Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep

The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 μg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P < 0.001). The efficiency of the extraction method was confirmed by the results obtained from qPCR which showed a Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.     

Apoptosis,cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Background

Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.

Methods

Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.

Results

The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.

Conclusions

In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.

     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Characterization of the Mediterranean Italian buffaloes melatonin receptor 1A (MTNR1A) gene and its association with reproductive seasonality

The aim of this study was to examine the polymorphism in MTNR1A gene and its relation to reproductive seasonality in Mediterranean Italian buffaloes reared in Sardinia. The mating period and calving of 100 multiparous buffalo-cows were recorded for three years (2005-2008). Genomic DNA was subjected to PCR for the amplification of the exon II, then 40 amplicons were sequenced. The obtained sequence was deposited in GeneBank database (accession number GU817415). PCR products were checked for the presence of HpaI restriction sites and assigned to genotypes “C/C”, “C/T” or “T/T”. Allelic frequency of C and T alleles was 0.44 and 0.56 and genotypic frequency was 26% for genotype C/C, 40% for C/T and 34% for T/T. In the three observed years the animals with C/C genotype showed the highest number of mating in the semester between August and January and their calving mainly occurred from August to September. On the other hand animals with T/T genotype showed mating mostly in the semester between February and July and calving occurred largely from March to May in all the three years. Heterozygous, in all the three years, showed about the same number of animals mated within each six-month period. The results of the present study provide for the first time a partial sequence as well as one polymorphic site of the MTNR1A receptor gene from buffaloes. Moreover our data showed an association between Single Nucleotide Polymorphism and seasonal reproductive activity in these animals.     

Polymer optoelectronic structures for retinal prosthesis

This commentary highlights the effectiveness of optoelectronic properties of polymer semiconductors based on recent results emerging from our laboratory, where these materials are explored as artificial receptors for interfacing with the visual systems. Organic semiconductors based polymer layers in contact with physiological media exhibit interesting photophysical features, which mimic certain natural photoreceptors, including those in the retina. The availability of such optoelectronic materials opens up a gateway to utilize these structures as neuronal interfaces for stimulating retinal ganglion cells. In a recently reported work entitled “A polymer optoelectronic interface provides visual cues to a blind retina,” we utilized a specific configuration of a polymer semiconductor device structure to elicit neuronal activity in a blind retina upon photoexcitation. The elicited neuronal signals were found to have several features that followed the optoelectronic response of the polymer film. More importantly, the polymer-induced retinal response resembled the natural response of the retina to photoexcitation. These observations open up a promising material alternative for artificial retina applications.     

Higher frequency of septic shock in septic patients with the 47C allele (rs4880) of the SOD2 gene

Aim

To analyze the effect of the two different versions of the manganese superoxide dismutase gene (SOD2) on sepsis. The SOD2 gene presents the 47C > T single nucleotide polymorphism (SNP; ID: rs4880) which produces MnSOD with different activities. The − 9Val MnSOD (47T allele) is less efficient than the − 9Ala version (47C allele). During sepsis there are abundance of ROS, high SOD2 expression and excess of H₂O₂ synthesis. High concentrations of H₂O₂ could affect the sepsis scenario and/or the sepsis outcome.

Methods

We determined the 47C > T single nucleotide polymorphism (SNP) frequencies in 529 critically ill patients with or without sepsis, facing outcome. To collect information on population frequencies, we obtained a pilot 47C > T genotypic and allelic frequencies in a random group of 139 healthy subjects.

Results

We compared the 47C allele carriers (47CC + 47CT genotypes) with 47TT homozygotes and noticed a significant association between 47C allele carriers and septic shock in septic patients (P = 0.025). With an adjusted binary multivariate logistic regression, incorporating 47C > T SNP and the main clinical predictors, we showed high SOFA scores [P < 0.001, OR = 9.107 (95% CI = 5.319–15.592)] and 47C allele [P = 0.011, OR = 2.125 (95% CI = 1.190–3.794)] were significantly associated with septic shock outcome. With this information we presented a hypothesis suggesting that this negative outcome from sepsis is possibly explained by effects on cellular stress caused by 47C allele.

Conclusion

In our population there was a significant higher frequency of septic shock in septic patients with the 47C allele of the SOD2 gene. This higher 47C allele frequency in septic patients with negative outcome could be explained by effects of higher activity MnSOD on cellular stress during the sepsis.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.