Cell Damage Threshold Research Articles

Balancing act: optimizing blue light for melanogenesis while minimizing cellular damage in primary human skin cells.

Recent findings show that visible light, particularly blue light, stimulates melanogenesis in human skin, though the underlying mechanisms remain debated. This study aimed to determine the cell damage threshold of non-ionizing blue light on keratinocytes while preserving their ability to stimulate melanogenesis. Human keratinocytes (N = 3) and melanocytes (N = 3) were isolated from skin samples of varying Fitzpatrick skin phototypes and irradiated with blue light (λpeak = 457nm) and UVA light (λpeak = 385nm). Cellular metabolic activity was assessed using the AlamarBlue HS assay, α-Melanocyte-Stimulating Hormone (α-MSH) production by keratinocytes was quantified using ELISA, and Western blotting was used to assess pro-melanogenic factor expression in melanocytes. High blue light intensity (50mW/cm2, 50J/cm2) and UVA light (15mW/cm2, 20J/cm2) significantly reduced cellular metabolic activity, with a 0.86 ± 0.055 and 0.60 ± 0.031 (mean ± SD) fold decrease compared to their respective sham by day 7. In contrast, moderate blue light intensities (5-15 mW/cm2, 10-20 J/cm2) preserved cellular metabolic activity while stimulating α-MSH production, with an optimal balance achieved at 10mW/cm2, 15J/cm2 (1.14 ± 0.046 fold increase relative to sham on day 7). Co-culture experiments confirmed that irradiated keratinocytes enhanced melanogenesis in melanocytes via paracrine signaling, increasing the expression of Tyrosinase and Dopachrome Tautomerase (DCT). Direct blue light irradiation on melanocytes also increased pigmentation without significant cellular damage. Moderate-intensity blue light at 10mW/cm2, 15J/cm2 effectively stimulates melanogenesis while maintaining cellular metabolic activity in both keratinocytes and melanocytes, offering a promising, safe approach for blue light therapies targeting pigmentation disorders.

Mecanismos fisiopatológicos de la tolerancia a la isquemia en el sistema nervioso central

El cerebro es uno de los órganos del cuerpo humano más sensibles a la isquemia, por lo que adquiere mecanismos endógenos para protegerse y recuperarse contra estímulos nocivos. La tolerancia isquémica constituye la adaptación del cerebro a la isquemia cuando se expone por primera vez a cualquier lesión que sea por debajo del umbral del daño celular. En las últimas décadas se han estudiado las vías moleculares y beneficios potenciales del pre y post condicionamiento, representando un objetivo terapéutico para pacientes con lesión cerebral aguda; sin embargo, las estrategias que se han experimentado no han demostrado seguridad en humanos y requieren un seguimiento de investigaciones sistemáticas para abordar su eficacia.

Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis.

Alpha-tocopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention.

A simple add-on microfluidic appliance for accurately sorting small populations of cells with high fidelity

Current advances in single cell sequencing, gene expression and proteomics require the isolation of single cells, frequently from a very small source population. In this work we describe the design and characterization of a manually operated microfluidic cell sorter that (1) can accurately sort single or small groups of cells from very small cell populations with minimal losses, (2) that is easy to operate and that can be used in any laboratory that has a basic fluorescent microscope and syringe pump, (3) that can be assembled within minutes, (4) that can sort cells in very short time (minutes) with minimum cell stress, (5) that is cheap and reusable. This microfluidic sorter is made from hard plastic material (PMMA) into which microchannels are directly milled with hydraulic diameter of 70 µm. Inlet and outlet reservoirs are drilled through the chip. Sorting occurs through hydrodynamic switching ensuring low hydrodynamic shear stresses, which were modeled and experimentally confirmed to be below the cell damage threshold. Manually operated, the maximum sorting frequencies were approximately 10 cells min−1. Experiments verified that cell sorting operations could be achieved in as little as 15 min, including the assembly and testing of the sorter. In only one out of ten sorting experiments the sorted cells were contaminated with another cell type. This microfluidic cell sorter represents an important capability for protocols requiring fast isolation of single cells from small number of rare cell populations.

Second-harmonic generation in lithium niobate nanowires for local fluorescence excitation

We study the nonlinear optical properties of lithium niobate (LiNbO(3)) nanowires (NWs) fabricated by a top-down ion beam enhanced etching method. First, we demonstrate generation and propagation of the second-harmonic (SH) light in LiNbO(3) NWs of typical rectangular cross-sections of 400 x 600 nm(2) and length from 10 to 50 μm. Then, we show local fluorescent excitation of 4',6-diamidino-2-phenylindole (DAPI) dye with the propagated SH signal in standard concentrations as for biological applications. By measuring the detected average power of the propagated fundamental harmonic (FH) and the SH signal at the output of the NWs, we directly prove the dominating role of the SH signal over possible two-photon excitation processes with the FH in the DAPI dye. We estimate that 63 ± 6 pW of the propagated SH average power is required for detectable dye excitation. Finally, we model the waveguiding of the SH light to determine the smallest NW cross-section (around 40x60 nm(2)) which is potentially able to excite fluorescence with a FH intensity below the cell damage threshold.

Mechanisms of nanoparticle-mediated photomechanical cell damage

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Photobioreactor scale-up for a shear-sensitive dinoflagellate microalga

Large scale culture of marine dinoflagellate microalgae has proved difficult owing to their extreme sensitivity to hydrodynamic forces in photobioreactors. This work discusses the scale up of Protoceratium reticulatum dinoflagellate culture from a successful 2 L stirred-tank photobioreactor operation to a 15 L stirred photobioreactor. Both bioreactors were equipped with internal spinfilters for cell retention. A semicontinuous perfusion culture in the 15 L photobioreactor proved to be more productive than fed-batch perfusion culture. Under the best operational conditions, the average cell productivity in the 15 L photobioreactor was 5228 cell mL −1 day −1, or nearly 3.7-fold greater than the best attainable value in static flask cultures, but similar to the results obtained in the 2 L stirred tank. At 9.16 μg L −1 day −1 the average volumetric productivity of yessotoxin in the scaled up operation in the semicontinuous perfusion mode was comparable to the results obtained in the small bioreactor. P. reticulatum has a cell damage threshold shear rate of as low as 0.1 s −1. Notwithstanding this extreme shear sensitivity, the results suggest a good potential for mass scale culture of this dinoflagellate in suitably designed photobioreactors.

Comparison of threshold irradiances and online dosimetry for selective retina treatment (SRT) in patients treated with 200 nanoseconds and 1.7 microseconds laser pulses

Selective retina therapy (SRT) solely affecting the RPE while sparing of the photoreceptors is usually performed with a train of repetitive laser pulses of 1.7 microseconds in duration. It was our purpose to evaluate the principle feasibility of SRT with shorter 200 nanoseconds laser pulses in patients. Nineteen patients with macular disorders [diabetic maculopathy (DMP), geographic atrophy (GA), drusen maculopathy and central serous chorioretinopathy (CSR)] were treated with a prototype of a SRT laser (Nd:YLF laser; 527 nm; 1.7 microseconds and 200 nanoseconds pulse duration; 30 pulses at 100 Hz; spot size: 200 microm). Test lesions (n = 175) with increasing energy were applied at the lower arcade to determine the individual angiographic and ophthalmoscopic threshold radiant exposures (therapeutic window) before applying the central treatment lesions within these ranges additionally guided by online optoacoustic measurements. Postoperatively RPE damage was visualized and confirmed by fluorescein angiographic leakage and correlated with optoacoustic results. Additionally ED(50) damage thresholds were calculated by probit analysis. None of the short repetitive 200 nanoseconds laser pulses led to retinal hemorrhages or retinal ruptures. Nearly all of the test- and treatment lesions could be visualized by angiography indicating desired RPE damage but were ophthalmoscopically invisible suggesting intact neurosensory retinal structures. ED(50) cell damage threshold energies were significantly lower using 200 nanoseconds (99.6 microJ; n = 122) instead of 1.7 microseconds (196.3 microJ; n = 53) laser pulses. Optoacoustic and angiographic visibility correlated in 83.7% (200 nanoseconds) and 87.5% (1.7 microseconds). Selective RPE effects can safely be achieved using shorter 200 nanoseconds laser pulses in patients without adverse effects to the neurosensory retina. The required pulse energy compared to the standard 1.7 microseconds regime was reduced by about a factor of 2 suggesting a reduced heat generation and flow into adjacent tissues during the shorter laser impact and thus possibly enhancing selectivity. Optoacoustics also seem to be a viable alternative in 200 nanoseconds treatment for a non-invasive online dosimetry control system.

Bioeffects of Low-Frequency Ultrasonic Gene Delivery and Safety on Cell Membrane Permeability Control

To develop a novel method of ultrasonic naked gene delivery (UNGD); to examine the relationship between optimal parameters of ultrasound exposure and cell membrane permeability, enzymes, and free radicals; and to find optimal control parameters that were realizable, reliable, and noncytotoxic for use in gene therapy. Suspensions of chicken, rabbit, and rat red blood cells and S180 cells were exposed to a calibrated ultrasonic field with different parameters in both the still and flowing states to obtain optimal parameters for UNGD. The optimal parameters then were used to implement UNGD. We examined morphologic characteristics, membrane permeability, enzymes, free radicals, naked gene expression efficiency, cell damage threshold, and cell viability by laser scanning confocal microscopy, fluorescent microscopy, flow cytometry, and spectrophotometry. Green fluorescent protein (GFP) as a reporter was delivered into S180 cells under the optimal parameters without cell damage or cytotoxicity. The transfection rate (mean +/- SD) was approximately 35.83% +/- 2.53% (n = 6) in viable cells, and cell viability was 90.17% +/- 1.47% (n = 6). The intensity of GFP expression with UNGD showed a higher fluorescent peak over both an adeno-associated virus vector-GFP group and a control group (P < .001). Additionally, malondialdehyde, hydroxyl free radicals, alkaline phosphatase, and acid phosphatase displayed an S-shaped growth model (r = 0.98 +/- 0.01) in response to permeability and morphologic alteration. Under optimal conditions, low-frequency ultrasound can safely deliver naked genes into cells without causing cell damage. The analytical results indicate that, except for subcavitation, free radical products are responsible for bioeffects in gene delivery. The constant E of energy deposition at 90% cell viability is the optimal control factor, and 80% viability represents the damage threshold. Optimal gene uptake by cells and safety depend on E. Constant E can be applied to control the gene delivery effect in combination with other parameters.

Selective photothermal interaction using an 805-nm diode laser and indocyanine green in gel phantom and chicken breast tissue.

Laser immunotherapy, a novel therapy for breast cancer, utilises selective photothermal interaction to raise the temperature of tumour tissue above the cell damage threshold. Photothermal interaction is achieved with intratumoral injection of a laser-absorbing dye followed by non-invasive laser irradiation. When tumour heating is used in combination with immunoadjuvant to stimulate an immune response, antitumour immunity can be achieved. In this study, the selective photothermal effect was investigated using gel phantom and chicken breast tissue. An 805-nm diode laser and indocyanine green (ICG) were used. An ICG-containing gelatin phantom was constructed to simulate targeted tumour tissue. The target gel was buried inside chicken breast tissue and the tissue-gel construct was irradiated by the laser. Temperatures at different locations in the construct were measured during the laser irradiation. For comparison, the thermal effect of an Nd:YAG laser on the tissue-gel construct was also investigated. Selective heating of target gel containing 0.27% ICG and buried 1 cm below the chicken tissue surface was achieved with the 805-nm diode laser using a power of 0.85 W and beam radius of 1 cm. The target gel experienced a temperature increase of more then 6 degrees C whereas the surrounding chicken breast tissue experienced only a minor temperature increase. The feasibility of this experimental set-up has been shown. It will be used in the future to optimise treatment parameters such as laser power, laser beam radius, and dye concentration.