<Emphasis Type="BoldItalic">In vivo</Emphasis> conditions influence the coding of stimulus features by bursts of action potentials

The functional role of burst firing (i.e. the firing of packets of action potentials followed by quiescence) in sensory processing is still under debate. Should bursts be considered as unitary events that signal the presence of a particular feature in the sensory environment or is information about stimulus attributes contained within their temporal structure? We compared the coding of stimulus attributes by bursts in vivo and in vitro of electrosensory pyramidal neurons in weakly electric fish by computing correlations between burst and stimulus attributes. Our results show that, while these correlations were strong in magnitude and significant in vitro, they were actually much weaker in magnitude if at all significant in vivo. We used a mathematical model of pyramidal neuron activity in vivo and showed that such a model could reproduce the correlations seen in vitro, thereby suggesting that differences in burst coding were not due to differences in bursting seen in vivo and in vitro. We next tested whether variability in the baseline (i.e. without stimulation) activity of ELL pyramidal neurons could account for these differences. To do so, we injected noise into our model whose intensity was calibrated to mimic baseline activity variability as quantified by the coefficient of variation. We found that this noise caused significant decreases in the magnitude of correlations between burst and stimulus attributes and could account for differences between in vitro and in vivo conditions. We then tested this prediction experimentally by directly injecting noise in vitro through the recording electrode. Our results show that this caused a lowering in magnitude of the correlations between burst and stimulus attributes in vitro and gave rise to values that were quantitatively similar to those seen under in vivo conditions. While it is expected that noise in the form of baseline activity variability will lower correlations between burst and stimulus attributes, our results show that such variability can account for differences seen in vivo. Thus, the high variability seen under in vivo conditions has profound consequences on the coding of information by bursts in ELL pyramidal neurons. In particular, our results support the viewpoint that bursts serve as a detector of particular stimulus features but do not carry detailed information about such features in their structure.     

A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for <Emphasis Type="Italic">Candida</Emphasis> species identification

Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

<Emphasis Type="Italic">Rhaponticum carthamoides</Emphasis> transformed root extract inhibits human glioma cells viability,induces double strand DNA damage,H2A.X phosphorylation,and PARP1 cleavage

Rhaponticum carthamoides transformed root extract induces double strand DNA damage by increasing the number of phosphorylated H2A.X- and cleaved PARP1-positive U87MG cells and patient-derived IV grade glioma cells. Furthermore, treatment of these cells with root extract causes down-regulation of UHRF1 and DNMT1. Transformed root extract is rich in caffeoylquinic acid derivatives, especially tricaffeoylquinic acid derivatives. Our findings demonstrate that the R. carthamoides transformed root extract may trigger apoptosis in glioma cells by induction of DNA damage, PARP cleavage and epigenetic modification.     

Effect of ionizing radiation on the DNA damage response in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The DNA damage response (DDR) is induced by various DNA damaging factors and maintains genome stability in all organisms. The Chlamydomonas reinhardtii genome contains putative homologous genes involved in DDR; however, little is known about the functions and responses of these genes to DNA damage. In this study, DDR by gamma radiation was determined in C. reinhardtii. Irradiation with 80, and 200 Gy gamma radiation caused death in approximately 47 and 97 % of C. reinhardtii cells, respectively. The absolute lethality of cells was at 300 Gy. The rate of DNA breaks was also determined using comet assays after exposure to different doses of gamma radiation. Irradiation with 80 and 400 Gy resulted in 17 and 34 % of nuclear degradation in C. reinhardtii cells, respectively. To identify the major DDR pathway of C. reinhardtii induced by gamma radiation, 24 putative DDR genes were selected from the Joint Genome Institute (JGI) database. Gamma radiation significantly affected expression of 15 genes among these. Therefore, these genes displaying expressional changes by gamma radiation are involved in DDR, which indicate that C. reinhardtii may possess a fundamental conserved DDR pathway with higher plants. Furthermore, radiation responsive proteins were identified by proteomic analysis, which are involved in metabolisms of carbohydrate, energy, and photosynthesis. This is the first report to describe the responses of DDR homologous genes to gamma radiation and to identify gamma radiation-responsive proteins in C. reinhardtii. Our data should provide molecular insights into gamma radiation responses including DNA damage in green algae.     

In vitro antifungal efficacy of <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9642 chitosan-AgNPs composite against post-harvest disease of citrus fruits

Green and blue mold postharvest diseases are the most vital negative components influencing the local market of citrus fruits. Citrus fruits were collected, and fungi were isolated. Among the fungal isolates identified, Penicillium digitatum and Penicillium italicum recorded the highest occurrence of 39.5 and 25.6%, respectively. In this work, we extracted chitosan from Aspergillum niger ATCC 9642. Fourier transform infrared spectroscopy was utilized to confirm the functional groups of the obtained compound, which exhibited the main characteristic bands of O–H stretching at 3302 cm^-1, and C–O–C band at 1125 cm^-1. A. niger ATCC 9642 chitosan had the degree of deacetylation of 88.5%, a molecular weight of 1.8 × 10⁵ Da, and viscosity of 7.3 centipoises; these values were comparable to those for standard shrimp chitosan. Ultraviolet-visible light spectra revealed the presence of A. niger ATCC 9642 chitosan-AgNPs composite. Using antifungal and spore germination assays, it was found that this composite exhibited effective antifungal action against P. digitatum and P. italicum compared with a chitosan standard. In a comet assay, the percentage of tail DNA was considered as a parameter that indicated DNA damage. The comet parameter increased significantly (P < 0.05) with A. niger ATCC 9642 chitosan–AgNPs composite, and the increase was dose-dependent. The increase in the DNA damage positively correlated with the inhibition performance of the A. niger ATCC 9642 chitosan–AgNPs composite.     

Dds20 operates in Cds1-independent mechanism of tolerance to UV-induced DNA damage in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> cells

Repair of DNA double-stranded breaks caused by ionizing radiation or cellular metabolization, homologous recombination, is an evolutionary conserved process controlled by RAD52 group genes. Genes of recombinational repair also play a leading role in the response to DNA damage caused by UV light. Cells with deletion in gene dds20 of recombinational repair were shown to manifest hypersensitivity to the action of UV light at lowered incubation temperature. Epistatic analysis revealed that dds20 ⁺ is not a member of the NER and UVER gene groups responsible for the repair of DNA damage induced by UV light. The Dds protein has functions in the Cds1-independent mechanism of UV damage tolerance of DNA.     

Detection of alien genetic introgressions in bread wheat using dot-blot genomic hybridisation

Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare, Secale cereale and Agropyron cristatum in the wheat background was also reported.     

Enzymatic depolymerization of <Emphasis Type="Italic">N</Emphasis>-succinylchitosan

A complex of chitinolytic enzymes of Streptomyces kurssanovii and also lysozyme and Celloviridin, an industrial cellulase preparation, were demonstrated to provide for an enzymatic hydrolysis of N-succinylchitosan. Our studies were carried out on a high-molecular N-succinylchitosan with M of 390 kDa and a substitution degree of 0.8 as a substrate. All the enzymatic preparations were shown to be suitable for the obtaining of low-molecular derivatives of N-succinylchitosan. The complex of enzymes from S. kurssanovii showed the greatest activity: they reduced the characteristic viscosity of initial solution of the substrate by 78% for 30 min. A biodegradation of N-succinylchitosan of various molecular masses was shown to proceed under the action of lysozyme, and the cleavage reaction was revealed to decelerate at a decrease of the polymer molecular mass. A use of N-succinylchitosan in a complex with drugs for a prolongation of their action in a live organism was presumed.     

Chromosome Replication in Toluenized <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cells

BIOCHEMICAL studies of chromosome replication have been hampered by the unavailability of an adequate in vitro system with the basic features of in vivo DNA replication. The criteria for such a system are: (1) semiconservative replication; (2) normal biological activity of newly synthesized DNA; (3) normal advancement of the original replication fork; (4) rate of DNA replication equivalent to in vivo; and (5) expected phenotypic behaviour of temperature-sensitive dna mutants. Systems in Escherichia coli, a membrane-DNA fraction¹, an agar-embedded cell lysate² and toluene-treated cells³ have met two or three of the requirements. Several laboratories have also reported the expected behaviour of ts-dna E. coli mutants in toluenized cells^3–5.     

Interactions Between the Circadian Clock and Heme Oxygenase in the Retina of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The Drosophila retina has an autonomous peripheral circadian clock in which the expression of the gene encoding heme oxygenase (HO) is under circadian control with the ho mRNA peaking at the beginning of the day and in the middle of the night. The function of HO in the retina is unknown, but we observed that it regulates the circadian clock and protects photoreceptors against DNA damage. The decline in HO level increases and decreases the expression of the canonical clock genes period (per) and Clock (Clk), respectively. The opposite result was observed after increasing HO expression. Among three products of HO activity—carbon monoxide (CO), ferrous ions, and biliverdin—the latter has no effect on per and Clk expressions, but CO exerts the same effect as the increase of ho expression. This suggests that HO action on the clock is mediated by CO, which may affect Clk expression during the day and the level of per expression. While ho expression is not stimulated by nitric oxide (NO), NO has the same effect on the clock as HO, increasing Clk expression and decreasing the expression of per.     

A revision of <Emphasis Type="Italic">Sphaeria pilosa</Emphasis> Pers. and re-evaluation of the Trichosphaeriales

Sphaeria pilosa, the basionym of the type of Trichosphaeria, T. pilosa, was insufficiently described by Persoon. The current interpretation of T. pilosa comes from Fuckel and is based on his own material published in the exsiccatal series Fungi Rhen. Exs. no. 946. The examination of the type and other authentic material of S. pilosa and T. pilosa revealed several different fungi. Fresh material of T. pilosa is not available and Fuckel’s historical specimens have never been sampled for DNA. In order to reconcile Persoon’s concept of S. pilosa with Fuckel’s concept of T. pilosa, we designate a neotype in our study. The species is a unitunicate ascomycete characterized by perithecial ascomata, persistent paraphyses, and cylindrical short-stipitate asci without visible apical annulus containing eight hyaline, ellipsoidal ascospores. The genus Trichosphaeria includes 87 described species and the Trichosphaeriales with the single family Trichosphaeriaceae recently accommodate 17 genera of apparently diverse phylogenetic affinity. Although the relationship of Trichosphaeria with other members of the Sordariomycetes is unknown, the family and order based on it are widely used to label incertae sedis clades inferred in phylogenetic analyses. Based on these findings, the Trichosphaeriales are re-evaluated and their use in phylogenetic studies is recommended to be abandoned until recognition of T. pilosa by molecular data.     

Communities of arbuscular mycorrhizal fungi under <Emphasis Type="Italic">Picconia azorica</Emphasis> in native forests of Azores

Arbuscular mycorrhizal fungi (AMF) from the rhizosphere of the endemic Laurisilva tree, Picconia azorica, were characterised at two sites in each of two Azorean islands (Terceira and São Miguel). Forty-six spore morphotypes were found, and DNA extraction was attempted from individual spores of each of these. DNA was obtained from 18 of the morphotypes, from which a 1.5 kb long fragment of the nuclear ribosomal RNA gene (SSU-ITS-LSU) was sequenced. A total of 125 AMF sequences were obtained and assigned to 18 phylotypes. Phylogenetic analysis revealed sequences belonging to the families, Acaulosporaceae, Archaeosporaceae, Claroideoglomeraceae, Gigasporaceae and Glomeraceae. Phylotype richness changed between islands and between sampling sites at both islands suggesting that geographical and historical factors are determinant in shaping AMF communities in native forest of Azores. Ecological analysis of the molecular data revealed differences in AMF community composition between islands. In Terceira, the rhizosphere of P. azorica was dominated by species belonging to Acaulosporaceae and Glomeraceae, while São Miguel was dominated by members of Glomeraceae and Gigasporaceae. This is the first molecular study of AMF associated with P. azorica in native forest of the Azores. These symbiont fungi are key components of the ecosystem. Further research is needed to develop their use as promoters of plant establishment in conservation and restoration of such sites.     

<Emphasis Type="Italic">Bruguiera hainesii</Emphasis>, a critically endangered mangrove species,is a hybrid between <Emphasis Type="Italic">B. cylindrica</Emphasis> and <Emphasis Type="Italic">B. gymnorhiza</Emphasis> (Rhizophoraceae)

Bruguiera hainesii (Rhizophoraceae) is one of the two Critically Endangered mangrove species listed in the IUCN Red List of Threatened Species. Although the species is vulnerable to extinction, its genetic diversity and the evolutionary relationships with other Bruguiera species are not well understood. Also, intermediate morphological characters imply that the species might be of hybrid origin. To clarify the genetic relationship between B. hainesii and other Bruguiera species, we conducted molecular analyses including all six Bruguiera species using DNA sequences of two nuclear genes (CesA and UNK) and three chloroplast regions (intergenic spacer regions of trnL-trnF, trnS-trnG and atpB-rbcL). For nuclear DNA markers, all nine B. hainesii samples from five populations were heterozygous at both loci, with one allele was shared with B. cylindrica, and the other with B. gymnorhiza. For chloroplast DNA markers, the two haplotypes found in B. hainesii were shared only by B. cylindrica. These results suggested that B. hainesii is a hybrid between B. cylindrica as the maternal parent and B. gymnorhiza as the paternal one. Furthermore, chloroplast DNA haplotypes found in B. hainesii suggest that hybridization has occurred independently in regions where the distribution ranges of the parental species meet. As the IUCN Red List of Threatened Species currently excludes hybrids (except for apomictic plant hybrids), the conservation status of B. hainesii should be reconsidered.     

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

Background

The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome’) strategy to expand our understanding of human gene regulation in vivo.

Results

In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear.

Conclusions

We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

     

Mechanism for the Action of λ Exonuclease in Genetic Recombination

Lambda exonuclease degrades in vitro redundant single stranded regions which probably result in λ DNA from genetic recombination in vivo.