Determination of thyreostat residues from bovine matrices using high-performance liquid chromatography

High-performance liquid chromatography (HPLC) methodologies were evaluated for the detection and quantification of thyreostatic drug residues in cattle serum and thyroid tissue. The paper details a protocol, using a simple ethyl acetate extraction for the determination of thiouracil, tapazole, methyl thiouracil, propyl thiouracil and phenyl thiouracil in thyroid tissue. Using two sequential HPLC injections, and quantitative analysis, in two steps, all five thyreostats were detectable at concentrations greater than 2.45–4.52 ng/g. Modifications to a published method for detection of thyreostatic residues in serum involving the addition of mercaptoethanol and a freezing step are described. The modifications improved sensitivity and allowed detection of the five thyreostats at levels greater than 16.98–35.25 ng/ml. Young bulls were treated with thyreostats to demonstrate the validity of the methodologies described. Administered thyreostats were not absorbed equally by the test animals and the compounds were not all detected in the serum samples removed at 7 days following drug withdrawal. These experiments indicate the necessity to be able to detect thyreostat residues in a variety of matrices.     

Structural design,synthesis and substituent effect of hydrazone-N-acylhydrazones reveal potent immunomodulatory agents

4-(Nitrophenyl)hydrazone derivatives of N-acylhydrazone were synthesized and screened for suppress lymphocyte proliferation and nitrite inhibition in macrophages. Compared to an unsubstituted N-acylhydrazone, active compounds were identified within initial series when hydroxyl, chloride and nitro substituents were employed. Structure-activity relationship was further developed by varying the position of these substituents as well as attaching structurally-related substituents. Changing substituent position revealed a more promising compound series of anti-inflammatory agents. In contrast, an N-methyl group appended to the 4-(nitrophenyl)hydrazone moiety reduced activity. Anti-inflammatory activity of compounds is achieved by modulating IL-1β secretion and prostaglandin E2 synthesis in macrophages and by inhibiting calcineurin phosphatase activity in lymphocytes. Compound SintMed65 was advanced into an acute model of peritonitis in mice, where it inhibited the neutrophil infiltration after being orally administered. In summary, we demonstrated in great details the structural requirements and the underlying mechanism for anti-inflammatory activity of a new family of hydrazone-N-acylhydrazone, which may represent a valuable medicinal chemistry direction for the anti-inflammatory drug development in general.     

Eyes,More Than Other Facial Features,Enhance Real-World Donation Behavior

Humans often behave more prosocially when being observed in person and even in response to subtle eye cues, purportedly to manage their reputation. Previous research on this phenomenon has employed the “watching eyes paradigm,” in which adults displayed greater prosocial behavior in the presence of images of eyes versus inanimate objects. However, the robustness of the effect of eyes on prosocial behavior has recently been called into question. Therefore, the first goal of the present study was to attempt to replicate this effect. Additionally, it remains unclear whether the watching-eyes effect is driven specifically by reputation management (owing to the monitoring function of the eyes) or whether any cues indexing human presence more generally also have a similar effect. To address these questions, the current study compared prosocial behavior in the presence of eyes versus inanimate objects as well as other human features. The study was conducted as a field experiment at a children’s museum. Each week, the donation signs were changed to show eyes, noses, mouths, or chairs. Total donation amount and number of patrons per week were recorded. Participants donated more when they were exposed to eyes than to inanimate objects (chairs). We thus replicated the previously reported watching-eyes effect. Moreover, more money was donated when individuals were exposed to eyes than to more general cues of human presence (nose and mouth). The current findings suggest that eyes play a special role in promoting cooperation in humans, likely by serving as cues of monitoring and thus eliciting reputation management behavior.     

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita,western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16 years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8⁺ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.     

Cheatgrass (<Emphasis Type="Italic">Bromus tectorum</Emphasis>) distribution in the intermountain Western United States and its relationship to fire frequency,seasonality, and ignitions

Cheatgrass (Bromus tectorum) is an invasive grass pervasive across the Intermountain Western US and linked to major increases in fire frequency. Despite widespread ecological impacts associated with cheatgrass, we lack a spatially extensive model of cheatgrass invasion in the Intermountain West. Here, we leverage satellite phenology predictors and thousands of field surveys of cheatgrass abundance to create regional models of cheatgrass distribution and percent cover. We compare cheatgrass presence to fire probability, fire seasonality and ignition source. Regional models of percent cover had low predictive power (34% of variance explained), but distribution models based on a threshold of 15% cover to differentiate high abundance from low abundance had an overall accuracy of 74%. Cheatgrass achieves ≥ 15% cover over 210,000 km² (31%) of the Intermountain West. These lands were twice as likely to burn as those with low abundance, and four times more likely to burn multiple times between 2000 and 2015. Fire probability increased rapidly at low cheatgrass cover (1–5%) but remained similar at higher cover, suggesting that even small amounts of cheatgrass in an ecosystem can increase fire risk. Abundant cheatgrass was also associated with a 10 days earlier fire seasonality and interacted strongly with anthropogenic ignitions. Fire in cheatgrass was particularly associated with human activity, suggesting that increased awareness of fire danger in invaded areas could reduce risk. This study suggests that cheatgrass is much more spatially extensive and abundant than previously documented and that invasion greatly increases fire frequency, even at low percent cover.     

Functional insights from proteome-wide structural modeling of <Emphasis Type="Italic">Treponema pallidum</Emphasis> subspecies <Emphasis Type="Italic">pallidum</Emphasis>, the causative agent of syphilis

Background

Syphilis continues to be a major global health threat with 11 million new infections each year, and a global burden of 36 million cases. The causative agent of syphilis, Treponema pallidum subspecies pallidum, is a highly virulent bacterium, however the molecular mechanisms underlying T. pallidum pathogenesis remain to be definitively identified. This is due to the fact that T. pallidum is currently uncultivatable, inherently fragile and thus difficult to work with, and phylogenetically distinct with no conventional virulence factor homologs found in other pathogens. In fact, approximately 30% of its predicted protein-coding genes have no known orthologs or assigned functions. Here we employed a structural bioinformatics approach using Phyre2-based tertiary structure modeling to improve our understanding of T. pallidum protein function on a proteome-wide scale.

Results

Phyre2-based tertiary structure modeling generated high-confidence predictions for 80% of the T. pallidum proteome (780/978 predicted proteins). Tertiary structure modeling also inferred the same function as primary structure-based annotations from genome sequencing pipelines for 525/605 proteins (87%), which represents 54% (525/978) of all T. pallidum proteins. Of the 175 T. pallidum proteins modeled with high confidence that were not assigned functions in the previously annotated published proteome, 167 (95%) were able to be assigned predicted functions. Twenty-one of the 175 hypothetical proteins modeled with high confidence were also predicted to exhibit significant structural similarity with proteins experimentally confirmed to be required for virulence in other pathogens.

Conclusions

Phyre2-based structural modeling is a powerful bioinformatics tool that has provided insight into the potential structure and function of the majority of T. pallidum proteins and helped validate the primary structure-based annotation of more than 50% of all T. pallidum proteins with high confidence. This work represents the first T. pallidum proteome-wide structural modeling study and is one of few studies to apply this approach for the functional annotation of a whole proteome.

     

Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

Introduction

Subcellular compartmentalization enables eukaryotic cells to carry out different reactions at the same time, resulting in different metabolite pools in the subcellular compartments. Thus, mutations affecting the mitochondrial energy metabolism could cause different metabolic alterations in mitochondria compared to the cytoplasm. Given that the metabolite pool in the cytosol is larger than that of other subcellular compartments, metabolic profiling of total cells could miss these compartment-specific metabolic alterations.

Objectives

To reveal compartment-specific metabolic differences, mitochondria and the cytoplasmic fraction of baker’s yeast Saccharomyces cerevisiae were isolated and subjected to metabolic profiling.

Methods

Mitochondria were isolated through differential centrifugation and were analyzed together with the remaining cytoplasm by gas chromatography–mass spectrometry (GC–MS) based metabolic profiling.

Results

Seventy-two metabolites were identified, of which eight were found exclusively in mitochondria and sixteen exclusively in the cytoplasm. Based on the metabolic signature of mitochondria and of the cytoplasm, mutants of the succinate dehydrogenase (respiratory chain complex II) and of the F_OF₁-ATP-synthase (complex V) can be discriminated in both compartments by principal component analysis from wild-type and each other. These mitochondrial oxidative phosphorylation machinery mutants altered not only citric acid cycle related metabolites but also amino acids, fatty acids, purine and pyrimidine intermediates and others.

Conclusion

By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations affecting the mitochondrial metabolism.

     

Molecular evidence and clinical importance of β‐arrestins expression in patients with acromegaly

β‐arrestins seem to have a role in endocytosis and desensitization of somatostatin receptor subtype 2 (sst2) and could be associated with the responsiveness to somatostatin receptor ligands (SRL) in patients with acromegaly. To investigate the in vivo correlation between β‐arrestins 1 and 2 with sst2, sst5 and dopamine receptor subtype 2 (D2) expressions, and the association of β‐arrestins with response to first‐generation SRL and invasiveness in somatotropinomas. β‐arrestins 1 and 2, sst2, sst5 and D2 mRNA expressions were evaluated by quantitative real‐time RT‐PCR on tumoral tissue of 96 patients. Moreover, sst2 and sst5 protein expressions were also evaluated in 40 somatotropinomas by immunohistochemistry. Response to SRL, defined as GH <1 μg/l and normal IGF‐I levels, was assessed in 40 patients. The Knosp‐Steiner criteria were used to define invasiveness. Median β‐arrestin 1, β‐arrestin 2, sst2, sst5 and D2 mRNA copy numbers were 478; 9375; 731; 156 ; and 3989, respectively. There was a positive correlation between β‐arrestins 1 and 2 (R = 0.444, P < 0.001). However, no correlation between β‐arrestins and sst2, sst5 (mRNA and protein levels) or D2 was found. No association was found between β‐arrestins expression and SRL responsiveness or tumour invasiveness. Although previous data suggest a putative correlation between β‐arrestins and sst2, our data clearly indicated that no association existed between β‐arrestins and sst2, sst5 or D2 expression, nor with response to SRL or tumour invasiveness. Therefore, further studies are required to clarify whether β‐arrestins have a role in the response to treatment with SRL in acromegaly.