Phyto-Photodynamic Therapy of Prostate Cancer Cells Mediated by Yemenite 'Etrog' Leave Extracts.

Cancer therapy, from malignant tumor inhibition to cellular eradication treatment, remains a challenge, especially regarding reduced side effects and low energy consumption during treatment. Hence, phytochemicals as cytotoxic sensitizers or photosensitizers deserve special attention. The dark and photo-response of Yemenite 'Etrog' leaf extracts applied to prostate PC3 cancer cells is reported here. An XTT cell viability assay along with light microscope observations revealed pronounced cytotoxic activity of the extract for long exposure times of 72 h upon concentrations of 175 µg/mL and 87.5 µg/mL, while phototoxic effect was obtained even at low concentration of 10.93 µg/mL and a short introduction period of 1.5 h. For the longest time incubation of 72 h and for the highest extract concentration of 175 µg/mL, relative cell survival decreased by up to 60% (below the IC50). In combined phyto-photodynamic therapy, a reduction of 63% compared to unirradiated controls was obtained. The concentration of extract in cells versus the accumulation time was inversely related to fluorescence emission intensity readings. Extracellular ROS production was also shown. Based on an ATR-FTIR analysis of the powdered leaves and their liquid ethanolic extract, biochemical fingerprints of both polar and non-polar phyto-constituents were identified, thereby suggesting their implementation as phyto-medicine and phyto-photomedicine.

Metastasis diagnosis using attenuated total reflection-Fourier transform infra-red (ATR-FTIR) spectroscopy.

The suitability of Fourier transform infrared spectroscopy as a metastasis prognostic tool has not been reported for some cancer types. Our main aim was to show spectroscopic differences between live un-preprocessed cancer cells of different metastatic levels. Spectra of four cancer cell pairs, including colon cancer (SW480, SW620); human melanoma (WM115, WM266.4); murine melanoma (B16F01, B16F10); and breast cancer (MCF7, MDA-MB-231); each pair having the same genetic background, but different metastatic level were analyzed in the regions 1400-1700 cm-1 and 3100-3500 cm-1 using Principal Component Analysis, curve fitting, multifractal dimension and receiver operating characteristic (ROC) curves. The results show spectral markers I1540/I1473, I1652/I1473, [Formula: see text], and multifractal dimension of the spectral images are significantly different for the cells based on their metastatic levels. ROC curve analysis showed good diagnostic performance of the spectral markers in separating cells based on metastatic degree, with areas under the ROC curves having 95% confidence interval lower limits greater than 0.5 for most instances. These spectral features can be important in predicting the probability of metastasis in primary tumors, providing useful guidance for treatment planning. Our markers are effective in differentiating metastatic levels without sample fixation or drying and therefore could be compactible for future use in in-vivo procedures involving spectroscopic cancer diagnosis.

Multifractal analysis of cellular ATR-FTIR spectrum as a method for identifying and quantifying cancer cell metastatic levels.

Cancer is a leading cause of mortality today. Sooner a cancer is detected, the more effective is the treatment. Histopathological diagnosis continues to be the gold standard worldwide for cancer diagnosis, but the methods used are invasive, time-consuming, insensitive, and still rely to some degree on the subjective judgment of pathologists. Recent research demonstrated that Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy can be used to determine the metastatic potential of cancer cells by evaluating their membrane hydration. In the current study, we demonstrate that the conversion of ATR-FTIR spectra using multifractal transformation generates a unique number for each cell line's metastatic potential. Applying this technique to murine and human cancer cells revealed a correlation between the metastatic capacity of cancer cells within the same lineage and higher multifractal value. The multifractal spectrum value was found to be independent of the cell concentration used in the assay and unique to the tested lineage. Healthy cells exhibited a smaller multifractal spectrum value than cancer cells. Further, the technique demonstrated the ability to detect cancer progression by being sensitive to the proportional change between healthy and cancerous cells in the sample. This enables precise determination of cancer metastasis and disease progression independent of cell concentration by comparing the measured spectroscopy derived multifractal spectrum value. This quick and simple technique devoid of observer bias can transform cancer diagnosis to a great extent improving public health prognosis worldwide.

A Poly-D-lysine-Coated Coralline Matrix Promotes Hippocampal Neural Precursor Cells' Differentiation into GFAP-Positive Astrocytes.

A major goal of regenerative medicine of the central nervous system is to accelerate the regeneration of nerve tissue, where astrocytes, despite their positive and negative roles, play a critical role. Thus, scaffolds capable of producing astrocytes from neural precursor cells (NPCs) are most desirable. Our study shows that NPCs are converted into reactive astrocytes upon cultivation on coralline-derived calcium carbonate coated with poly-D-lysine (PDL-CS). As shown via nuclei staining, the adhesion of neurospheres containing hundreds of hippocampal neural cells to PDL-CS resulted in disaggregation of the cell cluster as well as the radial migration of dozens of cells away from the neurosphere core. Migrating cells per neurosphere averaged 100 on PDL-CS, significantly higher than on uncoated CS (28), PDL-coated glass (65), or uncoated glass (20). After 3 days of culture on PDL-CS, cell migration plateaued and remained stable for four more days. In addition, NPCs expressing nestin underwent continuous morphological changes from round to spiky, extending and elongating their processes, resembling activated astrocytes. The extension of the process increased continuously during the maturation of the culture and doubled after 7 days compared to day 1, whereas bifurcation increased by twofold during the first 3 days before plateauing. In addition, nestin positive cells' shape, measured through the opposite circularity level correlation, decreased approximately twofold after three days, indicating spiky transformation. Moreover, nestin-positive cells co-expressing GFAP increased by 2.2 from day 1 to 7, reaching 40% of the NPC population on day 7. In this way, PDL-CS promotes NPC differentiation into reactive astrocytes, which could accelerate the repair of neural tissue.

Optimizing the average distance between a blue light photosensitizer and a harmonic nanoparticle for effective infrared photodynamic therapy.

Photodynamic therapy can be significantly improved by techniques utilizing light windows of higher tissue penetration depths with optimally matched photoactive agents to provide deep interstitial treatment. Classical blue light photosensitizers were photodynamically activated using infrared light via coupled harmonic nanoparticles with optimized intermediary distances using spacers. Upon 800 nm pulsed laser irradiation perovskite nanoparticles with optimized coupling to either curcumin or protoporphyrin IX reduced the viability of MCF7 breast cancer cells by 73 percent and 64 percent, respectively, while exhibiting negligible dark toxicity. The findings pave the way for clinical adaptation of ease-of-synthesis photodynamically active preparations operable under deep tissue penetrating infrared lights using commonly available otherwise infrared inactive classical blue light photosensitizers.

Second harmonic generation nanoparticles enables Near-Infrared Photodynamic Therapy from visible light reactive photosensitizer conjugates.

Combination of photosensitizers (PS) with nanotechnology can improve the therapeutic efficiency of clinical Photodynamic Therapy (PDT) by converting visible light reactive PSs into Near-Infrared (NIR) light responsive molecules using Harmonic Nanoparticles (HNP). To test the PDT efficiency of HNP-PS conjugates, pathogenic S. aureus cell cultures were treated with perovskite (Barium Titanate) Second Harmonic Generation (SHG) nanoparticles conjugated to photosensitizers (PS) (we compared both FDA approved Protoporphyrin IX and Curcumin) and subjected to a femtosecond pulsed Near-Infrared (NIR) laser (800 nm, 232-228 mW, 12-15 fs pulse width at repetition rate of 76.9 MHz) for 10 minutes each. NIR PDT using Barium Titanate (BT) conjugated with Protoporphyrin IX as HNP-PS conjugate reduced the viability of S. aureus cells by 77.3 ± 9.7% while BT conjugated with Curcumin did not elicit any significant effect. Conventional PSs reactive only to visible spectrum light coupled with SHG nanoparticles enables the use of higher tissue penetrating NIR light to generate an efficient photodynamic effect, thereby overcoming low light penetration and tissue specificity of conventional visible light PDT treatments.

Automatic Identification of Dendritic Branches and their Orientation.

The structure of neuronal dendritic trees plays a key role in the integration of synaptic inputs in neurons. Therefore, characterization of the morphology of dendrites is essential for a better understanding of neuronal function. However, the complexity of dendritic trees, both when isolated and especially when located within neuronal networks, has not been completely understood. We developed a new computational tool, SOA (Segmentation and Orientation Analysis), which allows automatic measurement of the orientation of dendritic branches from fluorescence images of 2D neuronal cultures. SOA, written in Python, uses segmentation to distinguish dendritic branches from the image background and accumulates a database on the spatial direction of each branch. The database is then used to calculate morphological parameters such as the directional distribution of dendritic branches in a network and the prevalence of parallel dendritic branch growth. The data obtained can be used to detect structural changes in dendrites in response to neuronal activity and to biological and pharmacological stimuli.

Targeted antiviral treatment using non-ionizing radiation therapy for SARS-CoV-2 and viral pandemics preparedness: Technique, methods and practical notes for clinical application.

OBJECTIVE: Pandemic outbreaks necessitate effective responses to rapidly mitigate and control the spread of disease and eliminate the causative organism(s). While conventional chemical and biological solutions to these challenges are characteristically slow to develop and reach public availability; recent advances in device components operating at Super High Frequency (SHF) bands (3-30 GHz) of the electromagnetic spectrum enable novel approaches to such problems. METHODS: Based on experimentally documented evidence, a clinically relevant in situ radiation procedure to reduce viral loads in patients is devised and presented. Adapted to the currently available medical device technology to cause viral membrane fracture, this procedure selectively inactivates virus particles by forced oscillations arising from Structure Resonant Energy Transfer (SRET) thereby reducing infectivity and disease progression. RESULTS: Effective resonant frequencies for pleiomorphic Coronavirus SARS-CoV-2 is calculated to be in the 10-17 GHz range. Using the relation y = -3.308x + 42.9 with x and y representing log10 number of virus particles and the clinical throat swab Ct value respectively; in situ patient-specific exposure duration of ~15x minutes can be utilized to inactivate up to 100% of virus particles in the throat-lung lining, using an irradiation dose of 14.5 ± 1 W/m2; which is within the 200 W/m2 safety standard stipulated by the International Commission on Non-Ionizing Radiation Protection (ICNIRP). CONCLUSIONS: The treatment is designed to make patients less contagious enhancing faster recoveries and enabling timely control of a spreading pandemic. ADVANCES IN KNOWLEDGE: The article provides practically applicable parameters for effective clinical adaptation of this technique to the current pandemic at different levels of healthcare infrastructure and disease prevention besides enabling rapid future viral pandemics response.

Higher pulse frequency of near-infrared laser irradiation increases penetration depth for novel biomedical applications.

BACKGROUND: The clinical efficiency of laser treatments is limited by the low penetration of visible light used in certain procedures like photodynamic therapy (PDT). Second Harmonic Generation (SHG) PDT is an innovative technique to overcome this limitation that enables the use of Near Infrared (NIR) light instead of visible light. NIR frequency bands present an optical window for deeper penetration into biological tissue. In this research, we compare the penetration depths of 405 and 808 nm continuous wave (CW) lasers and 808 nm pulsed wave (PW) laser in two different modes (high and low frequency). METHODS: Increasing thicknesses of beef and chicken tissue samples were irradiated under CW and PW lasers to determine penetration depths. RESULTS: The 808 nm CW laser penetrates 2.3 and 2.4 times deeper than the 405 nm CW laser in beef and chicken samples, respectively. 808 nm PW (pulse frequency-500 Hz) penetrates deeper than CW laser at the same wavelength. Further, increasing the pulse frequency achieves higher penetration depths. High frequency 808 nm PW (pulse frequency-71.4 MHz) penetrates 7.4- and 6.0-times deeper than 405 nm CW laser in chicken and beef, respectively. CONCLUSIONS: The results demonstrate the higher penetration depths of high frequency PW laser compared to low frequency PW laser, CW laser of the same wavelength and CW laser with half the wavelength. The results indicate that integrating SHG in the PDT process along with pulsed NIR light may allow the treatment of 6-7 times bigger tumours than conventional PDT using blue light.

Aragonite-Polylysine: Neuro-Regenerative Scaffolds with Diverse Effects on Astrogliosis.

Biomaterials, especially when coated with adhesive polymers, are a key tool for restorative medicine, being biocompatible and supportive for cell adherence, growth, and function. Aragonite skeletons of corals are biomaterials that support survival and growth of a range of cell types, including neurons and glia. However, it is not known if this scaffold affects neural cell migration or elongation of neuronal and astrocytic processes, prerequisites for initiating repair of damage in the nervous system. To address this, hippocampal cells were aggregated into neurospheres and cultivated on aragonite skeleton of the coral Trachyphyllia geoffroyi (Coral Skeleton (CS)), on naturally occurring aragonite (Geological Aragonite (GA)), and on glass, all pre-coated with the oligomer poly-D-lysine (PDL). The two aragonite matrices promoted equally strong cell migration (4.8 and 4.3-fold above glass-PDL, respectively) and axonal sprouting (1.96 and 1.95-fold above glass-PDL, respectively). However, CS-PDL had a stronger effect than GA-PDL on the promotion of astrocytic processes elongation (1.7 vs. 1.2-fold above glass-PDL, respectively) and expression of the glial fibrillary acidic protein (3.8 vs. and 1.8-fold above glass-PDL, respectively). These differences are likely to emerge from a reaction of astrocytes to the degree of roughness of the surface of the scaffold, which is higher on CS than on GA. Hence, CS-PDL and GA-PDL are scaffolds of strong capacity to derive neural cell movements and growth required for regeneration, while controlling the extent of astrocytic involvement. As such, implants of PDL-aragonites have significant potential as tools for damage repair and the reduction of scar formation in the brain following trauma or disease.

Gliosis of astrocytes cultivated on coral skeleton is regulated by the matrix surface topography.

Astrogilosis is the response of astrocytes to brain trauma which manifest opposite roles on brain injury repair. On the one hand, astrocytes undergoing astrogliosis inhibit tissue regeneration by forming scar tissue, but, on the other hand, they enhance damage repair through secretion of neuro-protecting and neurotrophic factors. Therefore, identifying means that regulate astrogliosis can provide a control over progression and repair of brain damage. We have previously shown that the calcium carbonate skeleton of corals upregulates two manifestations of astrogliosis in astrocytes in culture-expression of the Glial Fibrillay Acidic Protein (GFAP), and shape conversion from non-spiky to reactive spiky cell morphology. Here, we investigated if the surface topography of the coralline skeleton plays a role in GFAP expression and the morphogenesis of reactive astrocytes. To address that, we utilized the non-porous exoskeleton of the coral Trachyphyllia geoffroyi, having three topographies of distinct heights on its surface: rough surface (made of <30 µm height bumps), protrusions (50-250 µm) and ridges (>250 µm). We observed that astrocytes reacted similarly to all three structures in terms of adhesion, acquisition of a spiky morphology and organization in networks. By contrast, the extent by which these cells expressed GFAP was structure-dependent. The expression was 2-fold higher on protrusions and ridges than on the rough surface and acquired. Accordingly, the distribution pattern of the GFAP overexpressing astrocytes followed that of the protrusions and ridges. Hence, fabricating coralline scaffolds with designed flatness/protrusions/ridges ratios can serve to control astrogliosis-derived regeneration in TBI wounds, and as a result improve the capacity to repair brain damage.

Using Attenuated Total Reflection-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy to distinguish between melanoma cells with a different metastatic potential.

The vast majority of cancer related deaths are caused by metastatic tumors. Therefore, identifying the metastatic potential of cancer cells is of great importance both for prognosis and for determining the correct treatment. Infrared (IR) spectroscopy of biological cells is an evolving research area, whose main aim is to find the spectral differences between diseased and healthy cells. In the present study, we demonstrate that Attenuated Total Reflection Fourier Transform IR (ATR-FTIR) spectroscopy may be used to determine the metastatic potential of cancer cells. Using the ATR-FTIR spectroscopy, we can identify spectral alterations that are a result of hydration or molecular changes. We examined two murine melanoma cells with a common genetic background but a different metastatic level, and similarly, two human melanoma cells. Our findings revealed that higher metastatic potential correlates with membrane hydration level. Measuring the spectral properties of the cells allows us to determine the membrane hydration levels. Thus, ATR-FTIR spectroscopy has the potential to help in cancer metastasis prognosis.

Microtubule minus end motors kinesin-14 and dynein drive nuclear congression in parallel pathways.

Microtubules (MTs) and associated motors play a central role in nuclear migration, which is crucial for diverse biological functions including cell division, polarity, and sexual reproduction. In this paper, we report a dual mechanism underlying nuclear congression during fission yeast karyogamy upon mating of haploid cells. Using microfluidic chambers for long-term imaging, we captured the precise timing of nuclear congression and identified two minus end-directed motors operating in parallel in this process. Kinesin-14 Klp2 associated with MTs may cross-link and slide antiparallel MTs emanating from the two nuclei, whereas dynein accumulating at spindle pole bodies (SPBs) may pull MTs nucleated from the opposite SPB. Klp2-dependent nuclear congression proceeds at constant speed, whereas dynein accumulation results in an increase of nuclear velocity over time. Surprisingly, the light intermediate chain Dli1, but not dynactin, is required for this previously unknown function of dynein. We conclude that efficient nuclear congression depends on the cooperation of two minus end-directed motors.

Effects antifreeze peptides on the thermotropic properties of a model membrane.

In this paper, we report on the effect of short segments of type I antifreeze protein (AFP I) on the thermotropic properties of a model membrane. Two different types of dimyristoylphosphatidylcholine model membranes were used, multilamellar vesicles and small unilamellar vesicles. The membrane properties were studied by differential scanning calorimetry (DSC) and fluorescence anisotropy. With the incorporation of AFP I and its short segments, the order of the model membrane increased both in the gel state and in the liquid crystalline state. The interaction of AFPs with the model membrane caused a shift in the phase transition to lower temperatures, which is accompanied by a broadening of the DSC thermogram. This preferential stabilization to a more ordered phase by the AFPs could be due to ordering the hydrophobic membrane core and separation into domains. Overall, this approach of employing short segments of AFP I simplifies the correlation between antifreeze protein characteristics and the effect of these parameters on the interaction mechanism of AFP with cell membranes. The success of this approach can lead to the identification of short peptides with high antifreeze activity.

The effect of solution electrolytes on the uptake of photosensitizers by liposomal membranes: a salting-out effect.

In this study we investigated, spectroscopically, the effect of electrolytes on the partitioning of hematoporphyrin IX (HP) and hypericin (Hy) into non-charged lipid vesicles. Our aim was to assess the salting-out effect of electrolytes on membrane-partitioning. We titrated aqueous solutions of HP and Hy with lecithin liposomes, at different concentrations of several monovalent and divalent electrolytes in the suspension. The partitioning constant of HP to lecithin liposomes increased from 3.3 (mL/mg) in water containing only 5mM buffer to 8.7 (mL/mg) at 0.36M KCl. KF had a similar effect. NaCl caused a 3-fold increase in the partitioning of Hy to liposomes. MgSO(4) and MgCl(2) also increased the partitioning of HP, by a factor of more than 4 and this occurred already at 0.03M concentration. We analyze the comparative effects of the electrolytes in relation to the Hofmeister series. The salting-out effect could be utilized to enhance the uptake of HP and Hy, and possibly other photosensitizers as well, by artificial and natural membranes.

Dithiaporphyrin derivatives as photosensitizers in membranes and cells.

We synthesized a series of analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-21,23-dithiaporphyrin (I) as potential photosensitizers for photodynamic therapy (PDT). The photosensitizers differ in the length of the side chains that bind the carboxyl to the phenol at positions 10 and 15 of the thiaporphyrin. The spectroscopic, photophysical, and biophysical properties of these photosensitizers are reported. The structural changes have almost no effect on the excitation/emission spectra with respect to I's spectra or on singlet oxygen generation in MeOH. All of the photosensitizers have a very high, close to 1.00, singlet oxygen quantum yield in MeOH. On the contrary, singlet oxygen generation in liposomes was considerably affected by the structural change in the photosensitizers. The photosensitizers possessing short side chains (one and three carbons) showed high quantum yields of around 0.7, whereas the photosensitizers possessing longer side chains showed smaller quantum yield, down to 0.14 for compound X (possessing side-chain length of 10 carbons), all at 1 microM. Moreover a self-quenching process of singlet oxygen was observed, and the quantum yield decreased as the photosensitizer's concentration increased. We measured the binding constant of I to liposomes and found Kb = 23.3 +/- 1.6 (mg/mL)-1. All the other photosensitizers with longer side chains exhibited very slow binding to liposomes, which prevented us from assessing their Kb's. We carried out fluorescence resonance energy transfer (FRET) measurements to determine the relative depth in which each photosensitizer is intercalated in the liposome bilayer. We found that the longer the side chain the deeper the photosensitizer core is embedded in the bilayer. This finding suggests that the photosensitizers are bound to the bilayer with their acid ends close to the aqueous medium interface and their core inside the bilayer. We performed PDT with the dithiaporphyrins on U937 cells and R3230AC cells. We found that the dark toxicity of the photosensitizers with the longer side chain (X, VI, V) is significantly higher than the dark toxicity of sensitizers with shorter side chains (I, III, IV). Phototoxicity measurements showed the opposite direction; the photosensitizers with shorter side chains were found to be more phototoxic than those with longer side chains. These differences are attributed to the relationship between diffusion and endocytosis in each photosensitizer, which determines the location of the photosensitizer in the cell and hence its phototoxicity.

Enhanced acidity, photophysical properties and liposome binding of perfluoroalkylated phthalocyanines lacking C-H bonds.

The acid-base, spectroscopic, photophysical and liposome-binding properties of the recently synthesized free base, 29H,31H,1,4,8,11,15,18,22,25-octafluoro-2,3,9,10,16,17,23, 24-octakisperfluoro(isopropyl) phthalocyanine, F64PcH2, are reported. The perfluoroalkylation of the phthalocyanine core renders the hydrogen atoms acidic, with a pK(a) = 6. The F64Pc(-2) dianion is detected already at pH 3, by singular-value decomposition analysis of electronic spectra. F64Pc(-2) generates 1O2 with quantum yields phi(delta) = 0.252 (in MeOH) and 0.019 in liposomes. Metallation of the Pc macrocycle to yield F64PcZn increases phi(delta) to 0.606 and 0.126 in MeOH and liposomes, respectively. Surprisingly, F64Pc(-2) (but not F64PcH2 or F64PcZn) binds strongly to liposomes, with a binding constant K(b) = 25 (mg/mL)(-1). The fully protonated F64PcH2, but not the zwitterionic F64Pc(-2), might favor hydrogen bonding, thus reducing its lipophilicity. Similarly, the Lewis acidity of Zn in F64PcZn, and thus its ability to bind water within a hydrophobic perfluoroalkyl pocket, is significantly enhanced by the fluorinated substituents.