with 5% CO2. Heat-inactivated hyperimmune pig serum collected after multiple inoculations with H. ducreyi, which has been shown to promote bactericidal activity against H. ducreyi, was used as a positive control (kindly provided by Thomas Kawula, University of North Carolina, Chapel Hill) [27]. Data were reported as percent survival in active NHS compared to that in heat-inactivated-NHS. Each experiment was repeated three times, and arithmetic mean and standard deviation of the percent survival were calculated.