Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models.

In all retroviruses, reverse transcription of viral genes is primed by a host‐cell tRNA whose 3′‐end 18 nucleotides are complementary to the viral primer binding site. For lentiviruses, including HIV, the natural primer is tRNALys3 which harbors several modified bases in its anticodon stem‐loop (ASL). Interaction of the fully modified ASL with the HIV RNA template is required for efficient reverse transcription of viral RNA, and continuation of viral life cycle. One of the modified nucleosides of the ASL of tRNALys3 is N6‐threonylcarbamoyl adenosine (t6A37), a universally conserved modification found at position 37 in all tRNAs decoding ANN codons. t6A37 stabilizes the ASL structure and prevents ribosomal frameshifting in the course of translation. Eukaryotic t6A37 biosynthesis is catalyzed by the enzyme Sua5 and the KEOPS complex, comprised of the four subunits Kae1, Pcc1, Bud32 and Cgi121.We hypothesize that t6A37 plays an important role in viral replication in vivo, by stabilizing ASL structure or by direct interaction with viral RNA. To test this hypothesis, we set out to suppress the biosynthesis of t6A37 in CD4+ cells by targeting its biosynthesis genes with siRNA and testing viral load upon HIV‐1 infection. Background expression levels of the core human t6A37 biosynthesis gene Osgep (Kae1 ortholog) were determined by qRT‐PCR. Treatment with anti‐OSGEP siRNA resulted in knockdown of Osgep expression by 60% 48 hrs post‐transfection and sustained for 72 hrs, 4 times longer than the reported half‐life of OSGEP protein in human cells.

In this study, sixteen unique staphylococcal enterotoxin B (SEB)-reactive nanobodies (nbs), including ten monovalent and six bivalent nbs, were developed. All characterized nbs were highly specific for SEB and did not cross-react with other staphylococcal enterotoxins (SE). Several formats of highly sensitive enzyme-linked immunosorbent assays (ELISAs) were established using SEB nbs and a polyclonal antibody (pAb). The lowest limit of detection (LOD) reached 50 pg/mL in PBS. When applied to an ELISA to detect SEB-spiked milk (a commonly contaminated foodstuff), a LOD as low as 190 pg/mL was obtained. The sensitivity of ELISA was found to increase concurrently with the valency of nbs used in the assay. In addition, a wide range of thermal tolerance was observed among the sixteen nbs, with a subset of nbs, SEB-5, SEB-9, and SEB-62, retaining activity even after exposure to 95 °C for 10 min, whereas the conventional monoclonal and polyclonal antibodies exhibited heat-labile properties. Several nbs demonstrated a long shelf-life, with one nb (SEB-9) retaining 93% of its activity after two weeks of storage at room temperature. In addition to their usage in toxin detection, eleven out of fifteen nbs were capable of neutralizing SEB’s super-antigenic activity, demonstrated by their inhibition on IL-2 expression in an ex vivo human PBMC assay. Compared to monoclonal and polyclonal antibodies, the nbs are relatively small, thermally stable, and easy to produce, making them useful in applications for sensitive, specific, and cost-effective detection and management of SEB contamination in food products.

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential; but the mechanism(s) regulating A. fumigatus germination in the airways are unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine lung based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named Lung Homogenate Evolved (LH-EVOL)). We identified a potential loss of function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decrease ability to induce the SakA-dependent stress pathway. In support of the whole genome variant analyses, sskA, sakA, or mpkC loss of function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA MAPK pathway and drives enhanced disease initiation and inflammation in the lungs.