Galloflavin prevents the binding of lactate dehydrogenase A to single stranded DNA and inhibits RNA synthesis in cultured cells.

Lactate dehydrogenase A (LDH-A) binds single stranded DNA (ssDNA) and stimulates cell transcription. Binding is prevented by NADH, suggesting that the coenzyme site is involved in the interaction LDHA/ ssDNA. We recently identified an inhibitor of LDH-A enzymatic activity (Galloflavin, GF) which occupies the NADH site. In the experiments reported here we studied whether GF can also hinder the binding of LDH-A to ssDNA and investigated its effects on RNA synthesis in cultured cells. Using a filter binding assay we observed that 4 uM GF inhibited the binding of human LDH-A to a single stranded [3H]DNA sample by 50%. After only 0.5\u20131 h, 50\u2013100 uM GF inhibited RNA synthesis in SW620 cells maintained in a medium in which galactose substituted glucose. In these culture conditions, SW620 cells did not produce lactic acid and effects caused by the inhibition of the enzymatic activity of LDH-A could be excluded. Novel LDH-A inhibitors which hinder aerobic glycolysis of cancer cells are at present actively searched. Our results suggest that: (i) inhibitors which bind the NADH site can exert their antiproliferative activity not only by blocking aerobic glycolysis but also by causing an inhibition of RNA synthesis independent from the effect on glycolysis; (ii) GF can be a useful tool to study the biological role of LDH-A binding to ssDNA

A Ligand of Retinoid X Receptor Overcome Multidrug Resistance in Human Colon Carcinoma LoVomdr Cell Line

Multidrug resistance (MDR) is one of the major causes of failure of tumoral chemotherapy and it is often present in colorectal cancer. Some retinoids may have antitumoral effects also in MDR cancer cell lines. We investigated the anticancer effect of the all-trans retinoic acid (RA) and the 6-OH-11-O-hydroxyphenantrene (IIF), a specific ligand of Retinoid X Receptor (RXR), on a colon carcinoma doxorubicin resistant cell line (LoVoMDR). IIF and RA treatment inhibited proliferation and vitality and induced apoptosis in a time and dose dependent manner. Interestingly, IIF was significantly more effective than RA and in particular, IIF increased Bax and decreased Bcl-2 protein expression, as well as it inducer caspase-9, caspase-3 and PARP-1 cleavage after 24 hours of exposure. Furthermore, we studied the effects of these retinoids on two plasma membrane proteins that are involved in the mechanism of drug-resistance: Multidrug Resistance Protein-1 (MRP-1) and P-glycoprotein (P-gp). Expression and activity of P-gp, but not of MRP-1, was decreased by retinoids. Matrix Metalloproteinases (MMPs) -2 and -9, involved in cell invasion and metastasis, and Extracellular Matrix Metalloproteinase Inducer (EMMPRIN), a glycoprotein able to activate MMPs, were significantly reduced after IIF treatment. In conclusion we can suggest that IIF could be used to overcome MDR in human colon carcinoma and it may be a powerful tool in the development of cancer therapies

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. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1\u3b2, TNF\u3b1, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1\u3b2) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1\u3b2, TNF\u3b1), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children

Shiga toxin 1 and ricin A chain bind to human polymorphonuclear leucocytes through a common receptor

The main cause of acute renal failure in children is HUS (haemolytic uraemic syndrome), a consequence of intestinal infections with Escherichia coli strains producing Stx (Shiga toxins). Stx released in the gut by the non-invasive bacteria reach the bloodstream and are targeted to cerebral and renal endothelium triggering HUS. PMN (polymorphonuclear leucocytes) seem to be involved in Stx delivery through an unidentified membrane receptor (Kd =10 nM; 200,000 binding sites) which does not allow internalization. Some experts in the field have defined the Stx\u2013PMN interaction as non-specific and of little biological significance. In the present study, we show that the A chain of ricin, the well-known plant RIP (ribosome-inactivating protein), interacts with PMN (Kd =1 nM; 200,000 binding sites) competing for the same receptor that recognizes Stx, whereas diphtheria toxin and several agonists of TLRs (Toll-like receptors) or the mannose receptor were ineffective. No toxic effects of ricin A chain on PMN were observed, as assessed bymeasuring protein synthesis and the rate of spontaneous apoptosis of leucocytes. Moreover, two single-chain RIPs (gelonin and saporin S6) had the same competing effect. Thus RIPs and Stx1 share structural similarities, the same enzymatic activity and a common receptor on PMN. These observations reveal that the Stx\u2013PMN interaction is specific, confirming that PMN recognize molecular patterns common to different foreign molecules

A deletion in the N gene may cause diagnostic escape in SARS-CoV-2 samples

Five SARS-CoV-2-positive samples showed N-gene drop-out with a RT-PCR multiplex test. WGS found all samples to harbor a deletion in the same region of the N gene, which is likely to impair the efficiency of amplification. This highlights the need for a continued surveillance of viral evolution and diagnostic test performance

Identification of TLR4 as the Receptor That Recognizes Shiga Toxins in Human Neutrophils.

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin\u2013producing infections is a worldwide healthproblem, as dramatically exempli\ufb01ed by the German outbreak occurred in summer 2011 and by a constant burden of cases inchildren. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globo-triaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstratedin vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulatingtoxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in differentstudies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identi\ufb01ed. Inthis study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respec-tively), we have identi\ufb01ed TLR4 as the receptor that speci\ufb01cally recognizes Stx1 and Stx2 in human neutrophils. Accordingly,these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed thesame pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminatingLPS in Stx-binding and activation of neutrophils. The identi\ufb01cation of the Stx-receptor on neutrophils provides additionalelements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development oftherapeutic strategie