Automated system for gene annotation and metabolic pathway reconstruction using general sequence databases

Despite the growing number of genomes published or currently being sequenced, there is a relative paucity of software for functional classification of newly discovered genes and their assignment to metabolic pathways. Available software for such analyses has a very steep learning curve and requires the installation, configuration, and maintenance of large amounts of complex infrastructure, including complementary software and databases. Many such tools are restricted to one or a few data sources and classification schemes. In this work, we report an automated system for gene annotation and metabolic pathway reconstruction (ASGARD), which was designed to be powerful and generalizable, yet simple for the biologist to install and run on centralized, commonly available computers. It avoids the requirement for complex resources such as relational databases and web servers, as well as the need for administrator access to the operating system. Our methodology contributes to a more rapid investigation of the potential biochemical capabilities of genes and genomes by the biological researcher, and is useful in biochemical as well as comparative and evolutionary studies of pathways and networks.     

A Probasin‐MerCreMer BAC allows inducible recombination in the mouse prostate

Tissue‐specific transgene expression in the prostate epithelium has previously been achieved using short prostate‐specific promoters, rendering transgenic mouse lines susceptible to integration site‐dependent effects. Here we demonstrate the applicability of bacterial artificial chromosome (BAC) technology to transgene expression in the prostate epithelium. We present mouse lines expressing an inducible Cre protein (MerCreMer) under the control of regulatory elements of the probasin gene on a BAC. These mouse lines show high organ specificity, high transgene expression in anterior, dorsal and lateral prostate lobes, no background Cre recombination using a reporter strain and adjustable amounts of Cre‐induced recombination upon tamoxifen induction. Together with two recently reported transgenic lines expressing the Cre‐ERT2 protein from small prostate‐specific promoters, these mouse lines will be useful in research focused on prostate‐specific disorders such as benign hyperplasia or cancer. genesis 47:757–764, 2009. © 2009 Wiley‐Liss, Inc.     

Antinociceptive activity of the volatile oils of Hyptis pectinata L. Poit. (Lamiaceae) genotypes

Hyptis pectinata L. Poit (Lamiaceae) is known popularly in Brazil as “sambacaita” or “canudinho” and is used in the treatment of inflammations, bacterial infections and ache. The antinociceptive activity of the volatile oils of six genotypes, at doses of 100, 200 and 400 mg/kg body wt., were investigated using abdominal writhe models induced by acetic acid and hot plate tests. The volatile oils of all the genotypes are composed mainly of sesquiterpenoids. All the genotypes showed antinociceptive effects in both models used; the SAM002 genotype showed the major inhibitory effect at dose of 100 mg/kg body wt. These results suggest that the volatile oil of H. pectinata has peripheral (writhe reduction) and central (time delay of thermal reaction) effects. These observations indicate that H. pectinata may be useful as an analgesic drug.     

Cytomegalovirus Infection in Inflammatory Bowel Disease Is Not Associated with Worsening of Intestinal Inflammatory Activity

Background

Cytomegalovirus is highly prevalent virus and usually occurs in immunocompromised patients. The pathophysiology and treatment of inflammatory bowel disease often induce a state of immunosuppression. Because this, there are still doubts and controversies about the relationship between inflammatory bowel disease and cytomegalovirus.

Aim

Evaluate the frequency of cytomegalovirus in patients with inflammatory bowel disease and identify correlations.

Methods

Patients with inflammatory bowel disease underwent an interview, review of records and collection of blood and fecal samples. The search for cytomegalovirus was performed by IgG and IgM blood serology, by real-time PCR in the blood and by qualitative PCR in feces. Results were correlated with red blood cell levels, C-reactive protein levels, erythrocyte sedimentation rates and fecal calprotectin levels for each patient.

Results

Among the 400 eligible patients, 249 had Crohn''s disease, and 151 had ulcerative colitis. In the group of Crohn''s disease, 67 of the patients had moderate or severe disease, but 126 patients presented with active disease, based on the evaluation of the fecal calprotectin. In patients with ulcerative colitis, only 21 patients had moderate disease, but 76 patients presented with active disease, based on the evaluation of the fecal calprotectin. A large majority of patients had positive CMV IgG. Overall, 10 patients had positive CMV IgM, and 9 patients had a positive qualitative detection of CMV DNA by PCR in the feces. All 400 patients returned negative results after the quantitative detection of CMV DNA in blood by real-time PCR. Analyzing the 19 patients with active infections, we only found that such an association occurred with the use of combined therapy (anti-TNF-alpha + azathioprine)

Conclusion

The findings show that latent cytomegalovirus infections are frequent and active cytomegalovirus infection is rare. We did not find any association between an active infection of CMV and inflammatory bowel disease activity.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

Effects of bound Ts3 on voltage dependence of sodium channel transitions to and from inactivation and energetics of its unbinding

Recently, we proposed a quantitative model to explain the molecular mechanism of action of the Tityus serrulatus Ts3 α-toxin on sodium channels. In this model, the toxin acts as a stop that prevents the segment S4 of domain IV from reaching its outermost position, thus impairing the normal fast inactivation without affecting activation. In the present work, we analyze the predictions of the proposed model with regard to the voltage-dependent transitions to and from inactivation. Our results show that the recovery from inactivation was significantly faster in Ts3-bound channels and that there was no significant voltage dependence. The transition to inactivated state from open state in Ts3-modified channels presented a small but significant voltage dependence, which may derive from an intrinsic voltage dependence of inactivation or by a short movement of IVS4 in the presence of bound Ts3. We also studied the thermodynamic parameters of the voltage-dependent displacement of Ts3 from its binding site. We have observed that the activation energy to remove the toxin is 27 kJ/mol, part of which derives from the imposed depolarizing potential and the movement of an equivalent electrical charge of 0.54 c ₀. These results support the proposed model.     

The Predictive Power of R 0 in an Epidemic Probabilistic System

An important issue in theoretical epidemiology is the epidemic thresholdphenomenon, which specify the conditions for an epidemic to grow or die out.In standard (mean-field-like) compartmental models the concept of the basic reproductive number, R ₀, has been systematically employed as apredictor for epidemic spread and as an analytical tool to study thethreshold conditions. Despite the importance of this quantity, there are nogeneral formulation of R ₀ when one considers the spread of a disease ina generic finite population, involving, for instance, arbitrary topology ofinter-individual interactions and heterogeneous mixing of susceptible andimmune individuals. The goal of this work is to study this concept in ageneralized stochastic system described in terms of global and localvariables. In particular, the dependence of R ₀ on the space ofparameters that define the model is investigated; it is found that near ofthe `classical' epidemic threshold transition the uncertainty about thestrength of the epidemic process still is significantly large. Theforecasting attributes of R ₀ for a discrete finite system is discussedand generalized; in particular, it is shown that, for a discrete finitesystem, the pretentious predictive power of R ₀ is significantlyreduced.     

Evidence of aerobic and anaerobic format dehydrogenase and aldehyde dehydrogenase immunological similarities shown by polyclonal antibodies raised against molybdoproteins

Antisera against metal(Mo)-containing dye-linked dehydrogenases from sulphate-reducing bacteria were used to screen for immunological similarities with NAD⁺-linked dehydrogenases detected in aerobic methanol-utilizing bacterial isolates. Out of eleven strains tested, the strains #5, 8, 9 and 11 were shown to have specific formate and aldehyde dehydrogenases displaying antibody cross-reaction against highly purified Mo-containing dye-linked dehydrogenases. The apparent molecular mass of the identified proteins observed during the antibody reaction correlated with the molecular mass of the dehydrogenases obtained after native PAGE electrophoresis. The strains #8 and 11 exhibited one formate dehydrogenase apparently of identical molecular mass 140–145 kDa, whereas strains #5, 9 and 11 synthesized aldehyde dehydrogenases with apparent molecular masses of about 110, 120 and 155 kDa (two forms) and 120 kDa, respectively. All these aerobic enzymes shared antigenic properties with the anaerobic metalloproteins, indicating the existence of structural similarities between those enzymes in spite of having different cofactor moieties.