The production of high‐quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents ...a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off‐pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.
The lack of symptoms at the early stages of clear cell renal cell carcinoma (ccRCC) allows the tumour to metastasize, leading to a dramatic reduction in patient survival. Therefore, we studied and ...set up a method based on urinary microRNAs (miRNAs) for the diagnosis of ccRCC. First, miRNA expression in ccRCC specimens and kidney tissues from healthy subjects (HSs) was investigated through analysis of data banks and validated by comparing expression of miRNAs in ccRCC and adjacent non-cancerous kidney tissue specimens by RT-qPCR. Subsequently, we developed an algorithm to establish which miRNAs are more likely to be found in the urine of ccRCC patients that indicated miR-122, miR-1271, and miR-15b as potential interesting markers. The evaluation of their levels and three internal controls in the urine of 13 patients and 14 HSs resulted in the development of a score (7p-urinary score) to evaluate the presence of ccRCC in patients. The resulting area under the Receiver Operating Characteristic (ROC) curve, sensitivity, and specificity were equal to 0.96, 100% (95% CI 75-100%), and 86% (95% CI 57-98%), respectively. In conclusion, our study provides a proof of concept that combining the expression values of some urinary miRNAs might be useful in the diagnosis of ccRCC.
Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher organisms, and that is involved in multiple plant processes, including germination, root growth, and ...fertility. Regulation of NO-levels is predominantly achieved by reaction of oxidation products of NO with glutathione to form
-nitrosoglutathione (GSNO), the principal bioactive form of NO. The enzyme
-nitrosoglutathione reductase (GSNOR) is a major route of NADH-dependent GSNO catabolism and is critical to NO homeostasis. Here, we performed a proteomic analysis examining changes in the total leaf proteome of an
GSNOR null mutant (
). Significant increases or decreases in proteins associated with chlorophyll metabolism and with redox and stress metabolism provide insight into phenotypes observed in
plants. Importantly, we identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9. Because specific AKRs have been linked to NO metabolism in mammals, we expressed and purified
AKR4C8 and 9 and close homologs AKR4C10 and 11 and determined that they have NADPH-dependent activity in GSNO and
-nitroso-coenzyme A (SNO-CoA) reduction. Further, we found an increase of NADPH-dependent GSNO reduction activity in
mutant plants. These data uncover a new, NADPH-dependent component of NO metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in plants.
Flexibility of plant metabolism is supported by redox regulation of enzymes via posttranslational modification of cysteine residues, especially in plastids. Here, the redox states of cysteine ...residues are partly coupled to the thioredoxin system and partly to the glutathione pool for reduction. Moreover, several plastid enzymes involved in reactive oxygen species (ROS) scavenging and damage repair draw electrons from glutathione. In addition, cysteine residues can be post-translationally modified by forming a mixed disulfide with glutathione (S-glutathionylation), which protects thiol groups from further oxidation and can influence protein activity. However, the evolution of the plastid glutathione-dependent redox network in land plants and the conservation of cysteine residues undergoing S-glutathionylation is largely unclear. We analysed the genomes of nine representative model species from streptophyte algae to angiosperms and found that the antioxidant enzymes and redox proteins belonging to the plastid glutathione-dependent redox network are largely conserved, except for lambda- and the closely related iota-glutathione S-transferases. Focussing on glutathione-dependent redox modifications, we screened the literature for target thiols of S-glutathionylation, and found that 151 plastid proteins have been identified as glutathionylation targets, while the exact cysteine residue is only known for 17% (26 proteins), with one or multiple sites per protein, resulting in 37 known S-glutathionylation sites for plastids. However, 38% (14) of the known sites were completely conserved in model species from green algae to flowering plants, with 22% (8) on non-catalytic cysteines. Variable conservation of the remaining sites indicates independent gains and losses of cysteines at the same position during land plant evolution. We conclude that the glutathione-dependent redox network in plastids is highly conserved in streptophytes with some variability in scavenging and damage repair enzymes. Our analysis of cysteine conservation suggests that S-glutathionylation in plastids plays an important and yet under-investigated role in redox regulation and stress response.
•MiRNAs are involved in key steps of prostate cancer development and progression.•MiRNAs act as oncogenic or oncosuppressor factor in cancerogenesis.•MiRNAs as potential biomarkers for diagnosis and ...prognosis of prostate cancer.•MiRNAs could play a relevant role in targeted therapy.
Many different genetic alterations, as well as complex epigenetic interactions, are the basis of the genesis and progression of prostate cancer (CaP). This is the reason why until now the molecular pathways related to development of this cancer were only partly known, and even less those that determine aggressive or indolent tumour behaviour. MicroRNAs (miRNAs) represent a class of about 22 nucleotides long, small non-coding RNAs, which are involved in gene expression regulation at the post-transcriptional level. MiRNAs play a crucial role in regulating several biological functions and preserving homeostasis, as they carry out a wide modulatory activity on various molecular signalling pathways. MiRNA genes are placed in cancer-related genomic regions or in fragile sites, and they have been proven to be involved in the main steps of carcinogenesis as oncogenes or oncosuppressors in many types of cancer, including CaP. We performed a narrative review to describe the relationship between miRNAs and the crucial steps of development and progression of CaP. The aims of this study were to improve the knowledge regarding the mechanisms underlying miRNA expression and their target genes, and to contribute to understanding the relationship between miRNA expression profiles and CaP.
Purpose
Urinary incontinence (UI) is one of the most bothersome surgical side effects after robot-assisted radical prostatectomy (RARP). Alteration of both smooth and striate urethral sphincter ...occurs after RARP. Since the contraction of perineal muscles is involved in the complex mechanism of urinary continence, the uroflow stop test (UST) seems to predict early continence recovery after RARP. The UST was added of perineal surface electromyography (EMG) evaluation to evaluate the latency time (LT) between muscular contraction and the complete stop of urine flow. Our aim was to present UST–EMG as a new tool in the investigation of UI, implying pelvic floor integrity, and to evaluate whether an early return (≤ 3 months) to preoperative LT could be associated with early recovery of UC (≤ 3 months).
Methods
After prospectively enrolling 137 patients who underwent RARP with a minimum follow-up of 6 months, we identified two groups: Group 1 (93 patients) with early (≤ 3 months) pre-surgical LT recovery and Group 2 (44 patients) with late > 3 months or not able to reach pre-surgical LT recovery.
Results
Between the two groups, there were significant differences in terms of different surgical technique (nerve-sparing versus non-nerve-sparing) and pads use at 3 and 6 months after RARP (
p
< 0.05). No differences were recorded neither in postoperative maximum flow-rate, nor in postoperative symptoms score.
Conclusions
UST with EMG can be considered as a noninvasive, reproducible and easily performable tool. LT is applicable in the clinical evaluation to predict urinary continence recovery after RARP.
Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational ...protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens.
While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation.
Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.
One sentence summary: Removal of class I GRX activity in the chloroplast stroma of P. patens kinetically uncouples GRX-dependent cysteine redox changes from the local glutathione redox potential, without an effect on non-photochemical quenching or photosynthetic carbon reactions.
Display omitted
•Stromal GRXC5 keeps target Cys near thermodynamic equilibrium with steady state EGSH.•Δgrxc5 lines reveal negligible cross-talk with metabolic regulation via the TRX system.•GRXC5 is not a major factor for phenotypes induced by lacking GSSG reduction.•Lack of GRXC5 is advantageous after thiol-specific oxidation.
The main benefits of dual clutch transmissions (DCTs) are: (i) a higher energy efficiency than automatic transmission systems with torque converters; and (ii) the capability to fill the torque gap ...during gear shifts to allow seamless longitudinal acceleration profiles. Therefore, DCTs are viable alternatives to automated manual transmissions (AMTs). For vehicles equipped with engines that can generate considerable torque, large clutch-slip energy losses occur during power-on gear shifts and, as a result, DCTs need wet clutches for effective heat dissipation. This requirement substantially reduces DCT efficiency because of the churning and ancillary power dissipations associated with the wet clutch pack. To the knowledge of the authors, this study is the first to analyse the detailed power loss contributions of a DCT with wet clutches, and their relative significance along a set of driving cycles. Based on these results, a novel hybridised AMT (HAMT) with a single dry clutch and an electric motor is proposed for the same vehicle. The HAMT architecture combines the high mechanical efficiency typical of AMTs with a single dry clutch, with the torque-fill capability and operational flexibility allowed by the electric motor. The measured efficiency maps of a case study DCT and HAMT are compared. This is then complemented by the analysis of the respective fuel consumption along the driving cycles, which is simulated with an experimentally validated vehicle model. In its internal combustion engine mode, the HAMT reduces fuel consumption by >9% with respect to the DCT.
Background: PD-L1 represents a crucial immune checkpoint molecule in the tumor microenvironment, identified as a key target for cancer immunotherapy. A correlation between PD-L1 and EMT-related genes ...expression in various human cancers has been suggested. Methods: By ScreenCell filtration, digital droplet PCR and confocal microscopy analysis, we aimed to investigate the expression of PD-L1 and EMT/invasive genes (TWIST1, ZEB1, VIMENTIN, TIMP2) in circulating tumor cells (CTCs) collected from the blood of non-muscle-invasive bladder cancer (NMIBC) patients, assessing the prognostic value of these biomarkers in the disease. Welchs’ test and Mann–Whitney U test, correlation index, Kaplan–Meier, Univariate and Multivariate Cox hazard proportional analysis were used. Results: Higher PD-L1, TIMP2 and VIM mRNA levels were found in pT1 compared to pTa NMIBC. As evaluated by Kaplan–Meier and Univariate and Multivariate Cox analysis, enhancement of PD-L1, TWIST1 and TIMP2 expression reduces the recurrent free survival in NMIBC patients. Conclusions: High PD-L1, TWIST1 and TIMP2 mRNAs mark the recurrent-NMIBC patients and by reducing the RFS represent negative prognostic biomarkers in these patients.
S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological ...level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.
PDF
