Advantages and limitations of various treatment chamber designs for reversible and irreversible electroporation in life sciences.

The fundamental mechanisms of pulsed electric fields on biological cells are not yet fully elucidated, though it is apparent that membrane electroporation plays a crucial role. Little is known about treatment-chamber-specific effects, and systematic studies are scarce. Thus, the present study evaluates the (dis-)advantages of various treatment chamber designs for liquid applications at differing scales. Three chambers, namely parallel plate microfluidic (V̇: 0.1 ml/min; titanium electrodes), co-linear meso (V̇: 5.0 ml/min; stainless steel electrodes), and co-linear macro (V̇: 83.3 ml/min; stainless steel electrodes) chambers, were studied. Electroporation effects on Escherichia coli in media with 0.1-10.0 mS/cm were evaluated by plate counts and flow cytometry at 8, 16, and 20 kV/cm. For the microfluidic chamber, predominantly irreversible electroporation (2.5 logs10 reductions) was seen at 0.1 mS/cm, while high irreversible electroporation (4.2 logs10 reductions) at 10.0 mS/cm was observed for the macro chamber. The meso chamber indicated a similar trend towards increased conductivity, even though only low inactivation levels were present. Variation in conductivity and electrode configuration or area likely induces effects resulting in distinct electroporation levels, as observed for the micro and macro chamber. Suitable application scenarios, depending on targeted electroporation effects, were suggested.

Capacitive coupling increases the accuracy of cell-specific tumour disruption by electric fields.

Irreversible electroporation holds great potential for cell-specific lysis due to the size-dependent susceptibility of cells to externally imposed electric fields. Previous attempts at selective cell lysis lead to significant overlap between affected populations and struggle with inconsistent biological outcome. We propose that charge transfer at the electrode-liquid interface is responsible by inducing multifactorial effects originating from both the electric field and electrochemical reactions. A promising remedy is the coating of electrodes with a high-k dielectric layer. The resulting capacitive coupling restores the selective potential of electric field mediated lysis in a microfluidic setup. Initial experiments show the consistent depletion of erythrocytes from whole blood while leaving leukocytes intact. The same is true for the reproducible and selective depletion of Jurkat and MCF-7 cells in a mixture with leukocytes. Unexpectedly, the observed order of lysis cannot be correlated with cell size. This implies that the cellular response to capacitive coupling features a selective characteristic that is different from conventional lysis configurations.

Controllable cell manipulation in a microfluidic pipette-tip design using capacitive coupling of electric fields.

Systems designed toward cell manipulation by electric fields are inherently challenged by energy dissipation along the electrode-electrolyte interface. A promising remedy is the introduction of high-k electrode passivation, enabling efficient capacitive coupling of electric fields into biological samples. We present the implementation of this strategy in a reusable pipette tip design featuring a 10 µl chamber volume for life science applications. Prototype validation and comparison to conductive gold-coated electrodes reveal a consistent and controllable biological effect that significantly increases the reproducibility of lysis events. The system provides precise descriptions of HEK-293 lysis dependency to variables such as field strength, frequency, and conductivity. Over 80% of cells were reversibly electroporated with minimal electrical lysis over a broad range of field settings. Successful transfection requires exponential decay pulses and showcases how modulating capacitive coupling can advance our understanding of fundamental mechanics in the field of electroporation.

Triggering receptor expressed on myeloid cells-2 fine-tunes inflammatory responses in murine Gram-negative sepsis.

During infections, TLR-mediated responses require tight regulation to allow for pathogen removal, while preventing overwhelming inflammation and immunopathology. The triggering receptor expressed on myeloid cells (TREM)-2 negatively regulates inflammation by macrophages and impacts on phagocytosis, but the function of endogenous TREM-2 during infections is poorly understood. We investigated TREM-2's role in regulating TLR4-mediated inflammation by studying wild-type and TREM-2(-/-) mice challenged with LPS and found TREM-2 to dampen early inflammation. Augmented early inflammation in TREM-2(-/-) animals was followed by an accelerated resolution and ultimately improved survival, associated with the induction of the negative regulator A20. Upon infection with Escherichia coli, the otherwise beneficial effect of an exaggerated early immune response in TREM-2(-/-) animals was counteracted by a 50% reduction in bacterial phagocytosis. In line with this, TREM-2(-/-) peritoneal macrophages (PMs) exhibited augmented inflammation following TLR4 stimulation, demonstrating the presence and negative regulatory functionality of TREM-2 on primary PMs. Significantly, we identified a high turnover rate because TREM-2 RNA is 25-fold down-regulated and the protein proteasomally degraded upon LPS encounter, thus ensuring a tightly regulated and versatile system that modulates inflammation. Our results illustrate TREM-2's effects on infection-triggered inflammation and identify TREM-2 as a potential target to prevent overwhelming inflammation while preserving antibacterial-effector functions.

Cross-talk between calcium signalling and protein phosphorylation at the thylakoid.

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.