The stromal‐vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots

Adipose tissue represents a complex tissue both in terms of its cellular composition, as it includes mature adipocytes and the various cell types comprising the stromal‐vascular fraction (SVF), and in relation to the distinct biochemical, morphological and functional characteristics according to its anatomical location. Herein, we have characterized the proteomic profile of both mature adipocyte and SVF from human visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) fat depots in order to unveil differences in the expression of proteins which may underlie the distinct association of VAT and SAT to several pathologies. Specifically, 24 proteins were observed to be differentially expressed between SAT SVF versus VAT SVF from lean individuals. Immunoblotting and RT‐PCR analysis confirmed the differential regulation of the nuclear envelope proteins lamin A/C, the membrane‐cytoskeletal linker ezrin and the enzyme involved in retinoic acid production, aldehyde dehydrogenase 1A2, in the two fat depots. In sum, the observation that proteins with important cell functions are differentially distributed between VAT and SAT and their characterization as components of SVF or mature adipocytes pave the way for future research on the molecular basis underlying diverse adipose tissue‐related pathologies such as metabolic syndrome or lipodystrophy.     

Import of the transfer RNase colicin D requires site-specific interaction with the energy-transducing protein TonB

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous tonB mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both tonB mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.     

Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi

Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens—Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum—secrete small pH‐affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC‐dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non‐preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high‐carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host‐dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.     

Modified lipoproteins provide lipids that modulate dendritic cell immune function

Both physiological and pathological situations can result in biochemical changes of low-density lipoproteins (LDL). Because they can deliver signals to dendritic cells (DC), these modified lipoproteins now appear as regulators of the immune response. Among these modified lipoproteins, oxidized LDL (oxLDL) that accumulate during inflammatory conditions have been extensively studied. Numerous studies have shown that oxLDL induce the maturation of DC, enhancing their ability to activate IFNγ secretion by T cells. LDL treated by secreted phospholipase A₂ also promote DC maturation. Among the bioactive lipids generated by oxidation or phospholipase treatment of LDL, lysophosphatidylcholine (LPC) and some saturated fatty acids induce DC maturation whereas some unsaturated fatty acids or oxidized derivatives have opposite effects. Among other factors, the nuclear receptor peroxisome-proliferator activated receptor γ (PPARγ) plays a crucial role in this regulation. Non-modified lipoproteins also contribute to the regulation of DC function, suggesting that the balance between native and modified lipoproteins, as well as the biochemical nature of the LDL modifications, can regulate the activation threshold of DC. Here we discuss two pathological situations in which the impact of LDL modifications on inflammation and immunity could play an important role. During atherosclerosis, modified LDL accumulating in the arterial intima may interfere with DC maturation and function, promoting a Th1 immune response and a local inflammation favoring the development of the pathology. In patients chronically infected, the hepatitis C virus (HCV) interferes with lipoprotein metabolism resulting in the production of infectious modified lipoproteins. These lipo-viral-particles (LVP) are modified low-density lipoproteins containing viral material that can alter DC maturation and affect specific toll-like receptor signaling. In conclusion, lipoprotein modifications play an important role in the regulation of immunity by delivering signals of danger to DC and modulating their function.     

SPECTROSCOPIC DETERMINATION OF THE TOXICITY,ABSORPTION, REDUCTION,AND TRANSLOCATION OF Cr(VI) IN TWO MAGNOLIOPSIDA SPECIES

Hexavalent chromium is a contaminant highly mobile in the environment that is toxic for plants at low concentrations. In this work, the physiological response of Convolvulus arvensis and Medicago truncatula plants to Cr(VI) treatments was compared. C. arvensis is a potential Cr hyperaccumulator well adapted to semiarid conditions that biotransform Cr(VI) to the less toxic Cr(III). M. truncatula is a model plant well adapted to semiarid conditions with a well studied genetic response to heavy metal stress. The results demonstrated that C. arvensis is more tolerant to Cr toxicity and has a higher Cr translocation to the leaves. The inductively coupled plasma optical emission spectroscopy results showed that C. arvensis plants treated with 10 mg Cr(VI) L^–1 accumulated 1512, 210, and 131 mg Cr kg^–1 in roots, stems, and leaves, respectively. While M. truncatula plants treated with the same Cr(VI) concentration accumulated 1081, 331, and 44 (mg Cr kg^–1) in roots, stems, and leaves, respectively. Enzymatic assays demonstrated that Cr(VI) decreased ascorbate peroxidase activity and increased catalase activity in M. truncatula, while an opposite response was found in C. arvensis. The x-ray absorption spectroscopy studies showed that both plant species reduced Cr(VI) to the less toxic Cr(III).     

Reproductive performance of multiparous rabbit lactating does: effect of lighting programs and PMSG use

This study aims to determine if Pregnant Mare Serum Gonadotrophin (PMSG), used for oestrous synchronization in multiparous lactating does, could be replaced by one of the following lighting schedules without impairing reproductive performance: (a) 12-h L (light)/12-h D (dark) or (b) 8-h L/16-h D, until day 6 before artificial insemination (AI), when in both cases photoperiod was changed to 16-h L/8-h D and maintained until the day of AI, and in the following 4 days post Al the light hours were progressively reduced to the initial schedules. Two groups of 20 does each were respectively submitted to one of the lighting schedules specified above. All does were artificially inseminated in 6 consecutive cycles at 42 days intervals. In the first, third and fifth AIs, PMSG (20 IU/doe via sc 48 h before AI) was used in the two groups of does, whereas in the second, fourth and sixth Als no hormonal treatment was used. Degree of oestrous synchronization (also referred in text as sexual receptivity) was estimated by the colour of the vulva at AI. Reproductive performance of does was evaluated based on fertility (kindling rates), prolificity, mortality at birth, mortality at 21 days post birth, weight of the litter at 21 days post birth and number of weaned rabbits. Oestrous was better synchronized when PMSG was used with any of the two lighting programs. Without using PMSG, a photoperiod of 12-h L/12-h D until 6 days before AI resulted in a better sexual receptivity of does than 8-h L/16-h D. Fertility, prolificity, mortality of young rabbits at 21 days, the weight of the litters at 21 days and the number of weaned rabbits did not vary with the lighting program and were not affected by the PMSG treatment. Mortality at birth, however, was higher (+1 dead kit per litter) in litters housed under a light program of 12-h L/12-h D. Global productivity (number of weaned rabbits per 100 inseminated does) was better when using PMSG, for both lighting schedules. When using a photoperiod of 12-h L/12-h D until 6 days before AI, and omitting the PMSG treatment, global productivity was scarcely reduced, however, it was considerably impaired when using a photoperiod of 8-h L/16-h D until 6 days before AI and no PMSG treatment.     

Identification of 99 novel mutations in a worldwide cohort of 1,056 patients with a nephronophthisis-related ciliopathy

Nephronophthisis-related ciliopathies (NPHP-RC) are autosomal-recessive cystic kidney diseases. More than 13 genes are implicated in its pathogenesis to date, accounting for only 40 % of all cases. High-throughput mutation screenings of large patient cohorts represent a powerful tool for diagnostics and identification of novel NPHP genes. We here performed a new high-throughput mutation analysis method to study 13 established NPHP genes (NPHP1–NPHP13) in a worldwide cohort of 1,056 patients diagnosed with NPHP-RC. We first applied multiplexed PCR-based amplification using Fluidigm Access-Array? technology followed by barcoding and next-generation resequencing on an Illumina platform. As a result, we established the molecular diagnosis in 127/1,056 independent individuals (12.0 %) and identified a single heterozygous truncating mutation in an additional 31 individuals (2.9 %). Altogether, we detected 159 different mutations in 11 out of 13 different NPHP genes, 99 of which were novel. Phenotypically most remarkable were two patients with truncating mutations in INVS/NPHP2 who did not present as infants and did not exhibit extrarenal manifestations. In addition, we present the first case of Caroli disease due to mutations in WDR19/NPHP13 and the second case ever with a recessive mutation in GLIS2/NPHP7. This study represents the most comprehensive mutation analysis in NPHP-RC patients, identifying the largest number of novel mutations in a single study worldwide.     

Isolation and characterization of seven tetranucleotide microsatellite loci from Atlantic northern bluefin tuna Thunnus thynnus thynnus

Microsatellite‐enriched genomic libraries were obtained from the Atlantic northern bluefin tuna, Thunnus thynnus thynnus and seven tetranucleotide markers were successfully isolated and characterized from this library. These markers were found to have between 1 and 17 alleles in Atlantic northern bluefin tuna and heterozygosity ranged from 0 to 0.85. No deviations from the expectation of Hardy‐Weinburg equilibrium were found for any marker. Several of these markers amplify reliably in other tuna species.