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The quality of research antibodies is an issue for decades. Although several papers have been published to improve the situation, their impact seems to be limited. This publication makes the effort to simplify the description of validation criteria in a way that the occasional antibody user is able to assess the validation level of an immunochemical reagent. A simple, 1-page checklist is supplied for the practical application of these criteria.
Homochirality is an obvious feature of life on Earth. On the other hand, extraterrestrial samples contain largely racemic compounds. The same is true for any common organic synthesis. Therefore, it has been a perplexing puzzle for decades how these racemates could have formed enantiomerically enriched fractions as a basis for the origin of homochiral life forms. Numerous hypotheses have been put forward as to how preferentially homochiral molecules could have formed and accumulated on Earth. In this article, it is shown that homochirality of the abiotic organic pool at the time of formation of the first self-replicating molecules is not necessary and not even probable. It is proposed to abandon the notion of a molecular ensemble and to focus on the level of individual molecules. Although the formation of the first self-replicating, most likely homochiral molecule, is a seemingly improbable event, on a closer look, it is almost inevitable that some homochiral molecules have formed simply on a statistical basis. In this case, the non-selective leap to homochirality would be one of the first steps in chemical evolution directly out of a racemic “ocean”. Moreover, most studies focus on the chirality of the primordial monomers with respect to an asymmetric carbon atom. However, any polymer with a minimal size that allows folding to a secondary structure would spontaneously lead to asymmetric higher structures (conformations). Most of the functions of these polymers would be influenced by this inherently asymmetric folding. Furthermore, a concept of physical compartmentalization based on rock nanopores in analogy to nanocavities of digital immunoassays is introduced to suggest that complex cell walls or membranes were also not required for the first steps of chemical evolution. To summarize, simple and universal mechanisms may have led to homochiral self-replicating systems in the context of chemical evolution. A homochiral monomer pool is deemed unnecessary and probably never existed on primordial Earth.
Homochirality is an obvious feature of life on Earth. On the other hand, extraterrestrial samples contain largely racemic compounds. The same is true for any common organic synthesis. Therefore, it has been a perplexing puzzle for decades how these racemates could have formed enantiomerically enriched fractions as a basis for the origin of homochiral life forms. Numerous hypotheses have been put forward as to how preferentially homochiral molecules could have formed and accumulated on Earth. In this article, it is shown that homochirality of the abiotic organic pool at the time of formation of the first self-replicating molecules is not necessary and not even probable. It is proposed to abandon the notion of a molecular ensemble and to focus on the level of individual molecules. Although the formation of the first self-replicating, most likely homochiral molecule is a seemingly improbable event, on a closer look, it is almost inevitable that homochiral molecules have formed simply on a statistical basis. In this case, the non-selective leap to homochirality would be one of the first steps in chemical evolution directly out of a racemic “ocean”. Moreover, most studies focus on the chirality of the primordial monomers with respect to an asymmetric carbon atom. However, any polymer with a minimal size that allows folding to a secondary structure, would spontaneously lead to asymmetric higher structures (conformations). Most of the functions of these polymers would be influenced by this inherently asymmetric folding. To summarize, simple and universal mechanisms may have led to homochiral self-replicating systems in the context of chemical evolution. A homochiral monomer pool is deemed unnecessary and probably never existed on primordial Earth.
A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.
A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 min. Due to the glass material’s excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nano-filtration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.
Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite.
For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs.
Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene.