Volume 8, Issue 5

The use of molecular methods is gaining popularity throughout the field of microbial community ecology studies thanks to their flexibility of application, which ranges from community structure to function and trait determination. Nonetheless, there are environmental microbiologists, who are new in the field and are just starting to get to grips with the genetic tool box. It is for them that this practitioner’s mini-review was compiled. The methods available for microbial community structure analysis are discussed, after which, the reader is introduced to sequencing, as this tool is the most appropriate and has seen the greatest development in recent years. A focus on the practical aspects of the methodology is maintained throughout. The sample preparation procedure from extraction to sequencing is described. Different applications and considerations of sequencing are briefly explained, including clone library sequencing vs. amplicon library sequencing, shotgun-metagenomics vs. metatranscriptomics and the ‘double RNA approach’.

The polarity and magnitude of primary electric charges carried by basidiospores in the airborne state were investigated in living fungal fruiting bodies under natural forest conditions using a portable experimental device designed by the author. The operating principle was the falling of spores in the homogeneous horizontal electric field. The vertical and horizontal components of the trajectories of the spores were determined according to their deposition sites on electrodes (vertical metal plates). Altogether 33 samples of spores were examined for polarity, 10 of these samples (with 104–106 spores per sample) also were used to calculate the mean spore charge-to-mass quotient and the mean spore charge. The detection limits of spore charge-to-mass quotient varied in the range from (4.9±2.3)×10−5 to (1.36±0.33)×10−4 C kg−1. Basidiospores (subglobose, smooth, diameter of 4–6 μm) of the closely related (sibling) species Phellinus alni, P. nigricans, P. populicola and P. tremulae (Hymenochaetales, Basidiomycota) carried positive electrical charges that have mean values from 48 to 305 elementary charges. The intraspecies variation of the spore charge could depend on the natural variation in spore size.

Luciferases are prominent reporters in molecular and cellular biology investigations including miRNA target studies and the determination of Internal Ribosome Entry Site (IRES) activities in bicistronic assays. A majority of the current bicistronic vectors contain a firefly luciferase reporter as the second cistron. One reason for this is the presence of cryptic transcription start sites inside the luciferase gene. We present here an experimental evaluation of the cryptic transcription within the latest version of the firefly luciferase gene, luc2. Using flow cytometric analysis, we observed a negligible amount of cryptic transcriptional activity that was only slightly above the background of untransfected cells. Nevertheless, quantitative reverse transcription PCR experiments revealed a six-to-nine-fold gradual increase of transcription along the coding region of the gene. The level of cryptic transcription from the coding region of the improved luc2 firefly luciferase gene is significantly lower when compared to the luc+ gene. In summary, the luc2 better fulfills the requirements of bicistronic assays than the previous luc+ version. The observed low cryptic transcription activity in luc2 could be limiting only in cases where weak IRESs are studied.

The challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.

Lithuanian wolves form part of the larger Baltic population, the distribution of which is continuous across the region. In this paper, we evaluate the genetic diversity of the Lithuanian wolf population using mitochondrial DNA analysis and 29 autosomal microsatellite loci. Analysis of the mtDNA control region (647 bp) revealed 5 haplotypes distributed among 29 individuals and high haplotype diversity (0.658). Two haplotypes were distributed across the country, whilst the others were restricted to eastern Lithuania. Analysis of microsatellites revealed high heterozygosity (HE=0.709) and no evidence for a recent bottleneck. Using detection of first generation migrants, four individuals appeared to assign better with populations genetically differentiated from those resident in Lithuania. These immigrants were males carrying rare mitochondrial haplotypes and were encountered in the eastern part of the country, this indicates that Lithuania is subject to immigration from differentiated populations. Additionally, we did not detect any signs of recent hybridisation with dogs.

We monitored bats hibernating in the Tatra Mountains during winters between 1997 and 2012. The Tatras are Central Europe’s second-highest massif after the Alps. Our winter censuses identified 14 species of bats hibernating in caves of the Polish Tatras. The most characteristic features of these winter bat assemblages were the dominance of Myotis mystacinus and high numbers of Eptesicus nilssonii. During the monitoring period, we noted qualitative and quantitative changes in the hibernating bat fauna. Two thermophilous species not recorded earlier and absent during the entire Holocene appeared: Rhinolophus hipposideros and Myotis emarginatus. The abundance of M. mystacinus, M. daubentonii, E. nilssonii and Plecotus auritus increased. We found no such changes in the abundance of M. myotis or M. nattereri. The Tatra Mountains are a key region for the occurrence of bats of the mystacinus group (particularly M. mystacinus sensu stricto) in Europe, and for E. nilssonii an important region in Central Europe.

The aim of this work was to examine H+-ATPase hydrolytic activity and the stability of transmembrane electrochemical gradients in membrane vesicles isolated from seedlings and leaf cells of an invasive Washington lupine (Lupinus polyphyllus Lindl.) and compare them with non-invasive yellow lupine (Lupinus luteus L.). Temperatures of 25 and 30°C, keeping in mind possible climate warming, were used. For harder stress conditions, short term cold treatment (−8°C in vivo) was used. Plasmalemma-, tonoplast-, and endoplasmic reticulum-enriched membrane fractions were obtained from a sucrose density gradient and identified. Differences in ATPase hydrolytic activity were significant only between lupine species and were more obvious in plasmalemma-enriched fractions. Preincubation of seedlings and leaves at −8°C for 15 min to 24 h showed that microsomic fraction membranes of invasive lupine were more stable (according to Na+-diffusive potential) at low temperature compared to yellow lupine ones. The level of transmembrane electrochemical potential, mainly evoked by ATP-dependent active proton transport, was almost equal in both lupine species. Supposedly, the cells of invasive lupine are able to maintain transmembrane electrochemical potential by the employment of lower hydrolytic activity of H+-ATPase, thus saving energy for growth.

The induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes mediated system was established. The frequency of genetic transformation varied from 4.8 to 12% depended on method of infection. The cefotaxime at concentration of 200 mg/l proved to suppress effectively the growth of A. rhizogenes after co-cultivation. Among the three tested nutrient media: Murashige and Skoog (MS), Gamborg’s (B5) and Schenk and Hildebrandt (SH), MS medium was superior for growth and high biomass production of transformed roots compared to other culture media. After culturing for 40 days the fresh weight of clone T4 increased 7.6 fold over the non-transformed roots. The transfer of rol A, rol B and rol C genes into Arnica genome was confirmed by PCR analysis. Established genetic transformation techniques in A. montana efficiently provided and generated a large number of transformed roots — an excellent system for studying gene function and could be used for the production of secondary metabolites synthesized in roots.

Alfalfa (Medicago sativa; =M. sativa ssp. sativa) in Lithuania is sown as albuminous forage for cattle due to favourable climatic condition. Over many generations, alfalfa plants have escaped from cultivation fields into natural ecosystems and established wild populations. We collected and analyzed individuals from seventeen wild populations of M. sativa. Using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses, 117 RAPD and 64 ISSR reproducible and highly polymorphic (90.8% for RAPD and 86.3% for ISSR) loci were established. AMOVA showed a high genetic differentiation of M. sativa populations for both types of DNA markers utilized. According to RAPD markers, the genetic variability among populations was 63.1% and 57.0% when ISSR markers were used. Taken together, these results demonstrate that wild populations of M. sativa possess a high potential of genetic variability, that could potentially result in colonization of natural ecosystems. The UPGMA cluster analysis also showed that the DNA markers discovered in this study can distinguish between M. sativa and M. falcata (=M. sativa ssp. falcata) populations and therefore may be used to study the genetic impact of M. sativa on the native populations of M. falcata.

Intergeneric cross was made between the cultivated sunflower inbred line HA89 and an accession of wild Verbesina encelioides tolerant to drought and high temperature. The line was a BC2F5 progeny. The most remarkable feature of the plants was their compact architecture due to short petiole length and also, rather specific bright-yellow inflorescences. Similar plant architecture did not exist in either the wild or the cultivated parent. For sunflower, it is considered as a favourable and potentially useful adaptive trait. The line was multi-branched of medium type branching and possessed good agronomic characteristics. The overall characteristics of HA-VERBENC line make it a useful plant material for research on wide hybridization.

Hydroponically grown wheat seedlings of two prominent Bulgarian cultivars (Katya and Prelom) were subjected to 48 h osmotic stress with PEG 8000 and were then rehydrated. The degree of stress was evaluated by monitoring relative water content, lipid peroxidation level, and accumulation of free proline and hydrogen peroxide in the leaves. Anatomy and ultrastructure of leaf tissue were observed under light microscopy. After imposition of stress, drought tolerant cultivar Katya displayed higher free proline content and significantly lower malondialdehyde and peroxide concentration in leaves than in the leaves of susceptible cultivar Prelom. After 24 h of rehydration Katya showed better ability to restore leaf water status and an apparent tendency towards recovery, whereas Prelom sustained higher levels of hydrogen peroxide, lipid peroxidation products and free proline and markedly low relative water content. Here, we have uncovered some of the characteristics displayed by cultivar Katya that enable it to survive and recover from severe osmotic stress. Interestingly, there was congruence between our results and the high level of cultivar Katya drought tolerance observed in the field.

The composition of essential oil isolated from Thymus algeriensis growing wild in Libya was analyzed by GC and GC-MS. The essential oil was characterized with thymol (38.50%) as the major component. The oil was screened for antioxidant activity using DPPH assay, and compared to thymol and carvacrol. Antioxidant activity was high, with the IC50 of 0.299 mg/ml, compared to 0.403 and 0.105 mg/ml for thymol and carvacrol, and 0.0717 mg/ml for BHA. In addition, antimicrobial activity was tested against eight bacteria and eight fungi. T. algeriensis oil showed inhibitory activity against tested bacteria at 0.001–0.05 mg/ml, while bactericidal activity (MBC) was achieved at 0.0025–0.05 mg/ml. For antifungal activity MICs ranged 0.0005–0.025 mg/ml and MFC 0.001–0.05 mg/ml. High antimicrobial activity against the fungi in particular suggests that the essential oil of Thymus algeriensis could have a useful practical application.