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The genome of Hepatitis E virus (HEV) is 7.2 kilobases long and has three open reading frames. The largest one is ORF1, encoding a non-structural protein involved in the replication process, and whose processing is ill-defined. The ORF1 protein is a multi-modular protein which includes a macro domain (MD). MDs are evolutionarily conserved structures throughout all kingdoms of life. MDs participate in the recognition and removal of ADP-ribosylation, and specifically viral MDs have been identified as erasers of ADP-ribose moieties interpreting them as important players at escaping the early stages of host-immune response. A detailed structural analysis of the apo and bound to ADP-ribose state of the native HEV MD would provide the structural information to understand how HEV MD is implicated in virus-host interplay and how it interacts with its intracellular partner during viral replication. In the present study we present the high yield expression of the native macro domain of HEV and its analysis by solution NMR spectroscopy. The HEV MD is folded in solution and we present a nearly complete backbone and sidechains assignment for apo and bound states. In addition, a secondary structure prediction by TALOS + analysis was performed. The results indicated that HEV MD has a α/β/α topology very similar to that of most viral macro domains.

Soluble guanylate cyclase (sGC) is considered as the primary NO receptor across several known eukaryotes. The main interest regarding the biological role and its function, focuses on the H-NOX domain of the β1 subunit. This domain in its active form bears a ferrous b type heme as prosthetic group, which facilitates the binding of NO and other diatomic gases. The key point that still needs to be answered is how the protein selectively binds the NO and how the redox state of heme and coordination determines H-NOX active state upon binding of diatomic gases. H-NOX domain is present in the genomes of both prokaryotes and eukaryotes, either as a stand-alone protein domain or as a partner of a larger polypeptide. The biological functions of these signaling modules for a wide range of genomes, diverge considerably along with their ligand binding properties. In this direction, we examine the prokaryotic H-NOX protein domain from Nostoc punctiforme (Npun H-NOX). Herein, we first report the almost complete NMR backbone and side-chain resonance assignment (1H, 13C, 15 N) of Npun H-NOX domain together with the NMR chemical shift-based prediction of the domain’s secondary structure elements.

SARS-CoV and MERS-CoV Macro Domains (MDs) exhibit topological and conformational features that resemble the nsP3b macro (or “X”) domain of SARS-CoV-2. Indeed, all the three domains (SARS-CoV-2, SARS-CoV and MERS-CoV MDs) fold in a three-layer α/β/α sandwich structure, as reported by crystallographic structural investigation of SARS-CoV MD and MERS-CoV MD. These viral MDs are able to bind ADP-ribose as many other MDs from different kingdoms. They have been characterized also as de-ADP-ribosylating enzymes. For this reason, these viral macrodomains recently emerged as important drug targets since they can counteract antiviral ADP-ribosylation mediated by poly-ADP-ribose polymerase (PARPs). Even in presence of the 3D structures of SARS-CoV MD and of MERS-CoV MD, we report herein the almost complete NMR backbone (1H, 13C, 15N) of SARS-CoV MD and MERS-CoV proteins in the free and ADPr bound forms, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will help to further understanding of the atomic-level conformational dynamics of these proteins and will allow an extensive screening of small molecules as potential antiviral drugs.

In this study, non-targeted 1 H NMR fingerprinting was used in combination with multivariate statistical analyses for the classification of Greek currants based on their geographical origins (Aeghion, Nemea, Kalamata, Zante, and Amaliada). As classification techniques, Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were carried out. To elucidate different components according to PDO (Protected Designation of Origin), products from Aeghion (Vostizza) were statistically compared with each one of the four other regions. PLS-DA plots ensure that currants from Kalamata, Nemea, Zante, and Amaliada are well classified with respect to the PDO currants, according to differences observed in metabolites. Results suggest that composition differences in carbohydrates, amino, and organic acids of currants are sufficient to discriminate them in correlation to their geographical origin. In conclusion, currants metabolites which mostly contribute to classification performance of such discriminant analysis model present a suitable alternative technique for currants traceability. The study results contribute information to the currants' metabolite fingerprinting by NMR spectroscopy and their geographical origin. PRACTICAL APPLICATION: This study presents an analytical approach for a high nutritional value Greek PDO product, Vostizza currant. A further research and implementation of this method in food industry, can be the key to food fraud incidents. Thus, application of this work opens up posibilities to "farm to table" mission.

Introduction Premature adrenarche (PA) for long time was considered a benign condition but later has been connected to various diseases in childhood and adulthood which remains controversial. Objective To investigate the effect of premature adrenarche on the metabolic phenotype, and correlate the clinical and biochemical data with the metabolic profile of children with PA. Methods Nuclear magnetic resonance (NMR)-based untargeted and targeted metabolomic approach in combination with multivariate and univariate statistical analysis applied to study the metabolic profiles of children with PA. Plasma, serum, and urine samples were collected from fifty-two children with Idiopathic PA and forty-eight age-matched controls from the division of Pediatric Endocrinology of the University Hospital of Patras were enrolled. Results Metabolomic results showed that plasma and serum glucose, myo-inositol, amino acids, a population of unsaturated lipids, and esterified cholesterol were higher and significantly different in PA children. In the metabolic profiles of children with PA and age-matched control group a gradual increase of glucose and myo-inositol levels was observed in serum and plasma, which was positively correlated their body mass index standard deviation score (BMI SDS) values respectively. Urine 1H NMR metabolic fingerprint of PA children showed positive correlation and a clustering-dependent relationship with their BMI and bone age (BA) respectively. Conclusion This study provides evidence that PA driven metabolic changes begin during the childhood and PA may has an inductive role in a BMI–driven increase of specific metabolites. Finally, urine may be considered as the best biofluid for identification of the PA metabolism as it reflects more clearly the PA metabolic fingerprint.

SUMMARYThe centriole duplication cycle must be tightly controlled and coordinated with the chromosome cycle. Aberrations in centriole biogenesis can lead to cancer, developmental disorders and ciliopathies. Here, we show that McIdas -previously implicated in cell cycle control and centriole amplification in multiciliated cells-is critical to maintain centriole numbers. Using expansion microscopy, we demonstrate that McIdas is present at the middle part of centrioles, where it exhibits a differential localization during the cell cycle. McIdas loss perturbs daughter centriole biogenesis and centrosomal SAS6 recruitment, whereas its overexpression induces centriole overduplication. Consistently, McIdas depletion reduces PLK4-induced centriole amplification. McIdas interacts with and is phosphorylated by PLK4 in multiple sites identified by mass spectrometry. Mutational analysis shows that McIdas phosphorylation is important for centriole number control. Overall, our results identify a novel, direct role of McIdas on centriole duplication that can link its previously characterized roles in the chromosome cycle and multiciliogenesis.

hPARP14 is a human ADP-ribosyl-transferase (ART) that belongs to the macroPARPs family, together with hPARP9 and hPARP15. It contains a tandem of three macro domains (MD) while each of them has different properties. The first one, namely MD1, has not been reported to exhibit a high binding affinity for ADP-ribose (ADPr) in contrast to the following two (MD2 and MD3). All three MDs exhibit an α/β/α sandwich-like fold as reported by the deposited crystallographic structures. MD2 and MD3 recognize mono-ADP-ribosylated (MARylated) but not poly-ADP-ribosylated (PARylated) substrates and thus they allow hPARP14 to bind its targets, which can be potentially MARylated by its catalytic domain (CD). hPARP14 participates in DNA damage repair process and immune response against viruses like SARS-CoV-2, which also harbors an MD fold. Furthermore, hPARP14 like the other two macroPARPs (hPARP9 and hPARP15), is implicated in numerous types of cancer, such as B-aggressive lymphoma and sarcoma, rendering its MDs as potential important drug targets. Herein, we report the complete NMR backbone and side chain assignment (1H, 13C, 15N) of hPARP14 MD2 in the free and ADPr bound states and the NMR chemical shift-based prediction of its secondary structure elements. This is the first reported NMR study of a hPARP macro domain, paving the way to screen by NMR chemical compounds which may alter the ability of hPARP14 to interact with its substrates affecting its function.

Arkadia is a positive regulator of the TGFβ-SMAD2/3 pathway, acting through its C-terminal RING-H2 domain and targeting for degradation of its negative regulators. Here we explore the role of regions outside the RING domain (non-RING elements) of Arkadia on the E2-E3 interaction. The contribution of the non-RING elements was addressed using Arkadia RING 68 aa and Arkadia 119 aa polypeptides. The highly conserved NRGA (asparagine-arginine-glycine-alanine) and TIER (threonine-isoleucine-glutamine-arginine) motifs within the 119 aa Arkadia polypeptide, have been shown to be required for pSMAD2/3 substrate recognition and ubiquitination in vivo. However, the role of the NRGA and TIER motifs in the enzymatic activity of Arkadia has not been addressed. Here, nuclear magnetic resonance interaction studies with the E2 enzyme, UBCH5B, C85S UBCH5B-Ub oxyester hydrolysis, and auto-ubiquitination assays were used to address the role of the non-RING elements in E2-E3 interaction and in the enzymatic activity of the RING. The results support that the non-RING elements including the NRGA and TIER motifs are required for E2-E3 recognition and interaction and for efficient auto-ubiquitination. Furthermore, while Arkadia isoform-2 and its close homologue Arkadia 2C are known to interact with free ubiquitin, the results here showed that Arkadia isoform-1 does not interact with free ubiquitin.

Among the proteins encoded by the SARS-CoV-2 RNA, nsP3 (non-structural Protein3) is the largest multi-domain protein. Its role is multifaceted and important for the viral life cycle. Nonetheless, regarding the specific role of each domain there are many aspects of their function that have to be investigated. SARS Unique Domains (SUDs), constitute the nsP3c region of the nsP3, and were observed for the first time in SARS-CoV. Two of them, namely SUD-N (the first SUD) and the SUD-M (sequential to SUD-N), exhibit structural homology with nsP3b (“X” or macro domain); indeed all of them are folded in a three-layer α/β/α sandwich. On the contrary, they do not exhibit functional similarities, like ADP-ribose binding properties and ADP-ribose hydrolase activity. There are reports that suggest that these two SUDs may exhibit a binding selectivity towards G-oligonucleotides, a feature which may contribute to the characterization of their role in the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral “components” with the host cell. While the structures of these domains of SARS-CoV-2 have not been determined yet, SUDs interaction with oligonucleotides and/or RNA molecules may provide a platform for drug discovery. Here, we report the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-N protein, and the NMR chemical shift-based prediction of the secondary structure elements. These data may be exploited for its 3D structure determination and the screening of chemical compounds libraries, which may alter SUD-N function.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.

The binding of p-coumaroyl glucose to glycogen phosphorylase (GP; a pharmaceutical target for the development of antihyperglycaemic drugs) has been studied by kinetics, and X-ray crystallography while its effect to HepG2 cells metabolism has been assessed by NMR metabolomics. p-Coumaroyl glucose is a potent inhibitor of human liver GP with a Ki value of 213 μΜ that binds at the active site of the enzyme. Comparative structural analysis with chemically similar GP inhibitors reveals the structural basis of its inhibitory potency. NMR metabolomics analysis revealed that HepG2 cells in the presence of p-coumaroyl glucose actively response to higher glucose uptake from their environment and a display an “insulin-sensitizing’’ state. Furthermore, NMR metabolomics analysis indicates an enhancement of gluconeogenesis towards lipid metabolism and glycerol-derived components.

Sea buckthorn berries (Hippophaë rhamnoides L.) (SB) are considered as a fruit with a high nutritional value with a plethora of bioactive ingredients. The present work focusses on the analysis of the whole NMR metabolic profile of SB berries grown in an organic orchard of Meteora/Greece. In parallel, this study validates/highlights qualitative characteristics of the osmotic processed berries according to the fresh fruit. The composition in bioactive metabolites of SB berries was elucidated through sophisticated high-resolution NMR spectroscopy. The lipophilic profile maintains the vitamins, flavonoid glycosides, phenolic esters and the essential lipid components of SB, while the polar profile reveals a variety of flavonoids, saccharides, organic acids, amino acids and esterified glycosides. This approach towards identification of SB bioactive ingredients may serve as basis for simultaneous profiling and quality assessment and may be applied to monitor fresh food quality regarding other food preservation methods.

Neural stem cells (NSCs) are important constituents of the nervous system, and they become constrained in two specific regions during adulthood: the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. The SVZ niche is a limited-space zone where NSCs are situated and comprised of growth factors and extracellular matrix (ECM) components that shape the microenvironment of the niche. The interaction between ECM components and NSCs regulates the equilibrium between self-renewal and differentiation. To comprehend the niche physiology and how it controls NSC behavior, it is fundamental to develop in vitro models that resemble adequately the physiologic conditions present in the neural stem cell niche. These models can be developed from a variety of biomaterials, along with different biofabrication approaches that permit the organization of neural cells into tissue-like structures. This review intends to update the most recent information regarding the SVZ niche physiology and the diverse biofabrication approaches that have been used to develop suitable microenvironments ex vivo that mimic the NSC niche physiology. ispartof: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY vol:9 ispartof: location:Switzerland status: published

Ubiquitination is a post-translational modification that regulates a plethora of processes in cells. Ubiquitination requires three type of enzyme: E1 ubiquitin (Ub) activating enzymes, E2 Ub conjugating enzymes and E3 ubiquitin ligases. The E2 enzymes perform a variety of functions, as Ub chain initiation, elongation and regulation of the topology and the process of chain formation. The E2 enzymes family is mainly characterized by a highly conserved ubiquitin conjugating domain (UBC), which comprises the binding region for the activated Ub, E1 and E3 enzymes. The E2 enzyme UbcH7 (UBE2L3) is a known interacting partner for different types of E3 Ub ligases such as HECT, RING and RBR. A structural analysis of the apo form of the native UbcH7 will provide the structural information to understand how this E2 enzyme is implicated in a wide range of diseases and how it interacts with its partners. In the present study we present the high yield expression of the native UbcH7 E2 enzyme and its preliminary analysis via solution NMR spectroscopy. The E2 enzyme is folded in solution and nearly a complete backbone assignment was achieved. Additionally, TALOS+ analysis was performed and the results indicated that UbcH7 adopts a αββββααα topology which is similar to that of the majority of E2 enzymes.

Neurotensin (NT) (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) exerts a dual function as a neurotransmitter/neuromodulator in the central nervous system and as a hormone/cellular mediator in periphery. This dual function of NT establishes a connection between brain and peripheral tissues that renders this peptide a central player in energy homeostasis. Many biological actions of NT are mediated through its interaction with three types of NT receptors (NTS receptors). Despite its role in energy homeostasis, NT has a short half-life that hampers further determination of the biological actions of this peptide and its receptors in brain and periphery. The short half-life of NT is due to the proteolytic degradation of its C-terminal side by several endopeptidases. Therefore, it is important to synthesize NT analogues with resistant bonds against metabolic deactivation. Based on these findings, we herein report the synthesis of ten linear, two cyclic and two dimeric analogues of NT with modifications in its structure that improve their metabolic stability, while retaining the ability to bind to NTS receptors. Modifications at position 11 (introduction of D-Tyrosine (OEthyl) [D-Tyr(Et)] or D-1-naphtylalanine [D-1-Nal] were combined with introduction of a L-Lysine or a D-Arginine at positions 8 or 9, and 1-[2-(aminophenyl)-2-oxoethyl]-1H-pyrrole-2-carboxylic acid (AOPC) at positions 7 or 8, resulting in compounds NT4-NT21. AOPC is an unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetic compounds. To biologically evaluate these analogues, we determined their plasma stability and their binding affinities to type 1 NT receptor (NTS1), endogenously expressed in HT-29 cells, Among the fourteen NT analogues, compounds, NT5, NT6, and NT8, which have D-Tyr(Et) at position 11, bound to NTS1 in a dose-response manner and with relatively high affinity but still lower than that of the natural peptide. Despite their lower binding affinities compared to NT, the NT5, NT6, and NT8 exhibited a remarkably higher stability, as a result of their chemistry, which provides protection from enzymatic activity. These results will set the basis for the rational design of novel NT molecules with improved pharmacological properties and enhanced enzymatic stability.

Simple Summary Cancer and inflammation share aberrant angiogenesis as a hallmark, and, thus, anti-angiogenetic strategies remain of key interest. Amifostine, which is already a drug on the market, may be of further benefit to patients also in the context of drug repurposing. To shed light on the anti-angiogenic properties of amifostine during human adult angiogenesis and grasp the early events of angiogenesis, we employed 3D cell untargeted metabolomics by liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy in the presence of vascular endothelial growth factor-A or deferoxamine (pro-angiogenic factors that exhibit distinct angiogenesis induction profiles). Our findings reveal mechanism-specific inhibitory profiles of amifostine against VEGF-A- and deferoxamine-induced angiogenesis. Amifostine may serve as a dual radioprotective and anti-angiogenic agent in radiotherapy patients. Abstract Aberrant angiogenesis is a hallmark for cancer and inflammation, a key notion in drug repurposing efforts. To delineate the anti-angiogenic properties of amifostine in a human adult angiogenesis model via 3D cell metabolomics and upon a stimulant-specific manner, a 3D cellular angiogenesis assay that recapitulates cell physiology and drug action was coupled to untargeted metabolomics by liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. The early events of angiogenesis upon its most prominent stimulants (vascular endothelial growth factor-A or deferoxamine) were addressed by cell sprouting measurements. Data analyses consisted of a series of supervised and unsupervised methods as well as univariate and multivariate approaches to shed light on mechanism-specific inhibitory profiles. The 3D untargeted cell metabolomes were found to grasp the early events of angiogenesis. Evident of an initial and sharp response, the metabolites identified primarily span amino acids, sphingolipids, and nucleotides. Profiles were pathway or stimulant specific. The amifostine inhibition profile was rather similar to that of sunitinib, yet distinct, considering that the latter is a kinase inhibitor. Amifostine inhibited both. The 3D cell metabolomics shed light on the anti-angiogenic effects of amifostine against VEGF-A- and deferoxamine-induced angiogenesis. Amifostine may serve as a dual radioprotective and anti-angiogenic agent in radiotherapy patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a β1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC “activators” aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules., Graphical abstract Image 1, Highlights • When the heme of Ns H-NOX is replaced by the sGC activators, the protein’s flexibility is significantly reduced. • Heme causes the conformational exchange of Ns H-NOX, as many residues around the heme adopt invisible conformation. • L-ascorbate prevents the proper action of BAY 58-2667 and BAY 60-2770 from forming a stable complex with the Ns H-NOX. • In A7r5 cells, L-ascorbate does not affect cGMP formation induced by BAY 58-2667 and it inhibits the effect of BAY 60-2770. • BAY molecules act on the H-NOX or the sGC in a bi-directional way, depending on the redox state of the heme.

In the present work a series of gadolinium porphyrin double deckers and the corresponding monoporphyrinate derivatives were investigated towards photocatalytic hydrogen evolution. The complexes were modified both on the meso- and on the beta- positions of the porphyrin macrocycle. The presence of the double decker was proved essential towards the photocatalytic activity since Gd(TPyP)2 exhibited the higher performance which was 13.8 mmolg-1h-1. A self-assembly investigation of the porphyrin derivatives revealed that different supramolecular architectures can be obtained by varying the solvent mixture and the peripheral substitution, however this did not affect the photocatalytic activity of the complexes.

The rapid progress achieved in the development of many biopharmaceuticals had a tremendous impact on the therapy of many metabolic/genetic disorders. This type of fruitful approach, called protein replacement therapy (PRT), aimed to either replace the deficient or malfunctional protein in human tissues that act either in plasma membrane or via a specific cell surface receptor. However, there are also many metabolic/genetic disorders attributed to either deficient or malfunctional proteins acting intracellularly. The recent developments of Protein Transduction Domain (PTD) technology offer new opportunities by allowing the intracellular delivery of recombinant proteins of a given therapeutic interest into different subcellular sites and organelles, such as mitochondria and other entities. Towards this pathway, we applied successfully PTD Technology as a protein therapeutic approach, in vitro, in SCO2 deficient primary fibroblasts, derived from patient with mutations in human SCO2 gene, responsible for fatal, infantile cardioencephalomyopathy and cytochrome c oxidase deficiency. In this work, we radiolabeled the recombinant TAT-L-Sco2 fusion protein with technetium-99 m to assess its in vivo biodistribution and fate, by increasing the sensitivity of detection of even low levels of the transduced recombinant protein. The biodistribution pattern of [99mTc]Tc-TAT-L-Sco2 in mice demonstrated fast blood clearance, significant hepatobiliary and renal clearance. In addition, western blot analysis detected the recombinant TAT-L-Sco2 protein in the isolated mitochondria of several mouse tissues, including heart, muscle and brain. These results pave the way to further consider this PTD-mediated Protein Therapy Approach as a potentially alternative treatment of genetic/metabolic disorders., Graphical abstract Unlabelled Image, Highlights • Radiolabeling of human recombinant mitochondrial TAT-L-Sco2 fusion protein with 99mTc for the first time. • [99mTc]Tc-TAT-L-Sco2 can be successfully transduced into the mitochondria of peripheral tissues upon injection into animals. • Protein Replacement Therapy, through PTD technology, can be a potential therapeutic approach for mitochondrial disorders.

Context Inhibitors of sodium-glucose cotransporters-2 have cardio- and renoprotective properties. However, the underlying mechanisms remain indeterminate. Objective To evaluate the effect of dapagliflozin on renal metabolism assessed by urine metabolome analysis in patients with type 2 diabetes. Design Prospective cohort study. Setting Outpatient diabetes clinic of a tertiary academic center. Patients Eighty patients with hemoglobin A1c > 7% on metformin monotherapy were prospectively enrolled. Intervention Fifty patients were treated with dapagliflozin for 3 months. To exclude that the changes observed in urine metabolome were merely the result of the improvement in glycemia, 30 patients treated with insulin degludec were used for comparison. Main Outcome Measure Changes in urine metabolic profile before and after the administration of dapagliflozin and insulin degludec were assessed by proton-nuclear magnetic resonance spectroscopy. Results In multivariate analysis urine metabolome was significantly altered by dapagliflozin (R2X = 0.819, R2Y = 0.627, Q2Y = 0.362, and coefficient of variation analysis of variance, P Conclusions Dapagliflozin significantly affects urine metabolome in patients with type 2 diabetes in a glucose lowering-independent way. Most of the observed changes can be considered beneficial and may contribute to the renoprotective properties of dapagliflozin.

Numerous molecular biological experiments performed throughout the world require the detection or quantification of a protein of interest. Western blotting is one of the most popular techniques used for this purpose and offers quantitative information with the aid of specialized software. However, its dependence on the picture that is captured, and the background and the absence of a common protocol prevent the technique from being completely quantitative. To overcome these obstacles, we present a simple and reliable assay that is similar to the regular technique, with the exception of the last stage of band visualization and quantification. We propose that small pieces of the blot that include the protein of interest can be cut and dipped in a small volume of 3,3',5,5'-tetramethylbenzidine solution, giving a colorimetric signal with linear dependence on the quantity of the protein. The reaction is stopped with H2 SO4 , and the signal is measured in a plate reader. This modification shows high linearity without additional costs and can be applied for both purified proteins and proteins found in a lysate. The results obtained with our proposed technique were compared with those obtained by the conventional method and proved to be more reliable.

La is an abundant phosphoprotein that protects polymerase III transcripts from 3'-5' exonucleolytic degradation and facilitates their folding. Consisting of the evolutionary conserved La motif (LAM) and two consecutive RNA Recognition Motifs (RRMs), La was also found to bind additional RNA transcripts or RNA domains like internal ribosome entry site (IRES), through sequence-independent binding modes which are poorly understood. Although it has been reported overexpressed in certain cancer types and depletion of its expression sensitizes cancer cells to certain chemotherapeutic agents, its role in cancer remains essentially uncharacterized. Herein, we study the effects of La overexpression in A549 lung adenocarcinoma cells, which leads to increased cell proliferation and motility. Expression profiling of several transcription and translation factors indicated that La overexpression leads to downregulation of global translation through hypophosphorylation of 4E-BPs and upregulation of IRES-mediated translation. Moreover, analysis of La localization after nutrition deprivation of the transfected cells showed a normal distribution in the nucleus and nucleoli. Although the RNA binding capacity of La has been primarily linked to the synergy between the conserved LAM and RRM1 domains which act as a module, we show that recombinant stand-alone LAM can specifically bind a pre-tRNA ligand, based on binding experiments combined with NMR analysis. We propose that LAM RNA binding properties could support the expanding and diverse RNA ligand repertoire of La, thus promoting its modulatory role, both under normal and pathogenic conditions like cancer.

Organotin(IV) derivatives of cholic acid (CAH) with the formulae R3Sn(CA) (R = Ph- (1), n-Bu- (2)) and R2Sn(CA)2 (R = Ph- (3), n-Bu- (4) and Me- (5)) were synthesized. The compounds were characterized in solid state by melting point, FT-IR, 119Sn Mossbauer, X-ray fluorescence (XRF) spectroscopy and in solution by 1H NMR, UV–Vis spectral data and by Electrospray Ionisation Mass spectrometry (ESI-MS), High Resolution Mass spectrometry (HRMS), and atomic absorption analysis. The in vitro bioactivity of 1–5 against human breast adenocarcinoma cancer cells MCF-7 (positive to hormone receptors) and MDA-MB-231 (negative to hormone receptors) reveal that triorganotin derivatives 1–2 exhibit significantly stronger activity than the corresponding diorganotin ones. Compound 5 is inactive against both cell lines at the concentrations tested. Triorganotins 1–2 inhibit selectively MCF-7 than MDA-MB-231 cells, suggesting hormone mimetic behavior of them. Organotins 1–4 inhibit both cancerous cell lines, stronger than cisplatin which rise up to 55-fold against MCF-7 and 170-fold against MDA-MB-231. The in vitro toxicity of 1–4 was evaluated on normal human fetal lung fibroblast cells (MRC-5), while their genotoxicity in vitro by micronucleus assay (MN). Moreover, the in vivo toxicity of 1–4 was tested by Artemia salina assay and their in vivo genotoxicity with Allium cepa test. The mechanism of action of 1–4 against MCF-7 was clarified in vitro by the means of cell morphology studies, cell cycle arrest, Acridine Orange/Ethidium Bromide (AO/EB) Staining, mitochondrial membrane permeabilization test and by their binding affinity toward the calf thymus (CT) DNA.

Soluble guanylate cyclase (sGC) enzyme is activated by the gaseous signaling agent nitric oxide (NO) and triggers the conversion of GTP (guanosine 5'-triphosphate) to cGMP (cyclic guanylyl monophosphate). It contains the heme binding H-NOX (heme-nitric oxide/oxygen binding) domain which serves as the sensor of NO and it is highly conserved across eukaryotes and bacteria as well. Many research studies focus on the synthesis of chemical compounds bearing possible therapeutic action, which mimic the heme moiety and activate the sGC enzyme. In this study, we report a preliminary solution NMR (Nuclear Magnetic Resonance) study of the H-NOX domain from Nostoc sp. cyanobacterium in complex with the chemical sGC activator cinaciguat (BAY58-2667). An almost complete sequence-specific assignment of its