RET fusions in a small subset of advanced colorectal cancers at risk of being neglected.

Background: Recognition of rare molecular subgroups is a challenge for precision oncology and may lead to tissue-agnostic approval of targeted agents. Here we aimed to comprehensively characterize the clinical, pathological and molecular landscape of RET rearranged metastatic colorectal cancer (mCRC). Patients and methods: In this case series, we compared clinical, pathological and molecular characteristics of 24 RET rearranged mCRC patients with those of a control group of 291 patients with RET negative tumors. RET rearranged and RET negative mCRCs were retrieved by systematic literature review and by taking advantage of three screening sources: (i) Ignyta's phase 1/1b study on RXDX-105 (NCT01877811), (ii) cohorts screened at two Italian and one South Korean Institutions and (iii) Foundation Medicine Inc. database. Next-generation sequencing data were analyzed for RET rearranged cases. Results: RET fusions were more frequent in older patients (median age of 66 versus 60 years, P = 0.052), with ECOG PS 1-2 (90% versus 50%, P = 0.02), right-sided (55% versus 32%, P = 0.013), previously unresected primary tumors (58% versus 21%, P < 0.001), RAS and BRAF wild-type (100% versus 40%, P < 0.001) and MSI-high (48% versus 7%, P < 0.001). Notably, 11 (26%) out of 43 patients with right-sided, RAS and BRAF wild-type tumors harbored a RET rearrangement. At a median follow-up of 45.8 months, patients with RET fusion-positive tumors showed a significantly worse OS when compared with RET-negative ones (median OS 14.0 versus 38.0 months, HR: 4.59; 95% CI, 3.64-32.66; P < 0.001). In the multivariable model, RET rearrangements were still associated with shorter OS (HR: 2.97; 95% CI, 1.25-7.07; P = 0.014), while primary tumor location, RAS and BRAF mutations and MSI status were not. Conclusions: Though very rare, RET rearrangements define a new subtype of mCRC that shows poor prognosis with conventional treatments and is therefore worth of a specific management.

A mutation in caspase-9 decreases the expression of BAFFR and ICOS in patients with immunodeficiency and lymphoproliferation.

Lymphocyte apoptosis is mainly induced by either death receptor-dependent activation of caspase-8 or mitochondria-dependent activation of caspase-9. Mutations in caspase-8 lead to autoimmunity/lymphoproliferation and immunodeficiency. This work describes a heterozygous H237P mutation in caspase-9 that can lead to similar disorders. H237P mutation was detected in two patients: Pt1 with autoimmunity/lymphoproliferation, severe hypogammaglobulinemia and Pt2 with mild hypogammaglobulinemia and Burkitt lymphoma. Their lymphocytes displayed defective caspase-9 activity and decreased apoptotic and activation responses. Transfection experiments showed that mutant caspase-9 display defective enzyme and proapoptotic activities and a dominant-negative effect on wild-type caspase-9. Ex vivo analysis of the patients' lymphocytes and in vitro transfection experiments showed that the expression of mutant caspase-9 correlated with a downregulation of BAFFR (B-cell-activating factor belonging to the TNF family (BAFF) receptor) in B cells and ICOS (inducible T-cell costimulator) in T cells. Both patients carried a second inherited heterozygous mutation missing in the relatives carrying H237P: Pt1 in the transmembrane activator and CAML interactor (TACI) gene (S144X) and Pt2 in the perforin (PRF1) gene (N252S). Both mutations have been previously associated with immunodeficiencies in homozygosis or compound heterozygosis. Taken together, these data suggest that caspase-9 mutations may predispose to immunodeficiency by cooperating with other genetic factors, possibly by downregulating the expression of BAFFR and ICOS.

Association among XRCC1, XRCC3, and BLHX gene polymorphisms and chromosome instability in lymphocytes from patients with endometriosis and ovarian cancer.

Endometriosis is a complex disease that has both benign and malignant characteristics. It affects 5-10% of women of reproductive age. Studies have demonstrated the existence of common genetic changes in endometriosis and ovarian cancer, suggesting a possible association between these 2 diseases. However, the mechanisms that lead to the development of cancer from endometriosis remain unknown. In this study, we evaluated 3 groups of women: 72 patients with endometriosis, 70 with ovarian cancer, and 70 healthy individuals (controls). Repair (XRCC1 codons 194 and 399, XPD codons 312 and 751, and XRCC3 codon 241)- and metabolism (BLHX codon 443)-related gene polymorphisms were analyzed using the polymerase chain reaction-restriction fragment length polymorphism technique; the efficiency of DNA damage repair was analyzed in vitro in lymphocytes exposed to bleomycin. The logistic regression model was used to evaluate key associations. The results showed an increased average of chromosome breakage in bleomycin-treated lymphocytes from patients with endometriosis and ovarian cancer compared with healthy women. We also detected significant association between XRCC1, XRCC3, and BLHX polymorphisms and a high frequency of chromosomal damage. Women with endometriosis or ovarian cancer may have an altered mechanism of DNA repair, and these defects may be related to a higher incidence of ovarian cancer.

Solid lipid nanoparticles of cholesteryl butyrate inhibit the proliferation of cancer cells in vitro and in vivo models.

BACKGROUND AND PURPOSE: Solid lipid nanoparticles containing cholesteryl butyrate (cholbut SLN) can be a delivery system for the anti-cancer drug butyrate. These nanoparticles inhibit adhesion of polymorphonuclear and tumour cells to endothelial cells and migration of tumour cells, suggesting that they may act as anti-inflammatory and anti-tumour agents. Here we have evaluated the effects of cholbut SLN on tumour cell growth using in vitro and in vivo models. EXPERIMENTAL APPROACH: Cholbut SLNs were incubated with cultures of four tumour cell lines, and cell growth was analysed by assessing viability, clonogenic capacity and cell cycle. Effects on intracellular signalling was assessed by Western blot analysis of Akt expression. The in vivo anti-tumour activity was measured in two models of PC-3 cell xenografts in SCID/Beige mice. KEY RESULTS: Cholbut SLN inhibited tumour cell line viability, clonogenic activity, Akt phosphorylation and cell cycle progression. In mice injected i.v. with PC3-Luc cells and treated with cholbut SLN, . in vivo optical imaging and histological analysis showed no metastases in the lungs of the treated mice. In another set of mice injected s.c. with PC-3 cells and treated with cholbut SLN when the tumour diameter reached 2 mm, analysis of the tumour dimensions showed that treatment with cholbut SLN substantially delayed tumour growth. CONCLUSION AND IMPLICATIONS: Cholbut SLN were effective in inhibiting tumour growth in vitro and in vivo. These effects may involve, in part, inhibition of Akt phosphorylation, which adds another mechanism to the activity of this multipotent drug.

Goat milk allergenicity as a function of αs1-casein genetic polymorphism.

Cow milk allergy is the most frequent allergy in the first years of life. Milk from other mammalian species has been suggested as a possible nutritional alternative to cow milk, but in several cases, the clinical studies showed a high risk of cross-reactivity with cow milk. In the goat species, αS1-casein (αS1-CN), coded by the CSN1S1 gene, is characterized by extensive qualitative and quantitative polymorphisms. Some alleles are associated with null (i.e., CSN1S1 0(1)) or reduced (i.e., CSN1S1 F) expression of the specific protein. The aim of this work was to obtain new information on goat milk and to evaluate its suitability for allergic subjects, depending on the genetic variation at αs1-CN. Individual milk samples from 25 goats with different CSN1S1 genotypes were analyzed by sodium dodecyl sulfate PAGE and immunoblotting, using monoclonal antibodies specific for bovine α-CN and sera from children allergic to cow milk. A lower reaction was observed to 2 goat milk samples characterized by the CSN1S1 0(1)0(1) and 0(1)F genotypes. Moreover, a fresh food skin prick test, carried out on 6 allergic children, showed the lack of positive reaction to the 0(1)0(1) milk sample and only one weak reactivity to the 0(1)F sample. The risk of cross-reactivity between cow and goat milk proteins suggests the need for caution before using goat milk for infant formulas. However, we hypothesize that it can be used successfully in the preparation of modified formulas for selected groups of allergic patients. The importance of taking the individual goat CN genetic variation into account in further experimental studies is evident from the results of the present work.

The Garfagnina goat: a zootechnical overview of a local dairy population.

Domestic livestock with a limited distribution are increasingly recognized in the action plans of the European Union as a reason for protecting rural land. The preservation and enhancement of the native germplasm and traits selected through the ages in different areas of farming is the first step in increasing typical products at a time when high quality products are increasingly in demand. This is the first time that a zootechnical overview has been performed on the Italian native goat population named "Garfagnina," which is registered on the Tuscan regional repertory of genetic resources at risk of extinction. The aim of the study was to give added value to this population by focusing on particular traits that could be used for promoting typical products. Data on the size of the local goats, zoometric measures, breeding system, milk quality, and genetic polymorphisms were collected to get insight into the current state of the population of this type of goat. The native goat population is reared in Tuscany in central Italy, mostly for its milk. The local goat farms considered in our study are located in the hills and mountains of the northwestern Tuscan Apennine area. For every farm we measured at least 10% of the reproductive females (273), randomly chosen, and all reproductive males (47) for a total of 320 subjects. Regarding the management of the animals and the feeding system, semi-extensive farming is practiced in all the flocks. From a morphological point of view the animals are relatively homogeneous, especially in terms of zoometric data, whereas they show a wider variability regarding coat. Milk gross and fatty acid composition were similar to that reported in the literature for bulk goat milk. Moreover, the average of somatic cell count and standard plate count found in Garfagnina goat milk indicated good hygienic farm management and correct milking practices, although milking is mainly manual. The average number of globules per milliliter found in Garfagnina goat milk was almost double compared with the literature, whereas the average diameter was lower. Milk coagulation properties were scarce, thus indicating poor cheesemaking aptitude of Garfagnina milk. Selecting haplotypes carrying alleles associated with a higher expression of the specific casein could help improve milk cheesemaking aptitude. Moreover, the rather high frequency of the faint CSN1S1*F allele and the occurrence of CSN2*0 might suggest that Garfagnina goat milk could be used, after an appropriate selection, for direct consumption of milk at low casein content for intolerant human subjects.

Technical note: simultaneous identification of CSN1S2 A, B, C, and E alleles in goats by polymerase chain reaction-single strand conformation polymorphism.

Most variability in goat caseins originates from the high number of genetic polymorphisms often affecting the specific protein expression, with strong effects on milk composition traits and technological properties. At least 7 alleles have been found in the goat alpha(S2)-CN gene (CSN1S2). Five of them (CSN1S2*A, CSN1S2*B, CSN1S2*C, CSN1S2*E, and CSN1S2*F) are widespread in most breeds, whereas the other 2 (CSN1S2*D and CSN1S2*0) are rarer alleles. Four different PCR-RFLP tests are needed to detect all of these variants at the DNA level. The objective of this study was to develop and validate a rapid method for typing 4 of the 5 most-common goat CSN1S2 alleles by means of PCR-single strand conformation polymorphism (SSCP). The method was validated by analyzing 37 goat samples at the protein and DNA level, respectively, by milk isoelectrofocusing and PCR-RFLP methods already described. The genotypes obtained using the PCR-SSCP approach were in full agreement with those obtained by the validation analyses. The newly developed PCR-SSCP approach provides an accurate and inexpensive assay highly suitable for genotyping goat CSN1S2.

Response of bacterial community during bioremediation of an oil-polluted soil.

AIM: To study the response of the bacterial community to bioremediation of a soil with an aged contamination of crude oil. METHODS AND RESULTS: The bacterial community in laboratory soil columns during a 72-day biostimulation treatment was followed by analysing the number of total cultivable hydrocarbon-degrading bacteria, soil respiratory activity and the 16S-23S rDNA internal transcribed spacer homoduplex heteroduplex polymorphisms (ITS-HHP) of total soil bacterial DNA. ITS-HHP permits an estimate of both length and sequence polymorphism in a 16S-23S rDNA spacer population, using to advantage the homoduplex and heteroduplex fragments that are generated during PCR. The treatment, made by air sparging and biostimulation with a mineral nutrient and surfactant solution, resulted in a 39.5% decrease of the total hydrocarbon content. Within 4 days of treatment onset the bacterial community underwent a first phase of activation that led to a substantial increase in the observable diversity. Subsequently, after a 12-day period of stability, another activation phase was observed with further shifts of the community structure and an increase in the abundance and diversity of catechol-2,3-dioxygenase (C23O) genes. CONCLUSIONS: The overall data suggest an important contribution of uncultivable bacteria to the soil bioremediation, since, during the second activation phase, the increases of the respiratory activity, bacterial diversity and C23O gene abundance and diversity were not accompanied by a corresponding increase of the cultivable bacteria number. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that successive phases of activation of bacterial populations occur during a bioremediation treatment of oil-polluted soil.

Laboratory-scale trials of electrolytic treatment on industrial wastewaters: microbiological aspects.

Animal, civil and industrial waste matter is a source of potential chemical, microbiological and air pollutants. In populated areas the presence of faecal bacteria and the production of malodorous compounds during waste storage and in the tanks of wastewater treatment plants, can cause concern. The general aim of the work was to study electrolytic waste treatment (recently applied on animal slurry) using low electric current across graphite and copper electrodes, determining its effect on the microflora of sludge, collected from the equalisation basin of an industrial aerobic wastewater treatment plant, and on odour emission abatement. Biochemical and enzymatic indicators like ATP content and a pool of 19 enzymatic activities were tested, comparing them with viable cell counts by traditional microbiological methods, to verify the validity of such indicators in monitoring the electrolytic treatment and to assess their correlation with odour reduction. The preliminary results of our laboratory-scale trials showed that in the presence of inert electrodes, such as graphite, metabolic activity is stimulated, whereas with copper electrodes the ATP content and some enzymatic activities are inhibited quite considerably after only four days, this being accompanied by a marked reduction in odour. Consideration was also given to the total copper released from the electrodes and its recovery using iron electrodes.

Survival and naphthalene-degrading activity of Rhodococcus sp. strain 1BN in soil microcosms.

AIMS: The survival and activity of Rhodococcus sp. strain 1BN, inoculated into naphthalene-contaminated sandy-loam soil microcosms, were studied using classical and molecular methods. METHODS AND RESULTS: The naphthalene-degrading activity of 1BN in microcosms was examined through viable counts, CO2 production and naphthalene consumption, while its survival after inoculation was monitored by detecting the contemporary presence of alkane and naphthalene degradative genes and by analysing the 16S rDNA specific restriction profile. The inoculation of 1BN did not significantly enhance naphthalene degradation in the naphthalene-contaminated native soil, where 1BN maintained its catabolic activity also when in the presence of indigenous microflora. Instead the rate of naphthalene degradation by the inoculated 1BN was greater in sterile naphthalene-contaminated soil. The level of 1BN was only slightly higher after inoculation regardless of whether indigenous naphthalene-degrading bacteria were present or not and 1BN remained viable even when the substrate was depleted. CONCLUSIONS: This study documents the colonization and growth of 1BN in a non-sterile, naphthalene-added, sandy-loam soil having an active indigenous naphthalene-degrading population. SIGNIFICANCE AND IMPACT OF THE STUDY: An active and well-established naphthalene-degrading bacterial population in the native soil did not hamper the survival of the introduced 1BN that, through its activity, enhanced the mineralization rate of naphthalene.

Ochratoxin A in cereal-based baby foods: occurrence and safety evaluation.

Ochratoxin A is a typical cereal contaminant with strong nephrotoxic activity. To estimate the quantity of ochratoxin A that can be taken in by a child in the weaning period, several samples of cereal-based baby foods were analysed. Although most samples analysed contained ochratoxin A in undetectable amounts or below the Italian legal limit of 0.5 microg kg(-1), some irregular products were found. In particular, the analyses of the 119 batches (338 samples) of baby foods considered indicated that: 20 batches (16.8%) contained detectable quantities of ochratoxin A and four of these (3.4% of the total) contained ochratoxin A above the Italian permitted value. All samples coming from agricultural practices based on integrated pest management contained undetectable amounts of ochratoxin A, while approximately 5% of batches coming from conventional and organic agricultural practices were above the legal limit. On the basis of the established provisional tolerable weekly intake (PTWI), there is no significant toxicological risk for a child who occasionally consumes a formula with ochratoxin concentration slightly above the permitted level. However, stricter controls have to be applied to reject the batches containing irregular concentrations of ochratoxin A.

Dynamic air-water exchange of polychlorinated biphenyls in the New York-New Jersey Harbor Estuary.

Simultaneous measurements of polychlorinated biphenyls (PCBs) in the air and water over Raritan Bay and New York Harbor were taken in July 1998, allowing the first determinations of air-water exchange fluxes for this heavily impacted system. Average gas-phase concentrations of sigmaPCBs were 1.0 ng m(-3) above Raritan Bay and 3.1 ng m(-3) above New York Harbor. A similar gradient was observed for dissolved water concentrations (1.6 and 3.8 ng L(-1), respectively). Shallow slopes of log K(oc) vs log K(ow) plots indicated a colloidal contribution to the dissolved concentrations, and a three-phase partitioning model was therefore applied. PCBs associated with colloids ranged from 6% to 93% for trichloro- to nonachlorobiphenyls, respectively. Air-water gas exchange fluxes of sigmaPCBs exhibited net volatilization for both Raritan Bay at +400 ng m(-2) day(-1) and New York Harbor at +2100 ng m(-2) day(-1). The correction for the colloidal interactions decreased the volatilization flux of sigmaPCBs by about 15%. Net air-water exchange fluxes of PCBs are expected to remain positive throughout the year due to the large water-air fugacity gradient and relatively constant seasonal water concentrations. The volatilization fluxes are approximately 40 times greater than atmospheric deposition of PCBs via both wet and dry particle deposition, suggesting that the estuary acts as a net source of PCBs to the atmosphere year-round.

Response of the bacteria and fungi of two soils to the sulfonylurea herbicide cinosulfuron.

Changes in aerobic bacteria and autotrophic nitrifier numbers, and in respiration and nitrification in two soils treated with cinosulfuron at 42 (field rate) and 4200 microg/kg were studied after 1 and 4 weeks of incubation under laboratory conditions. Only nitrification at 1 week was slightly inhibited by the cinosulfuron treatment, even at the field rate. In vitro toxicity tests carried out in agar media on representative aerobic bacteria, fungi and Azotobacter strains isolated from the two soils, as well as on nine collection soil bacteria, showed that only a very high cinosulfuron concentration (100 mg/l) can have negative effects on the growth of a limited number of soil heterotrophic microorganisms, under conditions similar to those of soil environment. The absence of three branched-chain amino acids increased bacterial sensitivity, thus showing the importance of the chemical conditions and suggesting acetolactate synthase enzyme blockage as the toxicity mechanism. It is concluded that cinosulfuron has a negative effect on only a few aspects of the microbial community in soil ecosystems, even at concentrations higher that those currently in use.

Differential effects of the herbicides bensulfuron and cinosulfuron on soil microorganisms.

Bensulfuron toxicity on soil microbes was evaluated by the methods used in a previous study on cinosulfuron; the effects of the two sulfonylureas were compared. Cinosulfuron and bensulfuron, at the normal field application rate and 100 times higher, had no effect on the total number of bacteria and nitrifiers, or on the respiration activity in the soil, but they did decrease the nitrification activity. In vitro toxicity tests carried out on representative soil microbial strains using bensulfuron at 50 mg l(-1) showed some inhibition of three of the 17 bacterial strains and strong inhibition of almost all the 12 fungal strains; cinosulfuron had had no effect on any of these strains in the previous study. It is concluded that, compared with cinosulfuron, bensulfuron is potentially more toxic on soil heterotrophic microorganisms, but only at very high concentrations that are nearly impossible to reach with the usual agricultural use of the herbicides. However, autotrophic nitrifiers were more sensitive to both sulfonylureas than the other microorganisms.

Influence of the herbicide bentazon on soil microbial community.

Changes in microbial numbers and activities in a soil in response to bentazon applied at 10 and 100 ppm were studied after 4 and 30 weeks of incubation in laboratory conditions. As regards the eight general and functional microbial groups studied (aerobic and anaerobic bacteria, fungi, aerobic and anaerobic N2-fixing bacteria, nitrifiers, aerobic and anaerobic cellulolytic microorganisms), only the number of anaerobic N2-fixing bacteria significantly decreased, in the presence of the highest herbicide concentration for 30 weeks. At both the incubation times, only the higher dose of bentazon markedly inhibited soil nitrification and CO2 emission. Methanogenesis was inhibited by 1000 ppm bentazon added to anaerobic liquid cultures containing 5% soil for at least 2 weeks. There was an incomplete recovery of the herbicide at the two incubation times: < 5% of 10 ppm after 4 weeks and about 30% of 100 ppm after 30 weeks. No biodegradation of the compound was observed in liquid cultures under aerobic or anaerobic conditions. It is concluded that a bentazon concentration no higher than the field rate distributed within a 2-cm layer of soil does not considerably affect the microflora even in the absence of microbial degradation.