Probiotic abilities of riboflavin-overproducing Lactobacillus strains: a novel promising application of probiotics

The probiotic potential of Lactobacillus plantarum and Lactobacillus fermentum strains, capable of overproducing riboflavin, was investigated. The riboflavin production was quantified in co-cultures of lactobacilli and human intestinal epithelial cells, and the riboflavin overproduction ability was confirmed. When milk and yogurt were used as carrier matrices, L. plantarum and L. fermentum strains displayed a significant ability to survive through simulated gastrointestinal transit. Adhesion was studied on both biotic and abiotic surfaces. Both strains adhered strongly on Caco-2 cells, negatively influenced the adhesion of Escherichia coli O157:H7, and strongly inhibited the growth of three reference pathogenic microbial strains. Resistance to major antibiotics and potential hemolytic activity were assayed. Overall, this study reveals that these Lactobacillus stains are endowed with promising probiotic properties and thus are candidates for the development of novel functional food which would be both enriched in riboflavin and induce additional health benefits, including a potential in situ riboflavin production, once the microorganisms colonize the host intestine.     

Large-Scale Assessment of Mediterranean Marine Protected Areas Effects on Fish Assemblages

Marine protected areas (MPAs) were acknowledged globally as effective tools to mitigate the threats to oceans caused by fishing. Several studies assessed the effectiveness of individual MPAs in protecting fish assemblages, but regional assessments of multiple MPAs are scarce. Moreover, empirical evidence on the role of MPAs in contrasting the propagation of non-indigenous-species (NIS) and thermophilic species (ThS) is missing. We simultaneously investigated here the role of MPAs in reversing the effects of overfishing and in limiting the spread of NIS and ThS. The Mediterranean Sea was selected as study area as it is a region where 1) MPAs are numerous, 2) fishing has affected species and ecosystems, and 3) the arrival of NIS and the northward expansion of ThS took place. Fish surveys were done in well-enforced no-take MPAs (HP), partially-protected MPAs (IP) and fished areas (F) at 30 locations across the Mediterranean. Significantly higher fish biomass was found in HP compared to IP MPAs and F. Along a recovery trajectory from F to HP MPAs, IP were similar to F, showing that just well enforced MPAs triggers an effective recovery. Within HP MPAs, trophic structure of fish assemblages resembled a top-heavy biomass pyramid. Although the functional structure of fish assemblages was consistent among HP MPAs, species driving the recovery in HP MPAs differed among locations: this suggests that the recovery trajectories in HP MPAs are likely to be functionally similar (i.e., represented by predictable changes in trophic groups, especially fish predators), but the specific composition of the resulting assemblages may depend on local conditions. Our study did not show any effect of MPAs on NIS and ThS. These results may help provide more robust expectations, at proper regional scale, about the effects of new MPAs that may be established in the Mediterranean Sea and other ecoregions worldwide.     

Expression of a highly differentiated phenotype and hepatic functionality markers in gilthead seabream (Sparus aurata L.) long-cultured hepatocytes: first morphological and functional in vitro characterization

The development of primary cultures and cell lines from aquatic organisms is a valuable tool for a wide range of research activities applied to aquaculture. Despite several efforts, derivation and long-term culturing of primary hepatocytes from marine vertebrates are still rare and unsuccessful. This is the first report to fully characterize long-term cultures of primary hepatocytes from the European seabream, Sparus aurata L. (Osteichthyes, Sparidae) (SaHePs). In this new model, hepatocyte cells were long-term viable, active proliferating, and fully retained liver function up to 3 weeks. SaHePs expressed a differentiated phenotype, owing to the reacquisition of the peculiar cytoarchitecture with the complete assembly of cytoskeletal and junctional network, as shown by the production and immunolocalization of several polarity markers and cytoskeletal proteins (MDR1, ZO-2, C-CAM1, Vimentin, Cadherin, ??-Tubulin, ??-Catenin, ??-Actin). Cytostructural analysis to identify polarized expression and bile canaliculi formation was performed by immunofluorescence and contrast phase microscopy. Long cultured SaHePs also demonstrated evidence of Albumin, ??1-Antitrypsin (AAT) and ??-Fetoprotein (AFP) synthesis, expression of the detoxifying metabolic enzyme cytochrome P-4501A (CYP 1A), and production of hepatocyte specific cytoskeleton proteins, such as Cytokeratin 8 (CK8) and Cytokeratin 18 (CK 18). The presence of specific markers for hepatic phenotype, detected by immunocytochemistry and Western blot analysis, is suggestive of the full maintenance of a highly differentiated phenotype and hepatic maturation. These data demonstrate that SaHePs can be long cultured without losing the hepatic functionality. This study provides a useful tool for innovative research applications in fish toxicological, pathological, and physiological studies, as one of the few hepatic, functionally active, in vitro model from marine fish.     

Reduced mechanical activity of perfused rat heart following morphine or enkephalin peptides administration

In the isolated and perfused rat heart, the addition of morphine, methionine-enkephalin or leucine-enkephalin to the coronary perfusate, significantly reduces the mechanical activity by negatively affecting both the heart rate and the developed tension. These effects are dose dependent and maximally evident with leucine-enkephalin. Furthermore all the opioids strongly reduce the activity of isoproterenol-stimulated hearts. The suggestion is made that opioid peptides directly influence the cardiac mechanical activity possibly by interacting with membrane-receptor systems.     

Sites in the AAV5 capsid tolerant to deletions and tandem duplications

Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >10⁵ members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands.     

Trop-2 Is a Determinant of Breast Cancer Survival

Trop-2 is a calcium signal transducer that drives tumor growth. Anti-Trop-2 antibodies with selective reactivity versus Trop-2 maturation stages allowed to identify two different pools of Trop-2, one localized in the cell membrane and one in the cytoplasm. Of note, membrane-localized/functional Trop-2 was found to be differentially associated with determinants of tumor aggressiveness and distinct breast cancer subgroups. These findings candidated Trop-2 states to having an impact on cancer progression. We tested this model in breast cancer. A large, consecutive human breast cancer case series (702 cases; 8 years median follow-up) was analyzed by immunohistochemistry with anti-Trop-2 antibodies with selective reactivity for cytoplasmic-retained versus functional, membrane-associated Trop-2. We show that membrane localization of Trop-2 is an unfavorable prognostic factor for overall survival (1+ versus 0 for all deaths: hazard ratio, 1.63; P = 0.04), whereas intracellular Trop-2 has a favorable impact on prognosis, with an adjusted hazard ratio for all deaths of 0.48 (high versus low; P = 0.003). A corresponding impact of intracellular Trop-2 was found on disease relapse (high versus low: hazard ratio, 0.51; P = 0.004). Altogether, we demonstrate that the Trop-2 activation states are critical determinants of tumor progression and are powerful indicators of breast cancer patients survival.     

Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus

 Callus induction, somatic embryogenesis and plant regeneration were obtained in six different citrus species [Citrus deliciosa Ten. (cv 'Avana'), C.limon (L.) Burm. (cv 'Berna'), C.madurensis Lour. (cv 'CNR P9'), C.medica L. (cv 'Cedro di Trabia'), C.tardiva Hort. ex Tan. (cv 'CNR P6'), C.sinensis (L.) Osb. (cv 'Ugdulena 7')] from cultures of pistil transverse thin cell layer explants [(t)TCL]. Explants were cultured on three different media: the nutrients and vitamins of Murashige and Skoog medium alone (MS) or MS supplemented with either 500 mg l^–1 malt extract (MS I) or 500 mg l^–1 malt extract and 13.3 μM 6-benzylaminopurine (MS II). Sucrose (146 mM) was used as the carbon source. Somatic embryos were visible 2–5 months after culture initiation. The different genotypes showed a different embryogenic frequency from stigma, style and ovary (t)TCL explants. All of the cultivars regenerated somatic embryos. Percentages of style (t)TCL explants producing somatic embryos ranged from 0% (C.deliciosa, C.madurensis, C.sinensis and C.tardiva on the three different media) to 5.2% (C.limon on MS II). Embryo formation in stigma (t)TCL explants ranged from 0% (C.madurensis on MS and MS I, C.sinensis on MS, C.deliciosa and C.tardiva on the three different media) to 42.4% (C.limon on MS II). Embryo formation in ovary (t)TCL explants ranged from 0% (C.deliciosa on MS, C.limon, C.medica, and C.sinensis on the three different media) to 9.3% (C.tardiva on MS I). After about 12 weeks somatic embryos developed into plantlets at a high frequency. Received: 22 September 1998 / Revision received: 6 November 1998 / Accepted: 23 November 1998