Applying a modified metabarcoding approach for the sequencing of macrofungal specimens from fungarium collections.

Premise: Fungaria are an underutilized resource for understanding fungal biodiversity. The effort and cost of producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor-efficient and cost-effective solution for sequencing the fungal DNA barcodes of hundreds of specimens at once. Methods: We applied a two-step PCR approach using nested, barcoded primers to sequence the fungal nrITS2 region of 766 macrofungal specimens using the Illumina platform. The specimens represent a broad taxonomic sampling of the Dikarya. Of these, 382 Lactarius specimens were analyzed to identify molecular operational taxonomic units (MOTUs) using a phylogenetic approach. The raw sequences were trimmed, filtered, assessed, and analyzed using the DADA2 amplicon de-noising toolkit and Biopython. The sequences were compared to the NCBI and UNITE databases and Sanger nrITS sequences from the same specimens. Results: The taxonomic identities derived from the nrITS2 sequence data were >90% accurate across all specimens sampled. A phylogenetic analysis of the Lactarius sequences identified 20 MOTUs. Discussion: The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections to advance fungal diversity identification and documentation.

Antioxidant treatment inhibits activation of myocardial nuclear factor kappa B and inhibits nitrosylation of myocardial heme protein in cardiac transplant rejection.

Nitric oxide production via inducible nitric oxide synthase (iNOS) is believed to play a role in cardiac allograft rejection. Previously, we showed that antioxidants can significantly prolong cardiac graft survival, but the nature of this protection is unknown. In the present study, we examined the protective effect of another antioxidant, dimethylthiourea (DMTU), in a model of cardiac allograft rejection. Specifically, we hypothesized that DMTU would prolong graft survival and decrease activation of nuclear factor-kappa B (NF-kappa B), an important redox-sensitive transcription factor necessary for iNOS gene expression. NF-kappa B was activated by twofold as early as postoperative day 2 in allografts. NF-kappa B activation in allografts progressed to a peak of ninefold by postoperative day and remained increased until postoperative day 6. No activation of NF-kappa B was observed in isografts for comparable time periods. Treatment with DMTU resulted in a significant prolongation of graft survival. This beneficial effect was associated with diminished activation of myocardial NF-kappa B. Treatment with DMTU also resulted in decreased formation of iron-nitrosylprotein complexes as evidenced by electron paramagnetic resonance spectroscopy. These studies provide evidence that reactive oxygen plays a significant role in signal transduction for activation via the transcription factor, NF-kappa B, thereby modulating distal actions and consequences of iNOS-derived nitric oxide.

A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge.

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150microg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150microg rPA, 150microg rPA+150microg of a conjugated 10-mer peptide representing the Bacillus anthracis capsule (conj), or 150microg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA+conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p=0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA+conj immunized groups (p=0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later.