No abstract

Most studies focusing on detecting microorganisms in air by polymerase chain reaction (PCR) have used a liquid impinger to sample bioaerosols, mainly because a liquid sample is easy to be processed by PCR analysis. Nevertheless, the use of multiple-hole impactors for the analysis of bioaerosols by PCR has not been reported despite its great utility in culture analysis. In this study we have modified the impaction onto an agar surface sampling method to impaction onto a liquid medium using the MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). To evaluate the recovery of airborne microorganisms of both sampling methods, a suspension containing Escherichia coli was artificially aerosolized and bioaerosols were collected onto Tergitol-7 agar and phosphate-buffered saline (PBS) with the MAS-100. A linear regression analysis of the results showed a strong positive correlation between both sampling methods (r = 0.99, slope 0.99, and y intercept 0.07). Afterwards, the method of impingement into a liquid medium was used to study airborne Legionella pneumophila by PCR. A total of 64 samples were taken at a wastewater treatment plant, a chemical plant, and an office building and analyzed by culture and PCR. Results showed that three samples were positive both by PCR and plate culture, and that nine samples negative by plate culture were positive by PCR, proving that L. pneumophila was present in bioaerosols from these three different environments. The results demonstrate the utility of this single-stage multiple-hole impactor for sampling bioaerosols, both by culture and by PCR.

No abstract

A large number of cytokines and growth factors support the development and subsequent maintenance of postnatal motor neurons. RegIII␤, also known as Reg2 in rat and HIP/PAP1 in humans, is a member of a family of growth factors found in many areas of the body and previously shown to play an important role in both the development and regeneration of subsets of motor neurons. It has been suggested that RegIII␤ expressed by motor neurons is both an obligatory intermediate in the downstream signaling of the leukemia inhibitory factor/ciliary neurotrophic factor (CNTF) family of cytokines, maintaining the integrity of motor neurons during development, as well as a powerful influence on Schwann cell growth during regeneration of the peripheral nerve. Here we report that in mice with a deletion of the RegIII␤ gene, motor neuron survival was unaffected up to 28 weeks after birth. However, there was no CNTF-mediated rescue of neonatal facial motor neurons after axotomy in KO animals when compared with wild-type. In mice, RegIII␤ positive motor neurons are concentrated in cranial motor nuclei that are involved in the patterning of swallowing and suckling. We found that suckling was impaired in RegIII␤ KO mice and correlated this with a significant delay in myelination of the hypoglossal nerve. In summary, we propose that RegIII␤ has an important role to play in the developmental fine-tuning of neonatal motor behaviors mediating the response to peripherally derived cytokines and growth factors and regulating the myelination of motor axons.Schwann cells ͉ suckling ͉ hypoglossal nerve ͉ LIF

Friedreich's ataxia (FRDA) is the most common form of hereditary ataxia caused by recessive mutations in the FXN gene. Recent results have indicated the presence of different frataxin isoforms due to alternative gene expression mechanisms. Our previous studies demonstrated the advantages of using high-capacity herpes simplex virus type 1 (HSV-1) amplicon vectors containing the entire FXN genomic locus (iBAC-FXN) as a gene-delivery vehicle capable of ensuring physiologically-regulated and long-term persistence. Here we describe how expression from the 135 kb human FXN genomic locus produces the three frataxin isoforms both in cultured neuronal cells and also in vivo. Moreover, we also observed the correct expression of these frataxin isoforms in patient-derived cells after delivery of the iBAC-FXN. These results lend further support to the potential use of HSV-1 vectors containing entire genomic loci whose expression is mediated by complex transcriptional and posttranscriptional mechanisms for gene therapy applications.

Most studies focusing on detecting microorganisms in air by polymerase chain reaction (PCR) have used a liquid impinger to sample bioaerosols, mainly because a liquid sample is easy to be processed by PCR analysis. Nevertheless, the use of multiple-hole impactors for the analysis of bioaerosols by PCR has not been reported despite its great utility in culture analysis. In this study we have modified the impaction onto an agar surface sampling method to impaction onto a liquid medium using the MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). To evaluate the recovery of airborne microorganisms of both sampling methods, a suspension containing Escherichia coli was artificially aerosolized and bioaerosols were collected onto Tergitol-7 agar and phosphate-buffered saline (PBS) with the MAS-100. A linear regression analysis of the results showed a strong positive correlation between both sampling methods (r = 0.99, slope 0.99, and y intercept 0.07). Afterwards, the method of impingement into a liquid medium was used to study airborne Legionella pneumophila by PCR. A total of 64 samples were taken at a wastewater treatment plant, a chemical plant, and an office building and analyzed by culture and PCR. Results showed that three samples were positive both by PCR and plate culture, and that nine samples negative by plate culture were positive by PCR, proving that L. pneumophila was present in bioaerosols from these three different environments. The results demonstrate the utility of this single-stage multiple-hole impactor for sampling bioaerosols, both by culture and by PCR.

Herpes simplex virus 1 (HSV-1)-derived amplicon vectors are unique in their ability to accommodate large DNA molecules allowing whole genomic loci to be included with all of their regulatory elements. Additional advantages of these amplicons include their minimal toxicity and ability to persist as episomes, with negligible risk of insertional mutagenesis, being particularly well-suited for gene therapy of neurological disorders due to their outstanding ability to deliver genes into neurons and other neural cells. However, extensive gene therapy application has been hindered by difficulties in vector production. This work improved HSV-1 amplicons production by genetic modification of the packaging cell line and optimization of the culture medium. A stably-transfected Vero 2-2 cell line overexpressing the anti-apoptotic Bcl-2 protein was generated, exhibiting an increased resistance to apoptosis, prolonged culture duration, and a significant improvement in viral vector production. Additionally, supplementation of the growth medium with antioxidants, polyamines, amino acids, and reduced glutathione further increased the yield of packaged amplicon vectors. With these modifications, HSV-1 amplicons could be isolated from culture supernatants instead of cell lysates, leading to vector preparations with higher titer and purity and paving the way for generation of stable cell lines that are capable of continuous herpesviral vector production.

Hemophilia A is caused by mutations in the gene encoding factor VIII (F8) and is an important target for gene therapy. The F8 gene contains 26 exons spread over approximately 186 kb and no work using the intact genomic locus has been carried out. We have constructed a 250-kb BAC carrying all 26 exons, the introns, and more than 40 kb of upstream and 20 kb of downstream DNA. This F8 BAC was further retrofitted with either the oriP/EBNA-1 elements from Epstein-Barr virus, which allow episomal maintenance in mammalian cells, or alphoid DNA, which allows human artificial chromosome formation in some human cell lines. Lipofection of the oriP/EBNA-1-containing version into mouse Hepa1-6 cells resulted in expression of F8 mRNA spanning the F8 gene. The >300-kb BAC carrying alphoid DNA was successfully delivered to 293A and HT1080 cells using bacterial delivery, resulting in greater than endogenous levels of F8 mRNA expression.