Paweł Olszewski

Wastewater-based epidemiology aims at measuring pathogens in wastewater as a means of deriving unbiased epidemiological information at a population scale, ranging from buildings and aircrafts to entire cities or countries. After gaining significant mainstream attention during the SARS-CoV-2 pandemic, the field holds significant promise as a continuous monitoring and early warning system tracking emerging viral variants or new pathogens with pandemic potential. To expand the current toolbox of analytical techniques for wastewater analysis, we explored the use of Hyperplex PCR (hpPCR) to analyse SARS-CoV-2 mutations in wastewater samples collected weekly in up to 22 sites across Sweden between October 2022 and December 2023. Approximately 900 samples were tested using a dynamic probe panel with a multiplexity ranging from 10-to 18-plex, continuously adapted within 1-2 weeks to quantify relevant mutations of concern over time. The panel simultaneously covered deletions, single nucleotide substitutions, as well as variable regions resorting to probe degeneracy. By analysing all samples in parallel resorting to gold standard methods including qPCR and two different NGS technologies, the performance of hpPCR is herein shown to bridge the gap between these methods by providing (1) systematic single nucleotide sensitivity with a simple probe design, (2) high multiplexity without panel re-optimization requirements and (3) 4-5-week earlier mutation detection compared to NGS with excellent quantitative linearity and a good correlation for mutation frequency (r=0.88). Based on the demonstrated performance, the authors propose the combined use of NGS and hpPCR for routine discovery and high-frequency monitoring of key pathogens/variants as a potential alternative to the current analysis paradigm.

Our study unveils the cellular and molecular dynamics in psoriasis epidermis, revealing diverse cell states and their interactions driving inflammation and altered developmental trajectories. Importantly, a previously overlooked plasmacytoid dendritic cell cluster with inflammatory properties was identified in the epidermis of chronic psoriasis lesions, suggesting a potential role for these cells in chronic psoriasis.

Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4+ T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY-cells (22%) in comparison to CD4+ T-lymphocytes and cytotoxic CD8+ T-lymphocytes. Using scRNA-seq LOY was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

Background: The COVID-19 pandemic, which has a prominent social and economic impact worldwide, shows a largely unexplained male bias for the severity and mortality of the disease. Loss of chromosome Y (LOY) is a risk factor candidate in COVID-19 due to its prior association with many chronic age-related diseases, and its impact on immune gene transcription.Methods: Publicly available scRNA-seq data of PBMC samples derived from male patients critically ill with COVID-19 were reanalyzed, and LOY status was added to the annotated cells. We further studied LOY in whole blood for 211 COVID-19 patients treated at intensive care units (ICU) from the first and second waves of the pandemic. Of these, 139 patients were subject to cell sorting for LOY analysis in granulocytes, low-density neutrophils (LDNs), monocytes, and PBMCs.Results: Reanalysis of available scRNA-seq data revealed LDNs and monocytes as the cell types most affected by LOY. Subsequently, DNA analysis indicated that 46%, 32%, and 29% of critically ill patients showed LOY above 5% cut-off in LDNs, granulocytes, and monocytes, respectively. Hence, the myeloid lineage that is crucial for the development of severe COVID-19 phenotype is affected by LOY. Moreover, LOY correlated with increasing WHO score (median difference 1.59%, 95% HDI 0.46% to 2.71%,p=0.025), death during ICU treatment (median difference 1.46%, 95% HDI 0.47% to 2.43%,p=0.0036), and history of vessel disease (median difference 2.16%, 95% HDI 0.74% to 3.7%,p=0.004), among other variables. In 16 recovered patients, sampled during ICU stay and 93–143 days later, LOY decreased significantly in whole blood and PBMCs. Furthermore, the number of LDNs at the recovery stage decreased dramatically (median difference 76.4 per 10,000 cell sorting events, 95% HDI 55.5 to 104,p=6e−11).Conclusions: We present a link between LOY and an acute, life-threatening infectious disease. Furthermore, this study highlights LOY as the most prominent clonal mutation affecting the myeloid cell lineage during emergency myelopoiesis. The correlation between LOY level and COVID-19 severity might suggest that this mutation affects the functions of monocytes and neutrophils, which could have consequences for male innate immunity.

COVID-19 shows an unexplained, strong male bias for severity and mortality. Loss of Y (LOY) in myeloid cells is a risk factor candidate in COVID-19 because of associations with many age-related diseases and its effect on transcription of immune genes. We report the highest levels of LOY in cells that are crucial for the development of severe COVID-19 phenotype, such as low-density neutrophils, granulocytes, and monocytes reaching 46%, 32%, and 29%, respectively, from men with critical COVID-19 (n=139). LOY in sorted subpopulations of leukocytes correlated with increased thrombocyte count, thromboembolic events, invasive mechanical ventilation and a history of vessel disease. In recovered patients, LOY decreased in whole blood and peripheral blood mononuclear cells. Moreover, sc-RNA-seq analysis of CD14+ monocytes from 30 COVID-19 males and 34 controls revealed pervasive transcriptional downregulation in LOY-cells, notably affecting HLA class I and II genes important for antigen presentation. The data support a link between LOY and emergency myelopoiesis as well as the role of LOY in modulation of COVID-19 severity. Our results might also be relevant for other viral infections showing similar male bias.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer’s disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a “genetic wasteland”, and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

Background: Phenotypically discordant monozygotic twins (PDMZTs) offer a unique opportunity to study post‐zygotic genetic variation and provide insights into the linkage between genotype and phenotype. We report a comprehensive analysis of a pair of PDMZTs.Methods: Dysmorphic features and delayed neuro‐motor development were observed in the proband, whereas her twin sister was phenotypically normal. Four tissues (blood, skin, hair follicles, and buccal mucosa) from both twins were studied using four complementary methods, including whole‐exome sequencing, karyotyping, array CGH, and SNP array.Results: In the proband, tetrasomy 18p affecting all studied tissues except for blood was identified. Karyotyping of fibroblasts revealed isochromosome 18p [i(18p)] in all metaphases. The corresponding analysis of the phenotypically normal sister surprisingly revealed low‐level mosaicism (5.4%) for i(18p) in fibroblasts.Conclusion: We emphasize that when mosaicism is suspected, multiple tissues should be studied and we highlight the usefulness of non‐invasive sampling of hair follicles and buccal mucosa as a convenient source of non‐mesoderm‐derived DNA, which complements the analysis of mesoderm using blood. Moreover, low‐level mosaic tetrasomy 18p is well tolerated and such low‐level mosaicism, readily detected by karyotyping, can be missed by other methods. Finally, mosaicism for low‐level tetrasomy 18p might be more common in the general population than it is currently recognized, due to detection limitations.

Marine bacteria display significant versatility in adaptation to variations in the environment and stress conditions, including temperature shifts. Shewanella baltica plays a major role in denitrification and bioremediation in the marine environment, but is also identified to be responsible for spoilage of ice-stored seafood. We aimed to characterize transcriptional response of S. baltica to cold stress in order to achieve a better insight into mechanisms governing its adaptation. We exposed bacterial cells to 8 °C for 90 and 180 min, and assessed changes in the bacterial transcriptome with RNA sequencing validated with the RT-qPCR method. We found that S. baltica general response to cold stress is associated with massive downregulation of gene expression, which covered about 70% of differentially expressed genes. Enrichment analysis revealed upregulation of only few pathways, including aminoacyl-tRNA biosynthesis, sulfur metabolism and the flagellar assembly process. Downregulation was observed for fatty acid degradation, amino acid metabolism and a bacterial secretion system. We found that the entire type II secretion system was transcriptionally shut down at low temperatures. We also observed transcriptional reprogramming through the induction of RpoE and repression of RpoD sigma factors to mediate the cold stress response. Our study revealed how diverse and complex the cold stress response in S. baltica is.

Rapid development of high-throughput sequencing methods and metagenomics revealed a diverse world of microbiota associated with multicellular organisms. Although recent discoveries indicate that freshwater invertebrates are hosts for specific bacteria, it is still unknown if this specificity is driven by host-derived factors or by the environment, especially in animals with diapause in ephemeral habitats, where parents and offspring are separated in time and space. In this work, using both low-throughput molecular approach and Next-generation sequencing of 16S ribosomal RNA gene, we present a taxonomic analysis of bacteria associated with two species of non-marine ostracods Sclerocypris tuberculata and Potamocypris mastigophora raised from diapausing eggs and co-cultured in laboratory conditions. Our analysis showed that despite sharing the same environment, each ostracod host developed distinct bacterial communities. The major difference was caused by the dominance of the family Comamonadaceae (Betaproteobacteria) in P. mastigophora and the Aeromonadaceae (Gammaproteobacteria) in S. tuberculata. Furthermore, prediction of metabolic pathways in metagenomes, revealed that microbiota of P. mastigophora exhibit higher number of sequences associated with the membrane transport and xenobiotics biodegradation and metabolism. Our study not only provides an insight into microbiota of non-marine ostracods but also shows that different ostracod species host functionally distinct bacterial communities.

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism^1,2,3,4,5, yet our knowledge of the causes and consequences of this is limited. Here, using a computational approach, we estimate that 20% of the male population represented in the UK Biobank study (n = 205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes that are involved in cell-cycle regulation and cancer susceptibility, as well as somatic drivers of tumour growth and targets of cancer therapy. We demonstrate that genetic susceptibility to LOY is associated with non-haematological effects on health in both men and women, which supports the hypothesis that clonal haematopoiesis is a biomarker of genomic instability in other tissues. Single-cell RNA sequencing identifies dysregulated expression of autosomal genes in leukocytes with LOY and provides insights into why clonal expansion of these cells may occur. Collectively, these data highlight the value of studying clonal mosaicism to uncover fundamental mechanisms that underlie cancer and other ageing-related diseases.