Small regulatory RNAs in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa is frequently associated with nosocomial infections, and can be life threatening in immunosuppressed, cancer and cystic fibrosis patients. Virulence in P. aeruginosa is combinatorial, and results from the activation of several genetic programs that regulate motility, attachment to the host epithelium as well as the synthesis of exotoxins. The pathogen has a high survival capacity in the host owing to its metabolic versatility, nutrient scavenging and resistance against both, antibiotics and immune defenses. Adaptive responses to various environmental stresses and stimuli are often regulated by small regulatory RNAs (sRNA). In this review, we summarize the current knowledge on the regulation and function of P. aeruginosa sRNAs that titrate regulatory proteins, base-pair with target mRNAs, and which are derived from CRISPR elements.     

Clinical and neurocognitive outcome in symptomatic isovaleric acidemia

Background

Approximately 20% of adrenoleukodystrophy (X-ALD) female carriers may develop clinical manifestations, typically consisting of progressive spastic gait, sensory deficits and bladder dysfunctions. A skewing in X Chromosome Inactivation (XCI), leading to the preferential expression of the X chromosome carrying the mutant ABCD1 allele, has been proposed as a mechanism influencing X-linked adrenoleukodystrophy (X-ALD) carrier phenotype, but reported data so far are conflicting.

Methods

To shed light into this topic we assessed the XCI pattern in peripheral blood mononuclear cells (PBMCs) of 30 X-ALD carriers. Since a frequent problem with XCI studies is the underestimation of skewing due to an incomplete sample digestion by restriction enzymes, leading to variable results, we developed a pyrosequencing assay to identify samples completely digested, on which to perform the XCI assay. Pyrosequencing was also used to quantify ABCD1 allele-specific expression. Moreover, very long-chain fatty acid (VLCFA) levels were determined in the same patients.

Results

We found severely (??90:10) or moderately (??75:25) skewed XCI in 23 out of 30 (77%) X-ALD carriers and proved that preferential XCI is mainly associated with the preferential expression of the mutant ABCD1 allele, irrespective of the manifestation of symptoms. The expression of mutant ABCD1 allele also correlates with plasma VLCFA concentrations.

Conclusions

Our results indicate that preferential XCI leads to the favored expression of the mutant ABCD1 allele. This emerges as a general phenomenon in X-ALD carriers not related to the presence of symptoms. Our data support the postulated growth advantage of cells with the preferential expression of the mutant ABCD1 allele, but argue against the use of XCI pattern, ABCD1 allele-specific expression pattern and VLCFA plasma concentration as biomarkers to predict the development of symptoms in X-ALD carriers.     

Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T₀ transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T₂ generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.     

Crystal structure of the intact archaeal translation initiation factor 2 demonstrates very high conformational flexibility in the alpha- and beta-subunits

In Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (α, β, and γ), is pivotal for binding of charged initiator tRNA to the small ribosomal subunit. The crystal structure of the full-sized heterotrimeric aIF2 from Sulfolobus solfataricus in the nucleotide-free form has been determined at 2.8-Å resolution. Superposition of four molecules in the asymmetric unit of the crystal and the comparison of the obtained structures with the known structures of the aIF2αγ and aIF2βγ heterodimers revealed high conformational flexibility in the α- and β-subunits. In fact, the full-sized aIF2 consists of a rigid central part, formed by the γ-subunit, domain 3 of the α-subunit, and the N-terminal α-helix of the β-subunit, and two mobile “wings,” formed by domains 1 and 2 of the α-subunit, the central part, and the zinc-binding domain of the β-subunit. High structural flexibility of the wings is probably required for interaction of aIF2 with the small ribosomal subunit. Comparative analysis of all known structures of the γ-subunit alone and within the heterodimers and heterotrimers in nucleotide-bound and nucleotide-free states shows that the conformations of switch 1 and switch 2 do not correlate with the assembly or nucleotide states of the protein.     

Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Comparison of Different Methods for the Evaluation of Treatment Effects from the Sleep EEG of Patients with Major Depression

In healthy subjects, sleep has a typical structure of three to five cyclic transitions between different sleep states. In major depression, this regular pattern is often destroyed but can be reestablished during successful treatment. The differences between healthy and abnormal sleep are generally assessed in a time-consuming process, which consists of determining the nightly variations of the sleep states (the hypnogram) based on visual inspection of the electroencephalogram (EEG), electrooculogram, and electromyogram. In this study, three different methods of sleep EEG analysis (spectrum, outlier, and recurrence analysis) have been examined with regard to their ability to extract information about treatment effects in patients with major depression. Our data suggest that improved sleep patterns during treatment with antidepressant medication can be identified with an appropriate analysis of the EEG. By comparing different methods, we have found that many treatment effects identified by spectrum analysis can be reproduced by the much simpler technique of outlier analysis. Finally, the cyclic structure of sleep and its modification by antidepressant treatment is best illustrated by a non-linear approach, the so-called recurrence method.     

Identification of androgen-selective androgen-response elements in the human aquaporin-5 and Rad9 genes

The AR (androgen receptor) is known to influence the expression of its target genes by binding to different sets of AREs (androgen-response elements) in the DNA. One set consists of the classical steroid-response elements which are partial palindromic repeats of the 5'-TGTTCT-3' steroid-receptor monomer-binding element. The second set contains motifs that are AR-specific and that are proposed to be partial direct repeats of the same motif. On the basis of this assumption, we used an in silico approach to identify new androgen-selective AREs in the regulatory regions of known androgen-responsive genes. We have used an extension of the NUBIScan algorithm to screen a collection of 85 known human androgen-responsive genes compiled from literature and database searches. We report the evaluation of the most promising hits resulting from this computational search by in vitro DNA-binding assays using full-size ARs and GRs (glucocorticoid receptors) as well as their isolated DBDs (DNA-binding domains). We also describe the ability of some of these motifs to confer androgen-, but not glucocorticoid-, responsiveness to reporter-gene expression. The elements found in the aquaporin-5 and the Rad9 (radiation-sensitive 9) genes showed selective AR versus GR binding in band-shift assays and a strong activity and selectivity in functional assays, both as isolated elements and in their original contexts. Our data indicate the validity of the hypothesis that selective AREs are recognizable as direct 5'-TGTTCT-3' repeats, and extend the list of currently known selective elements.     

Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values

The application of unusual high pH-values within enzymatic cyanohydrin synthesis has been investigated. Usually enzymatic cyanohydrin synthesis in two-phase systems requires low pH-values within the aqueous phase to suppress the non-enzymatic side reaction. In contrast, we investigated the usage of pH-values above pH 6 by using the highly enantioselective (S)-selective hydroxynitrile lyase from Manihot esculenta. With these unusual reaction conditions also the unfavorable substrate 3-phenoxy-benzaldehyde can be converted by the wild type enzyme with excellent conversion and enantiomeric excess yielding pure (S)-3-phenoxy-benzaldehyde cyanohydrin with an enantiomeric excess of 97%. Although the variant MeHNL–W128A shows a higher activity with respect to this reaction, the enantioselectivity was reduced (85% e.e.(S)). Additionally, a new continuous spectroscopic cyanohydrin assay monitoring the formation of 3-phenoxy-benzaldehyde cyanohydrin was developed. Dedicated to Prof. Dr. Christian Wandrey on the occasion of his 65th birthday.