SARS-CoV-2 variant introduction following spring break travel and transmission mitigation strategies.

BACKGROUND: University spring break carries a two-pronged SARS-CoV-2 variant transmission risk. Circulating variants from universities can spread to spring break destinations, and variants from spring break destinations can spread to universities and surrounding communities. Therefore, it is critical to implement SARS-CoV-2 variant surveillance and testing strategies to limit community spread before and after spring break to mitigate virus transmission and facilitate universities safely returning to in-person teaching. METHODS: We examined the SARS-CoV-2 positivity rate and changes in variant lineages before and after the university spring break for two consecutive years. 155 samples were sequenced across four time periods: pre- and post-spring break 2021 and pre- and post-spring break 2022; following whole genome sequencing, samples were assigned clades. The clades were then paired with positivity and testing data from over 50,000 samples. RESULTS: In 2021, the number of variants in the observed population increased from four to nine over spring break, with variants of concern being responsible for most of the cases; Alpha percent composition increased from 22.2% to 56.4%. In 2022, the number of clades in the population increased only from two to three, all of which were Omicron or a sub-lineage of Omicron. However, phylogenetic analysis showed the emergence of distantly related sub-lineages. 2022 saw a greater increase in positivity than 2021, which coincided with a milder mitigation strategy. Analysis of social media data provided insight into student travel destinations and how those travel events may have impacted spread. CONCLUSIONS: We show the role that repetitive testing can play in transmission mitigation, reducing community spread, and maintaining in-person education. We identified that distantly related lineages were brought to the area after spring break travel regardless of the presence of a dominant variant of concern.

Characterization of Glucokinases from Pathogenic Free-Living Amoebae.

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

SARS-CoV-2 variants of concern Alpha and Delta show increased viral load in saliva.

BACKGROUND: Higher viral loads in SARS-CoV-2 infections may be linked to more rapid spread of emerging variants of concern (VOC). Rapid detection and isolation of cases with highest viral loads, even in pre- or asymptomatic individuals, is essential for the mitigation of community outbreaks. METHODS AND FINDINGS: In this study, we analyze Ct values from 1297 SARS-CoV-2 positive patient saliva samples collected at the Clemson University testing lab in upstate South Carolina. Samples were identified as positive using RT-qPCR, and clade information was determined via whole genome sequencing at nearby commercial labs. We also obtained patient-reported information on symptoms and exposures at the time of testing. The lowest Ct values were observed among those infected with Delta (median: 22.61, IQR: 16.72-28.51), followed by Alpha (23.93, 18.36-28.49), Gamma (24.74, 18.84-30.64), and the more historic clade 20G (25.21, 20.50-29.916). There was a statistically significant difference in Ct value between Delta and all other clades (all p.adj<0.01), as well as between Alpha and 20G (p.adj<0.05). Additionally, pre- or asymptomatic patients (n = 1093) showed the same statistical differences between Delta and all other clades (all p.adj<0.01); however, symptomatic patients (n = 167) did not show any significant differences between clades. Our weekly testing strategy ensures that cases are caught earlier in the infection cycle, often before symptoms are present, reducing this sample size in our population. CONCLUSIONS: COVID-19 variants Alpha and Delta have substantially higher viral loads in saliva compared to more historic clades. This trend is especially observed in individuals who are pre- or asymptomatic, which provides evidence supporting higher transmissibility and more rapid spread of emerging variants. Understanding the viral load of variants spreading within a community can inform public policy and clinical decision making.

SARS-CoV-2 variants of concern Alpha and Delta show increased viral load in saliva.

BACKGROUND: Higher viral loads in SARS-CoV-2 infections may be linked to more rapid spread of emerging variants of concern (VOC). Rapid detection and isolation of cases with highest viral loads, even in pre- or asymptomatic individuals, is essential for the mitigation of community outbreaks. METHODS AND FINDINGS: In this study, we analyze Ct values from 1297 SARS-CoV-2 positive patient saliva samples collected at the Clemson University testing lab in upstate South Carolina. Samples were identified as positive using RT-qPCR, and clade information was determined via whole genome sequencing at nearby commercial labs. We also obtained patient-reported information on symptoms and exposures at the time of testing. The lowest Ct values were observed among those infected with Delta (median: 22.61, IQR: 16.72-28.51), followed by Alpha (23.93, 18.36-28.49), Gamma (24.74, 18.84-30.64), and the more historic clade 20G (25.21, 20.50-29.916). There was a statistically significant difference in Ct value between Delta and all other clades (all p.adj<0.01), as well as between Alpha and 20G (p.adj<0.05). Additionally, pre- or asymptomatic patients (n=1093) showed the same statistical differences between Delta and all other clades (all p.adj<0.01); however, symptomatic patients (n=167) did not show any significant differences between clades. Our weekly testing strategy ensures that cases are caught earlier in the infection cycle, often before symptoms are present, reducing this sample size in our population. CONCLUSIONS: COVID-19 variants Alpha and Delta have substantially higher viral loads in saliva compared to more historic clades. This trend is especially observed in individuals who are pre- or asymptomatic, which provides evidence supporting higher transmissibility and more rapid spread of emerging variants. Understanding the viral load of variants spreading within a community can inform public policy and clinical decision making.

Zinc homeostasis in the secretory pathway in yeast.

It is estimated that up to 10% of proteins in eukaryotes require zinc for their function. Although the majority of these proteins are located in the nucleus and cytosol, a small subset is secreted from cells or is located within an intracellular compartment. As many of these compartmentalized metalloproteins fold to their native state and bind their zinc cofactor inside an organelle, cells require mechanisms to maintain supply of zinc to these compartments even under conditions of zinc deficiency. At the same time, intracellular compartments can also be the site for storing zinc ions, which then can be mobilized when needed. In this review, we highlight insight that has been obtained from yeast models about how zinc homeostasis is maintained in the secretory pathway and vacuole.

The Loz1 transcription factor from Schizosaccharomyces pombe binds to Loz1 response elements and represses gene expression when zinc is in excess.

In Schizosaccharomyces pombe, the expression of the zrt1 zinc uptake gene is tightly regulated by zinc status. When intracellular zinc levels are low, zrt1 is highly expressed. However, when zinc levels are high, transcription of zrt1 is blocked in a manner that is dependent upon the transcription factor Loz1. To gain additional insight into the mechanism by which Loz1 inhibits gene expression in high zinc, we used RNA-seq to identify Loz1-regulated genes, and ChIP-seq to analyze the recruitment of Loz1 to target gene promoters. We find that Loz1 is recruited to the promoters of 27 genes that are also repressed in high zinc in a Loz1-dependent manner. We also find that the recruitment of Loz1 to the majority of target gene promoters is dependent upon zinc and the motif 5'-CGN(A/C)GATCNTY-3', which we have named the Loz1 response element (LRE). Using reporter assays, we show that LREs are both required and sufficient for Loz1-mediated gene repression, and that the level of gene repression is dependent upon the number and sequence of LREs. Our results elucidate the Loz1 regulon in fission yeast and provide new insight into how eukaryotic cells are able to respond to changes in zinc availability in the environment.

Zinc-dependent activation of the Pho8 alkaline phosphatase in Schizosaccharomyces pombe.

Genome-wide analyses have revealed that during metal ion starvation, many cells undergo programmed changes in their transcriptome or proteome that lower the levels of abundant metalloproteins, conserving metal ions for more critical functions. Here we investigated how changes in cellular zinc status affect the expression and activity of the zinc-requiring Pho8 alkaline phosphatase from fission yeast (Schizosaccharomyces pombe). In S. pombe, Pho8 is a membrane-tethered and processed glycoprotein that resides in the vacuole. Using alkaline phosphatase activity assays along with various biochemical analyses, we found that Pho8 is active when zinc is plentiful and inactive when zinc is limited. Although Pho8 activity depended on zinc, we also found that higher levels of pho8 mRNAs and Pho8 protein accumulate in zinc-deficient cells. To gain a better understanding of the inverse relationship between pho8 mRNA levels and Pho8 activity, we examined the effects of zinc on the stability and processing of the Pho8 protein. We show that Pho8 is processed regardless of zinc status and that mature Pho8 accumulates under all conditions. We also noted that alkaline phosphatase activity is rapidly restored when zinc is resupplied to cells, even in the presence of the protein synthesis inhibitor cycloheximide. Our results suggest that S. pombe cells maintain inactive pools of Pho8 proteins under low-zinc conditions and that these pools facilitate rapid restoration of Pho8 activity when zinc ions become available.

The gluconate shunt is an alternative route for directing glucose into the pentose phosphate pathway in fission yeast.

Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1 By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1 We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase.

Zinc sensing and regulation in yeast model systems.

The Zap1 transcription factor of Saccharomyces cerevisiae and the Loz1 transcription factor of Schizosaccharomyces pombe both play a central role in zinc homeostasis by controlling the expression of genes necessary for zinc metabolism. Zap1 activates gene expression when cells are limited for zinc, while Loz1 is required for gene repression when zinc is in excess. In this review we highlight what is known about the underlying mechanisms by which these factors are regulated by zinc, and how transcriptional activation and repression in eukaryotic cells can be finely tuned according to intracellular zinc availability.