MAR elements as tools to increase protein production by CHO cells

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection, analysis and characterization of the numerous clones required to identify one with the desired characteristics. Various boundary elements, matrix attachment regions, and locus control regions were screened for for their ability to augment the expression of heterologous genes in CHO and other cells. The 5'-matrix-attachment region (MAR) of the chicken lysozyme gene was found to significantly increase stable gene expression, in culture dishes and in bioreactors. These MAR elements can be easily combined with various existing expression systems, as they can be added in trans (i.e. on a separate plasmid) in co-transfections with previously constructed expression vectors. Using cell population analysis, we found that the use of the MAR increases the proportion of high-producing CHO cell clones, thus reducing the number of cell lines that need to be screened while increasing maximal productivity. Random cDNA cloning and sequencing indicated that over 12% of the ESTs correspond to the transgene. Thus, productivity is no longer limited by transcriptional events in such MAR-containing cell lines. The identification of small and more convenient active MAR portions will also be summarized. Finally, we will show examples of how MAR elements can be combined with short term expression to increase the simultaneous synthesis of many proteins in parallel by CHO cells. Overall, we conclude that the MAR sequence is a versatile tool to increase protein expression in short and long term production processes.

Wound infection after excision and primary midline closure for pilonidal disease: risk factor analysis to improve patient selection.

BACKGROUND: Excision and primary midline closure for pilonidal disease (PD) is a simple procedure; however, it is frequently complicated by infection and prolonged healing. The aim of this study was to analyze risk factors for surgical site infection (SSI) in this context. METHODS: All consecutive patients undergoing excision and primary closure for PD from January 2002 through October 2008 were retrospectively assessed. The end points were SSI, as defined by the Center for Disease Control, and time to healing. Univariable and multivariable risk factor analyses were performed. RESULTS: One hundred thirty-one patients were included [97 men (74%), median age = 24 (range 15-66) years]. SSI occurred in 41 (31%) patients. Median time to healing was 20 days (range 12-76) in patients without SSI and 62 days (range 20-176) in patients with SSI (P < 0.0001). In univariable and multivariable analyses, smoking [OR = 2.6 (95% CI 1.02, 6.8), P = 0.046] and lack of antibiotic prophylaxis [OR = 5.6 (95% CI 2.5, 14.3), P = 0.001] were significant predictors for SSI. Adjusted for SSI, age over 25 was a significant predictor of prolonged healing. CONCLUSION: This study suggests that the rate of SSI after excision and primary closure of PD is higher in smokers and could be reduced by antibiotic prophylaxis. SSI significantly prolongs healing time, particularly in patients over 25 years.

Selection of patients with germline MLH1 mutated Lynch syndrome by determination of MLH1 methylation and BRAF mutation.

Lynch syndrome is one of the most common hereditary colorectal cancer (CRC) syndrome and is caused by germline mutations of MLH1, MSH2 and more rarely MSH6, PMS2, MLH3 genes. Whereas the absence of MSH2 protein is predictive of Lynch syndrome, it is not the case for the absence of MLH1 protein. The purpose of this study was to develop a sensitive and cost effective algorithm to select Lynch syndrome cases among patients with MLH1 immunohistochemical silencing. Eleven sporadic CRC and 16 Lynch syndrome cases with MLH1 protein abnormalities were selected. The BRAF c.1799T> A mutation (p.Val600Glu) was analyzed by direct sequencing after PCR amplification of exon 15. Methylation of MLH1 promoter was determined by Methylation-Sensitive Single-Strand Conformation Analysis. In patients with Lynch syndrome, there was no BRAF mutation and only one case showed MLH1 methylation (6%). In sporadic CRC, all cases were MLH1 methylated (100%) and 8 out of 11 cases carried the above BRAF mutation (73%) whereas only 3 cases were BRAF wild type (27%). We propose the following algorithm: (1) no further molecular analysis should be performed for CRC exhibiting MLH1 methylation and BRAF mutation, and these cases should be considered as sporadic CRC; (2) CRC with unmethylated MLH1 and negative for BRAF mutation should be considered as Lynch syndrome; and (3) only a small fraction of CRC with MLH1 promoter methylation but negative for BRAF mutation should be true Lynch syndrome patients. These potentially Lynch syndrome patients should be offered genetic counselling before searching for MLH1 gene mutations.

Eumelanin-based coloration and fitness parameters in birds: a meta-analysis

Although melanin is the most common pigment in animal integuments, the adaptive function of variation in melanin-based coloration remains poorly understood. The individual fitness returns associated with melanin pigments can be variable across species as these pigments can have physical and biological protective properties and genes involved in melanogenesis may vary in the intensity of pleiotropic effects. Moreover, dark and pale coloration can also enhance camouflage in alternative habitats and melanin-based coloration can be involved in social interactions. We investigated whether darker or paler individuals achieve a higher fitness in birds, a taxon wherein associations between melanin-based coloration and fitness parameters have been studied in a large number of species. A meta-analysis showed that the degree of melanin-based coloration was not significantly associated with laying date, clutch size, brood size, and survival across 26 species. Similar results were found when restricting the analyses to non-sexually dimorphic birds, colour polymorphic and monomorphic species, in passerines and non-passerines and in species for which inter-individual variation in melanism is due to colour intensity. However, eumelanic coloration was positively associated with clutch and brood size in sexually dimorphic species and those that vary in the size of black patches, respectively. Given that greater extent of melanin-based coloration was positively associated with reproductive parameters and survival in some species but negatively in other species, we conclude that in birds the sign and magnitude of selection exerted on melanin-based coloration is species- or trait-specific.

The application of chemometrics on Infrared and Raman spectra as a tool for the forensic analysis of paints

The aim of this work is to evaluate the capabilities and limitations of chemometric methods and other mathematical treatments applied on spectroscopic data and more specifically on paint samples. The uniqueness of the spectroscopic data comes from the fact that they are multivariate - a few thousands variables - and highly correlated. Statistical methods are used to study and discriminate samples. A collection of 34 red paint samples was measured by Infrared and Raman spectroscopy. Data pretreatment and variable selection demonstrated that the use of Standard Normal Variate (SNV), together with removal of the noisy variables by a selection of the wavelengths from 650 to 1830 cm−1 and 2730-3600 cm−1, provided the optimal results for infrared analysis. Principal component analysis (PCA) and hierarchical clusters analysis (HCA) were then used as exploratory techniques to provide evidence of structure in the data, cluster, or detect outliers. With the FTIR spectra, the Principal Components (PCs) correspond to binder types and the presence/absence of calcium carbonate. 83% of the total variance is explained by the four first PCs. As for the Raman spectra, we observe six different clusters corresponding to the different pigment compositions when plotting the first two PCs, which account for 37% and 20% respectively of the total variance. In conclusion, the use of chemometrics for the forensic analysis of paints provides a valuable tool for objective decision-making, a reduction of the possible classification errors, and a better efficiency, having robust results with time saving data treatments.

Inconsistency between different measures of sexual selection.

Measuring the intensity of sexual selection is of fundamental importance to the study of sexual dimorphism, population dynamics, and speciation. Several indices, pools of individuals, and fitness proxies are used in the literature, yet their relative performances are strongly debated. Using 12 independent common lizard populations, we manipulated the adult sex ratio, a potentially important determinant of the intensity of sexual selection at a particular time and place. We investigated differences in the intensity of sexual selection, as estimated using three standard indices of sexual selection-the standardized selection gradient (β'), the opportunity of selection (I), and the Bateman gradient (βss)--calculated for different pools of individuals and different fitness proxies. We show that results based on estimates of I were the opposite of those derived from the other indices, whereas results based on estimates of β' were consistent with predictions derived from knowledge about the species' mating system. In addition, our estimates of the strength and direction of sexual selection depended on both the fitness proxy used and the pool of individuals included in the analysis. These observations demonstrate inconsistencies in distinct measures of sexual selection and underscore the need for caution when comparing studies and species.

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection.

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor approximately 18-25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

Altruism - a philosophical analysis

Altruism is a malleable notion that is understood differently in various disciplines. The common denominator of most definitions of altruism is the idea of unidirectional helping behaviour. However, a closer examination reveals that the term altruism sometimes refers to the outcomes of a helping behaviour for the agent and its neighbours - i.e. reproductive altruism - and sometimes to what motivates the agent to help others - i.e. psychological altruism. Since these perspectives on altruism are crucially different, it is important to use a clear terminology to avoid confusion. In particular, we show that the notion of altruism used by biologists profoundly differs from the ones used by philosophers, psychologists and economists in cross-disciplinary debates about human altruism.

Split sex ratios in the social Hymenoptera: a meta-analysis

The study of sex allocation in social Hymenoptera (ants, bees, and wasps) provides an excellent opportunity for testing kin-selection theory and studying conflict resolution. A queen-worker conflict over sex allocation is expected because workers are more related to sisters than to brothers, whereas queens are equally related to daughters and sons. If workers fully control sex allocation, split sex ratio theory predicts that colonies with relatively high or low relatedness asymmetry (the relatedness of workers to females divided by the relatedness of workers to males) should specialize in females or males, respectively. We performed a meta-analysis to assess the magnitude of adaptive sex allocation biasing by workers and degree of support for split sex ratio theory in the social Hymenoptera. Overall, variation in relatedness asymmetry (due to mate number or queen replacement) and variation in queen number (which also affects relatedness asymmetry in some conditions) explained 20.9% and 5% of the variance in sex allocation among colonies, respectively. These results show that workers often bias colony sex allocation in their favor as predicted by split sex ratio theory, even if their control is incomplete and a large part of the variation among colonies has other causes. The explanatory power of split sex ratio theory was close to that of local mate competition and local resource competition in the few species of social Hymenoptera where these factors apply. Hence, three of the most successful theories explaining quantitative variation in sex allocation are based on kin selection.

Functional analysis and structural modeling of human APOBEC3G reveal the role of evolutionarily conserved elements in the inhibition of human immunodeficiency virus type 1 infection and Alu transposition.

Retroelements are important evolutionary forces but can be deleterious if left uncontrolled. Members of the human APOBEC3 family of cytidine deaminases can inhibit a wide range of endogenous, as well as exogenous, retroelements. These enzymes are structurally organized in one or two domains comprising a zinc-coordinating motif. APOBEC3G contains two such domains, only the C terminal of which is endowed with editing activity, while its N-terminal counterpart binds RNA, promotes homo-oligomerization, and is necessary for packaging into human immunodeficiency virus type 1 (HIV-1) virions. Here, we performed a large-scale mutagenesis-based analysis of the APOBEC3G N terminus, testing mutants for (i) inhibition of vif-defective HIV-1 infection and Alu retrotransposition, (ii) RNA binding, and (iii) oligomerization. Furthermore, in the absence of structural information on this domain, we used homology modeling to examine the positions of functionally important residues and of residues found to be under positive selection by phylogenetic analyses of primate APOBEC3G genes. Our results reveal the importance of a predicted RNA binding dimerization interface both for packaging into HIV-1 virions and inhibition of both HIV-1 infection and Alu transposition. We further found that the HIV-1-blocking activity of APOBEC3G N-terminal mutants defective for packaging can be almost entirely rescued if their virion incorporation is forced by fusion with Vpr, indicating that the corresponding region of APOBEC3G plays little role in other aspects of its action against this pathogen. Interestingly, residues forming the APOBEC3G dimer interface are highly conserved, contrasting with the rapid evolution of two neighboring surface-exposed amino acid patches, one targeted by the Vif protein of primate lentiviruses and the other of yet-undefined function.

Analysis of stop-gain and frameshift variants in human innate immunity genes.

Loss-of-function variants in innate immunity genes are associated with Mendelian disorders in the form of primary immunodeficiencies. Recent resequencing projects report that stop-gains and frameshifts are collectively prevalent in humans and could be responsible for some of the inter-individual variability in innate immune response. Current computational approaches evaluating loss-of-function in genes carrying these variants rely on gene-level characteristics such as evolutionary conservation and functional redundancy across the genome. However, innate immunity genes represent a particular case because they are more likely to be under positive selection and duplicated. To create a ranking of severity that would be applicable to innate immunity genes we evaluated 17,764 stop-gain and 13,915 frameshift variants from the NHLBI Exome Sequencing Project and 1,000 Genomes Project. Sequence-based features such as loss of functional domains, isoform-specific truncation and nonsense-mediated decay were found to correlate with variant allele frequency and validated with gene expression data. We integrated these features in a Bayesian classification scheme and benchmarked its use in predicting pathogenic variants against Online Mendelian Inheritance in Man (OMIM) disease stop-gains and frameshifts. The classification scheme was applied in the assessment of 335 stop-gains and 236 frameshifts affecting 227 interferon-stimulated genes. The sequence-based score ranks variants in innate immunity genes according to their potential to cause disease, and complements existing gene-based pathogenicity scores. Specifically, the sequence-based score improves measurement of functional gene impairment, discriminates across different variants in a given gene and appears particularly useful for analysis of less conserved genes.

Mechanisms governing lentivirus integration site selection.

An essential step of the life cycle of retroviruses is the stable insertion of a copy of their DNA genome into the host cell genome, and lentiviruses are no exception. This integration step, catalyzed by the viral-encoded integrase, ensures long-term expression of the viral genes, thus allowing a productive viral replication and rendering retroviral vectors also attractive for the field of gene therapy. At the same time, this ability to integrate into the host genome raises safety concerns regarding the use of retroviral-based gene therapy vectors, due to the genomic locations of integration sites. The availability of the human genome sequence made possible the analysis of the integration site preferences, which revealed to be nonrandom and retrovirus-specific, i.e. all lentiviruses studied so far favor integration in active transcription units, while other retroviruses have a different integration site distribution. Several mechanisms have been proposed that may influence integration targeting, which include (i) chromatin accessibility, (ii) cell cycle effects, and (iii) tethering proteins. Recent data provide evidence that integration site selection can occur via a tethering mechanism, through the recruitment of the lentiviral integrase by the cellular LEDGF/p75 protein, both proteins being the two major players in lentiviral integration targeting.

Evolution of a cellular immune response in Drosophila: a phenotypic and genomic comparative analysis.

Understanding the genomic basis of evolutionary adaptation requires insight into the molecular basis underlying phenotypic variation. However, even changes in molecular pathways associated with extreme variation, gains and losses of specific phenotypes, remain largely uncharacterized. Here, we investigate the large interspecific differences in the ability to survive infection by parasitoids across 11 Drosophila species and identify genomic changes associated with gains and losses of parasitoid resistance. We show that a cellular immune defense, encapsulation, and the production of a specialized blood cell, lamellocytes, are restricted to a sublineage of Drosophila, but that encapsulation is absent in one species of this sublineage, Drosophila sechellia. Our comparative analyses of hemopoiesis pathway genes and of genes differentially expressed during the encapsulation response revealed that hemopoiesis-associated genes are highly conserved and present in all species independently of their resistance. In contrast, 11 genes that are differentially expressed during the response to parasitoids are novel genes, specific to the Drosophila sublineage capable of lamellocyte-mediated encapsulation. These novel genes, which are predominantly expressed in hemocytes, arose via duplications, whereby five of them also showed signatures of positive selection, as expected if they were recruited for new functions. Three of these novel genes further showed large-scale and presumably loss-of-function sequence changes in D. sechellia, consistent with the loss of resistance in this species. In combination, these convergent lines of evidence suggest that co-option of duplicated genes in existing pathways and subsequent neofunctionalization are likely to have contributed to the evolution of the lamellocyte-mediated encapsulation in Drosophila.