Microphytobenthos and chironomid larvae attenuate nutrient recycling in shallow‐water sediments

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A randomized placebo-controlled study on high-dose oral algal docosahexaenoic acid supplementation in children with cystic fibrosis

Low plasma concentrations of docosahexaenoic acid (DHA) are reported in unsupplemented cystic fibrosis (CF) patients. Forty-one CF patients aged from 6 to 12 years were randomized to receive high-dose DHA (100 mg/kg/day in the first month and 1 g per day thereafter through a 12-month supplementation) or placebo (germ oil). Primary outcome was percentage change in plasma AA:DHA ratio. Secondary outcomes were changes in the number of pulmonary exacerbations compared to previous year, lung function, BMI, skinfold thicknesses, and body composition assessed by DXA and in serum concentrations of C-reactive protein, cytokines and vitamin (α-tocopherol and retinol). Compared to the control group plasma AA:DHA ratio decreased in the intervention group after 6 months (median percentage changes: ?73% in the intervention group vs. ?10% in the control group, P=0.001). No differences were detected between groups for secondary outcomes. Despite a decrease of the AA/DHA ratio, DHA supplementation for one year did not induce any significant biochemical and clinical improvement in CF patients.     

Next Generation Semiconductor Based-Sequencing of a Nutrigenetics Target Gene (GPR120) and Association with Growth Rate in Italian Large White Pigs

The GPR120 gene (also known as FFAR4 or O3FAR1) encodes for a functional omega-3 fatty acid receptor/sensor that mediates potent insulin sensitizing effects by repressing macrophage-induced tissue inflammation. For its functional role, GPR120 could be considered a potential target gene in animal nutrigenetics. In this work we resequenced the porcine GPR120 gene by high throughput Ion Torrent semiconductor sequencing of amplified fragments obtained from 8 DNA pools derived, on the whole, from 153 pigs of different breeds/populations (two Italian Large White pools, Italian Duroc, Italian Landrace, Casertana, Pietrain, Meishan, and wild boars). Three single nucleotide polymorphisms (SNPs), two synonymous substitutions and one in the putative 3′-untranslated region (g.114765469C > T), were identified and their allele frequencies were estimated by sequencing reads count. The g.114765469C > T SNP was also genotyped by PCR-RFLP confirming estimated frequency in Italian Large White pools. Then, this SNP was analyzed in two Italian Large White cohorts using a selective genotyping approach based on extreme and divergent pigs for back fat thickness (BFT) estimated breeding value (EBV) and average daily gain (ADG) EBV. Significant differences of allele and genotype frequencies distribution was observed between the extreme ADG-EBV groups (P < 0.001) whereas this marker was not associated with BFT-EBV.     

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

Anadromous trout threatened by whitefish gill‐net fisheries in the northern Baltic Sea

We estimated the effect of the gill‐net fisheries targeted at whitefish (Coregonus sp.) on anadromous sea trout, Salmo trutta, in the Gulf of Bothnia, Baltic Sea using separate data for fish species. The analysis of sea trout captures was based on tagging and recapture data collected in 1998–2011, while whitefish data were derived from individual samples of commercial fisheries from the same period. The mesh sizes used in gill‐net fishing and the seasonal and temporal distributions of recaptured sea trout and sampled whitefish were compared in the northern and southern Gulf of Bothnia. The trout had typically spent 1–2 years at sea, and they were mainly immature with a median body length of 40–43 cm at the time of recapture in gill nets. Despite the increase in the minimum permitted landing size from 40 to 50 cm in 2008, the median length of recaptured trout remained unchanged during the study period. Most (59%) of the gillnetted trout were caught in the southern Gulf of Bothnia in gill nets with mesh sizes of 40–45 mm, which were also used in the whitefish fishery (72%). In the northern Gulf of Bothnia, nets with a smaller mesh size of 25–39 mm took 83% of the whitefish catch and 39% from recaptured trout. In both areas, the overlap in mesh sizes used to gill‐net catch whitefish and sea trout increased during the study period. There were clear seasonal and areal differences in the relative probability of sea trout being captured in gill nets, suggesting that carefully tailored spatial and temporal restrictions on gill‐net fisheries could provide a tool to protect young sea trout without causing intolerable difficulties for the fisheries targeting other species.     

Inheritance of a mutant histocompatibility gene and a new mammary tumor virus genome in the B6.KH-84 mouse strain

The potential association between integration or deletion of mouse mammary tumor virus (MMTV) retroviral sequences and the appearance of non-H-2 histocompatibility (H) antigen mutations was investigated. Genomic blots from inbred strains carrying 22 loss, gain-loss, and gain mutations on the BALB/c and C57BL/6 backgrounds were hybridized with probes homologous to the long terminal repeat (LTR) and envelope (env) regions of MMTV. Twenty-one mutants were identical in restriction patterns to the respective background strains with all tested restriction enzymes and both probes. However, genomic blots of one gain mutant, B6.C-KH-84, exhibited restriction fragments which were not exhibited by either of the parental strains, C57BL/6 or BALB/c. An additional 5.5 kb Eco RI fragment was observed with the env probe and additional 9.2 kb and 5.5 kb fragments were observed with the LTR probe. These observations were substantiated by hybridization of these two probes with genomic blots generated with additional restriction enzymes. Assuming that the new provirus contains a single, internal Eco RI site as has been observed for other MMTV proviral sequences, it is presumed that the new provirus includes both 5 and 3 LTRs in addition to the env region. Based on the unique sizes of the observed restriction fragments relative to other identified MMTV proviral sequences, this provirus has been designated Mtv-22. The potential role of Mtv-22 in the genesis of the gained histocompatibility antigen in B6.C-KH-84 is discussed.On leave of absence from Istituto Nazionale per to Studio e la Cura dei Tumori, Milano, Italy     

Mda-9/syntenin is expressed in uveal melanoma and correlates with metastatic progression

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.     

The effects of an ideal beta-turn on beta-2 microglobulin fold stability

Beta-2 microglobulin (β2m) is the light chain of Class I major histocompatibility complex (MHC-I) complex. β2m is an intrinsically amyloidogenic protein capable of forming amyloid fibrils in vitro and in vivo. β2m displays the typical immunoglobulin-like fold with a disulphide bridge (Cys25-Cys80) cross-linking the two β-sheets. Engineering of the loop comprised between β-strands D and E has shown that mutations in this region affect protein structure, fold stability, folding kinetics and amyloid aggregation properties. Such overall effects have been related to the DE loop backbone structure, which presents a strained conformation in the wild-type (wt) protein, and a type I β-turn in the W60G mutant. Here, we report a biophysical and structural characterization of the K58P-W60G β2m mutant, where a Pro residue has been introduced in the type I β-turn i + 1 position. The K58P-W60G mutant shows improved chemical and temperature stability and faster folding relative to wt β2m. The crystal structure (1.25 ? resolution) shows that the Cys25-Cys80 disulphide bridge is unexpectedly severed, in agreement with electrospray ionization-mass spectrometry (ESI-MS) spectra that indicate that a fraction of the purified protein lacks the internal disulphide bond. These observations suggest a stabilizing role for Pro58, and stress a crucial role for the DE loop in determining β2m biophysical properties.