Leszek Lipinski

High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells

Mammalian genes are highly heterogeneous with respect to their nucleotide composition, but the functional consequences of this heterogeneity are not clear. In the previous studies, weak positive or negative correlations have been found between the silent-site guanine and cytosine (GC) content and expression of mammalian genes. However, previous studies disregarded differences in the genomic context of genes, which could potentially obscure any correlation between GC content and expression. In the present work, we directly compared the expression of GC-rich and GC-poor genes placed in the context of identical promoters and UTR sequences. We performed transient and stable transfections of mammalian cells with GC-rich and GC-poor versions of Hsp70, green fluorescent protein, and IL2 genes. The GC-rich genes were expressed several-fold to over a 100-fold more efficiently than their GC-poor counterparts. This effect was not due to different translation rates of GC-rich and GC-poor mRNA. On the contrary, the efficient expression of GC-rich genes resulted from their increased steady-state mRNA levels. mRNA degradation rates were not correlated with GC content, suggesting that efficient transcription or mRNA processing is responsible for the high expression of GC-rich genes. We conclude that silent-site GC content correlates with gene expression efficiency in mammalian cells.

Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice

Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.

Isotopic and other influences on the realization of the triple point of hydrogen

Within an international collaboration of the eight metrological institutes represented by the authors, the dependence of the triple-point temperature of equilibrium hydrogen on the deuterium content at low concentrations has been precisely determined so that the uncertainty in realizing the triple point as a temperature fixed point might be reduced by nearly one order of magnitude. To investigate the thermodynamic properties of the hydrogen–deuterium mixtures and to elucidate the factors that influence the melting temperature, 28 sealed fixed-point cells have been filled and measured, and some of these have been compared with an open-cell system. Hydrogen gas with a deuterium content ranging from 27.2 µmol D/mol H to 154.9 µmol D/mol H was studied using cells containing five different types of spin-conversion catalyst, with different catalyst-to-liquid volume ratios (a few per cent to more than 100%) and of different designs. The latter consideration is especially influential in determining the thermal behaviour of the cells and, thus, the temperature-measurement errors. The cells were measured at the eight participating institutes in accordance with a detailed protocol that facilitates a direct comparison of the results. Through analysis of the measurements, significant inter-institute deviations due to different measurement facilities and methods have been ruled out with respect to the determination of both the melting temperatures and the thermal parameters of the cells. The uncertainty estimates for the determination of the deuterium content have been verified by including isotopic analysis results from four different sources. The slope of the dependence of the triple-point temperature of equilibrium hydrogen isotopic mixtures on the deuterium content has been deduced from the melting temperatures of those sample portions not in direct contact with the catalysts. Evaluation of the data using different mathematical methods has yielded an average value of 5.42 µK per µmol D/mol H, with an upper bound of the standard uncertainty of 0.31 µK per µmol D/mol H. This is close to the literature value of 5.6 µK per µmol D/mol H that was obtained at higher deuterium concentrations. (Some figures in this article are in colour only in the electronic version)

Characterization of testis-specific serine–threonine kinase 3 and its activation by phosphoinositide-dependent kinase-1-dependent signalling

The family of testis‐specific serine–threonine kinases (TSSKs) consists of four members whose expression is confined almost exclusively to testis. Very little is known about their physiological role and mechanisms of action. We cloned human and mouse TSSK3 and analysed the biochemical properties, substrate specificity and in vitro activation. In vitro TSSK3 exhibited the ability to autophosphorylate and to phosphorylate test substrates such as histones, myelin basic protein and casein. Interestingly, TSSK3 showed maximal in vitro kinase activity at 30 °C, in keeping with it being testis specific. Sequence comparison indicated the existence of a so‐called ‘T‐loop’ within the TSSK3 catalytic domain, a structure present in the AGC family of protein kinases. To test if this T‐loop is engaged in TSSK3 regulation, we mutated the critical threonine residue within the T‐loop to alanine (T168A) which resulted in inactivation of TSSK3 kinase. Furthermore, Thr168 is phosphorylated in vitro by the T‐loop kinase phosphoinositide‐dependent protein kinase‐1 (PDK1). PDK1‐induced phosphorylation increased in vitro TSSK3 kinase activity, suggesting that TSSK3 can be regulated in the same way as AGC kinase family members. Analysis of peptide sequences identifies the peptide sequence RRSSSY containing Ser5 that is a target for TSSK3 phosphorylation, as an efficient and specific substrate for TSSK3.

Linear diode thermometer in the 4–300 K temperature range

Abstract The thermometric properties of Zener diodes in the temperature range 4.2–300 K are presented in this paper. It is expected that a change of doping concentration in Zener diodes of a few orders of magnitude, which influences their characteristics in the reverse direction, can also influence the characteristics in the forward direction. Zener diodes of type BZAP 83 manufactured in a variety of ways with different values of Zener voltage from 3.3 to 33 V have been tested. Their forward voltage versus temperature characteristics are monotonie functions, but for thermometric purposes the BZAP 83 diodes having a Zener voltage of 7.5 V are noteworthy because of their linear V = f ( T ) characteristic at I = constant over the whole temperature range from 4.2 to 300 K.

Physiological and Metagenomic Analyses of Microbial Mats Involved in Self-Purification of Mine Waters Contaminated with Heavy Metals.

Two microbial mats found inside two old (gold and uranium) mines in Zloty Stok and Kowary located in SW Poland seem to form a natural barrier that traps heavy metals leaking from dewatering systems. We performed complex physiological and metagenomic analyses to determine which microorganisms are the main driving agents responsible for self-purification of the mine waters and identify metabolic processes responsible for the observed features. SEM and energy dispersive X-ray microanalysis showed accumulation of heavy metals on the mat surface, whereas, sorption experiments showed that neither microbial mats were completely saturated with heavy metals present in the mine waters, indicating that they have a large potential to absorb significant quantities of metal. The metagenomic analysis revealed that Methylococcaceae and Methylophilaceae families were the most abundant in both communities, moreover, it strongly suggest that backbones of both mats were formed by filamentous bacteria, such as Leptothrix, Thiothrix, and Beggiatoa. The Kowary bacterial community was enriched with the Helicobacteraceae family, whereas the Zloty Stok community consist mainly of Sphingomonadaceae, Rhodobacteraceae, and Caulobacteraceae families. Functional (culture-based) and metagenome (sequence-based) analyses showed that bacteria involved in immobilization of heavy metals, rather than those engaged in mobilization, were the main driving force within the analyzed communities. In turn, a comparison of functional genes revealed that the biofilm formation and heavy metal resistance (HMR) functions are more desirable in microorganisms engaged in water purification than the ability to utilize heavy metals in the respiratory process (oxidation-reduction). These findings provide insight on the activity of bacteria leading, from biofilm formation to self-purification, of mine waters contaminated with heavy metals.

Novel molecular markers for the detection of methanogens and phylogenetic analyses of methanogenic communities.

Methanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures

Abstract Plasmids are mobile genetics elements that play an important role in the environmental adaptation of microorganisms. Although plasmids are usually analyzed in cultured microorganisms, there is a need for methods that allow for the analysis of pools of plasmids (plasmidomes) in environmental samples. To that end, several molecular biology and bioinformatics methods have been developed; however, they are limited to environments with low diversity and cannot recover large plasmids. Here, we present PlasFlow, a novel tool based on genomic signatures that employs a neural network approach for identification of bacterial plasmid sequences in environmental samples. PlasFlow can recover plasmid sequences from assembled metagenomes without any prior knowledge of the taxonomical or functional composition of samples with an accuracy up to 96%. It can also recover sequences of both circular and linear plasmids and can perform initial taxonomical classification of sequences. Compared to other currently available tools, PlasFlow demonstrated significantly better performance on test datasets. Analysis of two samples from heavy metal-contaminated microbial mats revealed that plasmids may constitute an important fraction of their metagenomes and carry genes involved in heavy-metal homeostasis, proving the pivotal role of plasmids in microorganism adaptation to environmental conditions.

The effect of the source of microorganisms on adaptation of hydrolytic consortia dedicated to anaerobic digestion of maize silage

The main aim of this study was to evaluate the effect of the source of microorganisms on the selection of hydrolytic consortia dedicated to anaerobic digestion of maize silage. The selection process was investigated based on the analysis of changes in the hydrolytic activity and the diversity of microbial communities derived from (i) a hydrolyzer of a commercial agricultural biogas plant, (ii) cattle slurry and (iii) raw sewage sludge, during a series of 10 passages. Following the selection process, the adapted consortia were thoroughly analyzed for their ability to utilize maize silage and augmentation of anaerobic digestion communities. The results of selection of the consortia showed that every subsequent passage of each consortium leads to their adaptation to degradation of maize silage, which was manifested by the increased hydrolytic activity of the adapted consortia. Biodiversity analysis (based on the 16S rDNA amplicon sequencing) confirmed the changes microbial community of each consortium, and showed that after the last (10th) passage all microbial communities were dominated by the representatives of Lactobacillaceae, Prevotellaceae, Veillonellaceae. The results of the functional analyses showed that the adapted consortia improved the efficiency of maize silage degradation, as indicated by the increase in the concentration of glucose and volatile fatty acids (VFAs), as well as the soluble chemical oxygen demand (sCOD). Moreover, bioaugmentation of anaerobic digestion communities by the adapted hydrolytic consortia increased biogas yield by 10-29%, depending on the origin of the community. The obtained results also indicate that substrate input (not community origin) was the driving force responsible for the changes in the community structure of hydrolytic consortia dedicated to anaerobic digestion.

The triple point of nitrogen

We present accurate measurements of the triple point of nitrogen. The quality of this triple point is once more confirmed, and its temperature value is found to be T90 = 63.152 6 K (expanded uncertainty U = 0.30 mK, reproducibility better than 0.1 mK). This is the first direct determination of this point on ITS-90 since the introduction of the scale. This point is useful as a non-uniqueness checkpoint.