Experimental demonstration of a new near-infrared spectroscopy technique based on optical dual-comb: DC-NIRS.

We present a novel near-infrared spectroscopy technique based on Dual-Comb optical interrogation (DC-NIRS) applied to dispersive media. The technique recovers the frequency response of the medium under investigation by sampling its spectral response in amplitude and phase. The DC-NIRS reference and sample signals are generated using electro-optic modulation which offers a cost-effective, integrable solution while providing high adaptability to the interrogated medium. A careful choice of both line spacing and optical span of the frequency comb ensures that the retrieved information enables the reconstruction of the temporal impulse response of the medium, known as the diffuse-time-of-flight (DTOF), to obtain its optical properties with a 70 µs temporal resolution and 32 ps photon propagation delay resolution. Furthermore, the DC-NIRS technique also offers enhanced penetration due to noiseless optical amplification (interferometric detection). The presented technique was demonstrated on a static bio-mimetic phantom of known optical properties reproducing a typical brain's optical response. The DTOF and optical properties of the phantom were measured, showing the capabilities of this new technique on the estimation of absolute optical properties with a deviation under 3%. Compared to current technologies, our DC-NIRS technique provides enhanced temporal resolution, spatial location capabilities, and penetration depth, with an integrable and configurable cost-effective architecture, paving the way to next-generation, non-invasive and portable systems for functional brain imaging, and brain-computer interfaces, among other. The system is patent pending PCT/ES2022/070176.

Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis.

BACKGROUND: Persistent inflammatory response in the brain can lead to tissue damage and neurodegeneration. In Alzheimer's disease (AD), there is an aberrant activation of inflammasomes, molecular platforms that drive inflammation through caspase-1-mediated proteolytic cleavage of proinflammatory cytokines and gasdermin D (GSDMD), the executor of pyroptosis. However, the mechanisms underlying the sustained activation of inflammasomes in AD are largely unknown. We have previously shown that high brain cholesterol levels promote amyloid-ß (Aß) accumulation and oxidative stress. Here, we investigate whether these cholesterol-mediated changes may regulate the inflammasome pathway. METHODS: SIM-A9 microglia and SH-SY5Y neuroblastoma cells were cholesterol-enriched using a water-soluble cholesterol complex. After exposure to lipopolysaccharide (LPS) plus muramyl dipeptide or Aß, activation of the inflammasome pathway was analyzed by immunofluorescence, ELISA and immunoblotting analysis. Fluorescently-labeled Aß was employed to monitor changes in microglia phagocytosis. Conditioned medium was used to study how microglia-neuron interrelationship modulates the inflammasome-mediated response. RESULTS: In activated microglia, cholesterol enrichment promoted the release of encapsulated IL-1ß accompanied by a switch to a more neuroprotective phenotype, with increased phagocytic capacity and release of neurotrophic factors. In contrast, in SH-SY5Y cells, high cholesterol levels stimulated inflammasome assembly triggered by both bacterial toxins and Aß peptides, resulting in GSDMD-mediated pyroptosis. Glutathione (GSH) ethyl ester treatment, which recovered the cholesterol-mediated depletion of mitochondrial GSH levels, significantly reduced the Aß-induced oxidative stress in the neuronal cells, resulting in lower inflammasome activation and cell death. Furthermore, using conditioned media, we showed that neuronal pyroptosis affects the function of the cholesterol-enriched microglia, lowering its phagocytic activity and, therefore, the ability to degrade extracellular Aß. CONCLUSIONS: Changes in intracellular cholesterol levels differentially regulate the inflammasome-mediated immune response in microglia and neuronal cells. Given the microglia-neuron cross-talk in the brain, cholesterol modulation should be considered a potential therapeutic target for AD treatment, which may help to block the aberrant and chronic inflammation observed during the disease progression.

Cell-free mitochondrial DNA deletions in idiopathic, but not LRRK2, Parkinson's disease.

Mitochondrial dysfunction happens in both idiopathic (iPD) and LRRK2-related Parkinson's disease (LRRK2-PD). Nonetheless, previous studies suggested that a different type of mitochondrial pathology underlies the neurodegeneration in these two disorders. To further explore this hypothesis, we developed a novel multiplex digital PCR assay that allows the absolute quantification of cell-free mitochondrial DNA (cf-mtDNA) copy number and deletion ratio directly in cerebrospinal fluid (CSF) by simultaneously measuring two opposed regions of the mtDNA circular molecule, one of them in the commonly deleted major arc. The results confirmed that the content of cf-mtDNA in CSF was statistically significantly different between iPD and LRRK2-PD patients. Moreover, we found high cf-mtDNA deletion levels in CSF from patients with iPD, but not LRRK2-PD. The high cf-mtDNA deletion frequency in iPD was validated in an independent cohort. These results indicated that the content and deletion ratio of cf-mtDNA may differentiate iPD from LRRK2-PD, and provides further evidence of the different mitochondrial pathophysiology between these two forms of the disease.

Upregulation of brain cholesterol levels inhibits mitophagy in Alzheimer disease.

Mitochondrial dysfunction is behind several neurodegenerative diseases, including Alzheimer disease (AD). Accumulation of damaged mitochondria is already observed at the early stages of AD and has been linked to impaired mitophagy, but the mechanisms underlying this alteration are still not fully known. In our recent study, we show that intracellular cholesterol enrichment can downregulate amyloid beta (Aß)-induced mitophagy. Mitochondrial glutathione depletion resulting from high cholesterol levels promotes PINK1 (PTEN induced kinase 1)-mediated mitophagosome formation; however, mitophagy flux is ultimately disrupted, most likely due to fusion deficiency of endosomes-lysosomes caused by cholesterol. Meanwhile, in APP-PSEN1-SREBF2 mice, an AD mouse model that overexpresses the cholesterol-related transcription factor SREBF2, cholesterol accumulation prompts an oxidative- and age-dependent cytosolic aggregation of the mitophagy adaptor OPTN (optineurin), which prevents mitophagosome formation despite enhanced PINK1-PRKN/parkin signaling. Hippocampal neurons from postmortem brain of AD individuals reproduce the progressive accumulation of OPTN in aggresome-like structures accompanied by high levels of mitochondrial cholesterol in advanced stages of the disease. Overall, these data provide new insights into the impairment of the PINK1-PRKN mitophagy pathway in AD and suggest the combination of mitophagy inducers with strategies focused on restoring the cholesterol homeostasis and mitochondrial redox balance as a potential disease-modifying therapy for AD.

Cholesterol alters mitophagy by impairing optineurin recruitment and lysosomal clearance in Alzheimer's disease.

BACKGROUND: Emerging evidence indicates that impaired mitophagy-mediated clearance of defective mitochondria is a critical event in Alzheimer's disease (AD) pathogenesis. Amyloid-beta (Aß) metabolism and the microtubule-associated protein tau have been reported to regulate key components of the mitophagy machinery. However, the mechanisms that lead to mitophagy dysfunction in AD are not fully deciphered. We have previously shown that intraneuronal cholesterol accumulation can disrupt the autophagy flux, resulting in low Aß clearance. In this study, we examine the impact of neuronal cholesterol changes on mitochondrial removal by autophagy. METHODS: Regulation of PINK1-parkin-mediated mitophagy was investigated in conditions of acute (in vitro) and chronic (in vivo) high cholesterol loading using cholesterol-enriched SH-SY5Y cells, cultured primary neurons from transgenic mice overexpressing active SREBF2 (sterol regulatory element binding factor 2), and mice of increasing age that express the amyloid precursor protein with the familial Alzheimer Swedish mutation (Mo/HuAPP695swe) and mutant presenilin 1 (PS1-dE9) together with active SREBF2. RESULTS: In cholesterol-enriched SH-SY5Y cells and cultured primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation triggered by Aß while impairing lysosomal-mediated clearance. Antioxidant recovery of cholesterol-induced mitochondrial glutathione (GSH) depletion prevented mitophagosomes formation indicating mitochondrial ROS involvement. Interestingly, when brain cholesterol accumulated chronically in aged APP-PSEN1-SREBF2 mice the mitophagy flux was affected at the early steps of the pathway, with defective recruitment of the key autophagy receptor optineurin (OPTN). Sustained cholesterol-induced alterations in APP-PSEN1-SREBF2 mice promoted an age-dependent accumulation of OPTN into HDAC6-positive aggresomes, which disappeared after in vivo treatment with GSH ethyl ester (GSHee). The analyses in post-mortem brain tissues from individuals with AD confirmed these findings, showing OPTN in aggresome-like structures that correlated with high mitochondrial cholesterol levels in late AD stages. CONCLUSIONS: Our data demonstrate that accumulation of intracellular cholesterol reduces the clearance of defective mitochondria and suggest recovery of the cholesterol homeostasis and the mitochondrial scavenging of ROS as potential therapeutic targets for AD.

Mitochondrial Glutathione: Recent Insights and Role in Disease.

Mitochondria are the main source of reactive oxygen species (ROS), most of them deriving from the mitochondrial respiratory chain. Among the numerous enzymatic and non-enzymatic antioxidant systems present in mitochondria, mitochondrial glutathione (mGSH) emerges as the main line of defense for maintaining the appropriate mitochondrial redox environment. mGSH's ability to act directly or as a co-factor in reactions catalyzed by other mitochondrial enzymes makes its presence essential to avoid or to repair oxidative modifications that can lead to mitochondrial dysfunction and subsequently to cell death. Since mitochondrial redox disorders play a central part in many diseases, harboring optimal levels of mGSH is vitally important. In this review, we will highlight the participation of mGSH as a contributor to disease progression in pathologies as diverse as Alzheimer's disease, alcoholic and non-alcoholic steatohepatitis, or diabetic nephropathy. Furthermore, the involvement of mitochondrial ROS in the signaling of new prescribed drugs and in other pathologies (or in other unmet medical needs, such as gender differences or coronavirus disease of 2019 (COVID-19) treatment) is still being revealed; guaranteeing that research on mGSH will be an interesting topic for years to come.

Hyperspectral terahertz imaging with electro-optic dual combs and a FET-based detector.

In this paper, a terahertz hyperspectral imaging architecture based on an electro-optic terahertz dual-comb source is presented and demonstrated. In contrast to single frequency sources, this multi-heterodyne system allows for the characterization of the whole spectral response of the sample in parallel for all the frequency points along the spectral range of the system. This hence provides rapid, highly consistent results and minimizes measurement artifacts. The terahertz illumination signal can be tailored (in spectral coverage and resolution) with high flexibility to meet the requirements of any particular application or experimental scenario while maximizing the signal-to-noise ratio of the measurement. Besides this, the system provides absolute frequency accuracy and a very high coherence that allows for direct signal detection without inter-comb synchronization mechanisms, adaptive acquisition, or post-processing. Using a field-effect transistor-based terahertz resonant 300 GHz detector and the raster-scanning method we demonstrate the two-dimensional hyperspectral imaging of samples of different kinds to illustrate the remarkable capabilities of this innovative architecture. A proof-of-concept demonstration has been performed in which tree leaves and a complex plastic fragment have been analyzed in the 300 GHz range with a frequency resolution of 10 GHz.

Recent Insights into the Mitochondrial Role in Autophagy and Its Regulation by Oxidative Stress.

Autophagy is a self-digestive process that degrades intracellular components, including damaged organelles, to maintain energy homeostasis and to cope with cellular stress. Autophagy plays a key role during development and adult tissue homeostasis, and growing evidence indicates that this catalytic process also has a direct role in modulating aging. Although autophagy is essentially protective, depending on the cellular context and stimuli, autophagy outcome can lead to either abnormal cell growth or cell death. The autophagic process requires a tight regulation, with cellular events following distinct stages and governed by a wide molecular machinery. Reactive oxygen species (ROS) have been involved in autophagy regulation through multiple signaling pathways, and mitochondria, the main source of endogenous ROS, have emerged as essential signal transducers that mediate autophagy. In the present review, we aim to summarize the regulatory function of mitochondria in the autophagic process, particularly regarding the mitochondrial role as the coordination node in the autophagy signaling pathway, involving mitochondrial oxidative stress, and their participation as membrane donors in the initial steps of autophagosome assembly.

Oxidative inactivation of amyloid beta-degrading proteases by cholesterol-enhanced mitochondrial stress.

Familial early-onset forms of Alzheimer's disease (AD) are linked to overproduction of amyloid beta (Aß) peptides, while decreased clearance of Aß is the driving force leading to its toxic accumulation in late-onset (sporadic) AD. Oxidative modifications and defective function have been reported in Aß-degrading proteases such as neprilysin (NEP) and insulin-degrading enzyme (IDE). However, the exact mechanisms that regulate the proteolytic clearance of Aß and its deficits are largely unknown. We have previously showed that cellular cholesterol loading, by depleting the mitochondrial GSH (mGSH) content, stimulates Αß-induced mitochondrial oxidative stress and promotes AD-like pathology in APP-PSEN1-SREBF2 mice. Here, using the same AD mouse model we examined whether cholesterol-enhanced mitochondrial oxidative stress affects NEP and IDE function. We found that brain extracts from APP-PSEN1-SREBF2 mice displayed increased presence of oxidatively modified forms of NEP and IDE, associated with impaired enzymatic activities. Both alterations were substantially recovered after an in vivo treatment with the cholesterol-lowering agent 2-hydroxypropyl-ß-cyclodextrin. The recovery of the proteolytic activity after treatment was accompanied with a significant reduction of Aß levels. Supporting these results, cholesterol-enriched SH-SY5Y cells were more sensitive to Aß-induced impairment of IDE and NEP function in vitro. The rise of cellular cholesterol also stimulated the extracellular release of IDE by an unconventional autophagy-coordinated mechanism. Recovery of depleted pool of mGSH in these cells not only prevented the detrimental effect of Aß on intracellular AßDPs activities but also had an impact on extracellular IDE levels and function, stimulating the extracellular Aß degrading activity. Therefore, changes in brain cholesterol levels by modifying the mGSH content would play a key role in IDE and NEP-mediated proteolytic elimination of Aß peptides and AD progression.

Astaxanthin exerts protective effects similar to bexarotene in Alzheimer's disease by modulating amyloid-beta and cholesterol homeostasis in blood-brain barrier endothelial cells.

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-ß peptides (Aß) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aß production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aß oligomers and ~80 kDa intracellular 6E10-reactive APP/Aß species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aß clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aß species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aß oligomers in mBCEC and Aß species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aß from cerebrovascular endothelial cells.

Electro-optic THz dual-comb architecture for high-resolution, absolute spectroscopy.

An absolute-frequency terahertz (THz) dual-frequency comb spectrometer based on electro-optic modulators for tunable, high-resolution, and real-time rapid acquisition is presented. An optical line of a master frequency comb (filtered via optical injection locking) serves as the seed to electro-optically generate a pair of new frequency combs (probe and local oscillator). Photomixing both combs with another coherent line from the same original master comb generates a narrow linewidth THz dual-comb with teeth frequencies that can be referenced to a radio-frequency standard. The system is validated with a proof-of-principle measurement of a microwave filter in the W-band.

Hypoxia modulates the antioxidant effect of hydroxytyrosol in MCF-7 breast cancer cells.

Although cancer is multifactorial, a strong correlation between this pathology and increased oxidative stress has long been stablished. Hypoxia, inherent to solid tumors, increases reactive oxygen species and should be taken into account when analyzing the response of tumor cells to antioxidants. The Mediterranean diet has been related to a lower incidence of cancer, and particularly of breast cancer. Given that hydroxytyrosol (HT) is largely responsible for the antioxidant properties of olive oil, we have performed a comprehensive and comparative study of its effect on the oxidative stress response of the human breast cancer cell line MCF-7 in hypoxia and normoxia. Our results demonstrate that the antioxidant action of HT is particularly effective in a hypoxic environment. Moreover, we have observed that this polyphenol modulates the transcription and translation of members of the PGC-1α/ERRα and PGC-1α/Nrf2 pathways. However, while the transcriptional effects of HT are similar in normoxic and hypoxic conditions, its translational action is less prominent and partially attenuated in hypoxia, and therefore cannot completely explain the antioxidant effect of HT. Consequently, our results underscore that the hypoxic environment of tumor cells should be considered when analyzing the effect of bioactive compounds. Besides, this study also points to the importance of assessing the regulatory role of HT at both mRNA and protein level to get a complete picture of its effects.

An All-Organic Flexible Visible Light Communication System.

Visible light communication systems can be used in a wide variety of applications, from driving to home automation. The use of wearables can increase the potential applications in indoor systems to send and receive specific and customized information. We have designed and developed a fully organic and flexible Visible Light Communication system using a flexible OLED, a flexible P3HT:PCBM-based organic photodiode (OPD) and flexible PCBs for the emitter and receiver conditioning circuits. We have fabricated and characterized the I-V curve, modulation response and impedance of the flexible OPD. As emitter we have used a commercial flexible organic luminaire with dimensions 99 × 99 × 0.88 mm, and we have characterized its modulation response. All the devices show frequency responses that allow operation over 40 kHz, thus enabling the transmission of high quality audio. Finally, we integrated the emitter and receiver components and its electronic drivers, to build an all-organic flexible VLC system capable of transmitting an audio file in real-time, as a proof of concept of the indoor capabilities of such a system.

Cholesterol impairs autophagy-mediated clearance of amyloid beta while promoting its secretion.

Macroautophagy/autophagy failure with the accumulation of autophagosomes is an early neuropathological feature of Alzheimer disease (AD) that directly affects amyloid beta (Aß) metabolism. Although loss of presenilin 1 function has been reported to impair lysosomal function and prevent autophagy flux, the detailed mechanism leading to autophagy dysfunction in AD remains to be elucidated. The resemblance between pathological hallmarks of AD and Niemann-Pick Type C disease, including endosome-lysosome abnormalities and impaired autophagy, suggests cholesterol accumulation as a common link. Using a mouse model of AD (APP-PSEN1-SREBF2 mice), expressing chimeric mouse-human amyloid precursor protein with the familial Alzheimer Swedish mutation (APP695swe) and mutant presenilin 1 (PSEN1-dE9), together with a dominant-positive, truncated and active form of SREBF2/SREBP2 (sterol regulatory element binding factor 2), we demonstrated that high brain cholesterol enhanced autophagosome formation, but disrupted its fusion with endosomal-lysosomal vesicles. The combination of these alterations resulted in impaired degradation of Aß and endogenous MAPT (microtubule associated protein tau), and stimulated autophagy-dependent Aß secretion. Exacerbated Aß-induced oxidative stress in APP-PSEN1-SREBF2 mice, due to cholesterol-mediated depletion of mitochondrial glutathione/mGSH, is critical for autophagy induction. In agreement, in vivo mitochondrial GSH recovery with GSH ethyl ester, inhibited autophagosome synthesis by preventing the oxidative inhibition of ATG4B deconjugation activity exerted by Aß. Moreover, cholesterol-enrichment within the endosomes-lysosomes modified the levels and membrane distribution of RAB7A and SNAP receptors (SNAREs), which affected its fusogenic ability. Accordingly, in vivo treatment with 2-hydroxypropyl-ß-cyclodextrin completely rescued these alterations, making it a potential therapeutic tool for AD.

Gain-switching injection-locked dual optical frequency combs: characterization and optimization.

In this work, the generation of dual optical frequency combs based on gain-switching and optical injection locking is experimentally examined. The study reveals that an effective process of optical injection can lead to optimized RF combs in terms of span and signal-to-noise ratio. The system also minimizes the overlap of lines and reduces the number of optical components involved, eliminating the need for any external modulator (electro-optic, acousto-optic). The validation of the system was performed as a dual-comb spectrometer, which allowed for determination of the absorption and dispersion profiles of the molecular transition of H13CN at 1538.523 nm.

Dual optical frequency comb architecture with capabilities from visible to mid-infrared.

In this paper, a new approach to dual comb generation based on well-known optical techniques (Gain-Switching and Optical Injection Locking) is presented. The architecture can be implemented using virtually every kind of continuous-wave semiconductor laser source (DFB, VCSEL, QCL) and without the necessity of electro-optic modulators. This way, a frequency-agile and adaptive dual-comb architecture is provided with potential implementation capabilities from mid-infrared to near ultraviolet. With a RF comb comprising around 70 teeth, the system is validated in the 1.5 µm region measuring the absorption feature of H13CN at 1538.523 nm with a minimum integration time of 10 µs.

Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma.

BACKGROUND: The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. METHODS: NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 µg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. RESULTS: The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. CONCLUSIONS: The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.

Visible light communication system using an organic bulk heterojunction photodetector.

A visible light communication (VLC) system using an organic bulk heterojunction photodetector (OPD) is presented. The system has been successfully proven indoors with an audio signal. The emitter consists of three commercial high-power white LEDs connected in parallel. The receiver is based on an organic photodetector having as active layer a blend of poly(3-hexylthiophene) (P3HT) and phenyl C61-butyric acid methyl ester (PCBM). The OPD is opto-electrically characterized, showing a responsivity of 0.18 A/W and a modulation response of 790 kHz at -6 V.