Molecular profiling of indoor microbial communities in moisture damaged and non-damaged buildings

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-effect relationship has not been well established and knowledge of the causative agents is incomplete. The present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identification is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the often inconsistent findings between objectively measured microbiological attributes and health. In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). The results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi. Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. The dominant sequence types of fungi, and also of bacteria were mainly affiliated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affiliated with the classes Dothideomycetes (mould-like filamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like filamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Tremellomycetes and the family Malasseziales (both yeast-like basidiomycetes). The most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affiliated with human-associated members within the phyla Actinobacteria and Firmicutes. Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation firm conclusions concerning the effect of moisture damage on the species diversity could not be made. The fungal communities in dust samples showed seasonal variation, which reflected the seasonal fluctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. The comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confirmed that culture based methods give both a qualitative and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specific qPCR was a more efficient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials. In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. The suitability of the screening method to different kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. The usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material.

Conformational Polymorphism in Telomeric Structures: Loop Orientation and Interloop Pairing in d(G4TnG4),

Sequence repeats constituting the telomeric regions of chromosomes are known to adopt a variety of unusual structures, consisting of a G tetraplex stem and short stretches of thymines or thymines and adenines forming loops over the stem. Detailed model building and molecular mechanics studies have been carried out for these telomeric sequences to elucidate different types of loop orientations and possible conformations of thymines in the loop. The model building studies indicate that a minimum of two thymines have to be interspersed between guanine stretches to form folded-back structures with loops across adjacent strands in a G tetraplex (both over the small as well as large groove), while the minimum number of thymines required to build a loop across the diagonal strands in a G tetraplex is three. For two repeat sequences, these hairpins, resulting from different types of folding, can dimerize in three distinct ways-i.e., with loops across adjacent strands and on same side, with loops across adjacent strands and on opposite sides, and with loops across diagonal strands and on opposite sides-to form hairpin dimer structures. Energy minimization studies indicate that all possible hairpin dimers have very similar total energy values, though different structures are stabilized by different types of interactions. When the two loops are on the same side, in the hairpin dimer structures of d(G(4)T(n)G(4)), the thymines form favorably stacked tetrads in the loop region and there is interloop hydrogen bonding involving two hydrogen bonds for each thymine-thymine pair. Our molecular mechanics calculations on various folded-back as well as parallel tetraplex structures of these telomeric sequences provide a theoretical rationale for the experimentally observed feature that the presence of intervening thymine stretches stabilizes folded-back structures, while isolated stretches of guanines adopt a parallel tetraplex structure

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.

Biocompatibility and biofunctionalization of mesoporous silicon particles

Several of the newly developed drug molecules experience poor biopharmaceutical behavior, which hinders their effective delivery at the proper site of action. Among the several strategies employed in order to overcome this obstacle, mesoporous silicon-based materials have emerged as promising drug carriers due to their ability to improve the dissolution behavior of several poorly water-soluble drugs compounds confined within their pores. In addition to improve the dissolution behavior of the drugs, we report that porous silicon (PSi) nanoparticles have a higher degree of biocompatibility than PSi microparticles in several cell lines studied. In addition, the degradation of the nanoparticles showed its potential to fast clearance in the body. After oral delivery, the PSi particles were also found to transit the intestines without being absorbed. These results constituted the first quantitative analysis of the behavior of orally administered PSi nanoparticles compared with other delivery routes in rats. The self-assemble of a hydrophobin class II (HFBII) protein at the surface of hydrophobic PSi particles endowed the particles with greater biocompatibility in different cell lines, was found to reverse their hydrophobicity and also protected a drug loaded within its pores against premature release at low pH while enabling subsequent drug release as the pH increased. These results highlight the potential of HFBII-coating for PSi-based drug carriers in improving their hydrophilicity, biocompatibility and pH responsiveness in drug delivery applications. In conclusion, mesoporous silicon particles have been shown to be a versatile platform for improving the dissolution behavior of poorly water-soluble drugs with high biocompatibility and easy surface modification. The results of this study also provide information regarding the biofunctionalization of the THCPSi particles with a fungal protein, leading to an improvement in their biocompatibility and endowing them with pH responsive and mucoadhesive properties.

Resistance of Norway spruce (Picea abies) to root and butt rot (Heterobasidion parviporum) in peatland and mineral soil

Root and butt rot is the most harmful fungal disease affecting Norway spruce in southern Finland. In approximately 90 % of cases the causal agent is Heterobasidion parviporum. Root and butt rot infections have not been reported in Finnish peatlands. However, the increase in logging operations in peatlands means there is a risk that the fungus will eventually spread to these areas. The aim of this study was to find out the impact of growing site on the resistance of Norway spruce to Heterobasidion parviporum infections. This was investigated by artificially inoculating H. parviporum to spruce trees in pristine mire, drained peatland and mineral soil and comparing the defence reactions. Additionally, the effect of genotype on resistance was studied by comparing the responses of spruce clones representing different geographic origins. The roots and stems of the trees to be sampled were wounded and inoculated with wood dowels pre-colonised by H. parviporum hyphae. The resulting necrosis around the point of inoculation was observed. It was presumed that increased length of necrosis indicates high susceptibility of the tree to the disease. The relationship between growth rate and host resistance was also studied. The results indicated that growing site does not have a statistically significant effect on host resistance. The average length of necrosis around the point of inoculation was 35 mm in pristine mire, 37 mm in drained peatland and 40 mm in mineral soil. It was observed that growth rate does not affect resistance, but that the genotype of the tree does have an effect. The most resistant spruce clone was the one with Russian origin. The results suggest that the spruce stands in peatlands are not more resistant to root and butt rot infections than those in mineral soil. These findings should be taken into consideration when logging peatland forests.

Thermophilic fungi: An assessment of their potential for growth in soil

An attempt has been made to forecast the potential of thermophilic fungi to grow in soil in the laboratory and in the field in the presence of a predominantly mesophilic fungal flora at usual temperature. The respiratory rate of thermophilic fungi was markedly responsive to changes in temperature, but that of mesophilic fungi was relatively independent of such changes. This suggested that in a thermally fluctuating environment, thermophilic fungi may be at a physiological disadvantage compared to mesophilic fungi. In mixed cultures in soil plates, thermophilic fungi outgrew mesophilic fungi under a fluctuating temperature regime only when the amplitude of the fluctuating temperatures was small and approached their temperature optima for growth. An antibody probe was used to detect the activity of native or an introduced strain of a thermophilic fungus, Thermomyces lanuginosus, under field conditions. The results suggest that although widespread, thermophilic fungi are ordinarily not an active component of soil microflora. Their presence in soil most likely may be the result of the aerial dissemination of propagules from composting plant material.

Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.

Behaviour of the germ cell specific lamin through mammalian spermatogenesis as probed with monoclonal antibodies.

We had earlier identified a 60 kDa nuclear lamin protein (lamin(g)) unique to the germ cells of rat testis which was subsequently shown to be antigenically conserved in germ cells of grasshopper, rooster, frog and plants. We have now obtained eight monoclonal antibodies in mouse against this lamin(g) antigen. While all the eight Mabs reacted with lamin(g) antigen in an immunoblot analysis, only three Mabs (A(11)C(7), A(11)D(4), C1F7) showed strong reactivity in the immunofluorescence analysis of the germ cells. The Mabs A(11)C(7) and A(11)D(4) showed a slight cross-reactivity with rat liver lamin B. Indirect immunofluorescence analysis of pre-meiotic, meiotic and post-meiotic germ cells with Mabs have shown that while the lamin(g) is localized in the lamina structures of spermatogonia and round spermatids, it is localized to the phase dense regions of pachytene spermatocytes which is in conformity with our previous observations using rabbit polyclonal antibodies. The localization of the antigen in the germ cells was also confirmed by immunohistochemical staining of the thin sections of seminiferous tubules. By immunostaining the surface spread pachytene spermatocytes, the antigen was further localized to the telomeric ends of the paired homologous chromosomes. Using anti-somatic lamin B antibodies, we have also demonstrated the absence of somatic lamins in meiotic and post-meiotic germ cells. The lamina structure of pre-meiotic spermatogonial nucleus contains both somatic lamin B and lamin(g) as evidenced by immunofluorescence studies with two differently fluorochrome labelled anti-lamin B and anti-lamin(g) antibodies. The selective retention of lamin(g) in the pachytene spermatocytes is probably essential for anchoring the telomeric ends of the paired chromosomes to the inner nuclear membrane.

Proposed role of w chromosome inactivation and the absence of dosage compensation in avian sex determination

Three features of avian sex chromosomes - female heterogamety (ZZ male, ZW female), the apparently inactive state of the W chromosome, and dose-dependent expression of Z-linked genes - are examined in regard to their possible relation to sex determination. It is proposed that the W chromosome is facultatively heterochromatic and that the Z and W chromosomes carry one or more homologous sex-determination genes. The absence of dosage compensation in ZZ embryos, and W inactivation in ZW embryos, would then bring about a 2n(ZZ)-n(ZW) inequality in the effective copy number of such genes. The absence of dosage compensation of Z-linked genes in ZZ embryos is viewed as a means by which two copies of Z-W homologous sex determination genes are kept active to meet the requirements of testis determination. W inactivation may promote ovarian development by reducing the effective copy number of these genes from 2n to n. If there is a W-specific gene for femaleness, spread of heterochromatization to this gene in cells forming the right gonadal primordium may explain the latter's normally undifferentiated state; reversal of heterochromatization may similarly explain the development of the right gonad into a testis following left ovariectomy.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus

Differential organisation of homologous chromosomes is related to both sex determination and genomic imprinting in coccid insects, the mealybugs. We report here the identification of two middle repetitive sequences that are differentially organised between the two sexes and also within the same diploid nucleus. These two sequences form a part of the male-specific nuclease-resistant chromatin (NRC) fraction of a mealybug Planococcus lilacinus. To understand the phenomenon of differential organisation we have analysed the components of NRC by cloning the DNA sequences present, deciphering their primary sequence, nucleosomal organisation, genomic distribution and cytological localisation, Our observations suggest that the middle repetitive sequences within NRC are functionally significant and we discuss their probable involvement in male-specific chromatin organisation.

The inactive X chromosome in the human female is enriched in 5-methylcytosine to an unusual degree and appears to contain more of this modified nucleotide than the remainder of the genome

By employing a procedure that combines ELISA and photoacoustic spectroscopy, we have examined the content of 5-methylcytosine (m(5)C) in DNA of individuals who differed from one another in the number of X chromosomes in their genomes. The results show that the human inactive X chromosome (Xi) contains very high amounts of this modified nucleotide. We estimate that in the 46,XX female there is more m(5)C in Xi (similar to3.6 x 10(7)) than in all the remaining chromosomes put together (similar to2.1 x 10(7)). Our results also suggest that nearly one-fifth of all cytosines in Xi are methylated and that, in addition to CpG methylation, there is extensive non-CpG methylation as well.

How does DNA break during chromosomal translocations?

Chromosomal translocations are one of the most common types of genetic rearrangements and are molecular signatures for many types of cancers. They are considered as primary causes for cancers, especially lymphoma and leukemia. Although many translocations have been reported in the last four decades, the mechanism by which chromosomes break during a translocation remains largely unknown. In this review, we summarize recent advances made in understanding the molecular mechanism of chromosomal translocations.

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFN gamma and TNF alpha levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

A tropical oviparous lizard, Calotes versicolor, exhibiting a potentially novel FMFM pattern of temperature-dependent sex determination

Among squamate reptiles, lizards exhibit an impressive array of sex-determining modes viz. genotypic sex determination, temperature-dependent sex determination, co-occurrence of both these and those that reproduce parthenogenetically. The oviparous lizard, Calotes versicolor, lacks heteromorphic sex chromosomes and there are no reports on homomorphic chromosomes. Earlier studies on this species presented little evidence to the sex-determining mechanism. Here we provide evidences for the potential role played by incubation temperature that has a significant effect (P<0.01) on gonadal sex and sex ratio. The eggs were incubated at 14 different incubation temperatures. Interestingly, 100% males were produced at low (25.5 +/- 0.5 degrees C) as well as high (34 +/- 0.5 degrees C) incubation temperatures and 100% females were produced at low (23.5 +/- 0.5 degrees C) and high (31.5 +/- 0.5 degrees C) temperatures, clearly indicating the occurrence of TSD in this species. Sex ratios of individual clutches did not vary at any of the critical male-producing or female-producing temperatures within as well as across the seasons. However, clutch sex ratios were female- or male-biased at intermediate temperatures. Thermosensitive period occurred during the embryonic stages 3033. Three pivotal temperatures operate producing 1:1 sex ratio. Histology of gonad and accessory reproductive structures provide additional evidence for TSD. The sex-determining pattern, observed for the first time in this species, that neither compares to Pattern I [Ia (MF) and Ib (FM)] nor to Pattern II (FMF), is being referred to as FMFM pattern of TSD. This novel FMFM pattern of sex ratio exhibited by C. versicolor may have an adaptive significance in maintaining sex ratio. J. Exp. Zool. 317:3246, 2012. (c) 2011 Wiley Periodicals, Inc.