Lactobacillus paracasei DTA81, a cholesterol-lowering strain having immunomodulatory activity, reveals gut microbiota regulation capability in BALB/c mice receiving high-fat diet.

AIMS: In-vitro/In-vivo evaluation of cholesterol-lowering probiotic strain Lactobacillus paracasei DTA81 and the possible connection with the gut microbiota modulation. METHODS AND RESULTS: In the present study, strain DTA81 has been evaluated for the possible influence on blood lipid and glucose concentrations, modulation of the immune system, gastrointestinal survivability and modulation of gut microbiota in BALB/c mice receiving a high-fat diet. After 6 weeks of treatment, a significant reduction of total cholesterol and fasting blood sugar (FBS) among animals treated with L. paracasei DTA81 has been recorded. Comparison of colon tissue levels of different cytokines revealed a significant reduction of the inflammatory cytokine interleukin-6. The comparison of gut microbiota using the 16S rRNA approach indicated that the treatment with L. paracasei DTA81 significantly increased the taxa Bacteroidetes and Coprococcus. Moreover, the genome of DTA81 was sequenced for the in-silico assessment, and the analysis indicated the presence of cholesterol assimilation-related genes as well as the absence of negative traits such as transmissible antibiotic resistance genes, plasmids and prophage regions. CONCLUSION: The outcome of this study revealed the in-vitro and in-vivo properties of L. paracasei DTA81 and the possible mechanism between consumption of this strain, the abundance of Bacteriodetes/Coprococcus taxa, immunomodulatory activity and the subsequent reduction of cholesterol/FBS in BALB/c mice. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus paracasei DTA81 as a non-pharmacological potential probiotic supplement can influence metabolic homeostasis in individuals, particularly those adopting high-fat diets, and it can contribute to reduce coronary heart disease.

New rapid PCR protocol based on high-resolution melting analysis to identify Saccharomyces cerevisiae and other species within its genus.

AIMS: Selection projects aiming at the identification of new Saccharomyces strains are always on going as the use of the suitable yeast can strongly improve fermented food production, particularly winemaking. They are mainly targeted on Saccharomyces cerevisiae, but other species in the Saccharomyces genus are of interest. For this reason, more and more efficient molecular techniques for yeast identification able to accelerate yeast selection process are always needed. Among the Saccharomyces genus, four yeasts are widespread in natural environments: S. cerevisiae; S. uvarum; S. kudriavzevii and S. paradoxus. Therefore, among the Saccharomyces species, their discrimination is of great interest. METHODS AND RESULTS: A two-step protocol is proposed. Firstly the Saccharomyces genus identification is achieved by multiplex PCR analysis. Then, the Saccharomyces species is determined by a new method based on high-resolution melting analysis (HRMA). CONCLUSIONS: For HRMA two primer pairs have been proposed. The first was able to achieve the simultaneous identification of the four widespread Saccharomyces species, the second was used for the unambiguous discrimination of S. cerevisiae within its taxonomical genus. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay allowed an easy, rapid and simultaneous discrimination of S. cerevisiae, S. uvarum and S. paradoxus during yeast selection programs.

Saccharomyces cerevisiae vineyard strains have different nitrogen requirements that affect their fermentation performances.

In this work the fermentation performances of seven vineyard strains, together with the industrial strain EC1118, have been investigated at three differing yeast assimilable nitrogen (YAN) concentrations (300 mg N l-1 , 150 mg N l-1 and 70 mg N l-1 ) in synthetic musts. The results indicated that the response to different nitrogen levels is strain dependent. Most of the strains showed a dramatic decrease of the fermentation at 70 mg N l-1 but no significant differences in CO2 production were found when fermentations at 300 mg N l-1 and 150 mg N l-1 were compared. Only one among the vineyard strains showed a decrease of the fermentation when 150 mg N l-1 were present in the must. These results contribute to shed light on strain nitrogen requirements and offer new perspectives to manage the fermentation process during winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected vineyard Saccharomyces cerevisiae strains can improve the quality and the complexity of local wines. Wine quality is also influenced by nitrogen availability that modulates yeast fermentation activity. In this work, yeast nitrogen assimilation was evaluated to clarify the nitrogen requirements of vineyard strains. Most of the strains needed high nitrogen levels to express the best fermentation performances. The results obtained indicate the critical nitrogen levels. When the nitrogen concentration was above the critical level, the fermentation process increased, but if the level of nitrogen was further increased no effect on the fermentation was found.

sTRAIL coupled to liposomes improves its pharmacokinetic profile and overcomes neuroblastoma tumour resistance in combination with Bortezomib.

Neuroblastoma (NB), the most common and deadly extracranial solid tumour of childhood, represents a challenging in paediatric oncology. Soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL) is a cancer cell-specific molecule exerting remarkable anti-tumour activities against paediatric malignancies both in vitro and in preclinical settings. However, due to its too fast elimination and to the undesired related side effects, the improvement of sTRAIL in vivo bioavailability and the specific delivery to the tumour is mandatory for increasing its therapeutic efficacy. In this manuscript, we developed an innovative pegylated liposomal formulation carrying the sTRAIL at the outer surface (sTRAIL-SL) with the intent to improve its serum half-life and increase its efficacy in vivo, while reducing side effects. Furthermore, the possibility to combine sTRAIL-SL with the proteasome inhibitor Bortezomib (BTZ) was investigated, being BTZ able to sensitize tumour cells toward TRAIL-induced apoptosis. We demonstrated that sTRAIL preserved and improved its anti-tumour activity when coupled to nanocarriers. Moreover, sTRAIL-SL ameliorated its PK profile in blood allowing sTRAIL to exert a more potent anti-tumour activity, which led, upon BTZ priming, to a statistically significant enhanced life spans in two models of sTRAIL-resistant NB-bearing mice. Finally, mechanistic studies indicated that the combination of sTRAIL with BTZ sensitized sTRAIL-resistant NB tumour cells to sTRAIL-induced cell death, both in vitro and in vivo, through the Akt/GSK3/ß-catenin axis-dependent mechanism. In conclusion, our results suggest that sTRAIL-SL might be an efficient vehicle for sTRAIL delivery and that its use in clinic, in combination with BTZ, might represent an adjuvant strategy for the treatment of stage IV, sTRAIL-resistant, NB patients.

Perifosine and sorafenib combination induces mitochondrial cell death and antitumor effects in NOD/SCID mice with Hodgkin lymphoma cell line xenografts.

The effects of the Akt inhibitor perifosine and the RAF/MEK/ERK inhibitor sorafenib were investigated using two CD30(+)Hodgkin lymphoma cell lines (L-540 and HDLM-2) and the CD30(-)HD-MyZ histiocytic cell line. The combined perifosine/sorafenib treatment significantly inhibited mitogen-activated protein kinase and Akt phosphorylation in two of the three cell lines. Profiling of the responsive cell lines revealed that perifosine/sorafenib decreased the amplitude of transcriptional signatures that are associated with the cell cycle, DNA replication and cell death. Tribbles homolog 3 (TRIB3) was identified as the main mediator of the in vitro and in vivo antitumor activity of perifosine/sorafenib. Combined treatment compared with single agents significantly suppressed cell growth (40-80%, P<0.001), induced severe mitochondrial dysfunction and necroptotic cell death (up to 70%, P<0.0001) in a synergistic manner. Furthermore, in vivo xenograft studies demonstrated a significant reduction in tumor burden (P<0.0001), an increased survival time (81 vs 45 days, P<0.0001), an increased apoptosis (2- to 2.5-fold, P<0.0001) and necrosis (2- to 8-fold, P<0.0001) in perifosine/sorafenib-treated animals compared with mice receiving single agents. These data provide a rationale for clinical trials using perifosine/sorafenib combination.

Patient selection for MitraClip therapy impaired left ventricular systolic function.

Mitral regurgitation (MR) is a disabling disease associated with poor prognosis and high incidence of clinical events if left untreated. To reduce the invasiveness of the surgical approach, different types of transcatheter procedures are becoming available. The MitraClip procedure (Abbott Vascular Inc. Menlo Park, CA, USA) is yet the only catheter-based procedure available in clinical practice at the moment. The device has been evaluated in a number of preclinical studies, registries and in FDA approved clinical trials. (EVEREST trial, ACCESS-EU trial). Indication and timing of intervention is a crucial step in the diagnostic-therapeutic pathway of patients with mitral regurgitation. The aim of this review is to clarify the potential of MitraClip in clinical practice, particularly focusing on patient selection for this novel therapy. Patient selection and overall decision making is strongly influenced by anatomical and clinical factors. Decision-making in degenerative MR (DMR) vs. functional (FMR) can be quite different. Generally, MitraClip is effective in treating either type II or IIIb dysfunction (at the moment FMR is the main indication for MitraClip in Europe, according to the ACCESS registry data). The relative role of MitraClip and surgery in the management of patients with MR is still unclear. From the global initial experience, MitraClip therapy could be complementary to surgery in those patients at high risk for surgery who have ideal anatomical characteristics for implantation. The procedure is quite predictable in patients with favorable anatomy. In patients with suboptimal anatomy, if the risk of surgery is too high, MitraClip could be still indicated sometimes. Our preliminary experience suggests that in patients with DMR, the EVEREST anatomical criteria are strong predictors of early and mid-term success. According to it, MitraClip therapy is appropriate in those DMR patients with high surgical risk and ideal anatomy for clip implantation according to the EVEREST criteria. In FMR refractory to medical therapy and resynchronization therapy, MitraClip could be considered as first option therapy, particularly in those patients with comorbidities, or advanced age, being the operative risk of surgery above 5% in this population. In the future, novel devices, improved knowledge, more efficient imaging and transcatheter mitral prosthetic valve implantation may expand the indications to those patients currently not treated by MitraClip for anatomical unsuitability, and may improve the results both in term of early efficacy and long term durability.

Effects of grape marcs acidification treatment on the evolution of indigenous yeast populations during the production of grappa.

AIMS: Grappa is a typical Italian product obtained from the distillation of grape marcs, the main by-product of grape crushing. One technological treatment frequently performed on marcs is their acidification, in order to contrast the development of unwanted spoilage bacteria during the storage period needed for alcoholic fermentation. A pilot-scale experiment was set-up to study the dynamics of yeast populations during a 30-day fermentation of acidified and nonacidified Prosecco grape pomace. METHODS AND RESULTS: Saccharomyces cerevisiae population, examined after 4 and 15 days of storage by mitochondrial DNA-RFLP analysis, resulted considerably different at strain level upon acidification. In particular, although the number of different strains rescued appeared particularly high in both kind of marcs compared with what happens in must fermentation, in the acidified material such number tends to moderately decrease during storage. CONCLUSIONS: Results obtained evidence that the acidification treatment did not influence yeast population neither in terms of number of cells nor in terms of biodiversity at species level. Therefore, such treatment can be used in distillery without negatively influencing ethanol production. SIGNIFICANCE AND IMPACT OF STUDY: Even though some data are available on the effects of technological treatments on the chemical composition of the distillate, no microbiological studies have been published so far on the consequence of these practices on composition, biodiversity and evolution of yeast population.

Haemoglobin E in pregnancy. A case report. Diagnosis, familiar study and counselling, follow up until delivery and new-born observation.

Haemoglobin E is a beta chain variant quite common in Southeastern Asia. The case of a gravid Thai woman with a microcytic anaemia is reported. The diagnosis of homozygous haemoglobin E was suspected on the basis of ethnic considerations when analysis of her haemoglobin showed the absence of normal HbA1 and about 100% of a variant Hb with electrophoretic mobility with HbC and HbA2. Identification of the haemoglobin variant was performed by using an association of alkaline electrophoresis on agarose gel, acid electrophoresis on agarose gel, haemoglobin isoelectrofocusing, high performance liquid chromatography. A study of haemoglobin pattern in the partner, parents and siblings was also performed. Pregnancy continued without any problems until the 40th week when a caesarean section was performed due to a difficult labour with foetal distress. The haemoglobin pattern of the new-born was studied at birth and after 1 year; as expected, it was quite normal at birth and a heterozygous condition for HbE was observed after 1 year. HbE, in even heterozygous and homozygous states, gives a mild clinical picture but its association with other haemoglobinopathies, such as a double heterozygous state (i.e. HbE/beta Thalassaemia) gives rise to a severe transfusion dependent thalassaemia syndrome. It is the authors' opinion that only a strict interaction between obstetricians and pathologists is the possible correct answer to the new diagnostic question proposed by a rapidly evolving inter-ethnic society.

Biological qualification of blood units: considerations about the effects of sample's handling and storage on stability of nucleic acids.

BACKGROUND: In transfusional setting introduction of nucleic amplification technique (NAT) for HBV-DNA, HCV-RNA and HIV-RNA in biological qualification of blood units suggest some problems. At first the opportunity to operate on mini-pool, at second the need to store the samples at +4 degrees C. The authors therefore have tried to estimate the impact of these conditions on the operativity of NAT testing in the transfusional setting. METHODS: The following parameters has been estimated: distribution of viral-load in untreated subjects, stability of nucleic acids during storage at +4 degrees C, stability of nucleic acids after repeated cycles of freezing and defrosting, robustness of the test to the cross-contamination, definition of the detection-limit (95%). Quantitative tests has been performed by using the following kits: Cobas Amplicor HBV Monitor, Cobas Amplicor HCV Monitor, Cobas Amplicor HIV Monitor; the qualitative tests has been performed by using the following kits: Ampliscreen HBV, Ampliscreen HCV 2,0, Ampliscreen HIV 1,5 all supplied by Roche Molecular System (Brancburg, NJ). RESULTS: Viral load in untreated subjects showed wide variation for HBV, HCV and HIV. HBV has been demonstrated much stable to the conservation +4 degrees C also until 168 h while for HCV and HIV a greater decrease of the viral-load was observed. For all and three virus the conservation to +4 degrees C until 72 h does not seem to involve meaningful fall in the viral-load. A remarkable reduction of the viral-load has been observed after five cycles of freezing and defrosting. All the tests showed a good robustness to cross-contamination. The detection-limit (95%) was 8 U/ml for HBV, 21 U/ml for HCV and 27 copy/ml for HIV. CONCLUSIONS: Samples for NAT testing, can be stored until 72 h to +4 degrees C without appreciable lowering of the viral-load. Repeated cycles of changes of state should be avoided. The tests showed a good robustness to cross-contamination. NAT tests for biological qualification of blood units had a minimal sensibility around 50 (copy/unit/ml). In our experience the detection-limit (95%) was 21 U/ml for HCV, 27 copies/ml for HIV, 8 U/ml for HBV. The availability of NAT test for HBV-DNA, HCV-RNA e HIV-RNA, sensitive and reliable, together with epidemiological data, suggest the opportunity to place side by side, in the biological qualification of the blood units, to add the tests for HBV-DNA and HIV-RNA to the test for HCV-RNA mandatory by low, in Italy in the biological qualification of blood units.

Assessment of in vitro platelet activation by Advia 120 platelet parameters.

The Advia 120 Hematology System provides new platelet parameters that have been proposed as useful markers of platelet activation. The aim of this study was to investigate platelet parameter variations after adenosine diphosphate (ADP) activation of platelet-rich plasma (PRP) in vitro, with particular interest in the mean platelet component (MPC), which was compared with two well-known degranulation antigens. Changes in platelet parameters that were induced by the activation of PRP with different concentrations of ADP were examined first. The time course of parameter values up to 60 minutes after maximal ADP activation and the relationships between the MPC and P-selectin and granulophysin expression as determined by flow cytometry were then investigated. After 10 minutes of ADP stimulation, the MPC presented a dose-dependent increase. At the maximal ADP concentration, the initial increase of the MPC was followed by a progressive decrease, leading the MPC to become significantly lower with respect to the baseline after 60 minutes of incubation. Significant variations in other parameters are also described. Finally, a negative correlation was found between the MPC change with respect to time 0 and both P-selectin and granulophysin expression. The present study suggests that platelet parameter variation, particular in the MPC, may be used to assess platelet activation in PRPs stimulated by ADP.

Field evaluation of a second-generation cytometer UF-100 in diagnosis of acute urinary tract infections in adult patients.

AIMS: The authors evaluated the analytical performance of the Sysmex UF-100 cytometer vs. the diagnosis of urinary tract infections (UTI). METHODS: We considered 2010 subjects, aged between 18 and 78, 870 males and 1140 females. The majority (90.2%) of the samples were voided urine specimens collected by using the midstream technique. Each sample was subjected to microbiological evaluation (culture + residual antibacterial activity), dipstick tests, UF-100 examination and microscopic observation. In order to obtain a final diagnosis of UTI these laboratory results were taken into consideration together with clinical data and patients' characteristics. The analytical performance of the laboratory tests was obtained by adopting this diagnosis as standard practice. RESULTS: Out of the total 2010 subjects considered a clinical diagnosis of UTI was obtained in 529 cases (26.32%). The UF-100-based screening had sensitivity, 0.94; specificity, 0.93; positive predictive value, 0.83; negative predictive value, 0.98; and correctly classified incidence, 0.93. CONCLUSIONS: In our experience the results of the UF-100-based screening show a very good correlation with the diagnosis of acute UTI in adults patients.

[Foetal-maternal alloimmunizations in the South-East area of the Venice province]. / Alloimmunizzazioni feto-materne nel Sud-Est del Veneto.

BACKGROUND: This study report the results obtained in a retrospective analysis of the foetal-maternal alloimmunizations observed from 1993 to 1999 in the South-East area of the Venice province. METHODS: The data concerning 17,000 pregnancy observed in this area from 1993-1999 have been collected. For each pregnancy data concerning maternal ABO, Rh, Kk and IAT as well as foetal ABO, Rh, Kk and DAT were available. Further data (mainly antibodies concentration and specificity) were available if a foetal-maternal alloimmunization was detected and if transfusional support was given after the birth. RESULTS: The authors observed 465 alloimmunizations (prevalence 2.7%): 381 (82%) of these were due to an ABO foetal-maternal incompatibility, 23 due to D incompatibility and the other 61 due to other blood groups antigens. Only 6 cases needed transfusional support: 5 exchange transfusion (a patient needed 2 exchanges) and a delayed transfusion. CONCLUSIONS: Foetal-maternal alloimmunizations are today a rare but not exceptional event (about 3% of pregnancy), the great majority of these alloimmunizations are due to an ABO incompatibility. Despite the prevention of alloimmunization in D negative women by using Rh immune globulin, anti-D alloimmunization is still observed. A great number of other blood groups antigens are involved in foetal-maternal alloimmunization mainly within the Rh system (CcEe, etc.). In the authors' experience the great majority of foetal-maternal alloimmunizations were clinically silent, only 6 cases (1.3% of patients with a positive DAT) needed transfusional therapy.

Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens.

The organization of the alginate gene cluster in Pseudomonas fluorescens was characterized. A bank of genomic DNA from P. fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (algJ::Tn501) in the alginate biosynthetic operon that rendered it non-mucoid. Phenotypic complementation in this heterologous host was observed, and a complementing clone containing 32 kb of P. fluorescens DNA was obtained. Southern hybridization studies showed that genes involved in alginate biosynthesis (e.g. algD, algG, and algA) were approximately in the same order and position as in P. aeruginosa. When the clone was mobilized to a P. aeruginosa algG mutant that produced alginate as polymannuronate due to its C5-epimerase defect, complementation was observed and the alginate from the recombinant strain contained L-guluronate as determined by proton nuclear magnetic resonance spectroscopy. A sequence analysis of the P. fluorescens DNA containing algG revealed sequences similar to P. aeruginosa algG that were also flanked by algE- and algX-like sequences. The predicted AlgG amino acid sequence of P. fluorescens was 67% identical (80% similar) to P. aeruginosa AlgG and 60% identical (76% similar) to Azotobacter vinelandii AlgG. As in P. aeruginosa, AlgG from P. fluorescens appeared to have a signal sequence that would localize it to the periplasm where AlgG presumably acts as a C5-epimerase at the polymer level. Non-polar algG knockout mutants of P. fluorescens were defective in alginate production, suggesting a potential role for this protein in polymer formation.

Platelet count and parameters determined by the Bayer ADVIA 120 in reference subjects and patients.

In order to study the behaviour of traditional and new platelet parameters determined by the ADVIA 120 Hematology System, five hundred samples from reference subjects, divided for sex and age, were processed. Significant variations on the basis of sex and age were found. Reference ranges as 95% confidence limits were therefore calculated for each age class, and platelet parameters proved to have specific variations during lifetime. Moreover, one hundred samples from thrombocytopenic patients were processed by the ADVIA 120 System. When compared with those of reference subjects matched for sex and age, all platelet parameters, except mean platelet component (MPC), showed significant differences.

Comparative strain typing of Rhizobium leguminosarum bv. viciae natural populations.

372 natural isolates of Rhizobium leguminosarum bv. viciae, rescued from nodules of pea plants grown in an agricultural field in northern Italy, were analyzed by different methods. Three DNA-based fingerprinting techniques were lined up to compare their relative degree of resolution and possible advantages of each approach. The methods included (i) Eckhardt gel plasmid profiles, (ii) pulsed-field gel electrophoresis (PFGE) of genomic large fragment digests, and (iii) random amplified polymorphic DNA (RAPD) profiles, generated with arbitrary primers. The scheme also involved the isolation of a number of different isolates per nodule to estimate the level of intra-nodular variability. It was therefore possible to evaluate the frequency of double and multiple occupancies, and the proportion of the alternative profiles sharing the same nodule, generally resulting in a numerically dominant, main representative accompanied by a secondary one with a slightly different fingerprint. This finding revealed that the different profiles within a nodule are normally due to bacteria derived from the same single invader following genetic alterations possibly occurred during infection, e.g., by plasmid loss. The analysis of 31 nodules revealed 16 different patterns, representing the most frequently occurring nodulation-proficient isolates of the natural soil examined, five of which were found with frequencies around 15%. The sensitivity of the methods in differentiating isolates was compared. The relatedness of the different natural rhizobial isolates was investigated by densitometrical gel analysis of the fingerprints, allowing a comparison of the results. One of the most interesting conclusions was that the degree of information yielded by the plasmid gel profiling alone, carried out by simple visual inspection without software-aided analyses, was surprisingly high, as it enabled a placement of the isolates, whose accuracy, in terms of relatedness, was subsequently confirmed by each of the two genomic methods.

Aspects of Marker/Reporter Stability and Selectivity in Soil Microbiology.

Based on several experiences of microbial release using genetically modified Rhizobium leguminosarum, we have highlighted a number of aspects related to the suitability of introduced markers such as resistance to mercury and b-galactosidase activity, the latter serving the function of high-expression level reporter gene obtained by the introduction of a synthetic promoter conferring strong inducible expression in Gram-negative bacteria. In vitro expression and in vivo performances of the chosen examples have been followed in model strains comparing gene dosage and expression levels. The technical possibility of unambiguously monitoring the marked GMM has been evaluated in medium- and long-term experiments carried out both in microcosms and soil, also including the presence of the plant symbiotic host. Marker stability, regardless the nature of the gene, was shown to be dependent on the location of the genetic modification and on its degree of gene expression regulation. Reporter strength was found to be an advantage allowing the distinction of marker-bearing bacteria while negatively affecting their genetic stability. Plasmid-borne regulated reporters were found to be stable up to the stages of rhizosphere colonization, but were more critically selected against upon symbiotic host invasion.

Nucleotide sequence and analysis of plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) involved in pediocin A production.

The complete sequence of the 19515-bp plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) has been determined. This plasmid is involved in Pediocin A production, a bacteriocin active against a wide range of gram-positive bacteria. It appears to replicate via a theta mechanism, with structures closely related to those of many lactococcal plasmids. Genes homologous to mobilization functions are also present, which are similar in sequence and arrangement to mobA, mobB, and mobC of some staphylococcal plasmids, although the last one contains a deletion in its central part. The region involved in bacteriocin activity has been limited to a 9.4-kb fragment, containing 10 open reading frames organized in a single operon. Since Pediocin A has a molecular weight of about 80 kDa (Piva and Headon, Microbiology, 140, 697-702, 1994), and a gene long enough to encode it is not present in pMD136, it is proposed that genes residing on the plasmid are responsible for the regulation of bacteriocinogenic activity. Gene arrangement and sequence homologies suggest the presence of a two-component-like regulatory mechanism.