Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae

A novel biosensor based on immobilised whole cell Chlorella vulgaris microalgae as a bioreceptor and interdigitated conductometric electrodes as a transducer has been developed and tested for alkaline phosphatase activity (APA) analysis. These sensors were also used for the detection of toxic compounds, namely cadmium ions, in aquatic habitats. Algae were immobilised inside bovine serum albumin (BSA) membranes cross-linked with glutaraldehyde vapours. The detection of the local conductivity variations caused by algae enzymatic reactions could be achieved. The inhibition of C. vulgaris microalgae Alkaline phosphatase activities in presence of cadmium ions was measured. These results were compared with measurements in bioassays. It finally appeared that conductometric biosensors using algae seemed more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for the first experiments was 1 ppb of Cd2+). The main advantages of these alkaline phosphatase biosensors consist of their high specificity in regard to the toxic compounds they enable to detect, but also on their high stability since contrary to enzymatic biosensors, they use whole algae cells with APs on their walls.     

A serpin regulates dorsal-ventral axis formation in the Drosophila embryo

Extracellular serine protease cascades have evolved in vertebrates and invertebrates to mediate rapid, local reactions to physiological or pathological cues. The serine protease cascade that triggers the Toll signaling pathway in Drosophila embryogenesis shares several organizational characteristics with those involved in mammalian complement and blood clotting. One of the hallmarks of such cascades is their regulation by serine protease inhibitors (serpins). Serpins act as suicide substrates and are cleaved by their target protease, forming an essentially irreversible 1:1 complex. The biological importance of serpins is highlighted by serpin dysfunction diseases, such as thrombosis caused by a deficiency in antithrombin. Here, we describe how a serpin controls the serine protease cascade, leading to Toll pathway activation. Female flies deficient in Serpin-27A produce embryos that lack dorsal-ventral polarity and show uniform high levels of Toll signaling. Since this serpin has been recently shown to restrain an immune reaction in the blood of Drosophila, it demonstrates that proteolysis can be regulated by the same serpin in different biological contexts.     

Replacement of sulfide by selenide in the [4Fe-4S] clusters of the ferredoxin fromClostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from $\text{C. pasteurianum}$ have been replaced by selenium. The optical absorption spectrum of the Se-ferredoxin is slightly different from the spectrum of the native protein, but it displays the characteristic features of [4Fe-4X] ( X = S, Se) clustors. The reduced Se-ferredoxin can reduce hydrogenase, and the oxidized Se-ferredoxin can be reduced by hydrogenase in the presence of molecular hydrogen. This is the first report of sulfide substitution by selenide in an iron-sulfur protein containing [4Fe-4S] active sites.     

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 1990¹ in a rat, then described in mice². Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.     

Laboratory-Based Surveillance of Neisseria meningitidis Isolates from Disease Cases in Latin American and Caribbean Countries, SIREVA II 2006-2010

Background

Published data on the epidemiology of meningococcal disease in Latin America and the Caribbean region is scarce and, when available, it is often published in Spanish and/or in non-peer-reviewed journals, making it difficult for the international scientific community to have access.

Methods

Laboratory data on 4,735 Neisseria meningitidis strains was collected and reported by the National Reference Laboratories in 19 Latin American countries and the Caribbean Epidemiology Centre (CAREC) between 2006 and 2010 as part of the work carried out by the SIREVA II network. Serogroup and MIC to penicillin, rifampin and chloramphenicol were determined.

Results

Isolates were mainly obtained from patients <5 years, but each year around 25% of isolates came from adult patients. Serogroup distribution was highly variable among countries. Serogroup C was the main cause of disease in Brazil; the majority of disease seen in the Southern cone was caused by serogroup B, but serogroup W135 strains have increased in recent years. In the Andean and Mexico, Central America and Caribbean regions, serogroups B and C were equally present, and serogroup Y was frequently isolated. Isolates were generally susceptible to chloramphenicol, penicillin and rifampin, but almost 60% of isolates characterized in Southern cone countries presented intermediate resistance to penicillin. Five rifampin-resistant isolates have been isolated in Uruguay and Brazil.

Conclusions

Serogroup distribution is highly variable among countries, but some geographic structuring can be inferred from these data. Epidemiological and laboratory data are scarce among Andean and Mexico, Central America and Caribbean countries. Evaluation and implementation of corrective measures on disease surveillance and reporting systems and the implementation of molecular diagnostic techniques and molecular characterization on meningococcal isolates are advised.     

Differential Interaction of Phencyclidine-Like Drugs with the Dopamine Uptake Complex In Vivo

The phencyclidine (PCP) derivative, [3H]N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine ([3H]BTCP), was used to label in vivo the dopamine uptake complex in mouse brain. The striatum accumulated the highest level of total and specific binding. Drugs which bind to the dopamine uptake site inhibited [3H]BTCP binding on an order similar to their in vitro affinities for the high-affinity [3H]BTCP site. Drugs which label selectively other monoamine uptake complexes. PCP, or sigma recognition sites were ineffective at doses up to 40 mg/kg. PCP bound to and dissociated from the dopamine uptake complex very rapidly. N-[1-(2-Thienyl)cyclohexyl]pideridine (TCP) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) had no effect at any time or at any dose. These results imply that the pharmacological effects of PCP are due to its simultaneous interaction with the dopamine uptake complex and the PCP receptor. Conversely, TCP and MK-801, which have the same behavioral properties as PCP, exert their action only through the interaction with the PCP receptor.     

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field.     

Protective or Deleterious Role of Scavenger Receptors SR-A and CD36 on Host Resistance to Staphylococcus aureus Depends on the Site of Infection

Staphylococcus aureus is a major human opportunistic pathogen responsible for a broad spectrum of infections ranging from benign skin infection to more severe life threatening disorders (e.g. pneumonia, sepsis), particularly in intensive care patients. Scavenger receptors (SR-A and CD36) are known to be involved in S. aureus recognition by immune cells in addition to MARCO, TLR2, NOD2 and α5β1 integrin. In the present study, we further deciphered the contribution of SR-A and CD36 scavenger receptors in the control of infection of mice by S. aureus. Using double SR-A/CD36 knockout mice (S/C-KO) and S. aureus strain HG001, a clinically relevant non-mutagenized strain, we showed that the absence of these two scavenger receptors was protective in peritoneal infection. In contrast, the deletion of these two receptors was detrimental in pulmonary infection following intranasal instillation. For pulmonary infection, susceptible mice (S/C-KO) had more colony-forming units (CFU) in their broncho-alveolar lavages fluids, associated with increased recruitment of macrophages and neutrophils. For peritoneal infection, susceptible mice (wild-type) had more CFU in their blood, but recruited less macrophages and neutrophils in the peritoneal cavity than resistant mice. Exacerbated cytokine levels were often observed in the susceptible mice in the infected compartment as well as in the plasma. The exception was the enhanced compartmentalized expression of IL-1β for the resistant mice (S/C-KO) after peritoneal infection. A similar mirrored susceptibility to S. aureus infection was also observed for MARCO and TLR2. Marco and tlr2 -/- mice were more resistant to peritoneal infection but more susceptible to pulmonary infection than wild type mice. In conclusion, our results show that innate immune receptors can play distinct and opposite roles depending on the site of infection. Their presence is protective for local pulmonary infection, whereas it becomes detrimental in the peritoneal infection.     

Alterations in hepatic glucagon receptor density and in Gsalpha and Gialpha2 protein content with diet-induced hepatic steatosis: effects of acute exercise

The present study was undertaken to test the hypothesis that a high-fat diet-induced liver lipid infiltration is associated with a reduction of hepatic glucagon receptor density (B(max)) and affinity (K(d)), and with a decrease in stimulatory G protein (G(s)alpha) content while enhancing inhibitory G protein (G(i)alpha(2)) expression. We also hypothesized that, under this dietary condition, a single bout of endurance exercise would restore hepatic glucagon receptor parameters and G protein expression to standard levels. Female Sprague-Dawley rats were fed either a standard (SD) or a high-fat diet (HF; 40% kcal) for 2 wk (n = 20 rats/group). Each dietary group was thereafter subdivided into a nonexercised (Rest) and an acute-exercised group (Ac-Ex). The acute exercise consisted of a single bout of endurance exercise on a treadmill (30 min, 26 m/min, and 0% slope) immediately before being killed. The HF compared with the SD diet was associated with significantly (P < 0.05) higher values in hepatic triglyceride concentrations (123%), fat pad weight, and plasma free fatty acid (FFA) concentrations. The HF diet also resulted in significantly (P < 0.05) lower hepatic glucagon receptor density (45%) and G(s)alpha protein content (75%), as well as higher (P < 0.05) G(i)alpha(2) protein content (27%), with no significant effects on glucagon receptor affinity. Comparisons of all individual liver triglyceride and B(max) values revealed that liver triglycerides were highly (P < 0.003) predictive of the decreased glucagon receptor density (R = -0.512). Although the 30-min exercise bout resulted in some typical exercise effects (P < 0.05), such as an increase in FFA (SD diet), a decrease in insulin levels, and an increase in plasma glucagon concentrations (SD diet), it did not change any of the responses related to liver glucagon receptors and G proteins, with the exception of a significant (P < 0.05) decrease in G(i)alpha(2) protein content under the HF diet. The present results indicate that the feeding of an HF diet is associated with a reduction in plasma membrane hepatic glucagon receptor density and G(s)alpha protein content, which is not attenuated by a 30-min exercise bout. It is suggested that liver lipid infiltration plays a role in reducing glucagon action in the liver through a reduction in glucagon receptor density and glucagon-mediated signal transduction.