Auto-regulation of DNA degrading bacteriocins: molecular and ecological aspects

Colicins, proteinaceous antibiotics produced by Escherichia coli, specifically target competing strains killing them through one of a variety of mechanisms, including pore formation and nucleic acid degradation. The genes encoding colicins display a unique form of expression, which is tightly regulated, involving the DNA damage response regulatory system (the SOS response system), confined to stressful conditions and released by degradation of the producing cell. Given their lethal nature, colicin production has evolved a sophisticated system for repression and expression. While exploring the expression of 13 colicins we identified a novel means of induction unique to strains that kill by DNA degradation: these colicinogenic strains mildly poison themselves inflicting DNA damage that induces their DNA repair system (the SOS system), and their own expression. We established that among the four known DNase colicins (E2, E7, E8 and E9), three act to induce their own production. Using different stresses we show that this form of self-regulation entails high cost when growth conditions are not optimal, and is not carried out by individual cells but is a population-mediated trait. We discuss this novel form of colicins’ regulation and expression, and its possible molecular mechanism and evolutionary implications.     

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC₅₀ values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.     

Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.     

Calcified gallstone in a 3 year-old boy: a case report

ABSTRACT: BACKGROUND: Gallstones are relatively rare in children. At-risk populations include patients suffering from hemolysis syndromes. Regardless of etiology, these patients usually will present with postprandial abdominal pain, and ultrasonography is the mainstay of diagnosis. However, some gallstones are radiopaque and can be visualized on plain abdominal radiography. CASE PRESENTATION: We present the uncommon but classic plain x-ray finding of a calcified gallstone in a 3 year-old Hispanic boy. He was treated with elective laparoscopic cholecystectomy. CONCLUSIONS: Cholelithiasis is rare in children, and calcified stones that will appear on plain abdominal x-rays are even rarer. If symptomatic, cholecystectomy by a pediatric surgeon is the treatment of choice. We discuss some of the recent developments in treatment of this condition in this patient population.     

Rodent phylogeny revised: analysis of six nuclear genes from all major rodent clades

Background  

Rodentia is the most diverse order of placental mammals, with extant rodent species representing about half of all placental diversity. In spite of many morphological and molecular studies, the family-level relationships among rodents and the location of the rodent root are still debated. Although various datasets have already been analyzed to solve rodent phylogeny at the family level, these are difficult to combine because they involve different taxa and genes.     

Evolutionary modeling of rate shifts reveals specificity determinants in HIV-1 subtypes

A hallmark of the human immunodeficiency virus 1 (HIV-1) is its rapid rate of evolution within and among its various subtypes. Two complementary hypotheses are suggested to explain the sequence variability among HIV-1 subtypes. The first suggests that the functional constraints at each site remain the same across all subtypes, and the differences among subtypes are a direct reflection of random substitutions, which have occurred during the time elapsed since their divergence. The alternative hypothesis suggests that the functional constraints themselves have evolved, and thus sequence differences among subtypes in some sites reflect shifts in function. To determine the contribution of each of these two alternatives to HIV-1 subtype evolution, we have developed a novel Bayesian method for testing and detecting site-specific rate shifts. The RAte Shift EstimatoR (RASER) method determines whether or not site-specific functional shifts characterize the evolution of a protein and, if so, points to the specific sites and lineages in which these shifts have most likely occurred. Applying RASER to a dataset composed of large samples of HIV-1 sequences from different group M subtypes, we reveal rampant evolutionary shifts throughout the HIV-1 proteome. Most of these rate shifts have occurred during the divergence of the major subtypes, establishing that subtype divergence occurred together with functional diversification. We report further evidence for the emergence of a new sub-subtype, characterized by abundant rate-shifting sites. When focusing on the rate-shifting sites detected, we find that many are associated with known function relating to viral life cycle and drug resistance. Finally, we discuss mechanisms of covariation of rate-shifting sites.     

Actigraphic motor activity during sleep from infancy to adulthood

A secondary analysis of longitudinal and cohort studies was carried out to quantitatively investigate the motor activity pattern, recorded through actigraphy, during the first six hours of nocturnal sleep. The first study was of longitudinal nature. Ten healthy participants (four females) were monitored three times, at baseline (T1) when they were infants (mean age 7.10 ± 0.32 months), at the first follow-up examination (T2) around 4 months later (mean age 11.20 ± 0.63 months) and at the second follow-up (T3) around three years later, when they were preschoolers (mean age 4.68 ± 0.14 years). At T1, T2 and T3 each participant wore the actigraph Basic Mini-Motionlogger (Ambulatory Monitoring, Inc., Ardsley, NY, USA) over at least two consecutive nycthemeral cycles, with the aim to measure the mean hourly motor activity count. Seven- and 11-month-old infants had a higher level of motor activity over the night compared to preschoolers. Furthermore, motor activity increased as the night progressed, with a pronounced increment at both T1 and T2, while at T3 such an increase was less marked. The second study was cross-sectional and aimed to explore the motor activity pattern, using actigraphy, during the first six hours of nocturnal sleep in multiple-age healthy groups, from infancy to adulthood. We assigned participants to eight groups according to age: 20 (five females) aged around 10 months old (mean age 10.65 ± 0.67 months); 13 (nine females) aged around 4 years (mean age 4.38 ± 0.51 years); 21 (10 females) aged around 10 years (mean age 9.67 ± 0.91 years); 21 (nine females) aged around 20 years (mean age 19.33 ± 2.44 years); 20 (10 females) aged around 30 years (mean age 29.80 ± 1.99 years); 20 (15 females) aged around 40 years (mean age 40.70 ± 1.26 years); 20 (11 females) aged around 50 years (mean age 50.15 ± 2.80 years) and 20 (nine females) aged around 60 years (mean age 59.25 ± 3.23 years). The participants aged between 10 and 60 years wore the actigraph Basic Mini-Motionlogger over seven consecutive nycthemeral cycles (infants and preschoolers wore the actigraph over at least two consecutive nycthemeral cycles), with the aim to measure the mean hourly motor activity count. The results indicated a significantly higher motor activity count in 10-month-old infants compared to all the remaining age groups. Moreover, the pattern of motor activity of 10-month-old infants was different from that of all other groups, with the highest motor activity counts from the second to the sixth hour of sleep. Considered as a whole, the results of both studies converge regarding the high motor activity detected among infants, which could be explained by the presence of a maturational process that has not yet been fully completed at this stage of life. In both studies, only the motor activity of infants was above the cutoff level established for normal adults, highlighting the need to establish a specific cutoff value for infants.     

Weight Loss Reduces Tissue Factor in Morbidly Obese Patients

Objective: To investigate the tissue factor (TF) pathway in clinical obesity and associated metabolic syndrome. Research Methods and Procedures: Thirty‐seven morbidly obese patients (4 men; BMI, 48 ± 7 kg/m²; range, 42 to 53 kg/m²), undergoing elective gastroplasty for the induction of weight loss, were examined for hemostatic, metabolic, and inflammatory parameters at baseline and 14 ± 5 months postoperatively. Results: Weight loss significantly reduced circulating plasma TF (314 ± 181 vs. 235 ± 113 pg/mL, p = 0.04), coagulation factor VII (130 ± 22% vs. 113 ± 19%, p = 0.023), and prothrombin fragment F1.2 (2.4 ± 3.4 vs. 1.14 ± 1.1 nM, p = 0.04) and normalized glucose metabolism in 50% of obese patients preoperatively classified as diabetic or of impaired glucose tolerance. The postoperative decrease in plasma TF correlated with the decrease of F1.2 (r = 0.56; p = 0.005), a marker of in vivo thrombin formation. In subgroup analysis stratified by preoperative glucose tolerance, baseline circulating TF (402.6 ± 141.6 vs. 176.2 ± 58.2, p < 0.001) and TF decrease after gastroplasty (ΔTF: 164.7 ± 51.4 vs. ?81 ± 31 pg/mL, p = 0.02) were significantly higher in obese patients with impaired glucose tolerance than in patients with normal glucose tolerance. Discussion: Procoagulant TF is significantly reduced with weight loss and may contribute to a reduction in cardiovascular risk associated with obesity.     

Critical role of costimulation in the activation of naive antigen-specific TCR transgenic CD8+ T cells in vitro.

The influence of costimulation on the activation of naive CD8+ T cells and thymocytes was studied in vitro using H-Y-specific TCR-transgenic mice and H-Y antigenic peptide. Using a variety of physiological APC types, the activation of naive CD8+ T cells depended strictly on costimulation, which could not be substituted by high epitope density. T cell activation is known to be regulated by the interactions between CD86/CD80 and CD28/CD152, although it remains unclear whether the B7 isoforms have distinct roles. Addition of soluble anti-CD86 Ab led to profound inhibition of T cell reactivity, further confirming the importance of costimulation in naive CD8+ T cell activation. Finally, TCR engagement in the absence of costimulation had no effect on the subsequent reactivity of peripheral naive transgenic CD8+ T cells, but induced nonresponsiveness in mature CD8+ transgenic thymocytes. Collectively, these results demonstrate the importance of costimulation for naive CD8+ T cell activation, suggest that CD80 and CD86 can mediate opposing effects, possibly due to differential interaction with CD152 and CD28, and indicate differences in the sensitivity of immature vs mature CD8+ T cells to the induction of nonresponsiveness following costimulation-deficient Ag presentation.