Space-time analysis of COVID-19 cases and SARS-CoV-2 wastewater loading: A geodemographic perspective.

Severe acute respiratory syndrome - coronavirus 2 (SARS-CoV-2) continues to effect communities across the world. One way to combat these effects is to enhance our collective ability to remotely monitor community spread. Monitoring SARS-CoV-2 in wastewater is one approach that enables researchers to estimate the total number of infected people in a region; however, estimates are often made at the sewershed level which may mask the geographic nuance required for targeted interdiction efforts. In this work, we utilize an apportioning method to compare the spatial and temporal trends of daily case count with the temporal pattern of viral load in the wastewater at smaller units of analysis within Austin, TX. We find different lag-times between wastewater loading and case reports. Daily case reports for some locations follow the temporal trend of viral load more closely than others. These findings are then compared to socio-demographic characteristics across the study area.

Quantification of genes and gene transcripts for microbial perchlorate reduction in fixed-bed bioreactors.

AIMS: Optimization of full-scale, biological perchlorate treatment processes for drinking water would benefit from knowledge of the location and quantity of perchlorate-reducing bacteria (PRB) and expression of perchlorate-related genes in bioreactors. The aim of this study was to quantify perchlorate removal and perchlorate-related genes (pcrA and cld) and their transcripts in bioreactors and to determine whether these genes or transcripts could serve as useful biomarkers for perchlorate treatment processes. METHODS AND RESULTS: Quantitative PCR (qPCR) assays targeting pcrA and cld were applied to two pilot-scale, fixed-bed bioreactors treating perchlorate-contaminated groundwater. pcrA and cld genes per microgram of DNA were two- to threefold higher and three- to fourfold higher, respectively, in the bioreactor showing superior perchlorate-removal performance. In a laboratory-scale bioreactor, quantities of pcrA and cld genes and transcripts were compared under two distinct performance conditions (c.60 and 20% perchlorate removal) for a 5-min empty bed contact time. cld genes per microgram of DNA were approximately threefold higher and cld transcripts per microgram of RNA were approximately sixfold higher under the higher perchlorate-removal condition. No differences in pcrA genes or transcripts per microgram of DNA or RNA, respectively, were detected between the c.60 and 20% perchlorate-removal conditions, possibly because these assays did not accurately quantify pcrA genes and transcripts in the mixed culture present. CONCLUSIONS: Quantities of cld genes and transcripts per microgram of DNA and RNA, respectively, were found to be higher when perchlorate removal was higher. However, quantities of pcrA and cld genes or transcripts were not found to directly correlate with perchlorate-removal rates. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this study represents the first application of qPCR assays to quantify perchlorate-related genes and transcripts in continuous-flow bioreactors. The results indicate that cld gene and transcript quantities can provide insights regarding the quantity, location and gene expression of PRB in bioreactors.

Evidence for associations between the purinergic receptor P2X(7) (P2RX7) and toxoplasmosis.

Congenital Toxoplasma gondii infection can result in intracranial calcification, hydrocephalus and retinochoroiditis. Acquired infection is commonly associated with ocular disease. Pathology is characterized by strong proinflammatory responses. Ligation of ATP by purinergic receptor P2X(7), encoded by P2RX7, stimulates proinflammatory cytokines and can lead directly to killing of intracellular pathogens. To determine whether P2X(7) has a role in susceptibility to congenital toxoplasmosis, we examined polymorphisms at P2RX7 in 149 child/parent trios from North America. We found association (FBAT Z-scores +/-2.429; P=0.015) between the derived C(+)G(-) allele (f=0.68; OR=2.06; 95% CI: 1.14-3.75) at single-nucleotide polymorphism (SNP) rs1718119 (1068T>C; Thr-348-Ala), and a second synonymous variant rs1621388 in linkage disequilibrium with it, and clinical signs of disease per se. Analysis of clinical subgroups showed no association with hydrocephalus, with effect sizes for associations with retinal disease and brain calcifications enhanced (OR=3.0-4.25; 0.004

The dependence of quorum sensing on the depth of a growing biofilm.

In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly. In this paper, a one-dimensional model of a growing Pseudomonas aeruginosa biofilm is examined. Quorum sensing has been included in the model through equations describing the production, degradation, and diffusion of the signaling molecules, acyl-homoserine lactones, in the biofilm. From this model, we are able to make some important observations about quorum sensing. First, in order for quorum sensing to initiate near the substratum, in accordance with experimental observations, the model suggests that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound. Second, the induction of quorum sensing is related to a critical biofilm depth; once the biofilm grows to the critical depth, quorum sensing is induced. Third, the critical biofilm depth varies with the pH of the surrounding fluid. Of particular interest is the prediction of a critical pH threshold, above which quorum sensing is not possible at any depth. These results highlight the importance of careful study of the relationship among metabolic activity of the bacterium, signal synthesis, and the chemistry of the surrounding environment.

Delivery of antimicrobials into parasites.

To eliminate apicomplexan parasites, inhibitory compounds must cross host cell, parasitophorous vacuole, and parasite membranes and cyst walls, making delivery challenging. Here, we show that short oligomers of arginine enter Toxoplasma gondii tachyzoites and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP reductase (ENR), conjugated to arginine oligomers enters extracellular tachyzoites, host cells, tachyzoites inside parasitophorous vacuoles within host cells, extracellular bradyzoites, and bradyzoites within cysts. We identify, clone, and sequence T. gondii enr and produce and characterize enzymatically active, recombinant ENR. This enzyme has the requisite amino acids to bind triclosan. Triclosan released after conjugation to octaarginine via a readily hydrolyzable ester linkage inhibits ENR activity, tachyzoites in vitro, and tachyzoites in mice. Delivery of an inhibitor to a microorganism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to sequestered parasites, a model system to characterize transport across multiple membrane barriers and structures, a widely applicable paradigm for treatment of active and encysted apicomplexan and other infections, and a generic proof of principle for a mechanism of medicine delivery.

A mathematical model of quorum sensing in a growing bacterial biofilm.

In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly. This paper describes a one-dimensional model of a growing Pseudomonas aeruginosa biofilm. Quorum sensing has been included in the model by the addition of equations describing the production, degradation, and diffusion of acyl-homoserine lactones in the biofilm. In order for quorum sensing to initiate near the substratum, in accordance with experimental observations, model results suggest that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound. This result highlights the importance of careful study of the relationship between metabolic activity of the bacterium and signal synthesis.

Water quality factors affecting bromate reduction in biologically active carbon filters.

Biological removal of the ozonation by-product, bromate, was demonstrated in biologically active carbon (BAC) filters. For example, with a 20-min EBCT, pH 7.5, and influent dissolved oxygen (DO) and nitrate concentrations 2.1 and 5.1 mg/l, respectively, 40% bromate removal was obtained with a 20 microg/l influent bromate concentration. In this study, DO, nitrate and sulfate concentrations, pH, and type of source water were evaluated for their effect on bromate removal in a BAC filter. Bromate removal decreased as the influent concentrations of DO and nitrate increased, but bromate removal was observed in the presence of measurable effluent concentrations of DO and nitrate. In contrast, bromate removal was not sensitive to the influent sulfate concentration, with only a slight reduction in bromate removal as the influent sulfate concentration was increased from 11.1 to 102.7 mg/l. Bromate reduction was better at lower pH values (6.8 and 7.2) than at higher pH values (7.5 and 8.2), suggesting that it may be possible to reduce bromate formation during ozonation and increase biological bromate reduction through pH control. Biological bromate removal in Lake Michigan water was very poor as compared to that in tapwater from a groundwater source. Bromate removal improved when sufficient organic electron donor was added to remove the nitrate and DO present in the Lake Michigan water, indicating that the poor biodegradability of the natural organic matter may have been limiting bromate removal in that water. Biological bromate removal was demonstrated to be a sustainable process under a variety of water quality conditions, and bromate removal can be improved by controlling key water quality parameters.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Measurement of the efficacy of vaccines and antimicrobial therapy against infection with Toxoplasma gondii.

To facilitate studies of vaccines and antimicrobial agents effective against Toxoplasma gondii infection, an assay system was developed to semi-quantitate parasitaemia using PCR amplification of T. gondii DNA obtained from the blood of mice infected with the parasite. A competitive internal standard DNA fragment of the B1 gene of T. gondii was generated and used in PCR so that the amplified product could be semi-quantitated and false negative results could be avoided. The PCR assay system was used to analyse the levels of parasitaemia in immunised and antimicrobial agent treated mice at various times after infection with T. gondii. The results of these studies indicate that this highly sensitive detection method is a rapid and reliable procedure that can be employed to assess the abilities of vaccines or antimicrobial agents to provide protection early following T. gondii infection.

Multiple TL-like loci in the grc-G/C region of the rat.

The grc-G/C region of the rat is homologous to the Q/TL region of the mouse, and deletions in this region are associated with fetal mortality, developmental defects, and decreased resistance to cancer. Several cosmids spanning approximately 45 kilobases of this region were analyzed for their class I loci, using a mouse general class I probe (pAG64c), grc-specific probes (pGRC1.4, pGRC1.7), and four probes derived from the TL-like locus RT1.N1. The results showed that TL-like genes other than RT1.N1 exist in the rat: a duplicated gene, RT1.N2, was identified, sequenced, and shown to be 99.3% similar to RT1.N1; and a third TL-like gene, RT1.N3, was isolated from a cDNA library, sequenced, and shown to be 92.8% similar to RT1.N1. These observations suggest that the rat TL-like loci are duplicated and that there is more than one cluster of these duplicated genes. The TL-like genes are transcribed predominantly in the thymus, except in grc- strains, and their level of transcription increases during fetal life and reaches its maximum at birth. Finally, a cosmid that appears to identify the end of the deletion in grc- strains was identified.

Genomic DNA sequence and organization of a TL-like gene in the grc-G/C region of the rat.

Genes in the grc-G/C region, which is linked to the rat major histocompatibility complex, influence the control of growth, development, and susceptibility to chemical carcinogens. As an initial approach to analyzing the structure and organization of these genes, a class I hybridizing fragment designated RT(5.8) was isolated from an R21 genomic DNA library and sequenced from overlapping restriction enzyme fragments. The RT(5.8) clone has 5788 base pairs and contains the eight exons characteristic of a class I gene. There are CAAT and TATA boxes upstream of the signal peptide, and the recognition sequence that precedes the site of polyadenylation is located downstream from the third cytoplasmic domain. Comparison of the RT(5.8) gene with representative class I genes from the rat and other species shows that the nucleotide sequences of RT(5.8) have a high level of similarity to those of TL region genes of several strains of mice. The peptide sequence deduced from the RT(5.8) clone is distinct from all previously published class I gene sequences, and at many positions there are amino acid residues that are unique to the RT(5.8) sequence. Probes have been isolated from the third exon and from the 5' and 3' flanking regions of the RT(5.8) clone, and Southern blot analysis with genomic DNA of various rat strains shows that these probes are specific for the RT(5.8) fragment. Northern blot analysis shows that the gene is transcribed in the thymus but not in the liver or spleen. The RT(5.8) sequence is more similar to some mouse TL genes (especially in the alpha 2 and cytoplasmic domains and in the 5' and 3' untranslated regions) than it is to other rat class I genes. Hence, TL-like genes are not restricted to the mouse.