Transposase concentration controls transposition activity: Myth or reality?

Deciphering the mechanisms underlying the regulation of DNA transposons might be central to understanding their function and dynamics in genomes. From results obtained under artificial experimental conditions, it has been proposed that some DNA transposons self-regulate their activity via overproduction inhibition (OPI), a mechanism by which transposition activity is down-regulated when the transposase is overconcentrated in cells. However, numerous studies have given contradictory results depending on the experimental conditions. Moreover, we do not know in which cellular compartment this phenomenon takes place, or whether transposases assemble to form dense foci when they are highly expressed in cells.     

miR-605-5p promotes invasion and proliferation by targeting TNFAIP3 in non–small-cell lung cancer

Lung cancer is an significant cause of death worldwide, and non–small-cell lung cancer (NSCLC) is the most common type of lung cancer. MicroRNAs (miRNAs) have been identified to play key roles in NSCLC development. Recently, it has been reported that miR-605-5p is a cancer-related miRNA in several types of tumors. In this study, we study the role of miR-605-5p in NSCLC cells. We find that miR-605-5p is upregulated in NSCLC cells. Overexpression of miR-605-5p significantly promotes lung cancer invasion and migration in H460 and H1299 cells. Besides this, miR-605-5p also promotes lung cancer cell carcinoma proliferation and metastasis in vivo. However, downregulation of miR-605-5p inhibits cell invasion and migration by inhibiting lung cancer cell carcinoma proliferation and metastasis. In addition, the luciferase report assay identifies 3′-untranslated region tumor necrosis factor α-induced protein 3 (TNFAIP3) as a target of miR-605-5p. Silencing of TNFAIP3 promotes invasion and proliferation in lung cancer. In addition, the knockdown of TNFAIP3 restores the significant decrease in invasion and proliferation in miR-605-5p-inhibitor–transfected lung cancer cells. In conclusion, miR-605-5p promotes invasion and proliferation by targeting TNFAIP3 in NSCLC, and may provide possible biomarkers for NSCLC therapy.     

Redesign of the PAK1 autoinhibitory domain for enhanced stability and affinity in biosensor applications

The inhibitory switch (IS) domain of p21-activated kinase 1 (PAK1) stabilizes full-length PAK1 in an inactive conformation by binding to the PAK1 kinase domain. Competitive binding of small guanosine triphosphatases to the IS domain disrupts the autoinhibitory interactions and exposes the IS domain binding site on the surface of the kinase domain. To build an affinity reagent that selectively binds the activated state of PAK1, we used molecular modeling to reengineer the isolated IS domain so that it was soluble and stable, did not bind to guanosine triphosphatases and bound more tightly to the PAK1 kinase domain. Three design strategies were tested: in the first and second cases, extension and redesign of the N-terminus were used to expand the hydrophobic core of the domain, and in the third case, the termini were redesigned to be adjacent in space so that the domain could be stabilized by insertion into a loop in a host cyan fluorescent protein (CFP). The best-performing design, called CFP-PAcKer, was based on the third strategy and bound the kinase domain of PAK1 with an affinity of 400 nM. CFP-PAcKer binds more tightly to a full-length variant of PAK1 that is stabilized in the “open” state (K_d = 3.3 μM) than to full-length PAK1 in the “closed” state (undetectable affinity), and binding can be monitored with fluorescence by placing an environmentally sensitive fluorescence dye on CFP-PAcKer adjacent to the binding site.     

Hypervariable loci that enhance the discriminatory ability of newly proposed 15-loci and 24-loci variable-number tandem repeat typing method on Mycobacterium tuberculosis strains predominated by the Beijing family

The newly proposed 15- and 24-loci mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem repeat (VNTR) typing method was evaluated for its ability to differentiate 181 Mycobacterium tuberculosis Beijing family strains. Compared with the original 12-loci MIRU-VNTR typing method, the 15-loci system dramatically improved the discriminatory power for Beijing strains; however, large clusters that could be further differentiated by IS6110 restriction fragment length polymorphism (RFLP) were still obtained. The clonal stability and allelic diversity of a total of 31 VNTR loci were evaluated. VNTRs 3232, 3820, and 4120 were identified as the effective hypervariable VNTR set for the second-line typing of clustered strains following the 15-loci based scheme. Consequently, the discriminatory power of the new scheme (18 loci) equaled that of IS6110 RFLP.     

Purifying selection and positive selection on the myxovirus resistance gene in mammals and chickens

The myxovirus resistance gene (Mx) expresses antiviral activity in many species, e.g. mouse, human and chicken. It is not clear if the antiviral activity of Mx has evolved in these species to inhibit a set of species-specific pathogens, nor what factors drive Mx evolution in different animal lineages. Therefore, it is important to determine the evolutionary pattern of Mx and positively selected sites which affect the antiviral activity of the Mx gene in mammals and birds. We used sequence comparisons among species to detect positively selected sites by conducting phylogenetic analysis. The two-ratio model was significantly better than the one-ratio model in four species (mouse, rat, chicken and duck, p<0.05). Although selection pressure varied among different lineages, Mx had strong purifying selection in mammals and positive selection in chicken and duck lineages. Relative rate test revealed that Mx evolved faster in chickens than in ducks (Tajima's relative rate test, chi(2)=7.17, p<0.01). In the further analysis using a branch-site model A test, 8 sites were positively selected in the chicken lineage while no positive selection signals were observed for any site in the other lineages. The branch-site model A test had a omega value of 4.374 for the chicken lineage (2Deltal=14.20, d.f.=1, p<0.001). Comparisons of all currently available Mx mRNA sequences showed that these predicted positively selected sites had been fixed in the chicken lineage, suggesting that the chicken Mx gene evolved within the species to resist newly challenging environments. There is an increased selection constraint leading to mammals, while positive selection has acted on the chicken Mx.     

Comparative analysis of the catechol 2,3-dioxygenase gene locus in thermoacidophilic archaeon Sulfolobus solfataricus strain 98/2

A catechol 2,3-dioxygenase (C23O) gene was found from Sulfolobus solfataricus strain 98/2. Heterologous thermophilic C23O expressed in Escherichia coli showed the highest activity against catechol and 4-chlorocatechol, and at neutral pH. The C23O gene located with a putative multicomponent monooxygenase (MM) gene cluster that exactly matched with the homologous region of S. solfataricus strain P2. Primary sequence comparison identified an insertion sequence (IS) element inserted into a putative MM protein A N-terminal fragment gene in strain 98/2. Both ends of the transposase gene in the IS element, ISC1234, were flanked by 19 bp inverted repeat and 4 bp direct repeat sequences which are typical features of mobile elements. Our analysis and the two geographically distant origins of strains 98/2 and P2 (USA and Italy, respectively) suggest that the two strains have evolved from a common ancestor.     

Isc1 regulates sphingolipid metabolism in yeast mitochondria

The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly purified mitochondria, and western blots using highly purified mitochondrial membrane fractions demonstrated that epitope-tagged Isc1p localized to the outer mitochondrial membrane as an integral membrane protein. Next, LC/MS was employed to determine the sphingolipid composition of highly purified mitochondria which were found to be significantly enriched in α-hydroxylated phytoceramides (21.7 fold) relative to the whole cell. Mitochondria, on the other hand, were significantly depleted in sphingoid bases. Compared to the parental strain, mitochondria from isc1Δ in the post-diauxic phase showed drastic reduction in the levels of α-hydroxylated phytoceramide (93.1% loss compared to WT mitochondria with only 2.58 fold enrichment in mitochondria compared to whole cell). Functionally, isc1Δ showed a higher rate of respiratory-deficient cells after incubation at high temperature and was more sensitive to hydrogen peroxide and ethidium bromide, indicating that isc1Δ exhibits defects related to mitochondrial function. These results suggest that Isc1p generates ceramide in mitochondria, and the generated ceramide contributes to the normal function of mitochondria. This study provides a first insight into the specific composition of ceramides in mitochondria.     

Biomolecular identification of ancient Mycobacterium tuberculosis complex DNA in human remains from Britain and continental Europe

Tuberculosis is known to have afflicted humans throughout history and re‐emerged towards the end of the 20th century, to an extent that it was declared a global emergency in 1993. The aim of this study was to apply a rigorous analytical regime to the detection of Mycobacterium tuberculosis complex (MTBC) DNA in 77 bone and tooth samples from 70 individuals from Britain and continental Europe, spanning the 1st–19th centuries AD. We performed the work in dedicated ancient DNA facilities designed to prevent all types of modern contamination, we checked the authenticity of all products obtained by the polymerase chain reaction, and we based our conclusions on up to four replicate experiments for each sample, some carried out in an independent laboratory. We identified 12 samples that, according to our strict criteria, gave definite evidence for the presence of MTBC DNA, and another 22 that we classified as “probable” or “possible.” None of the definite samples came from vertebrae displaying lesions associated with TB. Instead, eight were from ribs displaying visceral new bone formation, one was a tooth from a skeleton with rib lesions, one was taken from a skeleton with endocranial lesions, one from an individual with lesions to the sacrum and sacroiliac joint and the last was from an individual with no lesions indicative of TB or possible TB. Our results add to information on the past temporal and geographical distribution of TB and affirm the suitability of ribs for studying ancient TB. Am J Phys Anthropol 153:178–189, 2014. © 2013 Wiley Periodicals, Inc.     

MiR-605 represses PSMD10/Gankyrin and inhibits intrahepatic cholangiocarcinoma cell progression

The aberrant expression of PSMD10 has important functions in various malignancies. This study showed that PSMD10 was highly expressed and inversely correlated with the expression of miR-605 in intrahepatic cholangiocarcinoma (ICC) specimens. MiR-605 directly targeted and repressed PSMD10 expression. In addition, over-expression of miR-605 inhibited ICC cell progression both in vitro and in vivo. This effect of miR-605 on ICC cells was similar to that of PSMD10 knock-down by RNAi. Moreover, restoration of PSMD10 could reverse the phenotypic alteration caused by miR-605 in ICC cells. These results suggest a new therapeutic strategy in ICC by restoring miR-605, which is regulated by p53.