Targeted access to the genomes of low-abundance organisms in complex microbial communities

Current metagenomic approaches to the study of complex microbial consortia provide a glimpse into the community metabolism and occasionally allow genomic assemblies for the most abundant organisms. However, little information is gained for the members of the community present at low frequencies, especially those representing yet-uncultured taxa, which include the bulk of the diversity present in most environments. Here we used phylogenetically directed cell separation by fluorescence in situ hybridization and flow cytometry, followed by amplification and sequencing of a fraction of the genomic DNA of several bacterial cells that belong to the TM7 phylum. Partial genomic assembly allowed, for the first time, a look into the evolution and potential metabolism of a soil representative from this group of organisms for which there are no species in stable laboratory cultures. Genomic reconstruction from targeted cells of uncultured organisms isolated directly from the environment represents a powerful approach to access any specific members of a community and an alternative way to assess the community's metabolic potential.     

Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.     

Spatial and temporal distribution of archaeal diversity in meromictic,hypersaline Ocnei Lake (Transylvanian Basin,Romania)

Saline, meromictic lakes with significant depth are usually formed as a result of salt mining activity. Ocnei Lake is one of the largest Transylvanian (Central Romania) neutral, hypersaline lake of man-made origin. We aimed to survey the seasonal dynamics of archaeal diversity in the water column of Ocnei Lake by employing microbiological methods as well as molecular techniques based on the sequence analysis of the 16S rRNA gene. We found that archaeal diversity in the water column increased with depth and salinity, with 8 OTUs being detected in the epilimnion compared to 21 found in the chemocline, and 32 OTUs in the monimolimnion. Down to 3.5 m depth, the archaeal community was markedly dominated by the presence of an unclassified archaeon sharing 93 % sequence identity to Halogeometricum spp. At the chemocline, the shift in archaeal community composition was associated with an increase in salinity, the main factor affecting the vertical distribution of archaeal assemblages. It appears that the microoxic and hypersaline monimolimnion is populated by several major haloarchaeal taxa, with minor fluctuations in their relative abundances throughout all seasons. The culturable diversity was reasonably correlated to the dominant OTUs obtained by molecular methods. Our results indicate that Ocnei Lake represents a relatively stable extreme habitat, accommodating a diverse and putatively novel archaeal community, as 30 % of OTUs could not be classified at the genus level.     

Human papillomavirus (HPV) 16 E6 variants in tonsillar cancer in comparison to those in cervical cancer in Stockholm, Sweden

Background

Human papillomavirus (HPV), especially HPV16, is associated with the development of both cervical and tonsillar cancer and intratype variants in the amino acid sequence of the HPV16 E6 oncoprotein have been demonstrated to be associated with viral persistence and cancer lesions. For this reason the presence of HPV16 E6 variants in tonsillar squamous cell carcinoma (TSCC) in cervical cancer (CC), as well as in cervical samples (CS), were explored.

Methods

HPV16 E6 was sequenced in 108 TSCC and 52 CC samples from patients diagnosed 2000–2008 in the County of Stockholm, and in 51 CS from young women attending a youth health center in Stockholm.

Results

The rare E6 variant R10G was relatively frequent (19%) in TSCC, absent in CC and infrequent (4%) in CS, while the well-known L83V variant was common in TSCC (40%), CC (31%), and CS (29%). The difference for R10G was significant between TSCC and CC (p = 0.0003), as well as between TSCC and CS (p = 0.009). The HPV16 European phylogenetic lineage and its derivatives dominated in all samples (>90%).

Conclusion

The relatively high frequency of the R10G variant in TSCC, as compared to what has been found in CC both in the present study as well as in several other studies in different countries, may indicate a difference between TSCC and CC with regard to tumor induction and development. Alternatively, there could be differences with regard to the oral and cervical prevalence of this variant that need to be explored further.     

The consequences of gustatory deafferentation on body mass and feeding patterns in the rat

The contribution of orosensory signals, especially taste, on body mass, and feeding and drinking patterns in the rat was examined. Gustatory deafferentation was produced by bilateral transection of the chorda tympani, glossopharyngeal, and greater superficial petrosal nerves. Total calories consumed from sweetened-milk diet and oil-chow mash by the nerve-transected rats significantly decreased relative to sham-operated controls, mostly attributable to decreases in bout number, but not size. Nevertheless, caloric intake steadily increased over the postsurgical observation period, but body mass remained below both presurgical baseline and control levels and did not significantly increase over this time. After the sweetened-milk diet/oil-chow mash phase, rats received a series of sucrose preference tests. Interestingly, the nerve-transected rats preferred sucrose, and intake did not differ from controls, likely due to the stimulus sharing some nontaste chemosensory properties with the sweetened-milk diet. The neurotomized rats initiated a greater number of sucrose-licking bouts that were smaller in size and slower in licking rate, compared with control rats, and, unlike in control rats, the latter two bout parameters did not vary across concentration. Thus, in the absence of gustatory neural input, body mass is more stable compared with the progressive trajectory of weight gain seen in intact rats, and caloric intake initially decreases but recovers. The consequences of gustatory neurotomy on processes that determine meal initiation (bout number) and meal termination (bout size) are not fixed and appear to be influenced by presurgical experience with food stimuli coupled with its nongustatory chemosensory properties.     

Antifungal Activities of Chelidonium majus Extract on Botrytis cinerea in vitro and Ultrastructural Changes in its Conidia

The extract from the aboveground parts of Chelidonium majus reduced the mycelial growth of Botrytis cinerea on Czapek agar medium. In minimum fungicidal concentration (60  μ l/ml), the extract induced irreversible ultrastructural changes in B. cinerea conidia.     

The quantitative characterization of free radical sources and traps by electromigration applications

Nowadays, very diverse human activities generate urgent demands for fast, sensitive reliable innovative tools capable of detecting major industrial, military, and other dangerous products. An important part of these compounds are free radicals. Capillary electrophoresis (CE), especially in its miniaturized format (lab-on-a-chip), and other electromigration methods offer special possibilities to resolve this problem. These measurements have a great opportuness because of very wide chemical and biological role of free radicals. Several compounds, e.g. monomers and some biologically important groups (as are nitrones) oppose oxidative challenges by virtue of their trap very rapidly oxygen- or carbon-centered radicals and generating other radical species which are stable and biochemically less harmful than the original ones. In many cases, conventionally, the relative trap capacity is measured against tert.-butylhydroperoxide (TBH). In this lecture are presented numerous important free radical species (active oxygen–, nitrogen- and carbon-centered ones, as HO, NO etc) and their adequate in vitro and in vivo applied bioanalytical methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence analysis. A simple and highly sensitive method is the capillary zone electrophoresis with amperometric detection (CZE-AD); It was introduced to determine indirectly OH by analysing its reaction products with salicylic and dihydroxybenzoic acids. Hydroxylated radical products of these acids are often used as a relative measurement in free radical research. Accurate determination of pK(a) values is important for proper characterization of newly synthesized molecules. CZE method was used for determination of their values. Are initiated new research fields as Fenton-, electro-Fenton and photoelectro-Fenton chemistry and foreseen their perspectives.

Nitric oxide is an important cell signaling molecule in physiology and pathophysiology. An indirect method for monitoring nitric oxide (NO) by determining nitrate and nitrite by microchip capillary electrophoresis (CE) with electrochemical (EC) detection has been developed. The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography). Were observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range. These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic media. Polarography is an adequate method also to quantitative kinetic study of the free radical activity and of the trapping capacity of different compounds. This method is based on measure of the catalytic polarografic current of Fe(III) in the presence of free radical sources (TBH, hydrogen-peroxydes), and their traps. Personal contribution of the authors in this field is discussed.     

Daily activity patterns of coyotes (Canis latrans) in a tropical deciduous forest of western Mexico

We studied the activity patterns of the coyote (Canis latrans) in a tropical deciduous forest in the Mexican Pacific coast over 3 years. Fifteen coyotes (six females, nine males) were fitted with radio-collars equipped with activity sensors to determine the influence of seasonality (dry vs. wet), gender (males vs. females) and diel intervals (dusk, night, dawn, and day) on activity patterns. We found differences in activity patterns between diel intervals, but the only pair of diel intervals that showed significant differences was dawn (more active) vs. day (less active). We found no differences due to sex or season on any of the four studied diel intervals. Coyote activity patterns in this tropical forest could be responding to prey availability, human avoidance or thermoregulation.