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The red seaweed, Kappaphycus sp. is cultivated extensively as a source of carrageenans which have been used for decades in food applications and are generally regarded as safe. A deep chemical investigation has been performed on carrageenans from two species of Kappaphycus, K. alvarezii and K. striatum, by means of NMR spectroscopy technique. The NMR spectra study suggested the presence for both samples of a mixture of prevailing ?- and ?-carrageenans. Results confirmed that NMR spectroscopy represents actually the preferred technique to determine and quantify the composition of carrageenan from different algae sources. Moreover, the acetylcholinesterase (AChE) inhibition activity of different extracts of K. alvarezii and K. striatus has have been evaluated by using two different assays, thin layer chromatography (TLC) assay with Fast Blue B salt as a reagent and a multiwell plate assay based on Ellman's method. Chloroform/methanol extracts of both samples showed the best AChE inhibition activity starting from 0.01 mg ml.

Retinal gene therapy based on adeno-associated viral (AAV) vectors is safe and efficient in humans. The low intrinsic DNA transfer capacity of AAV has been expanded by dual vectors where a large expression cassette is split in two halves independently packaged in two AAV vectors. Dual AAV transduction efficiency, however, is greatly reduced compared to that obtained with a single vector. As AAV intracellular trafficking and processing are negatively affected by phosphorylation, we set to identify kinase inhibitors that can increase dual AAV vector transduction. By high throughput screening of a kinase inhibitors library, we identified 3 compounds that increase AAV transduction in vitro, one of which has higher effect on dual than on single AAV vectors. Importantly, the transduction enhancement is exerted on various AAV serotypes and is not transgene-dependent. As kinase inhibitors are promiscuous, we performed siRNA-mediated silencing of targeted kinases and identified AURKA and B, PLK1 and PTK2 among those involved in the increase of AAV transduction levels, and we show that kinase inhibitors administration reduces AAV2 capsid phosphorylation and increases the activity of DNA-repair pathways involved in AAV DNA processing. Importantly, the kinase inhibitor PF-00562271 improves dual AAV8 transduction in photoreceptors following subretinal delivery in mice. Our study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.

Marine sponges have been intensively studied by organic chemists, biologists and pharmacologists during last 60 years, due to great biodiversity of their secondary metabolites. A couple of very promising bioactivities of these compounds have been reported. Indeed, some of these metabolites are considered as the leads for the future drugs targeting tumours, viral and bacterial diseases, malaria and inflammations. Herein we briefly report key bioactivities of avarol, the main secondary metabolite of the marine sponge Dysidea avara (Schmidt, 1862).

This work extends in vitro screening of antimicrobial activity of the sesquiterpene hydroquinone avarol, a main secondary metabolite of the Mediterranean sponge species Dysidea avara. The antimicrobial activity was in part evaluated by microdilution method against selected bacterial and fungal strains. Additionally, the screening included evaluation of anti-quorum sensing (anti-QS) effects. At a different extent avarol was proven to be active against all the microorganisms tested (MIC 0.002-0.008 mg/mL and MBC 0.004-0.016 mg/mL; MIC 0.004-0.015 mg/mL and MFC 0.008-0.030 mg/mL; respectively). This marine natural product also showed moderate anti-QS effects, reducing Pseudomonas aeruginosa PAO1 biofilm formation (75%), its twitching and protrusions motilities as well as pyocianin production (39%). According to the best of our knowledge, this is the first report both on avarol anti Gram-negative bacterial activity and anti-QS effects.

The cytotoxicity of 40-leucine-avarone, amino derivative of the sponge Dysidea avara secondary metabolite avarone, was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide assay in vitro against seven human solid tumours for the first time. The compound tested induced dose-dependent cytotoxic response in all cancer cells showing better activity towards the lung A-549 and colon HT-29 cell lines (IC50 7.40mM and 9.62mM, respectively) than towards the breast adenocarcinoma ER positive MCF-7 and ERnegativeMDA-MB-231 cells (IC50 11.64mMand 17.31mM, respectively), the prostate adenocarcinoma PC-3 and epiteloid cervix carcinoma HeLa cells (IC50 14.24mM and 15.54mM, respectively). No toxicity was found towards the foetal lung fibroblast MRC-5 cell line at the concentrations used. According to experimental data obtained, the sesquiterpenoid quinone structure of avarone may inspire development of new drug-like substances with improved cytotoxicity on lung cancer in humans

Although the second biggest terrestrial group of plants, bryophytes remain poorly known chemically compared to the angiosperms. In this article, the sugars of the moss Rhodobryum ontariense, an unstudied representative of the medicinally known genus, are reported. The chemical analysis revealed the usual plant sugar sucrose, and a new sugar, fructooligosaccharide 1- kestose, which is reported first not only for the genus Rhodobryum, but also for mosses. The trisaccharides have been scantily reported in bryophytes hitherto. This gives more significance to this study for further investigation of its role in the moss species. The health-promoting effect of 1-kestose is also briefly discussed.

To define whether age modifies the prognosis of patients with chronic kidney disease (CKD) on nephrology care, we prospectively followed patients with CKD who have been receiving nephrology care in a clinic for 1 year or more. The incidence of end-stage renal disease (ESRD), defined by the occurrence of dialysis or transplant, or death without ESRD was estimated by a competing-risk approach, and interactions between age and risk factors tested in Cox models over a median follow-up period of 62.4 months. Of 1248 patients with stage III-V CKD, 481 were younger than 65, 410 were between 65 and 75, and 357 were over 75 years old. Within each age class, the mean estimated glomerular filtration rate (eGFR) was 31, 32, and 29 ml/min per 1.73 m2, respectively. There were 394 ESRD events and 353 deaths. The risk of ESRD was higher than the risk of death without ESRD for ages 60 years, and independent of eGFR. The ESRD risk diminished with aging but still prevailed for eGFRs of 25-35 in patients between 65 and 75 years and with an eGFR below 15 in those up to 85 years old. Proteinuria significantly increased the risk of ESRD with advancing age. Surprisingly, the unfavorable effects of cardiovascular disease on ESRD and of diabetes on survival significantly decreased with increasing age. Male gender, higher phosphate, lower body mass index, and hemoglobin were age-independent predictors for ESRD, while cardiovascular disease, lower hemoglobin, higher proteinuria and uric acid, and ESRD also predicted death. Thus, in older patients on nephrology care, the risk of ESRD prevailed over mortality even when eGFR was not severely impaired. Proteinuria increases ESRD risk, while the predictive role of other modifiable risk factors was unchanged compared with younger patients. © 2012 International Society of Nephrology.

Gene therapy of Usher syndrome type 1B (USH1B) due to mutations in the large Myosin VIIA (MYO7A) gene is limited by the packaging capacity of adeno-associated viral (AAV) vectors. To overcome this, we have previously developed dual AAV8 vectors which encode human MYO7A (dual AAV8.MYO7A). Here we show that subretinal administration of 1.37E+9 to 1.37E+10 genome copies of a good-manufacturing-practice-like lot of dual AAV8.MYO7A improves the retinal defects of a mouse model of USH1B. The same lot was used in non-human primates at doses 1.6× and 4.3× the highest dose proposed for the clinical trial which was based on mouse efficacy data. Long-lasting alterations in retinal function and morphology were observed following subretinal administration of dual AAV8.MYO7A at the high dose. These findings were modest and improved over time in the low-dose group, as also observed in other studies involving the use of AAV8 in non-human primates and humans. Biodistribution and shedding studies confirmed the presence of vector DNA mainly in the visual pathway. Accordingly, we detected human MYO7A mRNA expression predominantly in the retina. Overall, these studies pave the way for the clinical translation of subretinal administration of dual AAV vectors in USH1B subjects.

Split intein-mediated protein trans-splicing expands AAV transfer capacity, thus overcoming the limited AAV cargo. However, non-mammalian inteins persist as trans-splicing by-products, and this could raise safety concerns for AAV intein clinical applications. In this study, we tested the ability of several degrons to selectively decrease levels of inteins after protein trans-splicing and found that a version of E. coli dihydrofolate reductase, which we have shortened to better fit into the AAV vector, is the most effective. We show that subretinal administration of AAV intein armed with this short degron is both safe and effective in a mouse model of Stargardt disease (STGD1), which is the most common form of inherited macular degeneration in humans. This supports the use of optimized AAV intein for gene therapy of both STGD1 and other conditions that require transfer of large genes., Graphical abstract, Intein-mediated protein trans-splicing (PTS) expands AAV gene transfer capacity. However, non-mammalian inteins persist as trans-splicing by-products. Here, we show that ecDHFR selectively degrades inteins after PTS and that AAV intein vectors armed with this degron are both safe and effective in the retina of a mouse model of genetic blindness.

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

To determine whether oogonial proliferation and oocyte recruitment are under control of hypophyseal and/or ovarian factors, we carried out a series of investigations using Podarcis sicula, a lizard inhabiting the temperate lowlands of Europe in which oocyte recruitment occurs throughout the year, as animal model. Germinal beds containing oogonia and oocytes in prefollicular stages were cocultured with different ovarian compartments in presence/absence of FSH, and the effects of different treatments were evaluated by counting the number of prelepto-leptotene oocytes. Results revealed that oocyte recruitment from the pool of oogonia is under the control of a factor released by follicle cells while FSH has an indirect effect on modulating oogonial proliferation. SDS-PAGE analyses carried out on media conditioned by follicles suggest that the factor involved in the control of oocyte recruitment may be a small protein (about 21 kDa) and that its release is dependent on the period of the ovarian cycle but apparently not on the circulating levels of FSH.