A physicochemical model for rationalizing SARS-CoV-2 concentration in sewage. Case study: The city of Thessaloniki in Greece.

Detection of SARS-CoV-2 in sewage has been employed by several researchers as an alternative early warning indicator of virus spreading in communities, covering both symptomatic and asymptomatic cases. A factor that can seriously mislead the quantitative measurement of viral copies in sewage is the adsorption of virus fragments onto the highly porous solids suspended in wastewater, making them inaccessible. This depends not only on the available amount of suspended solids, but also on the amount of other dissolved chemicals which may influence the capacity of adsorption. On this account, the present work develops a mathematical framework, at various degrees of spatial complexity, of a physicochemical model that rationalizes the quantitative measurements of total virus fragments in sewage as regards the adsorption of virus onto suspended solids and the effect of dissolved chemicals on it. The city of Thessaloniki in Greece is employed as a convenient case study to determine the values of model variables. The present data indicate the ratio of the specific absorption (UV254/DOC) over the dissolved oxygen (DO) as the parameter with the highest correlation with viral copies. This implies a strong effect on viral inaccessibility in sewage caused (i) by the presence of humic-like substances and (ii) by virus decay due to oxidation and metabolic activity of bacteria. The present results suggest days where many fold corrections in the measurement of viral copies should be applied. As a result, although the detected RNA load in June 2020 is similar to that in April 2020, virus shedding in the city is about 5 times lower in June than in April, in line with the very low SARS-CoV-2 incidence and hospital admissions for COVID-19 in Thessaloniki in June.

In vitro three-dimensional modelling of human ovarian surface epithelial cells.

OBJECTIVES: Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three-dimensional (3D) cultures. MATERIALS AND METHODS: OSE cells were cultured as multicellular spheroids (MCS) (i) in a rotary cell culture system (RCCS) and (ii) on polyHEMA-coated plastics. The MCSs were examined by electron microscopy and compared to OSE from primary tissues and cells grown in 2D. Annexin V FACS analysis was used to evaluate apoptosis and expression of extracellular matrix (ECM) proteins was analysed by immunohistochemical staining. RESULTS: On polyHEMA-coated plates, OSE spheroids had defined internal architecture. RCCS MCSs had disorganized structure and higher proportion of apoptotic cells than polyHEMA MCSs and the same cells grown in 2D culture. In 2D, widespread expression of AE1/AE3, laminin and vimentin were undetectable by immunohistochemistry, whereas strong expression of these proteins was observed in the same cells grown in 3D culture and in OSE on primary tissues. CONCLUSIONS: Physiological and biological features of OSE cells grown in 3D culture more closely resemble characteristics of OSE cells in vivo than when grown by classical 2D approaches. It is likely that establishing in vitro 3D OSE models will lead to greater understanding of the mechanisms of neoplastic transformation in epithelial ovarian cancers.

Three-dimensional in vitro cell biology models of ovarian and endometrial cancer.

OBJECTIVES: This study aims to establish three-dimensional (3D) cell culture models of human ovarian and endometrial cancers and to compare biological and morphological characteristics of these models with those of two-dimensional (2D) models of the same cell lines and the primary tumours. METHODS: 3D models of ovarian and endometrial cancer cell cultures were established using a Rotary Cell Culture System. Immunohistochemical profiling and differential proteomics were used to characterize biological characteristics of multicellular spheroids (MCS) formed from these cultures. These were compared to characteristics of the same cells established in 2D and of the primary tumours from which the cell lines were derived. RESULTS: MCSs from 3D cell cultures appeared histologically similar to the primary tumours. Immunohistochemical profiling of multiple markers, including CA125, BCL2 and p53, showed that patterns of protein expression in MCSs resemble those of the primary tumours. Proteomic profiling identified several differentially expressed protein markers between 2D and 3D cultures. These included prohibitin, which was down-regulated in 3D cultures suggesting cells proliferate less compared to 2D cultures; and VDAC1 and annexin 4, which were up-regulated in 3D cultures suggesting greater levels of apoptosis in 3D compared to 2D models. CONCLUSION: Establishing 3D models of cancer cell lines is likely to be of value for studying the molecular and biological mechanisms of ovarian/endometrial tumour progression and for testing novel molecular targets for cancer therapy.

Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression.

OBJECTIVE: Cell immortalization is considered to be a prerequisite status for carcinogenesis. Normal human ovarian surface epithelial (OSE) cells, which are thought to be the origin of most of human ovarian carcinomas, have a very limited lifespan in culture. Establishment of immortalized OSE cell lines has, in the past, required inactivation of pRb and p53 functions. However, this often leads to increased chromosome instability during prolonged culture. MATERIALS AND METHODS: In this study, we have used a retroviral infection method to overexpress human telomerase reverse transcriptase (hTERT) gene, in primary normal OSE cells, under optimized culture conditions. RESULTS: In vitro and in vivo analysis of hTERT-immortalized cell lines confirmed their normal epithelial characteristics. Gene expression profiles and functional analysis of p16(INK4A), p15(INK4B), pRb and p53 confirmed the presence of their intact functions. Our study suggests that inactivation of pRb and p53 is not necessary for OSE immortalization. Furthermore, down-regulation of p15(INK4B) in the immortalized cells may indicate a functional role for this protein in them. CONCLUSION: These immortal OSE cell lines are likely to be an important tool for studying human OSE biology and carcinogenesis.