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Market Research Group

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Lucas Watson
Lucas Watson

Bs 8002 Pdfl

The 8002 Nitro-Fuzer plastic welding system combines a simplified analog nitrogen welder with a traditional airless plastic welder. By combining these welders into one package, it allows you to make nitrogen welds economically to virtually any automotive plastic.

Bs 8002 Pdfl

Unlike some competitive products, the 8002 features precision flow control so the user can match the flow and temperature to substrate types and thicknesses. The flow and temperature can be reduced to a very low point in order to weld thin, fragile substrates, something others cannot do. When welding very thick substrates, the temperature and flow can be increased greatly to allow more heat to be transferred to the weld. The analog flow gauge gives the welder a repeatable reference over how much gas is used in the welding process. To extend heating element lifespan, a low-pressure safety switch is incorporated into the system, shutting off the power to the heating element if the air flow drops below a safe level.

In order to reduce the cost of the 8002, the welder and cart will be shipped in two separate boxes. The welder is fully assembled and ready to go, but the cart will have to be assembled. We have detailed instructions in the box, as well as a video, to help you assemble the cart. The Welding Cart Assembly instructions, Setup and Use video, and Quick-Start Guide can be found below. The 8002 is packaged with one welding rod organizer case filled with a selection of welding rods, a hand seamer, and aluminum tape. It is covered by a two-year "bumper-to-bumper" warranty covering all parts except the heating elements and misuse.

BS 8004:2015 and BS 8002:2015 have also been brought up-to-date with Eurocode 7. BS 8004 provides recommendations for foundations design, covers spread and pile foundations, helical steel pile foundations and underpinning. Whilst BS 8002 provides recommendations for gravity, semi-gravity, and embedded retaining walls.

DCP-8002 - Designing Scholarly Research Semester Credits: 3 Weeks: 12Prerequisites: You will enroll in this course after successfully completing DCP-8001 - Scholarly Writing and Synthesis of Literature You will explore research designs and analytical techniques you may plan to use with your envisioned dissertation research. You will review the skills and tools necessary for data collection, interpretation and application of quantitative, qualitative, and mixed methods research. You also will complete the required CITI ethics training. This course is designed to build upon prior doctoral-level research coursework.

It has been previously shown that the expression of MCT4 results in reduced antitumor efficacy of MCT1 inhibitors (14, 15). However, little is known about additional factors that may predict sensitivity or resistance as well as potential mechanisms of acquired resistance. In this study, we used BAY-8002 to investigate resistance mechanisms and to identify sensitive tumor subindications. We identified several genes whose expression correlates with response to MCT1 inhibition and show that absence of MCT4 expression is not sufficient as a single marker to predict response to treatment. In vivo, blockade of MCT1 resulted in modulation of both intratumor lactate and pyruvate levels. Consequently, the growth of tumors decreased, yet, no tumor regression was observed. Re-expression of MCT isoforms as well as increased energy generation via oxidative phosphorylation was identified as acquired resistance mechanism to MCT1 inhibition.

DLD-1, WSU-DLCL2, SH-SY5Y, Colo320DM, and 786-O cells were obtained from the American Type Culture Collection, and EVSA-T, Raji, Daudi, and SU-DHL-4 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). All cell lines were authenticated by fingerprint techniques at DSMZ after two to four passages of the original stock from the providers. The cells were maintained in RPMI-1640 (Raji, DLD-1, Daudi WSU-DLCL2, Colo320DM, SU-DHL-4), DMEM (EVSA-T, SH-SY5Y), or DMEM/Ham's F12 (786-O) medium containing 10% heat-inactivated FCS. BAY-8002 [2-([2-chloro-5-(phenylsulfonyl)phenyl]-carbonylamino)benzoic acid; Fig. 1A] was identified and synthesized at Bayer AG as described in the Supplementary Methods. BAY-876 was synthesized at Bayer AG as described previously (16). AZD3965 was published previously (14) and synthesized according to the procedure described in the literature (described in WO 2004/065394, Example 10. Intermediates are described in WO 2003/011868) at WuXi AppTec Co. [3H]BAY-8002 and [3H]AZD3965 were both prepared by catalytic exchange reaction with tritium gas as described in the Supplementary Methods. For in vitro studies, a 10 mmol/L stock solution of BAY-8002 or AZD3965 in dimethyl sulfoxide (DMSO) was prepared.

In order to identify inhibitors of MCT1-dependent lactate transport, we performed a cell-based high-throughput screen using the Bayer proprietary screening deck of approximately 3 million compounds. As a read-out for MCT1 activity, lactate import-dependent intracellular acidification was measured based on the fluorescent pH indicator SNARF-5. All resulting hits were counter-screened against activity in MCT4-expressing EVSA-T cells. Performing our hit-to-lead process led to the identification of BAY-8002 (Fig. 1A), which inhibited cellular SNARF-5 fluorescence change with an IC50 of 85 (6) nmol/L in MCT1-expressing DLD-1 cells and displayed excellent selectivity against MCT4 (IC50 > 50 μmol/L in EVSA-T cells, Fig. 1B and C). In order to rule out the possibility that the change in intracellular pH might be modulated by mechanisms other than lactate transport, we measured the cellular uptake of [14C]-L-lactate into the cells. The uptake of [14C]-L-lactate was efficiently blocked with an IC50 in the nanomolar range in MCT1-expressing cells, but not in MCT4-expressing cells (Fig. 1D). Because MCT1 facilitates bidirectional lactate transport, we next tested the effect of BAY-8002 on lactate extrusion. BAY-8002 inhibited the accumulation of lactate in the cell supernatant (Fig. 1E) and decreased the ECAR (Fig. 1F). In both assays, we observed only 60% efficacy in blocking the export of lactate. This effect was observed in different cell lines and with AZD3965 (Supplementary Fig. S1) and may be due to the free diffusion at lower pH of the undissociated lactic acid across the cell membrane (20). The inhibition of lactate transport resulted in efficient proliferation inhibition of MCT1-expressing Raji and Daudi Burkitt lymphoma cells (Fig. 1B and G). We did not observe any significant off-target activity in a panel of 68 channels, transporters, and other proteins (Supplementary Table S1).

For its use in in vivo efficacy and toxicity experiments, we tested the species cross-reactivity of BAY-8002 by measuring radiolabeled lactate uptake in murine and rat cells. The IC50s in these cell lines were in the range of the IC50s determined in the human cell lines, indicating that the compound equally inhibits the human and the rodent orthologs (Fig. 1H). MCT2 is structurally very similar to MCT1 and AZD3965, and its analogs have been reported to be dual inhibitors of MCT1 and MCT2 (21). Therefore, we analyzed the effect of BAY-8002 on this isoform by determining the inhibition of radiolabeled uptake in X. laevis oocytes expressing recombinant human MCT1, MCT2, or MCT4. BAY-8002 inhibited lactate uptake in MCT2-expressing oocytes, with approximately 5-fold lower potency compared with the MCT1 isoform (Fig. 1I). No inhibition in MCT4-expressing oocytes was observed, confirming the specificity against this isoform.

Because no crystal structure of MCT1 is available to date, the determination of the binding site of MCT1 inhibitors is challenging. We therefore set up a radioactive-binding assay in order to assess whether BAY-8002 and AZD3965 bind to the same or overlapping sites on MCT1. First, we prepared tritium-labeled BAY-8002 and AZD3965 and measured specific binding to cell membranes. Both [3H]-BAY-8002 and [3H]-AZD3965 bound to membranes of MCT1-expressing DLD-1 and Raji cells, but not to the membranes of MCT4-expressing EVSA-T cells, confirming the specificity of the inhibitors (Fig. 2A). The observed dissociation constants (KD) of the inhibitors were in the same low nanomolar range as the IC50s in the lactate transport assays ([3H]-AZD3965: KD = 5.5 nmol/L; [3H]-BAY-8002: KD = 7.9 nmol/L, Supplementary Fig. S2). [3H]-AZD and [3H]-BAY-8002 were fully displaced by both AZD3965 and BAY-8002 with Ki values in the 1-digit nmol/L range (Fig. 2B and C). No displacement was observed when using the Glut1-specific inhibitor BAY-876 (16) as control (Fig. 2B). Although allosteric inhibition cannot be entirely excluded, these results suggest overlapping binding sites for AZD and BAY-8002. Consistently, in cellular lactate transport assays, BAY-8002 and AZD3965 showed comparable low nanomolar activities. In contrast to AZD3965, BAY-8002 displayed very high protein binding. Therefore, in proliferation assays where the cells are cultivated with 10% FCS, BAY-8002 showed much higher IC50s than AZD3965 (Supplementary Table S2). When corrected for protein binding, the IC50s were again in the same range (Supplementary Table S2).

In order to identify markers of sensitivity to MCT1 inhibition as well as cancer types that would be particularly good responders to MCT1 inhibition, we profiled a set of 246 cancer cell lines for their antiproliferative response to BAY-8002. In addition, we explored the antiproliferative capacity of AZD3965. The sensitivity pattern observed for both compounds was highly similar, providing further evidence for the lack of off-target activities of both compounds, as observed in the selectivity panel described above. We found that 11% of the tested cell lines responded to MCT1 inhibitor treatment. The IC50s of the cell lines showed a bimodal distribution pattern, with two populations of either highly sensitive or resistant cells to MCT1 inhibitor treatment (Supplementary Fig. S3A). More than half (59%) of the sensitive cell lines belonged to various hematopoietic malignancies (Fig. 3A). The highest response rate was observed for diffuse large B-cell lymphoma (DLBCL) with 6 of 9 cell lines responding (Supplementary Fig. S3B). In order to test whether basal metabolic preferences of cancer cells determine sensitivity to MCT1 inhibition, we measured basal OCRs of a set of 13 cell lines. However, we observed no correlation with response to MCT1 inhibitor treatment (Supplementary Fig. S3C). 350c69d7ab


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