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A prototype ejector chiller with nominal cooling power of 40 kW, designed according to the “CRMC” criterion, has been tested by our research group since 2011. The prototype has undergone several refinements and finally has been upgraded to R1233zd(E), which is non-flammable, has low-GWP and favourable thermodynamic properties, i.e. “dry-expansion” and moderate pressure at generator. In terms of saturation pressure curve, the new fluid is similar to R245fa, which was previously used in the prototype. Hence, R1233zd(E) has been basically used as a “drop-in” replacement. The ejector chiller was tested at different evaporation temperatures (2.5 to 10°C), typical of air conditioning applications. Saturation temperatures at generator from 95 to 105°C have been selected as representative of waste-heat recovery. The experimental findings prove that the CRMC ejector has a satisfactory performance with the new working fluid.

This paper numerically investigates the performance of a high capacity, industrial ejector chiller working with the non-flammable, low GWP refrigerant R1233zd(E). The test procedures adopted to acquire the experimental data are explained in part 1 of this work. Different turbulence models were examined in order to accurately predict the ejector off-design transition and critical pressure. A peculiar difference has been noticed between the numerical results obtained when initializing the CFD simulation from the solution of a case with lower or higher condenser pressure. This difference may partly explain the difficulties in the accurate predictions of the critical conditions reported by this and other works in the literature. A detailed discussion is presented on the nature and possible cause of this phenomenon, which is identified as typical of non-linear fluid dynamics.

Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we produced and characterized a recombinant peptibody capable of recognizing and depleting murine MDSCs. Using SOE-PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. The purified peptibody appeared as a 70 kDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P

An impulse facility for analysis of water vapour nozzle flows using both shock tunnel and Ludwieg tube operating modes has been developed and tested at the University of Southern Queensland. Unique high-speed flow visualisation of the water vapour condensation shock has been acquired in the throat region of a nominally two-dimensional convergent-divergent nozzle with a 120 mm2 throat area. This paper presents the facility performance and the time-resolved visualisation results for the nozzle flow, and the results are analysed with the aid of image post-processing tools and quasi-one-dimensional (Q1D) thermodynamic calculations. The experiments have produced qualitative and quantitative data on the flow conditions at four different relative humidity values from 25% to 100%, and over a range of nozzle supply temperatures between 293 K and 343 K. The direct measurement of the pressures and wave speeds within the tunnel resulted in a good agreement with the ideal gas Q1D calculations, particularly for the first incident shocks and expansion waves. Differences progressively increased with the number of reflections, due to the effect of viscous dissipation and the non-ideal end-wall interaction caused by the presence of the nozzle inlet. Moreover, flow visualizations of the location and orientation of both the weak disturbance waves and the condensation shock enabled the investigation of the supersonic water vapour condensation within the nozzle of this impulse facility. Based on these measurements, the nozzle flow downstream of the condensation shock was demonstrated to have a lower Mach number relative to the dry nitrogen flow case. This phenomenon is due to the phase change heat release, which appeared to be more significant when the condensation shock intensity was higher. Although further development of the apparatus is possible, the impulse test-rig configuration that we have developed permits a relatively high flow rate of test gas for a short period of time, leading to significantly reduced operating expenses relative to a continuous flow facility with the same mass flow rate. It is therefore suggested that future studies further improve and test the impulse facility techniques for studying condensation phenomena as a potential alternative to high-power, continuous flow facilities.

AIM Placenta-specific 1 (PLAC1) is among recently-discovered placental antigens which exerts fundamental role in placental function and development. Increasing body of literature shows that PLAC1 is frequently activated and expressed in a wide variety of human cancers and promote cancer progression. However, no data is available regarding the expression of mouse orthologue, plac1, in murine cancer cell lines. Materials and Methods: We investigated the expression of plac1 in a series of murine cell lines from different histological origins, mammary carcinoma (4T1), melanoma (B16F10), colorectal carcinoma (CT26), renal carcinoma (Renca), glioma (GL26), B-cell lymphoma (A20 and BCL1) and also two fibroblast cell lines (NIH3T3 and L929), using RT-PCR, Western blotting and flow cytometry. Results: Our data demonstrated that plac1 transcript and plac1 protein were expressed in all examined cell lines, as judged by RT-PCR and Western blot, respectively. The molecular weight of mouse plac1 was experimentally observed to be approximately 24 kD. Flow cytometric analysis showed surface expression of plac1 in aforesaid cell lines ranging from 2% to 42.5%. Conclusion: Based on the ubiquitous expression of plac1, the investigated cancer cell lines or immortalized cell lines can be used to examine the role of plac1 in the process of immortalization.

Research activity on ejectors is ongoing at the University of Florence since the late nineties. The most important achievement is a 40 kW ejector chiller designed according to the “CRMC” criterion. The experimentally validated CFD simulations have given some hints about some possible improvements, i.e. refine the surface finish of the ejector, study the effect of heat transfer and improve the final part of the diffuser, which in its present shape does not produce a measurable compression. The prototype has been recently filled with low-GWP refrigerant R1233zd, as a drop-in replacement of previously used R245fa. Both fluids are “dry-expanding” and hence significantly easier to model in CFD simulations. Synthetic low-GWP refrigerants may be an option for ejector chillers, due to their ability to reach below-zero temperature and high volumetric refrigerant capacity. Some lessons learned with synthetic refrigerants can be transferred to the project of a steam ejector chiller, which remains one of our future targets. Herein we resume the principal findings gathered by means of experimental and numerical activity on our prototype and propose a few ideas for the future research.

The emergence of immunotherapy has revolutionized medical oncology with unprecedented advances in cancer treatment over the past two decades. However, a major obstacle in cancer immunotherapy is identifying appropriate tumor-specific antigens to make targeted therapy achievable with fewer normal cells being impaired. The similarity between placentation and tumor development and growth has inspired many investigators to discover antigens for effective immunotherapy of cancers. Placenta-specific 1 (PLAC1) is one of the recently discovered placental antigens with limited normal tissue expression and fundamental roles in placental function and development. There is a growing body of evidence showing that PLAC1 is frequently activated in a wide variety of cancer types and promotes cancer progression. Based on the restricted expression of PLAC1 in testis, placenta and a wide variety of cancers, we have designated this molecule with new terminology, cancer–testis–placenta (CTP) antigen, a feature that PLAC1 shares with many other cancer testis antigens. Recent reports from our lab provide compelling evidence on the preferential expression of PLAC1 in prostate cancer and its potential utility in prostate cancer immunotherapy. PLAC1 may be regarded as a potential CTP antigen for targeted cancer immunotherapy based on the available data on its promoting function in cancer development and also its expression in cancers of different histological origin. In this review, we will summarize current data on PLAC1 with emphasis on its association with cancer development and immunotherapy.

Our recent findings strongly support the idea of PLAC1 being as a potential immunotherapeutic target in prostate cancer (PCa). Here, we have generated and evaluated an anti-placenta-specific1 (PLAC1)-based antibody drug conjugate (ADC) for targeted immunotherapy of PCa. Prostate cancer cells express considerable levels of PLAC1. The Anti-PLAC1 clone, 2H12C12, showed high reactivity with recombinant PLAC1 and selectivity recognized PLAC1 in prostate cancer cells but not in LS180 cells, the negative control. PLAC1 binding induced rapid internalization of the antibody within a few minutes which reached to about 50% after 15 min and almost completed within an hour. After SN38 conjugation to antibody, a drug-antibody ratio (DAR) of about 5.5 was achieved without apparent negative effect on antibody affinity to cell surface antigen. The ADC retained intrinsic antibody activity and showed enhanced and selective cytotoxicity with an IC50 of 62 nM which was about 15-fold lower compared to free drug. Anti-PLAC1-ADC induced apoptosis in human primary prostate cancer cells and prostate cell lines. No apparent cytotoxic effect was observed in in vivo animal safety experiments. Our newly developed anti-PLAC1-based ADCs might pave the way for a reliable, efficient, and novel immunotherapeutic modality for patients with PCa.

The coproduction of acrylate and hydrogen from 1,3-propandiol is achieved by electrochemical reforming using a nanoparticle Pd on mixed Vulcan XC-72 carbon and ceria electrocatalyst (Pd/C-CeO2). Electrolysis cell parameters (potential, temperature, and flow conditions) are tuned to favor the formation of acrylate with respect to the other oxidation products (3-hydroxypropanoate and malonate). Using high cell temperature combined with a low cell voltage favors the formation of acrylate, the selectivity for which is further enhanced by flowing the 1,3-propandiol solution in single-pass mode rather than recycling through the electrolyzer. Hence, at a cell temperature of 80 °C and at a fixed cell potential of 400 mV using a one pass continuous flow of 1,3-propandiol, 77% of selectivity for acrylate is obtained.

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.

‎Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem ‎cells (HSCs) and the small-‎vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a ‎marker for diagnosis ‎and classification of leukemia. Anti CD34 antibodies are used for isolation and ‎purification ‎of HSCs from bone marrow, peripheral blood and cord blood.To characterize a newly produced monoclonal antibody against a human CD34 peptide.Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line.‎ELISA experiment revealed that the antibody recognized CD34 peptide. Western ‎blot analysis on TF1 ‎cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.‎‎Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. Materials and Methods: In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Results: Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Conclusion: Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.

We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

e22198 Background: The receptor tyrosine kinase ROR1 has important functions during normal embryogenesis. It is overexpressed and of importance for the survival of different malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. There is however little information on the expression and function of ROR1 in melanoma. Here, we investigated ROR1 expression, phosphorylation and the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on seven melanoma cell lines. Methods: ROR1 expression and phosphorylation was analysed in a series of malignant melanoma cell lines using RT-PCR, immunocytochemistry, flow cytometry and western blot. Annexin V/PI staining, MTT assay, PARP and caspase 8 and 9 cleavage as well as MCL-1 protein were used for detection of apoptosis induced by anti-ROR1 mAbs. The cytotoxic effects of anti-ROR1 mAbs were evaluated in the absence or presence of complement (CDC) or immune effector cells (ADCC). Transfection of melanoma cell lines using ROR1 siRNA was used for gene silencing. Results: ROR1 was shown to be overexpressed to a varying degree at protein level. ROR1 was phosphorylated at tyrosine and serine residues. Three out of four anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in the presence of complement or immune effector cells. ESTDAB081 and 094 cell lines were resistant to direct apoptosis of the four anti-ROR1 mAbs alone compared to the other melanoma cell lines, but not to complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC). ROR1 siRNA transfection induced down-regulation of the ROR1 expression both at the mRNA and protein levels proceeded by a significant apoptosis of the melanoma cells. Conclusions: The results indicate that ROR1 may play an important role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable targeted for mAb therapy.

Background Opticin (OPTC) is a member of the small leucine-rich proteoglycan (SLRP) family and is localized particularly in certain extracellular matrices. We have previously reported the unique expression of another SLRP, fibromodulin (FMOD) in the leukemic cells of patients with chronic lymphocytic leukemia (CLL). OPTC is located in the same region as FMOD on chromosome 1 (1q32.1). Cluster up-regulation of genes may be observed in malignancies and the aim of the present study was to analyze the expression of OPTC in CLL cells. Methods The expression of OPTC was tested by RT-PCR and realtime qPCR in PBMC from CLL patients, other hematological malignancies and healthy controls. The presence of OPTC protein, and its subcellular localization, was investigated using fractionation methods where the obtained lysate fractions were analyzed by Western blotting. Deglycosylation experiments were performed to investigate the glycosylation status of the CLL OPTC. Results OPTC was expressed at the gene level in all patients with CLL (n = 90) and in 2/8 patients with mantle cell lymphoma (MCL) but not in blood mononuclear cells of healthy control donors (n = 20) or in tumor samples from nine other types of hematological malignancies. OPTC was detected by Western blot in all CLL samples analyzed (n = 30) but not in normal leukocytes (n = 10). Further analysis revealed a CLL-unique unglycosylated 37 kDa core protein that was found to be located preferentially in the cell nucleus and endoplasmic reticulum (ER) of the CLL cells. Conclusions A 37 kDa unglycosylated OPTC protein was detected in ER and in the nucleus of CLL cells and not in healthy control donors. The function of this OPTC core protein remains unclear but its CLL-specific expression and subcellular localization warrants further investigations in the pathobiology of CLL.