Draft genome sequence of two "Candidatus Intestinicoccus colisanans" strains isolated from faeces of healthy humans.

OBJECTIVES: In order to provide a better insight into the functional capacity of the human gut microbiome, we isolated a novel bacterium, "Candidatus Intestinicoccus colisanans" gen. nov. sp. nov., and performed whole genome sequencing. This study will provide new insights into the functional potential of this bacterium and its role in modulating host health and well-being. We expect that this data resource will be useful in providing additional insight into the diversity and functional potential of the human microbiome. DATA DESCRIPTION: Here, we report the first draft genome sequences of "Candidatus Intestinicoccus colisanans" strains MH27-1 and MH27-2, recovered from faeces collected from healthy human donors. The genomes were sequenced using short-read Illumina technology and whole-genome-based comparisons and phylogenomics reconstruction indicate that "Candidatus Intestinicoccus colisanans" represents a novel genus and species within the family Acutalibacteraceae. Both genomes were estimated to be > 98% completed and to range in size from 2.9 to 3.3 Mb with a G + C content of approximately 51%. The gene repertoire of "Candidatus Intestinicoccus colisanans" indicate it is likely a saccharolytic gut bacterium.

Critical evaluation of faecal microbiome preservation using metagenomic analysis.

The ability to preserve microbial communities in faecal samples is essential as increasing numbers of studies seek to use the gut microbiome to identify biomarkers of disease. Here we use shotgun metagenomics to rigorously evaluate the technical and compositional reproducibility of five room temperature (RT) microbial stabilisation methods compared to the best practice of flash-freezing. These methods included RNALater, OMNIGene-GUT, a dry BBL swab, LifeGuard, and a novel method for preserving faecal samples, a Copan FLOQSwab in an active drying tube (FLOQSwab-ADT). Each method was assessed using six replicate faecal samples from five participants, totalling 180 samples. The FLOQSwab-ADT performed best for both technical and compositional reproducibility, followed by RNAlater and OMNIgene-GUT. LifeGuard and the BBL swab had unpredictable outgrowth of Escherichia species in at least one replicate from each participant. We further evaluated the FLOQSwab-ADT in an additional 239 samples across 10 individuals after storage at -20 °C, RT, and 50 °C for four weeks compared to fresh controls. The FLOQSwab-ADT maintained its performance across all temperatures, indicating this method is an excellent alternative to existing RT stabilisation methods.

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers.

BACKGROUND: Wastewater-based epidemiology (WBE) for 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 minimize 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: Aircraft and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 using two virus concentration methods, adsorption-extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative qPCR 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 minimized false-negative results. Representative qPCR amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among molecular 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 detection 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.

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community.

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 among 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 viral 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.

A Natural History of Actinic Keratosis and Cutaneous Squamous Cell Carcinoma Microbiomes.

Cutaneous squamous cell carcinoma (SCC) is the second-most-common cancer in Australia. The majority of SCCs progress from premalignant actinic keratosis (AK) lesions that form on chronically sun-exposed skin. The role of skin microbiota in this progression is not well understood; therefore, we performed a longitudinal microbiome analysis of AKs and SCCs using a cohort of 13 SCC-prone immunocompetent men. The majority of variability in microbial profiles was attributable to subject, followed by time and lesion type. Propionibacterium and Malassezia organisms were relatively more abundant in nonlesional photodamaged skin than in AKs and SCCs. Staphylococcus was most commonly associated with lesional skin, in particular, sequences most closely related to Staphylococcus aureus Of 11 S. aureus-like operational taxonomic units (OTUs), six were significantly associated with SCC lesions across seven subjects, suggesting their specific involvement with AK-to-SCC progression. If a causative link exists between certain S. aureus-like OTUs and SCC etiology, therapeutic approaches specifically targeting these bacteria could be used to reduce SCC.IMPORTANCE 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.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development: a possible role for AHCYL1 in inositol phospholipid signaling.

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with approximately 50% 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.

The CMRF58 antibody recognizes a subset of CD123hi dendritic cells in allergen-challenged mucosa.

CD123(hi) CD11c(-) dendritic cells (CD123(hi) DC) are a distinct subset of human DC present in bone marrow, blood, lymphoid organs, and peripheral tissues. Pathogen stimulation, cytokine, or CD40 ligation induces CD123(hi) DC maturation, involving a shift from their innate immune to cognate antigen-presenting functions. In this study, we revealed that blood CD123(hi) DC in the presence of cytokine (granulocyte macrophage-colony stimulating factor and interleukin-3) undergo progressive, step-wise maturation through an "early" stage, delineated by expression of the antigen detected by the new monoclonal antibody CMRF58 (CD123(hi)CMRF58(+)CD40(-)CD86(-)CD83(-)) to the "late" stage with costimulatory antigen expression (CD123(hi)CMRF58(+)CD40(+)CD86(+)CD83(+/-)). In this early stage, cytokine-maintained CD123(hi) DC do not display changes in their morphology, no longer produce interferon-alpha (IFN-alpha) in response to bacteria, and develop the capacity to induce proliferation and polarization of allogeneic T cells. CD123(hi)CMRF58(+) DC, phenotypically similar to in vitro cytokine-maintained CD123(hi) DC, were not detected in tonsil but are present in allergen-challenged nasal mucosa of allergic individuals. Thus, CD123(hi) DC in certain tissue environments such as allergen-challenged nasal mucosa share a common CD123(hi)CMRF58(+) phenotype with in vitro cytokine-maintained blood CD123(hi) DC characterized by lack of IFN-alpha production.

Hodgkin's lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1.

Classic Hodgkin's lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3'-rapid amplification of cDNA ends (RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines (L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5'-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34-35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.

Identification of an S-adenosylhomocysteine hydrolase-like transcript induced during dendritic cell differentiation.

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.