Clinical evaluation of tear substitute utility after anti-VEGF intravitreal injection.

PURPOSE: The purpose of this study is to evaluate the role of a polyethylene glycol 400 - propylene glycol - hydroxypropyl guar - hyaluronic acid eye drops in relieving the symptoms of dry eye syndrome after anti-VEGF intravitreal injection. METHODS: In this randomized, parallel-group study, patients were randomized to receive standard therapy alone or study eye drops plus standard therapy. Patients enrolled were older than 50 with exudative age-related macular degeneration, who have never had intravitreal injections, and without severe dry eye condition or severe ocular or systemic conditions. Patients had baseline preoperative evaluation (V0), and successively after 15 (V1) and 30 (V2) days. At VO and V1 T-BUT, Schirmer's test, fluorescein staining, DEQ5 and OSDI questionnaires were performed and at V2 (30) the OSDI questionnaire was administered. RESULTS: 80 patients were included in the study, of them 40 received standard therapy plus study eye drops. DEQ5 questionnaire showed an increase in the control group between the values at V0 and V1, while in the study group a decrease was observed. No changes in T-BUT, Schirmer, and fluorescein staining values between V0 and V1 were observed in both groups. OSDI data in the control group showed no statistically significant differences, while in the study group they showed a statistically significant difference. CONCLUSIONS: In our study patients undergoing for the first time intravitreal treatment presented dry eye symptoms in the postoperative period: in this group of patients the use of a tears substitute reduces postoperative ocular discomfort.

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

MAIN CONCLUSION: The complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp. The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

Impact of High Light Intensity and Low Temperature on the Growth and Phenylpropanoid Profile of Azolla filiculoides.

Exposure to high light intensity (HL) and cold treatment (CT) induces reddish pigmentation in Azolla filiculoides, an aquatic fern. Nevertheless, how these conditions, alone or in combination, influence Azolla growth and pigment synthesis remains to be fully elucidated. Likewise, the regulatory network underpinning the accumulation of flavonoids in ferns is still unclear. Here, we grew A. filiculoides under HL and/or CT conditions for 20 days and evaluated the biomass doubling time, relative growth rate, photosynthetic and non-photosynthetic pigment contents, and photosynthetic efficiency by chlorophyll fluorescence measurements. Furthermore, from the A. filiculoides genome, we mined the homologs of MYB, bHLH, and WDR genes, which form the MBW flavonoid regulatory complex in higher plants, to investigate their expression by qRT-PCR. We report that A. filiculoides optimizes photosynthesis at lower light intensities, regardless of the temperature. In addition, we show that CT does not severely hamper Azolla growth, although it causes the onset of photoinhibition. Coupling CT with HL stimulates the accumulation of flavonoids, which likely prevents irreversible photoinhibition-induced damage. Although our data do not support the formation of MBW complexes, we identified candidate MYB and bHLH regulators of flavonoids. Overall, the present findings are of fundamental and pragmatic relevance to Azolla's biology.

Whole-Transcriptome Analysis Unveils the Synchronized Activities of Genes for Fructans in Developing Tubers of the Jerusalem Artichoke.

Helianthus tuberosus L., known as the Jerusalem artichoke, is a hexaploid plant species, adapted to low-nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 expressed sequence tags (ESTs) from an H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T0), at maximum tuber elongation rate (T3), and at tuber physiological maturity (Tm), in "VR" and "K8-HS142"clones. The former was selected as an early tuberizing and the latter as a late-tuberizing clone. We quantified inulin and starch levels, and qRT-PCR confirmed the expression of critical genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T0 and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady-state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

The R2R3-MYB TT2b and the bHLH TT8 genes are the major regulators of proanthocyanidin biosynthesis in the leaves of Lotus species.

MAIN CONCLUSION: By exploiting interspecific hybrids and their progeny, we identified key regulatory and transporter genes intimately related to proanthocyanidin biosynthesis in leaves of Lotus spp. Proanthocyanidins (PAs), known as condensed tannins, are polymeric flavonoids enriching forage legumes of key nutritional value to prevent bloating in ruminant animals. Unfortunately, major forage legumes such as alfalfa and clovers lack PAs in edible tissues. Therefore, engineering the PA trait in herbage of forage legumes is paramount to improve both ecological and economical sustainability of cattle production system. Progresses on the understanding of genetic determinants controlling PA biosynthesis and accumulation have been mainly made studying mutants of Arabidopsis, Medicago truncatula and Lotus japonicus, model species unable to synthesize PAs in the leaves. Here, we exploited interspecific hybrids between Lotus corniculatus, with high levels of PAs in the leaves, and Lotus tenuis, with no PAs in these organs, and relative F2 progeny, to identify among candidate PA regulators and transporters the genes mainly affecting this trait. We found that the levels of leaf PAs significantly correlate with the expression of MATE1, the putative transporter of glycosylated PA monomers, and, among the candidate regulatory genes, with the expression of the MYB genes TT2a, TT2b and MYB14 and the bHLH gene TT8. The expression levels of TT2b and TT8 also correlated with those of all key structural genes of the PA pathways investigated, MATE1 included. Our study unveils a different involvement of the three Lotus TT2 paralogs to the PA trait and highlights differences in the regulation of this trait in our Lotus genotypes with respect to model species. This information opens new avenues for breeding bloat safe forage legumes.

The R2R3MYB VvMYBPA1 from grape reprograms the phenylpropanoid pathway in tobacco flowers.

MAIN CONCLUSION: This work shows that, in tobacco, the ectopic expression of VvMYBPA1 , a grape regulator of proanthocyanidin biosynthesis, up- or down-regulates different branches of the phenylproanoid pathway, in a structure-specific fashion. Proanthocyanidins are flavonoids of paramount importance for animal and human diet. Research interest increasingly tilts towards generating crops enriched with these health-promoting compounds. Flavonoids synthesis is regulated by the MBW transcriptional complex, made of R2R3MYB, bHLH and WD40 proteins, with the MYB components liable for channeling the complex towards specific branches of the pathway. Hence, using tobacco as a model, here, we tested if the ectopic expression of the proanthocyanidin regulator VvMYBPA1 from grape induces the biosynthesis of these compounds in not-naturally committed cells. Here, we show, via targeted transcriptomic and metabolic analyses of primary transgenic lines and their progeny, that VvMYBPA1 alters the phenylpropanoid pathway in tobacco floral organs, in a structure-specific fashion. We also report that a modest VvMYBPA1 expression is sufficient to induce the expression of both proanthocyanidin-specific and early genes of the phenylpropanoid pathway. Consequently, proanthocyanidins and chlorogenic acids are induced or de novo synthetised in floral limbs, tubes and stamens. Other phenylpropanoid branches are conversely induced or depleted according to the floral structure. Our study documents a novel and distinct function of VvMYBPA1 with respect to other MYBs regulating proanthocyanidins. Present findings may have major implications in designing strategies for enriching crops with health-promoting compounds.

11ß-hydroxysteroid dehydrogenase type 1 and pregnancy: Role in the timing of labour onset and in myometrial contraction.

Glucocorticoids play a primary role in the maturation of fetal organs and may contribute to the onset of labour. Glucocorticoid activity depends on the 11ß-hydroxysteroid dehydrogenase family (11ß-HSDs), catalysing the interconversion between "active" cortisol into inactive cortisone. No definitive study exists on 11ß-HSD expression profile in human decidua and myometrium during pregnancy. We investigated the implications of 11ß-HSD1 in the regulation of uterine activity in pregnancy, examining its role on contraction of a myocyte cell line and murine 11ß-hsd1 levels in utero. Murine 11ß-hsd1 mRNA and protein levels in utero progressively increased until the last day of gestation and significantly decreased at the onset of labour (P < 0.0001) (n = 3 to 5 in the various gestational days analysed). Experiments on human myometrial samples confirm the significant fall in 11ß-hsd1 mRNA levels at labour, compared to end pregnancy samples (n = 5 to 8). In vitro experiments showed that human myometrial contraction is inhibited by using a non-selective inhibitor of 11ß-HSD1. The present study shows the temporal localisation of 11ß-HSD1 in uterus, highlighting its importance in the timing of gestation and suggesting its contribution in the myometrium contraction.

Expression and regulation of 11ß-hydroxysteroid dehydrogenase type 1 in first trimester human decidua cells: Implication in preeclampsia.

Glucocorticoids are implicated in successful blastocyst implantation, whereas alterations in glucocorticoid levels are associated with various pregnancy disorders including preeclampsia. Tissue concentration of active glucocorticoids depends on the expression of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). This study investigated the contribution of first trimester decidua to glucocorticoid availability at the fetal-maternal interface by assessing the expression and regulation of 11ß-HSD in human first trimester decidual tissues and cells and by evaluating 11ß-HSD levels in preeclamptic vs. gestational age-matched decidua. 11ß-HSD1 was the predominant isoform in first trimester decidua. In vitro, decidual cell 11ß-HSD1 levels and enzymatic activity were up-regulated by ovarian steroids and inflammatory cytokines. Higher levels of 11ß-HSD1 were found in preeclamptic decidua compared to controls. The present study indicates the predominance of 11ß-HSD oxoreductase isoform in early decidua. Observations that ovarian hormones and inflammatory cytokines up-regulate 11ß-HSD1, together with increased 11ß-HSD1 expression in preeclampsia, highlight a role for decidual cells in controlling biologically active glucocorticoids in early pregnancy.

Simple and fast orotracheal intubation procedure in rats.

INTRODUCTION: Endotracheal intubation in the rat is difficult because of the extremely small size of anatomical structures (oral cavity, epiglottis and vocal cords), small inlet for an endotracheal tube and the lack of proper technical instruments. MATERIAL AND METHODS: In this study we used seventy rats weighting 400-500 g. The equipment needed for the intubation was an operating table, a longish of cotton, a cotton tip, orotracheal tube, neonatal laryngoscope blades, KTR4 small animal ventilator and isoflurane for inhalation anaesthesia. Premedication was carried out by medetomidine hydrochloride 1 mg/mL; then, thanks to a closed glass chamber, a mixture of oxygen and isoflurane was administered. By means of a neonatal laryngoscope the orotracheal tube was advanced into the oral cavity until the wire guide was visualized trough the vocal cords; then it was passed through them. The tube was introduced directly into the larynx over the wire guide; successively, the guide was removed and the tube placed into the trachea. Breathing was confirmed using a glove, cut at the end of a finger, simulating a small balloon. RESULTS: We achieved a fast and simple orotracheal intubation in all animals employed. CONCLUSIONS: We believe that our procedure is easier and faster than those previously reported in scientific literature.

Validation of a modified model of TNBS-induced colitis in rats. How to induce a chemical colitis in rats.

BACKGROUND: There is no standard practice in the induction of colitis by 2,4,6-trinitrobenzene sulfonic (TNBS) acid. Usually, the repeated administration of TNBS is preferred, because it will result in a local Th1 response that has the characteristics of Crohn`s disease. MATERIALS AND METHODS: A total of 30 rats were randomized into two groups, consisting of a saline control group of ten rats and a TNBS groups of 20 rats. After the animals were anesthetized, 0.5 ml of either 0.9% saline (controls) or TNBS 50 mg/kg dissolved in 50% ethanol were instilled into the colon through a rubber catheter. The experiment was repeated weekly for four weeks, then, the rats were killed at day 40, and the distal colon removed. RESULTS: At day 40, the bowel wall was basically normal in the control group. In the TNBS group, the bowel lumen became narrow with thickened wall, and the mucosal surface presented adherent membrane with brown black, linear ulcers, proliferous lymphocyte tissue, inflammatory granulomas and submucosal neutrophil infiltration. The median score of the severity of the colonic damage was 0 in the control group, and 4,75 (range 4-5) in the TNBS group; the mean weight of the rats was 180+35 g in the TNBS group, while it was 215+25 in the control group. CONCLUSIONS: The presented experiment is a cost-effective and safe method to induce Crohn-like colonic damage using a lower dose of TNBS, thus avoiding the risk of a massive loss of rats. This model is rather suitable for the assessment of the effects of potential therapeutic agents. (www.actabiomedica.it).

Lotus tenuis x L. corniculatus interspecific hybridization as a means to breed bloat-safe pastures and gain insight into the genetic control of proanthocyanidin biosynthesis in legumes.

BACKGROUND: Proanthocyanidins (PAs) are secondary metabolites that strongly affect plant quality traits. The concentration and the structure of these metabolites influence the palatability and nutritional value of forage legumes. Hence, modulating PAs in the leaves of forage legumes is of paramount relevance for forage breeders worldwide. The lack of genetic variation in the leaf PA trait within the most important forage species and the difficulties in engineering this pathway via the ectopic expression of regulatory genes, prompted us to pursue alternative strategies to enhance this trait in forage legumes of agronomic interest. The Lotus genus includes forage species which accumulate PAs in edible organs and can thus be used as potential donor parents in breeding programs. RESULTS: We recovered a wild, diploid and PA-rich population of L. corniculatus and crossed with L. tenuis. The former grows in an alkaline-salty area in Spain while the latter is a diploid species, grown extensively in South American pastures, which does not accumulate PAs in the herbage. The resulting interspecific hybrids displayed several traits of outstanding agronomic relevance such as rhizome production, PA levels in edible tissues sufficient to prevent ruminal bloating (around 5 mg of PAs/g DW), biomass production similar to the cultivated parent and potential for adaptability to marginal lands. We show that PA levels correlate with expression levels of the R2R3MYB transcription factor TT2 and, in turn, with those of the key structural genes of the epicatechin and catechin biosynthetic pathways leading to PA biosynthesis. CONCLUSIONS: The L. tenuis x L. corniculatus hybrids, reported herein, represent the first example of the introgression of the PA trait in forage legumes to levels known to provide nutritional and health benefits to ruminants. Apart from PAs, the hybrids have additional traits which may prove useful to breed forage legumes with increased persistence and adaptability to marginal conditions. Finally, our study suggests the hybrids and their progeny are an invaluable tool to gain a leap forward in our understanding of the genetic control of PA biosynthesis and tolerance to stresses in legumes.

Refractive error after cataract surgery combined with descemet stripping automated endothelial keratoplasty.

PURPOSE: To assess refractive error after cataract surgery combined with Descemet stripping automated endothelial keratoplasty using adjusted keratometric (K) readings to calculate intraocular lens (IOL) power. DESIGN: Prospective, interventional case series. METHODS: One eye of 39 consecutive patients with Fuchs endothelial dystrophy and cataract were included. To calculate IOL power before surgery, the Javal steep and flat K readings (Haag-Streeit) were adjusted by reducing their value by -1.19 diopters (D); the axial length was measured by immersion biometry, and the standard A-constant was used. Surgery included phacoemulsification, IOL (Acrysof SN60AT; Alcon) implantation within the capsular bag, and Descemet stripping automated endothelial keratoplasty using posterior lamella prepared with a 300-µm head microkeratome (Moria). The absolute prediction error (absolute difference between predicted and achieved refraction) was assessed 6 months after surgery. RESULTS: The mean power of the implanted IOL was 23.22 ± 2.90 D. The mean predicted and achieved refractions were -0.27 ± 0.26 D and -0.23 ± 0.73 D, respectively. The mean absolute prediction error was 0.59 ± 0.42 D (range, 0.05 to -1.52 D). The postoperative spherical equivalent fell within ±0.50 D, ±1.00 D, and ±2.00 D of the predicted refraction in 55.5%, 83.3%, and 100% of cases, respectively. Had the IOL power been calculated without adjusting the K readings, the mean absolute prediction error would have been significantly higher (0.86 ± 0.62 D; P = .04). CONCLUSIONS: In this study, in which posterior lamellae were prepared using a 300-µm head microkeratome, adjusting preoperative K readings by -1.19 D led to accurate IOL power calculation and highly predictable refractive error after cataract surgery combined with Descemet stripping automated endothelial keratoplasty.

Argentum-quarz solution in the treatment of anorectal fistulas: is it possible a conservative approach?

Patients suffering from chronic intestinal diseases (Crohn's disease, Ulcerative Colitis, Indeterminate Colitis) are prone to the development of pyogenic complications. These complications are most commonly in the form of perianal or intraabdominal abscesses and/or fistulas. The treatment of these complications are managed differently but, after an initial treatment based on medical or minimally invasive management, the solution of the pathological condition is always achieved by a surgical procedure. In the last few years prospective studies have proposed an alternative conservative therapeutic approach based on application of fibrin glue in the healing of patients with fistulas-in-ano. In this paper we suggest and discuss the therapeutic potential of silver and quarz in the conservative treatment of anorectal fistulas pointing out their role in modulating particular steps of the pathogenetic process which characterizes this pathological condition.

The strawberry transcription factor FaMYB1 inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves.

Proanthocyanidins (PAs) are agronomically important biopolymers in higher plants composed primarily of catechin and epicatechin units. The biosynthesis of these natural products is regulated by transcription factors including proteins of the R2R3MYB class. To gain insight into the genetic control of the catechin and epicatechin branches of the PA pathway in forage legumes, here the effects of the expression of FaMYB1, a flavonoid R2R3MYB repressor from strawberry, in Lotus corniculatus (birdsfoot trefoil), were tested. It was found that in leaves of T(0) transgenic lines the degree of PA inhibition correlated with the level of FaMYB1 expression. These effects were heritable in the transgene-positive plant T(1) generation and were tissue specific as the suppression of proanthocyanidin biosynthesis was most pronounced in mesophyll cells within the leaf, whereas other flavonoid and phenolic compounds were substantially unaltered. The data suggest that FaMYB1 may counter-balance the activity of the endogenous transcriptional MYB-bHLH-WD40 (MBW) complex promoting proanthocyanidin biosynthesis via the catechin and epicatechin branches and that FaMYB1 does not interfere with the expression levels of a resident R2R3MYB activator of PAs. It is proposed that in forage legumes leaf cell commitment to synthesize proanthocyanidins relies on the balance between the activity of activator and repressor MYBs operating within the MBW complex.

Effects of titanium surface roughness on mesenchymal stem cell commitment and differentiation signaling.

PURPOSE: Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces. MATERIALS AND METHODS: hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium. RESULTS: The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins. CONCLUSION: These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface.

[Microbiological quality of haemodialysis water in various hospitals and private clinics in the Lazio region (Italy): 2000-2004]. / La qualità microbiologica delle acque utilizzate in emodialisi in vari ospedali e case di cura del Lazio sulla base di controlli effettuati nell'arco di cinque anni.

The aim of this study was to evaluate the quality of dialysis water used in dialysis units of various hospitals and private clinics in Rome and the Lazio region (Italy). The study was performed between January 2000 and December 2004. A total of 507 samples were analysed, 205 of which obtained from public hospitals and 302 from privately-owned medical facilities. Microbiological analysis was performed as specified by the 1995 Decree of the Lazio Region n degrees 1650, which also sets threshold values for bacterial contamination and minimum frequency of inspections. The decree also specifies the modalities and frequency of disinfection of dialysis water treatment and distribution systems. The findings show a superior microbiological quality of dialysis water in privately-owned dialysis units. This may be due to several factors but especially to a more regular maintenance and disinfection of the water treatment systems. Overall, over half of the samples analysed, in both types of facilities, showed bacterial loads well below the set limits. In our view, however, bacterial limits for dialysis water are too high especially considering the fact that they are the same limits set by previous regulations for drinkable water. Haemodialysis patients are exposed to large volumes of water used for the production of dialysis fluids during a treatment session. It is therefore clearly important that more stringent limits for bacterial contamination be introduced.

Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus.

Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves.

The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families.

[Testing for Pseudomonas aeruginosa: an additional parameter for assessing microbiological quality of dialysis water]. / Ricerca di Pseudomonas Aeruginosa nelle acque di dialisi quale ulteriore parametro microbiologico di qualità.

In 1995 the regional council of the Lazio region passed Decree N.1650 which regulates the maintenance of dialysis equipment and sets limiting values for microbiological and chemical parameters used to assess the quality of water used for dialysis fluids. As regards the microbiological parameters, limiting values were set for total bacterial count and endotoxins. However, in recent literature, serious health effects following dialysis have been described; these suggest the advisability of including testing for more specific microorganisms and of testing greater samples of dialysis water. In this study, conducted over 2 years, in addition to normal microbiological parameters, dialysis fluids were tested for the presence of bacteria of the Pseudomonas species. Results show that samples which met the standards for dialysis water set by legislation were actually found to be contaminated by Pseudomonas species.