ALBERT

Description: Through Hole Technology (THT) is a method in electronic printed circuit board (PCB) mounting technology by which components are mounted on the PCB board and it is later sent for Integrated circuit testing (ICT). The in-process material movement is the real culprit for decreased production rates. Here we discuss the various identified reasons for the same and calculated effective possible maximum production rates and improvise the process with the help of an automated material transfer system and validate the same using production simulation software called Tecnomatix. Each of the system i.e. the manual and the automated system was studied, and appropriate corrective decision was taken to improvise the overall productivity.

Description: Introduction: A plasma cell malignancy called multiple myeloma (MM) is an incurable cancer in which a majority of patients with refractory disease become resistant to therapy. The introduction of carfilzomib, a proteasome inhibitor, significantly improved the clinical outcome of the MM treatment. However, MM patients develop resistance to carfilzomib and relapse. In this study, we investigated the role of hypoxia and P-gp in the carfilzomib-resistance in MM cells in vitro and in vivo, and its therapeutic translational potential using a HIF inhibitor, PX-478. Methods: In vitro, MM cell lines were treated with carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions and cell survival was analyzed by MTT assay. The activity of P-gp was assessed by testing the efflux of a known P-gp substrate, RhodamineB (RhoB), in the hypoxic and normoxic conditions by measuring the intracellular RhoB content using flow cytometry. Moreover, we have tested the effect of HIF inhibition using PX-478 on the P-gp activity as well as the response to carfilzomib in hypoxic and normoxic cells. In vivo, we tested the effect of HIF inhibition on tumor initiation, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice, which were treated instantaneously with PX-478 (10mg/kg) three times a week. Furthermore, we tested the effect of PX-478 on MM tumor response to carfilzomib (measuring tumor size and mice survival). MM1s-Luc-GFP cells were injected IV into SCID mice, tumors grew for 3 weeks and the mice were then randomly divided into 4 groups treated with (1) vehicle (Captisol), (2) PX-478 (10mg/kg) alone, (3) carfilzomib (5mg/kg) alone, or (4) a combination of PX-478 (10mg/kg) and carfilzomib (5mg/kg). PX-478 or vehicle were administered by oral gavage three times a week (day 1, day 3, day 5), while vehicle and carfilzomib were injected IV twice a week (day 4 and day 5). Tumor size was imaged using bioluminescence imaging (BLI) and mice survival was followed for 70 days. Results: In vitro, we found that hypoxia induced resistance to carfilzomib in five MM cell lines. Moreover, hypoxia also increased activity of P-gp by causing decreased intracellular RhoB content in hypoxic MM cells. The HIF inhibitor, PX-478, as well as the P-gp inhibitor, tariquidar, reversed the activation of P-gp in hypoxic cells, while the combination of PX-478 and tariquidar did not induce further inhibition of P-gp activity. Furthermore, the combination of PX-478 or tariquidar with carfilzomib reversed the hypoxia-induced resistance in MM. However, tariquidar and other P-gp inhibitors have shown low selectivity and high toxicity in clinical trials; therefore, for our in vivo experiments we chose to inhibit HIF in order to reverse the hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells. In vivo, in the MM tumor-initiation model, our study revealed that the HIF inhibitor, PX-478, significantly delayed the tumor progression and extended survival in which all control mice died between 42-52 days, while the experiment was stopped at 70 days, with all PX-478-treated mice still alive. In the established in vivo tumor model, low dose carfilzomib alone delayed the progression by BLI but did not improve survival (vehicle and carfilzomib-treated mice died between 21-28 days after treatment). Despite the fact that PX-478 did not decrease tumor progression as shown by BLI compared to the vehicle-treated mice, it significantly extended the survival of the mice (animals died between 38-48 days). The combination of carfilzomib and PX-478 significantly decreased the proliferation of tumor shown by BLI (less than 5% of the growth at day 28), as well as considerable increase in survival (the experiment was stopped at 70 days with 100% of the group alive). Conclusions: We identified a novel resistance mechanism to carfilzomib in MM, in which hypoxia induces P-gp-mediated resistance to carfilzomib. Inhibition of the hypoxic response in MM cells by the HIF inhibitor reduced hypoxia-induced P-gp-mediated resistance to carfilzomib in MM cells in vitro, and delayed tumor progression significantly improving survival and response to carfilzomib in MM-bearing mice in vivo. Disclosures Azab: Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Description: Introduction Multiple myeloma (MM) is a malignant neoplastic cancer of plasma cells that involves the bone marrow. Generally, patients will respond to treatment initially, but they later become resistant to therapy, and this is ultimately due to a change in the biology of the tumor. Multi-drug-resistance transporter proteins were shown to play a role in drug resistance in MM patients; P-glyco-protein (P-gp) is the most studied of the multi-drug resistance proteins, and it becomes up-regulated in response to many chemotheries. Hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. Tumor-hypoxia was shown todevelop many kinds of solid tumors and hematologic malignancies. Specifically, hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. In this study, we examinned the effect of hypoxia on the expression and activity of P-gp in MM and its contributing to drug resistance to therapies used in MM. Methods and Results We tested the effect of hypoxia on the activity of P-gp in MM lines. We incubated MM cells under hypoxic and normoxic conditions, and we tested their ability to pump out Rhodamine (Rh) by measuring Rh content in the cells by fluorescent reader. First, we optimized the concentration of Rh and the time of incubation with the cells. We found that at all concentrations tested (0.1, 0.5, 1, 5 and 10 ug/ml) and at all incubation time of cells with Rh with MM cells (0.25, 0.5, 1, 2, 4, 6, 8 and 24hrs) , hypoxia increased the efflux of Rh. The most significant efflux was achieved when incubating the cells for 1hr with Rh 1ug/ml. We found that hypoxia increased the efflux of Rh in all MM cell lines tested. Incubation of RPMI cells under hypoxic for 24hrs and 48hrs decreased the Rh content of the cells by about 40% and 65%, respectively. Carfilzomib was previously reported to be a substrate of P-gp, we tested the effect of carfilzomib on the efflux of Rd in the MM cells. Hypoxic and normoxic MM cells were treated for 5hrs with carfilzomib (5 nM) and then incubated for 1hrs with Rh (1ug/ml). We tested the Rh content of the cells by fluorescent reader and found that carfilzomib competed with Rh on the P-gp and decreased the efflux of Rh induced by hypoxic. We tested the effect of carfilzomib on induction of P-gp in hypoxic and normoxic MM cells by treating RPMI cells with a low dose of carfilzomib (0.25nM) for 48hrs under hypoxic or normoxic conditions, and tested the cells ability to efflux Rh. We found that carfilzomib increased P-gp expression and induced efflux of about 30% of the Rh in non-treated normoxic cells.  Hypoxia induced efflux of about 65% of normoxic cells, but no effect was observed with the treatment of carfilzomib. Furthermore, we tested the hypoxia-induced P-gp expression in MM on the sensitivity of MM cells to carfilzomib. We incubated MM cells for 24hrs in hypoxic and normoxic conditions, and cells were treated with carfilzomib (0 or 5nM) for additional 24hrs. We found that while carfilzomib induced the death of about 40% of the cells under normoxic condition, it had no significant effect on the survival of MM cell under hypoxic conditions. Conclusion Hypoxia induced a significant up-regulation of P-gp in MM cells, and increased MM drug resistance to carfilzomib. These results provide mechanistic evidence for drug resistance to carfilzomib in MM, and suggest hypoxia as a novel therapeutic to prevent upregulation of P-gp and drug resistance. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is still incurable. The PI3K pathway is activated and correlated with drug resistance in MM. Pan-inhibition of the PI3K pathway resulted in serious side effects, therefore, we focused on specific inhibition of the PI3K-alpha isoform. We investigated the role of PI3K- alpha in the progression and drug resistance of MM, by inhibiting the PI3K- alpha with a novel specific inhibitor BYL719. This is the first study to describe the preclinical effect of BYL719 on MM in particular and in hematologic malignancies in general. Methods and Results We analyzed the gene expression of PI3K isoforms in MM patients based on published datasets from the Gene Expression Omnibus by Zhan et al and found that PI3K-alpha and beta isoforms were highly expressed in MM. However, the fold-change of expression of the alpha isoform in MM patients was higher than the beta isoform, compared to healthy subjects. BYL719 inhibited the survival of MM cells isolated from three MM patients by MTT, at an IC50 around 1uM, but had no effect on normal PBMCs. Furthermore, BYL719 inhibited survival of all MM cell lines in a different manner. In silico-predicted activity of PI3K-alpha in the cell lines was exponentially correlated with the killing induced by the PI3K inhibitor BYL719 in vitro. BYL719 significantly decreased the activation of the PI3K signaling related proteins (pAKT, pS6R, and pGSK) by western blotting. Moreover, BYL719 inhibited cell cycle of MM cells detected by PI staining showed induction of G1-phase cell cycle arrest. BYL719 inhibited MM proliferation of MM1s cells in a dose dependent manner, through decreasing the levels of pCyclin-E1 and pRb, and increasing of P27 levels. The results were in agreement with the in silico studies that predicted the inhibition of other cell-cycle proteins including CDK4-Cyclin D complex, Myc-Max complex and CDK1-Cyclin B complex by BYL719 in a dose dependent manner. Moreover, BYL719 (0-2.5uM) increased the fraction of apoptotic MM cells in a dose depended manner, as detected by Annexin/PI staining. BYL719 induced apoptosis signaling by inducing the cleavage of Caspase-3, Caspase-9 and PARP by western blotting, in a dose dependent manner. We tested the effect of the combination of BYL719 with other drugs (Bortezomib and Carfilzomib) on survival of MM cells by MTT. We found that the combination of the two drugs decreased the surviving fraction of MM cells more than each of the drugs alone. Mechanistically, Bortezomib increased pAKT and pS6R as a resistance mechanism, and BYL719 abolished the Bortezomib-induced increase of pAKT and pS6R. Moreover, BYL719 enhanced the activation of pJNK induced by Carfilzomib and the combination of each drug increased cleavage of PARP, caspase-3 and caspase-9 more than each of the drugs alone. We tested the effect of BYL719 on the interaction of MM cells with BM stromal cells (BMSCs), and it was found that BYL719 decreased the adhesion of MM cells to BMSCs in a dose-dependent manner. Mechanistically, BYL719 decreased the activation of adhesion signaling such as pFAK, pSRC and pCofilin in a dose dependent manner. The in silico studies predicted the inhibition of the small GTPases Rho, Rac and Cdc42. To test the effect of BYL719 on drug resistance induced by the BM stroma, MM cells were co-Cultured with BMSCs, treated with BYL719 in combination with Bortezomib or Carfilzomib. It was found that co-culture with BMSCs increased the surviving fraction of MM cells after treatment with Bortezomib and Carfilzomib, as a drug resistance mechanism. The combination of the two drugs with BYL719 overcame the resistance induced by the stroma and reduced the surviving fraction to the values observed for treatment without presence of stroma. Conclusion This is the first study to describe preclinical effect of BYL719 on MM in particular and hematologic malignancies in general. The PI3K-alpha isoform plays a major role in the progression and drug resistance in MM cells, and it's inhibition with BYL719 reduces proliferation, inhibits cell cycle and induces apoptosis in MM cells. Moreover, it showed that BYL719 synergizes with Bortezomib and Carfilzomib, and overcomes drug resistance induced by BM stroma. Our findings provide a preclinical basis of future clinical trial of BYL719 in MM as a single agent or in combination with other drugs. Disclosures: Vali: Cellworks Group Inc., San Jose, CA, USA: Employment. Abbasi:Cellworks Group Inc: Employment.

Description: Autism Spectrum Disorder (ASD) is a set of heterogeneous neurodevelopmental conditions defined by impairments in social communication and restricted, repetitive behaviors, interests or activities. Only a minority of ASD cases are determined to have a definitive etiology and the pathogenesis of most ASD is poorly understood. We hypothesized that a global analysis of the proteomes of human ASD vs. control brain, heretofore not done, would provide important data with which to better understand the underlying neurobiology of autism. In this study, we characterized the proteomes of two brain regions, Brodmann area 19 (BA19) and posterior inferior cerebellum (CB), from carefully selected idiopathic ASD cases and matched controls using label-free HPLC-tandem mass spectrometry. The data revealed marked differences between ASD and control brain proteomes for both brain regions. Unlike earlier transcriptomic analyses using frontal and temporal cortex, however, our proteomic analysis did not support ASD attenuating regional gene expression differences. Bioinformatic analyses of the differentially expressed proteins between cases and controls highlighted canonical pathways involving glutamate receptor signaling and glutathione-mediated detoxification in both BA19 and CB; other pathways such as Sertoli cell signaling and fatty acid oxidation were specifically enriched in BA19 or CB, respectively. Network analysis of both regions of ASD brain showed up-regulation of multiple pre- and post-synaptic membrane or scaffolding proteins including glutamatergic ion channels and related proteins, up-regulation of proteins involved in intracellular calcium signaling, and down-regulation of neurofilament proteins, with DLG4 and MAPT as major hub proteins in BA19 and CB protein interaction networks, respectively. Upstream regulator analysis suggests neurodegeneration-associated proteins drive the differential protein expression for ASD in both BA19 and CB. Overall, the proteomic data provide support for shared dysregulated pathways and upstream regulators for two brain regions in human ASD brain, suggesting a common ASD pathophysiology that has distinctive regional expression.