Agaricus bisporus (Lange) Imbach cultivation, scant attention has been given to the importance of biological properties of casing materials. In the present study, an attempt was made to characterize the bacterial flora in casing layer, namely, Farm Yard Manure (FYM) and Spent Mushroom Substrate/spent compost (SMS/SC)  and FYM and Vermi Compost (VC) , employing partial 16S rDNA sequencing. Available data showed a significant variety of organisms that included Acinetobacter and Pseudomonas of the γ-proteobacteria, that were the most frequently encountered genera. This is the first preliminary report on the microbial diversity of casing soils and demonstrates the presence of Acinetobacter spp. that has not been previously described in casing material.Despite evaluation of large number of agroindustrial wastes for their use as casing material for  Agaricus bisporus (Lange) Imbach, is a biotechnological process that recycles lignino-cellulosic wastes (spent substrates), and the spent substrates can be used in different ways [A. bisporus (Lange) Imbach, is the most widely cultivated species of edible mushrooms and it is the most popular cultivar among the artificially grown fungi of the world that contributes about 31.8% to the global mushroom cultivation and 85% of the total produce in India [A. bisporus requires two different substrates to form the fruit bodies, that is, the compost for nutrition on which it grows vegetatively and the nutrient deficient casing soil in which the suitable physicochemical/biological conditions stimulate the initiation process of pin head formation for fruit body production [ Cultivation of button mushroom, ent ways , 2. ConsA. bisporus cultivation, scant attention has been given to the importance of biological properties of the casing layer [A. bisporus production. It supports beneficial microbial populations that release growth stimulating substances, which are reportedly involved in stimulating the initiation of pin heads. Several reports are available on the beneficial effects of casing soil microbes, especially, Pseudomonas putida and Alcaligenes faecalis on A. bisporus [ The casing layer is one of the important growing parameters and source of variation in production, quality, and uniformity of commercial cropping. A variety of casing materials have been used worldwide, among these, use of farm yard manure (FYM), as a casing medium for mushroom cultivation, has been in vogue in Indian subcontinent because of its easy availability and nonavailability of peat moss generally used for casing in Europe and USA. The advantages of using FYM as a casing material over other agroindustrial wastes have been highlighted in recent reports , 5. Spenng layer . Bacteribisporus . A. bisporus. There are two production-related issues requiring attention of researchers in casing soil microbiology, namely, (i) the composition of microorganisms present in casing soil and (ii) their influence on mushroom production in situ. Several methods and approaches are now available to generate information on microorganisms that reside in casing layer, which allow better assessment of microbial flora, wherein molecular tools for the identification of microorganisms are now in common use, and 16S rRNA gene analysis is intensively used in phylogenetic investigations. Furthermore, 16S rDNA sequence typing approach now permits identification of the surviving and culturable bacterial species, based on employing highly conserved 16S rDNA oligonucleotide primers for the eubacteria with an intervening hypervariable gene sequence, which could be used as signature sequence to aid in species identification [ Not much information is available in the literature about the role of associated microflora in the casing layer and how the resident microflora in casing layer interacts with the vegetative mycelium of fication , 10. TheIn the present study casing material included spent mushroom substrate/spent compost (SMS/SC) and vermicompost (VC). Both the materials were amended with farm yard manure (FYM) in the ratio of 3 : 1. These casing materials were collected from Mushroom Research and Training Centre (MRTC), GB Pant University of Agriculture & Technology, Pantnagar, India. Samples were drawn from successive stages of each casing, namely, 0-day casing , mycelium impregnated stage (MIS) of casing, casing at primordial stage (PS), and harvesting stage (HS) casing.Processed casings  were used for physicochemical analysis using methods suggested by various workers: bulk density , water hμL) to 900 μL of 0.85% normal saline in each Eppendorf tube, and appropriate dilution (10−4) of this suspension (100 μL) was spread plated in triplicate, on King's B medium [Casing sample (10 g) was suspended in 90 mL of 0.85% normal saline (pH 7.0) and shaken vigorously at 150 rpm at 18°C for 1 h. The resulting slurry was serially diluted (100 B medium . Culturehttp://www.bergeys.org/).A total of 38 isolates were randomly selected, morphologically from all the four successive stages of casing soil. Cellular morphology and biochemical characteristics were determined based upon Bergey's manual  of DNA extract, 1 U of Taq polymerase (Genei), 1 X buffer  (Genei), 10 mM dNTPs (Genei), and 0.25 mM of each primer (Genei). Initial DNA-denaturation and enzyme activation steps were performed at 95°C for 7 min, followed by 25 cycles of denaturation at 94°C for 1 min, annealing at 51°C for 1 min and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The presence and yield of specific PCR product (16S rRNA gene) were monitored on 0.8% agarose (wt./vol.) (Life Technologies Inc.); gel electrophoresis was carried out at 100 V for 30 min in 1 X Tris-acetate-EDTA buffer and visualization by ethidium bromide staining and viewing on a UV transilluminator (Biosystematica).Total DNA, from bacterial isolates, was prepared following the procedure outlined by Bazzicalupo and Fani . Extract primers : fDI (5′http://www.ncbi.nlm.nih.gov/BLAST). Multiple sequence alignment of retrieved sequences was done by EBI ClustalW server http://www.ebi.ac.uk/Tools/msa/clustalw2/. Homology tree was constructed by using MEGA 4.0.2 software with bootstrap values in cluster algorithm, Phylip format, and topological algorithm. The sequences determined in this study have been submitted in the GenBank database of NCBI and discussed under accession numbers: AY961042-61, AY 967718-967724, and DQ074744-53/58.PCR products obtained from 38 bacterial strains were purified with an EXO-SAP. Components were supplemented with gold buffer (Applied Biosystem) and sequenced on an Applied Biosystem 310 Genetic analyzer , using big dye terminator cycle sequencing Ready Kit (Lab India). The partial sequences, amplified by the fDI primer, were used to determine the similarities. The homology analysis for bacterial strains, based on partial sequences, was compared with the sequences from the DNA databases, and similar sequences showing above 95% were retrieved by nucleotide Basic Local Alignment Search Tool (BLAST) program at the National Center for Biotechnology Information (NCBI) BLAST server (3), while the casing FYM+VC (3 : 1) showed a BD value of 0.68 g/cm3. The porosity was significantly high (92.0%) for FYM+SMS. Water holding capacity appears directly related to porosity and bulk density and may directly affect microbial build up and yield of A. bisporus. It was high (191.19%) for FYM+SMS. A significant increase in electrical conductivity (EC) was observed for FYM+VC (570.33 deci-simen−), whereas low value (398.00 deci-simen−) was obtained for FYM+SMS. Water holding capacity appears directly related to porosity and bulk density. It was maximum (191.19%) for FYM + SMS (3 : 1) (The casing sample FYM+SMS (3 : 1) had minimum bulk density (BD) (0.60 g/cm (3 : 1) .Agaricus bisporus was grown on wheat-straw-based mushroom compost (pasteurized/long method). Two casing mixtures were prepared from three casing materials: Farm Yard Manure (FYM), Spent Mushroom Substrate (SMS), and Vermi Compost (VC) were evaluated for their nutritional status. In order to assess the nutritional status of casing mixtures  during various stages of cropping, status of availability macro- and micronutrients were analyzed  among casing layers FYM+SMS and FYM+VC tried at successive stages of cropping (There was little variation (6.02 ± 0.81 to 5.87 ± 0.81) in total bacterial population counts (logcropping . The cashttp://www.ncbi.nlm.nih.gov/BLAST). Most strains were grouped together and were close to the genus Pseudomonas and Acinetobacter of the family Pseudomonadaceae and Moraxellaceae, respectively, besides, others showed 55% similarity with reference strain Sphingobacterium , out of 38, exhibited close proximity with the genus Pseudomonas. All the 11 strains showed 95–99% similarity with reference strain Pseudomonas putida (AY785244). Large numbers of strains were distributed in family Moraxellaceae and they exhibited close proximity to genus Acinetobacter and showed Gram stain negative and exhibited NO3 reductase and catalase activity along with citrate utilization.Partial 16S rDNA sequences were determined for 38 isolates and compared with the sequences from the DNA databases and similar sequences showing above 95% were retrieved by nucleotide Basic Local Alignment Search Tool (BLAST) program at the National Center for Biotechnology Information (NCBI) BLAST server (acterium . This baA. bisporus. It was maximum (191.19%) for FYM+SMS (3 : 1). In general increase in electrical conductivity (EC) is almost proportional to decrease in number of pinheads [Considering the significance of mushroom production in the country and, yet, limited availability of information on casing soil microflora, an exhibitive analysis of bacterial diversity characterization of this unique material was undertaken. The casing material was selected that its nutritional status was very low compared to that of compost and was thus expected to create conditions of nutritional stress. If the casing material is rich in organic matter, it is likely to disturb the selectivity of compost and thus provide continued support to the vegetative growth of the fungus and thus discourage fruiting. Characteristically, microorganisms bring about physical and chemical changes to their habitats . Water hpinheads . Data ofpinheads . In addipinheads . The macpinheads . γ-proteobacteria group and other isolates were bacilli. A single isolate in this study was found to belong to the genus Sphingobacterium. Two genera, Acinetobacter and Pseudomonas, were dominant and were sole representative of γ-proteobacteria. Dominance of the genus Acinetobacter was of significance since this has not earlier been reported from the mushroom casing ecosystem. Presence of Acinetobacter as a dominant genus was not totally surprising since this bacterium is an important component of the natural ecosystem, notably involved with the degradation of lignin containing compounds and xenobiotics and its scavenging property for other noxious compounds. Based on partial 16S rRNA gene amplification and sequencing, Watabe et al. [Bacillus licheniformis, B. subtilis, Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum, and Stenotrophomonas sp. in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas.Molecular tools for the identification of casing soil bacteria were used and 16S rRNA gene analysis was intensively used to understand the homology relationships. Comparative analysis with BLAST sequences revealed that 85% of the bacterial isolates belonged to e et al.  examinedSphingobacterium sp. and Acinetobacter sp. that has not been previously described in casing material.In this preliminary study, 16S-rDNA PCR and direct automatic sequencing of the amplicons were only carried out on culturable organisms isolated on the nonselective King's medium B (KMB). In conclusion, this is the first preliminary report on the microbial diversity of casing soils and that demonstrates the presence of


Lignosus rhinocerus, has been used to treat variety of ailments by local and indigenous communities in Malaysia. The aim of this study was to investigate the potential of the most valuable part of L. rhinocerus, the sclerotium, on neurite outgrowth activity by using PC-12Adh cell line. Differentiated cells with one thin extension at least double the length of the cell diameter were scored positive. Our results showed that aqueous sclerotium L. rhinocerus extract induced neurite outgrowths of 24.4% and 42.1% at 20 μg/mL (w/v) of aqueous extract alone and a combination of 20 μg/mL (w/v) aqueous extract and 30 ng/mL (w/v) of NGF, respectively. Combination of NGF and sclerotium extract had additive effects and enhanced neurite outgrowth. Neuronal differentiation was demonstrated by indirect immunofluorescence of neurofilament protein. Aqueous sclerotium extract contained neuroactive compounds that stimulated neurite outgrowth in vitro. To our knowledge this is the first report on neurite-stimulating activities of L. rhinocerus.A national treasure mushroom,  Formation of central nervous system (CNS) network requires active axonal elongation and systematic explorations activity of environment by growing of axons in order to be directed to their correct target . Neuronain vitro model system for investigation of neuronal differentiation. Epidermal growth factor (EGF) and nerve growth factor (NGF) both activate extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) [Extension and remodeling of neurites play essential roles in development and neuronal plasticity regulated by neuroserpin . Culturee (MAPK) . But PC-Of the many diseases that threaten humans, neurodegenerative diseases can be very traumatic as one ages. Neurohealth is the concern for the predicted silver tsunami to hit humans—the aging tsunami is projected to be 80–90 million of 65-plus population in 2050 [Many of the drugs in the market only delay further deterioration and do not reverse the damage done to cognitive functions. The search is now for small molecules that can cross the brain-blood and induce the production of nerve growth factor (NGF), a family of proteins responsible for maintenance, survival, and regeneration of neurons during adult life. It has been shown that NGF absence causes an Alzheimer's like symptoms in the adult brain of mice. We may have to turn to nature to prevent or reduce the severity of nerve-related diseases as we age. Prevention will be definitely better than cure. Many plants and spices such as turmeric are said to help. For example, in India, it is noted that Alzheimer among the older generation is not in alarming numbers. The regular consumption of spices including turmeric and pepper may be the reason and currently this is actively been studied. Currently mushrooms are also being investigated as sources of NGF stimulators.Mushroom, a macrofungus that has either culinary or medicinal properties, has a visible fruiting body. Culinary mushrooms have been known as famous appetizing and nutritious food all over the world. Historically, medicinal mushrooms used by different tribes are best documented in the eastern world.Lignosus rhinocerus has been singled out as one of the most potent mushrooms for medicinal purposes dating to the 1700s by Tuan Haji Mat Yusop, a Malay in Pahang [Lignosus rhinocerus is a unique “National Treasure” that can only be found in a small geographic region encompassing South China, Thailand, Malaysia, Indonesia, Philippines, Papua New Guinea, New Zealand, and Australia [Lignosus rhinocerus (Cooke) Ryvarden is also known as “cendawan susu rimau” in Malay language or Tiger's Milk mushroom in English. The medicinal properties recorded by ethno-mycological surveys are yet to be validated scientifically.In Malaysia, several macrofungi are used by the Malays, Chineses, and indigenous communities for treatment of variety ailments. n Pahang , 7. Lignustralia . LignosuLignosus rhinocerus has more than 15 medicinal uses. Different natives use it as an antipruritic, antipyretic, general tonic, starve off hunger, cancer, food poisoning, swollen breasts [ breasts , fever,  breasts . Althoug breasts  and the L. rhinocerus sclerotium that can stimulate neurite outgrowth in PC-12 and the enhancement of neurite outgrowth with the combination of NGF and aqueous extract in PC-12 cells. Quantitative morphological methods and neurofilament formation were used to study the differentiation effects on neurite outgrowth activities in PC-12 cell line. To our knowledge, this is the first report on the application of sclerotium of L. rhinocerus in neurite outgrowth stimulation activity.The aim of the study was to assess the presence of active agents in aqueous extract of Lignosus rhinocerus sclerotium freeze-dried powder was from Dr. Tan Chon Seng of Malaysian Agricultural Research and Development Institute (MARDI). The aqueous extraction method was modified by Wong et al. [g et al. . SclerotPC-12 cell line, derived from transplantable rat pheochromocytoma, has been widely used as neuronal model because it proliferates in growth medium, and stop proliferating and differentiate into neuron-like cells as they respond to NGF . The cel2 humidified incubator at 37 ± 2°C [PC-12Adh was chosen for this study because it is NGF-dependent and does not synthesize epinephrine. PC-12 cell line was purchased from American Type Culture Collection (Cat #: CRL-1721.1) and only early passage cells were used in this study. The cells were cultured in ATCC-formulated F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) supplemented with 2.5% (v/v) fetal bovine serum and 15% (v/v) horse serum with final pH 6.8–7.2 and incubated in 5% CO37 ± 2°C . For sto37 ± 2°C .4 cells per well in complete F-12K medium with concentrations 1 to 500 μg/mL (w/v) of sclerotium aqueous extract. Concentrations of NGF-7S from murine submaxillary gland  ranging from 10 ng/mL (w/v) to 100 ng/mL (w/v) were tested to examine the optimum concentration for neurite stimulation activity. The optimum concentration was used as positive control for the following assays. Cells in complete F-12K medium without treatment served as negative control. Freeze-dried aqueous extracts were diluted to various concentration with sterilized distilled water. After the preliminary test, optimum concentration of the sclerotium aqueous extract in combination with NGF ranging from 10 ng/mL (w/v) to 50 ng/mL (w/v) was tested to evaluate synergistic interaction, if any, between sclerotium aqueous extract and NGF. Assay plates were incubated at 37 ± 2°C in a 5% CO2-humidified incubator. Differentiation activity of cells in terms of neurite outgrowth and branching was observed after 48 hr of incubation at 37 ± 2°C in 5% CO2-humidified incubator.Cells were cultured for 2 to 3 days until 60–70% confluent prior to assay. They were plated into 12-well plates at cell density of 5 × 10A cell was scored positive for bearing neurites if it has a thin neurite extension that double the length of the cell body diameter. Morphology of the cells is polygonal. Cells with irregular patterns such as sheet-like spreading cells, rare radially oriented possess, and apparently arising by “shrink-age” were excluded , 12 and 2-humidified incubator. On the day of experiment cells were 20% to 50% confluent and were fixed with 4% parafamaldehyde at room temperature for 20 min. The cells were then incubated with primary antibody, anti-neurofilament 200 antibody produced in rabbit  at room temperature for 1 hr, washed with washing buffer, and followed by further reaction with the secondary antibody, Fluorophore-conjugated secondary antibody, Anti-Rabbit IgG-Fluorescein isothiocyanate (FITC) antibody produced in sheep  at room temperature for 1 to 2 hr in dark. After the same washing procedure, cells were mounted onto slides with aqueous mounting medium, with 4′-6-Diamidino-2-phenylindole (DAPI). DAPI is used to stain nuclei. Finally, slides were observed under Nikon Eclipse 80i microscope under fluorescence illumination using FITC and DAPI filters and images were captured with Nikon's Imaging Software, NIS-Elements.Immunofluorescence staining of neurofilament protein was carried out according to the method of Schimmelpfeng et al. . Cells wP < 0.001) [Statistical analysis was performed using the computing environment R  . Permutation test was used to examine the statistical significance of the differences . Confide< 0.001) .L. rhinocerus was observed after 48 hr of incubation. The uniqueness of PC-12 cell line is that it responds to NGF with a drastic change in its phenotype and cause proliferation, extension of neurite [Neurite outgrowth stimulatory of NGF and aqueous extract of  neurite .  FigureNGF concentration plot  determinLignosus rhinocerus showed visible neurite outgrowth activity of PC-12 after 48 hr of incubation (μg/mL (w/v) with an increase of 15% (95% CI: 13–16) compared to negative control and 2.5% (95% CI: 1–4) higher compared to positive control. Increasing the concentration showed minimal or even negative effect on the number of neurite-bearing cells. There were no significant differences between negative control and addition of extract at concentration of 80 μg/mL (w/v) and 90 μg/mL (w/v). At 100 μg/mL (w/v) when compared to a negative control, a reduction of 1.3% (95% CI: (−2)–(−0.3)) of percentage of neurite-bearing cells was recorded.Aqueous extract of cubation . Maximalμg/mL (w/v) of aqueous extract of L. rhinocerus was selected as the optimum concentration for neurite outgrowth activity. The percentage of cells treated with various concentrations of NGF and optimum concentration of aqueous extract at 20 μg/mL (w/v) showed increase in number of neurite-bearing cells compared to cells treated with optimum concentration of aqueous extract alone  and incubated for 48 hr. Figures L. rhinocerus extracts obvious enhancement of neurite outgrowth with extension that double the length of cell body diameter can be observed.Differentiation of cells is most often assessed by semiquantitative or quantitative morphological methods such as determination of cell size, number of extensions over number of cells, and extent of neurite growth or neurite length . Neurofiβ-glucan and water soluble components. Studies showed that aqueous extraction was employed to extract water-soluble polysaccharide from edible medicinal mushrooms. However, bioactive compounds from medicinal plants extracted with ethanol performed better activities than aqueous extractions [Cold water extraction has been reported to yield lower amount of extract than hot water extraction but it is usually applied in traditional medicine preparation . Most ofractions  but aqueractions .L. rhinocerus displayed NGF-like properties due to obvious morphological alteration shown by PC-12 cells. Further work on fractionation of L. rhinocerus extract that possesses NGF-like properties needs to be explored.Wong et al. reported that freeze drying may be the best method for long-term preservation of bioactive compounds in mushroom responsible for neurite outgrowth stimulation activity. Inactivation of bioactive compound via oxidation could cause short cellular outgrowth of neurite and insufficient elongation to be scored as neurite . Lower cNGF is a signaling molecule that plays an important role in the differentiation and survival of peripheral sensory and sympathetic neurons . When PCin vivo and in vitro differentiation). When neurosecretory cells, a type of neuron, were exposed to neurotrophins, peptides, and adhesion proteins, such as glycoproteins [Lignosus rhinocerus extract that possessed NGF-like properties may be responsible for the activation able to activate Akt which is required in combination with activated MAPK cascade proteins for neurite outgrowth.Neurite outgrowth takes place in neuron  whereas showed a weaker activity at combination with 50 μg/mL (w/v). Combination of aqueous extract and NGF neurite showed outgrowth activity.Additive effect occurs when there is a combination of two or more drugs with same therapeutic effect and the result is the sum of the drug's effects . NGF actTricholoma sp. [Termitomyces albuminosus [Dictyophora indusiata [Grifola frondosa was found to induce activation of MAP kinase cascade in PC-12 cells and resulted in neuronal differentiation and suppression of serum deprivation-induced apoptosis [Tremella fuciformis crude aqueous extract contains polysaccharides, including glycosaminoglycan, promoted neuritogenesis, peripheral nerve regeneration, and muscle reinnervation following a sciatic nerve lesion [Hericium erinaceus were shown to enhance the growth and neuronal differentiation of rat pheochromocytoma cells (PC-12) [H. erinaceus known as erinacines extracted from mycelium have been well investigated and proven as strong stimulators of NGF [via JNK signaling by inducing phosphorylation of JNK and its downstream substrate c-Jun and increased c-fos expression [Researchers found that some compounds extracted from mushrooms, for instance, tricholomalides from loma sp. , termitouminosus , and dicndusiata , 38, werpoptosis . Tremelle lesion –42. Park (PC-12) . Nonpolas of NGF , 45. It pression .Coptis chinensis, induced the growth of axon and dendrite and showed a sustained neurite outgrowth for a longer period whereas NGF-induced neurite did not maintain a fully differentiated state after 14 days in culture [Centella asiatica, known as asiatic acid showed significant neurite outgrowth activity on human SH-SY5Y cells [Water extracts of the rhizome of a traditional herb,  culture . The non5Y cells .L. rhinocerus contained compound/s that have potent NGF-addtive effect. It will be important to discover the compounds in L. rhinocerus that stimulate neurite outgrowth activity. As this mushroom is currently being investigated to validate traditional uses, the chemistry is not well known. Aqueous extracts are said to certain high molecular weight polysaccharides that have anti-inflammatory properties [L. rhinocerus. With further development and investigation, L. rhinocerus and its bioactive compounds may be developed as mushroom nutraceutical to enhance process of neurite outgrowth activity in an aging population of humans.Our results indicated that aqueous extract of operties . To our L. rhinocerus not only for nerve regeneration but also as a therapeutic agent for neurodegenerative diseases.Further detailed investigations are needed to ascertain clinical use of the extract in treating any neurodegenerative diseases and bioactive compounds that are responsible for neurite outgrowth activity have to be elucidated. This mushroom has been used as traditional medicine by indigenous folks of Malaysia for more than 15 diseases. The ethnopharmacological knowledge has to be scientifically validated to enable use of this mushroom as a nutraceutical or pharmaceutical. The findings warrant further studies of L. rhinocerus sclerotium contained NGF-like compound/s that enhanced neurite outgrowth activity. There was an enhanced stimulation of 17% of neurite outgrowth when a combination of 20 μg/mL (w/v) aqueous extract and 30 ng/mL (w/v) NGF was added to PC-12 compared to the aqueous extract alone.Aqueous extract of


Background. Serum free light chains (sFLC), the most commonly detected paraprotein in CLL, were recently proposed as useful tools for the prognostication of CLL patients. Objective. To investigate the prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients' series. Patients and Methods. We studied 143 CLL patients of which 18 were symptomatic and needed treatment, while 37 became symptomatic during follow-up. Seventy-two percent, 18%, and 10% were in Binet stage A, B and C, respectively. Median patients' followup was 32 months (range 4–228). Results. Increased involved (restricted) sFLC (iFLC) was found in 42% of patients, while the summated FLC-kappa plus FLC-lambda was above 60 mg/dL in 14%. Increased sFLC values as well as those of summated FLC above 60 were related to shorter time to treatment  and overall survival . They also correlated with β2-microglobulin , serum albumin (P = 0.009 for summated sFLC), hemoglobin (P < 0.001), abnormal LDH , Binet stage (P < 0.05) and with the presence of beta symptoms (P = 0.004 for summated sFLC). Conclusion. We confirmed the prognostic significance of sFLC in CLL regarding both time to treatment and survival and showed their relationship with other parameters. Chronic lymphocytic leukemia is the most common type of leukemia in the Western world accounting for 40% of all leukemias. It affects mainly elderly patients as the median age of diagnosis is about 72 years and the male to female ratio is 2 : 1. So far, Rai and Binet staging systems are used for predicting CLL patients' outcome. Other prognostic markers, which have been established but mainly concern symptomatic CLL patients, are lymphocyte CD38 expression, presence of ZAP-70, immunoglobulin (Ig) heavy gene mutation status, and cytogenetic profile . In sympImmunoglobulins (Igs) are produced by terminally differentiated B cells (either plasma cells or long lived memory cells), with the capacity to produce antibodies with high affinity for the immunizing antigen which are composed by 2 heavy and 2 light chains . During Recent data have shown that serum free light chains and their ratio may constitute prognostic factors in CLL –15. SeruIt is of great importance to identify simple prognostic markers that could be widely applied in clinical practice and with low cost, in order to predict the group of asymptomatic patients that will shortly require therapy . We therFrozen sera from patients that fulfilled the 1996 CLL criteria  were drain situ hybridization (FISH) studies for del p17 and del q11, were tested in a subset of patients only . Patients' standard workup at diagnosis included physical examination and whole body CT scanning for the evaluation of eventual lymph node swelling and organomegaly, complete blood counts and cell morphology evaluation on blood smears, bone marrow aspiration and biopsy, blood or marrow lymphocyte immunophenotype, and biochemical background including serum lactate dehydrogenase (LDH) and serum protein electrophoresis, while serum beta-2-microglobulin and fluorescent Light chain restriction was established by flow cytometry or bone marrow biopsy immunohistochemistry. The patients' characteristics can be seen in TM, the Binding Site Birmingham, UK) in frozen sera drawn at diagnosis. Serum free light chain values were retrospectively determined by nephelometry  cutoff value and showed it was 60.6 mg/mL , (3) we additionally tested arbitrarily a cutoff of 50 mg/L, because few early stage CLL patients had a summated FLC above 60 mg/L.We evaluated the prognostic significance of the summated sFLC, using as cut off values (1) their median level, (2) the value of 60 mg/L proposed by Morabito et al.  that perStatistical analysis was performed using SPSS v15.0. Correlations between disease variables derived from patients' standard workout at diagnosis and sFLC values were evaluated by the chi square test when assessed as categorical variables and by the Kendall's test if one of them was categorical. With regard to survival and time to first treatment (TFT), the prognostic significance of abnormal sFLC and ratios were determined by univariate Cox regression analysis. Kaplan Meier method was used for pictorial representation of survival and TFT. Sixty-nine percent of patients presented with kappa light chain restriction and 31% with lambda monoclonality. Kappa sFLC ranged from 2.54 to 196 mg/L (median 19.2 mg/L) and lambda from 9.19 to 121 mg/L (median 20 mg/L) in kappa-and lambda-restricted patients, respectively. Increased involved (restricted) sFLC (iFLC), either kappa or lambda, were found in 42% of patients. sFLC ratio (FLCR) ranged from 0.02 to 496 (median 0.95). Abnormal FLCR values, suggesting light chain paraprotein presence, were present in 30% of patients. The summated FLC-kappa plus FLC-lambda ranged from 9.4 to 217.4 mg/L ; it was higher than 60 mg/L in 14%.P = 0.039 and P = 0.02, resp.), the presence of lymphadenopathy , hemoglobin level (negatively), , white blood cells counts (P < 0.01 both), lymphocyte absolute number (P < 0.01 both), serum immunoglobulin IgG level , abnormal LDH (P = 0.037 and 0.001 resp.), beta-2-microglobulin , serum albumin (P = 0.009 only for FLC kappa + lambda > 60), and with the presence of beta-symptoms (P = 0.004 only for summated sFLC > 60) (iFLC and summated sFLC kappa + lambda > 60 significantly correlated with Binet stage (LC > 60) . No correlations were found with the presence of spleen enlargement and CD38 expression while the number of cases tested for del p17, del q11, and IGVH status are too small to reach conclusions. FLCR did not show any significant correlation with disease variables.P = 0.0005 and P = 0.000003, resp.) as shown in Increased involvment sFLC (iFLC) values as well as values of summated sFLC kappa + lambda > 60 were related to shorter TFT  as shown in Increased iFLC and summated sFLC above 60 correlated with shorter OS . Abnormal  LDH correlated with OS (P = 0.001) but not with TFT.Binet stage correlated with both TFT and OS (P < 0.001) but not with OS. Serum albumin correlated neither with TFT nor with OS.Beta-2-microglobulin above 3.5 mg/L correlated with TFT  highly depends on the time to first treatment (TFT). The most frequent paraprotein produced in CLL is serum free light chain in almost up to 50% of the patients. Accordingly, we also found increased involved FLC in more than 40% of patients and an abnormal FLCR, thus, confirming the monoclonal nature of sFLC in 30% of cases. β2-microglobulin, M-IgVH, and abnormal sFLC ratio. Patients with CLL with an abnormal sFLC ratio were significantly more likely to have U-IgVH, a Zap-70 positivity, a lymphocyte doubling time less than 12 months, and a high β2-microglobulin. In a similar way, in a study involving 84 patients with CLL, Perdigao et al. [κ chain. In a separate group of 120 patients with CLL (serum samples collected before initiation or 6 months after cessation of treatment), 71 patients (59%) had abnormal FLCR. In addition to improving M-protein detection in CLL, it was shown that abnormal low FLCR (indicating lambda FLC involvement) was associated with worse outcome. In addition, no correlation was found between sFLC and other prognostic factors  implying, according to the authors, that sFLC is an independent prognostic factor in patients with CLL [It has recently been shown that sFLC and their sum above 60.6 mg/L may contribute usefully to prognosis , mainly o et al.  showed ao et al.  an abnoratients 5% had abnκ or λ FLC and abnormal sFLCr , type 2 with elevated kappa or lambda FLC and normal sFLCr , and type 3 with normal range kappa and lambda FLC and abnormal FLCR . Patients were followedup for a median of 47 months. Forty-nine percent of patients with sFLC abnormalities had a worse TFT and overall survival than those with normal sFLC. The authors concluded that sFLC is an important prognostic factor and is maintained after adjustment for Rai staging, with different types of sFLC abnormality affecting prognosis to various degrees. In their recent study, Maurer et al.  classifiIn 2010, Yegin et al.  in theirThe finding that increased polyclonal sFLC also constituted an adverse marker for time to first treatment (TFT) in CLL was firstly reported by Maurer et al.  and almoβ2-microglobulin, serum albumin, negatively with hemoglobin, white blood cell and lymphocyte counts, abnormal LDH, Rai and Binet stage, and with the presence of lymphadenopathy and beta-symptoms. The relationship with IgG is very interesting and its significance remains to be fully evaluated. The observed correlations of sFLC with β2-microglobulin, albumin, and IgG are reported here for the first time and their interpretation remains to be further studied. In our study, increased sFLC were found in 42% of the patients, while the summated FLC-kappa plus FLC-lambda was higher than 60 mg/dL in 14%. Increased involvement of sFLC (iFLC) values and values of summated sFLC above 60 were related to shorter TFT and OS, in agreement with the aforementioned publications; we additionally tested the prognostic potential of the summated FLC using as cutoff the median value and confirmed that the value of 60 mg/L was a better cutoff. We also found significant correlations with disease variables. Likewise, iFLC and/or summated sFLC above 60 correlated with In the present study, Binet stage was, as expected, of prognostic relevance with regard to TFT and OS, while abnormal serum LDH was predictive of a worse outcome and increased beta-2-microglobulin of a shorter TFT. However, the number of patients is too small to build prognostic models associating iFLC or summated FLC with other variables of adverse outcome. This should be done in larger series.The results of our study confirmed the significance of sFLC in CLL with regard to TFT and OS, showed their relationship with adverse prognostic clinical and laboratory parameters and suggested, in accordance with others, that these tests should be included in CLL patients' initial workout, as they offer additional prognostic information.


ABCC1 overexpression has been shown to contribute to drug resistance in the CHL cell line, KMH2.The mechanisms responsible for chemoresistance in patients with refractory classical Hodgkin lymphoma (CHL) are unknown. ATP-binding cassette (ABC) transporters confer multidrug resistance in various cancers and We analyzed for expression of five ABC transporters ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2 using immunohistochemistry in 103 pre-treatment tumor specimens obtained from patients with CHL. All patients received first-line standard chemotherapy with doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens. ABCC1 was expressed in Hodgkin and Reed-Sternberg (HRS) cells in 16 of 82 cases (19.5%) and ABCG2 was expressed by HRS cells in 25 of 77 cases (32.5%). All tumors were negative for ABCB1, ABCC2 and ABCC3. ABCC1 expression was associated with refractory disease (p = 0.01) and was marginally associated with poorer failure-free survival (p = 0.06). Multivariate analysis after adjusting for hemoglobin and albumin levels and age showed that patients with CHL with HRS cells positive for ABCC1 had a higher risk of not responding to treatment .Expression of ABCC1 by HRS cells in CHL patients predicts a higher risk of treatment failure and is marginally associated with poorer failure-free survival using standard frontline chemotherapy regimens. Classical Hodgkin lymphoma (CHL) is largely a curable disease using the widely accepted current standard first-line chemotherapy regimen of doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens, with or without consolidation radiotherapy . HoweverThe development of chemotherapy resistance by cancer cells is multifactorial . ATP binABCC1. Prompted by this finding, we assessed for expression of five ABC transporters, ABCG2, ABCB1, ABCC1, ABCC2, and ABCC3, in untreated CHL tumor specimens. We also investigated the potential prognostic value of expression of these ABC transporters in CHL.Steidl et al. recently showed overexpression of the ABC transporter, ABCC1  in the therapy-resistant CHL-derived cell line, KMH2 . They fuThe overall clinical and pathologic features of the study group are summarized in Table 3), hemoglobin (< or > 105 g/L), lymphocytes (< and ≥ 600 per mm3 or < and ≥ 8% of WBC), gender, International Prognostic Score (IPS) (< and ≥ 3), and age (< and ≥ 45 years). Multivariate Cox proportional hazards models including variables with p value < 0.15 in univariate analysis were fitted to evaluate the association of survival with demographic and clinical factors. Variables with p values < 0.05 were considered statistically significant. S plus software 8.04  and SAS software  were used for statistical analysis.Fisher’s exact test was used to evaluate the association of clinical response with categorical variables. The Kaplan-Meier method and log rank test were used for survival analysis. The following variables were evaluated in univariate analysis: disease stage (IV vs. I/II/III), chemotherapy , radiation therapy (yes and no), bone marrow metastasis (positive and negative), serum albumin (< and > 40 g/L), WBC . Six of 16 patients (37.5%) with ABCC1 expression versus 6 of 66 patients (9.1%) without ABCC1 expression were primary refractory (p = 0.01). This finding supports the results of Steidl and colleagues in the KMH2 cell line [We sought to determine if there was an association between expression of either ABCC1 or ABCG2 and clinical endpoints, such as response to treatment (refractory disease vs non-refractory disease), overall survival (OS), and failure free survival (FFS). Admittedly, the numbers are relatively small hampering this analysis. In this study FFS was defined as lack of disease progression, recurrence or death. Refractory disease was defined as patients with only a partial response to therapy, or recurrence within the first 18 months of initial therapy ,20. The ell line  and suggin vitro [Expression of ABCG2 by HRS cells was not significantly associated with OS, FFS or initial response to treatment. The lack of association of ABCG2 expression with treatment refractoriness, in contrast to ABCC1, is not fully explained, and relatively little is known about the differential substrate profiles of these two proteins. However, some authors have shown that certain drugs that are poor ABCC1 substrates, such as mitoxantrone (a type 2 topoisomerase inhibitor), are associated with overexpression of ABCG2 in vitro ,21, and In summary, ABCC1 and ABCG2 are expressed by HRS cells in a subset of CHL tumors. Univariate and multivariate analyses showed that expression of ABCC1 by HRS cells is associated with an increased risk of tumor progression, treatment resistance or death in CHL patients. Our findings corroborate those published by Steidl and colleagues  in the KCHL: Classical Hodgkin lymphoma; ABVD: Adriamycin, bleomycin, vinblastine, and dacarbazine; HRS: Hodgkin Reed-Sternberg cells; MMP11: Matrix metalloproteinase 11; ABC: ATP binding cassette; MDR: Multidrug resistance; CVPP: Cyclophosphamide, vinblastine, procarbazine, and prednisone; NOVP: Novantrone, vincristine, vinblastine, and prednisone; TMA: Tissue microarrays; WBC: White blood count; IPS: International prognostic index; FFS: Failure free survival; OS: Overall survival.The authors indicated no potential conflicts of interest.WG carried out data analysis and interpretation and wrote the manuscript. LX and BSE carried out data analysis and interpretation and performed statistical analysis. AY participated in the provision of clinical data and patient samples. KSD and CSL participated constructing the tissue microarrays. KK performed the immunohistochemical studies. LJM participated in providing of patient samples, data analysis, and the writing of the manuscript. FV conceived of the study, performed data analysis and interpretation, and wrote the manuscript. Final approval of the manuscript: All the co-authors.Table S1. Fisher’s exact test to compare clinical factors between ABCC1 unknown and ABCC1 known groups.Click here for file


To the Editor: Until 1999, influenza B virus was considered to infect humans only. However, more recent data proved that harbor seals (Phoca vitulina) and gray seals  also can be infected . The samples had been collected upon the animal’s admission to the Seal Rehabilitation and Research Centre (SRRC) in Pieterburen, the Netherlands, from seals living in Dutch coastal waters during 2002–2012. We tested these samples for influenza B virus–specific antibodies with a previously described hemagglutination inhbition (HI) assay, using the following influenza B virus strains as antigens: B/Seal/Netherlands/1/1999, B/Jiangsu/010/2003, B/Yamanashi/166/1998, and B/Malaysia/2506/2004 , whereas lower antibody titers were detected against all other viruses, including influenza B/Seal/Netherlands/1/99, which was isolated from seals in the Dutch coastal waters in 1999 . These results suggest that the seals had been infected with an influenza B virus similar to B/Yamanashi/166/98, which is antigenically different from B/Seal/Netherlands/1/1999. . The obvious remaining question is whether the influenza B virus that had infected these seals in 2009–2010 is a drifted variant of influenza B/Seal/1/99 or the result of another introduction of a human influenza B virus several years ago. No evidence for transmission of influenza B virus from seals to humans was found when strains circulating in humans in the Netherlands were compared with those circulating in seals during the observation period (On the basis of serum antibody titers against influenza B virus antigens tested and those of respective homologs of ferret antiserum samples, an antigenic map was prepared as described (


We evaluated LGV prevalence and predictors in a high risk population attending a STI Outpatients Clinic in the North of Italy.Chlamydia trachomatis (CT) DNA detection by Versant CT/GC DNA 1.0 Assay . RFLP analysis was used for CT molecular typing.A total of 108 patients (99 MSM and 9 women), with a history of unsafe anal sexual intercourses, were enrolled. Anorectal swabs and urine samples were tested for P = 0.0046).L2 CT genotype was identified in 13/108 (12%) rectal swabs. All LGV cases were from MSM, declaring high-risk sexual behaviour and complaining anorectal symptoms. Patients first attending the STI Outpatient Clinic received a significant earlier LGV diagnosis than those first seeking care from general practitioners or gastroenterologists (P = 0.0008) and presented more STIs (P = 0.0023) than LGV negative ones, in particular due to syphilis (P < 0.001), HIV (P < 0.001) and HBV (P = 0.001).LGV prevalence and characteristics found in our population are in agreement with international reports. Statistical analysis showed that LGV positive patients were older (P = 0.001 and P = 0.010).Multivariate logistic regression analysis revealed that HIV and syphilis infections are strong risk factors for LGV presence (respectively, Even if our results do not provide sufficient evidence to recommend routine screening of anorectal swabs in high-risk population, they strongly suggest to perform CT NAAT tests and genotyping on rectal specimens in presence of ulcerative proctitis in HIV and/or syphilis-positive MSM. In this context, CT DNA detection by Versant CT/GC DNA 1.0 Assay, followed by RFLP analysis for molecular typing demonstrated to be an excellent diagnostic algorithm for LGV identification. Chlamydia trachomatis (CT) serovars L1- L3 [Lymphogranuloma venereum (LGV) is a systemic sexually transmitted infection caused by In 2003, a new outbreak of LGV proctitis was described in the Netherlands, mainly in men who have sex with men (MSM) [The first Italian LGV case was observed in Milan in 2006 and only few cases of LGV proctitis have been described in Italy  so far.In this study we assess LGV prevalence and predictors in a high-risk population attending a STI Outpatients Clinic of a University Hospital in the North of Italy.Neisseria gonorrhoeae (GC).From January 2012 to April 2013, all the patients attending the STI Outpatients Clinic of St. Orsola University Hospital of Bologna and reporting unsafe anal sexual intercourses have been asked to carry out a clinical examination. An anorectal swab, a pharyngeal swab  and an urine sample were collected from each patient for DNA detection of CT and Chlamydia antibodies by immunoenzimatic assays  were performed in all patients. Diagnosis of genital warts was made by visual inspection. Furthermore, personal data and information about urogenital and rectal disorders, sexual behaviour, number of sexual partners in the last 6 months and history of previous STIs were recorded from each patient. Three months after antibiotic treatment for LGV, patients were re-evaluated.Microbiological investigations for the main STDs  and a serological screening for anti-A written consent was obtained by all the patients and the study protocol was reviewed by the Ethics committee of St. Orsola Hospital.omp1 gene semi-nested PCR, followed by RFLP analysis was performed as previously described [porA pseudogene [Urine specimens, anorectal and pharyngeal swabs were processed by Versant CT/GC DNA 1.0 Assay , a Real-Time PCR test simultaneously detecting the presence of CT and/or GC DNA . In caseescribed -7. GC reeudogene .P value <0.05 was considered significant. Statistical tests were performed using SPSS 13.0 for Windows computer software .Analyses of the differences between the groups were performed with χ2 test. Univariate and multiple logistic regression analyses were performed to evaluate the influence of the different variables on the outcomes. A During the study period, a total of 108 patients met our admission criteria. In particular, 99 MSM  and 9 heterosexual women . Three of the women and 29 of the MSM complained about various rectal symptoms.Nineteen rectal swabs resulted positive only for CT and 10 only for GC, whereas 4 were simultaneously scored positive for CT and GC. Thanks to molecular genotyping, in 10 cases we found non-LGV serovars CT , while in 13 cases L2 serovar was identified, coming to the final diagnosis of LGV proctitis. The total prevalence of LGV infection was 12% (13/108).Four urine samples were positive for non-LGV serovars CT, whereas 12 pharyngeal swabs and three urine specimens were positive for GC. No urine samples nor pharyngeal swabs were found positive for LGV-serovars CT. Finally, it is noteworthy to underline that in the high risk population of this study 38.3% of MSM (38/99) and 37.5% of the women (3/8) had at least one specimen scored CT or GC positive.Detailed results are shown in Figure Based on rectal swab findings, patients with positive LGV results were defined as LGV-positive, while all the others  as LGV-negative.All LGV cases were detected in MSM reporting unsafe receptive anal intercourses in the last 6 months.In Table P < 0.001).All patients suffering from LGV proctitis were symptomatic, complaining about anal pain (13/13), anal discharge (11/13), change in bowel habit (7/13), tenesmus (9/13) and inguinal adenopathy (5/13). In contrast, patients with non-LGV CT or GC proctitis were asymptomatic or complained about slight symptoms. In particular, only 30% CT and 40% GC positive patients were symptomatic respectively, in contrast to 100% LGV positive patients (P = 0.0008), as well as comparing LGV cases with non LGV-CT positive patients .When considering the mean age of LGV positive patients, we noticed a significant difference between LGV and non-LGV group . Moreover, it is interesting to add that patients infected by non-LGV CT genotypes showed similar results to CT-negative patients (P = 0.102). Anti-Chlamydia IgA values were negative or only slight increased with no significant differences between studied groups.All LGV positive patients showed altered anti-All LGV positive patients but one suffered from other STIs; in particular, 9 were HIV-positive. Six of them were under HAART-therapy with a well control of the infection .P = 0.001).When evaluating the distribution of other STIs in our population, we noticed that LGV positive patients presented more coinfections than LGV negative subjects (P =0.0046). First diagnostic hypothesis included inflammatory bowel diseases (IBD), enteritis and other STIs with rectal localization such as syphilis or gonorrhoea.Finally, considering LGV cases, patients first attending the STI Outpatients Clinic (4/13) received an earlier diagnosis compared to patients who first sought help from general practitioners or gastroenterologists. In particular, in the former group, time taken for LGV diagnosis was 2 weeks (range: 10–18 days), in contrast with the median of 8 weeks (4–92 weeks) of the latter group .In agreement with international literature -15, we oet al. found some cases of concurrent urethral LGV in a group of MSM with anorectal infection [Although patients reported active and passive anal intercourses, a LGV urethral infection has not been identified in our population. This is in concordance with previously reported studies ,9 even infection .As observed in our cases, LGV proctitis can be easily misdiagnosed at the first investigations due to the unawareness of general practitioners and shame of patients in seeking care . The mosHigh-risk sexual behaviours, including high number of sexual partners, unsafe intercourses, fisting and sex toys sharing are reported by most of LGV infected patients ,19. CommP = 0.0023); in particular among LGV patients higher rates of HIV (P < 0.001), syphilis (P < 0.001) and HBV (P = 0.001) infections were found. Logistic regression analysis in our population showed that HIV and syphilis represent the strongest risk factors for LGV infection (P < 0.001).Our data highlighted a significant difference between LGV-positive and LGV-negative subjects regarding coinfections due to other STIs  indeed present critical issues.Chlamydia serology can support LGV diagnosis in the appropriate clinical context, but the diagnostic utility of serological methods other than complement fixation and microimmunofluorescence procedures has not been well established yet [Chlamydia IgG by an immunoenzimatic assay in LGV-positive patients, suggesting a potential role of IgG testing for LGV diagnosis.shed yet ,23. In tOn the other hand, NAATs have not been FDA cleared or CE marked yet on rectal specimens.For that reason, many laboratories have performed their own validation studies in order to provide results for clinical management . MoreoveIn our experience, even if Versant CT/GC DNA 1.0 Assay has been not licensed for extragenital use, it showed good performances in CT/GC DNA detection on rectal and pharyngeal swabs. In addition, RFLP analysis of CT positive samples seems to be a reliable, inexpensive and time-saving method for CT genotyping.It follows that our suggested algorithm  could be an excellent choice for laboratory LGV diagnosis.Even if our results do not provide sufficient evidence to recommend routine screening of anorectal swabs in MSM population, they confirm, in agreement with European guidelines, the indication to perform CT NAAT tests and genotyping on rectal specimens in presence of ulcerative proctitis or in sexual contacts of LGV positive people ,24.In particular, our results suggest to test for rectal LGV all MSM complaining of anorectal symptoms, especially if other STIs (HIV and/or syphilis) are identified. Moreover, these subjects should be presumptively treated for LGV infection until CT molecular genotyping is known.On the strength of our epidemiological and statistical results, testing for LGV infection should be recommended to all HIV and/or syphilis-positive MSM complaining of anorectal symptoms. For this purpose, CT DNA detection by Versant CT/GC DNA 1.0 Assay, followed by RFLP analysis for molecular typing demonstrated to be an excellent diagnostic algorithm for LGV identification.The authors declare that they have no competing interests.AM, AD, RC conceived and designed the study; CF, PN, MC, SB, AF performed the clinical visit and sample testing; CF, AM, LBR analyzed the data; CF, AM, RC wrote the paper; all authors read and approved the final manuscript.


We present a case of a 34-year-old male who presented to the emergency ward with fever and abdominal pain. The diagnosis of Takayasu's arteritis and also antiphospholipid syndrome was made during an imaging workup of deep-vein thrombosis. A spontaneous coronary artery dissection was revealed in coronary CT angiography requested for chest pain and dyspnea. The patient was treated medically and discharged on close followup. The concurrence of spontaneous coronary artery dissection with antiphospholipid syndrome and Takayasu's arteritis has not been reported in the previous literature. The possibility of a spontaneous coronary artery dissection should be considered in patients presenting with both diseases. A 34-year-old Turkish male presented to the emergency ward with a one-week history fever and abdominal pain originated from the epigastric region. History revealed a spiking fever of 38°C accompanied by chills and shivering. The patient was admitted to the Infectious diseases clinic for further investigation of the etiology of fever. Physical examination revealed an oriented and cooperative patient. Blood pressure was 115/80 mmHg, pulse 85/min, respiratory rate 20/min, and body temperature 37,8°C. Cardiopulmonary and abdominal examinations were unremarkable.9/L, platelet count 40 × 109/L, erythrocyte sedimentation rate (ESR) 49 mm/h, and C-reactive protein (CRP) 421 mg/L. On peripheral blood smear, thrombocyte count was 50 × 109/L, and there were no atypical cells. Renal and hepatic functions were normal. Microbiological analyses were negative. Since fever increased to 38.5°C on the day after admission, the patient was treated with an empirical antibiotic therapy consisting of piperacillin-tazobactam. Laboratory results were as follows: hemoglobin 11.4 gr/dL, white blood cell count (WBC) 12.7 × 10The patient reported a medical history of deep-vein thrombosis (DVT) and pulmonary thromboembolism three years ago, which had been diagnosed by pulmonary CT angiography and lower-extremity indirect CT venography. There was no information regarding further evaluation of thrombophilia during that period. The patient had been treated with warfarin 5 mg per day for 6 months, which led to resolution of the deep-vein thrombus confirmed by the Doppler ultrasound. Further investigation of the etiology of secondary thrombophilia showed strong positivity for lupus anticoagulant at 2.93 , positive anticardiolipin IgG of  28.9 GPL , and a positive anti-beta2-glycoprotein 1 (B2GP1) IgG of 9.3 U/mL . Antinuclear antibody and anti-ds-DNA antibody were negative. The diagnosis of systemic lupus erythematosus (SLE) was ruled out. The patient was treated with acetylsalicylic acid (300 mg/d), hydroxychloroquine (200 mg/d), and enoxaparin  with a possible diagnosis of primary antiphospholipid syndrome. CT imaging demonstrated bilateral diffuse wall thickening of the common carotid arteries (CCAs) , internaThe patient was subsequently put on 1 mg/kg/day methylprednisolone with the diagnosis of Takayasu's arteritis. No difference greater than 10 mmHg between blood pressures measured on both arms and limbs was noted. On the third day of corticosteroid therapy, the patient suffered from chest pain and dyspnea. Electrocardiography appeared normal; however, troponin I and CK-MB levels were elevated. He was treated as non-ST segment-elevation myocardial infarction (NSTEMI) with clopidogrel, metoprolol, atorvastatin, and ramipril in addition to his previous therapeutic line of ASA and enoxaparin. Echocardiography demonstrated inferior basal-mid wall hypokinesia. Two days later, when troponin level was further elevated, the patient was referred for an inpatient coronary angiogram, which surprisingly demonstrated a spontaneous dissection extending from the acute marginal branch of the right coronary artery (RCA) to the ostium of the posterior descending artery (PDA) , with normal left anterior descending (LAD) and circumflex arteries (CX) . Percutaneous coronary intervention was not considered because the dissection did not obstruct the blood flow. During the followup at the intensive care unit, chest pain subsided and troponin level fell gradually. A dosage of 15 mg/week methotrexate and folic acid was added to the patient's treatment regimen.On the fourteenth day under corticosteroid therapy, a repeat CT angiography of the aorta demonstrated a decrease in vessel wall thickness and resolution of mesenteric fatty infiltration Figures . A control coronary angiogram performed fifteen days after the first one demonstrated persistence of the dissection found at the distal RCA . 9/L on a 1-week control checkup. The patient was discharged on oral methylprednisolone and methotrexate and advised for follow-up visit for coronary artery dissection. The patient was considered to be in remission for Takayasu's arteritis and planned for a life-long anticoagulation.Laboratory findings showed that ESR was 4 mm/h, CRP was below 0.8 mg/dL, and the thrombocyte count was 280 × 10Spontaneous coronary artery dissection (SCAD) is an uncommon but clinically relevant cause of acute myocardial infarction in young otherwise healthy patients. The mean age at presentation is 35 to 40 years with a striking predilection for females within this age group. The precise etiology of SCAD remains uncertain. However, reported cases in the literature have focused mainly on female patients in the peripartum or postpartum period, suggesting a potential role of hormonal changes and hemodynamic stress. There have also been cases that have reported connective tissue diseases as potential risk factors for SCAD. Lepper et al. reported a 43-year-old woman who was diagnosed with Churg-Strauss syndrome and died of dissection of all three coronary arteries . An unusThe antiphospholipid syndrome is characterized by thrombotic and obstetric complications in the presence of antiphospholipid antibodies. It occurs in an isolated form or in association with systemic connective tissue diseases, mainly SLE. According to the revised 2006 Clinical Guidelines, a diagnosis of antiphospholipid syndrome requires that the patient has at least one clinical and one laboratory criterion . ClearlyThis is the first case in the literature that has reported the occurrence of a coronary artery dissection in the presence of both Takayasu arteritis and antiphospholipid syndrome. This case highlights the importance of considering the possibility of spontaneous coronary artery dissection in patients who present with multiple inflammatory vascular diseases.


Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen.Leishmania species identification was performed by PCR-based assays of positive specimens.We compared standard invasive procedures including biopsy histopathology, biopsy PCR, and leishmanin skin test (LST) to a novel, non-invasive, cytology-brush based PCR for the diagnosis of ML in Lima, Peru. Consensus reference standard was 2/4 tests positive, and outcome measures were sensitivity and specificity. L. (V). braziliensis (n = 4), and L. (V). peruviana (n = 3).Twenty-eight patients were enrolled, 23 of whom fulfilled criteria for a diagnosis of ML. Sensitivity and specificity of biopsy with histopathology were 21.7% [95% CI 4.9–38.5%] and 100%; 69.6% [95% CI 50.8–88.4%] and 100% for LST; 95.7% [95% CI 87.4–100%] and 100% for biopsy PCR; and 95.7% [95% CI 87.4–100%] and 90% [95% CI 71.4–100%] for cytology brush PCR using both Cervisoft® and Histobrush® cervical cytology brushes. Represented species identified by PCR-RFLP included: Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted. Leishmania including L. (Viannia) braziliensisMucosal leishmaniasis (ML) is a severe and stigmatizing chronic sequela of infection with predominantly New World species of Traditional methods of diagnosing ML, such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen Leishmania (Viannia) subgenus can cause mucosal disease.We herein compared several ‘traditional’ methods for diagnosing ML including biopsy with histopathology, biopsy PCR, and leishmanin skin test (LST) to the novel, non-invasive method of cytology brush PCR using 2 different commercial grade cervical cytology brushes. In addition, we performed species identification using PCR-based assays of clinical specimens, which is important in countries like Peru where several members of the This study was approved by the Institutional Review Boards of Hospital Nacional Cayetano Heredia (HNCH) and the University of Toronto. All patients provided written informed consent for the study procedures prior to enrolment.Leishmania Clinic of the Instituto de Medicina Tropical “Alexander Von Humboldt” and HNCH, in Lima, Peru, between January and December 2010. The Institute houses a large outpatient clinic for the diagnosis and management of American tegumentary leishmaniasis, with an average of 30–40 new cases diagnosed per month The study was conducted at the Leishmania Clinic for the evaluation of mucosal  and/or skin lesions were approached to participate in this study, and screened for eligibility criteria. All patients were interviewed and examined by a clinic physician. Direct anterior rhinoscopy and oropharyngoscopy were performed on all patients (including those referred for cutaneous ulcers), to determine if mucosal abnormalities such as erythema, infiltration, or ulceration were present. We included patients who were referred to the Leishmania Clinic for suspected ML or CL; had one or more mucosal lesions with a clinical indication for mucosal biopsy; and were able to give informed consent for the diagnostic procedures. We excluded patients undergoing active treatment for ML or CL, and those with any contraindication to mucosal biopsy.Consecutive patients presenting to the After removing any overlying scab or crust with moistened gauze, and cleansing the mucosal lesion with isopropyl alcohol, sterile and duplicate CerviSoft®  and Histobrush®  cervical cytology brushes  were rolAfter collection of the cytology brush specimens and cleansing the lesion again with isopropyl alcohol, mucosal lesions were anesthetized with 20 mg/mL lidocaine spray. Two small biopsy specimens were then obtained from lesions using sterile nasal or ethmoid biopsy forceps. The tissue was then stored in 1.5-mL Eppendorf tubes containing 700 µL 100% ethanol at −20°C for qualitative PCR testing, or placed in 10% formalin for histopathology with hematoxylin and eosin, Ziehl-Neelsen, and Giemsa staining. Sterile gauze was applied with pressure to the mucosal lesion until hemostasis was achieved.Leishmanin skin tests were applied and read as described Prior to DNA extraction, samples were centrifuged at 3000 g for 5 min and ethanol was discarded. Biopsied tissues were disaggregated with a sterile scalpel. Disaggregated tissue and cytology brushes were processed for DNA isolation using the High Pure PCR Template Preparation Kit®  according to manufacturer's instructions.Leishmania kDNA PCR was performed using the HotStar Taq Plus DNA Polymerase kit  and conditions were as described Leishmania sub-genus Viannia species including L. (V.) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis, the principal causative species in Peru, were used for the species identification following initial kDNA PCR. PCR assays were performed using the HotStar Taq Plus DNA Polymerase kit  as previously described Four PCR assays targeting different sequences specific to L. (V.) peruviana from L. (V.) braziliensis and L. (V.) guyanensis, as previously described Cpb) gene, employed primers which distinguish between L. (V.) braziliensis and non-L. (V.) braziliensis species as previously described Cpb PCR conditions were as described hsp70), employed primers which distinguish between L. (V.) guyanensis and non-L. (V.) guyanensis species as previously described Hsp70 PCR conditions were as described The first assay, targeting the mannose phosphate isomerase gene (MPI), consisted of two separate reactions employing allele-specific reverse primers, which distinguish Leishmania glycoprotein of 63 kDa (gp63), with the following primer sequences: MUS (fwd) 5′- GTGGGTGTCATCAACATCCC – 3′ and MUSA3 (rev) 5′- CTGCTGCCGTACACCTGGAC – 3′Gp63 PCR conditions were as follows: 95°C for 5 min, followed by 45 cycles of denaturation at 94°C for 30 s; primer annealing at 63°C for 60 s; extension at 72°C for 60 s, and a final extension step at 72°C for 6 min . All PCR products were visualized on 1.5% agarose gels  and stained with ethidium bromide.A fourth and final PCR assay was used to confirm species on those samples that yielded weak bands or lack of amplification products in previous assays. The PCR target was a 870 bp fragment of cpb, hsp70 and gp63 PCR amplification as above, products were separately digested overnight at 65°C for the cpb assay, or 37°C for the hsp70 and gp63 assay, in a total volume of 20 µL, with 5 U of each restriction enzyme. The following enzymes were used in each reaction: TaqI (cpb) and HaeIII (hsp70) . For gp63, products were digested in duplicate. One reaction was digested with HincII and the second with SalI restriction enzyme . Restriction fragments were then analyzed separately using 2.5% agarose gels for cpb and gp63 or 4% agarose gels for hsp70 , and stained with ethidium bromide. When weak amplification product was observed after PCR, restriction fragments were separated using 12% polyacrylamide gel electrophoresis using the MiniProtean III system , and stained with silver stain . Mpi PCR distinguishes L. (V.) peruviana, while Cpb PCR-RFLP distinguishes L. (V.) braziliensis, and hsp70 PCR-RFLP differentiates L. (V.) guyanensis from L. (V.) lainsoni.Following We defined a lesion as ML when any 2 of 4 tests were positive, where tests refer to biopsy with histopathology; biopsy PCR; LST; or cytology brush PCR. These 4 tests served as the composite reference standard against which each individual diagnostic test was compared. Assessors of LST, histopathology, and PCR were blind to the results of the other assays.Based on existing literature Descriptive statistics  were calculated for continuous variables, and differences were compared using 2-tailed t-testing. Categorical variables were quantitated by proportions, and differences between the groups were compared using Yate's corrected Chi-square analysis. Differences in sensitivities and specificities were compared using the z-test. Statistical analyses were performed using SigmaStat 2.03 software . Level of significance was set at p<0.05.Twenty-eight patients were enrolled in the study: 23 males and 5 females. Of 28 patients enrolled, 23 were referred for suspicious mucosal lesions only, and 5 were referred for evaluation of cutaneous lesions who were then noted to have mucosal involvement on examination. Clinical and demographic characteristics of the cohort are summarized in Performance characteristics of each tested assay are summarized in Subjective tolerability of the CerviSoft® brush was superior to that of the Histobrush®, as it was reported to cause less discomfort and was softer on the mucosa, particularly in the nose. All healthy control volunteers had negative CerviSoft® and Histobrush® cytology brush PCR.L. (V.) braziliensis, 4 patients; and L. (V.) peruviana, 3 patients. There were no cases of L. (V.) guyanensis or L. (V.) lainsoni identified  kDNA can be detected in non-invasive cytology brush specimens for the diagnosis of ML. PCR of non-invasive cytology brush specimens had a comparable performance to PCR of biopsy specimens, and was superior to either conventional biopsy with histopathology or LST. Compared to biopsy, non-invasive cytology brushes are easier to obtain, require no technical expertise or anesthesia, cause no discomfort to the patient, do not carry risks of bleeding or infection, and obviate the need for sharps, sharps biosafety disposal, or concerns regarding needle stick injuries in the health care worker. Unlike biopsies, cytology brush specimens can be easily collected in the field and transported back to a reference center for testing. Thus, our data represent an advance in the approach to diagnostic testing in ML that will benefit the patient and health care worker alike.We have demonstrated that Paracoccidioides braziliensis, but also had a buccal mucosa biopsy and cytology brushes that were positive for L. (Viannia) kDNA by PCR. It is therefore possible that this patient had detectable amounts of persistent parasite DNA in the mucosa and new infection with Paracoccidioides braziliensis. Alternatively, the patient may have had active infection with both paracoccidioidomycosis and leishmaniasis. In any case, this patient highlights the need for non-invasive multiplex assays that can differentiate between common causes of mucosal lesions in the tropics. Although the patient fulfilled reference criteria for a diagnosis of ML, the uncertainty surrounding the diagnosis given the histopathology also suggests that further refinements to the cytology brush method, possibly including a quantitative component, are warranted.Although PCR of biopsy and brush specimens were highly sensitive and specific, there may have been 1 patient with known previously treated CL (1990) and ML (1992), and new isolated involvement of the buccal mucosa with no other focus of infection, who was a biological false positive. This patient had PAS-positive bodies on histopathology suspicious for Leishmania kDNA has been detected in the normal mucosa of Latin American patients with CL L. Viannia subgenus are co-endemic and portend different prognoses and response to therapy L. (V). braziliensis is historically the most well represented causative species in ML L. (V). peruviana are implicated in mucosal disease as well. Case-report level data implicating non-braziliensis sub-species in ML is also mounting Species identification is important in countries like Peru where several members of the In summary, we have demonstrated that cytology brush PCR using CerviSoft® and Histobrush® cervical cytology brushes is adequate for the diagnosis of ML though identification of causative species requires further optimization. We have further demonstrated its superior performance to the gold standard biopsy with histopathology. Cytology brush PCR offers numerous practical advantages over biopsy PCR including simplicity, tolerability, and cost efficiency due to the lack of need for highly trained personnel to collect the specimen, anesthesia, sterile biopsy instruments, and sharps biohazard disposal and precautions. At just 30–50 cents (US) per cytology brush, this novel diagnostic specimen is practical and comparatively affordable. It can be transported easily to a reference center for diagnostic testing and is likely appropriate for field situations. Future field studies are indicated, as are studies that aim to differentiate between common causes of mucosal lesions in the tropics using this simple non-invasive specimen.


Pseudomonas strains, under conditions of reduced fertilization, increases anthocyanin concentration in the fruits of strawberry.Anthocyanins are a group of common phenolic compounds in plants. They are mainly detected in flowers and fruits, are believed to play different important roles such as in the attraction of animals and seed dispersal, and also in the increase of the antioxidant response in tissues directly or indirectly affected by biotic or abiotic stress factors. As a major group of secondary metabolites in plants commonly consumed as food, they are of importance in both the food industry and human nutrition. It is known that arbuscular mycorrhizal (AM) fungi can influence the plant secondary metabolic pathways such as the synthesis of essential oils in aromatic plants, of secondary metabolites in roots, and increase flavonoid concentration. Plant Growth-Promoting Bacteria (PGPB) are able to increase plant growth, improving plant nutrition and supporting plant development under natural or stressed conditions. Various studies confirmed that a number of bacterial species living on and inside the root system are beneficial for plant growth, yield and crop quality. In this work it is shown that inoculation with AM fungi and/or with selected and tested  Anthocyanins are a group of widespread natural phenolic compounds in plants. They are mainly detected in flowers and fruits  and are responsible for bright colours such as orange, red and blue . In straAnthocyanins are glycosides and acylglycosides (even if strawberry anthocyanins are reported to be non-acylated ) of anthMedicago sativa L.), barrel medic (Medicago truncatula), red clover (Trifolium pratense) and soybean (Glycine max L.) AM colonization increases flavonoid concentration [Glomus intraradices colonization increase cyanidin-3-glucoside concentration [It is known that beneficial soil microorganisms, like bacteria and arbuscular mycorrhizal (AM) fungi can influence the plant secondary metabolic pathways . AM fungntration –20. In sntration . In addintration  tomato [ntration ,24 and mntration , increasntration . IncreasPlant Growth-Promoting Bacteria (PGPB) improve plant nutrition, and support plant development under natural or stressed environments. This beneficial activity on plant growth can be exploited in order to realize cultivation protocols based on reduced amount of fertilizers and pesticides –29.2 and phosphate solubilization); the synthesis of siderophores, antibiotics, enzymes and fungicidal molecules and niche competition, resulting in antagonistic activity against phytopathogenic microorganisms [The mechanisms responsible for the stimulation of plant growth activated by PGPB can be both direct and indirect and involve: the biosynthesis of plant signaling molecules; the reduction of stress-ethylene in plants; the improvement of nutrient uptake  induced an increased concentration of anthocyanins in strawberry fruits.Therefore, the aim of this work was to demonstrate that the use of a consortium of AM fungi in combination with selected and tested The high performance liquid chromatography (HPLC) chromatogram displayed six peaks. Of these, five were identified as anthocyanins  on the bThe five anthocyanins identified in the strawberry fruits were cyanidin 3-glucoside (peak 1), pelargonidin 3-glucoside (peak 2), pelargonidin 3-rutinoside (peak 3), pelargonidin malonyl glucoside (peak 4), and pelargonidin acetyl glucoside (peak 5). In general, pelargonidin 3-glucoside was by far the most abundant identified anthocyanin, followed by pelargonidin 3-rutenoside.Pseudomonas sp. 5Vm1K was the most effective of all microorganisms: when inoculated alone cyanidin concentration was three times that of controls, while in combination with AM fungi it promoted the doubling of this anthocyanin.Any inoculation increased the concentration of cyanidin 3-glucoside compared to CFD , but strThe effect of bacteria and AM fungi was similar for pelargonidin 3-glucoside and pelargonidin 3-rutinoside: 5Vm1k, AM fungi and Pf4 + AM fungi significantly increased the concentration of the two anthocyanins compared to CFD and CRD  and 4. IP. fluorescens Pf4 and 4.1 ± 0.7 for Pseudomonas sp. 5Vm1K.At the end of the experiment the extent of AM colonization (M%) ranged between 2% and 4%. Traces of mycorrhizal colonization  were detected in uninoculated plants. Roots of plants inoculated only with the AM fungi were colonized at a low extent (M% 3.0 ± 0.6). Co-inoculation AM fungi and the two bacterial strains resulted in similar values: 2.0 + 0.4 for Low nutrient supply resulted in a significant reduction of nitrogen concentration in leaves but not in the other organs of uninoculated plants .In roots, reduction of the nitrogen concentration was observed only in plants inoculated with AM fungi, alone or in combination with the strain 5Vm1K. Strain Pf4 and AM fungi increased leaf nitrogen concentration compared to that measured in CRD plants, while the strain 5Vm1K induced lower nitrogen concentration in leaves compared to CDF plants. Co-inoculation of each pseudomonad with AM fungi lead to a reduced amount of nitrogen in leaves compared to that recorded in plants cultivated following the standard fertilization protocol.The reduced fertilization resulted in a decrease of phosphorus concentration in all plant organs of uninoculated plants . InoculaAM fungi are known to affect the plant secondary metabolism in roots and in shoots. For instance, in roots the flavonoid concentration is increased by AM symbiosis –18,20, wAzospirillum sp. is able to modulate the phenolic compounds in rice [Exiguobacterium oxidotolerans increases the concentration of bacoside-A in Bacopa monnieri [Arabidopsis, Pseudomonas fluorescens SS101 is able to trigger defense responses promoting the biosynthesis of camalexing and glucosinolates [Also PGPB can impact plant secondary metabolism. For example, it has been shown that  in rice , and thamonnieri . In Arabinolates .Artemisia annua it has been found that double inoculation with the AM fungus Glomus mosseae and Bacillus subtilis strain Daz26 can modulate artemisin content with synergistic effects [In  effects .et al. [The effect of AM colonization on the concentration of anthocyanins was previously measured in strawberry fruits by Castellanos-Morales et al. , who shoet al. ), we couet al.  are muchet al.  and withet al. ; in addiet al. .et al. [et al. [et al. [et al. [However, Crespo et al.  reported [et al.  analysed [et al.  observed [et al.  detectedCo-inoculation with AM fungi and PGPB can result in synergistic effects on plants ,44. In tStrawberries are a particularly good source of antioxidants; it was found that strawberries have an antioxidant capacity 10-fold greater than that of many other fruits, including oranges, kiwi, grapefruit, grapes and mangos, among others . AnthocyA certain correlation between diets rich in fruits and vegetables and a lower incidence of some major human chronic diseases, including several forms of cancer and cardiovascular disease, among others, has been observed in a wide variety of epidemiological studies . These bAnthocyanins are also responsible for the red and blue colours of many fruits and berries such as chokeberry, black currant and strawberry. The quantitative analysis of anthocyanins in strawberries is important not only to assess their degree of maturity, but also due to the fact that they are responsible for their colour, and this attribute does influence a great deal of the consumer’s preferences .Higher anthocyanin concentration following AM colonization might depend on the activation of a defence response in the plant. Indeed, pathogens and insects increase the concentration of anthocyanins and antioxidants in strawberry fruits as defence mechanisms in fruits . HoweverPhosphorus, in plants, has multifunctional roles as a constituent of nucleic acids or biomembranes. Furthermore, it is highly involved in the energy metabolism of cells and is therefore required for the biosynthesis of primary and secondary metabolites . In tomaThe low levels of mycorrhizal colonization observed in this study might depend on two factors. On the one hand, the plants were grown at N and P concentrations that, even when fertilization was reduced, were not limiting for the plant growth, nor for the fruit yield . It is wThe levels of mycorrhizal colonization observed in this study were low and this might depend on two factors. On the one hand, the plants were grown at N and P concentrations which, even when fertilization was reduced, were not limiting for the growth, nor for the fruit yield , of uninFragaria × ananassa var. Selva) with continuous flowering habit were used.Frigo plantlets of a commercial variety of strawberry (Glomus), collected from an agricultural soil where potatoes and carrots were grown, was produced and provided by Mybasol s.r.l. .A mycorrhizal inoculum consisting of sporocarps, spores, hyphae, and root fragments colonized by a number of AM fungi  was isolated from a woody soil located in Sassello . Pf4 synthesizes siderophores, is able to solubilize phosphates at neutral, acid and alkaline pH and produces the phytohormone IAA as reported in Berta et al. [Two bacterial strains were used to inoculate the plants, alone or in combination with the mycorrhizal inoculum. a et al. .Pseudomonas sp. 5Vm1K (abbreviated: 5Vm1K) was isolated from the rhizosphere of Vaccinium myrtillus and grown in a larch woodland located in Bellino  and characterized as described by Bona et al. [a et al. .Greenhouse assays were conducted to evaluate the effect of the microorganisms on plant growth, flower and fruit production, and quality.Each strawberry frigo plantlet was transplanted into a 3 l plastic pot filled with mineral substrate “Terra Mediterranea”  containing low concentrations of phosphorus and nitrogen.4 0.1 M , washed twice and finally resuspended in MgSO4·7H2O 0.1 M. Bacterial density of the suspension was assessed using a calibration curve assessed by turbidity (λ = 600 nm) and adjusted to 109 CFU·mL−1. 5 mL of the bacterial suspensions were used to inoculate each treated plant at transplanting, and the inoculation was repeated after 20 days. The same amount of buffer was provided to uninoculated plants.For plant inoculation, bacteria were grown on tryptic soy agar  at 28 °C for 48 h. Bacterial cells were scraped from the medium and suspended in MgSOMycorrhizal plants were inoculated with 100 mL of the above-described AM inoculums mixed with the substrate at transplanting.2O5 used in conventional practise , except one set of plants that was grown at the full dose of nutrients ; all plants received the following micronutrient: B 0.113 g/pt; Cu 0.225 g/pt; Fe 2.925 g/pt; Mn 0.900 g/pt; Mo 0.011 g/pt; Zn 0.675 g/pt. Fertilizers were provided by Greenhas Italia . On alternate days plants were fed with tap water. Therefore, the experimental design included seven treatments, each one consisting of 10 plants: CFD: uninoculated (control) plants fertilized according to the conventional practise; CRD: uninoculated (control) plants with reduced fertilization; Pf4: plants inoculated with P. fluorescens strain Pf4 and grown with reduced fertilization; 5Vm1K: plants inoculated with Pseudomonas sp. strain 5Vm1K and grown with reduced fertilization; Myc: plants inoculated with the AM consortium and grown with reduced fertilization; Pf4-Myc: plants inoculated with P. fluorescens strain Pf4 and the AM consortium, and grown with reduced fertilization; 5Vm1K-Myc: plants inoculated with Pseudomonas strain 5Vm1K and the AM consortium, and grown with reduced fertilization.All plants were fertilized weekly with a solution that contained about 70% of N and PThe experiment was performed between April and September 2011, in greenhouse and lasted 22 weeks. Temperature and humidity were monitored continuously by means of a thermo-hygrograph.Fruits were harvested from April to September when most of their surface reached a full red colour, from April to September. Sepals were discarded and only fruit flesh was used for further analyses . HarvestAfter 22 weeks of growth, the plants were harvested. The roots were separated from the aerial part and washed with deionised water. Roots from each plant were stored in 70% ethanol for mycorrhizal assay.et al. [Forty randomly chosen 1 cm-long pieces were cut from each root system, fixed in 70% ethanol, and then stored at 4 °C until analysis. Root pieces were cleared in 10% KOH for 45 min at 60 °C, stained with 1% methyl blue in lactic acid and mounted on a slide. Mycorrhizal colonization was estimated according to Trouvelot et al. .The total fruit production of each treatment was divided into three parts according to the harvest time . The concentration of two anthocyanins (pelargonidin and cyanidin) was measured on fruit homogenates.et al. [v/v formic acid in HPLC-grade water) and a solvent B (2.5% v/v formic acid in HPLC-grade methanol). The following linear elution gradient was employed: from the beginning to 5 min, 85% solvent A and 15% Solvent B; from 5 to 20 min, solvent A increased up to 65% and held constant until 25 min; from 25 to 35 min solvent A decreased to 50% and then was kept constant until 45 min; from 45 to 50 min solvent A decreased to 34%; finally, the columns were washed and restored to 85% solvent A. The total run time was 80 min. Anthocyanins were detected at 510 nm (injection volume 20 μL).Anthocyanins were extracted and separated according to Comandini et al.  with somet al. [Each anthocyanin was quantified according to Chandra et al. . CommercThe amounts of each anthocyanin measured in the samples were expressed as A × DF × CF/SWT where A = amount of anthocyanin expressed as external standard equivalent from the calibration curve (mg/mL); DF = dilution factor; CF = molecular white correction factor to convert individual anthocyanin calculated as the external standard equivalents to their respective forms; SWT = initial sample white. The concentration of anthocyanins was referred to the fruit fresh weight.The anthocyanin extract was also used for nano-LC/ESI-MS (liquid chromatography/electron spray ionization-mass spectrometry) analysis. Fractions  analysis. After injection the sample was pre-concentrated and washed onto a trapping cartridge  using a Famos autosampler  at 40 μL/min of 0.1% formic acid.Using an Ultimate solvent delivery system , a linear gradient of acetonitrile (0.1% formic acid) from 20% to 95% over 85 min at flow rate of ~300 nL/min was used.Quadrupole time of flight mass spectrometry (Q-TOF-MS) and quadrupole time of flight mass spectrometry/mass spectrometry (Q-TOF-MS/MS) analyses were performed using a hybrid quadruple-time of flight QStar XL  equipped with a nanospray source operating in the positive ion mode. The needle voltage was 1800V. Data were processed using Analyst-QS V2.0 software provided with the spectrometer.m/z for MS/MS.Key parameter settings were as follows: ionspray voltage (IS) = 1900 V, curtain gas (CUR) = 25, declustering potential (DP) = 80 V, focusing potential (FP) = 280 V, collision gas setting (CAD) = 3 for nitrogen gas, DP2 = 25. Data were acquired using information-dependent acquisition (IDA) mode with Analyst QS software (AB-SCIEX). Mass range 100–1500 m/z for MS and 10–1800 Phosphorus and nitrogen concentrations in strawberry shoot and roots were determined. Aliquots (0.5 g dry weight) of each sample were digested in 6 mL of 65% nitric acid (Sigma-Aldrich) using a MARS 5 microwave oven . Digested samples were analysed by inductively coupled plasma-optical emission  and inductively coupled plasma-MS . Certified standards of analysed metal and acid blanks were run with all sample series for quality control. The phosphorus and nitrogen concentrations are given as percentage of dry weight.p < 0.05 was used to assess differences between treatments.Statistical analyses were performed with StatView 4.5 . Data were analyzed by two-way ANOVA using “fungus”  and “bacterium”  as factors. A one-way ANOVA, using “treatment” as factor, followed by Fisher’s probable least-squares difference test with cut-off significance at Inoculation of strawberry with AM fungi and PGPB resulted in increased concentration of the two main anthocyanins in the fruit produced by plants cultivated under conditions of reduced fertilization. In addition, three forms of pelargonidin were identified. Overall, our results suggest that the common fertilization practise provides nutrients in excess. Lower concentration of nutrients and inoculation with soil microorganisms can result in healthier fruits, with a higher concentration of antioxidant molecules, consistent with the improved nutritional quality of vegetables, as previously reported ,24,67).