Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome

The bacteriophage P1 Cre—lox site-specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant lox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild-type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre—lox site-specific recombination system is useful for the single-copy integration of DNA into a chromosomal lox site.     

Identification of two poorly prognosed ovarian carcinoma subtypes associated with CHEK2 germ-line mutation and non-CHEK2 somatic mutation gene signatures

High-grade serous ovarian cancer (HG-SOC), a major histologic type of epithelial ovarian cancer (EOC), is a poorly-characterized, heterogeneous and lethal disease where somatic mutations of TP53 are common and inherited loss-of-function mutations in BRCA1/2 predispose to cancer in 9.5–13% of EOC patients. However, the overall burden of disease due to either inherited or sporadic mutations is not known.

We performed bioinformatics analyses of mutational and clinical data of 334 HG-SOC tumor samples from The Cancer Genome Atlas to identify novel tumor-driving mutations, survival-significant patient subgroups and tumor subtypes potentially driven by either hereditary or sporadic factors.

We identified a sub-cluster of high-frequency mutations in 22 patients and 58 genes associated with DNA damage repair, apoptosis and cell cycle. Mutations of CHEK2, observed with the highest intensity, were associated with poor therapy response and overall survival (OS) of these patients (P = 8.00e-05), possibly due to detrimental effect of mutations at the nuclear localization signal. A 21-gene mutational prognostic signature significantly stratifies patients into relatively low or high-risk subgroups with 5-y OS of 37% or 6%, respectively (P = 7.31e-08). Further analysis of these genes and high-risk subgroup revealed 2 distinct classes of tumors characterized by either germline mutations of genes such as CHEK2, RPS6KA2 and MLL4, or somatic mutations of other genes in the signature.

Our results could provide improvement in prediction and clinical management of HG-SOC, facilitate our understanding of this complex disease, guide the design of targeted therapeutics and improve screening efforts to identify women at high-risk of hereditary ovarian cancers distinct from those associated with BRCA1/2 mutations.     

Mutants of satellite bacteriophage P4 that are defective in the suppression of transcriptional polarity

The satellite bacteriophage P4 relies on a helper such as P2 to supply the gene products necessary for virion assembly and cell lysis (Six, 1975). P4 has the unique capacity to activate the late genes of P2 by a mechanism that differs from the one normally used by P2 itself. This process has been termed transactivation (Calendar et al., 1977). In addition, P4 is able to suppress the strong polarity associated with certain P2 amber mutations. The isolation of P4 mutants solely defective in polarity suppression (psu^?) demonstrates that the ability of P4 to suppress polarity is non-essential for P4 growth. In particular, polarity suppression plays no essential role in either transactivation or head size determination. The product of the P4 psu gene has been identified as a 19,900 M_r P4 late protein.     

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle.     

Current trends in high throughput proteomics in cyanobacteria

Advancements in genome sequencing and high throughput proteomics of cyanobacterial strains led to 13 published reports, from a small number of laboratories. These successful studies focused on Synechocystis, Nostoc and Anabaena strains, prochlorococcus, and halotolerant Euhalothece. The implications of emerging quantitative aspects developed and applied in these large-scale studies are assessed in the wake of advanced cyanobacterial research. Furthermore, contributions from traditional and early high throughput analysis of cyanobacterial proteomics are compared and summarised. Finally, opinions are provided to link both the trends and the future challenges. This review aims to push the synergy between proteomics and cyanobacterial research to improve both the technical and biological significance.     

Site-specific deletions in the tomato genome by the CinH-<Emphasis Type="Italic">RS2</Emphasis> and ParA-<Emphasis Type="Italic">MRS</Emphasis> recombination systems

We have tested the CinH-RS2 and ParA-MRS site-specific deletion systems in tomato (Solanum lycopersicum L.). The ParA-MRS system is derived from the broad-host-range plasmid RK2, where the 222 aa ParA recombinase recognizes a 133 bp multimer resolution site (MRS). The CinH-RS2 system is derived from Acinetobacter plasmids pKLH2 and pKLH204, where the 188 amino acid CinH recombinase recognizes a 113-bp recombination site known as RS2. In this study, target lines containing a DNA segment flanked by recombination sites were crossed to recombinase-expressing lines producing CinH or ParA recombinase. CinH-mediated recombination of RS2 substrates was detected in 2 of 3 F₁ plants that harbor both the target and recombinase loci. On the other hand, recombination mediated by ParA was not detected among F₁ plants, but was found among 13 of 47 F₂ plants. These data show that both systems can mediate site-specific DNA deletion in the tomato genome, and, upon further refinement, can provide additional molecular tools for tomato improvement through precise genome manipulation. As the target construct also contains additional recombination sites for site-specific integration by other recombination systems, these tomato lines could be used for future testing of gene stacking through site-specific integration.     

Measurement of plasma volume using fluorescent silica-based nanoparticles

Plasma volume (PV) is an important determinant of cardiovascular function and organ perfusion, and it is the target of infusion and diuretic therapies in daily clinical practice. Despite its fundamental importance PV is not commonly measured because available methods of tracer dilution are reliant on dye substances that suffer from numerous drawbacks including binding plasma proteins, spectral changes, and clearance kinetics that complicate analysis and interpretation. To address these issues, we have tested the utility of fluorescent nanoparticles comprised of a dye-rich silica core and polyethylene glycol-coated shell. Photophysical and visual analysis showed discrete size-gradated nanoparticle populations could be synthesized within a distribution tolerance of ±4 nm, which were optically unaffected in the presence of plasma/albumin. In normal mice, the cutoff for renal filtration of nanoparticles from blood into urine was ≤11 nm. A linear relationship between body weight and PV was readily determined in mice administered far red fluorescent nanoparticles sized either 20 or 30 nm. PV measurements using nanoparticles were correlated to values obtained with Evans blue dye. Induced expansion or contraction of PV was demonstrated with albumin or furosemide administration, respectively, in mice. Longitudinal experiments >30 min required matched untreated control mice to correct for nanoparticle loss (≈30%) putatively to the reticuloendothelial/phagocyte system. Collectively, the findings support a nanotechnology-based solution to methodological problems in measure of PV, notably in clinical settings where information on hemodynamic changes may improve treatment of injury and disease.