New 4H-chromen-4-one and 2H-chromene derivatives as anti-picornavirus capsid-binders

Substituted (E)-3-styryl-4H-chromen-4-ones 1a–d, 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones 2a–d, (E)-3-styryl-2H-chromenes 3a–d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes 4a–d were designed and synthesized to improve the anti-picornavirus activity of previously tested analogues. The new compounds were evaluated in vitro against human rhinovirus (HRV) serotypes 1B and 14 and enterovirus (EV) 71. All the compounds interfered with the replication of picornaviruses, although considerable differences were observed in the sensitivity of viruses to each compound. Generally, both HRVs were more susceptible than EV71 and their sensitivity was dependent upon the linker chain length as well as upon the oxidation state of the heterocyclic ring. (E)-3-Styryl-2H-chromene (3a) emerged as the most effective inhibitor of both HRVs showing IC₅₀ values of 0.20 μM and 1.38 μM towards serotype 1B and 14, respectively. The potent activity was also coupled with low cytotoxicity resulting in high therapeutic indexes (250 and 36, respectively). Mechanism of action studies indicated that 3a, like structurally related compounds, behaves as a capsid binder interfering with the early stages of rhinovirus infection, probably at the adsorption and/or uncoating level.     

Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999     

Dietary Total Antioxidant Capacity and Colorectal Cancer in the Italian EPIC Cohort

Background

Colorectal cancer is the third most common cancer worldwide. Diet has been hypothesized as involved in colorectal cancer etiology, but few studies on the influence of total dietary antioxidant intake on colorectal cancer risk have been performed.

Methods

We investigated the association between colorectal cancer risk and the total antioxidant capacity (TAC) of the diet, and also of intake of selected antioxidants, in 45,194 persons enrolled in 5 centers (Florence, Naples, Ragusa, Turin and Varese) of the European Prospective Investigation into Cancer and Nutrition (EPIC) Italy study. TAC was estimated by the Trolox equivalent antioxidant capacity (TEAC) assay. Hazard ratios (HRs) for developing colorectal cancer, and colon and rectal cancers separately, adjusted for confounders, were estimated for tertiles of TAC by Cox modeling, stratifying by center.

Results

Four hundred thirty-six colorectal cancers were diagnosed over a mean follow-up of 11.28 years. No significant association between dietary TAC and colorectal cancer incidence was found. However for the highest category of TAC compared to the lowest, risk of developing colon cancer was lower (HR: 0.63; 95% CI: 0.44–0.89, P trend: 0.008). By contrast, increasing TAC intake was associated with significantly increasing risks of rectal cancer (2nd tertile HR: 2.09; 95%CI: 1.19–3.66; 3rd tertile 2.48 95%CI: 1.32–4.66; P trend 0.007). Intakes of vitamin C, vitamin E, and ß-carotene were not significantly associated with colorectal cancer risk.

Conclusions

Further prospective studies are needed to confirm the contrasting effects of high total antioxidant intake on risk of colon and rectal cancers.     

MeCP2 Affects Skeletal Muscle Growth and Morphology through Non Cell-Autonomous Mechanisms

Rett syndrome (RTT) is an autism spectrum disorder mainly caused by mutations in the X-linked MECP2 gene and affecting roughly 1 out of 10.000 born girls. Symptoms range in severity and include stereotypical movement, lack of spoken language, seizures, ataxia and severe intellectual disability. Notably, muscle tone is generally abnormal in RTT girls and women and the Mecp2-null mouse model constitutively reflects this disease feature. We hypothesized that MeCP2 in muscle might physiologically contribute to its development and/or homeostasis, and conversely its defects in RTT might alter the tissue integrity or function. We show here that a disorganized architecture, with hypotrophic fibres and tissue fibrosis, characterizes skeletal muscles retrieved from Mecp2-null mice. Alterations of the IGF-1/Akt/mTOR pathway accompany the muscle phenotype. A conditional mouse model selectively depleted of Mecp2 in skeletal muscles is characterized by healthy muscles that are morphologically and molecularly indistinguishable from those of wild-type mice raising the possibility that hypotonia in RTT is mainly, if not exclusively, mediated by non-cell autonomous effects. Our results suggest that defects in paracrine/endocrine signaling and, in particular, in the GH/IGF axis appear as the major cause of the observed muscular defects. Remarkably, this is the first study describing the selective deletion of Mecp2 outside the brain. Similar future studies will permit to unambiguously define the direct impact of MeCP2 on tissue dysfunctions.     

A novel LMNA mutation (R189W) in familial dilated cardiomyopathy: evidence for a 'hot spot' region at exon 3: a case report

Background

To assess the reliability of fetal heart volume measurement by three-dimensional sonography (3DUS) using the eXtended Imaging Virtual Organ Computer-aided AnaLysis (XI VOCAL) method.

Methods

This reliability study enrolled 30 pregnant women with singleton healthy pregnancies between 19 and 34 weeks of gestation. All volume acquirements were performed with a convex volumetric transducer (C3-7ED) coupled to an Accuvix XQ sonography device (Medison, Korea). The XI VOCAL 10 planes was the method of choice for volumetric measurement. 3D datasets were analyzed by two observers (EQSB and HJFM); fetal heart volume was measured twice by the first and once by the second observer to calculate intra and interobserver reproducibility. Statistical analysis used pareated Student's t test (p) and calculated Intraclass correlation coefficients (ICC). Bland-Altman plots were also constructed.

Results

We observed an excellent intra- and interobserver reliability for fetal cardiac volume assessed by XI VOCAL. For the intraobserver the ICC was 0.998 (95% CI: 0.997; 0.999), with mean of differences of 0.12 cm³ (95% limits of agreement: -0.84; +0.84; p = 0.130). For interobserver the ICC was 0.899 (95%CI: 0.996; 0.998), mean of differences 0.05 cm³ (95% limits of agreement: -0.84; +0.84; p = 0.175).

Conclusion

Fetal cardiac volume assessed by 3DUS using XI VOCAL method is highly reproducible between 19 to 34 gestational weeks.     

The peculiar binding properties of 4''-deoxy,4''-iododoxorubicin to isolated DNA and 175 bp nucleosomes.

The thermodynamic parameters governing the interaction of 4'-deoxy,4'-iododoxorubicin (4'-IAM) to double stranded DNA or 175 bp nucleosomes have been evaluated at different ionic strength and temperature conditions by means of fluorescence techniques. The iodo-anthracycline exhibits quite different characteristics from the parent compounds adriamycin (AM) and daunomycin (DM) and other second generation derivatives. In fact, the contribution of electrostatic interactions to the total free energy of binding is rather poor and the changes in enthalpy, usually high and negative, are low and eventually positive. Unlike other members of its family, 4'-IAM exhibits preference for the nucleosomal structure. In addition, its binding to isolated DNA is remarkably cooperative. Circular dichroism studies show changes in the geometry of the intercalation complex when the drug binds to nucleosomes. The possibility for the iodo-sugar moiety to act as an alkylating or free-radical producing species was also considered as an alternative mechanism of action. However, no evidence was obtained to support these hypotheses. Thus the major differences observed in DNA-binding in comparison to parent anthracyclines appear to be mostly related to the lower pKa and higher lipophilicity exhibited by 4'-IAM.     

Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.     

The Saccharomyces cerevisiae PDS1 and RAD9 checkpoint genes control different DNA double-strand break repair pathways

In response to DNA damage, the Saccharomyces cerevisiae securin Pds1 blocks anaphase promotion by inhibiting ESP1-dependent degradation of cohesins. PDS1 is positioned downstream of the MEC1- and RAD9-mediated DNA damage-induced signal transduction pathways. Because cohesins participate in postreplicative repair and the pds1 mutant is radiation sensitive, we identified DNA repair pathways that are PDS1-dependent. We compared the radiation sensitivities and recombination phenotypes of pds1, rad9, rad51 single and double mutants, and found that whereas pds1 rad9 double mutants were synergistically more radiation sensitive than single mutants, pds1 rad51 mutants were not. To determine the role of PDS1 in recombinational repair pathways, we measured spontaneous and DNA damage-associated sister chromatid exchanges (SCEs) after exposure to X rays, UV and methyl methanesulfonate (MMS) and after the initiation of an HO endonuclease-generated double-strand break (DSB). The rates of spontaneous SCE and frequencies of DNA damage-associated SCE were similar in wild type and pds1 strains, but the latter exhibited reduced viability after exposure to DNA damaging agents. To determine whether pds1 mutants were defective in other pathways for DSB repair, we measured both single-strand annealing (SSA) and non-homologous end joining (NHEJ) in pds1 mutants. We found that the pds1 mutant was defective in SSA but efficient at ligating cohesive ends present on a linear plasmid. We therefore suggest that checkpoint genes control different pathways for DSB repair, and PDS1 and RAD9 have different roles in recombinational repair.     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)