Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol

Bacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200–300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block

Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication

BACKGROUND: Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication. METHODOLOGY/PRINCIPAL FINDINGS: We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro. CONCLUSIONS/SIGNIFICANCE: We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins

Regional spread of HIV-1 M subtype B in middle-aged patients by random env-C2V4 region sequencing

A transmission cluster of HIV-1 M:B was identified in 11 patients with a median age of 52 (range 26–65) in North-East Germany by C2V4 region sequencing of the env gene of HIV-1, who—except of one—were not aware of any risky behaviour. The 10 male and 1 female patients deteriorated immunologically, according to their information made available, within 4 years after a putative HIV acquisition. Nucleic acid sequence analysis showed a R5 virus in all patients and in 7 of 11 a crown motif of the V3 loop, GPGSALFTT, which is found rarely. Analysis of formation of this cluster showed that there is still a huge discrepancy between awareness and behaviour regarding HIV transmission in middle-aged patients, and that a local outbreak can be detected by nucleic acid analysis of the hypervariable env region

Advancing the public health applications of Chlamydia trachomatis serology.

Genital Chlamydia trachomatis infection is the most commonly diagnosed sexually transmitted infection. Trachoma is caused by ocular infection with C trachomatis and is the leading infectious cause of blindness worldwide. New serological assays for C trachomatis could facilitate improved understanding of C trachomatis epidemiology and prevention. C trachomatis serology offers a means of investigating the incidence of chlamydia infection and might be developed as a biomarker of scarring sequelae, such as pelvic inflammatory disease. Therefore, serological assays have potential as epidemiological tools to quantify unmet need, inform service planning, evaluate interventions including screening and treatment, and to assess new vaccine candidates. However, questions about the performance characteristics and interpretation of C trachomatis serological assays remain, which must be addressed to advance development within this field. In this Personal View, we explore the available information about C trachomatis serology and propose several priority actions. These actions involve development of target product profiles to guide assay selection and assessment across multiple applications and populations, establishment of a serum bank to facilitate assay development and evaluation, and development of technical and statistical methods for assay evaluation and analysis of serological findings. The field of C trachomatis serology will benefit from collaboration across the public health community to align technological developments with their potential applications

Resistance to FasL and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in Sezary syndrome T-cells associated with impaired death receptor and FLICE-inhibitory protein expression

Because of the low proliferative potential of tumor cells in patients with Sézary syndrome (SzS), their accumulation has been suggested to be due to defective regulation of apoptosis. We analyzed the sensitivity to soluble Fas-ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), 2 members of the TNF superfamily in peripheral blood leukocytes (PBL) from patients with SzS. Compared with healthy donors, CD4(+) cells from patients with SzS were completely resistant to FasL in 9 of 16 cases. Of these 9 FasL-resistant cases, 4 revealed a loss in Fas (CD95) expression, whereas the remaining 5 exhibited normal or enhanced Fas expression. In the latter 5 cases, the apoptosis inhibitor cFLIP was overexpressed in CD4(+)/CD26(-) tumor cells compared with CD4(+)/CD26(-) cells from Fas-expressing FasL-sensitive patients and healthy donors. Furthermore, resistance to TRAIL and tumor cell-restricted loss of TRAIL-receptor 2 were observed in 16 of 16 SzS PBLs. It is noteworthy that resistance to FasL could be overcome by the use of a hexameric FasL or upon exposure of SzS cells to interferon-alpha (IFN-alpha) or IFN-gamma, the latter by an increase of Fas expression. Our data on primary SzS lymphocytes reveal frequent resistance to apoptosis induced by FasL and TRAIL, which may contribute to their accumulation in patients with SzS and be relevant at a therapeutic level

Lipoprotein and Metabolic Profiles Indicate Similar Cardiovascular Risk of Liver Steatosis and NASH

$\textit {Background and Aim}$: Nonalcoholic fatty liver disease (NAFLD) affects about 25% of the global population, with no reliable noninvasive tests to diagnose nonalcoholic steatohepatitis (NASH) and to differentiate between NASH and nonalcoholic fatty liver (NAFL) (steatosis alone). It is unclear if NAFL and NASH differ in cardiovascular risk for patients. Here, we compared obese NAFLD patients with a healthy cohort to test whether cholesterol compounds could represent potential noninvasive markers and to estimate associated risks. $\it Method:$ Serum samples of 46 patients with histologically confirmed NAFLD (17 NAFL, 29 NASH) who underwent bariatric surgery were compared to 32 (9 males, 21 females) healthy controls (HCs). We analyzed epidemiological data, liver enzymes, cholesterol and lipid profile, and amino acids. The latter were analyzed by nuclear magnetic resonance spectroscopy. $\it Results:$ Total serum and high-density lipoprotein (HDL) cholesterol were significantly lower in the NAFLD group than in HCs, with a stronger reduction in NASH. Similar observations were made for sub-specification of HDL-p, HDL-s, SHDL-p, and LHDL-p cholesterols. Low-density lipoprotein (LDL)-s and LLDL-p cholesterol were significantly reduced in NAFLD groups. Interestingly, SLDL-p cholesterol was significantly higher in the NAFL group with a stronger elevation in NASH than in HCs. The amino acids alanine, leucin, and isoleucine were significantly higher in the NAFL and NASH groups than in HCs. $\it Conclusion:$ We show in this study that cholesterol profiles, apolipoproteins, and amino acids could function as a potential noninvasive test to screen for NAFLD or even NASH in larger populations. However, few differences in cholesterol profiles were identified between the NAFL and NASH groups, indicating similar cardiovascular risk profiles

Advancing the public health applications of Chlamydia trachomatis serology

Genital Chlamydia trachomatis infection is the most commonly diagnosed sexually transmitted infection. Trachoma is caused by ocular infection with C trachomatis and is the leading infectious cause of blindness worldwide. New serological assays for C trachomatis could facilitate improved understanding of C trachomatis epidemiology and prevention. C trachomatis serology offers a means of investigating the incidence of chlamydia infection and might be developed as a biomarker of scarring sequelae, such as pelvic inflammatory disease. Therefore, serological assays have potential as epidemiological tools to quantify unmet need, inform service planning, evaluate interventions including screening and treatment, and to assess new vaccine candidates. However, questions about the performance characteristics and interpretation of C trachomatis serological assays remain, which must be addressed to advance development within this field. In this Personal View, we explore the available information about C trachomatis serology and propose several priority actions. These actions involve development of target product profiles to guide assay selection and assessment across multiple applications and populations, establishment of a serum bank to facilitate assay development and evaluation, and development of technical and statistical methods for assay evaluation and analysis of serological findings. The field of C trachomatis serology will benefit from collaboration across the public health community to align technological developments with their potential applications.</p