castellanii cells were performed four hours after infection with fluorescein-labelled T. equigenitalis (Figure 2A) or T. asinigenitalis check details (Figure 2B). For both taylorellae, we observed exclusively intracellular bacteria, mainly grouped in clusters. No bacterium was observed attached to the cell surface of the amoeba. Our data show that the persistent amoeba-associated taylorellae are located within the cytoplasm of A. castellanii. Figure 2 Location of T. equigenitalis and T. asinigenitalis within A. castellanii . Confocal laser scanning micrographs of A. castellanii

cells at 4 h post-infection with fluorescein-labelled T. equigenitalis (A) or T. asinigenitalis (B). Similar results were observed in two independent

experiments. Actin polymerisation and phosphoinositide 3-kinase play a key role in taylorellae internalisation To investigate the uptake mechanism involved in taylorellae internalisation, two chemical inhibitors were used: Cytochalasin D (CytoD), a potent inhibitor of actin polymerisation, and Wortmannin (Wort), an inhibitor of phosphoinositide 3-kinases (PI3K). Bacterial uptake in amoebae was measured by trypan blue quenching of fluorescein-labelled T. equigenitalis, T. asinigenitalis, E. coli or L. pneumophila. Fluorescein-labelled Navitoclax in vitro bacteria were used to infect A. castellanii when CytoD or Wort were present, as indicated. After contact, trypan blue was added to quench the fluorescence of non-internalised bacteria and the fluorescence, which was representative of bacterial internalisation by amoebae, was measured (Figure 3). For the four selleck products tested bacterial species, amoebae exposed to CytoD and Wort show a decrease in fluorescence compared to untreated amoebae. The decrease in fluorescence was comparable for all four bacterial species and for both phagocytosis inhibitors. These results suggest that taylorellae are internalised by an uptake mechanism such as phagocytosis, which is dependent upon actin polymerisation and PI3K. Figure 3 Taylorellae are actively phagocytised by A. castellanii . Bacterial uptake assay by trypan blue

quenching. Acanthamoeba castellanii cells were infected with fluorescein-labelled E. coli, T. equigenitalis, T. asinigenitalis or L. pneumophila at an MOI of 50, in the presence, when indicated, of either 10 μM of cytochalasin D—an actin polymerization inhibitor (+CytoD)—or 2 μM of Wortmanin—a PI3K inhibitor (+Wort). After 30 min of incubation, the medium was replaced by trypan blue solution to quench the fluorescence of non-internalised bacteria. The fluorescence of internalised bacteria was measured using an excitation level of 485 nm and an emission of 530 nm. Fluorescence data were corrected for differences in labelling efficiency between the tested strains. Each bar represents the mean of triplicate wells and error bars represent the standard deviations.

1983; de Groot et al. 1985). For the ET from QA to QB a spin-catalytic Gefitinib in vitro role of the non-heme iron to facilitate spin-selective ET has been proposed

(Ivanov et al. 1999). In this concept ISC accelerated by the spin-catalytic active non-heme iron promotes the indirect ET from the triplet radical pair 3[QA −QB −] and therefore the product formation to 1[QAQB 2−]. One may assume that the phenomenon of the solid-state photo-CIDNP effect could be rationalized in terms of nuclear observer spins, on the one hand obtaining nuclear polarization, on the other hand providing a spin-catalyst for ET. Under natural conditions, however, the primary radical pair lives 200 ps, by far too short to allow for hf interaction. Hence, the effect cannot be the cause of the efficiency, but the assumed correlation between the parallel occurrence of effect and high efficiency may be based on common principles. There may be some until now unknown fundamental principles of photosynthetic charge separation and stabilization that leading to both phenomena. In that case, photo-CIDNP MAS NMR would be useful for studies Selleck LY2157299 in artificial photosynthesis for three reasons: (i) as an analytical tool, (ii) as heuristic guide based on the strength of the effect, and (iii) by the possibility for exploration of the fundamental principles.

These fundamental principles may be related to highly optimized constraints in geometry and ET kinetics as chosen

and conserved by nature. It has been pointed out cAMP that both the solid-state photo-CIDNP effect and the efficient light-induced ET require optimized overlap of the wavefunctions (Jeschke and Matysik 2003) corresponding to moderate electron–electron coupling parameters. A clear picture of the required architecture of orbitals, however, is still missing. Such concept of overlapping static orbitals of the cofactors would be sufficient for the microscopic description of both the ET and the coherent origin of the solid-state photo-CIDNP effect. On the other hand, understanding of both processes on the protein level would allow for including the dynamic role of energy dissipation and entropy production in the transfer of electrons and polarization. It is possible that both ET and the solid-state photo-CIDNP effect require optimized dissipation channels. The relevance of protein relaxation for photosynthetic ET has been stressed (Cherepanov et al. 2001). Under conditions of irreversible thermodynamics, self-organized ET, in which improved entropy management allows for active coupling of the ET to a matrix with non-linear response, may lead to negative friction and gating (Tributsch and Pohlmann 1998; Tributsch 2006). Hence, experiments mapping light-induced changes at the atomic resolution may provide the empirical basis for the determination of the origin of the parallel transfer of electrons and of electron polarization to nuclei.

Further information on this topic is provided by the results of the SAILING study that evaluated the use of RAL vs. DTG in a context in which previously treatment-experienced patients had received therapy with many other types of drugs but not with INSTIs. Moreover, the patients in this trial had

developed resistance against many of the compounds that were used in prior therapy. Accordingly, almost all of them had compromised background regimens that involved the use of the various antiretroviral compounds that were employed. The results of the SAILING study show clearly that DTG outperformed RAL in terms of percentage of patients who achieved significant drops in viral load [46]. This is important, as it suggests that find more DTG is a more potent compound than RAL when either of these drugs is used in a salvage setting for patients who have previously failed traditional drug regimens that did not include an INSTI. At the Small molecule library mouse same time, patients in the RAL arm of the trial who developed resistance against the latter compound did so due to development of mutations that are associated with the latter drug.

In contrast, patients in the DTG arm of the trial developed resistance in very few cases. Two individuals developed the R263K mutation [72] that had earlier been shown to be of potential significance for DTG on the basis of tissue culture selection studies [73]. Accordingly, it appears that resistance to DTG in the clinic may be very difficult to develop, even in the case of patients who have previously failed other drug regimens and who are currently being treated with DTG, almost in the context of functional monotherapy. This suggests that it may be very difficult to develop resistance against DTG under circumstances in which this compound is used as part of a first-line INSTI regimen. This may be because the mutations that develop against DTG, when the latter is used in first-line therapy, are ones that

significantly diminish viral replication capacity [73, 74]. In contrast, the use of DTG as part of a second-line INSTI regimen may be more laden with problems, given the fact that mutations at positions 148, 140, and elsewhere within the viral genome, that are associated Arachidonate 15-lipoxygenase with resistance to RAL and EVG, may interfere with the ability of DTG to perform well. Moreover, the use of DTG to treat previously INSTI-experienced patients, with resistance to RAL and/or EVG, may lead to the selection of additional mutations that may further compromise therapy and cause cross-resistance [71]. Notably, in vitro studies suggest that the very rare individuals who may fail DTG treatment following emergence of the R263K mutation may still be treatable with RAL but not with EVG [74]. As stated, the results of the VIKING studies showed that many patients who possessed mutations at positions 148 and 140 within integrase did not respond well to DTG [71].

coli and bezylpenicillin (1500 μg ml-1), kanamycin (50 μg ml-1), or streptomycin (200 μg Ceritinib ic50 ml-1) for P. putida. Selection strategy of phenol tolerant mutants in colR-deficient P. putida strain For identification of genes affecting phenol sensitivity, the colR-deficient strain was subjected to mutagenesis by Tn5 based mini-transposon containing streptomycin resistance marker. A mini-transposon-carrying plasmid mTn5SSgusA40 [21] was conjugatively transferred from E. coli CC118 λpir [16] into a P. putida colR-deficient strain with the aid of a helper plasmid pRK2013 [18]. Transconjugants with random chromosomal insertions of the mini-transposon were first selected on glucose minimal

plates supplemented with kanamycin and streptomycin. After colonies were grown for three days at 30°C, they were replicated onto glucose minimal plates containing 8 mM phenol. Although a single Z-VAD-FMK mw colR-deficient cell could not form a colony on these plates, replication of big and closely located colonies of colR-deficient bacteria enabled their growth on replica plates. After another three days, growth of replicated clones in the presence of phenol was evaluated. About 150 transconjugants out of approximately 9000 transposon mutants grew better than colR-deficient P. putida and they were subjected to secondary assay of phenol tolerance. In order to avoid spontaneous phenol tolerant mutants, the clones

of interest were picked up from glucose

plates of initial selection. The secondary screen yielded 34 clones with higher phenol tolerance than the parental colR-deficient strain. Finally, siblings were eliminated through analysis of clones by arbitrary PCR and sequencing, resulting in 27 independent transposon insertion mutants with elevated phenol tolerance. Arbitrary PCR To identify chromosomal loci interrupted by insertion of mini-transposon in selected clones arbitrary PCR and sequencing were used. PCR products were generated by two rounds of amplifications as described elsewhere [22]. In the first round, a primer specific for the Sm gene (Smsaba – 5′-GAAGTAATCGCAACATCCGC-3′) or for the gusA gene (Gus2 CYTH4 – 5′-ACTGATCGTTAAAACTGCCTGG) and an arbitrary primer were used (Arb6 – 5′-GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC-3′). Second-round PCR was performed with primers Smsaba or Gus2 and Arb2 (5′-GGCCACGCGTCGACTAGTAC-3′). Cloning procedures and construction of bacterial strains To inactivate the ttgC gene in both wild-type and colR-deficient backgrounds the ttgC gene was first amplified using oligonucleotides ttgCalgus (5′-GAAGAATTCGTCACCCCTGAAAATCC-3′) and ttgClopp (5′-CCGAATTCGGTGGGCTTTCTGCTTTT-3′) and inserted into EcoRI-opened pUCNotKm (R. Teras). For disruption of the ttgC gene in pUC/ttgC, a central 315-bp Eco255I fragment of ttgC was replaced with Smr gene from the pUTmini-Tn5Sm/Sp [23].

In turn, biology has long exploited similar iterative strategies in biochemical synthetic pathways; one Selleckchem MK 2206 example is provided by fatty acid biosynthesis [39] (Figure 4). Figure 4 Cascade reaction sequences developed for the synthesis of ‘non-skid-chain like’ polyazamacrocyclic compounds [40] . The synthesis of dendrimers follows

either a divergent or convergent approach Dendrimers can be synthesized by two major approaches. In the divergent approach, used in early periods, the synthesis starts from the core of the dendrimer to which the arms are attached by adding building blocks in an exhaustive and step-wise manner. In the convergent approach, synthesis starts from the exterior, beginning with the molecular structure that ultimately becomes the outermost arm of the final dendrimer. In this strategy, the final generation number is pre-determined, necessitating

the synthesis of branches of a variety of requisite sizes beforehand for each generation [41] (Figure 5). Figure 5 Approaches for the synthesis if dendrimers. (A) Divergent approach: synthesis of radially symmetric polyamidoamine (PAMAM)dendrimers using ammonia as the trivalent core; the generations are added at each synthetic cycle (two steps), leading to an exponential increase in the number of surface functional groups [37]. (B) Convergent approach: synthesis of dendrons or wedges or branches that will become the periphery of Small molecule library chemical structure the dendrimer when coupled to a multivalent core in the last step of the synthesis [13]. Properties of dendrimers When comparing dendrimers with other nanoscale synthetic structures (e.g., traditional polymers, Isotretinoin buck balls, or carbon nanotubes), these are either highly non-defined or have limited structural diversity. Pharmacokinetic properties Pharmacokinetic properties are one of the most significant aspects that need to be considered for the successful biomedical application of dendrimers, for instance, drug delivery, imaging, photodynamic therapy, and neutron capture therapy. The diversity of potential applications of dendrimers in medicine results

in increasing interest in this area. For example, there are several modifications of dendrimers’ peripheral groups which enable to obtain antibody-dendrimer, peptide-dendrimer conjugates or dendritic boxes that encapsulate guest molecules [42]. Covalent conjugation strategies The strategy of coupling small molecules to polymeric scaffolds by covalent linkages to improve their pharmacological properties has been under experimental test for over three decades [43–46]. In most cases, however, the conjugated dendritic assembly functions as ‘pro-drug’ where, upon internalization into the target cell, the conjugate must be liberated to activate the drug (Figure 6). Figure 6 Requirements for dendrimer-based, cancer-targeted drug delivery.

(C) Densitometric anaysis of the blots showing the ratios of Beclin-1 and LC3-II to β-actin in Figure 10A. * and ** denote p < 0.05 and p < 0.01 respectively in Figure 10B and 10C (vs. control); # and ## denote p < 0.05 and p < 0.01 respectively in Figure 10B and 10C (vs. LPS). (D) Graph represents percentage of remaining E.coli at different time points in each group treated as described above. Data are mean values ± SD (n ≥3). * and ** denote p < 0.05 and p < 0.01 respectively (LPS vs. control); # and ## denote p < 0.05 and

p < 0.01 respectively (LPS + TLR4 siRNA vs. Selleckchem Adriamycin LPS). Discussion Although aberrant autophagy is observed in many bacterial infectious diseases, the role of autophagy in PD-related peritonitis remains unknown. Our study has investigated the role of autophagy in PMCs against intracellular E.coli. We demonstrated that LPS could induce autophagy in HMrSV5 cells. LPS enhanced the intracellular bactericidal activity of HMrSV5 cells and promoted the co-localization of E.coli (K12-strain) with autophagosomes. Moreover, treatment with microtubule-disrupting agents such as 3-MA or Wm or Beclin-1 siRNA, markedly attenuated the Selleckchem MK 2206 intracellular bactericidal activity of HMrSV5 cells and the co-localization of E. coli with autophagosomes induced by LPS treatment. Furthermore, knockdown of TLR4 vanished LPS-induced autophagy and bactericidal activity. These data collectively suggest

that autophagy activated by LPS via TLR4 represents an innate defense mechanism for inhibiting intracellular E. Rucaparib ic50 coli replication. Autophagy is a process traditionally known to contribute to cellular cleaning

via the removal of intracellular components in lysosomes [26]. Recently, our colleagues reported that LPS stimulation led to autophagy in cultured peritoneal mesothelial cells [27]. In keeping with their reports, our data revealed that LPS induced accumulation of LC3-II in a time- and dose-dependent manner in HMrSV5 cells, as indicated by an increased aggregation of GFP-LC3 puncta and a higher number of autophagosome-like MDC-labeled vacuoles. Furthermore, HMrSV5 cells pretreated with 3-MA, Wm or Beclin-1 siRNA displayed defective autophagy induction in response to LPS. These results indicate that LPS is a general stimulant of autophagic activity in PMCs. In addition, our study showed the viability of LPS-treated cells had no significant difference compared to the control group. It has been demonstrated that exposure of PMCs to LPS resulted first in autophagy and later, apoptosis [27]. Apoptosis was only observed under higher concentrations of LPS (5 to 10 μg/ml) exposure for 48 hours in HMrSV5 cells [27]. We could not detect apoptosis in HMrSV5 cells following the incubation with lower doses of LPS (0-5 μg/ml) for shorter time periods (0-24 h) in present study, which was consistent with the previous report [27].

Three other operons containing uptake systems of unknown substrates are also present. Other regions of difference between TIGR4 and AP200 include the presence in the latter of a DpnII restriction system and a double glycin-type bacteriocin gene (Additional file 1). The extent and type of genomic variation between AP200

and TIGR4 is in line with the genetic diversity found within this species by other studies comparing a series of pneumococcal genomes [21, 25, 26]. Comparison of the AP200 genome with TIGR4 revealed also a large chromosomal inversion of approximately 163 kb across the selleck chemical replication axis and involving the termination site (Figure 2). Large-scale inversions are typically driven by homologous recombination among repeated regions. The AP200 inversion borders fall within the coding sequences of PhtB and PhtD, two proteins which are part of the histidine-triad proteins family, characterized by the repeated histidine HxxHxH triad motif [27]. This family is composed of 4 proteins (PhtA,

PhtB, PhtD, and PhtE) showing high sequence similarity. PhtB and PhtD, which are involved in selleck chemicals AP200 chromosomal inversion, reach approximately 87% amino acids identity. Figure 2 Genome alignment of S. pneumoniae strains TIGR4, AP200, CGSP14, Taiwan 19F-14 and TCH8431/19A. Each sequence of identically colored blocks represents a collinear set of matching regions linked across genomes. Regions that are inverted are shifted below a genome’s center axis. Figure generated by Mauve, free/open-source software available from http://​gel.​ahabs.​wisc.​edu/​mauve. Chromosomal inversions are thought to be implicated in the rebalance of the chromosomal architecture when it is affected by insertions of large DNA regions, such as transposons, IS elements or prophages. In particular, it has been speculated that the chromosomal imbalance could be caused when large

DNA fragments are inserted in one side of the replication axis [28], as in the case of AP200 genome, where the large exogenous filipin elements resided in right of the replication axis. To date, the only pneumococcal genome described to carry a large chromosomal inversion is CGSP14 [28]. Also in CGSP14 the inversion occurs across the termination site but involves a different region (Figure 2). Inversions are present also in 2 recently sequenced pneumococcal genomes, Taiwan 19F-14 [GenBank: NC_012469] and TCH8431/19A [GenBank: NC_014251], although they have not been described (Figure 2). In these strains, the chromosomal inversions involve much larger regions. These observations suggest that the synteny of pneumococcal genome is not always conserved. A striking feature of pneumococcal genomes is the over-distribution of IS elements [23, 29]. AP200 contains 63 transposases and inactivated derivatives thereof http://​www-is.​biotoul.​fr/​is.​html.

There were no significant differences (ANOVA, p > 0.05). C) pH of bile measured at 37°C as a function of state. Values represent means ± SE from T (n = 4), IBA (n =

4), SA (n = 10), and AB squirrels (n = 4). All values are significantly different except between T and IBA (ANOVA, p > 0.05). D) Total protein concentration in bile as a function of state. Values represent means ± SE from T (n = 3), IBA (n = 3), SA (n = 5), and AB squirrels (n = 4). There were no significant differences between T and X-396 concentration IBA or between SA and AB. All other values are significantly different (ANOVA, p < 0.05). Discussion The winter season for a hibernator is marked by extended anorexia [2]. Given the liver's role in fueling metabolism, we hypothesized that there might be changes in liver function as a function of hibernation and associated anorexia.

We present here the first data on the effects of hibernation on gallbladder bile constituents. Although there were no significant differences in bile constituents between torpid and aroused winter squirrels, a few differences were found between the winter squirrels and summer squirrels. Except for [bilirubin], these differences did not involve critical indicators of metabolic function. Finally, we examined bile in winter animals that failed to enter torpor and found that they had significantly lower [bile acids] and [lecithin] as compared to all other groups. We discuss below the implications of these findings. To satisfy its energetic demands during Nivolumab order winter, a hibernator relies on stored lipids. Changes in lipid pools have dramatic effects on the ability of a hibernator to successfully employ torpor; increased dietary poly-unsaturated fatty acids increase torpor bout usage, length, and depth [15]. A major function of the liver, and more specifically Cediranib (AZD2171) bile, is to facilitate digestion and absorption of lipids from the intestine. However, what happens to hepatobiliary function when there are no foodstuffs in the gut? The anorexia of hibernation allows for an examination of an extended (months long) anorexia not available

with almost any other mammalian system except denning bears. In our study, we were surprised by the few changes in the bile constituents between summer and normal winter squirrels (both IBA and T); important indicators of metabolic function such as biliary [bile acids], [cholesterol], [free fatty acids], and [lecithin] were unchanged despite the months long anorexia experienced by winter squirrels (Figs. 2, 3). Although biliary changes as a function of season were found for [bilirubin], spectral characteristics, pH, and [total protein], the roles that most of these other factors have as indicators of hepatobiliary function seems less robust (Figs. 1, 2, 3). For instance, biliary pH is known to be quite variable [e.g., [16]].

The extraordinary Barasertib research buy conservation of 16S rRNA in cyanobacteria seems to indicate that concerted evolution is a more likely explanation. To verify this suggestion we examined variation in the internal

transcribed spacer region, located between the 16S and 23S rRNA gene. Though previous studies have suggested conservation of some regions in the ITS sequence, several regions should not be affected by selection and evolve neutrally. If the entire ITS sequence showed the same degree of conservation as does the 16S gene sequence, then purifying selection —which would only act on the functional parts— could be rejected as a driving force. However, the strong conservation found in cyanobacterial 16S rRNA gene sequences could not be confirmed for the ITS-regions of four cyanobacterial

taxa (Additional file 9). For cyanobacteria and the eubacterial phyla studied here, both concerted evolution and strong purifying selection, appear to be the main contributing factors. Although, cyanobacteria are assumed to be an ancient phylum which presumably raised oxygen levels in the atmosphere more than 2.3 billion years ago [54], variation in 16S rRNA copies is extremely low. Indeed, phylogenetic tree reconstructions check details for 16S rRNA result in relatively short estimated branch lengths within this phylum, compared to other eubacterial phyla (Figure 2). Short evolutionary distances for 16S rRNA sequences are Carbachol consistent with a pattern that has been found for morphological characters in cyanobacteria before. In 1994, J.W. Schopf compared the tempo and mode of evolution in cyanobacteria from the Precambrian, to evolutionary patterns observed in fossils during the Phanerozoic. The latter have been described by G.G. Simpson in his book “The tempo and mode of evolution” [55]. Schopf found that evolutionary predictions which Simpson made for metazoan fossils from the Phanerozoic, can also be applied to cyanobacteria. Morphologically, cyanobacteria seem to evolve not only at a “bradytelic”, but “hypobradytelic” mode, meaning at exceedingly low

evolutionary rates. Fossils from the Precambrian strongly resemble present morphotypes. The oldest undisputed cyanobacterial fossils date back circa 2.0 billion years [18, 19]. Morphological appearance of these microfossils already suggests the presence of at least four of the morphological sections described by Castenholz [20]. It seems that cyanobacteria reached their maximum morphological complexity two billion years ago, and many of today’s species could be described as so-called ‘living fossils’. It remains to be seen whether the low evolutionary rates as seen in 16S rRNA sequences and morphological features, is also seen at the genomic and metabolic level. This question can be further resolved as further genomic sequences become available for the cyanobacteria.

fortuitum may represent an evolutionary intermediate stage between saprophytic mycobacteria like M. smegmatis and the highly pathogenic slow-growing mycobacteria. Conclusion Our study provides detailed information about porin genes of the mspA class in M. fortuitum and their importance for the

CHIR-99021 concentration growth rate and susceptibility to antibiotics. Our future studies will concentrate on the elucidation of the role of PorM1 and PorM2 in survival and replication of phagocytosed M. fortuitum. Methods Bacterial strains, cell lines and plasmids Mycobacterial strains (Table 3) were grown in Middlebrook 7H9 medium (BD Biosciences, Heidelberg, Germany), supplemented with 0.05% Tween 80 learn more and either ADC (BD Biosciences) or DC (2 g glucose, 0.85 g NaCl, in 100 ml H2O) at 37°C without shaking, or on Mycobacteria 7H11 agar (BD Biosciences), supplemented with ADC (BD Biosciences). For selection of recombinant mycobacteria, media were supplemented when required with 25 to 100 μg ml-1 kanamycin or 100 μg ml-1 hygromycin B.

E. coli DH5α was grown in LB medium at 37°C [35]. Media were supplemented with 100 μg ml-1 kanamycin or 200 μg ml-1 hygromycin B for selection of recombinant E. coli. All plasmids used in this study are described in Table 4. Table 3 Mycobacterial strains used in this work. Strains Characteristics Reference M. smegmatis SMR5 M. smegmatis mc2155 derivative, SMR [42] M. smegmatis ML10 SMR5 derivative, ΔmspA and ΔmspC [4] M. fortuitum DSM 46621 Type strain; HYGR   M. fortuitum 10851/03 Human patient isolate This study M. fortuitum 10860/03 Human patient isolate; HYGR This study M. bovis BCG Copenhagen Vaccine strain   HYG: hygromycin; SM: streptomycin Measurement of growth rates in broth culture To compare the growth rates of M. fortuitum strains, Middlebrook 7H9/DC medium was inoculated with preparatory cultures to obtain an initial OD600 of 0.02. During 16 ID-8 days, the optical

densities of the cultures were measured daily. Growth of the strains was monitored by quantification of the ATP content of the cultures with the luminescence-based kit BacTiter-Glo™ Microbial Cell Viability Assay (Promega). The luminescence was reported as relative light units (RLU) with the microplate luminometer LB96V (EG&G Berthold) [36]. Molecular biology techniques and in silico analysis Common molecular biology techniques were carried out according to standard protocols [35] or according to the recommendations of the manufacturers of kits and enzymes. Transformation of E. coli was performed according to the method of Hanahan [37]. PCR reactions were performed with the following kits: Taq DNA Polymerase (MBI Fermentas, St. Leon-Roth, Germany), BC Advantage GC Polymerase Mix (Takara Bio Europe S.A., Gennevilliers, France), BIO-X-ACT Short Mix and BIOTAQ DNA Polymerase (Bioline GmbH, Luckenwalde, Germany).