Bioelectroanalysis of pharmaceutical compounds

Recent developments in the bioelectroanalysis of pharmaceutical compounds are reviewed, concentrating particularly on the development of electrode materials and measurement strategies and on their application. The advantages of electroanalytical techniques as alternatives to other analytical procedures such as rapid response, sensitivity and low detection limits are highlighted and illustrated. Particular emphasis is given to carbon-based materials for voltammetric electroanalysis; new potentiometric sensors and electrochemical biosensors are also reviewed.     

Comparison of linkage disequilibrium and haplotype diversity on macro- and microchromosomes in chicken

Background

Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.

Results

The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.

Conclusion

The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^th amino acid position in LRR motif (TLR1–LRR10) and 4^th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.     

Phylogenetic composition and structure of tree communities shed light on historical processes influencing tropical rainforest diversity

The Neotropics, Afrotropics and Madagascar have different histories which have influenced their respective patterns of diversity. Based on current knowledge of these histories, we developed the following predictions about the phylogenetic structure and composition of rainforest tree communities: (Hypothesis 1) isolation of Gondwanan biotas generated differences in phylogenetic composition among biogeographical regions; (H2) major Cenozoic extinction events led to lack of phylogenetic structure in Afrotropical and Malagasy communities; (H3) greater angiosperm diversification in the Neotropics led to greater phylogenetic clustering there than elsewhere; (H4) phylogenetic overdispersion is expected near the Andes due to the co‐occurrence of magnoliids tracking conserved habitat preferences and recently diversified eudicot lineages. Using abundance data of tropical rainforest tree species from 94 communities in the Neotropics, Afrotropics and Madagascar, we computed net relatedness index (NRI) to assess local phylogenetic structure, i.e. phylogenetic clustering vs. overdispersion relative to regional species pools, and principal coordinates of phylogenetic structure (PCPS) to assess variation in phylogenetic composition across communities. We observed significant differences in phylogenetic composition among biogeographical regions (agreement with H1). Overall phylogenetic structure did not differ among biogeographical regions, but results indicated variation from Andes to Amazon. We found widespread phylogenetic randomness in most Afrotropical and all Malagasy communities (agreement with H2). Most of central Amazonian communities were phylogenetically random, although some communities presented phylogenetic clustering (partial agreement with H3). We observed phylogenetic overdispersion near the Andes (agreement with H4). We were able to identify how differences in lineage composition are related to local phylogenetic co‐occurrences across biogeographical regions that have been undergoing different climatic and orographic histories during the past 100 Myr. We observed imprints of the history following Gondwana breakup on phylobetadiversity and local phylogenetic structure of rainforest tree communities in the Neotropics, Afrotropics and Madagascar.     

Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.     

Renal Function in Hepatosplenic Schistosomiasis – An Assessment of Renal Tubular Disorders

Background

Renal involvement in Schistosoma mansoni infection is not well studied. The aim of this study is to investigate the occurrence of renal abnormalities in patients with hepatosplenic schistosomiasis (HSS), especially renal tubular disorders.

Methods

This is a cross-sectional study with 20 consecutive patients with HSS followed in a medical center in Maceió, Alagoas, Brazil. Urinary acidification and concentration tests were performed using calcium chloride (CaCl₂) after a 12-h period of water and food deprivation. The biomarker monocyte chemoattractant protein 1 (MCP-1) was quantified in urine. Fractional excretion of sodium (FE_Na+), transtubular potassium gradient (TTKG) and solute-free water reabsorption (TcH₂O) were calculated. The HSS group was compared to a group of 17 healthy volunteers.

Results

Patients'' mean age and gender were similar to controls. Urinary acidification deficit was found in 45% of HSS patients. Urinary osmolality was significantly lower in HSS patients (588±112 vs. 764±165 mOsm/kg, p = 0,001) after a 12-h period of water deprivation. TcH₂O was lower in HSS patients (0.72±0.5 vs. 1.1±0.3, p = 0.04). Urinary concentration deficit was found in 85% of HSS patients. The values of MCP-1 were higher in HSS group than in control group (122±134 vs. 40±28 pg/mg-Cr, p = 0.01) and positively correlated with the values of microalbuminuria and proteinuria.

Conclusions

HSS is associated with important kidney dysfunction. The main abnormalities found were urinary concentrating ability and incomplete distal acidification defect, demonstrating the occurrence of tubular dysfunction. There was also an increase in urinary MCP-1, which appears to be a more sensitive marker of renal damage than urinary albumin excretion rate.     

Evidence of Unique and Generalist Microbes in Distantly Related Sympatric Intertidal Marine Sponges (Porifera: Demospongiae)

The diversity and specificity of microbial communities in marine environments is a key aspect of the ecology and evolution of both the eukaryotic hosts and their associated prokaryotes. Marine sponges harbor phylogenetically diverse and complex microbial lineages. Here, we investigated the sponge bacterial community and distribution patterns of microbes in three sympatric intertidal marine demosponges, Hymeniacidon perlevis, Ophlitaspongia papilla and Polymastia penicillus, from the Atlantic coast of Portugal using classical isolation techniques and 16S rRNA gene clone libraries. Microbial composition assessment, with nearly full-length 16S rRNA gene sequences (ca. 1400 bp) from the isolates (n = 31) and partial sequences (ca. 280 bp) from clone libraries (n = 349), revealed diverse bacterial communities and other sponge-associated microbes. The majority of the bacterial isolates were members of the order Vibrionales and other symbiotic bacteria like Pseudovibrio ascidiaceiocola, Roseobacter sp., Hahellaceae sp. and Cobetia sp. Extended analyses using ecological metrics comprising 142 OTUs supported the clear differentiation of bacterial community profiles among the sponge hosts and their ambient seawater. Phylogenetic analyses were insightful in defining clades representing shared bacterial communities, particularly between H. perlevis and the geographically distantly-related H. heliophila, but also among other sponges. Furthermore, we also observed three distinct and unique bacterial groups, Betaproteobactria (∼81%), Spirochaetes (∼7%) and Chloroflexi (∼3%), which are strictly maintained in low-microbial-abundance host species O. papilla and P. penicillus. Our study revealed the largely generalist nature of microbial associations among these co-occurring intertidal marine sponges.     

Variability of the Mitochondrial SSU rDNA of <Emphasis Type="Italic">Nomuraea</Emphasis> Species and Other Entomopathogenic Fungi from Hypocreales

Hypocrealean arthropod pathogenic fungi have profound impact on the regulation of agricultural and medical pests. However, until now the genetic and phylogenetic relationships among species have not been clarified, such studies could clarify host specificity relationships and define species boundaries. Our purpose was to compare the sequences of the mitochondrial SSU rDNA fragments from several mitosporic entomopathogenic Hypocreales to infer relationships among them and to evaluate the possibility to use these sequences as species diagnostic tool in addition to the more commonly studied sequences of nuclear SSU rDNA. The SSU mt-rDNA proved to be useful to help in differentiation of species inside several genera. Clusters obtained with Parsimony, Bayesian, and Maximum Likelihood analyses were congruent with a new classification of the Clavicipitaceae (Sung et al. Stud Mycol. 2007;57:5-59) in which the anamorphic genera Nomuraea and Metarhizium species remain in the Clavicipitaceae and Isaria species sequenced here are assigned to the family Cordycipitaceae. Mitochondrial genomic information indicates the same general pattern of relationships demonstrated by nuclear gene sequences.     

Diphenyl Diselenide Reduces Mechanical and Thermal Nociceptive Behavioral Responses After Unilateral Intrastriatal Administration of 6-Hydroxydopamine in Rats

Parkinson’s disease (PD) patients, in addition to motor dysfunction, also present alterations in pain sensation. The present study characterized the antinociceptive effects of diphenyl diselenide ((PhSe)₂) in a model of nociception induced by unilateral, intrastriatal 6-hydroxydopamine (6-OHDA) injection in rats. Male adult Wistar rats received 20 μg/3 μl of 6-OHDA (in saline solution containing 0.02 % of ascorbic acid) or 3 μl of vehicle into the right striatum (1.0 mm anterior, 3.0 mm lateral, and 5.0 mm ventral—with respect to the bregma). Thirty days after injection, rats received (PhSe)₂ intragastrically at a dose of 10 mg/kg 1 h before behavioral tests (von Frey hairs, hot plate, tail immersion, formalin, and open field). Our results demonstrated that 6-OHDA injection to rats augmented the response frequency of von Frey hairs (VHF) stimulation, besides reducing the thermal withdrawal latency in the hot plate test. Importantly, the (PhSe)₂ treatment decreased the mechanical allodynia measured by the response frequency of VHF stimulation and diminished the thermal nociception in the hot plate test in 6-OHDA-injected rats. In conclusion, these results revealed that a single oral administration of (PhSe)₂ 1 h prior to the accomplishment of the behavioral tests was effective to attenuate the increased mechanical and thermal nociception caused by a single intrastriatal 6-OHDA injection to rats. Furthermore, other clarifying studies are warranted to improve the evidence bases for future clinical use of (PhSe)₂ as a new alternative therapy for the treatment of painful symptoms associated to PD.     

Influence of Toxoplasma gondii Acute Infection on Cholinesterase Activities of Wistar Rats

Several studies have shown the mechanisms and importance of immune responses against Toxoplasma gondii infection and the notable role of cholinesterases in inflammatory reactions. However, the association between those factors has not yet been investigated. Therefore, the aim of this study was to evaluate the acetylcholinesterase (AChE) activity in blood and lymphocytes and the activity of butyrylcholinesterase (BChE) in serum of rats experimentally infected with T. gondii during the acute phase of infection. For that, an in vivo study was performed with evaluations of AChE and BChE activities on days 5 and 10 post-infection (PI). The activity of AChE in blood was increased on day 5 PI, while in lymphocytes its activity was enhanced on days 5 and 10 PI (P<0.05). No significant difference was observed between groups regarding to the activity of BChE in serum. A positive (P<0.01) correlation was observed between AChE activity and number of lymphocytes. The role of AChE as an inflammatory marker is well known in different pathologies; thus, our results lead to the hypothesis that AChE has an important role in modulation of early immune responses against T. gondii infection.