Peptides inhibiting the assembly of monomeric human l-lactate dehydrogenase into catalytically active homotetramer decrease the synthesis of lactate in cultured cells.

The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A).

Detection of Cleaved Stx2a in the Blood of STEC-Infected Patients.

Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently.

Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells.

Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome.

Shiga toxin (Stx)-producing is the predominant offending agent of post-diarrheal hemolytic uremic syndrome (HUS), a rare disorder of microvascular thrombosis and acute kidney injury possibly leading to long-term renal sequelae. We previously showed that C3a has a critical role in the development of glomerular damage in experimental HUS. Based on the evidence that activation of C3a/C3a receptor (C3aR) signaling induces mitochondrial dysregulation and cell injury, here we investigated whether C3a caused podocyte and tubular injury through induction of mitochondrial dysfunction in a mouse model of HUS.

Allosteric transitions of rabbit skeletal muscle lactate dehydrogenase induced by pH-dependent dissociation of the tetrameric enzyme.

Among the functions exerted by eukaryotic lactate dehydrogenases, it is of importance the generation of lactate in muscles subjected to fatigue or to limited oxygen availability, with both these conditions triggering a decrease of cellular pH. However, the mutual dependence between lactate dehydrogenase (LDH) catalytic action and lactic acidosis is far from being fully understood. Here we show that the tetrameric LDH from rabbit skeletal muscle undergoes allosteric transitions as a function of pH, i.

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g.

Plasmonic Metasurfaces for Specific SERS Detection of Shiga Toxins.

The interest in the development of nanoscale plasmonic technologies has dramatically increased in recent years. The photonic properties of plasmonic nanopatterns can be controlled and tuned via their size, shape, or the arrangement of their constituents. In this work, we propose a 2D hybrid metallic polymeric nanostructure based on the octupolar framework with enhanced sensing property.

Bloody Diarrhea and Shiga Toxin-Producing Escherichia coli Hemolytic Uremic Syndrome in Children: Data from the ItalKid-HUS Network.

Objective: To analyze the results of an enhanced laboratory-surveillance protocol for bloody diarrhea aimed at identifying children with Shiga toxin-producing Escherichia coli (STEC) infection early in the course of the disease toward the early identification and management of patients with hemolytic uremic syndrome (HUS).

Study Design: The study (2010-2019) involved a referral population of 2.3 million children.

Extracellular Vesicles and Renal Endothelial Cells: A Fatal Attraction in Hemolytic Uremic Syndrome.

This review focuses on typical hemolytic uremic syndrome (HUS), a life-threatening sequela of human infections caused, particularly in children, by Shiga toxin-producing Escherichia coli strains. Thrombotic microangiopathy of the brain and the kidney is the end point of toxin action, resulting in the hallmarks of HUS (ie, thrombocytopenia, anemia, and acute renal failure). A growing body of evidence points to the role of extracellular vesicles released in the blood of patients by toxin-challenged circulating cells (monocytes, neutrophils, and erythrocytes) and platelets, as a key factor in the pathogenesis of HUS.

Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum.

The pathogenesis of -induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum proteins) whose interaction is crucial for the immediate outcome and for the development of this life-threatening sequela.

Is Shigatoxin 1 protective for the development of Shigatoxin 2-related hemolytic uremic syndrome in children? Data from the ItalKid-HUS Network.

Background: Shigatoxin (Stx)-producing Escherichia coli (STEC) are the most common causes of hemolytic uremic syndrome (STEC-HUS). The aim of our study is to compare the risk of developing STEC-HUS in relation to the type of Stx genes (Stx1, Stx2, or both).

Methods: This is a prospective, observational, multicenter study involving 63 pediatric units in Northern Italy (ItalKid-HUS Network).

Particulate Shiga Toxin 2 in Blood is Associated to the Development of Hemolytic Uremic Syndrome in Children.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear.

Deuterium Incorporation Protects Cells from Oxidative Damage.

In the cold environments of the interstellar medium, a variety of molecules in which a hydrogen (H) atom has been replaced by its heavier isotope deuterium (D) can be found. From its emergence, life had to counteract the toxic action of many agents, which posed a constant threat to its development and propagation. Oxygen-reactive species are archaic toxicants that lead to protein damage and genomic instability.

Hemidesmus indicus induces apoptosis via proteasome inhibition and generation of reactive oxygen species.

Proteasome inhibition represents an important anticancer strategy. Here, we studied the mechanisms at the basis of the pro-apoptotic activity of the standardized decoction of Hemidesmus indicus, a plant evoking a complex anticancer activity, and explored its inhibition of proteasome activity in human leukemia cells. Additionally, we preliminary tested the cytotoxicity of some H.

The structure of the Shiga toxin 2a A-subunit dictates the interactions of the toxin with blood components.

Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)-producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A-subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities.

Soluble Toll-Like Receptor 4 Impairs the Interaction of Shiga Toxin 2a with Human Serum Amyloid P Component.

Shiga toxin 2a (Stx2a) is the main virulence factor produced by pathogenic strains (Stx-producing , STEC) responsible for hemorrhagic colitis and the life-threatening sequela hemolytic uremic syndrome in children. The toxin released in the intestine by STEC targets the globotriaosylceramide receptor (Gb3Cer) present on the endothelial cells of the brain and the kidney after a transient blood phase during which Stx2a interacts with blood components, such as neutrophils, which, conversely, recognize Stx through Toll-like receptor 4 (TLR4). Among non-cellular blood constituents, human amyloid P component (HuSAP) is considered a negative modulating factor that specifically binds Stx2a and impairs its toxic action.

Human monocytes stimulated by Shiga toxin 1a via globotriaosylceramide release proinflammatory molecules associated with hemolytic uremic syndrome.

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells.

The inhibition of lactate dehydrogenase A hinders the transcription of histone 2B gene independently from the block of aerobic glycolysis.

Most cancer cells use aerobic glycolysis to fuel their growth and many efforts are made to selectively block this metabolic pathway in cancer cells by inhibiting lactate dehydrogenase A (LDHA). However, LDHA is a moonlighting protein which exerts functions also in the nucleus as a factor associated to transcriptional complexes. Here we found that two small molecules which inhibit the enzymatic activity of LDHA hinder the transcription of histone 2B gene independently from the block of aerobic glycolysis.

Cap-independent protein synthesis is enhanced by betaine under hypertonic conditions.

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged.

A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes.

We describe a cell-free translation system for evaluating the activity of ribosomes stringently purified from human cells. This system is based on in vitro reconstitution of the cellular translation machinery, in which a ribosome-free rabbit reticulocyte lysate (RRL) is reassembled with human ribosomes and in vitro-transcribed reporter mRNAs. The protocol describes the preparation of the RRL-derived fractions, purification of ribosomes devoid of detectable nonribosomal-associated factors, and assembly of the reactions to evaluate ribosomal translational efficiency and fidelity using appropriate reporter transcripts.

The Antibiotic Polymyxin B Impairs the Interactions between Shiga Toxins and Human Neutrophils.

Hemolytic uremic syndrome (HUS) is the life-threatenig sequela of intestinal infections by Shiga toxin (Stx)-producing Escherichia coli (STEC) in children. Human neutrophils specifically bind Stx through TLR4, the receptor of LPS. The binding could be considered protective (Stx sequestration) or harmful (toxin delivery to target organs).

Early Volume Expansion and Outcomes of Hemolytic Uremic Syndrome.

Background: Hemolytic uremic syndrome associated with Shiga toxin-producing Escherichia coli (STEC-HUS) is a severe acute illness without specific treatment except supportive care; fluid management is concentrated on preventing fluid overload for patients, who are often oligoanuric. Hemoconcentration at onset is associated with more severe disease, but the benefits of volume expansion after hemolytic uremic syndrome (HUS) onset have not been explored.

Methods: All the children with STEC-HUS referred to our center between 2012 and 2014 received intravenous infusion targeted at inducing an early volume expansion (+10% of working weight) to restore circulating volume and reduce ischemic or hypoxic tissue damage.

A rapid and sensitive method to measure the functional activity of Shiga toxins in human serum.

Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera.