Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within ...resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Apoptosis can be measured by number of methods by taking advantage of the morphological, biochemical, and molecular changes undergoing in a cell during this process. The best recognized biochemical ...hallmark of both early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals. Most common assays for examining caspase-3 activation include immunostaining, immunoblotting for active caspase-3, colorimetric assays using fluorochrome substrates, as well as employing the fluorescein-labeled CaspaTag pan-caspase in situ detection kit.
The dissolution and subsequent repassivation behavior of a lightweight and single-phase multi-principal element alloy (MPEA) AlTiVCr, have been investigated in a quiescent 0.6 M NaCl. The alloy ...exhibits excellent passivity and repassivation ability in spite of transpassive dissolution of V and Cr during cyclic potentiodynamic polarisation. Elemental-resolved dissolution analysis of the alloy using inline inductively coupled plasma mass spectroscopy (ICP-MS), together with composition analysis of the passive film using X-ray photoelectron spectroscopy (XPS) reveal that the alloy promptly recovers after each transpassive dissolution due to rapid growth of stable TiO2 in the film, which led to a wide passive window. Moreover, the excellent repassivation behavior was found to be due to significant enrichment of Ti on the alloy surface during anodic polarisation. The results, corresponding to the dissolution and compositional evolution of the passive film, were in good agreement with the predicted E-pH diagram of the alloy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Non-metallic inclusions originate mainly during secondary steelmaking due to deoxidation and other exogenous sources. Additional inclusions form during cooling and subsequent freezing of liquid ...steel. Rejection of solutes by the solidifying dendrites causes segregation of solutes in the interdendritic liquid with consequent build-up of their thermodynamic supersaturation. The work reported in the present paper was undertaken to develop a computation procedure for prediction of inclusion compositions formed during cooling and solidification of liquid steel. The model has been applied to an inclusion sensitive grade of steel. Segregation of various solutes with progress of freezing has been calculated using the Clyne–Kurz microsegregation equation. A sequential computation procedure involving segregation equation and thermodynamic equilibrium calculations by the Factsage thermodynamic software has been developed. Compositions of inclusions at various solid fractions have been determined. Model predictions have been compared with literature as well as with inclusion compositions determined in continuously cast billet samples using SEM-EDS. Reasonably good correspondence between model predictions and observed inclusions have been obtained.
•The ‘true’ corrosion rate of AM-316L was lower than that of the W-316L in 0.6 M NaCl.•The fraction of Cr2O3 was relatively higher in the passive film of AM-316L than that of the W-316L.•The Mn(Si, ...Al) rich oxide nano-particles present in AM-316L dissolved preferentially in 0.6 M NaCl.•Surface composition of the surrounding area changed during propagation of the pit in both the W-316L and AM-316L specimens.
The dissolution and passivation behavior of additively manufactured stainless steel 316L (AM-316L) prepared via selective laser melting was studied in quiescent 0.6 M NaCl. In a detailed comparison with the wrought stainless steel 316L (W-316L), it was determined via mass spectroelectrochemistry and x-ray photoelectron spectroscopy (XPS), that AM-316L exhibited a unique passive film relative to W-316L, resulting in a lower dissolution rate. The dissolution and enrichment of all alloying elements at open circuit potential and during polarisation were studied and elaborated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Morphology and centerline macrosegregation in continuously cast high carbon steel billet samples were investigated in order to establish the casting behavior of a six strand curved mold billet caster ...without electro-magnetic stirring. Several billet samples were collected from the continuous casting shop of Tata Steel, India and experimental observations were correlated with the operating parameters of the caster. Macrostructural examination revealed predominantly columnar structure associated with high degree of segregation and porosities in all billets cast above 21°C of tundish superheat. Centerline porosity was practically absent in the billet cast below 21°C. These billets showed prominent V-segregation and less prominent centerline segregation. Transition from U-segregation to V-segregation was observed around 21°C superheat. An attempt has been made to study the effectiveness of secondary cooling by measurements of the secondary dendrite arm spacing at various locations in billet samples. Finally, degree of segregation of constituent solute elements were correlated among themselves and applicability of one of the simple segregation models to the centerline macrosegregation has been tested.
Label-free quantification of shotgun LC–MS/MS data is the prevailing approach in quantitative proteomics but remains computationally nontrivial. The central data analysis step is the detection of ...peptide-specific signal patterns, called features. Peptide quantification is facilitated by associating signal intensities in features with peptide sequences derived from MS2 spectra; however, missing values due to imperfect feature detection are a common problem. A feature detection approach that directly targets identified peptides (minimizing missing values) but also offers robustness against false-positive features (by assigning meaningful confidence scores) would thus be highly desirable. We developed a new feature detection algorithm within the OpenMS software framework, leveraging ideas and algorithms from the OpenSWATH toolset for DIA/SRM data analysis. Our software, FeatureFinderIdentification (“FFId”), implements a targeted approach to feature detection based on information from identified peptides. This information is encoded in an MS1 assay library, based on which ion chromatogram extraction and detection of feature candidates are carried out. Significantly, when analyzing data from experiments comprising multiple samples, our approach distinguishes between “internal” and “external” (inferred) peptide identifications (IDs) for each sample. On the basis of internal IDs, two sets of positive (true) and negative (decoy) feature candidates are defined. A support vector machine (SVM) classifier is then trained to discriminate between the sets and is subsequently applied to the “uncertain” feature candidates from external IDs, facilitating selection and confidence scoring of the best feature candidate for each peptide. This approach also enables our algorithm to estimate the false discovery rate (FDR) of the feature selection step. We validated FFId based on a public benchmark data set, comprising a yeast cell lysate spiked with protein standards that provide a known ground-truth. The algorithm reached almost complete (>99%) quantification coverage for the full set of peptides identified at 1% FDR (PSM level). Compared with other software solutions for label-free quantification, this is an outstanding result, which was achieved at competitive quantification accuracy and reproducibility across replicates. The FDR for the feature selection was estimated at a low 1.5% on average per sample (3% for features inferred from external peptide IDs). The FFId software is open-source and freely available as part of OpenMS (www.openms.org).
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Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which ...cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.
► Quantitative proteomic strategy reveals dynamics of cell-cycle protein interactions ► Cyclins confer biochemical specificity to cyclin/cdk interaction networks ► Cyclin A phosphorylates Sld3/c15orf42 and Wee1 to promote S phase and mitosis ► Cyclins A and B interact sequentially with proteins in G2 phase and mitosis
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution ...of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Exposure to genotoxic agents, such as irradiation produces DNA damage, the toxicity of which is augmented when the DNA repair is impaired. Poly (ADP-ribose) polymerase (PARP) inhibitors were found to ...be "synthetic lethal" in cells deficient in BRCA1 and BRCA2 that impair homologous recombination. However, since many tumors, including prostate cancer (PCa) rarely have on such mutations, there is considerable interest in finding alternative determinants of PARP inhibitor sensitivity. We evaluated the effectiveness of radiation in combination with the PARP inhibitor, rucaparib in PCa cells. The combination index for clonogenic survival following radiation and rucaparib treatments revealed synergistic interactions in a panel of PCa cell lines, being strongest for LNCaP and VCaP cells that express ETS gene fusion proteins. These findings correlated with synergistic interactions for senescence activation, as indicated by β--galactosidase staining. Absence of PTEN and presence of ETS gene fusion thus facilitated activation of senescence, which contributed to decreased clonogenic survival. Increased radiosensitivity in the presence of rucaparib was associated with persistent DNA breaks, as determined by χ-H2AX, p53BP1, and Rad51 foci. VCaP cells, which harbor the TMPRSS2-ERG gene fusion and PC3 cells that stably express a similar construct (fusion III) showed enhanced sensitivity towards rucaparib, which, in turn, increased the radiation response to a similar extent as the DNA-PKcs inhibitor NU7441. Rucaparib radiosensitized PCa cells, with a clear benefit of low dose-rate radiation (LDR) administered over a longer period of time that caused enhanced DNA damage. LDR mimicking brachytherapy, which is used successfully in the clinic, was most effective when combined with rucaparib by inducing persistent DNA damage and senescence, leading to decreased clonogenic survival. This combination was most effective in the presence of the TMPRSS2-ERG and in the absence of PTEN, indicating clinical potential for brachytherapy in patients with intermediate and high risk PCa.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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