Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 ...(MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.
In this study, a combined computational and experimental particle tracking investigation was performed for a solid-state additive manufacturing and repair process, Additive Friction Stir Deposition ...(AFSD). Specifically, smoothed particle hydrodynamics (SPH) simulations of AFSD were conducted in-order to elucidate deposition mechanics. The particle tracking of the SPH AFSD simulations was validated using experimental depositions of two feedstock varieties, including anodized AA6061-T6 feedstock to track external particles and AA6061-T6 copper wire core feedstock to track internal particles, to represent flow behavior from different regions of the feedstock. The X-Ray computed tomography (CT) experimental results revealed that the anodized oxides on the outside of the feedstock flowed to the retreating side, whereas the copper wire in the center of the feedstock migrated to the advancing side. Particle tracking results from the SPH simulations showed that, in general, particle movement is limited to directly beneath the feedstock. The rotational, radial, and traverse flow interactions visualized by AFSD simulations explained the advancing and retreating side biases experienced by the internal copper wire and surface oxides on the anodized feedstock. This work demonstrates the ability to predict AFSD material distributions, which has a significant impact on as-deposited material quality.
Graphical abstract
While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. ...Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.
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In this work, Additive Friction Stir Deposition (AFSD) was employed for ballistic repair of AA7075-T6511 plates. After penetration with 7.62 × 51 mm FMJ rounds, the AA7075-T6511 plates were repaired ...by AFSD using the same AA7075-T6511 feedstock material. The repaired plates were impacted and penetrated with the same 7.62 × 51 mm FMJ rounds, and the surface damage characteristics including the initial and residual velocities were compared against the control wrought plates. The AFSD process successfully repaired the damaged control plates with the same alloy, without any observable defects such as large cracks or pores prior to impact tests. Although the surface appeared pristine other than milling marks, the surface damage characteristics of the repaired plates were significantly different than the control plates. The increase of spalling and petalling with the repaired material can be attributed to the thermomechanical processing of AFSD, which would alter the control T6511 temper of the feedstock due to coarsening of strengthening precipitates. A cross-sectioned repaired plate was analyzed using microhardness plots and optical microscopy to illustrate the effectiveness of the AFSD process for ballistic repair by depositing the same material into the damaged area. Despite the surface damage discrepancy, the repaired plates performed similarly to the control plates with respect to initial and residual velocities.
Graphical Abstract
The human ABCA2 transporter gene encodes a member of a large family of ATP‐binding proteins that transport a variety of macromolecules across biological membranes. We have performed luciferase ...reporter gene assays with promoter constructs comprising the 5′‐flanking region to identify cis‐regulatory DNA elements and have mapped the minimal promoter region to 321 bp upstream of the translation start site. We have discovered a functional role for two GC‐boxes located in the proximal promoter of the ABCA2 gene that contain overlapping sites for the EGR‐1 and Sp1 transcription factors. We observed that oligonucleotides containing overlapping EGR‐1/Sp1 sites bind the Sp1, Sp3 and Sp4 transcription factors. When BE(2)‐M17 cells were treated with phorbol 12‐myristate 13‐acetate, we observed inducible expression and binding of the EGR‐1 transcription factor to the two GC‐boxes. Transfection of Sp1, Sp3 or Sp4 expression constructs into Drosophila S2 induced a dose‐dependent increase in transcriptional activation of the ABCA2 promoter, but transfection of EGR‐1 alone failed to activate transcription. When increasing amounts of EGR‐1 were transfected into the BE(2)‐M17 neuroblastoma cells we observed a dose‐dependent decrease in expression of the ABCA2 promoter, although expression of the endogenous ABCA2 gene increased following transfection of EGR‐1.
ABSTRACT
With the use of a novel method for detecting differential gene expression, alterations in functional gene clusters related to transport or oxidative stress response and β‐amyloid (Aβ) ...peptide metabolism were identified in a HEK293 cell line engineered to overexpress the human ATP binding cassette transporter ABCA2. These included fatty acid binding protein, phospholipid binding protein, phospholipid synthesis protein, transporter cofactors, seladin‐1, Aβ precursor protein (APP), vimentin, and low‐density lipoprotein receptor‐related protein. ABCA2 was highly expressed in neuroblastoma cells and colocalized with Aβ and APP. Additionally, increased APP protein levels were detected within ABCA2/APP double‐transfected cells, and increased Aβ was detected in the media of ABCA2‐transfected cells relative to controls. The transporter was abundant in the temporal and frontal regions of both normal and Alzheimer's disease (AD) brain but was detected at lower concentrations in the parietal, occipital, and cerebellar regions. The ABCA2 transfected cell line expressed resistance to a free radical initiator, confirming involvement in protection against reactive oxygen species and suggesting a further possible link to AD.
We have isolated the full-length cDNA for human ATP-binding cassette, sub-family A, member 2 transporter (ABCA2). The ORF of this cDNA encodes a protein consisting of 2436 amino acids with apparent ...molecular weight of M(r) 270,000. Accordingly, ABCA2 is the largest known mammalian ABC transporter described thus far. Analysis of mRNA expression levels indicated that ABCA2 is highest in human brain and has a broad expression pattern in a panel of tumor cell lines. Using specific antibodies to ABCA2 and various organelle marker proteins, ABCA2 was found to colocalize with the lysosomal/endosomal marker LAMP1, forming discrete, punctate intracellular vesicles. In ABCA2-transfected cells, the transporter also colocalized with a fluorescently labeled steroid analogue, estramustine. The sequestration of the steroid into the lysosomal/endosomal compartment indicates a potential substrate specificity for ABCA2. Furthermore, the presence of a lipocalin signature motif in the ABCA2 sequence suggests a possible broad role for this protein in the transport of steroids, lipids, and related molecules.
Amplified differential gene expression (ADGE) is a novel technique, designed to profile gene expression of the whole transcriptome or to compare expression of a set of genes between two samples. ADGE ...employs hybridization to quadratically amplify the ratio of an expressed gene between control and tester samples before displaying. The subtle structures of adapters and primers are designed for displaying the amplified ratio of an expressed gene between two samples. Four selective nucleotides at the 3' end of primers are used to increase PCR efficiency for targeted molecules and to improve detection of PCR products. Double PCR with the same pair of primers expands the detection range, especially for genes of low abundance. Integration of these steps makes ADGE sensitive and accurate. Application to drug resistant human tumor cell lines showed that ADGE accurately profiled expression levels for induced, repressed or unchanged genes. The qualitative expression patterns for ADGE were verified with RT-PCR.
The aminothiol WR-1065 (the active form of amifostine) protects normal tissues from the toxic effects of certain cancer drugs, while leaving their antitumor effects unchanged. The present data ...address the mechanism of action of this dichotomous effect. (35)S-Labeled WR-1065 bound directly to the transcription factors nuclear factor-kappaB, activator protein-1, and p53, resulting in enhanced binding of these proteins to target regulatory DNA sequences and subsequent transactivation of a number of downstream genes. Since other small molecular thiols could mimic WR-1065, the redox potential of the sulfhydryl is an important determinant of its activity. In nontransformed cells, WR-1065 protected cells from the cytotoxic effects of paclitaxel in a p53-dependent manner. However, in a transformed human tumor cell line, there was no cytoprotectivity by WR-1065, consistent with the premise that p53-dependent growth arrest is the basis for the protective effect of this compound, and that this pathway is abrogated in human tumors. The combined data support the principle that the cellular effects of the aminothiol WR-1065 are mediated through an impact on transcriptional regulation and are not only a consequence of radical scavenging.
Although Th2 driven inflammation is present in COPD, it is not clearly elucidated which COPD patients are affected. Since periostin is associated with Th2 driven inflammation and inhaled ...corticosteroid (ICS)-response in asthma, it could function as a biomarker in COPD. The aim of this study was to analyze if serum periostin is elevated in COPD compared to healthy controls, if it is affected by smoking status, if it is linked to inflammatory cell counts in blood, sputum and endobronchial biopsies, and if periostin can predict ICS-response in COPD patients.Serum periostin levels were measured using Elecsys Periostin immunoassay. Correlations between periostin and inflammatory cell count in blood, sputum and endobronchial biopsies were analyzed. Additionally, the correlation between serum periostin levels and treatment responsiveness after 6 and 30 months was assessed using i.e. ΔFEV1% predicted, ΔCCQ score and ΔRV/TLC ratio. Forty-five COPD smokers, 25 COPD past-smokers, 22 healthy smokers and 23 healthy never-smokers were included. Linear regression analysis of serum periostin showed positive correlations age (B = 0.02, 95%CI 0.01-0.03) and FEV1% predicted (B = 0.01, 95%CI 0.01-0.02) in healthy smokers, but not in COPD patients In conclusion, COPD -smokers and -past-smokers have significantly higher periostin levels compared to healthy smokers, yet periostin is not suitable as a biomarker for Th2-driven inflammation or ICS-responsiveness in COPD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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