Horseradish Peroxidase Combined With Oxidase Enzymes a Valuable Bioanalytical Tool: Lactate Oxidase – A Case Study

Enzymatic biosensors have been extensively investigated for real‐time bioprocess monitoring and other online analysis. However, implementation of biosensors has been strongly hindered by their limited stability. This work reports a significant improvement of the stability of the immobilized oxidases by in situ reduction of the harmful H₂O₂. Thus, stabilized oxidases can serve as the basis for ethanol, glucose, and lactate sensors, with the ability to operate for long periods of time with virtually no change in activity. As an example, a lactate sensor, containing lactate oxidase aimed for bioprocess monitoring, has been described and characterized. Operational stabilities that allow up to 8 h continuous lactate conversion with virtually no activity loss have been achieved. The described system based on the in situ stabilization strategy is a promising new tool for the development of online analyses.     

Three‐dimensional cell culture microarray for high‐throughput studies of stem cell fate

We have developed a novel three‐dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high‐throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox‐1, whose levels were also measured in situ using a GFP reporter system. In addition, the high‐throughput capacity of the platform was tested using a dual‐slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor‐4 (FGF‐4) on the pluripotency of mouse ES cells. This high‐throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.     

Nitric oxide modulates sodium vitamin C transporter 2 (SVCT-2) protein expression via protein kinase G (PKG) and nuclear factor-κB (NF-κB)

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.     

Molecular sexing of birds: A comparative review of polymerase chain reaction (PCR)-based methods

Accurate identification of sex in birds is important for the management and conservation of avian wildlife in several ways, namely in the development of population, behavioral and ecological studies, as well as in the improvement of ex situ captive breeding programs. In general, nestlings, juveniles and adult birds of a wide number of sexually monomorphic species cannot be sexed based on phenotypic traits. The development of molecular methodologies for avian sexing overcame these difficulties, allowing a reliable gender differentiation for these species. The polymerase chain reaction (PCR)-based methods have been widely applied in molecular sexing of birds, using a large diversity of sex-linked markers. During the last 15 yrs, there was a continuous improvement in the PCR-based protocols for bird sexing, increasing the accuracy, speed and high-throughput applicability of these techniques. The recent advances in real-time PCR platforms and whole genome analysis methods provided new resources for the detection and analysis of novel specific markers and protocols. This review presents a comparative guide of classical and recent advances in PCR-based methods for avian molecular sexing, highlighting its strengths and limitations. Future research opportunities in this field are also addressed.     

Glomerella truncata: another Glomerella species with an atypical mating system

In the genus Glomerella all species studied to date do not fit the usual mating system of heterothallic ascomycetes. This study investigated the mating system of G. truncata (anamorph Colletotrichum truncatum), a pathogen responsible for lentil anthracnose. Twenty-two field isolates from the Canadian prairies were crossed in all possible combinations, including selfings. All isolates also were screened for the presence of the MAT1-1 and MAT1-2 idiomorphs by targeting small conserved areas of the MAT genes (the alpha domain and the high mobility group HMG box) with degenerate primers, and a pair of G. truncata-specific HMG primers (CT21HMG) were designed. The results of the classical mating study suggested that G. truncata is heterothallic. Isolates fell into two incompatibility groups, which is consistent with a bipolar mating system but different from what has been described in other Glomerella species. Molecular screening showed that the HMG box used as a marker for the MAT1-2 idiomorph was present in both partners of fertile crosses in G. truncata, unlike in the typical ascomycete system, but as previously described for two other Glomerella species. G. truncata therefore appears to share unusual mating system characteristics with the other Glomerella species studied to date.     

Structuring of bacterioplankton diversity in a large tropical bay

Structuring of bacterioplanktonic populations and factors that determine the structuring of specific niche partitions have been demonstrated only for a limited number of colder water environments. In order to better understand the physical chemical and biological parameters that may influence bacterioplankton diversity and abundance, we examined their productivity, abundance and diversity in the second largest Brazilian tropical bay (Guanabara Bay, GB), as well as seawater physical chemical and biological parameters of GB. The inner bay location with higher nutrient input favored higher microbial (including vibrio) growth. Metagenomic analysis revealed a predominance of Gammaproteobacteria in this location, while GB locations with lower nutrient concentration favored Alphaproteobacteria and Flavobacteria. According to the subsystems (SEED) functional analysis, GB has a distinctive metabolic signature, comprising a higher number of sequences in the metabolism of phosphorus and aromatic compounds and a lower number of sequences in the photosynthesis subsystem. The apparent phosphorus limitation appears to influence the GB metagenomic signature of the three locations. Phosphorus is also one of the main factors determining changes in the abundance of planktonic vibrios, suggesting that nutrient limitation can be observed at community (metagenomic) and population levels (total prokaryote and vibrio counts).     

Cruzipain Promotes Trypanosoma cruzi Adhesion to Rhodnius prolixus Midgut

Background

Trypanosoma cruzi is the etiological agent of Chagas'' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction.

Methodology/Principal Findings

Here, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain.

Conclusions/Significance

These data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host.     

Terrorist Radiological Dispersive Device (RDD) Scenario and Cancer Risk Assessment

Terrorist attacks using radiological dispersal devices (RDD) are important threats that can lead to major environmental, economic, and public health concerns. In a simulation using a Gaussian model, we sought to determine dose curves as a function of distance from the explosion core site and from these data to determine the relative risk of developing tumors as well as the probability of causation (i.e., the statistical correlation between occurrence of malignancies and previous exposure to radiation). Calculation confirmed the cause dependence on age at exposure and the probability of tumor causation. This study aimed at emphasizing the importance of responding rapidly and efficiently, with a user-friendly user methodology that initially may help to guide the response from basic actions to the complete decision process.     

Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications.

Human clinical trial of gene therapy with nonviral vectors demands large amounts of pharmaceutical-grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the development of processing methodologies for the large-scale production and purification of plasmids. This work describes several studies that were undertaken during the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine technique for monitoring plasmid purity in process streams. The use of RNase or high temperatures during alkaline lysis was proved unnecessary. Instead, RNA could be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusion chromatography. Also, clarification of streams by precipitation was independent of the chaotropic salt used. Furthermore, by proceeding directly from cell lysis to chromatography it was possible to obtain plasmid with purity/quality identical to that of the one obtained when clarification and concentration were included in the process. This strategy has the advantage of increasing the overall process yield to 38%. The plasmid thus purified was depleted of RNA, chromosomal DNA, and proteins. Additionally, no animal-derived enzymes, alcohols, or toxic solvents were used, rendering validation potentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without affecting plasmid purity.