Efficient and Stable All-Polymer Solar Cells Enabled by Dual Working Mechanism.

Ternary strategy with integration characteristics and adaptability is a simple and effective method for blooming of the performance of photovoltaic devices. Herein, a novel wideband gap polymer donor PBB2-Hs is synthesized as the guest component to optimize all-polymer solar cells (all-PSCs). High-energy photon absorption and long exciton lifetime of PBB2-Hs constitute efficient energy transfer. Good miscibility and cascade energy levels promote the formation of alloy-like structure between PBB2-Hs and host system. The dual working mechanisms greatly improve photon capture and charge transfer in active layers. Additionally, the introduction of PBB2-Hs also optimizes the ordered molecular stacking of acceptors and suppresses molecular peristalsis. Upon adding 15 wt% PBB2-Hs, the ternary all-PSC achieved a champion efficiency of 17.66%, and can still maintain 82% photostability (24 h) and 91% storage stability (1000 h) of the original PCE. Moreover, the strong molecular stacking and entanglement between PBB2-Hs and the host material increased the elongation at break of ternary blend film by 1.6 times (16.2%), allowing the flexible device to maintain 83% of the original efficiency after 800 bends (R = 5 mm). This work highlights the effectiveness of guest polymer on simultaneously improving photovoltaic performance, photostability and mechanical stability in all-PSCs.

Inhibitory Peptide of Soluble Guanylyl Cyclase/Trx1 Interface Blunts the Dual Redox Signaling Functions of the Complex.

Soluble guanylyl cyclase (GC1) and oxido-reductase thioredoxin (Trx1) form a complex that mediates two NO signaling pathways as a function of the redox state of cells. Under physiological conditions, reduced Trx1 (rTrx1) supports the canonical NO-GC1-cGMP pathway by protecting GC1 activity from thiol oxidation. Under oxidative stress, the NO-cGMP pathway is disrupted by the S-nitrosation of GC1 (addition of a NO group to a cysteine). In turn, SNO-GC1 initiates transnitrosation cascades, using oxidized thioredoxin (oTrx1) as a nitrosothiol relay. We designed an inhibitory peptide that blocked the interaction between GC1 and Trx1. This inhibition resulted in the loss of a) the rTrx1 enhancing effect of GC1 cGMP-forming activity in vitro and in cells and its ability to reduce the multimeric oxidized GC1 and b) GC1's ability to fully reduce oTrx1, thus identifying GC1 novel reductase activity. Moreover, an inhibitory peptide blocked the transfer of S-nitrosothiols from SNO-GC1 to oTrx1. In Jurkat T cells, oTrx1 transnitrosates procaspase-3, thereby inhibiting caspase-3 activity. Using the inhibitory peptide, we demonstrated that S-nitrosation of caspase-3 is the result of a transnitrosation cascade initiated by SNO-GC1 and mediated by oTrx1. Consequently, the peptide significantly increased caspase-3 activity in Jurkat cells, providing a promising therapy for some cancers.

Soluble guanylyl cyclase mediates noncanonical nitric oxide signaling by nitrosothiol transfer under oxidative stress.

Soluble guanylyl cyclase (GC1) is an α/ß heterodimer producing cGMP when stimulated by nitric oxide (NO). The NO-GC1-cGMP pathway is essential for cardiovascular homeostasis but is disrupted by oxidative stress, which causes GC1 desensitization to NO by heme oxidation and S-nitrosation (SNO) of specific cysteines. We discovered that under these conditions, GC1-α subunit increases cellular S-nitrosation via transfer of nitrosothiols to other proteins (transnitrosation) in cardiac and smooth muscle cells. One of the GC1 SNO-targets was the oxidized form of Thioredoxin1 (oTrx1), which is unidirectionally transnitrosated by GC1 with αC610 as a SNO-donor. Because oTrx1 itself drives transnitrosation, we sought and identified SNO-proteins targeted by both GC1 and Trx1. We found that transnitrosation of the small GTPase RhoA by SNO-GC1 requires oTrx1 as a nitrosothiol relay, suggesting a SNO-GC1→oTrx1→RhoA cascade. The RhoA signaling pathway, which is antagonized by the canonical NO-cGMP pathway, was alternatively inhibited by GC1-α-dependent S-nitrosation under oxidative conditions. We propose that SNO-GC1, via transnitrosation, mediates adaptive responses triggered by oxidation of the canonical NO-cGMP pathway.

Selective cysteines oxidation in soluble guanylyl cyclase catalytic domain is involved in NO activation.

Nitric oxide (NO) binds to soluble guanylyl cyclase (GC1) and stimulates its catalytic activity to produce cGMP. Despite the key role of the NO-cGMP signaling in cardiovascular physiology, the mechanisms of GC1 activation remain ill-defined. It is believed that conserved cysteines (Cys) in GC1 modulate the enzyme's activity through thiol-redox modifications. We previously showed that GC1 activity is modulated via mixed-disulfide bond by protein disulfide isomerase and thioredoxin 1. Herein we investigated the novel concept that NO-stimulated GC1 activity is mediated by thiol/disulfide switches and aimed to map the specific Cys that are involved. First, we showed that the dithiol reducing agent Tris (2-carboxyethyl)-phosphine reduces GC1 response to NO, indicating the significance of Cys oxidation in NO activation. Second, using dibromobimane, which fluoresces when crosslinking two vicinal Cys thiols, we demonstrated decreased fluorescence in NO-stimulated GC1 compared to unstimulated conditions. This suggested that NO-stimulated GC1 contained more bound Cys, potentially disulfide bonds. Third, to identify NO-regulated Cys oxidation using mass spectrometry, we compared the redox status of all Cys identified in tryptic peptides, among which, ten were oxidized and two were reduced in NO-stimulated GC1. Fourth, we resorted to computational modeling to narrow down the Cys candidates potentially involved in disulfide bond and identified Cys489 and Cys571. Fifth, our mutational studies showed that Cys489 and Cys571 were involved in GC1'response to NO, potentially as a thiol/disulfide switch. These findings imply that specific GC1 Cys sensitivity to redox environment is critical for NO signaling in cardiovascular physiology.

Identification of osteoporosis markers through bioinformatic functional analysis of serum proteome.

Osteoporosis is a severe chronic skeletal disorder that increases the risks of disability and mortality; however, the mechanism of this disease and the protein markers for prognosis of osteoporosis have not been well characterized. This study aims to characterize the imbalanced serum proteostasis, the disturbed pathways, and potential serum markers in osteoporosis by using a set of bioinformatic analyses. In the present study, the large-scale proteomics datasets (PXD006464) were adopted from the Proteome Xchange database and processed with MaxQuant. The differentially expressed serum proteins were identified. The biological process and molecular function were analyzed. The protein-protein interactions and subnetwork modules were constructed. The signaling pathways were enriched. We identified 209 upregulated and 230 downregulated serum proteins. The bioinformatic analyses revealed a highly overlapped functional protein classification and the gene ontology terms between the upregulated and downregulated protein groups. Protein-protein interactions and pathway analyses showed a high enrichment in protein synthesis, inflammation, and immune response in the upregulated proteins, and cell adhesion and cytoskeleton regulation in the downregulated proteins. Our findings greatly expand the current view of the roles of serum proteins in osteoporosis and shed light on the understanding of its underlying mechanisms and the discovery of serum proteins as potential markers for the prognosis of osteoporosis.

Comprehensive identification of protein disulfide bonds with pepsin/trypsin digestion, Orbitrap HCD and Spectrum Identification Machine.

Disulfide bonds (SS) are post-translational modifications important for the proper folding and stabilization of many cellular proteins with therapeutic uses, including antibodies and other biologics. With budding advances of biologics and biosimilars, there is a mounting need for a robust method for accurate identification of SS. Even though several mass spectrometry methods have emerged for this task, their practical use rests on the broad effectiveness of both sample preparation methods and bioinformatics tools. Here we present a new protocol tailored toward mapping SS; it uses readily available reagents, instruments, and software. For sample preparation, a 4-h pepsin digestion at pH 1.3 followed by an overnight trypsin digestion at pH 6.5 can maximize the release of SS-containing peptides from non-reduced proteins, while minimizing SS scrambling. For LC/MS/MS analysis, SS-containing peptides can be efficiently fragmented with HCD in a Q Exactive Orbitrap mass spectrometer, preserving SS for subsequent identification. Our bioinformatics protocol describes how we tailored our freely downloadable and easy-to-use software, Spectrum Identification Machine for Cross-Linked Peptides (SIM-XL), to minimize false identification and facilitate manual validation of SS-peptide mass spectra. To substantiate this optimized method, we've comprehensively identified 14 out of 17 known SS in BSA. SIGNIFICANCE: Comprehensive and accurate identification of SS in proteins is critical for elucidating protein structures and functions. Yet, it is far from routine to accomplish this task in many analytical or core laboratories. Numerous published methods require complex sample preparation methods, specialized mass spectrometers and cumbersome or proprietary software tools, thus cannot be easily implemented in unspecialized laboratories. Here, we describe a robust and rapid SS mapping approach that utilizes readily available reagents, instruments, and software; it can be easily implemented in any analytical core laboratories, and tested for its impact on the research community.

Bioinformatic analysis reveals the key pathways and genes in early-onset breast cancer.

Early-onset breast cancer is the most prevalent cancer in the female. To identify the differentially expressed genes and the key signaling pathways in early-onset breast cancer, we have carried out the bioinformatic analysis of an RNA array dataset in the GEO database, GSE109169, which was acquired from early-onset breast cancer patient. A total of 118 differentially expressed genes in early-onset breast cancer were significantly changed compared with that in adjacent normal tissues. Most of these genes are classified into three categories: signaling molecule, enzyme modulator, and hydrolase. Gene ontology terms reveal that most of these genes are involved in cellular and metabolic processes, biological regulation, binding and catalytic activities, and receptor regulation. Protein-protein interaction network was constructed and has two highly enriched modules: one with up-regulated genes and the other with down-regulated genes. The singling pathways are mainly enriched in the cellular immune system, lipid metabolism and other types of metabolic pathways. Finally, we have plotted the Kaplan-Meier curves of two up-regulated and two down-regulated genes for the overall survival prediction in breast cancer. These results greatly expand the current view of early-onset breast cancer and shed light on the discovery of drug candidates and the improvement for the prognosis.

Biotin Switch Processing and Mass Spectrometry Analysis of S-Nitrosated Thioredoxin and Its Transnitrosation Targets.

S-Nitrosation is a key posttranslational modification in regulating proteins in both normal physiology and diverse human diseases. To identify novel therapies for human diseases linked to oxidative and nitrosative stress, understanding how cells control S-nitrosation specificity could be critical. Among the enzymes known to control S-nitrosation of proteins, thioredoxin 1 (Trx1), a conserved disulfide reductase, transnitrosates and denitrosates distinct sets of target proteins. To recognize the function of Trx1 in both normal and dysfunctional cells, S-nitrosation targets of Trx1 in different cells need to be identified. However, S-nitrosation is usually too labile to be detected directly by mass spectrometry (MS). Here we present two optimized MS techniques to identify S-nitrosated Trx1 and its transnitrosation targets, using both direct and indirect MS methods.

Identification of novel S-nitrosation sites in soluble guanylyl cyclase, the nitric oxide receptor.

Soluble Guanylyl Cyclase (sGC) is the main receptor for nitric oxide (NO). NO activates sGC to synthesize cGMP, triggering a plethora of signals. Recently, we discovered that NO covalently modifies select sGC cysteines via a post-translational modification termed S-nitrosation or S-nitrosylation. Earlier characterization was conducted on a purified sGC treated with S-nitrosoglutathione, and identified three S-nitrosated cysteines (SNO-Cys). Here we describe a more biologically relevant mapping of sGC SNO-Cys in cells to better understand the multi-faceted interactions between SNO and sGC. Since SNO-Cys are labile during LC/MS/MS, MS analysis of nitrosation typically occurs after a biotin switch reaction, in which a SNO-Cys is converted to a biotin-Cys. Here we report the identification of ten sGC SNO-Cys in rat neonatal cardiomyocytes using an Orbitrap MS. A majority of the SNO-Cys identified is located at the solvent-exposed surface of the sGC, and half of them in the conserved catalytic domain, suggesting biological significance. These findings provide a solid basis for future studies of the regulations and functions of diverse sGC S-nitrosation events in cells.

Master redox regulator Trx1 upregulates SMYD1 & modulates lysine methylation.

Thioredoxin 1 (Trx1) is а antioxidant protein that regulates protein disulfide bond reduction, transnitrosylation, denitrosylation and other redox post-translational modifications. In order to better understand how Trx1 modulates downstream protective cellular signaling events following cardiac ischemia, we conducted an expression proteomics study of left ventricles (LVs) after thoracic aortic constriction stress treatment of transgenic mice with cardiac-specific over-expression of Trx1, an animal model that has been proven to withstand more stress than its non-transgenic littermates. Although previous redox post-translational modifications proteomics studies found that several cellular protein networks are regulated by Trx1-mediated disulfide reduction and transnitrosylation, we found that Trx1 regulates the expression of a limited number of proteins. Among the proteins found to be upregulated in this study was SET and MYND domain-containing protein 1 (SMYD1), a lysine methyltransferase highly expressed in cardiac and other muscle tissues and an important regulator of cardiac development. The observation of SMYD1 induction by Trx1 following thoracic aortic constriction stress is consistent with the retrograde fetal gene cardiac protection hypothesis. The results presented here suggest for the first time that, in addition to being a master redox regulator of protein disulfide bonds and nitrosation, Trx1 may also modulate lysine methylation, a non-redox post-translational modification, via the regulation of SMYD1 expression. Such crosstalk between redox signaling and a non-redox PTM regulation may provide novel insights into the functions of Trx1 that are independent from its immediate function as a protein reductase.