Behavior of PBTC, HEDP, and aminophosphonates in the process of wastewater treatment

Ten times at intervals of 1–2 months, individual treatment stages of two wastewater treatment plants (WWTPs) were analyzed for the five quantitatively most widely used phosphonates. The total dissolved concentration of the investigated phosphonates in the influents was between 131 µg/L and 384 µg/L. The nitrogen-free phosphonates 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC) and 1-hydroxyethylidene(1,1-diphosphonic acid) (HEDP) accounted for an average proportion of 83–85%. Diethylenetriaminepenta(methylene phosphonic acid) (DTPMP) contributed with 13–14%, whereas aminotris(methylphosphonic acid) (ATMP) (≤15 µg/L) and ethylenediaminetetra(methylene phosphonic acid) (EDTMP) (≤11 µg/L) contents detected in the WWTP influents were comparatively low. The application of new analytical methods allowed the quantification of phosphonates in the solid fraction of the WWTP influents for the first time. High loads of phosphonates were determined (223–2555 mg/kg), indicating that 20%–80% of the phosphonates are present in the adsorbed state. The removal of total dissolved phosphonate by secondary clarification was between 69.7% and 92.4% (medians: 90.7% and 87.7%). In both WWTPs, HEDP (medians: 89.2% and 86.4%) was slightly better eliminated than PBTC (medians: 87.2% and 82.5%). In the sand filtration stage of a WWTP, the average removal was not further improved. In contrast, an additional removal of dissolved phosphonates could be achieved by activated carbon treatment (median: 96.4%). The proportion of phosphonate-P in the dissolved unreactive phosphorus fraction was consistently between 10% and 40% throughout all treatment stages

Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a “cytokine window” defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed

Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a “cytokine window” defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed

Untersuchungen zur Substrattoleranz von Sactisynthasen für die Generierung Thioether‐verbrückter Sactipeptide mit neuen Eigenschaften

Sactipeptide sind ribosomal synthetisierte Peptide, die eine einzigartige Verknüpfung von Schwefel und α‐Kohlenstoffen enthalten. Die Bildung von Thioetherbrücken wird in diesen Molekülen durch Sactisynthasen katalysiert. Diese spezielle Art der Verknüpfung verleiht Sactipeptiden eine erhöhte strukturelle, thermische und proteolytische Stabilität, was sie zu attraktiven Gerüsten für die Entwicklung neuer Biotherapeutika macht. In diesem Artikel berichten wir über eine Studie zur Substrattoleranz der Sactisynthase AlbA, die die Bildung von Thioetherbrücken im Sactipeptid Subtilosin A katalysiert. Wir haben eine Modifikationsstelle innerhalb dieses Sactipeptids identifiziert, die ein Peptid‐Engineering ohne Beeinträchtigung der Bildung von Thioetherbrücken ermöglicht. Eine Reihe von natürlichen und hybriden Sactipeptidkonstrukten wurde hergestellt, um die AlbA vermittelte Bildung von Thioetherbrücken zu untersuchen und diese massenspektrometrisch zu identifizieren. In einer Proof‐of‐Principle‐Studie haben wir Subtilosin A mit einer neue Funktion ausgestattet, ein Thioether‐verbrücktes Streptavidin‐bindendes Peptid generiert und damit die Tür für das funktionelle Engineering von Sactipeptiden weiter geöffnet

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether‐Bond‐Forming Sactisynthase

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α‐carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in‐depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA‐mediated formation of thioether bridges, which were identified mass‐spectrometrically. In a proof‐of‐principle study, we re‐engineered subtilosin A to a thioether‐bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides

Self-decorating cells via surface-initiated enzymatic controlled radical polymerization

Through the innovative use of surface-displayed horseradish peroxidase, this work explores the enzymatic catalysis of both bioRAFT polymerization and bioATRP to prompt polymer synthesis on the surface of Saccharomyces cerevisiae cells, with bioATRP outperforming bioRAFT polymerization. The resulting surface modification of living yeast cells with synthetic polymers allows for a significant change in yeast phenotype, including growth profile, aggregation characteristics, and conjugation of non-native enzymes to the clickable polymers on the cell surface, opening new avenues in bioorthogonal cell-surface engineering

Influence of wastewater discharge on the occurrence of PBTC, HEDP, and aminophosphonates in sediment, suspended matter, and the aqueous phase of rivers

Sediment, suspended matter (SM), and water of a large river (Neckar; River1) and a small river (Körsch; River2) were analyzed for the phosphonates 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC), 1-hydroxyethylidene (1,1-diphosphonic acid) (HEDP), aminotris (methylphosphonic acid) (ATMP), ethylenediaminetetra (methylene phosphonic acid) (EDTMP), and diethylenetriaminepenta (methylene phosphonic acid) (DTPMP). Ten samplings were performed at intervals of one to two months during one year, each covering the relevant matrices before and behind the discharge point of a wastewater treatment plant (WWTP). In River1, the total concentration of dissolved phosphonate did not change significantly (2.4–5.8 µg/L before vs. 2.5–6.6 µg/L behind WWTP; p = 0.9360). In River2, it increased significantly from 2000 mg/kg phosphonate loads. In general, the nitrogen-free phosphonates PBTC and HEDP were most predominant in both dissolved and adsorbed form, of which HEDP had the highest adsorption affinity

A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast

The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor. We hence developed a predictable and modular, versatile expression control system. The selection of a reporter gene set up combining a gene of interest (GOI) with a fluorophore by the ribosomal skipping T2A sequence allows to adapt the system to any gene of interest without losing reporter function. In order to obtain a better understanding of the underlying principles and the functioning of our system, we backed our experimental findings with the development of a mathematical model and single-cell analysis

DataSheet_1_Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.pdf

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a “cytokine window” defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.</p

Table_1_Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.xlsx

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a “cytokine window” defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.</p