Corneal keratocytes retain neural crest progenitor cell properties

Corneal keratocytes have a remarkable ability to heal the cornea throughout life. Given their developmental origin from the cranial neural crest, we asked whether this regenerative ability was related to the stem cell-like properties of their neural crest precursors. To this end, we challenged corneal stromal keratocytes by injecting them into a new environment along cranial neural crest migratory pathways. The results show that injected stromal keratocytes change their phenotype, proliferate and migrate ventrally adjacent to host neural crest cells. They then contribute to the corneal endothelial and stromal layers, the musculature of the eye, mandibular process, blood vessels and cardiac cushion tissue of the host. However, they fail to form neurons in cranial ganglia or branchial arch cartilage, illustrating that they are at least partially restricted progenitors rather than stem cells. The data show that, even at late embryonic stages, corneal keratocytes are not terminally differentiated, but maintain plasticity and multipotentiality, contributing to non-neuronal cranial neural crest derivatives.     

Biochemical and genetic analysis of the four DNA ligases of mycobacteria

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.     

Phytochemical composition of Potentilla anserina L. analyzed by an integrative GC-MS and LC-MS metabolomics platform

Potentilla anserina L. (Rosaceae) is known for its beneficial effects of prevention of pre-menstrual syndrome (PMS). For this reason P. anserina is processed into many food supplements and pharmaceutical preparations. Here we analyzed hydroalcoholic reference extracts and compared them with various extracts of different pharmacies using an integrative metabolomics platform comprising GC-MS and LC-MS analysis and software toolboxes for data alignment (MetMAX Beta 1.0) and multivariate statistical analysis (COVAIN 1.0). Multivariate statistics of the integrated GC-MS and LC-MS data showed strong differences between the different plant extract formulations. Different groups of compounds such as chlorogenic acid, kaempferol 3-O-rutinoside, acacetin 7-O-rutinoside, and genistein were reported for the first time in this species. The typical fragmentation pathway of the isoflavone genistein confirmed the identification of this active compound that was present with different abundances in all the extracts analyzed. As a result we have revealed that different extraction procedures from different vendors produce different chemical compositions, e.g. different genistein concentrations. Consequently, the treatment may have different effects. The integrative metabolomics platform provides the highest resolution of the phytochemical composition and a mean to define subtle differences in plant extract formulations.     

Serum proteomic signature of human chagasic patients for the identification of novel potential protein biomarkers of disease

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.     

In situ analysis of native microbial communities in complex samples with high particulate loads

In the present study a procedure combining a cell extraction method and Fluorescence In Situ Hybridization (FISH) for molecular monitoring and quantification of bacteria in soil and aquifer samples is presented. FISH was applied to bacterial cells extracted from the matrix by density gradient centrifugation. This separation method was applied to soil and aquifer samples and produced high cell recovery of 76.5%+/-4.4 and 78.0%+/-3.2, respectively. FISH, performed on the harvested cells, permitted a perfect visualization and quantification of bacteria. This approach is therefore promising for in situ detection of indigenous bacterial communities in complex samples.     

CYP116B5: a new class VII catalytically self‐sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N‐ and C‐terminal domains suggests an evolutionary model involving a plasmid‐mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer–Villiger monooxygenase, allowed the recombinant bacteria to grow on long‐ and medium‐chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium‐chain alkanes overlapping AlkB and in the first step of sub‐terminal oxidation of long‐chain alkanes. It was also demonstrated that CYP116B5 is a self‐sufficient cytochrome P450 consisting of a heme domain (aa 1–392) involved in the oxidation step of n‐alkanes degradation, and its reductase domain (aa 444–758) comprising the NADPH‐, FMN‐ and [2Fe2S]‐binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.