Replication methods and tools in high-throughput cultivation processes - recognizing potential variations of growth and product formation by on-line monitoring

Background  

High-throughput cultivations in microtiter plates are the method of choice to express proteins from recombinant clone libraries. Such processes typically include several steps, whereby some of them are linked by replication steps: transformation, plating, colony picking, preculture, main culture and induction. In this study, the effects of conventional replication methods and replication tools (8-channel pipette, 96-pin replicators: steel replicator with fixed or spring-loaded pins, plastic replicator with fixed pins) on growth kinetics of Escherichia coli SCS1 pQE-30 pSE111 were observed. Growth was monitored with the BioLector, an on-line monitoring technique for microtiter plates. Furthermore, the influence of these effects on product formation of Escherichia coli pRhotHi-2-EcFbFP was investigated. Finally, a high-throughput cultivation process was simulated with Corynebacterium glutamicum pEKEx2-phoD-GFP, beginning at the colony picking step.     

An Interactive SAR Approach to Discover Novel Hybrid Thieno Probes as Ligands for D2‐Like Receptors with Affinities in the Subnanomolar Range

A series of [(phenylpiperazinyl)alkyl]‐isoindole‐1,3‐dione derivatives was synthesized to serve as probes for dopaminergic receptors. Among this series, compound 6a showed the highest affinity towards D4 and D3 receptors with K_i values in the low nanomolar range, and D2/D4‐ and D2/D3‐selectivity indices of 72 and 20, respectively. Optimization rounds were adopted and led to the D4‐selective ligand thiophene‐2‐carboxamide 9a with a K_i(D4) value of 0.62 nM , and to its butyl analog, 10a , with K_i(D4) and K_i(D3) values of 0.03 and 0.26 nM , respectively. Docking experiments revealed the importance of the unique D4 residue Arg186 in manipulating the ligands' D4‐subtype‐receptor selectivity.     

ipso-Hydroxylation and Subsequent Fragmentation: a Novel Microbial Strategy To Eliminate Sulfonamide Antibiotics

Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso-hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino-N-phenylbenzenesulfonamide, and N-(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso-hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para-position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us.     

Unraveling epistasis with triple testcross progenies of near-isogenic lines

Libraries of near-isogenic lines (NILs) are a powerful plant genetic resource to map quantitative trait loci (QTL). Nevertheless, QTL mapping with NILs is mostly restricted to genetic main effects. Here we propose a two-step procedure to map additive-by-additive digenic epistasis with NILs. In the first step, a generation means analysis of parents, their F₁ hybrid, and one-segment NILs and their triple testcross (TTC) progenies is used to identify in a one-dimensional scan loci exhibiting QTL-by-background interactions. In a second step, one-segment NILs with significant additive-by-additive background interactions are used to produce particular two-segment NILs to test for digenic epistatic interactions between these segments. We evaluated our approach by analyzing a random subset of a genomewide Arabidopsis thaliana NIL library for growth-related traits. The results of our experimental study illustrated the potential of the presented two-step procedure to map additive-by-additive digenic epistasis with NILs. Furthermore, our findings suggested that additive main effects as well as additive-by-additive digenic epistasis strongly influence the genetic architecture underlying growth-related traits of A. thaliana.     

Analysis of residuals from enzyme kinetic and protein folding experiments in the presence of correlated experimental noise

Experimental data from continuous enzyme assays or protein folding experiments often contain hundreds, or even thousands, of densely spaced data points. When the sampling interval is extremely short, the experimental data points might not be statistically independent. The resulting neighborhood correlation invalidates important theoretical assumptions of nonlinear regression analysis. As a consequence, certain goodness-of-fit criteria, such as the runs-of-signs test and the autocorrelation function, might indicate a systematic lack of fit even if the experiment does agree very well with the underlying theoretical model. A solution to this problem is to analyze only a subset of the residuals of fit, such that any excessive neighborhood correlation is eliminated. Substrate kinetics of the HIV protease and the unfolding kinetics of UMP/CMP kinase, a globular protein from Dictyostelium discoideum, serve as two illustrative examples. A suitable data-reduction algorithm has been incorporated into software DYNAFIT [P. Kuzmi?, Anal. Biochem. 237 (1996) 260-273], freely available to all academic researchers from http://www.biokin.com.     

Cross-talk of membrane lipids and Alzheimer-related proteins

Alzheimer’s disease (AD) is neuropathologically characterized by the combined occurrence of extracellular β-amyloid plaques and intracellular neurofibrillary tangles in the brain. While plaques contain aggregated forms of the amyloid β-peptide (Aβ), tangles are formed by fibrillar forms of the microtubule associated protein tau. All mutations identified so far to cause familial forms of early onset AD (FAD) are localized close to or within the Aβ domain of the amyloid precursor protein (APP) or in the presenilin proteins that are essential components of a protease complex involved in the generation of Aβ. Mutations in the tau gene are not associated with FAD, but can cause other forms of dementia. The genetics of FAD together with biochemical and cell biological data, led to the formulation of the amyloid hypothesis, stating that accumulation and aggregation of Aβ is the primary event in the pathogenesis of AD, while tau might mediate its toxicity and neurodegeneration.The generation of Aβ involves sequential proteolytic cleavages of the amyloid precursor protein (APP) by enzymes called β-and γ-secretases. Notably, APP itself as well as the secretases are integral membrane proteins. Thus, it is very likely that membrane lipids are involved in the regulation of subcellular transport, activity, and metabolism of AD related proteins.Indeed, several studies indicate that membrane lipids, including cholesterol and sphingolipids (SLs) affect Aβ generation and aggregation. Interestingly, APP and other AD associated proteins, including β-and γ-secretases can, in turn, influence lipid metabolic pathways. Here, we review the close connection of cellular lipid metabolism and AD associated proteins and discuss potential mechanisms that could contribute to initiation and progression of AD.     

Mapping the membrane proteome of Corynebacterium glutamicum

In order to avoid the specific problems with intrinsic membrane proteins in proteome analysis, a new procedure was developed which is superior to the classical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method in terms of intrinsic membrane proteins. For analysis of the membrane proteome from Corynebacterium glutamicum, we replaced the first separation dimension, i.e., the isoelectric focusing step, by anion-exchange chromatography, followed by sodium dodecyl sulfate (SDS)-PAGE in the second separation dimension. Enrichment of the membrane intrinsic subproteome was achieved by washing with 2.5 M NaBr which removed more than 35% of the membrane-associated soluble proteins. For the extraction and solubilization of membrane proteins, the detergent amidosulfobetaine 14 (ASB-14) was most efficient in a detailed screening procedure and proved also suitable for chromatography. 356 gel bands were spotted, and out of 170 different identified proteins, 50 were membrane-integral. Membrane proteins with one up to 13 transmembrane helices were found. Careful analysis revealed that this new procedure covers proteins from a wide pI range (3.7-10.6) and a wide mass range of 10-120 kDa. About 50% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, protein translocation, and proteolysis while for the others a function is not yet known, indicating the potential of the developed method for elucidation of membrane proteomes in general.     

Articular Joint Lubricants during Osteoarthritis and Rheumatoid Arthritis Display Altered Levels and Molecular Species

Background

Hyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA).

Methods

We used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis.

Results

Compared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs.

Conclusions

The levels, composition, and MW distribution of all currently known lubricants in SF—HA, lubricin, PLs—vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction.     

Species richness and trait composition of butterfly assemblages change along an altitudinal gradient

Species richness patterns along altitudinal gradients are well-documented ecological phenomena, yet very little data are available on how environmental filtering processes influence the composition and traits of butterfly assemblages at high altitudes. We have studied the diversity patterns of butterfly species at 34 sites along an altitudinal gradient ranging from 600 to 2,000 m a.s.l. in the National Park Berchtesgaden (Germany) and analysed traits of butterfly assemblages associated with dispersal capacity, reproductive strategies and developmental time from lowlands to highlands, including phylogenetic analyses. We found a linear decline in butterfly species richness along the altitudinal gradient, but the phylogenetic relatedness of the butterfly assemblages did not increase with altitude. Compared to butterfly assemblages at lower altitudes, those at higher altitudes were composed of species with larger wings (on average 9 %) which laid an average of 68 % more eggs. In contrast, egg maturation time in butterfly assemblages decreased by about 22 % along the altitudinal gradient. Further, butterfly assemblages at higher altitudes were increasingly dominated by less widespread species. Based on our abundance data, but not on data in the literature, population density increased with altitude, suggesting a reversed density–distribution relationship, with higher population densities of habitat specialists in harsh environments. In conclusion, our data provide evidence for significant shifts in the composition of butterfly assemblages and for the dominance of different traits along the altitudinal gradient. In our study, these changes were mainly driven by environmental factors, whereas phylogenetic filtering played a minor role along the studied altitudinal range.