Journal Pre-proof J o u r n a l P r e -p r o o f 2 ABSTRACT Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections amongst the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.Journal Pre-proof de Roda Husman, 2020;Medema et al., 2020;Wu et al., 2020). Here, we report the first evidence for the presence of SARS-CoV-2 RNA in wastewater in Australia. Our preliminary findings demonstrate the applicability of WBE for COVID-19 surveillance as a potential tool for public health monitoring at the community level. Journal Pre-proof conditions ranging from 3 × 20 s at 8,000 rpm at a 10 s interval. From here on RNA was extracted using RNeasy Power Microbiome kit as per manufacturer's instruction. Method B began with centrifugation of wastewater samples (100-200 mL) at 4,750 g for 30 mins. Supernatant was then removed carefully without disturbing the pellet and Journal Pre-proof 4 /reaction) of Oncorhynchus keta (O. keta) was added in the DNAse and RNAse free water and the Cq value obtained acted as a reference point. If the Cq value of a wastewater sample increases compared to the reference Cq value, the sample is considered to have PCR inhibitors. Wastewater samples with a 2-Cq (quantification cycle) delay was considered to have RT-qPCR inhibition (Staley et al., 2012). All RNA samples were Journal Pre-proof average quality of 15 (SLIDINGWINDOW:4:15). Reads were cropped to 120bp (CROP:120), with any less than 120bp in length discarded (MINLEN:120). Overlapping forward and reverse reads were merged using bbmerge from the BBMap suite (ver. 38.41, https://sourceforge.net/projects/bbmap/). Quality-controlled, merged reads were then mapped Journal Pre-proof describing it, through the model 10,000 times. For each estimate of infected persons, the corresponding prevalence was calculated by dividing the number of persons infected by the number of persons in the catchment. Sensitivity of the estimated number o...

Background Wastewater-based epidemiology (WBE) for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimise SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool. Methods Airline and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 RNA using two virus concentration methods, adsorption-extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative amplicons from positive samples were sequenced to confirm assay specificity. Results SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however, concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimised false-negative results. Representative amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among assays and concentration methods. Conclusions The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.

Actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) are two of the most common dermatologic conditions in Western countries and cause substantial morbidity worldwide. The role of human papillomaviruses under these conditions has been well studied yet remains inconclusive. One PCR-based study has investigated bacteria in the etiology of these conditions; however, no study has investigated the microbiomes of AK and SCC more broadly. We longitudinally profiled the microbiomes of 112 AK lesions, profiled cross sections of 32 spontaneously arising SCC lesions, and compared these to matching nonlesional photodamaged control skin sites. We identified commonly occurring strains of Propionibacterium and Malassezia at higher relative abundances on nonlesional skin than in AK and SCC lesions, and strains of Staphylococcus aureus were relatively more abundant in lesional than nonlesional skin. These findings may aid in the prevention of SCC.

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Dendritic cells (DC) are the professional antigen-presenting cells that initiate immune responses. While DC take up antigen, migrate to lymph nodes and present processed antigen to T lymphocytes, little is known of the intracellular biochemical pathways controlling these events. Using the differential display technique, employing the activated blood DC-like cell line L428, we isolated a cDNA induced during DC differentiation likely to have a regulatory function. This cDNA encoded a putative 530-amino-acid (aa) protein consisting of a unique hydrophilic domain (106 aa) and a domain (424 aa) similar to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (AHCY). Therefore, this molecule was termed DC-expressed AHCY-like molecule (DCAL). DCAL mRNA was expressed moderately in fresh blood DC, but was not detectable in other peripheral blood mononuclear cells. DCAL mRNA increased markedly during activation of blood and skin DC (Langerhans cells), but was diminished in terminally differentiated tonsil DC. Cultured monocytes expressed little DCAL mRNA, but levels increased markedly when differentiated into DC by cytokines GM-CSF and IL-4. The DCAL gene [Chromosome (Chr) 1] and another previously identified DCAL-like molecule KIAA0828 (Chr 7) differed from the AHCY gene (Chr 20) in gene organization. Thus, DCAL may have a role in controlling critical events in DC differentiation and belong to a novel family of AHCY-like molecules.

Classic Hodgkin's lymphoma (HL)1 is a common malignant lymphoma characterized by the presence of a small population (Ͻ1%) of putative malignant cells, the morphologically distinct Hodgkin and Reed-Sternberg (HRS) cells. Recent advances in cell isolation techniques and molecular biology has identified Ig gene rearrangements within the majority of individual HRS cells, suggesting their B cell origin (1). These are surrounded by a large population of apparently non-malignant lymphocytes and histiocytes, whose proliferation is likely to be mediated by the wide range of cytokines and chemokines released by the HRS cells (reviewed in Refs. 2 and 3).HRS cells have many characteristics in common with antigen-presenting cells (APCs) such as activated B cells and dendritic cells (DCs) (4). Indeed, the HRS cell lines (L428, HDLM-2, and/or KM-H2) express cell surface molecules required for costimulation/proliferation of T cells (major histocompatibility complex class II, CD40, CD80, and CD86) (5-7), cell adhesion molecules involved in DC-T cell interactions (LFA-1, CD11c, and ICAM-1-3) (8, 9), and the DC-associated molecules (CD83 and fascin) (6, 10). They also produce inflammatory cytokines (e.g. tumor necrosis factor-␣ and lymphotoxin) (11), non-inflammatory cytokines (e.g. granulocyte macrophage-colony stimulating factor and interleukins 5 and 13) (12, 13), and chemokines (e.g. TARC) (14), which are associated with APCs. L428 cells have been used successfully in our laboratory to produce monoclonal antibodies (mAb) against DC differentiation antigens such as CMRF-44 (15) and and to clone the DC-associated molecules such as DEC-205 type I transmembrane multilectin receptor (17) and the adenosylhomocysteine hydrolase-like molecule DCAL/AHCYL-1 (18).We have investigated cell surface molecules on HRS cell lines with a view to identifying novel molecules related to APC function. These molecules might also be candidate targets for antibody-based HL immunotherapy. Indeed, CD20, CD25, and CD30 reagents (markers for B cells and activated lymphocytes) have been investigated in this regard (19 -21), but molecules more restricted to HRS cells might be preferred as targets for more specific therapeutics.During the cloning of DEC-205 from the L428 cell line by 3Ј-rapid amplification of cDNA ends (RACE) (17), we discovered an alternatively spliced novel DEC-205 mRNA. This mRNA encodes the intact DEC-205 ectodomain but included unique sequences encoding for an additional carbohydrate recognition domain (CRD) and a transmembrane (TM) and a cytoplasmic (CP) domain derived from a newly identified type I transmembrane C-type lectin DCL-1. A partial cDNA sequence

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with ϳ50% protein identity to adenosylhomocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs (zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.We discovered human adenosylhomocysteine hydrolase-like protein 1 (hAHCYL1 2 ; previously termed dendritic cell-expressed AHCY-like protein, or DCAL) by differential display from the Hodgkin disease-derived cell line L428 (1). The hAH-CYL1 protein (ϳ60 kDa, 530 amino acids) consists of a novel N-terminal hydrophilic domain (106 amino acids) and a C-terminal domain (424 amino acids), which is homologous (51% protein identity) to the methylation pathway enzyme, AHCY (EC 3.3.1.1, ϳ46 kDa). AHCY is the evolutionarily conserved and ubiquitously expressed enzyme that catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine, a byproduct of the S-adenosyl-L-homomethionine-dependent methyltransferase reaction, into adenosine and homocysteine using NAD ϩ as a cofactor (2). Although AHCYL1 conserves the cysteines required for a tight globular structure of AHCY and the NAD ϩ binding motif, AHCYL1 lacks some binding sites for S-adenosyl-L-homocysteine, suggesting that AHCYL1 has a different function from AHCY. The N-terminal hydrophilic domain contains 79 polar or charged amino acids, including a cluster of Ser, Thr, and Tyr (potential phosphorylation sites), and this domain is likely to regulate AHCYL1 function (1). AHCYL1 is highly conserved among species, and the human and mouse orthologs have 100% protein identity, suggesting a highly conserved function yet to be understood. The other AHCY-like prote...

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