Novel mechanism of drug resistance triggered by tumor-associated macrophages through Heat Shock Factor-1 activation.

Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism.

High-sensitivity and high-speed measurements of ultrashort pulses as short as 74 fs at 1.9 µm using a GRENOUILLE device.

Ultrashort laser pulse sources in the wavelength range of 1.8 to 2 µm have many potential applications including medicine, materials processing, and sensing. In the use of such lasers, a crucial task is to measure their pulse's temporal intensity and phase. Such measurement devices are most useful when they are simple to build and operate and also have high speed and high sensitivity. The GRENOUILLE measurement device with few components, no moving parts, sensitivity of hundreds of picojoules, and measurement speed of hundreds of milliseconds, is commonly used to solve this problem at other wavelengths. In this paper, the measurement of ultrashort pulses by a GRENOUILLE device, developed using a silicon matrix sensor, for pulses in the wavelength range of 1.8 to 2 µm has been demonstrated. It is shown that ultrashort pulses with durations of 74 to 900 fs and a maximum spectral FWHM of 85 nm can be measured with this device. The recently developed ultra-reliable RANA approach was used for pulse retrieval from the measured traces. The device's performance was validated by comparing its measurements with those obtained by the robust FROG technique.

Laser induced damage threshold of GaSe with antireflection microstructures at a wavelength of 5 µm.

Large GaSe crystals were grown and various antireflection microstructures (ARMs) were fabricated on their cleaved surfaces using optimized femtosecond laser ablation, which provided the antireflection effect in a wide wavelength range of 4-16 µm. The influence of ARMs created on the GaSe surface on the change of the laser-induced damage threshold (LIDT) of the crystal at a wavelength of 5 µm was evaluated. The 5-µm Fe:ZnMgSe laser with the pulse duration of 135 ns was used for the LIDT test in conditions close to single pulse exposure. The measured values of LIDT of 56 ± 6 MW/cm2 and 51 ± 9 MW/cm2 for two GaSe substrates, respectively, were comparable with the known data of single pulse LIDT of GaSe. The average LIDT intensities of 54 ± 6 MW/cm2 and 52 ± 7 MW/cm2 for the ARMs at two GaSe plates, respectively, were close to LIDT intensities for the corresponding GaSe substrates. The ARMs with lower structural quality had lower LIDT (50-52 MW/cm2) in comparison with the high-quality ARMs (58-60 MW/cm2). High LIDT for high-quality ARMs can be caused by increased selenium content in the ARMs. In any case, all the tested ARMs on the GaSe plates with different surface quality are workable for development of widely tunable mid-infrared nonlinear optical converters.

Antireflection microstructures fabricated on the surface of a LiGaSe2 nonlinear crystal.

LiGaSe2 is a propitious material for nonlinear parametric conversion in the mid-infrared (mid-IR) range. Its refractive index of n = 2.25 in the 2-12 µm wavelength range results in significant losses due to Fresnel reflection. However, the conventional method of increasing the transmittance with antireflection coatings (ARCs) significantly reduces the damage threshold of the material. Fabrication of the antireflection microstructures (ARMs) is an alternative approach for increasing the surface transmittance. In this work, ARMs were fabricated on the surface of a LiGaSe2 crystal using a single-pulse femtosecond laser ablation method. An average transmittance of 97.2% in the 2-8 µm spectral range and the maximum transmittance of 98.6% at 4.1 µm were achieved.

Indolylazine Derivative Induces Chaperone Expression in Aged Neural Cells and Prevents the Progression of Alzheimer's Disease.

The risk of progression of most sporadic neurodegenerative diseases, including Alzheimer's disease, increases with age. Traditionally, this is associated with a decrease in the efficiency of cell protection systems, in particular, molecular chaperones. Thus, the development of small molecules able to induce the synthesis of chaperones is a promising therapeutic approach to prevent neural diseases associated with ageing. Here, we describe a new compound IA-50, belonging to the class of indolylazines and featured by a low size of topological polar surface area, the property related to substances with potentially high membrane-penetrating activity. We also estimated the absorption, distribution, metabolism and excretion characteristics of IA-50 and found the substance to fit the effective drug criteria. The new compound was found to induce the synthesis and accumulation of Hsp70 in normal and aged neurons and in the hippocampi of young and old mice. The transgenic model of Alzheimer's disease, based on 5xFAD mice, confirmed that the injection of IA-50 prevented the formation of ß-amyloid aggregates, loss of hippocampal neurons and the development of memory impairment. These data indicate that this novel substance may induce the expression of chaperones in neural cells and brain tissues, suggesting its possible application in the therapy of ageing-associated disorders.

Speed characterization of p-i-n photodiode based on GaSb/GaInAsSb/GaAlAsSb heterostructure with frontal bridge contact at 1.9 µm.

We report a study of the response function parameters (amplitude and rise/fall time) of a high-speed GaSb/GaInAsSb/GaAlAsSb photodiode operating at 1.9 µm as a function of optical input power and reverse bias voltage. The experimental measurement results yield the optimal pulse energy and optimal reverse bias voltage for the photodiode. The 44 ps minimal rise time of the response function and 3.6 GHz bandwidth are achieved under a 3 V reverse bias voltage and pulse energy in the 0.27-2.5 pJ range.

Disruption of the Complex between GAPDH and Hsp70 Sensitizes C6 Glioblastoma Cells to Hypoxic Stress.

Hypoxia, which commonly accompanies tumor growth, depending on its strength may cause the enhancement of tumorigenicity of cancer cells or their death. One of the proteins targeted by hypoxia is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and we demonstrated here that hypoxia mimicked by treating C6 rat glioblastoma cells with cobalt chloride caused an up-regulation of the enzyme expression, while further elevation of hypoxic stress caused the enzyme aggregation concomitantly with cell death. Reduction or elevation of GAPDH performed with the aid of specific shRNAs resulted in the augmentation of the tumorigenicity of C6 cells or their sensitization to hypoxic stress. Another hypoxia-regulated protein, Hsp70 chaperone, was shown to prevent the aggregation of oxidized GAPDH and to reduce hypoxia-mediated cell death. In order to release the enzyme molecules from the chaperone, we employed its inhibitor, derivative of colchicine. The compound was found to substantially increase aggregation of GAPDH and to sensitize C6 cells to hypoxia both in vitro and in animals bearing tumors with distinct levels of the enzyme expression. In conclusion, blocking the chaperonic activity of Hsp70 and its interaction with GAPDH may become a promising strategy to overcome tumor resistance to multiple environmental stresses and enhance existing therapeutic tools.

Fabrication of antireflection microstructures on the surface of GaSe crystal by single-pulse femtosecond laser ablation.

GaSe crystals are promising as nonlinear optical converters in the mid- and far-IR ranges. However, it is challenging to increase the GaSe surface transmittance of 77% with conventional antireflection coatings because of poor surface quality, leading to coating adhesion problems. Antireflection microstructures (ARMs) offer an alternative way of increasing surface transmittance. In this work, ARMs were fabricated on the surface of a GaSe plate by single-pulse femtosecond laser ablation. An average GaSe surface transmittance of 94% in the 7-11 µm range and a maximum transmittance of 97.8% at 8.5 µm were obtained. The proposed method can be used to increase the efficiency of GaSe-based nonlinear converters.

High-efficiency continuous-wave single-mode room-temperature operation of Cr:CdSe single-crystal laser with output power of 2.3 W.

We report on the study of quenching and thermal lensing based on simple effective lens approximation in a Cr2+:CdSe active medium, including detailed research on the medium's luminescence lifetime dependence on temperature in the 236-391 K range. This work has allowed us to partially overcome the limitations associated with thermal effects in the medium and build a laser system that allowed power scalability to be realized for the Cr2+:CdSe laser. Longitudinal pumping using a continuous-wave Tm-doped fiber laser at 1.908 µm produced an output of 2.3 W at 2.65 µm with an absorbed pump power slope efficiency of 47.6%, which, to the best of our knowledge, is the highest output power achieved in Cr:CdSe continuous-wave lasers.

GAPDH-targeted therapy - A new approach for secondary damage after traumatic brain injury on rats.

Massive neuronal death caused by a neurodegenerative pathology or damage due to ischaemia or traumatic brain injury leads to the appearance of cytosolic proteins in the extracellular space. We found that one of the most abundant cellular polypeptides, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), appearing in the medium of dying cells or body fluids is able to form aggregates that are cytotoxic to adjacent cells. Since we previously showed that the hydrocortisone derivative RX624 can inhibit the ability of GAPDH to transport the enzyme complex with polyglutamine and reduce the cytotoxicity of the complex, we explored the effects of GAPDH on SH-SY5Y neuroblastoma cells. We found that the latter treated with particular forms of GAPDH molecules die with a high efficiency, suggesting that the exogenous enzyme does kill adjacent cells. RX624 prevented the interaction of exogenous GAPDH with the cell membrane and reduced the level of death by more than 10%. We also demonstrated the efficiency of RX624 treatment in a rat model of traumatic brain injury. The chemical blocked the formation of GAPDH aggregates in the brain, inhibited the cytotoxic effects of cerebrospinal fluid and rescued the motor function of injured rats. Importantly, RX624 treatment of rats had a similar effect as the intracranial injection of anti-GAPDH antibodies.

Etoposide-Induced Apoptosis in Cancer Cells Can Be Reinforced by an Uncoupled Link between Hsp70 and Caspase-3.

The Hsp70 chaperone binds and inhibits proteins implicated in apoptotic signaling including Caspase-3. Induction of apoptosis is an important mechanism of anti-cancer drugs, therefore Hsp70 can act as a protective system in tumor cells against therapeutic agents. In this study we present an assessment of candidate compounds that are able to dissociate the complex of Hsp70 with Caspase-3, and thus sensitize cells to drug-induced apoptosis. Using the PASS program for prediction of biological activity we selected a derivative of benzodioxol (BT44) that is known to affect molecular chaperones and caspases. Drug affinity responsive target stability and microscale thermophoresis assays indicated that BT44 bound to Hsp70 and reduced the chaperone activity. When etoposide was administered, heat shock accompanied with an accumulation of Hsp70 led to an inhibition of etoposide-induced apoptosis. The number of apoptotic cells increased following BT44 administration, and forced Caspase-3 processing. Competitive protein⁻protein interaction and immunoprecipitation assays showed that BT44 caused dissociation of the Hsp70⁻Caspase-3 complex, thus augmenting the anti-tumor activity of etoposide and highlighting the potential role of molecular separators in cancer therapy.

A hydrocortisone derivative binds to GAPDH and reduces the toxicity of extracellular polyglutamine-containing aggregates.

Huntington's disease (HD) has been recently shown to have a horizontally transmitted, prion-like pathology. Thus, the migration of polyglutamine-containing aggregates to acceptor cells is important for the progression of HD. These aggregates contain glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which increases their intracellular transport and their toxicity. Here, we show that RX624, a derivative of hydrocortisone that binds to GAPDH, prevents the formation of aggregates of GAPDH-polyglutamine excreted into the culture medium by PC-12 rat cells expressing mutant huntingtin. RX624 was previously shown to be unable to penetrate cells and, thus, its principal therapeutic action might be the inhibition of polyglutamine-GAPDH complex aggregation in the extracellular matrix. The administration of RX624 to SH-SY5Y acceptor cells that incubated in conditioned medium from PC-12 cells expressing mutant huntingtin caused an approximately 20% increase in survival. This suggests that RX624 might be useful as a drug against polyglutamine pathologies, and that is could be administered exogenously without affecting target cell physiology. This protective effect was validated by the similar effect of an anti-GAPDH specific antibody.

Glyceraldehyde 3-phosphate dehydrogenase augments the intercellular transmission and toxicity of polyglutamine aggregates in a cell model of Huntington disease.

The common feature of Huntington disease is the accumulation of oligomers or aggregates of mutant huntingtin protein (mHTT), which causes the death of a subset of striatal neuronal populations. The cytotoxic species can leave neurons and migrate to other groups of cells penetrating and damaging them in a prion-like manner. We hypothesized that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), previously shown to elevate the aggregation of mHTT, is associated with an increased efficiency of intercellular propagation of mHTT. GAPDH, on its own or together with polyglutamine species, was shown to be released into the extracellular milieu mainly from dying cells as assessed by a novel enzyme immunoassay, western blotting, and ultrafiltration. The conditioned medium of cells with growing GAPDH-polyQ aggregates was toxic to naïve cells, whereas depletion of the aggregates from the medium lowered this cytotoxicity. The GAPDH component of the aggregates was found to increase their toxicity by two-fold in comparison with polyQ alone. Furthermore, GAPDH-polyQ complexes were shown to penetrate acceptor cells and to increase the capacity of polyQ to prionize its intracellular homolog containing a repeat of 25 glutamine residues. Finally, inhibitors of intracellular transport showed that polyQ-GAPDH complexes, as well as GAPDH itself, penetrated cells using clathrin-mediated endocytosis. This suggested a pivotal role of the enzyme in the intercellular transmission of Huntington disease pathogenicity. In conclusion, GAPDH occurring in complexes with polyglutamine strengthens the prion-like activity and toxicity of the migrating aggregates. Aggregating polygluatmine tracts were shown to release from the cells over-expressing mutant huntingtin in a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The enzyme enhances the intracellular transport of aggregates to healthy cells, prionization of normal cellular proteins and finally cell death, thus demonstrating the pivotal role of GAPDH in the horizontal transmission of neurodegeneration.

Hsp70 chaperone rescues C6 rat glioblastoma cells from oxidative stress by sequestration of aggregating GAPDH.

The Hsp70 chaperone is known to elicit cytoprotective activity and this protection has a negative impact in anti-tumor therapy. In cancer cells subjected to oxidative stress Hsp70 may bind damaged polypeptides and proteins involved in apoptosis signaling. Since one of the important targets of oxidative stress is glyceraldehyde-3-phospate dehydrogenase (GAPDH) we suggested that Hsp70 might elicit its protective effect by binding GAPDH. Microscopy data show that in C6 rat glioma cells subjected to hydrogen peroxide treatment a considerable proportion of the GAPDH molecules are denatured and according to dot ultrafiltration data they form SDS-insoluble aggregates. Using two newly developed assays we show that Hsp70 can bind oxidized GAPDH in an ATP-dependent manner. Pharmacological up- or down-regulation of Hsp70 with the aid of U133 echinochrome or triptolide, respectively, reduced or increased the number of C6 glioma cells containing GAPDH aggregates and dying due to treatment with hydrogen peroxide. Using immunoprecipitation we found that Hsp70 is able to sequester aggregation-prone GAPDH and this may explain the anti-oxidative power of the chaperone. The results of this study led us to conclude that in cancer cells constantly exposed to conditions of oxidative stress, the protective power of Hsp70 should be abolished by specific inhibitors of Hsp70 expression.

High-energy, sub-100 fs, all-fiber stretched-pulse mode-locked Er-doped ring laser with a highly-nonlinear resonator.

We report on ultra-short stretched pulse generation in an all-fiber erbium-doped ring laser with a highly-nonlinear germanosilicate fiber inside the resonator with a slightly positive net-cavity group velocity dispersion (GVD). Stable 84 fs pulses were obtained with a 12 MHz repetition rate at a central wavelength of 1560 nm with a 48.1 nm spectral pulse width (full width at half maximum, FWHM) and 30 mW average output power; this corresponds to the 29.7 kW maximum peak power and 2.5 nJ pulse energy obtained immediately from the oscillator.

Conservative method for vertical electrooculogram attenuation based on local suppression of ongoing EEG artifact templates.

Eye movement during blinking can be a significant artifact in Event-Related Potentials (ERP) analysis. Blinks produce a positive potential in the vertical electrooculogram (VEOG), spreading towards the posterior direction. Two methods are frequently used to suppress VEOGs: linear regression to subtract the VEOG signal from the electroencephalogram (EEG) and Independent Component Analysis (ICA). However, some information is lost in both. The present algorithm (1) statistically identifies the position of VEOGs in the frontopolar channels; (2) performs EEG averaging for each channel, which results in 'blink templates'; (3) subtracts each template from the respective EEG at each VEOG position, only when the linear correlation index between the template and the segment is greater than a chosen threshold L. The signals from twenty subjects were acquired using a behavioral test and were treated using FilterBlink for subsequent ERP analysis. A model was designed to test the method for each subject using twenty copies of the EEG signal from the subject's mid-central channel (with nearly no VEOG) representing the EEG channels and their respective blink templates. At the same 200 equidistant time points (marks), a signal (2.5 sinusoidal cycles at 1050 ms emulating an ERP) was mixed with each model channel and the respective blink template of that channel, between 500 to 1200 ms after each mark. According to the model, VEOGs interfered with both ERPs and the ongoing EEG, mainly on the anterior medial leads, and no significant effect was observed on the mid-central channel (Cz). FilterBlink recovered approximately 90% (Fp1) to 98% (Fz) of the original ERP and EEG signals for L = 0.1. The method reduced the VEOG effect on the EEG after ERP and blink-artifact averaging in analyzing real signals. The method is straightforward and effective for VEOG attenuation without significant distortion in the EEG signal and embedded ERPs.

Novel mechanism of Hsp70 chaperone-mediated prevention of polyglutamine aggregates in a cellular model of huntington disease.

The key feature of polyglutamine aggregates accumulating in the course of Huntington disease (HD) is their resistance to protein denaturants, and to date only chaperones are proved to prevent mutant protein aggregation. It was suggested that expanded polyglutamine chains (polyQ) of mutant huntingtin are cross-linked to other proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Here we clarify the roles of GAPDH and molecular chaperone Hsp70 in the formation of sodium dodecyl sulfate (SDS)-insoluble polyQ aggregates. First, the addition of pure GAPDH was found to enhance the aggregation of polyQ in a cell-free model of HD. Secondly, the immunodepletion of GAPDH dose-dependently decreased polyQ aggregation. Finally, siRNA-mediated inhibition of GAPDH protein in SK-N-SH neuroblastoma cells has also reduced the aggregation of cellular polyQ. Regulated over-expression of Hsp70 decreased the amount of GAPDH associated with SDS-insoluble polyQ aggregates. Physical association of Hsp70 and GAPDH in SK-N-SH cells was shown by reciprocal immunoprecipitation and confocal microscopy. Pure Hsp70 dose-dependently inhibited the formation of polyQ aggregates in cell-free model of HD by sequestering both GAPDH and polyQ. We demonstrated that Hsp70 binds to polyQ in adenosine triphosphate-dependent manner, which suggests that Hsp70 exerts a chaperoning activity in the course of this interaction. Binding of Hsp70 to GAPDH was nicotinamide adenine dinucleotide-dependent suggesting another type of association. Based on our findings, we conclude that Hsp70 protects cells in HD by removing/sequestering two intrinsic components of protein aggregates: the polyQ itself and GAPDH. We propose that GAPDH might be an important target for pharmacological treatment of HD and other polyglutamine expansion-related diseases.

Protein Interactome of Amyloid-ß as a Therapeutic Target.

The amyloid concept of Alzheimer's disease (AD) assumes the ß-amyloid peptide (Aß) as the main pathogenic factor, which injures neural and other brain cells, causing their malfunction and death. Although Aß has been documented to exert its cytotoxic effect in a solitary manner, there is much evidence to claim that its toxicity can be modulated by other proteins. The list of such Aß co-factors or interactors includes tau, APOE, transthyretin, and others. These molecules interact with the peptide and affect the ability of Aß to form oligomers or aggregates, modulating its toxicity. Thus, the list of potential substances able to reduce the harmful effects of the peptide should include ones that can prevent the pathogenic interactions by specifically binding Aß and/or its partners. In the present review, we discuss the data on Aß-based complexes in AD pathogenesis and on the compounds directly targeting Aß or the destructors of its complexes with other polypeptides.

Diffuse reflectance spectroscopy of the cartilage tissue in the fourth optical window.

Studies of the optical properties of biological tissues in the infrared range have demonstrated significant potential for diagnostic tasks. One of the insufficiently explored ranges for diagnostic problems at the moment is the fourth transparency window, or short wavelength infrared region II (SWIR II). A Cr2+:ZnSe laser with tuning capability in the range from 2.1 to 2.4 µm was developed to explore the possibilities in this region. The capability of diffuse reflectance spectroscopy to analyze water and collagen content in biosamples was investigated using the optical gelatin phantoms and the cartilage tissue samples during their drying process. It was demonstrated that decomposition components of the optical density spectra correlated with the partial content of the collagen and water in the samples. The present study indicates the possibility of using this spectral range for the development of diagnostic methods, in particular, for observation of the changes in the content of cartilage tissue components in degenerative diseases such as osteoarthritis.