The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

Prevalencia de sobrepeso y obesidad en niños y adolescentes de 2 a 16 años

ObjectivesTo estimate the prevalence of obesity and overweight in children and adolescents in our city and to investigate the associated factors.Subjects and methodsA cross-sectional study of 1317 children and adolescents aged 2-16 years. Multistage probability sampling was used to select three groups of subjects: 411 aged 12 to 16 years, 504 aged 6 to 12 years, and 402 aged 2 to 6 years. Body mass index was calculated, and obesity and overweight were diagnosed using the threshold levels of the International Obesity Task Force for children and adolescents. Parents were asked about eating habits, health, social, and demographic aspects. Results are given as percentages (95% confidence interval). The relationship between obesity and overweight and the different variables was studied using multiple logistic regression. The adjusted odds ratio (OR) was calculated.ResultsAmong children and adolescentes aged 2-16 years, 9.5% (8.0%-11.0%) were obese and 22.4% (23.3%-24.6%) were overweight. Of subjects aged 12-16 years, 8.5% (5.9%-11.2%) were obese and 20.5% (16.7%-24.3%) were overweight. In the groups aged 6-12 years and 2-6 years, rates of obesity and overweight were 11.6% (8.9% -14.3%) and 31.0% (27.0-35.0) and 8.0% (5.4%-10.6%) and 13.6% (10.3%-16.9%) respectively. Obesity or overweight was associated to age (OR 1.21; P< 0.001), maternal obesity (OR 10.99; P= 0.008), a birthweight higher than 4 kg (OR 2.91; p 0.002), and formula feeding (OR 1.82; P= 0.005).ConclusionObesity and overweight in children and adolescents are highly prevalent problems in our city.     

Aportaciones de los tests de supresión de cortisol al conocimiento de los trastornos psiquiátricos: revisión narrativa de la literatura

Activity of the hypothalamic-pituitary-adrenal axis had been studied for the past half century, when some researchers noted that some patients with Cushing's syndrome and severe mood disorders had high baseline cortisol levels, which resulted in an inhibited response in the 1 mg dexamethasone suppression test. Altered dexamethasone suppression test results were subsequently found in many psychiatric diseases, including anorexia nervosa, obsessive-compulsive disorder, degenerative dementia, bipolar disorders, and schizophrenia. The relationship between high baseline cortisol levels and stress has also been studied. Some researches on the genesis of borderline personality disorder focused on traumatic childhood backgrounds. Other investigations aimed at elucidating the relationship between traumatic backgrounds and some psychiatric disorders noted that patients with post-traumatic stress disorder and borderline personality disorder showed an enhanced cortisol suppression with low cortisol doses (0.5 mg). Recent studies showed that use of an ultra-low dose of cortisol during the dexamethasone suppression test may be helpful for deteting disorders with hyperactivity of the hypothalamic-pituitary-adrenal axis.Recent advances in neuroimaging support the existence of hyperactivity of the hypothalamic-pituitary-adrenal axis in patients with borderline personality disorder, relating a decreased pituitary gland volume to major traumatic backgrounds and suicidal attempts. The purpose of this paper is to make a narrative review of research using dexamethasone suppression test in psychiatric disorders, in order to ascertain its value as a supplemental diagnostic test or as a prognostic marker.     

1H, 13C and 15N chemical shift assignments of unliganded Bcl-xL and its complex with a photoresponsive Bak-derived peptide

Here we report the ¹H, ¹³C and ¹⁵N resonance assignments of free Bcl-x_L and of Bcl-x_L in complex with an azobenzene-modified peptide derived from the BH3 domain of the pro-apoptotic Bak. The spectra suggest predominantly folded proteins; chemical shift difference analysis provides a detailed view of the reorganization occurring on peptide binding.     

Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes

Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.     

Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time.     

An Enhanced Heterologous Virus-Like Particle for Human Papillomavirus Type 16 Tumour Immunotherapy

Cervical cancer is caused by high-risk, cancer-causing human papillomaviruses (HPV) and is the second highest cause of cancer deaths in women globally. The majority of cervical cancers express well-characterized HPV oncogenes, which are potential targets for immunotherapeutic vaccination. Here we develop a rabbit haemorrhagic disease virus (RHDV) virus-like particle (VLP)-based vaccine designed for immunotherapy against HPV16 positive tumours. An RHDV-VLP, modified to contain the universal helper T cell epitope PADRE and decorated with an MHC I-restricted peptide (aa 48–57) from the HPV16 E6, was tested for its immunotherapeutic efficacy against the TC-1 HPV16 E6 and E7-expressing tumour in mice. The E6-RHDV-VLP-PADRE was administered therapeutically for the treatment of a pre-existing TC-1 tumour and was delivered with antibodies either to deplete regulatory T cells (anti-CD25) or to block T cell suppression mediated through CTLA-4. As a result, the tumour burden was reduced by around 50% and the median survival time of mice to the humane endpoint was almost doubled the compared to controls. The incorporation of PADRE into the RHDV-VLP was necessary for an E6-specific enhancement of the anti-tumour response and the co-administration of the immune modifying antibodies contributed to the overall efficacy of the immunotherapy. The E6-RHDV-VLP-PADRE shows immunotherapeutic efficacy, prolonging survival for HPV tumour-bearing mice. This was enhanced by the systemic administration of immune-modifying antibodies that are commercially available for use in humans. There is potential to further modify these particles for even greater efficacy in the path to development of an immunotherapeutic treatment for HPV precancerous and cancer stages.     

TLR and NKG2D Signaling Pathways Mediate CS-Induced Pulmonary Pathologies

Long-term exposure to cigarette smoke (CS) can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR) signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure.     

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa,Cry1Ab,Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests,Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with ¹²⁵I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.