•Methane addition delays onset of both low- and high-temperature heat release in dual fuel combustion.•Methane trapped in crevices is a major source of unburned hydrocarbons at early injection ...timings.•Local temperature stratification influences the formation of nitrogen oxide emissions.•The extent of high reactivity fuel stratification significantly influences combustion evolution.
The present work focuses on the development and validation of a CFD simulation setup of diesel-methane dual fuel combustion in a single-cylinder research engine (SCRE). The validated setup is used to provide insight about dual fuel combustion at low-load operation. The computational campaign consisted of evaluating three different diesel injection timings of 310, 320, and 330 CAD at a methane percent energy substitution (PES) of 80%, 5.1 bar gross IMEP, 500 bar diesel injection pressure, and 1.5 bar manifold air pressure where 360 CAD corresponds to firing top dead center. The computational setup ability to capture combustion, performance, and emissions trends accurately is demonstrated by good agreement with experimental data. The validated setup is further utilized to provide insights into the nature of dual fuel combustion, particularly, the effect of methane on diesel autoignition. Analysis of the computational results showed that the onset of both low-temperature heat release and high-temperature heat release of n-dodecane (the chemical surrogate used for diesel) is delayed by the presence of methane in the system. For early diesel injection in a dual-fuel engine at low-load, initial high-temperature combustion arises from the burning of n-dodecane followed by methane combustion. Most of the methane present in the piston compression ring crevices, areas near the piston top and the liner, remained unreacted after combustion is done. The effect of diesel and methane fuel amount on engine performance at low-load was also explored.
In bulk materials, defects are usually considered to be unwanted since deviations from perfect lattices may degrade device performance. Interestingly, the presence of defects throws open new ...possibilities in the case of nanostructures due to the properties related to their limited size scale. Defects and disorders which alter the electronic structure of nanostructures can significantly influence their electronic, magnetic and nonlinear optical properties. Here, we show that defect engineering is an effective strategy for tailoring the nonlinear optical (NLO) properties of carbon and ZnO nanostructures. The effects of surface states, lattice disorders, polycrystalline interfaces and heterogeneous dopants on the nonlinear absorption behaviour of these nanostructures are discussed in detail. Realistic tunable NLO features achieved by controlling such defects enhance the scope of these nanostructures in device applications such as optical limiting, optical switching, pulse shaping, pulse compression and optical diode action.
In bulk materials, defects are usually considered to be unwanted since deviations from perfect lattices may degrade device performance.
Nanostructured carbons are posited to offer an alternative to silicon and lead to further miniaturization of photonic and electronic devices. Here, we report the experimental realization of the first ...all-carbon solid-state optical diode that is based on axially asymmetric nonlinear absorption in a thin saturable absorber (graphene) and a thin reverse saturable absorber (C60) arranged in tandem. This all-optical diode action is polarization independent and has no phase-matching constraints. The nonreciprocity factor of the device can be tuned by varying the number of graphene layers and the concentration or thickness of the C60 coating. This ultracompact graphene/C60 based optical diode is versatile with an inherently large bandwidth, chemical and thermal stability, and is poised for cost-effective large-scale integration with existing fabrication technologies.
In this article we present a perspective on the current state of the art in the photoionization of atomic clusters in few-cycle near-infrared laser pulses. Recently, several studies have reported ...intriguing phenomena associated with the photoionization of clusters by pulses as short as ∼10 fs which approach the natural timescales of collective electronic motion in such nanoscale aggregates. In contrast to the dynamics occurring on few- and sub-picosecond timescales where ionic motion sets in and plays a key role marked by resonant plasmon oscillations, the few-cycle limit precludes cluster expansion due to the nuclear motion of ionic constituents. Thus, pulses lasting just a few optical cycles explore a new "impulsive" regime for the first time in cluster nanoplasmas wherein ions essentially remain "frozen". Along with the perspective on this new regime, we present first measurements of photoelectron distributions and temperatures.
In this article we present a perspective on the current state of the art in the photoionization of atomic clusters in few-cycle near-infrared laser pulses.
Abstract
Background: DNA damage response (DDR) is critical for the maintenance of genome stability and serves as an anti-cancer barrier. Deregulation of key components of the DDR pathway such as p53 ...and ATM is associated with breast cancer progression. Recent studies implicated Alternative Non-homologous End-Joining (A-NHEJ) pathway in hormonal therapy resistance. Expression of proline, glutamic acid, leucine rich protein 1 (PELP1), a proto-oncogene, is an indicator of poor prognosis and therapy resistance in ER+ breast cancer. PELP1 was recently identified as a novel substrate of DNA damage kinases such as ATM. The objective of this study is to determine the role of PELP1 in the regulation of DNA repair and to study its effects on therapy resistance
Methods: We have used ER+ therapy sensitive MCF7, MCF7-Aro, ZR75 and therapy resistant MCF7-Tam, MCF7-LTLT, MCF7-HER2 model cells in our assays. UV-laser micro-irradiation, immunofluorescence staining and confocal microscopy were used to examine Ser-1033 phospho-PELP1 localization upon DNA damage. Homologous recombination (HR) reporter cell lines and NHEJ pathway reporter plasmids were used to determine the role of PELP1 in DNA repair pathways. U2OS-EJ2-GFP reporter cell line was used to evaluate the role of PELP1 in A-NHEJ pathway. MEFs lacking PELP1 and PELP1 specific siRNA were used to determine the role of PELP1 in DNA repair. Immunoprecipitation and western blotting were used to determine PELP1 interacting proteins. Yeast based peptide library screen was performed to identify PELP1 binding inhibitory peptides. MTT and clonogenic assays were used to determine the therapeutic effect of PELP1 inhibitor on therapy resistant breast cancer model cells.
Results: Results from this study showed that Ser-1033 phospho PELP1 was recruited to the site of DSB and co-localize with γ-H2A.X in breast cancer cells. Using reporter plasmid assays that distinguish various DNA repair pathways, we found that PELP1 regulates the NHEJ pathway and not the HR pathway. Further, using U2OS-EJ2-GFP reporter cell lines, we demonstrated that PELP1 is important for A-NHEJ mediated double strand break repair. Mechanistic studies revealed PELP1 interacts with E3ubiquitin ligase, SNF2 Histone Linker PHD RING Helicase (SHPRH, a yeast homolog of Rad5p). We have developed cell permeable peptide inhibitor (PIP3) that bind PELP1 and interferes with its interactions with SHPRH. Accordingly, treatment of U2OS-EJ2-GFP reporter cell lines with the (PIP3) peptide reduced the A-NHEJ repair frequency. Further, therapy resistant breast cancer cells which are addicted to A-NHEJ pathway were highly sensitive to PIP3 treatment.
Conclusions: These data provide novel insights into role of PELP1 in DNA repair, underscore the importance of PELP1 in the A-NHEJ pathway of DNA repair and demonstrates the significance of targeting PELP1 to sensitize therapy resistant breast cancer cells.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-12.
Abstract
BACKGROUND: Estrogen contribute to the progression of breast cancer via estrogen receptor 1 (ESR1) and current therapies involve either antiestrogens or aromatase inhibitors. However, most ...patients develop resistance to these drugs. Critically, therapy-resistant tumors retain ESR1-signaling. Mechanisms of therapy resistance involve the activation of ESR1 in the absence of ligand or mutations in ESR1 that allow interaction between the ESR1 and coregulators leading to sustained ESR1 signaling and proliferation. For patients with therapy-resistant breast cancers, there is a critical unmet need for novel agents to disrupt ESR1 signaling by blocking ESR1 interactions with its coregulators.
METHODS: Using rational design, we synthesized and evaluated a small organic molecule (ESR1 coregulator binding inhibitor, ECBI) that mimics the ESR1 coregulator nuclear receptor box motif. Using in vitro cell proliferation and apoptosis assays, we tested the effect of ECBI on several breast cancer and therapy-resistant model cells. Mechanistic studies were conducted using established biochemical assays, reporter gene assays, RT-qPCR and RNA-Seq analysis. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (IPA). ESR1 positive (MCF7 and ZR75) xenografts were used for preclinical evaluation and toxicity. The efficacy of ECBI was tested using ex vivo cultures of freshly extirpated primary human breast tissues.
RESULTS: In estrogen induced proliferation assays using several ESR1 positive model cells, ECBI significantly inhibited growth and promoted apoptosis. Importantly, ECBI showed little or no activity on ESR1 negative cells. Further, ECBI also reduced the proliferation of several ESR1 positive hormonal therapy resistant cells. Mechanistic studies showed that ECBI interacts with ESR1, efficiently blocks ESR1 interactions with coregulators and reduces the ESR1 driven ERE reporter gene activity. Further, ECBI directly interacted with mutant-ESR1 with high affinity and significantly inhibited mutant-ESR1 driven oncogenic activity. RNA sequencing analysis revealed that ECBI blocks multiple ESR1 driven pathways, likely representing the ability of a single ECBI compound to block multiple ESR1-coregulator interactions. Treatment of ESR1-positive xenograft tumors with ECBI (10 mg/kg/day/oral) significantly reduced the tumor volume compared to control. Further, ECBI also significantly reduced the tumor growth of coregulator-overexpressed breast cancer cells in xenograft model. Using human primary breast tissue ex vivo cultures, we have provided evidence that ECBI has potential to dramatically reduce proliferation of human breast tumors.
CONCLUSIONS: The ECBI is a novel agent that targets ESR1 with a unique mechanism of action. ECBI has distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Remarkably, ECBI block both native and mutant forms of ESR1 and have activity against therapy resistant breast cancer cell proliferation both in vitro and in vivo and against primary human tumor tissues ex vivo. Thus development of ECBI represents a quantum leap in therapies to target ESR1.
Citation Format: Vadlamudi RK, Sareddy GR, Viswanadhapalli S, Lee T-K, Ma S-H, Lee WR, Mann M, Krishnan SR, Gonugunta V, Strand DW, Tekmal RR, Ahn J-M, Raj GV. ESR1 coregulator binding inhibitor (ECBI) as a novel therapeutic to target hormone therapy resistant metastatic breast cancer. abstract. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S3-04.
We address the problem of high-resolution reconstruction in frequency-domain optical-coherence tomography (FDOCT). The traditional method employed uses the inverse discrete Fourier transform, which ...is limited in resolution due to the Heisenberg uncertainty principle. We propose a reconstruction technique based on zero-crossing (ZC) interval analysis. The motivation for our approach lies in the observation that, for a multilayered specimen, the backscattered signal may be expressed as a sum of sinusoids, and each sinusoid manifests as a peak in the FDOCT reconstruction. The successive ZC intervals of a sinusoid exhibit high consistency, with the intervals being inversely related to the frequency of the sinusoid. The statistics of the ZC intervals are used for detecting the frequencies present in the input signal. The noise robustness of the proposed technique is improved by using a cosine-modulated filter bank for separating the input into different frequency bands, and the ZC analysis is carried out on each band separately. The design of the filter bank requires the design of a prototype, which we accomplish using a Kaiser window approach. We show that the proposed method gives good results on synthesized and experimental data. The resolution is enhanced, and noise robustness is higher compared with the standard Fourier reconstruction.
Abstract
Triple negative breast cancer (TNBC) comprises approximately 15% of all breast cancers, lacks expression of ER, PR, and HER2 and is clinically aggressive with shorter disease free survival. ...TNBC patients do not benefit from antiestrogen and herceptin-based therapies. Evolving evidence suggests that overexpression of mutant p53 is significantly associated with TNBC progression. DNA damage response (DDR) is critical for the maintenance of genome stability and serves as an anti-cancer barrier during tumorigenesis. However, the role of DDR in tumor progression and metastasis is less known. Recent studies suggest that the ATM kinase is hyperactive in late stage breast tumor tissues with lymph node metastasis. Our ongoing studies have identified proline, glutamic acid, leucine rich protein 1 (PELP1), a proto-oncogene overexpressed in breast cancer, as a novel substrate of ATM. The objective of this study is to determine the mechanism and significance of ATM mediated phosphorylation of PELP1 and its crosstalk with the p53 pathway in TNBC cells. To test this we have used three ER-negative mutant p53 breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT20), with ER-positive (ZR-75 and MCF7) cell lines as controls. Using PELP1 specific shRNAs, we generated model cells that have stable expression of either control or PELP1 shRNA. Our results with PELP1 knockdown models indicate that PELP1 promotes stability of p53 and functions as a co-regulator of p53. PELP1 has the potential to modulate CBP/p300 mediated acetylation of the Lysine 382 residue of p53. Immunoprecipitation and chromatin immunoprecipitation (ChIP) assays were performed to examine PELP1 interaction with p53 and its recruitment to p53 target genes respectively. ChIP assays revealed that PELP1 knockdown significantly reduces the recruitment of p53 to the target genes. The p53 regulatory role of PELP1 is mediated by the phosphorylation of PELP1 at the conserved SQ motif by ATM. We generated model cells that overexpress WT or mutant (S1033A) PELP1 and demonstrated the significance of ATM mediated phosphorylation in PELP1 mediated p53 co-activation functions. Based on the sequence of the site of phosphorylation, we have developed a novel cell permeable peptide (TAT-1033PELP1 inhibitor) as well as a phospho-PELP1 antibody that uniquely recognizes S1033 phosphorylated PELP1. We confirmed ATM mediated phosphorylation of PELP1 in vivo using phospho PELP1 antibody (1033p-PELP1). The TAT 1033 inhibitor peptide significantly reduced ATM mediated phosphorylation of endogenous PELP1. Treatment of ER− positive breast cancer cells with this peptide resulted in resistance to genotoxic stress compared to cells that are treated with control TAT peptide. However, in TNBC cells that has a mutant p53, PELP1 knockdown or treatment with TAT-PELP1 inhibitor resulted in significant loss of cell viability and increase in apoptosis in response to genotoxic stress. IHC analysis of tumor tissue array (n = 100) revealed increased PELP1 phosphorylation in advanced ER-negative tumors and its status correlated with ATM. Collectively, our results suggest that hyperactive ATM-PELP1-p53 pathway contributes to TNBC progression and that the TAT-1033PELP1 inhibitor represents a novel therapeutic to block PELP1 oncogenic functions in TNBC.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD09-07.
Abstract
BACKGROUND: Growth factor induced activation of MAPK pathway is suggested to play a critical role both in invasive and hormone therapy resistant breast cancer via ligand independent ...activation of ERα. Proline Glutamic acid Leucine Rich Protein (PELP1) is a well established ER-coregulator that plays a critical role in ER's nuclear and extra-nuclear functions. PELP1 functions as a proto-oncogene and deregulation of PELP1 expression is linked to increased proliferation, metastasis and therapy resistance. The objective of this study is to examine, whether growth factor signaling modulates PELP1 mediated ER coactivation functions via MAPK pathway.
METHODS: Bioinformatics and phosphopeptide specific antibodies were employed in this study. In vitro kinase assays and MAPK inhibitors were used to confirm MAPK phosphorylation of PELP1. Site directed mutants and ERE reporter gene assays were used to demonstrate the significance of MAPK site in PELP1. Growth factor and estrogen regulation of PELP1 phosphoryaltion at Ser991 was validated in therapy sensitive (MCF7, ZR75) and resistant (MCF-7 HER2, MCF7-Tam and MCF-7Ca-LTLT cells using PELP1 specific phosphoantibody. Breast cancer model cells stably expressing PELP1 mutant or PELP1 peptides encompassing MAPK phosphorylation was used to test the significance of this MAPK site in PELP1 using proliferation, migration, and invasion assays. IHC analysis using PELP1 phospho antibodies was performed using murine xenograft tumors and human breast tumor arrays (TMA).
RESULTS: Growth factors (EGF, Heregulin) induced Ser991 phosphorylation of PELP1 in murine mammary epithelial cells and also in human breast cancer cell lines. Growth factor mediated Ser991PELP1 phosphorylation was abrogated by ERK1/2 MAPK pathway inhibitors including PD98059 and U0126. In ERE-reporter luciferase assays using ZR75 cells, PELP1 (WT) but not the ser991Ala mutant of PELP1 corroborated growth factor-ER crosstalk. Therapy resistant model cells (MCF-7-Tam, MCF-7-LTLT-ca and MCF-7-HER2) exhibited enhanced Ser991PELP1 phosphoryation compared to parental MCF7. TAT-Peptide encompassing Ser991 inhibited growth factor mediated phosphorylation of PELP1 and revealed a potent ability to inhibit the growth of therapy resistant cells. Xenograft tumors of hormone therapy model cell lines showed increased Ser991PELP1 phosphorylation and enhanced nuclear localization compared to hormonal sensitive xenograft tumors. IHC analysis of human breast invasive TMA revealed enhanced PELP1 ser991 phosphorylation in advanced metastatic tumors with prominent nuclear/nucleolar localization compared to normal and benign tumors. CONCLUSIONS: In this study, we identified that growth factor-MAPK pathway modulate ER coregulator PELP1 functions via phosphorylation and demonstrated that Ser991 phosphorylation of PELP1 has the potential to govern the growth Factor-ER cross talk leading to therapy resistance and metastatic phenotype of breast cancer. Peptides encompassing Ser991PELP1 site and Phosphoantibodies targeting Ser991 represent a novel therapeutic target and diagnostic markers in breast cancer. Supported by DOD Fellowship W81XWH-09-1-0010 (BCN) and DOD W81XWH-08-1-0604 (RKV)
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-08.
Mature human erythrocyte membranes contained specific, high affinity (Kd 3.3 X 10(-11) M) folate binding moieties. Folate binding was pH, time- and temperature-dependent, saturable, and was much ...greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging.
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