Rapid and precise tracking of water influx and efflux across cell membranes induced by a pulsed electric field.

Quantitative measurements of water content within a single cell are notoriously difficult. In this work, we introduce a single-shot optical method for tracking the intracellular water content, by mass and volume, of a single cell at video rate. We utilize quantitative phase imaging and a priori knowledge of a spherical cellular geometry, leveraging a two-component mixture model to compute the intracellular water content. We apply this technique to study CHO-K1 cells responding to a pulsed electric field, which induces membrane permeabilization and rapid water influx or efflux depending upon the osmotic environment. The effects of mercury and gadolinium on water uptake in Jurkat cells following electropermeabilization are also examined.

600-ns pulsed electric fields affect inactivation and antibiotic susceptibilities of Escherichia coli and Lactobacillus acidophilus.

Cell suspensions of Escherichia coli and Lactobacillus acidophilus were exposed to 600-ns pulsed electric fields (nsPEFs) at varying amplitudes (Low-13.5, Mid-18.5 or High-23.5 kV cm-1) and pulse numbers (0 (sham), 1, 5, 10, 100 or 1000) at a 1 hertz (Hz) repetition rate. The induced temperature rise generated at these exposure parameters, hereafter termed thermal gradient, was measured and applied independently to cell suspensions in order to differentiate inactivation triggered by electric field (E-field) from heating. Treated cell suspensions were plated and cellular inactivation was quantified by colony counts after a 24-hour (h) incubation period. Additionally, cells from both exposure conditions were incubated with various antibiotic-soaked discs to determine if nsPEF exposure would induce changes in antibiotic susceptibility. Results indicate that, for both species, the total delivered energy (amplitude, pulse number and pulse duration) determined the magnitude of cell inactivation. Specifically, for 18.5 and 23.5 kV cm-1 exposures, L. acidophilus was more sensitive to the inactivation effects of nsPEF than E. coli, however, for the 13.5 kV cm-1 exposures E. coli was more sensitive, suggesting that L. acidophilus may need to meet an E-field threshold before significant inactivation can occur. Results also indicate that antibiotic susceptibility was enhanced by multiple nsPEF exposures, as observed by increased zones of growth inhibition. Moreover, for both species, a temperature increase of ≤ 20 °C (89% of exposures) was not sufficient to significantly alter cell inactivation, whereas none of the thermal equivalent exposures were sufficient to change antibiotic susceptibility categories.

Nanosecond pulsed electric field exposure does not induce the unfolded protein response in adult human dermal fibroblasts.

Cell-circuit models have suggested that nanosecond pulsed electric fields (nsPEFs) can disrupt intracellular membranes including endoplasmic reticulum (ER), mitochondria, and/or nucleus thereby inducing intrinsic apoptotic pathways. Therefore, we hypothesized that the unfolded protein response (UPR) would be activated, due to the fluctuations of ionic concentrations, upon poration of the ER membrane. Quantitative real-time polymerase chain reaction was utilized to measure changes in messenger RNA (mRNA) expression of specific ER stress genes in adult human dermal fibroblast (HDFa) cells treated with tunicamycin (TM) (known ER stress inducer) and cells exposed to nsPEFs (100, 10-ns pulses at 150 kV/cm delivered at a repetition rate of 1 Hz). For HDFa cells, results showed time-dependent UPR activation to TM; however, when HDFa cells were exposed to nsPEFs, no significant changes in mRNA expression of ER stress genes, and/or caspase gene were observed. These results indicate that although cell death can be observed under these exposure parameters, it is most likely not initiated through activation of the UPR. Bioelectromagnetics. 2018;39:491-499, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Adult human dermal fibroblasts exposed to nanosecond electrical pulses exhibit genetic biomarkers of mechanical stress.

BACKGROUND: Exposure of cells to very short (<1 µs) electric pulses in the megavolt/meter range have been shown to cause a multitude of effects, both physical and molecular in nature. Physically, nanosecond electrical pulses (nsEP) can cause disruption of the plasma membrane, cellular swelling, shrinking and blebbing. Molecularly, nsEP have been shown to activate signaling pathways, produce oxidative stress, stimulate hormone secretion and induce both apoptotic and necrotic death. We hypothesize that studying the genetic response of primary human dermal fibroblasts exposed to nsEP, will gain insight into the molecular mechanism(s) either activated directly by nsEP, or indirectly through electrophysiology interactions. METHODS: Microarray analysis in conjunction with quantitative real time polymerase chain reaction (qRT-PCR) was used to screen and validate genes selectively upregulated in response to nsEP exposure. RESULTS: Expression profiles of 486 genes were found to be significantly changed by nsEP exposure. 50% of the top 20 responding genes coded for proteins located in two distinct cellular locations, the plasma membrane and the nucleus. Further analysis of five of the top 20 upregulated genes indicated that the HDFa cells' response to nsEP exposure included many elements of a mechanical stress response. CONCLUSIONS: We found that several genes, some of which are mechanosensitive, were selectively upregulated due to nsEP exposure. This genetic response appears to be a primary response to the stimuli and not a secondary response to cellular swelling. GENERAL SIGNIFICANCE: This work provides strong evidence that cells exposed to nsEP interpret the insult as a mechanical stress.

Action potential block in neurons by infrared light.

Short infrared laser pulses (SILP) have many physiological effects on cells, including the ability to stimulate action potentials (APs) in neurons. Here, we show that SILPs can also reversibly block APs. Reversible AP block in hippocampal neurons was observed following SILP (0.26 to [Formula: see text]; 1.37 to 5.01 ms; 1869 nm) with the block persisting for more than 1 s with exposures greater than [Formula: see text]. AP block was sustained for 30 s with SILPs pulsed at 1 to 7 Hz. Full recovery of neuronal activity was observed 5 to 30 s post SILP exposure. These results indicate that SILP can be used for noncontact, reversible AP block. Due to the high spatial precision and noncontact manner of infrared light delivery, AP block by SILP (infrared neural inhibition) has the potential to transform medical care for sustained pain inhibition and suppression of unwanted nerve activity.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.