Neoadjuvant weekly paclitaxel and carboplatin with trastuzumab and pertuzumab in HER2-positive breast cancer: a Brown University Oncology Research Group (BrUOG) study.

PURPOSE: In HER2-positive breast cancer (HER2+ BC), neoadjuvant chemotherapy (NACT) with dual HER2-targeted therapy achieves high pathologic complete response (pCR) rates. Anthracycline-free NACT regimens avoid toxicities associated with anthracyclines, but every 3-week TCHP also has substantial side effects. We hypothesized that a weekly regimen might have equivalent efficacy with less toxicity; we also investigated whether poorly responding patients would benefit from switching to AC. METHODS: Patients with clinical stage II-III HER2+ BC received weekly paclitaxel 80 mg/m2 and carboplatin AUC2 with every 3-week trastuzumab and pertuzumab (wPCbTP), with the option of splitting the pertuzumab loading dose. After 12 weeks, responding patients continued wPCbTP for another 6 weeks, while non-responders switched to AC. Dose modifications and post-op therapy were at investigator discretion. RESULTS: In 30 evaluable patients, the pCR rate was 77% (95% CI 58-90%); 12/14 (86%) in ER-negative and 11/16 (69%) in ER-positive. Only two patients transitioned to AC for non-response, of which one achieved pCR. There were no episodes of febrile neutropenia or grade ≥ 3 peripheral neuropathy, though several patients who continued wPCbTP stopped before week 18. Split-dose pertuzumab was associated with less grade ≥ 2 diarrhea (40%) than the standard loading dose (60%). CONCLUSION: pCR rates with our regimen were as high as reported with TCHP with fewer grade ≥ 3 toxicities, though diarrhea remains a concern. Too few patients had a suboptimal response to adequately test switching to AC. The wPCbTP regimen should be considered an alternative to TCHP as neoadjuvant therapy for HER2+ BC. TRAIL REGISTRATION: ClinicalTrials.gov identifier: NCT02789657.

[Expression of Fermintin family homologous protein 2 in non-small cell lung cancer and its clinical significance].

Objective: To investigate the expression of Fermintin family homologous protein 2 (FERMT2) in non-small cell lung cancer and its clinical significance. Methods: Seventy-two patients with non-small cell lung cancer were collected at Xinxiang Central Hospital, Henan Province, from January 2015 to January 2017.There were 48 male and 24 female patients, the age ranged from 37 to 78 years (mean 58 years). The expression of FERMT2 in tumor samples and para-cancerous tissues were detected by immunohistochemistry. Protein and mRNA expression of FERMT2 were detected by Western blot and real-time fluorescence quantitative PCR, respectively. Western blot method was also used to detect integrin-related protein expression, including integrin beta 1 (CD29), vascular cell adhesion molecule (VCAM1), and mobile related protein-1 (MRP1). Results: Immunohistochemistry showed that the positive rates of FERMT2 expression were 81.9%(59/72)in carcinoma tissue and 15.4%(11/72) in para-cancerous tissues, and the difference was statistically significant (P<0.01). Positive FERMT2 expression was different in tumors at different tumor stages: 11/17 at stage Ⅰ, 16/20(80.0%)at stage Ⅱ, 17/20(85.0%)at stage Ⅲ, and 15/15 at stage Ⅳ, and there was a significant difference between each stage (P<0.01). By real-time PCR and Western blot, the expression of FERMT2 in non-small cell lung cancer tissues was significantly higher than that of para-cancerous tissue (P<0.01). The expression levels of integrin related proteins (integrin ß1, VCAM1 and MRP1) in tumor tissues were significantly higher than those in para-cancerous tissues, and positively correlated with the expression of FERMT2 (r=0.531, P<0.01; r=0.483, P<0.01; r=0.612, P<0.01). Conclusions: FERMT2 is highly expressed in non-small cell lung carcinomas. Its expression is closely correlated with the tumor clinical stage. It is hypothesized that FERMT2 may promote tumor metastasis through interactions with integrin-like protein.

Ductile bulk metallic glass by controlling structural heterogeneities.

A prerequisite to utilize the full potential of structural heterogeneities for improving the room-temperature plastic deformation of bulk metallic glasses (BMGs) is to understand their interaction with the mechanism of shear band formation and propagation. This task requires the ability to artificially create heterogeneous microstructures with controlled morphology and orientation. Here, we analyze the effect of the designed heterogeneities generated by imprinting on the tensile mechanical behavior of the Zr52.5Ti5Cu18Ni14.5Al10 BMG by using experimental and computational methods. The imprinted material is elastically heterogeneous and displays anisotropic mechanical properties: strength and ductility increase with increasing the loading angle between imprints and tensile direction. This behavior occurs through shear band branching and their progressive rotation. Molecular dynamics and finite element simulations indicate that shear band branching and rotation originates at the interface between the heterogeneities, where the characteristic atomistic mechanism responsible for shear banding in a homogeneous glass is perturbed.

Mitochondrial inhibitor sensitizes non-small-cell lung carcinoma cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-X(L)/p53-mediated amplification mechanisms.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-X(L) overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-X(L) downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-X(L) and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-X(L) and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.

Proteomic analysis of short- and intermediate-term memory in Hermissenda.

Changes in cellular and synaptic plasticity related to learning and memory are accompanied by both upregulation and downregulation of the expression levels of proteins. Both de novo protein synthesis and post-translational modification of existing proteins have been proposed to support the induction and maintenance of memory underlying learning. However, little is known regarding the identity of proteins regulated by learning that are associated with the early stages supporting the formation of memory over time. In this study we have examined changes in protein abundance at two different times following one-trial in vitro conditioning of Hermissenda using two-dimensional difference gel electrophoresis (2D-DIGE), quantification of differences in protein abundance between conditioned and unpaired controls, and protein identification with tandem mass spectrometry. Significant regulation of protein abundance following one-trial in vitro conditioning was detected 30 min and 3 h post-conditioning. Proteins were identified that exhibited statistically significant increased or decreased abundance at both 30 min and 3 h post-conditioning. Proteins were also identified that exhibited a significant increase in abundance only at 30 min, or only at 3 h post-conditioning. A few proteins were identified that expressed a significant decrease in abundance detected at both 30 min and 3 h post-conditioning, or a significant decrease in abundance only at 3 h post-conditioning. The proteomic analysis indicates that proteins involved in diverse cellular functions such as translational regulation, cell signaling, cytoskeletal regulation, metabolic activity, and protein degradation contribute to the formation of memory produced by one-trial in vitro conditioning. These findings support the view that changes in protein abundance over time following one-trial in vitro conditioning involve dynamic and complex interactions of the proteome.

Proteomic analysis of post-translational modifications in conditioned Hermissenda.

Post-translational modifications of proteins are a major determinant of biological function. Phosphorylation of proteins involved in signal transduction contributes to the induction and maintenance of several examples of cellular and synaptic plasticity. In this study we have identified phosphoproteins regulated by Pavlovian conditioning in lysates of Hermissenda nervous systems using two-dimensional electrophoresis (2DE) in conjunction with (32)P labeling, fluorescence based phosphoprotein in-gel staining, and mass spectrometry. Modification of protein phosphorylation regulated by conditioning was first assessed by densitometric analysis of (32)P labeled proteins resolved by 2DE from lysates of conditioned and pseudorandom control nervous systems. An independent assessment of phosphorylation regulated by conditioning was obtained from an examination of 2D gels stained with Pro-Q Diamond phosphoprotein dye. Mass spectrometric analysis of protein digests from phosphoprotein stained analytical gels or Coomassie Blue stained preparative gels provided for the identification of phosphoproteins that exhibited statistically significant increased phosphorylation in conditioned groups as compared to pseudorandom controls. A previously identified cytoskeletal related protein, Csp24 (24 kDa conditioned stimulus pathway phosphoprotein), involved in intermediate-term memory exhibited significantly increased phosphorylation detected 24 h post-conditioning. Our results show that proteins involved in diverse cellular functions such as transcriptional regulation, cell signaling, cytoskeletal regulation, metabolic activity, and protein degradation contribute to long-term post-translational modifications associated with Pavlovian conditioning.

NFKB1-94ins/del polymorphism is not associated with lung injury after cardiopulmonary bypass.

Nuclear factor (NF)-kappaB (NFKB1)-94ins/del is an important polymorphism that affects promoter activity of the NFKB1 gene and is potentially associated with several inflammatory diseases. We investigated the association of this polymorphism with lung injury after cardiac surgery and cardiopulmonary bypass in a prospective cohort study of 283 patients. Genotyping was performed by high resolution melting analysis; analysis indicated no association of NFKB1 with postoperative lung injury (p = 0.064). Relative risks of the del allele and the del/del genotype were 1.34 (95% CI 1.02-1.75) and 1.74 (95% CI 1.00-3.05) respectively. Logistic regression analysis (with factors including age, peripheral vascular disease and surgical duration as risk factors of lung injury after cardiac surgery with cardiopulmonary bypass) also failed to confirm that the NFKB1 genotype is influential for lung injury (p = 0.113). We conclude that, contrary to some other evidence, the NFKB1-94ins/del polymorphism is not associated with lung injury after cardiac surgery with cardiopulmonary bypass.

One-trial in vitro conditioning regulates an association between the beta-thymosin repeat protein Csp24 and actin.

One-trial conditioning in Hermissenda results in enhanced intrinsic cellular excitability of sensory neurons in the conditioned stimulus pathway, and the phosphorylation of several proteins. Previous results demonstrated that the development of enhanced intrinsic excitability was dependent on the expression of conditioned stimulus pathway phosphoprotein-24 (Csp24), an intracellular protein containing four repeated beta-thymosin homology domains. Consistent with this, antisense oligonucleotide-mediated inhibition of Csp24 expression prevents the reduction in amplitude of the A-type transient K+ current (I(A)) and the depolarized shift in the steady-state activation curve normally produced by one-trial in vitro conditioning of isolated photoreceptors. One-trial conditioning also regulates Csp24 phosphorylation. We now show that purified recombinant Csp24 sequesters G-actin in vitro with an approximate K(d) value of 2.8 microM. We also observed a significant increase in the coprecipitation of actin with Csp24 after one-trial in vitro conditioning using antibodies directed toward either Csp24 or phospho-Csp24. Preincubation with protein kinase C (PKC) selective inhibitors attenuated the increase in Csp24 phosphorylation and coprecipitated actin observed after one-trial conditioning. Our findings indicate that the PKC signaling pathway contributes to the phosphorylation of Csp24 after one-trial conditioning, and that PKC activity modulates an association between Csp24 and actin. These data suggest Csp24 may influence intrinsic excitability by regulating cytoskeletal dynamics.

One-trial in vitro conditioning regulates a cytoskeletal-related protein (CSP24) in the conditioned stimulus pathway of Hermissenda.

Hermissenda CSP24 (cytoskeletal-related protein 24) is a 24 kDa beta-thymosin-like protein that is associated with intermediate memory. We showed previously that one-trial conditioning resulted in a significant increase in the phosphorylation of CSP24 detected in lysates of the pathway supporting the conditioned stimulus (CS). Here we report the association of the protein with the actin cytoskeleton and the distribution of CSP24-immunoreactive neurons in two sensory structures and the circumesophageal nervous system. Identified photoreceptors, hair cells, and neurons in the cerebropleural and pedal ganglia were immunoreactive for CSP24. Immunoprecipitation experiments with 32PO4-labeled lysates of the circumesophageal nervous system identified a 44 kDa protein band (consistent with actin) that coprecipitates with CSP24. An analysis of immunoprecipitates on Western blots probed with anti-actin antibody also showed that actin coprecipitates with CSP24. Laser confocal microscopy of photoreceptors costained with fluorescently labeled anti-actin antibody and anti-CSP24 antibody, or fluorescent phalloidin and anti-CSP24 antibody showed that CSP24 is localized with actin in the cytosol of photoreceptor cell bodies and colocalized with presumed G-actin, but not F-actin, in regions adjacent to the plasma membrane. Although CSP24 is widely distributed in the Hermissenda nervous system, its regulation by one-trial conditioning was observed only in the CS pathway. Our findings suggest that CSP24 may interact with components of the actin cytoskeleton that contribute to structural changes underlying the formation and maintenance of enduring forms of memory.

Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium-dependent protein kinase C.

The mitogen-activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal-regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one-trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5-hydroxytryptamine (5-HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by 'upstream' signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5-HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA-dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5-HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA-AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca(2+)-dependent PKC activation contributes to ERK phosphorylation, although a PKC-independent pathway is also involved in 5-HT-dependent ERK phosphorylation and activation.

Identification of a 24 kDa phosphoprotein associated with an intermediate stage of memory in Hermissenda.

A requirement for protein synthesis is a critical feature in dissociating different phases of memory. However, in examples of cellular and synaptic plasticity in which an early or intermediate requirement for protein synthesis has been implicated, specific proteins have not been identified. Here we report the identification of a 24 kDa phosphoprotein (CSP24) associated with an intermediate stage of memory, distinct from short-term memory, detected after one-trial conditioning of Hermissenda. CSP24, initially identified from (32)PO(4)-labeled proteins resolved by two dimensional (2-D) PAGE, was excised from multiple Coomassie blue-stained 2-D gels and subjected to reverse phase HPLC and automated sequence analysis. The sequenced peptides exhibited a homology to the beta-thymosin family of actin-binding protein. Anti-CSP24 antibody recognized CSP24 on 1- and 2-D gels by Western blot analysis. Labeled CSP24 immunoprecipitated with anti-CSP24 antibody revealed that significantly more (32)PO(4) was incorporated in preparations that received one-trial conditioning compared with unpaired controls. In contrast, labeled CSP24 immunoprecipitated with anti-CSP24 from conditioned and unpaired control preparations receiving a procedure that only produced short-term enhanced excitability did not exhibit differences in (32)PO(4) incorporation into the immunoprecipitates. These results show that a specific identified phosphoprotein is associated with an intermediate stage of memory for one-trial conditioning in Hermissenda.

Protein synthesis-dependent and mRNA synthesis-independent intermediate phase of memory in Hermissenda.

The conditioned stimulus pathway in Hermissenda has been used to examine the time-dependent mechanisms of memory consolidation following one-trial conditioning. Here we report an intermediate phase of memory consolidation following one-trial conditioning that requires protein synthesis, but not mRNA synthesis. In conditioned animals, enhanced excitability normally expressed during an intermediate phase of memory was reversed by the protein synthesis inhibitor anisomycin, but not by the mRNA synthesis inhibitor 5, 6-dichloro-1-beta-D-ribobenzimidazole (DRB). Associated with the intermediate phase of memory is an increase in the phosphorylation of a 24-kDa protein. Anisomycin present during the intermediate phase blocked the increased phosphorylation of the 24-kDa phosphoprotein, but did not block the increased phosphorylation of other proteins associated with conditioning or significantly change their baseline phosphorylation. DRB did not reverse enhanced excitability or decrease protein phosphorylation expressed during the intermediate phase of memory formation, but it did reverse enhanced excitability 3.5 h after conditioning. Phosphorylation of the 24-kDa protein may support enhanced excitability during the intermediate phase, in the transition period between short- and long-term memory.

Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.

The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.