Genetic and Epigenetic Variation across Genes Involved in Energy Metabolism and Mitochondria of Chinese Hamster Ovary Cell Lines

The increasingdemandfor biopharmaceutical products drives the search for efficient cell factories that are able to sustainably support rapid growth, high productivity, and product quality. As these depend on energy generation, here the genomic variation in nuclear genes associated with mitochondria and energy metabolism and the mitochondrial genome of 14 cell lines is investigated. The variants called enable reliable tracing of lineages. Unique sequence variations are observed in cell lines adapted to grow in protein‐free media, enriched in signaling pathways or mitogen‐activated protein kinase 3. High‐producing cell lines bear unique mutations in nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND2 and ND4) and in peroxisomal acyl‐CoA synthetase (ACSL4), involved in lipid metabolism. As phenotypes are determined not only by functional mutations, but also by the exquisite regulation of expression patterns, it is not surprising that ≈50% of the genes investigated here are found to be differentially methylated and thus epigenetically controlled, enabling a clear distinction of high producers, and cells adapted to a minimal, glutamine (Gln)‐free medium. Similar pathways are enriched as those identified by genome variation. This strengthens the hypothesis that these phenomena act together to define cell behavior.     

Intra‐thallus differentiation of fatty acid and pigment profiles in some temperate Fucales and Laminariales

Intra‐thallus variation in fatty acid and pigment contents and profiles was investigated in five species of Laminariales (Alaria esculenta, Laminaria digitata, Laminaria hyperborea, Saccharina latissima, and Saccorhiza polyschides), and three Fucales (Ascophyllum nodosum, Fucus serratus, and Himanthalia elongata). Significant variation occurred across all species and compounds examined. Total fatty acids were generally higher in the fronds, with highest levels and largest variability observed in A. nodosum (1.5% of dry weight (DW) in the base, 6.3% of DW in frond tips). Percentages of the omega‐3 fatty acids 18:4 n‐3 and 20:5 n‐3 were generally higher in more distal parts, while 20:4 n‐6 exhibited a contrasting pattern, with higher levels in basal structures and holdfasts. Trends for pigments were similar to those for fatty acids in Laminariales. In the Fucales, highest levels were detected in the mid‐fronds, with lower concentrations in meristematic areas. Highest levels and greatest variability in pigments (e.g., chl a) was observed in F. serratus (1.07 mg · g^?1 DW in the base, 3.04 mg · g^?1 DW in the mid frond). Intra‐thallus variability was attributed to physiological functions of the respective thallus sections, e.g., photosynthetic activity, meristematic tissue, and to variations in physical attributes of the structures investigated. Regarding potential commercial nutritional applications, fronds appeared to represent most suitable source materials, due to higher levels of pigments, polyunsaturated fatty acids, and more preferable omega‐3/omega‐6 ratios.     

Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit 'bizarre' secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading 'pseudogenes', even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.     

Systems-wide Analysis of a Phosphatase Knock-down by Quantitative Proteomics and Phosphoproteomics

Signal transduction in metazoans regulates almost all aspects of biological function, and aberrant signaling is involved in many diseases. Perturbations in phosphorylation-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific antibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data. SILAC-based quantitation after phosphatase knock-down showed that apart from the phosphatase, the proteome was minimally affected whereas 288 of 6,478 high-confidence phosphorylation sites changed significantly. Responses at the phosphotyrosine level included the already described Ptp61F substrates Stat92E and Abi. Our analysis highlights a connection of Ptp61F to cytoskeletal regulation through GTPase regulating proteins and focal adhesion components.Information processing in biological systems relies heavily on activation and inactivation of proteins by phosphorylation. This key post-translational modification is involved in the regulation of most cellular processes and mediates many rapid responses as well as long-term gene expression changes in response to stimuli. Protein kinases and protein phosphatases coordinately regulate this highly dynamic and reversible modification. Phosphorylation is usually studied in a candidate-based approach by in vitro kinase assays or by immune techniques employing phospho-specific antibodies. Despite the success of this reductionist approach, it does not afford a systems-wide observation of the effects upon perturbations of signaling networks.Recent advances in MS-based¹ proteomics now allow the identification of thousands of phosphorylation sites from complex protein mixtures (1–3). Most large-scale phosphoproteomics studies have been qualitative rather than quantitative; however, isotope-based methods enable precise quantitation of phosphorylation sites between two or more cellular states (4–6). Our group has applied the metabolic labeling technology termed stable isotope labeling by amino acids in cell culture (SILAC) (7) for the quantitative comparison of phosphoproteomes. For example, we quantified phosphorylation dynamics in response to epidermal growth factor stimulation. Out of a measured phosphoproteome of several thousand sites only a minority (about 10%) was regulated by the signal, highlighting the importance of quantitation in pinpointing specific systems responses (8).Drosophila is a well established model system to study key players in cell signaling and development. Genetic studies have been performed for decades whereas more recently also RNA interference (RNAi) has been employed for gene function studies using a highly efficient silencing protocol (9). A further advantage of Drosophila as a model system is the lower degree of functional redundancy compared with higher vertebrates while maintaining a high level of conservation of human genes linked to disease (10).Two large-scale, non-quantitative Drosophila phosphoproteome studies were carried out in embryonic Kc167 cells (11) and embryos (12). Both studies identified more than 10,000 sites of the Drosophila phosphoproteome.We have recently adapted the SILAC methodology for quantitative proteomics to Drosophila. Schneider line 2 (SL2) cells were treated with either mock dsRNA or dsRNA against ISWI, a component of chromatin remodeling complexes. The combination of RNAi and SILAC allows the unbiased “phenotypization” of the gene knock-down directly at the proteome level (13).Here we determined a high-quality basal phosphoproteome in SL2 cells and characterized its structural and evolutionary properties. We compared kinase substrate motives between Drosophila and human and trained a Drosophila phosphorylation site predictor.To explore the potential of quantitative phosphoproteomics in a systems-wide manner, we focused on the Drosophila non-transmembrane tyrosine phosphatase Ptp61F. This phosphatase is the ortholog of mammalian PTB1B, which is thought to be involved in type 2 diabetes, obesity, and cancer (14), and which is the target of several ongoing drug development projects (15). Ptp61F is a negative regulator of JAK/STAT signaling (16, 17) and, together with the Ableson kinase (Abl), involved in the regulation of the Abl interacting protein (Abi) and lamella formation (18). Both PTP1B and Ptp61F are among the best studied protein tyrosine phosphatases in their respective organisms; however the characterization of their substrates is still far from complete. Two recent mass spectrometric studies employed substrate trapping to identify direct substrates of PTP1B and Ptp61F (19, 20). The PTP1B study was combined with phosphotyrosine peptide enrichment, which led to site-specific detection of potential PTP1B targets. PTP1B function was additionally investigated by quantitative phosphotyrosine proteomics comparing wild type and PTP1B-deficient fibroblasts. In contrast, the Ptp61F study identified potential substrates without site-specific information. One of these was PVR, the Drosophila homolog of VEGFR and PDGFR, suggesting that Ptp61F - like its mammalian counterpart - counteracts receptor tyrosine kinase signaling. Apart from Abi, further components of the SCAR/WAVE complex as well as its regulatory kinase Abl were identified as potential Ptp61F substrates. This supports an involvement of Ptp61F in the regulation of actin reorganization and remodeling.To study the role of Ptp61F in a global and unbiased approach we combined global quantitative phosphoproteome analysis with RNA interference. We profiled tyrosine, serine and threonine phosphorylation changes upon ablation of Ptp61F by RNAi. In parallel, we quantified changes in the proteome, which allowed us to normalize changes in phosphorylation sites to corresponding changes at the protein level. Interestingly, we observed increased tyrosine phosphorylation of the protein tyrosine kinase Abl which suggests an enhanced Abl activity upon Ptp61F RNAi. We additionally detected up-regulated phosphotyrosine sites on GTPase regulating proteins (like RhoGAP15B and Vav) and constituents of focal adhesions (like Paxillin and Lasp) which expand the proposed involvement of Ptp61F in the regulation of cytoskeleton organization. Our work represents proof-of-principle that the combination of large-scale phosphoproteomics and a loss-of-function approach can contribute significantly to elucidating the role of key players in phosphorylation-dependent signaling. Importantly, this systems-wide approach measures the net effect of the perturbation on the entire signaling network, without the need to define specific substrate-kinase or substrate -phosphatase relationships or other direct functional mechanisms.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

Gender Differences in Survival among Adult Patients Starting Antiretroviral Therapy in South Africa: A Multicentre Cohort Study

Background

Increased mortality among men on antiretroviral therapy (ART) has been documented but remains poorly understood. We examined the magnitude of and risk factors for gender differences in mortality on ART.

Methods and Findings

Analyses included 46,201 ART-naïve adults starting ART between January 2002 and December 2009 in eight ART programmes across South Africa (SA). Patients were followed from initiation of ART to outcome or analysis closure. The primary outcome was mortality; secondary outcomes were loss to follow-up (LTF), virologic suppression, and CD4+ cell count responses. Survival analyses were used to examine the hazard of death on ART by gender. Sensitivity analyses were limited to patients who were virologically suppressed and patients whose CD4+ cell count reached >200 cells/µl. We compared gender differences in mortality among HIV+ patients on ART with mortality in an age-standardised HIV-negative population.Among 46,201 adults (65% female, median age 35 years), during 77,578 person-years of follow-up, men had lower median CD4+ cell counts than women (85 versus 110 cells/µl, p<0.001), were more likely to be classified WHO stage III/IV (86 versus 77%, p<0.001), and had higher mortality in crude (8.5 versus 5.7 deaths/100 person-years, p<0.001) and adjusted analyses (adjusted hazard ratio [AHR] 1.31, 95% CI 1.22–1.41). After 36 months on ART, men were more likely than women to be truly LTF (AHR 1.20, 95% CI 1.12–1.28) but not to die after LTF (AHR 1.04, 95% CI 0.86–1.25). Findings were consistent across all eight programmes. Virologic suppression was similar by gender; women had slightly better immunologic responses than men. Notably, the observed gender differences in mortality on ART were smaller than gender differences in age-standardised death rates in the HIV-negative South African population. Over time, non-HIV mortality appeared to account for an increasing proportion of observed mortality. The analysis was limited by missing data on baseline HIV disease characteristics, and we did not observe directly mortality in HIV-negative populations where the participating cohorts were located.

Conclusions

HIV-infected men have higher mortality on ART than women in South African programmes, but these differences are only partly explained by more advanced HIV disease at the time of ART initiation, differential LTF and subsequent mortality, and differences in responses to treatment. The observed differences in mortality on ART may be best explained by background differences in mortality between men and women in the South African population unrelated to the HIV/AIDS epidemic. Please see later in the article for the Editors'' Summary.     

High-Pressure-Mediated Survival of Clostridium botulinum and Bacillus amyloliquefaciens Endospores at High Temperature

Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120°C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110°C) accelerated the inactivation of C. botulinum spores. However, incubation at 100°C, 110°C, or 120°C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120°C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120°C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.