Launching the dialogue: Safety and innovation as partners for success in advanced manufacturing.

Emerging and novel technologies, materials, and information integrated into increasingly automated and networked manufacturing processes or into traditional manufacturing settings are enhancing the efficiency and productivity of manufacturing. Globally, there is a move toward a new era in manufacturing that is characterized by: (1) the ability to create and deliver more complex designs of products; (2) the creation and use of materials with new properties that meet a design need; (3) the employment of new technologies, such as additive and digital techniques that improve on conventional manufacturing processes; and (4) a compression of the time from initial design concept to the creation of a final product. Globally, this movement has many names, but "advanced manufacturing" has become the shorthand for this complex integration of material and technology elements that enable new ways to manufacture existing products, as well as new products emerging from new technologies and new design methods. As the breadth of activities associated with advanced manufacturing suggests, there is no single advanced manufacturing industry. Instead, aspects of advanced manufacturing can be identified across a diverse set of business sectors that use manufacturing technologies, ranging from the semiconductors and electronics to the automotive and pharmaceutical industries. The breadth and diversity of advanced manufacturing may change the occupational and environmental risk profile, challenge the basic elements of comprehensive health and safety (material, process, worker, environment, product, and general public health and safety), and provide an opportunity for development and dissemination of occupational and environmental health and safety (OEHS) guidance and best practices. It is unknown how much the risk profile of different elements of OEHS will change, thus requiring an evolution of health and safety practices. These changes may be accomplished most effectively through multi-disciplinary, multi-sector, public-private dialogue that identifies issues and offers solutions.

Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.

BACKGROUND: KRAS mutations in NSCLC are associated with a lack of response to epidermal growth factor receptor inhibitors. Selumetinib (AZD6244; ARRY-142886) is an oral selective MEK kinase inhibitor of the Ras/Raf/MEK/ERK pathway. PATIENTS AND METHODS: Advanced nonsmall-cell lung cancer (NSCLC) patients failing one to two prior regimens underwent KRAS profiling. KRAS wild-type patients were randomized to erlotinib (150 mg daily) or a combination of selumetinib (150 mg daily) with erlotinib (100 mg daily). KRAS mutant patients were randomized to selumetinib (75 mg b.i.d.) or the combination. The primary end points were progression-free survival (PFS) for the KRAS wild-type cohort and objective response rate (ORR) for the KRAS mutant cohort. Biomarker studies of ERK phosphorylation and immune subsets were carried out. RESULTS: From March 2010 to May 2013, 89 patients were screened; 41 KRAS mutant and 38 KRAS wild-type patients were enrolled. Median PFS in the KRAS wild-type arm was 2.4 months [95% confidence interval (CI) 1.3-3.7] for erlotinib alone and 2.1 months (95% CI 1.8-5.1) for the combination. The ORR in the KRAS mutant group was 0% (95% CI 0.0% to 33.6%) for selumetinib alone and 10% (95% CI 2.1% to 26.3%) for the combination. Combination therapy resulted in increased toxicities, requiring dose reductions (56%) and discontinuation (8%). Programmed cell death-1 expression on regulatory T cells (Tregs), Tim-3 on CD8+ T cells and Th17 levels were associated with PFS and overall survival in patients receiving selumetinib. CONCLUSIONS: This study failed to show improvement in ORR or PFS with combination therapy of selumetinib and erlotinib over monotherapy in KRAS mutant and KRAS wild-type advanced NSCLC. The association of immune subsets and immune checkpoint receptor expression with selumetinib may warrant further studies.

A phase I/II study of sepantronium bromide (YM155, survivin suppressor) with paclitaxel and carboplatin in patients with advanced non-small-cell lung cancer.

BACKGROUND: This phase I/II study examined the safety and efficacy of Sepantronium Bromide (S), a small-molecule selective survivin suppressant, administered in combination with carboplatin (C) and paclitaxel (P). PATIENTS AND METHODS: Forty-one patients were treated on study. Twenty-two patients received escalating doses of S (3.6-12 mg/m(2)) and 19 with untreated stage IV non-small-cell lung cancer (NSCLC) were treated with the maximum tolerated dose of 10 mg/m(2) in combination with standard doses of C (AUC6) and P (200 mg/m(2)) for six cycles. S was administered as a continuous intravenous infusion (CIVI) over 72 h in 21-day treatment cycles. Study end points included safety and toxic effect, response rate, progression-free and overall survival (PFS and OS), as well as exploratory pharmacodynamic correlates. RESULTS: Treatment with S was well tolerated, and toxic effects were mostly hematological in the phase II study. Two (11%) partial responses were observed with a median PFS of 5.7 months and median OS 16.1 months. Pharmacodynamic analysis did not demonstrate an association with response. CONCLUSION: The combination of S (10 mg/m(2)/day 72-h CIVI) administered with C and P every 3 weeks exhibited a favorable safety profile but failed to demonstrate an improvement in response rate in advanced NSCLC. CLINICAL TRIAL NUMBER: NCT01100931.

Sprouty 2 binds ESCRT-II factor Eap20 and facilitates HIV-1 gag release.

The four ESCRT (endocytic sorting complexes required for transport) complexes (ESCRT-0, -I, -II, and -III) normally operate sequentially in the trafficking of cellular cargo. HIV-1 Gag trafficking and release as virus-like particles (VLPs) require the participation of ESCRTs; however, its use of ESCRTs is selective and nonsequential. Specifically, Gag trafficking to release sites on the plasma membrane does not require ESCRT-0 or -II. It is known that a bypass of ESCRT-0 is achieved by the direct linkage of the ESCRT-I component, Tsg101, to the primary L domain motif (PTAP) in Gag and that bypass of ESCRT-II is achieved by the linkage of Gag to ESCRT-III through the adaptor protein Alix. However, the mechanism by which Gag suppresses the interaction of bound ESCRT-I with ESCRT-II is unknown. Here we show (i) that VLP release requires the steady-state level of Sprouty 2 (Spry2) in COS-1 cells, (ii) that Spry2 binds the ESCRT-II component Eap20, (iii) that binding Eap20 permits Spry2 to disrupt ESCRT-I interaction with ESCRT-II, and (iv) that coexpression of Gag with a Spry2 fragment that binds Eap20 increases VLP release. Spry2 also facilitated release of P7L-Gag (i.e., release in the absence of Tsg101 binding). In this case, rescue required the secondary L domain (YPX(n)L) in HIV-1 Gag that binds Alix and the region in Spry2 that binds Eap20. The results identify Spry2 as a novel cellular factor that facilitates release driven by the primary and secondary HIV-1 Gag L domains.

Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release.

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.

HIV-1 capsid protein forms spherical (immature-like) and tubular (mature-like) particles in vitro: structure switching by pH-induced conformational changes.

The viral genome and replicative enzymes of the human immunodeficiency virus are encased in a shell consisting of assembled mature capsid protein (CA). The core shell is a stable, effective protective barrier, but is also poised for dissolution on cue to allow transmission of the viral genome into its new host. In this study, static light scattering (SLS) and dynamic light scattering (DLS) were used to examine the entire range of the CA protein response to an environmental cue (pH). The CA protein assembled tubular structures as previously reported but also was capable of assembling spheres, depending on the pH of the protein solution. The switch from formation of one to the other occurred within a very narrow physiological pH range (i.e., pH 7.0 to pH 6.8). Below this range, only dimers were detected. Above this range, the previously described tubular structures were detected. The ability of the CA protein to form a spherical structure that is detectable by DLS but not by electron microscopy indicates that some assemblages are inherently sensitive to perturbation. The dimers in equilibrium with these assemblages exhibited distinct conformations: Dimers in equilibrium with the spherical form exhibited a compact conformation. Dimers in equilibrium with the rod-like form had an extended conformation. Thus, the CA protein possesses the inherent ability to form metastable structures, the morphology of which is regulated by an environmentally-sensitive molecular switch. Such metastable structures may exist as transient intermediates during the assembly and/or disassembly of the virus core.

Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag).

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55(Gag) structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55(Gag) and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55(Gag). The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.

Activation of prostate-specific antigen precursor (pro-PSA) by prostin, a novel human prostatic serine protease identified by degenerate PCR.

A novel serine protease was found in human prostate by degenerate oligonucleotide PCR amplification and cloned. The zymogen form of this enzyme, named prostinogen, is composed of 240 amino acid residues with an amino-terminal propiece of 5 residues and a 235-residue mature enzyme. The transcript has a signal peptide of 15 amino acid residues. The mature enzyme has 41% sequence identity with prostate specific antigen (PSA). Prostinogen was expressed in Escherichia coli and refolded from inclusion bodies. The zymogen, with a molecular mass of 28 kDa, was readily activated by agarose-immobilized trypsin to generate prostin, a serine protease, which cleaves the chromogenic substrate (N-benzoyl-L-Ile-L-Glu-L-Gly-L-Arg-p-nitroaniline hydrochloride) (S-2222). Recombinant prostin readily activates the precursor of PSA (pro-PSA) by cleavage of the amino terminal Arg(7)-Ile(8) peptide bond. These results indicate that prostin may be a physiological activator of pro-PSA following its own proteolytic activation, as part of a cascade system involving a series of serine protease precursor proteins in the prostate.

Both extraneuronal monoamine transporter and O(6)-methylguanine-DNA methyltransferase expression influence the antitumor efficacy of 2-chloroethyl-3-sarcosinamide- 1-nitrosourea in human tumor xenografts.

We previously have found that 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) is a selective cytotoxin that enters cells via the extraneuronal transporter for monoamine transmitters (EMT). Both in vitro and in vivo studies demonstrated that SarCNU was more effective than BCNU against human gliomas. To clarify whether EMT expression correlates with antitumor efficacy of SarCNU, we determined human EMT (EMTh) and O(6)-methylguanine-DNA methyltransferase (MGMT) expression in nine human xenograft models using semiquantitative reverse-transcription polymerase chain reaction. These results were compared with the antitumor effects of SarCNU and the standard chloroethylnitrosourea antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). There was no significant correlation between EMTh expression and antitumor efficacy of SarCNU or BCNU. Also, there was no significant correlation between MGMT expression and SarCNU efficacy. However, a significant correlation was found between MGMT expression and BCNU antitumor efficacy. Interestingly, multiple regression analysis demonstrated a significant correlation between SarCNU efficacy and EMTh plus MGMT expression, whereas there was no correlation between BCNU efficacy and MGMT plus EMTh expression. Thus, the absence of a linear correlation between SarCNU efficacy and EMTh expression appears to be due, at least in part, to the presence of DNA repair, specifically, MGMT, in these xenograft models. These studies suggest that MGMT expression alone correlates with BCNU activity, whereas both EMTh and MGMT expression are important determinants of SarCNU activity against human tumor xenograft models. SarCNU is in clinical trials and these results may have important clinical implications.

Formation and Reactivity of the Osmium(IV)-Cyanoimido Complex [Os(IV) (bpy)(Cl)3 (NCN)](-).

Oxo-like reactivity exists for a new series of osmium complexes such as [Os(IV) (bpy)(Cl)3 (NCN)](-) (bpy=2,2'-bipyridine, see structure) containing the cyanoimido ligand. This ligand is formed directly at the metal center by the reaction of Os(VI) -nitrido precursors with tetraethylammonium cyanide. In the cyanoimido complexes there is an extensive electron-transfer chemistry at the metal center and an extensive functional-group chemistry based on the ligand.

[Expression of extraneuronal monoamine transporter gene and DNA repair gene vis-à-vis with antitumor efficacy of SarCNU in human tumor xenografts].

OBJECTIVE: To clarify if there are any correlations between extraneuronal monoamine transmitters (EMT), DNA repair gene expressions and SarCNU antitumor efficacy. METHODS: EMT, DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) and excision repair cross-complementing rodent repair deficiency gene (ERCC1-6) expressions in 9 human xenograft tumor models were determined by RT-PCR. The results were compared with the antitumor effects of SarCNU on these tumor xenografts. RESULTS: Multiple regression analysis revealed significant correlations of SarCNU antitumor activity with different combinations of gene expression. The most significant correlation was observed with all of the 4 genes expressed. CONCLUSION: The results suggest that expression of both EMT and DNA repair genes, specifically, MGMT, ERCC2 and ERCC4, are important determinants of SarCNU activity against human tumors. While DNA repair decreases SarCNU's activity by repairing damaged DNA, EMT appears to enhance its antitumor efficacy.

A newly characterized human endometrial adenocarcinoma cell line (CAC-1) differentiates in response to retinoic acid treatment.

A new cell line of poorly differentiated human endometrial adenocarcinoma cells termed "CAC-1" cells has been established. These cells are epithelial, as indicated by positive cytokeratin and negative vimentin staining. They are rounded and possess a high nuclear-to-cytoplasmic ratio, desmosomes, surface microvilli, intercelular lumens, and pleomorphic nuclei containing multiple nucleoli. These cells have been in long-term culture for 2 years. Our previous studies demonstrated that moderately differentiated (RL95-2) cells differentiated in response to retinoic acid treatment, illustrated by their reorganization of actin filaments and cell enlargement (Carter et al., 1996; Anticancer Res. 16, 17-24). CAC-1 cells exhibited a similar response because they also organized actin filaments and enlarged in response to retinoic acid treatment. Concurrently, retinoic acid treatment caused a 40% decrease in cell detachment in an in vitro detachment assay compared to controls. A slight lag in cell growth was observed when CAC-1 cells were treated with 1 microM 13-cis or all-trans retinoic acid during a 12-day growth curve. In addition, we examined the effects of retinoic acid on protein kinase C-alpha (PKC-alpha) and myristoylated alanine-rich C-kinase substrate (MARCKS). Treatment with retinoic acid caused cytoplasmic PKC-alpha to increase concomitant with a decrease in PKC-alpha in the membrane. In contrast, MARCKS increased in the membrane in response to retinoic acid treatment. These data indicate that retinoid treatment causes inactivation of PKC-alpha, allowing MARCKS to relocalize to the membrane, where it can cross-link actin filaments. CAC-1 cells represent an ideal model for investigating the effects of retinoids on differentiation induction concomitant with actin reorganization.

The efficacy of the social norms approach to substance abuse prevention applied to fraternity men.

Students tend to overestimate the amount of alcohol consumed among their peers and often drink to that imaginary level. The social norms strategy, designed to correct norm misperceptions, has been correlated with a decrease in reported consumption in the general college population. However, it has had little or no impact among Greek students, the group that consumes the most alcohol. The authors investigated and subsequently found three possible flaws in the application of the social norms strategy that may account for the failure to decrease binge drinking among fraternity men: there is no predominant, healthy drinking norm in this population; students are influenced more by people within their network(s) than by others; and binge drinking is the norm in this group and may serve to perpetuate the problem. The findings, though preliminary, provide the first step in developing interventions beyond the social norms approach to address binge drinking among fraternity men.

Ascorbic acid promotes recovery of cellular functions following toxicant-induced injury.

We have shown that renal proximal tubular cells (RPTC) recover cellular functions following sublethal injury induced by the oxidant t-butylhydroperoxide but not by the nephrotoxic cysteine conjugate dichlorovinyl-L-cysteine (DCVC). This study investigated whether L-ascorbic acid phosphate (AscP) promotes recovery of RPTC functions following DCVC-induced injury. DCVC exposure (200 microM; 100 min) resulted in 60% RPTC death and loss from the monolayer at 24 h independent of physiological (50 microM) or pharmacological (500 microM) AscP concentrations. Likewise, the DCVC-induced decrease in mitochondrial function (54%), active Na(+) transport (66%), and Na(+)-K(+)-ATPase activity (77%) was independent of the AscP concentration. Analysis of Na(+)-K(+)-ATPase protein expression and distribution in the plasma membrane using immunocytochemistry and confocal laser scanning microscopy revealed the loss of Na(+)-K(+)-ATPase protein from the basolateral membrane of RPTC treated with DCVC. DCVC-injured RPTC cultured in the presence of 50 microM AscP did not proliferate nor recover their physiological functions over time. In contrast, RPTC cultured in the presence of 500 microM AscP proliferated, recovered all examined physiological functions, and the basolateral membrane expression of Na(+)-K(+)-ATPase by day 4 following DCVC injury. These results demonstrate that pharmacological concentrations of AscP do not prevent toxicant-induced cell injury and death but promote complete recovery of mitochondrial function, active Na(+) transport, and proliferation following toxicant-induced injury.

Retinoic acid affects the EGF-R signaling pathway during differentiation induction of human endometrial adenocarcinoma cells.

We have shown that moderately differentiated endometrial adenocarcinoma (RL95-2) cells differentiate in response to retinoic acid treatment, illustrated by their reorganization of actin filaments and cell enlargement (Carter et al., Anticancer Res. 16, 17-24, 1996). Tyrphostin, an inhibitor of epidermal growth factor receptor (EGF-R)-associated protein tyrosine kinases, caused a dramatic reorganization of actin filaments in RL95-2 cells, similar to retinoic-acid-treated cells (Carter and Bellido, J. Cell. Physiol. 178, 320-332, 1999). We evaluated the possibility that the differentiating effects of retinoids are due to retinoic-acid-induced decreases in phosphorylation of EGF-R and changes in downstream effector proteins. Retinoic acid caused a decrease in tyrosine phosphorylation of EGF-R. Retinoic acid treatment induced a dramatic actin filament reorganization and cell enlargement. Treatment with EGF reversed this effect, because cells treated with retinoic acid followed by EGF only possessed disrupted actin aggregates and appeared small, thus resembling medium controls. Retinoic acid induced a relocalization and decrease in the amount of Shc protein, another actin-binding protein which is an adaptor protein for EGF-R signaling. In addition, retinoic acid induced a relocalization of gelsolin from the plasma membrane to the cytoplasm. Retinoic acid decreased cell detachment in detachment assays; one-half as many retinoic-acid-treated cells detached as in controls. These results are consistent with the idea that retinoic acid induces differentiation of RL95-2 cells by interfering with the EGF-R signaling pathway.

Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy.

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

Protein kinase C as a drug target: implications for drug or diet prevention and treatment of cancer.

Protein kinase C (PKC) isoforms are serine/threonine kinases involved in signal transduction pathways that govern a wide range of physiological processes including differentiation, proliferation, gene expression, brain function, membrane transport and the organization of cytoskeletal and extracellular matrix proteins. PKC isoforms are often overexpressed in disease states such as cancer. In this review, PKC in a variety of cancers is discussed along with some specific cell biological mechanisms by which PKC exerts its function(s). The PKC family consists of several isoforms comprising three groups: classical, novel and atypical. Although PKC has been investigated for around 2 decades, only recently has the specific function of each isoform started to be elucidated and the isoforms evaluated for use as targets of drug action. Phorbol esters such as the tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol (DAG) activate classical and novel PKC isoforms. Naturally occurring retinoids, antisense oligonucleotides against specific PKC isoforms and specific PKC inhibitors can block this activation. Beta carotene and retinoid derivatives act as anticarcinogenic agents and can antagonize some of the biological actions of phorbol esters and oxidants. Another important area of investigation is the use of antisense oligonucleotides to inhibit specific PKC isoforms. These compounds have proven effective in reducing specific types of cancer in rodents and humans and are currently used in clinical trials. This review examines PKC isoforms as a target of drug action with special emphasis on their use in cancer therapy.