Time to revisit the endpoint dilution assay and to replace the TCID50 as a measure of a virus sample’s infection concentration

The endpoint dilution assay’s output, the 50% infectious dose (ID50), is calculated using the Reed-Muench or Spearman-Kärber mathematical approximations, which are biased and often miscalculated. We introduce a replacement for the ID50 that we call Specific INfection (SIN) along with a free and open-source web-application, midSIN (https://midsin.physics.ryerson.ca) to calculate it. midSIN computes a virus sample’s SIN concentration using Bayesian inference based on the results of a standard endpoint dilution assay, and requires no changes to current experimental protocols. We analyzed influenza and respiratory syncytial virus samples using midSIN and demonstrated that the SIN/mL reliably corresponds to the number of infections a sample will cause per mL. It can therefore be used directly to achieve a desired multiplicity of infection, similarly to how plaque or focus forming units (PFU, FFU) are used. midSIN’s estimates are shown to be more accurate and robust than the Reed-Muench and Spearman-Kärber approximations. The impact of endpoint dilution plate design choices (dilution factor, replicates per dilution) on measurement accuracy is also explored. The simplicity of SIN as a measure and the greater accuracy provided by midSIN make them an easy and superior replacement for the TCID50 and other in vitro culture ID50 measures. We hope to see their universal adoption to measure the infectivity of virus samples.

Enantiomeric purity deviations of radiolabelled amino acids obtained from chiral columns

Enantiomeric purity (EP) is an important value which denotes the relative percentage of the L-isomer with respect to the D-isomer. For 11C and 18F-labelled amino acid (AA) radiopharmaceutical (RP) production, EP represents a quality control parameter specified in European and national monographs for particular RPs. In most instances, EP value of greater then 90 or 95% (depending on AA type) is required as part of the quality control (QC) value of a RP following radiosynthesis. In common practice, two chromatographic columns are used for the EP determination of RPs: Crownpak CR(+) (Daicel), which contains a crown ether stationary phase or Chirobiotic T (Astec), which contains silica-bound glycoproteins as the stationary phase. The application of column Crownpak CR(+) requires that only perchloric acid solution (with pH 1–2) may be used, as the retention capability of the stationary phase is greatly reduced using organic solvents. This work intends to identify which chromatographic system is more accurate and reliable for EP determination as part of QC. We performed a series of parallel injections of the same batch of the widely used AA RPs [11C]MET and [18F]FET on the two aforementioned columns. The EP determination using column Crownpak CR(+) consistently provided a lower EP value compared to the Chirobiotic T column; the EP deviation between the respective columns was found to range from 2.4–4.0% for the same RP sample. Furthermore, the EP value was influenced by a sample’s dilution factor, e.g. the EP was observed to increase up to 1.5% when the radioactive sample had a fivefold dilution factor. This phenomenon was consistent for both Crownpak CR(+) and Chirobiotic T columns. Finally, a series of standard solutions of non-radioactive methionine with various ratios of L-and D-isomers was analyzed. The data obtained for non-radioactive methionine confirmed that column Crownpak CR(+) incorrectly provided a higher D-enantiomer concentration, whereas Chirobiotic T was found to provide a lower D-enantiomer concentration of the same sample. The deviation from the theoretical EP value was between 0.67 and 1.92%.

Global distribution of wastewater treatment plants and their released effluents into rivers and streams

The main objective of wastewater treatment plants (WWTPs) is to remove contaminants such as pathogens, nutrients, and organic and other pollutants from wastewaters using physical, biological and/or chemical processes prior to discharge into receiving waterbodies. However, since WWTPs cannot remove all contaminants, they inevitably represent concentrated point sources of residual contaminant loads into surface waters. To understand the severity and extent of the impact of wastewater discharges from such facilities into rivers and lakes, as well as to identify opportunities of improved management, detailed information about WWTPs is required, including (1) their explicit geospatial locations to identify the waterbodies affected; and (2) individual plant characteristics such as population served, flow rate of effluents, and level of treatment of processed wastewaters. These characteristics are especially important for contaminant fate models that are designed to assess the distribution of substances that are not typically included in environmental monitoring programs, such as contaminants of emerging concern. Although there are several regional datasets that provide information on WWTP locations and characteristics, data are still lacking at a global scale, especially in developing countries. Here we introduce HydroWASTE, a location-explicit global database of 58,502 WWTPs and their characteristics. This database was developed by combining national and regional datasets with auxiliary information to derive or complete missing WWTP characteristics, including the amount of people served. A high-resolution river network with streamflow estimates was used to georeference WWTP outfall locations and calculate each plant’s dilution factor (i.e., the ratio of the natural discharge of the receiving waterbody to the WWTP effluent discharge). The utility of this information was demonstrated in an assessment of the distribution of wastewaters at a global scale. Results show that 1.2 million kilometers of the global river network receive wastewater input from upstream WWTPs, of which more than 90,000 km are downstream of WWTPs that offer only primary treatment. Wastewater ratios originating from WWTPs exceed 10 % in over 72,000 km of rivers, mostly in areas of high population densities in Europe, USA, China, India, and South Africa. In addition, 2,533 plants show a dilution factor of less than 10, which represents a common threshold for environmental concern.

Preparation of Single Cell Suspension from Human Lung Tissue v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from human lung sections of about two grams. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Isolation of Nucleated Cells from Whole Blood v1

This protocol describes a method for the isolation of pan-lymphocytes and pan-myeloid cells from human whole blood. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Lymph Node Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human lymph node tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from the epithelial layer and the lamina propria of human gut sections of about one gram of tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples. This protocol can be used for any section of the intestinal tract from duodenum to distal colon.

Preparation of Single Cell Suspension from Human Spleen Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human spleen tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of a Single Cell Suspension from Bronchoalevolar Lavage v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from lavage fluid collected from human lung. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Isolation of Nucleated Cells from Bone Marrow Aspirate v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human bone marrow aspirate. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.