C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism

C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.     

Multilocus microsatellite typing (MLMT) of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group

Background

New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic.

Methodology/Principal Findings

The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey.

Conclusion

The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region.     

Quantitation of folates and their catabolites in blood plasma, erythrocytes, and urine by stable isotope dilution assays

New stable isotope dilution assays were developed for the simultaneous quantitation of the folates 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid, and folic acid as well as for their catabolites para-aminobenzoylglutamate (pABG) and acetyl-para-aminobenzoylglutamate (ApABG) in clinical samples. The methods were based on cleanup by strong anion exchange followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. Deuterated analogues of the folates and [¹³C₅]-labeled isotopologues of the catabolites were applied as the internal standards in stable isotope dilution assays. Extraction in 4-morpholineethanesulfonic acid (MES) buffer at pH 5.0 ensured the optimum stability of folates and, in combination with solid-phase extraction (SPE) based on strong anion exchange, resulted in higher recoveries compared with other combinations of extraction buffers and SPE. The method was sensitive enough to detect pABG in plasma generally and unmetabolized folic acid in the plasma of a volunteer after oral dosage of an aqueous folic acid solution. The sum of folate catabolites increased by a factor of 2 in the urine of the latter volunteer, compared with that resulting when only water was dosed.     

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (⁸⁹Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of ⁸⁹Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of ⁸⁹Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg ⁸⁹Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent ⁸⁹Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of ⁸⁹Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of ⁸⁹Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, ⁸⁹Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.     

A dry lab for medical engineers?

Post-operatory wound infections are a very uncommon finding after thyroidectomy. For these reasons international guidelines do not routinely recommend systemic antibiotic prophylaxis. The benefits of this antibiotic prophylaxis is not supported by clinical evidence in the literature. We have conducted a multicentric randomized double-blind trial on 500 patients who had undergone thyroidectomy for goitre or thyroid carcinoma. The 500 patients enrolled in the study (mean age 47 years) were randomized in two subgroups of 250 patients. 250 patients were treated with standard antibiotic prophylaxis with sulbactam/ampicillin 1 fl (3 gr.) 30 min before surgery. No antibiotic prophylaxis was instituted in the remainder 250 patients. Our RCT showed that prophylactic antibiotic treatment is not beneficial in patients younger than eighty years old, with no concomitant metabolic, infective and hematologic disease, with no cardiac valvulopathies, not under steroidal or immunosuppressive treatment, and not severely obese. Our study should be regarded only as a preliminary RCT, and should be followed by a study in which a larger number of patients should be enrolled so that statistically significant data can be obtained.     

Vegetable microbiomes: is there a connection among opportunistic infections,human health and our ‘gut feeling'?

The highly diverse microbiomes of vegetables are reservoirs for opportunistic and emerging pathogens. In recent years, an increased consumption, larger scale production and more efficient distribution of vegetables together with an increased number of immunocompromised individuals resulted in an enhanced number of documented outbreaks of human infections associated with the consumption of vegetables. Here we discuss the occurrence of potential pathogens in vegetable microbiomes, the impact of farming and processing practices, and plant and human health issues. Based on these results, we discuss the question if vegetables can serve as a source of infection for immunocompromised individuals as well as possible solutions to avoid outbreaks. Moreover, the potentially positive aspects of the vegetables microbiome for the gut microbiota and human health are presented.     

Competition for grooming partners and interference in affiliation among female mandrills

Grooming is the most common primate affiliative behaviour, and primates compete for accessing grooming partners. We studied a captive group of mandrills (Mandrillus sphinx) to evaluate the role of different types of competitive interactions in shaping the distribution of grooming among females. Mandrill females preferentially groomed high‐ranking individuals, but low‐ranking females were less able to do so. Interference in others’ grooming and a (consequent) reluctance of low‐ranking females to access dominant group mates occurred frequently and contributed to the observed pattern of grooming distribution, while takeovers of grooming partners was relatively rare. Interference in others’ affiliation was possibly used to prevent the formation of revolutionary alliances. Difficulties in accessing individuals already engaged in grooming exerted a strong but rank‐independent effect on grooming interactions. These results highlight the role of competition in determining access to preferred social partners.     

Assessing the Risk of Biological Control Agents on the Indigenous Microbial Communities: <Emphasis Type="Italic">Serratia plymuthica</Emphasis> HRO-C48 and <Emphasis Type="Italic">Streptomyces</Emphasis> sp. HRO-71 as Model Bacteria

The phytopathogenic fungus Verticillium dahliae Kleb. causes high yield losses in strawberry production. As effective chemical control of this fungus is no longer available, biological control based on natural antagonists might provide new control strategies. The aim of this study was to assess the impact of the two biological control agents S. plymuthica HRO-C48 and Streptomyces sp. HRO-71 on the rhizosphere community of the Verticillium host plant strawberry in field trials at two different sites in Germany. Therefore, we determined the abundances of culturable bacteria and investigated the community structure of the total rhizosphere microbiota by PCR-single strand conformation polymorphism analysis of the 16S rRNA and fungal ITS1 region. The abundances of culturable rhizobacteria on R2A medium as well as the proportion of in vitro Verticillium antagonists did not differ significantly. Additionally, no treatment specific differences were obtained in the composition of species of the non-target antagonistic bacteria in the rhizospheres. The culture-independent analysis revealed only transient differences between the bacterial communities not due to the treatments rather than to the plant growth stage. Fungal and bacterial community fingerprints showed the development of a microbiota, specific for a field site. However, no sustainable impact of the bacterial treatments on the indigenous microbial communities was found using culture-dependent and -independent methods.     

Transgenic Pm3 multilines of wheat show increased powdery mildew resistance in the field

Resistance (R) genes protect plants very effectively from disease, but many of them are rapidly overcome when present in widely grown cultivars. To overcome this lack of durability, strategies that increase host resistance diversity have been proposed. Among them is the use of multilines composed of near-isogenic lines (NILs) containing different disease resistance genes. In contrast to classical R-gene introgression by recurrent backcrossing, a transgenic approach allows the development of lines with identical genetic background, differing only in a single R gene. We have used alleles of the resistance locus Pm3 in wheat, conferring race-specific resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici), to develop transgenic wheat lines overexpressing Pm3a, Pm3c, Pm3d, Pm3f or Pm3g. In field experiments, all tested transgenic lines were significantly more resistant than their respective nontransformed sister lines. The resistance level of the transgenic Pm3 lines was determined mainly by the frequency of virulence to the particular Pm3 allele in the powdery mildew population, Pm3 expression levels and most likely also allele-specific properties. We created six two-way multilines by mixing seeds of the parental line Bobwhite and transgenic Pm3a, Pm3b and Pm3d lines. The Pm3 multilines were more resistant than their components when tested in the field. This demonstrates that the difference in a single R gene is sufficient to cause host-diversity effects and that multilines of transgenic Pm3 wheat lines represent a promising strategy for an effective and sustainable use of Pm3 alleles.