Protein multiple sequence alignment by hybrid bio-inspired algorithms

This article presents an immune inspired algorithm to tackle the Multiple Sequence Alignment (MSA) problem. MSA is one of the most important tasks in biological sequence analysis. Although this paper focuses on protein alignments, most of the discussion and methodology may also be applied to DNA alignments. The problem of finding the multiple alignment was investigated in the study by Bonizzoni and Vedova and Wang and Jiang, and proved to be a NP-hard (non-deterministic polynomial-time hard) problem. The presented algorithm, called Immunological Multiple Sequence Alignment Algorithm (IMSA), incorporates two new strategies to create the initial population and specific ad hoc mutation operators. It is based on the 'weighted sum of pairs' as objective function, to evaluate a given candidate alignment. IMSA was tested using both classical benchmarks of BAliBASE (versions 1.0, 2.0 and 3.0), and experimental results indicate that it is comparable with state-of-the-art multiple alignment algorithms, in terms of quality of alignments, weighted Sums-of-Pairs (SP) and Column Score (CS) values. The main novelty of IMSA is its ability to generate more than a single suboptimal alignment, for every MSA instance; this behaviour is due to the stochastic nature of the algorithm and of the populations evolved during the convergence process. This feature will help the decision maker to assess and select a biologically relevant multiple sequence alignment. Finally, the designed algorithm can be used as a local search procedure to properly explore promising alignments of the search space.     

Artificial seeding of the green seaweed Monostroma for cultivation

In Japan, the green seaweed Monostroma is an important source of humanfood. Monostroma nitidum Wittrock (Japanese name: hitoegusa) is cultivated in brackish waters and estuaries of central to southern Japan. The green seaweed Monostroma grows in the brackish water area in the upper part of the intertidal zone in the warm waters. Artificial seed culture began with the collection of many gametes in April. The resultant zygotes were allowed to adhere to plastic settlement boards (20 cm long and 10 cm wide). The zygoteboards were then cultured in tanks (1 ×2 ×0.5 m) with fertiliser in a controlled growth room (10–87 μmol photon m^-2s^-1). The cultivated zygotes on the board in the indoor tanks gradually increased in size from 10 to 40 μm in diameter during May to early August. Zygote growth became slowed at the end of August. The zygotesmatured in early September, and the plates were transferred into culture tanks in a dark room for dark treatment. Maturation of the zygote was promoted by providing dark conditions for two weeks. The production of a concentrated zoospore solution from the mature blades was achieved by adding fresh water at temperature 2–3 °C above that of the seeding vats. Zoospores were released in large numbers when exposed to strong irradiance of 100 μmol photon m^-2 s^-1 for 30 min. The zygotes produced flat unicellular fronds at the germling stage. The technology of artificial seed culture and zoospore release from the zygotes is based mainly on these experiments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

Lectin-like binding and antibiotic sensitivity of enterococci from wild herbivores

Fifty eight enterococcal isolates from wild herbivores were tested for their antibiotic sensitivity pattern and lectin-like binding of extracellular matrix (ECM) and serum proteins. Kanamycin resistance was very frequent; many multiresistant strains were also isolated. All isolates were sensitive to rifampicin. Resistance to gentamicin, novobiocin, and tetracycline was widely distributed in the microflora of wild herbivores breeded in zoological garden in Košice.

No autoaggregating strains were detected among these 58 enterococcal isolates. Various degrees of binding of mucins, fetuin, heparin, fibrinogen, and fibronectin were observed in individual strains. However, bovine lactoferrin binding by enterococci from deers and chamoises was either negative (0) or strongly positive (3).

With regard to influence of growth media, TH agar was found to be better for the expression of lectin-like binding than blood agar, TH broth and Nutrient broth.

A significant effect (P < 0.001 or P < 0.05) of proteolytic treatment was observed in six selected strains. However, there is a difference between the effect of trypsin and pronase P.Pronase treatment more effectively decreased binding of some strains (1H, 6A, EF 1111, EC 1292), while trypsin treatment decreased more binding of other enterococcal strains (EF953 and 1E). Significant (P < 0.001) influence of metaperiodate, which cleaves the C-C bond between vicinal groups of sugars, on collagen I binding by three selected strains (1E, 1H, 6A) and bovine lactoferrin binding (by EF 1111, EC 1292, EF 953) was also observed. However, its influence was very different. In two strains (1H and EC 1292), ECM binding was decreased, while in four other strains (1E, 6A, EF 1111, EF 953) it was increased.     

Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2

Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the beta-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.     

The Avoidance Response in Phycomyces

Phycomyces sporangiophores grow away from stationary objects, a phenomenon known as the avoidance response. Evidence is presented suggesting that a growth-stimulating gas is emitted from the sporangiophore and is then swept to the leeward side by air currents resulting in higher gas concentration on that side. The presence of a stationary barrier decreases the passive movement of the gas away from the leeward side. It is proposed that an increase of this gas on one side causes that side to grow faster. Indirect evidence suggests that the gas is water vapor.     

S100A13-lipid interactions-role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM As-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM An-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ.