Inactivation of bacterial endospores on surfaces by plasma processed air.

AIMS: In case of biological hazards and pandemics, personal protective equipment of rescue forces is currently manually decontaminated with harmful disinfectants, primarily peracetic acid. To overcome current drawbacks regarding supply, handling and disposal of chemicals, the use of plasma processed air (PPA) represents a promising alternative for surface decontamination on site. In this study, the sporicidal efficiency of a portable plasma system, designed for field applications, was evaluated. METHODS AND RESULTS: The developed plasma device is based on a dielectric barrier discharge (DBD) and operated with ambient air as process gas. PPA from the plasma nozzle was flushed into a treatment chamber (volume: 300 l) and bacterial endospores (Bacillus subtilis and Bacillus atrophaeus) dried on different surfaces were treated under variable conditions. Reductions in spores by more than 4 log10 were found within 3 min of PPA exposure. However, the presence of endospores in agglomerates or in an organic matrix as well as the complexity of the respective surface microstructure negatively affected the inactivation efficiency. When endospores were embedded in a dried protein matrix, mechanical wiping with swabs during exposure to PPA increased the inactivation effect significantly. Gaseous ozone alone did not provide a sporicidal effect. Significant spore inactivation was only obtained when water vapour was injected into the PPA stream. CONCLUSION: The results show that endospores dried on surfaces can be reduced by several orders of magnitude within few minutes in a treatment chamber which is flushed with PPA from of a DBD plasma nozzle. SIGNIFICANCE AND IMPACT OF THE STUDY: Plasma processed air generated on site by DBD plasma nozzles could be a suitable alternative for the disinfection of various surfaces in closed rooms.

Comparison of pre- and post-implantation development following the application of three artificial activating stimuli in a mouse model with round-headed sperm cells deficient for oocyte activation.

STUDY QUESTION: Does the application of three different artificial activating stimuli lead to a difference in pre- and post-implantation embryo development in the wobbler mouse, a mouse model with oocyte activation deficient round-headed sperm cells similar to human globozoospermia? SUMMARY ANSWER: No gross differences were found between strontium chloride, electrical pulses or ionomycin with respect to the pre- and post-implantation development in the wobbler mouse. WHAT IS KNOWN ALREADY: Fertilization failure following intra-cytoplasmic sperm injection (ICSI) occurs in 1-3% of the ICSI cycles in human assisted reproduction technology (ART) and has been successfully overcome by different artificial activating stimuli. No comparison has been made yet in terms of their efficiency and safety. STUDY DESIGN, SIZE, DURATION: Calcium release and embryo development were compared between oocytes fertilized by wobbler and wild-type (WT) sperm following ICSI with or without three different artificial activating agents. Preimplantation development was assessed on 70 injected oocytes on average per group. On average, 10 foster mothers were used per activating group to compare post-implantation development. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used the wobbler mouse model that possesses oocyte activation deficient round-headed sperm cells. First, the calcium release following ICSI using wobbler sperm was compared with that of WT sperm. Outcome measures were the percentage of oocytes that showed calcium release and their mean amount of calcium rises. Secondly, the pre- and post-implantation development was assessed following ICSI with wobbler sperm plus artificial oocyte activation using either: (i) strontium chloride (Wob-Sr), (ii) electrical pulses (Wob-E) or (iii) ionomycin (Wob-I). Outcome measures were the activation, cleavage and blastocyst rates and the assessment of blastocyst quality by differential staining. Following mouse embryo transfer, pregnancy and birth rates as well as mean litter sizes were examined. Finally, pups were followed up until 8 weeks of age and then mated with fertile controls to assess their fertility. MAIN RESULTS AND THE ROLE OF CHANCE: The percentage of oocytes showing calcium rises as well as the number of calcium rises per oscillating oocyte were significantly lower in the wobbler group when compared with the WT group (9.3 versus 96% and 2.1 calcium rises versus 31 calcium rises) (P < 0.001). The fertilization rate was significantly lower in the wobbler group (11.4%) when compared with the WT group (92.1%) and the artificial activation groups (strontium chloride: 99%, electrical pulses: 99% and ionomycin: 81%, respectively) (P < 0.001). Post-implantation development did not differ significantly between the WT and artificial activation groups, with pregnancy rates in favor of strontium chloride and electrical pulses. The weight of the male pups did not differ between the study groups, whereas the weight of the female pups originating from Wob-Sr embryos was significantly lower at weeks 2, 3 and 4 when compared with female pups originating from WT embryos. However, the latter difference was not observed at later time points, nor in the other artificial activating groups. All offspring mated successfully with fertile controls. LIMITATIONS, REASONS FOR CAUTION: Results in animal models should be extrapolated with caution to a subfertile human population. Also, ionomycin is currently the most widely used artificial oocyte activating agent in human ART. WIDER IMPLICATIONS OF THE FINDINGS: The low frequency of observed calcium rises and the low activation rate make the wobbler mouse a highly suitable model to study oocyte activation deficiency. Strontium chloride and electrical pulses were more efficient means to restore fertilization rates and to support pre- and post-implantation embryonic development than ionomycin. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Flemish foundation of Scientific Research (FWO-Vlaanderen) (aspirant clinical research mandate to F.V.M., fundamental clinical research mandate to P.D.S.); and Ghent University grant (KAN-BOF E/01321/01 to B.H.). The authors have no competing interests to declare.

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Alpha-crystallins are involved in specific interactions with the murine gamma D/E/F-crystallin-encoding gene.

The promoter of the murine gamma E-crystallin (gamma E-Cry) encoding gene (gamma E-cry) was analyzed for specific interactions with lenticular proteins in a gel-retardation assay. A 21-bp fragment immediately downstream of the transcription initiation site (DOTIS) is demonstrated to be responsible for specific interactions with lens extracts. The DOTIS-binding protein(s) accept only the sense DNA strand as target; anti-sense or double-stranded DNA do not interact with these proteins. The DOTIS sequence element is highly conserved among the murine gamma D-, gamma E- and gamma F-cry and is present at comparable positions in the orthologous rat genes. Only a weak or even no protein-binding activity is observed if a few particular bases are changed, as in the rat gamma A-, gamma C- and gamma E-cry elements. DOTIS-binding proteins were found in commercially available bovine alpha-Cry preparations. The essential participation of alpha-Cry in the DNA-binding protein complex was confirmed using alpha-Cry-specific monoclonal antibody. The results reported here point to a novel function of alpha-Cry besides the structural properties in the lens.

Conservation of the 3'-untranslated region of the Rab1a gene in amniote vertebrates: exceptional structure in marsupials and possible role for posttranscriptional regulation.

The YPT1/RAB1 protein, a key regulator of the intracellular vesicle transport in eukaryotes, is highly conserved in function and amino acid sequence. Here we report that the most highly conserved nucleotide sequence of the Rab1a gene of amniote vertebrates corresponds to the 3'-untranslated region (3'-UTR) of the mRNA. Sequences of 27 species ranging from mammals to sauropsida are >91% identical in this region. Secondary structure prediction procedures applied to the 3'-UTR sequences between positions 750 and 984 and 1428 (mouse cDNA: Y00094), respectively, of the RAB1a mRNAs revealed families of alternative structures around nucleotide position 800 as recurrent features. The two hairpin loops are also predicted for marsupials, despite of their exceptional extension of the A-rich sequence in between. Yet, sequence conservation is much higher than required to conserve secondary structure. Implications for posttranscriptional regulation and protein binding are discussed.

Spatiotemporal progression of neurodegeneration and glia activation in the wobbler neuropathy of the mouse.

The wobbler mouse (phenotype WR; genotype wr/wr) has been investigated as a model for neurodegenerative diseases like SMA and ALS. A new diagnostic marker based on a polymorphism in the closely linked chaperonine gene Cct4 enabled us to diagnose the allelic status at the wr locus within the original background strain C57BL/6. Using this marker, we investigated the spatiotemporal progression of neuropathology in WR mice from postnatal day (d.p.n.) 10 to 60. Neurodegeneration starts at 13 d.p.n. in the thalamus (N. ventralis), in deep cerebellar nuclei, brain stem (N. vestibularis) and spinal cord interneurons. The motor nuclei of spinal nerves and motoneurons degenerate from 15 d.p.n. onward. Reactive astrocytes are observed around 17 d.p.n. in the white and grey matter of the spinal cord. Microgliosis occurs only from 23 d.p.n. onward. Our data demonstrate that in the WR disease, neurodegeneration in thalamus, cerebellum, and brain stem precedes motoneuron degeneration, astrogliosis and microgliosis.

Promoter constructions for efficient secretion expression in Streptomyces lividans.

Promoters from different Streptomyces genes were cloned in front of the Tendamistat gene from S. tendae, in order to study secretion-expression in S. lividans using a pIJ702 plasmid vector system. Besides the promoters we cloned a transcriptional terminator downstream of the Tendamistat gene to improve transcription efficiency. The promoters we selected were: (1) a synthetic Escherichia coli-like consensus promoter; (2) the aphI promoter of the neomycin resistance gene from S. fradiae; (3) an ermE-up promoter mutant from Saccharopolyspora erythraea; (4) the melC promoter of the tyrosinase operon from Streptomyces antibioticus. In addition, we tested the thiostrepton-inducible tipA promoter from S. lividans in our Tendamistat secretion system. The promoters were cloned upstream of the Tendamistat ribosome binding site in order to conserve the original translation initiation. The Tendamistat secretion mediated by the different promoter constructions above varied dramatically in up to 10 mg/l in the case of the synthetic promoter and the aph promoter, and up to 500 mg/l mediated by the ermE-up promoter. The melC promoter allowed about 200 mg/l Tendamistat secretion and the tipA promoter proved to be inducible from less than 0.5 mg/l up to 40 mg/l of Tendamistat secretion. Based on the amount of secreted Tendamistat and on the analysis of mRNA levels, we conclude that transcriptional activity regulates the efficiency of our secretion-expression system.

Cat3vl and Cat3vao cataract mutations on mouse chromosome 10: phenotypic characterization, linkage studies and analysis of candidate genes.

Cat3vl and Cat3vao are two allelic, dominant cataract mutations that arose independently in the F1 generation after gamma-irradiation of male mice. The cataracts are already present at birth. Examination of the eyes with a slit lamp revealed completely vacuolated lenses in Cat3vl mutants and anteriorly located opacity in Cat3vao mutants. The appearance of the opacities does not differ between the individuals or between heterozygotes and homozygotes. Penetrance of the mutations is complete. Viability and fertility of the mutants are normal except in the case of the Cat3vl homozygotes. Cat3vao was assigned to the distal part of mouse chromosome 10, 3.2 +/- 0.9 cM away from the visible marker Steel (SlgbH). Using polymorphic markers the following locus order was found: D10Mit230-(0.2 +/- 0.1 cM)-Cat3vao-(2.5 +/- 0.6 cM)-D10Mit70. No recombinants were found between Cat3vao and the markers D10Mit4l and D10Mit95 among 921 offspring. The results exclude allelism of Cat3vao with CatLop or To2, which also map to chromosome 10. Candidate genes were tested by examination of their expression in the eye of newborn mice and by analysis of cDNA sequences. So far, negative results have been obtained for the genes encoding the proteoglycans lumican and decorin, the nuclear orphan receptor Tr2-11 and the transcription factor Elk3. Based on syntenic homology of the Cat3 region to the human chromosome 12q, the Cat3 mutants are discussed as mouse models for cornea plana congenita in man. The recovery of the Cat3 mutations demonstrates the importance of the corresponding locus for proper eye development.

Close linkage of the dominant cataract mutations (Cat-2) with Idh-1 and cryge on mouse chromosome 1.

The murine dominant gene Cat-2 was located on chromosome 1 between the loci of fuzzy and leaden. Subsequent linkage analysis revealed one recombinant between Cat-2t and isocitrate dehydrogenase-1, and one between Cat-2t and gamma E-crystallin among 338 offspring in three-point backcrosses. The resulting genetic distance between the loci is 0.3 +/- 0.3 cM. The very close linkage between the Cat-2 and the gamma-crystallin gene cluster together with the finding of reduced gamma-crystallin transcripts in mutant lenses suggest strongly that the gamma-crystallin genes may be candidate genes for the Cat-2 mutations.

Integrated radiation hybrid map of human chromosome 2p13: possible involvement of dynactin in neuromuscular diseases.

The genes for the human neuromuscular diseases limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy are located on chromosome 2p13-p14, and two neuromuscular mutations of the mouse have been mapped to regions homologous to human chromosome 2p13 by conserved synteny, wobbler (wr) on proximal Chr 11 and motor neuron degeneration 2 (mnd2) on Chr 6. Neither one is a mouse homologue of LGMD2B. Recently the gene DCTN1, coding for the large subunit of the cytoskeletal protein dynactin, was shown by FISH to be located in this region and therefore should be considered a candidate for all these disease genes. Here we present mapping data based on radiation hybrid and physical mapping that more precisely define the location of nine genetic markers in the critical region and the homology relationship of human chromosome 2p with mouse proximal Chr 11 and Chr 6. The human dynactin gene was mapped between markers TGFA and D2S1394, implying that the mouse dynactin gene Dctn1 is located on Chr 6, distal to mnd2. Thus DCTN1/Dctn1 is a candidate for LGMD2B but not for mnd2 or wr.

Isolation and characterization of the novel popeye gene family expressed in skeletal muscle and heart.

We identified a novel gene family in vertebrates which is preferentially expressed in developing and adult striated muscle. Three genes of the Popeye (POP) family were detected in human and mouse and two in chicken. Chromosomal mapping indicates that Pop1 and Pop3 genes are clustered on mouse chromosome 10, whereas Pop2 maps to mouse chromosome 16. We found evidence that POP1 and POP3 in chicken may also be linked and multiple transcript isoforms are generated from this locus. The POP genes encode proteins with three potential transmembrane domains that are conserved in all family members. Individual POP genes exhibit specific expression patterns during development and postnatally. Chicken POP3 and mouse Pop1 are first preferentially expressed in atrium and later also in the subepicardial compact layer of the ventricles. Chicken POP1 and mouse Pop2 are expressed in the entire heart except the outflow tract. All three Pop genes are expressed in heart and skeletal muscle of the adult mouse and lower in lung. Pop1 and Pop2 expression is upregulated in uterus of pregnant mice. Like the mouse genes, human POP genes are predominantly expressed in skeletal and cardiac muscle. The strong conservation of POP genes during evolution and their preferential expression in heart and skeletal muscle suggest that these novel proteins may have an important function in these tissues in vertebrates.

Homology between human chromosome 2p13.3 and the wobbler critical region on mouse chromosome 11: comparative high-resolution mapping of STS and EST loci on YAC/BAC contigs.

Human Chr 2p13-14 and homologous regions on mouse Chrs 6 and 11 have been subjects of previous studies because they comprise the loci for several neuromuscular diseases. Here we report on high-resolution mapping of 55 STS and EST loci on human Chr 2p13.3 and of 47 markers on the corresponding region on proximal mouse Chr. 11. The maps comprise several known genes, MEIS1/Meis1, RAB1a/Rab1a, MDH1/Mor2, OTX1/Otx1, and REL on human 2p13.3 and mouse Chr 11, respectively, as well as the wobbler (wr) critical region of the mouse. Whereas a perfect correspondence was found in most of the 4-Mb region, a small rearrangement was discovered around the OTX1/Otx1 locus. The detailed STS and EST transcript maps of these regions and a further narrowing down of the mouse wr critical region to the interval between D11Mit79 and D11Mit19 allow for the selection of positional candidate genes for wr, and the exclusion of others.