Genome Annotation of Bacteriophages Allegro and Moostard

During the spring semester of the one-year-long HHMI SEA-PHAGES experience, we annotated the sequenced genomes of two novel Mycobacterium smegmatis bacteriophages, Allegro and Moostard. Allegro is a member of the Mycobacterium Cluster B and a member of the sub-cluster B2. Allegro’s genome is 67,439 bp in length and has approximately 92 predicted genes. Moostard is a member of Mycobacterium Cluster L and a member of the sub-cluster L3. Its genome is 69,480 bp in length and has approximately 108 predicted genes. Using DNA Master, Phamerator, and Starterator, along with homology comparisons to databases at HHPred and NCBI, the putative open reading frames were assessed for designated location and function. Multiple students called each gene allowing for an independent assessment of the reading frames. The data from all students were merged and assessed. Moostard encoded for integrase proteins which suggest a temperate lifestyle. Allegro did not contain an integrase protein but did encode for a putative immunity suppressor protein. The comparative analysis of phage genomes helps expand our understanding of the genomic diversity of bacteriophages found in this region of South Carolina and successfully introduces a genuine research experience to students in their first year of undergraduate studies at Winthrop University

Genome Annotation of Bacteriophages Allegro and Moostard

During the spring semester of the one-year-long HHMI SEA-PHAGES experience, we annotated the sequenced genomes of two novel Mycobacterium smegmatis bacteriophages, Allegro and Moostard. Allegro is a member of the Mycobacterium Cluster B and a member of the sub-cluster B2. Allegro’s genome is 67,439 bp in length and has approximately 92 predicted genes. Moostard is a member of Mycobacterium Cluster L and a member of the sub-cluster L3. Its genome is 69,480 bp in length and has approximately 108 predicted genes. Using DNA Master, Phamerator, and Starterator, along with homology comparisons to databases at HHPred and NCBI, the putative open reading frames were assessed for designated location and function. Multiple students called each gene allowing for an independent assessment of the reading frames. The data from all students were merged and assessed. Moostard encoded for integrase proteins which suggest a temperate lifestyle. Allegro did not contain an integrase protein but did encode for a putative immunity suppressor protein. The comparative analysis of phage genomes helps expand our understanding of the genomic diversity of bacteriophages found in this region of South Carolina and successfully introduces a genuine research experience to students in their first year of undergraduate studies at Winthrop University

Isolating, Purifying, and Characterizing Mycobacterium Bacteriophages Collected from Soil on the Winthrop University Campus

This was Winthrop University’s sixth year as part of the national HHMI sponsored SEA-PHAGES program. As freshman undergraduate students we isolated, purified, and characterized 4 bacteriophages found in the soil in and around Winthrop University, Rock Hill, South Carolina. COVID-19 limited the number of students in the laboratory at any given time, but working in pairs and eventually teams, we were able to isolate and culture four soil bacteriophages. Bacteriophages are viruses that infect bacterial host cells. They require a bacterial cell to reproduce and are specific to a particular host cell. Each phage was purified and amplified in the bacterial host Mycobacterium smegmatis mc2155. Following isolation, the phages were characterized following DNA extraction using restriction enzyme digests and gel electrophoresis. Calendula, Grindelwald, Allegro, and Moostard high-titer lysates were archived for future study. Two phages, Allegro and Moostard, were chosen to be sequenced at the University of Pittsburgh. The genome sequences will be returned to Winthrop for annotation. This research not only adds to the increasing knowledge of novel phages being discovered in this area of South Carolina but also allows Winthrop University and its students as members of an internationally renowned research program

Isolating, Purifying, and Characterizing Mycobacterium Bacteriophages Collected from Soil on the Winthrop University Campus

This was Winthrop University’s sixth year as part of the national HHMI sponsored SEA-PHAGES program. As freshman undergraduate students we isolated, purified, and characterized 4 bacteriophages found in the soil in and around Winthrop University, Rock Hill, South Carolina. COVID-19 limited the number of students in the laboratory at any given time, but working in pairs and eventually teams, we were able to isolate and culture four soil bacteriophages. Bacteriophages are viruses that infect bacterial host cells. They require a bacterial cell to reproduce and are specific to a particular host cell. Each phage was purified and amplified in the bacterial host Mycobacterium smegmatis mc2155. Following isolation, the phages were characterized following DNA extraction using restriction enzyme digests and gel electrophoresis. Calendula, Grindelwald, Allegro, and Moostard high-titer lysates were archived for future study. Two phages, Allegro and Moostard, were chosen to be sequenced at the University of Pittsburgh. The genome sequences will be returned to Winthrop for annotation. This research not only adds to the increasing knowledge of novel phages being discovered in this area of South Carolina but also allows Winthrop University and its students as members of an internationally renowned research program

Immunogenicity and efficacy of vaccine boosters against SARS-CoV-2 Omicron subvariant BA.5 in male Syrian hamsters

Abstract The SARS-CoV-2 Omicron subvariant BA.5 rapidly spread worldwide and replaced BA.1/BA.2 in many countries, becoming globally dominant. BA.5 has unique amino acid substitutions in the spike protein that both mediate immune escape from neutralizing antibodies produced by immunizations and increase ACE2 receptor binding affinity. In a comprehensive, long-term (up to 9 months post primary vaccination), experimental vaccination study using male Syrian hamsters, we evaluate neutralizing antibody responses and efficacy against BA.5 challenge after primary vaccination with Ad26.COV2.S (Janssen) or BNT162b2 (Pfizer/BioNTech) followed by a homologous or heterologous booster with mRNA-1273 (Moderna) or NVX-CoV2373 (Novavax). Notably, one high or low dose of Ad26.COV2.S provides more durable immunity than two primary doses of BNT162b2, and the NVX-CoV2373 booster provides the strongest augmentation of immunity, reduction in BA.5 viral replication, and disease. Our data demonstrate the immunogenicity and efficacy of different prime/boost vaccine regimens against BA.5 infection in an immune-competent model and provide new insights regarding COVID-19 vaccine strategies

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive

Vaccination Decreases the Infectious Viral Load of Delta Variant SARS-CoV-2 in Asymptomatic Patients

The Delta variant of SARS-CoV-2 has caused many breakthrough infections in fully vaccinated individuals. While vaccine status did not generally impact the number of viral RNA genome copies in nasopharyngeal swabs of breakthrough patients, as measured by Ct values, it has been previously found to decrease the infectious viral load in symptomatic patients. We quantified the viral RNA, infectious virus, and anti-spike IgA in nasopharyngeal swabs collected from individuals asymptomatically infected with the Delta variant of SARS-CoV-2. Vaccination decreased the infectious viral load, but not the amount of viral RNA. Furthermore, vaccinees with asymptomatic infections had significantly higher levels of anti-spike IgA in their nasal secretions compared to unvaccinated individuals with asymptomatic infections. Thus, vaccination may decrease the transmission risk of Delta, and perhaps other variants, despite not affecting the amount of viral RNA measured in nasopharyngeal swabs

The NSP3 protein of SARS-CoV-2 binds fragile X mental retardation proteins to disrupt UBAP2L interactions

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome

Positivity of fecal samples harvested from ten rabid <i>Parastrellus hesperus</i> carcasses and tested at time 0 and after 24 hours at ambient conditions.

Positivity of fecal samples harvested from ten rabid Parastrellus hesperus carcasses and tested at time 0 and after 24 hours at ambient conditions.</p