Cesario Z. Cerna

Identification of microRNAs associated with hyperthermia-induced cellular stress response

MicroRNAs (miRNAs) are a class of small RNAs that play a critical role in the coordination of fundamental cellular processes. Recent studies suggest that miRNAs participate in the cellular stress response (CSR), but their specific involvement remains unclear. In this study, we identify a group of thermally regulated miRNAs (TRMs) that are associated with the CSR. Using miRNA microarrays, we show that dermal fibroblasts differentially express 123 miRNAs when exposed to hyperthermia. Interestingly, only 27 of these miRNAs are annotated in the current Sanger registry. We validated the expression of the annotated miRNAs using qPCR techniques, and we found that the qPCR and microarray data was in well agreement. Computational target-prediction studies revealed that putative targets for the TRMs are heat shock proteins and Argonaute-2—the core functional unit of RNA silencing. These results indicate that cells express a specific group of miRNAs when exposed to hyperthermia, and these miRNAs may function in the regulation of the CSR. Future studies will be conducted to determine if other cells lines differentially express these miRNAs when exposed to hyperthermia.

Development of a compact terahertz time-domain spectrometer for the measurement of the optical properties of biological tissues

Terahertz spectrometers and imaging systems are currently being evaluated as biomedical tools for skin burn assessment. These systems show promise, but due to their size and weight, they have restricted portability, and are impractical for military and battlefield settings where space is limited. In this study, we developed and tested the performance of a compact, light, and portable THz time-domain spectroscopy (THz-TDS) device. Optical properties were collected with this system from 0.1 to 1.6 THz for water, ethanol, and several ex vivo porcine tissues (muscle, adipose, skin). For all samples tested, we found that the index of refraction (n) decreases with frequency, while the absorption coefficient (μ(a)) increases with frequency. Muscle, adipose, and frozen/thawed skin samples exhibited comparable n values ranging between 2.5 and 2.0, whereas the n values for freshly harvested skin were roughly 40% lower. Additionally, we found that the freshly harvested samples exhibited higher μ(a) values than the frozen/thawed skin samples. Overall, for all liquids and tissues tested, we found that our system measured optical property values that were consistent with those reported in the literature. These results suggest that our compact THz spectrometer performed comparable to its larger counterparts, and therefore may be a useful and practical tool for skin health assessment.

Terahertz Radiation: A Non-contact Tool for the Selective Stimulation of Biological Responses in Human Cells

Collective motions of water and of many biological macromolecules have characteristic time scales on the order of a picosecond. As a result, these biomolecules can strongly absorb terahertz (THz) radiation. Due to this absorption, THz radiation can exert a diverse range of effects on biological structures. For example, THz radiation has been shown to impact the structure, functional activity, and dynamics of macromolecules such as DNA and proteins. THz radiation can affect several gene expression pathways and, consequently, can alter various biochemical and physiological processes in cells. Indeed, THz radiation has been shown to influence the expression of several genes within different cell types. However, a complete view of the global transcriptional responses and the intracellular canonical pathways specifically triggered by THz radiation has not been elucidated. In this study, we performed a global profiling of transcripts in human cells exposed to 2.52 THz radiation and compared the exposure responses to a thermally-matched bulk-heating (BH) protocol. Our results show that both THz radiation and BH induce a significant change in the expression of numerous mRNAs and microRNAs. The data also show that THz radiation triggers specific intracellular canonical pathways that are not affected in the BH-exposed cells. This study implies that THz radiation may be a useful, non-contact tool for the selective control of specific genes and cellular processes.

Noninvasive Techniques for the Determination of Burn Severity in Real Time.

Visual diagnosis of second-degree burns has proven inadequate for determining the appropriate treatment regimen. Although multiple noninvasive imaging techniques have shown promise for providing information about burn wound severity, the ideal technology to aid burn wound excision would provide real-time readouts. Herein, the authors examine a high-resolution infrared (IR) camera (thermography) and a multiprobe adapter system (MPAS-6; transepidermal evaporative water loss, colorimetry) to assess their usefulness in predicting burn severity. Contact burn wounds of increasing severity were created in a porcine model. Wounds were assessed for 4 days with an IR camera and MPAS-6. In addition, each day, the burn wounds were biopsied for histological analysis to determine burn depth for correlation with noninvasive measures. Surface temperatures decreased with increasing burn severity, which was associated with increasing transepidermal evaporative water loss. Melanin content correlated with the depth of collagen coagulation and was bimodal, with superficial and full-thickness burns having higher values than deep partial thickness wounds. Erythema content was highest in superficial burns and negatively correlated with necrosis (high-mobility group box protein 1 expression). Importantly, surface temperature taken on every single day after injury was predictive of all histologically determined measurements of burn depth (ie, collagen coagulation, apoptosis, necrosis, vascular occlusion). The results indicate that IR imaging and skin quality probes can be used to support the diagnosis of burn severity. Most importantly, IR measurements gave insight into both the zone of coagulation and the zone of stasis on every postburn day studied.

Temporal Gene Expression Kinetics for Human Keratinocytes Exposed to Hyperthermic Stress

The gene expression kinetics for human cells exposed to hyperthermic stress are not well characterized. In this study, we identified and characterized the genes that are differentially expressed in human epidermal keratinocyte (HEK) cells exposed to hyperthermic stress. In order to obtain temporal gene expression kinetics, we exposed HEK cells to a heat stress protocol (44 °C for 40 min) and used messenger RNA (mRNA) microarrays at 0 h, 4 h and 24 h post-exposure. Bioinformatics software was employed to characterize the chief biological processes and canonical pathways associated with these heat stress genes. The data shows that the genes encoding for heat shock proteins (HSPs) that function to prevent further protein denaturation and aggregation, such as HSP40, HSP70 and HSP105, exhibit maximal expression immediately after exposure to hyperthermic stress. In contrast, the smaller HSPs, such as HSP10 and HSP27, which function in mitochondrial protein biogenesis and cellular adaptation, exhibit maximal expression during the “recovery phase”, roughly 24 h post-exposure. These data suggest that the temporal expression kinetics for each particular HSP appears to correlate with the cellular function that is required at each time point. In summary, these data provide additional insight regarding the expression kinetics of genes that are triggered in HEK cells exposed to hyperthermic stress.

Effects of different terahertz frequencies on gene expression in human keratinocytes

In recent years, a surge in the development of many terahertz (THz) sensing and imaging technologies occurred leading to increased use in military and civil operations. Therefore, understanding the biological effects associated with exposures to this radiation is becoming increasingly important. Previous studies have speculated that cells exposed to different frequencies of THz radiation may exhibit differential responses. However, empirical studies to confirm such differences have not been performed. The question of whether cells exposed to different THz frequencies exhibited specific biological responses remains unclear. In this study, we exposed human keratinocytes to a THz laser tuned to several different THz frequencies using our recently developed THz exposure system. This system consists of an optically pumped molecular gas THz laser source coupled to a modified cell culture incubator permitting THz radiation exposures under controlled standard tissue culture conditions. For all frequencies, we matched the THz exposure duration and irradiance. During THz exposure, we monitored the power as DC voltage-logged values (LabVIEW™ IV log). To determine the temperature changes by THz exposure, we collected temperature readings from the unexposed and THz-exposed cells using thermocouples. We assessed cellular viability after exposure using MTT colorimetric assays. We compared the changes in gene expression profiles using messenger RNA (mRNA) microarrays, and we identified the THz-induced signaling pathways for each frequency using bioinformatics. Our data provide valuable new insights that give a comparative picture of the genes and intracellular signaling pathways triggered in cells exposed to THz radiation at different frequencies.

Investigation of a direct effect of nanosecond pulse electric fields on mitochondria

The unique cellular response to nanosecond pulsed electric field (nsPEF) exposure, as compared to longer pulse exposure, has been theorized to be due to permeabilization of intracellular organelles including the mitochondria. In this investigation, we utilized a high-throughput oxygen and pH sensing system (Seahorse® XF24 extracellular flux analyzer) to assess the mitochondrial activity of Jurkat and U937 cells after nsPEF. The XF Analyzer uses a transient micro-chamber of only a few μL in specialized cell culture micro-plates to enable oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to be monitored in real-time. We found that for nsPEF exposures of 10 pulses at 10-ns pulse width and at 50 kV/cm e-field, we were able to cause an increase in OCR in both U937 and Jurkat cells. We also found that high pulse numbers (>100) caused a significant decrease in OCR. Higher amplitude 150 kV/cm exposures had no effect on U937 cells and yet they had a deleterious effect on Jurkat cells, matching previously published 24 hour survival data. These results suggest that the exposures were modulating metabolic activity in cells possibly due to direct effects on the mitochondria themselves. To validate this hypothesis, we isolated mitochondria from U937 cells and exposed them similarly and found no significant change in metabolic activity for any pulse number. In a final experiment, we removed calcium from the buffer solution that the cells were exposed in and found that no significant enhancement in metabolic activity was observed. These results suggest that direct permeabilization of the mitochondria is unlikely a primary effect of nsPEF exposure and calcium-mediated intracellular pathway activation is likely responsible for observed pulse-induced mitochondrial effects.

State-of-the-art exposure chamber for highly controlled and reproducible THz biological effects studies

Terahertz (THz) imaging and sensing technologies are increasingly being used at international airports for security screening purposes and at major medical centers for cancer and burn diagnosis. The emergence of new THz applications has directly resulted in an increased interest regarding the biological effects associated with this frequency range. Knowledge of THz biological effects is also desired for the safe use of THz systems, identification of health hazards, and development of empirically-based safety standards. In this study, we developed a state-of-the-art exposure chamber that allowed for highly controlled and reproducible studies of THz biological effects. This innovative system incorporated an industry grade cell incubator system that permitted a highly controlled exposure environment, where temperatures could be maintained at 37 °C ± 0.1 °C, carbon dioxide (CO2) levels at 5% ± 0.1%, and relative humidity (RH) levels at 95% ± 1%. To maximize the THz power transmitted to the cell culture region inside the humid incubator, a secondary custom micro-chamber was fabricated and incorporated into the system. This micro-chamber shields the THz beam from the incubator environment and could be nitrogen-purged to eliminate water absorption effects. Additionally, a microscope that allowed for real-time visualization of the live cells before, during, and after THz exposure was integrated into the exposure system.