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The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Background Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of health-care associated infections, particularly bloodstream infections (BSI). Concomitant colonization of the anterior nares has been shown to have implications on persistence, recurrence, and transmission events. We performed genomic comparisons between paired nasal and blood MRSA isolates obtained from the same patient to elucidate the genetic differences that may contribute to within-host invasion and virulence. Methods Patients hospitalized with MRSA bloodstream infection were swabbed in the bilateral nares. Paired blood and nasal isolates underwent DNA extraction and spa typing. Nasal and blood isolates from the same patients with matching spa types were sequenced by Illumina and PacBio respectively. For genomic analysis, PathoSPOT and Snippy were used to compare genomes of nasal and blood isolate pairs to evaluate single nucleotide polymorphisms, insertions and deletions. Results Of 121 patients with MRSA BSI, 87 patients (72%) had concomitant colonization of the anterior nares with MRSA. Spa type of t002 predominated in both blood and nasal isolates. From the 76 pairs that underwent genomic analysis, several mutated genes were identified across all pairwise comparisons, 8 of which recurred in more than one patient pair. These included genes involved in conferring antibiotic resistance, increased mutability, immune evasion, and adaptive metabolism. Conclusion In patients with MRSA BSI and concomitant MRSA nasal colonization, mutations identified by genomic analysis in blood-nasal pairs are implicated in contributing to invasion and within-host adaptability. Disclosures All Authors: No reported disclosures.

Objectives As part of an active MRSA surveillance programme in our neonatal ICU, we identified nares surveillance cultures from two infants that displayed heterogeneity in methicillin resistance between isolated subclones that lacked mecA and mecC. Methods The underlying mechanism for the modified Staphylococcus aureus (MODSA) methicillin-resistance phenotype was investigated by WGS. Results Comparison of finished-quality genomes of four MODSA and four MSSA subclones demonstrated that the resistance changes were associated with unique truncating mutations in the gene encoding the cyclic diadenosine monophosphate phosphodiesterase enzyme GdpP or a non-synonymous substitution in the gene encoding PBP2. Conclusions These two cases highlight the difficulty in identifying non-mecA, non-mecC-mediated MRSA isolates in the clinical microbiology laboratory, which leads to difficulties in implementing appropriate therapy and infection control measures.

Modified mRNA (modRNA) is a gene-delivery platform for transiently introducing a single gene or several genes of interest to different cell types and tissues. modRNA is considered to be a safe vector for gene transfer, as it negligibly activates the innate immune system and does not compromise the genome integrity. The use of modRNA in basic and translational science is rising, due to the clinical potential of modRNA. We are currently using modRNA to induce cardiac regeneration post-ischemic injury. Major obstacles in using modRNA for cardiac ischemic disease include the need for the direct and single administration of modRNA to the heart and the inefficient translation of modRNA due to its short half-life. Modulation of the 5′ untranslated region (5′ UTR) to enhance translation efficiency in ischemic cardiac disease has great value, as it can reduce the amount of modRNA needed per delivery and will achieve higher and longer protein production post-single delivery. Here, we identified that 5′ UTR, from the fatty acid metabolism gene carboxylesterase 1D (Ces1d), enhanced the translation of firefly luciferase (Luc) modRNA by 2-fold in the heart post-myocardial infarction (MI). Moreover, we identified, in the Ces1d, a specific RNA element (element D) that is responsible for the improvement of modRNA translation and leads to a 2.5-fold translation increment over Luc modRNA carrying artificial 5′ UTR, post-MI. Importantly, we were able to show that 5′ UTR Ces1d also enhances modRNA translation in the liver, but not in the kidney, post-ischemic injury, indicating that Ces1d 5′ UTR and element D may play a wider role in translation of protein under an ischemic condition., Graphical Abstract, The use of modified mRNA (modRNA) in basic and translational science is rising. Major obstacles in using modRNA are its inefficient translation and short half-life. Here, we identified that 5′ UTR from the Ces1d gene enhanced the translation of modRNA by 2-fold in the heart or liver post-ischemic injury.

The COVID-19 pandemic sparked rapid development of SARS-CoV-2 diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines RT-PCR and MALDI-TOF mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified dataset of 1,262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 through April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly-specific for the alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Active surveillance in our neonatal intensive care unit identified Staphylococcus aureus cultures from two infants with heterogeneity in methicillin resistance between isolated subclones lacking mecA and mecC. Whole-genome analysis of 4 modified (MODSA) and 4 methicillin-susceptible (MSSA) subclones for each culture identified either truncating mutations in the cyclic diadenosine monophosphate phosphodiesterase enzyme (GdpP), or a nonsynonymous substitution in penicillin binding protein 2 (PBP2). These cases highlight the difficulty in identifying non-mecA/non-mecC-mediated methicillin-resistance in clinical laboratories.

Blighted Gotham Deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in New York City (NYC) during the spring of 2020 have vastly exceeded those reported in China and many other countries. What were the early events that led to such a severe outbreak? Gonzalez-Reiche et al. sampled some of the early patients seeking assistance in February and March of 2020 at the Mount Sinai Health System. Phylogenetic analysis of virus sequences in these people, who were drawn from across NYC, showed that the virus had been independently introduced many times from Europe and elsewhere in the United States. Subsequent clusters of community transmission occurred. The focus of infection in NYC is a marker of the role this city plays as a two-way hub for human movement. Science this issue p. 297

Synthetic mRNA is an attractive vehicle for gene therapies because of its transient nature and improved safety profile over DNA. However, unlike DNA, broadly applicable methods to control expression from mRNA are lacking. Here we describe a platform for small-molecule-based regulation of expression from modified RNA (modRNA) and self-replicating RNA (replicon) delivered to mammalian cells. Specifically, we engineer small-molecule-responsive RNA binding proteins to control expression of proteins from RNA-encoded genetic circuits. Coupled with specific modRNA dosages or engineered elements from a replicon, including a subgenomic promoter library, we demonstrate the capability to externally regulate the timing and level of protein expression. These control mechanisms facilitate the construction of ON, OFF, and two-output switches, with potential therapeutic applications such as inducible cancer immunotherapies. These circuits, along with other synthetic networks that can be developed using these tools, will expand the utility of synthetic mRNA as a therapeutic modality.