Expression of anti-inflammatory markers IL-2, IL-10, TGF-ß1, ßDEF-2, ßDEF-3 and Cathelicidin LL37 in dairy cattle milk with different health status of the udder.

Great economic losses to the dairy industry are associated with bovine mastitis, which results in poor milk quality and high treatment costs. Anti-inflammatory proteins play an important role in the suppression of the immune response against invading pathogenic microorganisms and are therefore being studied for possible use in the early diagnosis of mastitis. In our study, we used milk samples from 15 cows of Holstein Friesian breed with different health status (5 healthy, 5 subclinical, and 5 clinical animals), and tested them using immunohistochemical (IHC) analysis to evaluate the presence of IL-2, IL-10, TGF-ß1, ßDEF-2, DEF-3, and Cathelicidin LL37 proteins. The calculation of positively and negatively stained cells for each biomarker was performed using the semiquantitative counting method. We found the presence of all factors with the exception of Cathelicidin LL37, which was almost absent in milk samples of all animal groups. The significant decrease of IL-10, ß-def2, and ß-def3 expression levels within the 3 days of sampling, found in the milk of animals with sub- and clinical mastitis, indicates the loss of antiinflammatory protection of the affected cow's udder. In contrast, the stable increase of IL-2 and TGF-ß1 positive cells observed in the milk of mastitis-affected cows, and the similar expression of these factors in the milk of healthy animals, indicate the possible lack of involvement of these cytokines at an early stage of udder inflammation.

Genome-level insights into the operation of an on-site biological wastewater treatment unit reveal the importance of storage time.

On-site wastewater treatment systems are gaining popularity in areas where centralized wastewater treatment is not available. In the current case study a domestic activated sludge system was investigated, where treated effluent was stored in a short-term (1 week turn-over time) and a long-term (over 2-3 months) storage tank and was then used for irrigation. This design provided a unique opportunity to assess the chemical and microbial changes of the effluent upon storage. Long-term storage greatly improved both the chemical quality and the degradation efficiency of most organic micropollutants examined, including petroleum hydrocarbons and the pesticide diethyltoluamide. Taxonomic profile of the core microbiome of the effluent was also influenced upon storage. Relative abundance values of the members of Azoarcus and Thauera genera, which are important in degrading polycyclic aromatic hydrocarbons compounds, clearly indicated the biodegrading activity of these microbes across samples. The abundance of xenobiotics degradation functions correlated with the observed organic micropollutant degradation values indicating efficient microbial decomposition of these contaminants. Functions related to infectious diseases also had the highest abundance in the short-term storage tank corresponding well with the relative abundance of indicator organisms and implying to the significance of storage time in the elimination of pathogens. Based on these results, small, on-site wastewater treatment systems could benefit from long-term storage of wastewater effluent.

Effect of light on growth and endogenous hormones in Chlorella minutissima (Trebouxiophyceae).

Plant growth regulators (PGRs) play an important role in mediating growth and stress responses in plants. Light influences PGRs concentrations in vascular plants. The effect of light on growth and endogenous PGR concentrations in microalgae was investigated in the present study. Chlorella minutissima MACC 360 was grown in 14:10 h light:dark (L:D), continuous dark (CD) and continuous dark with the addition of 5 g L(-1) glucose (CD + G) for 48 h. Cultures were synchronized in the L:D cultures, increasing in size during the light period and dividing during the dark period. C. minutissima cells did not increase in size or undergo cell division in CD cultures. In CD + G conditions, the cultures were no longer synchronized but did continue to increase in cell size and constantly underwent cell division although fewer cells divided than in the L:D cultures. Endogenous auxin and cytokinin concentrations increased and gibberellin concentrations decreased over time in the actively growing cultures (L:D and CD + G) but did not increase in the CD cultures. The largest increase in indole content was in the CD + G cultures while the L:D cultures had the largest cytokinin increase. Brassinosteroid concentrations decreased over time in all the cultures including those grown in CD conditions. Abscisic acid (ABA) concentrations were low and only increased in the CD cultures. These results show that endogenous PGRs were affected by the light regime and/or culture growth.

Environment-responsive fluorescent nucleoside analogue probe for studying oligonucleotide dynamics in a model cell-like compartment.

The majority of fluorescent nucleoside analogue probes that have been used in the in vitro study of nucleic acids are not suitable for cell-based biophysical assays because they exhibit excitation maxima in the UV region and low quantum yields within oligonucleotides. Therefore, we propose that the photophysical characterization of oligonucleotides labeled with a fluorescent nucleoside analogue in reverse micelles (RM), which are good biological membrane models and UV-transparent, could provide an alternative approach to studying the properties of nucleic acids in a cell-like confined environment. In this context, we describe the photophysical properties of an environment-sensitive fluorescent uridine analogue (1), based on the 5-(benzo[b]thiophen-2-yl)pyrimidine core, in micelles and RM. The emissive nucleoside, which is polarity- and viscosity-sensitive, reports the environment of the surfactant assemblies via changes in its fluorescence properties. The nucleoside analogue, incorporated into an RNA oligonucleotide and hybridized to its complementary DNA and RNA oligonucleotides, exhibits a significantly higher fluorescence intensity, lifetime, and anisotropy in RM than in aqueous buffer, which is consistent with the environment of RM. Collectively, our results demonstrate that nucleoside 1 could be utilized as a fluorescent label to study the function of nucleic acids in a model cellular milieu.

Heavy atom containing fluorescent ribonucleoside analog probe for the fluorescence detection of RNA-ligand binding.

Although numerous biophysical tools have provided effective systems to study nucleic acids, our current knowledge on how RNA structure complements its function is limited. Therefore, development of robust tools to study the structure­function relationship of RNA is highly desired. Toward this endeavor, we have developed a new ribonucleoside analog, based on a (selenophen-2-yl)pyrimidine core, which could serve as a fluorescence probe to study the function of RNA in real time and as an anomalous scattering label (selenium atom) for the phase determination in X-ray crystallography. The fluorescent selenophene-modified uridine analog is minimally perturbing and exhibits probe-like properties such as sensitivity to microenvironment and conformation changes. Utilizing these properties and amicability of the corresponding ribonucleotide analog to enzymatic incorporation, we have synthesized a fluorescent bacterial ribosomal decoding site (A-site) RNA construct and have developed a fluorescence binding assay to effectively monitor the binding of aminoglycoside antibiotics to the A-site. Our results demonstrate that this simple approach of building a dual probe could provide new avenues to study the structure­function relationship of not only nucleic acids, but also other biomacromolecules.

Synthesis, photophysical characterization, and enzymatic incorporation of a microenvironment-sensitive fluorescent uridine analog.

The synthesis of a microenvironment-sensitive base-modified fluorescent ribonucleoside analog based on a 5-(benzo[b]thiophen-2-yl)pyrimidine core, enzymatic incorporation of its corresponding triphosphate into RNA oligonucleotides, and photophysical characterization of fluorescently modified oligoribonucleotides are described.

Anaerobic regulation of hydrogenase transcription in different bacteria.

Hydrogen metabolism is closely related to other important metabolic and energetic processes of bacterial cells, such as photosynthesis, anaerobic respiration and sulphur metabolism. Even small environmental changes influence these networks through different regulatory systems. The presence or absence of oxygen is one of the most important signals; how the cascades evolved to transmit this signal in different bacteria is summarized. In many instances, hydrogen is released only under anoxic conditions, because of bioenergetic considerations. Most [NiFe] hydrogenases are inactivated by oxygen, but many of them can be re-activated under reducing conditions. In addition to direct inactivation of the hydrogenases, oxygen can also regulate their expression. The global regulatory systems [FNR (fumarate and nitrate reduction regulator), ArcAB (aerobic respiratory control) and RegAB], which respond to alterations in oxygen content and redox conditions of the environment, have an important role in hydrogenase regulation of several bacteria. FNR-like proteins were shown to be important for the regulation of hydrogenases in Escherichia coli, Thiocapsa roseopersicina and Rhizobium leguminosarum, whereas RegA protein modulates the expression of hupSL genes in Rhodobacter capsulatus.

The hydrogenases of Thiocapsa roseopersicina.

The purple sulphur phototrophic bacterium, Thiocapsa roseopersicina BBS, contains several NiFe hydrogenases. One of these enzymes (HynSL) is membrane associated, remarkably stable and can be used for practical applications. HupSL is also located in the photosynthetic membrane, its properties are similar to other known Hup-type NiFe hydrogenases. A third hydrogenase activity was located in the soluble fraction and was analogous to the NAD-reducing hydrogenases of cyanobacteria. The hoxEFUYH genes are transcribed together. HoxE is needed for the in vivo electron flow to and from the soluble hydrogenase. Some of the accessory genes were identified using random mutagenesis, and sequencing of the T. roseopersicina genome is in progress. The HupD, HynD and HoxW gene products corresponded to the proteases processing the C-termini of the three NiFe hydrogenases respectively. HypF and HupK mutants displayed significant in vivo H(2) evolution, which could be linked to the nitrogenase activity for the DeltahypF and to the bidirectional Hox activity in the DeltahupK strain. Both HypC proteins are needed for the biosynthesis of each NiFe hydrogenase. The hydrogenase expression is regulated at the transcriptional level through distinct mechanisms. The expression of hynSL is up-regulated under anaerobic conditions with the participation of an FNR (fumarate and nitrate reduction regulator)-type protein, FnrT. Although the genes encoding a typical H(2) sensor (hupUV) and a two-component regulator (hupR and hupT) are present in T. roseopersicina, the system is cryptic in the wild-type BBS strain. The hupR gene was identified in the gene cluster downstream from hupSL. Introduction of actively expressed hupT repressed the hupSL gene expression as expected by analogy with other bacteria.