A tripartite SNARE-K+ channel complex mediates in channel-dependent K+ nutrition in Arabidopsis.

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K(+) channel subunit that assembles with different inward-rectifying K(+) channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K(+) channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K(+) channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K(+) current in Arabidopsis root epidermal protoplasts as well as K(+) acquisition and growth in seedlings when channel-mediated K(+) uptake was limiting. That SYP121 should be important for gating of a K(+) channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE-ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K(+) uptake coordinately with membrane expansion for cell growth.

Singlet oxygen luminescence detector based on low-cost InGaAs avalanche photodiode.

Molecular oxygen excited to singlet state (Singlet oxygen, 1O2) becomes highly reactive and cytotoxic chemical. 1O2 is commonly generated by photoexcitation of dyes (photosensitizers), including the photodynamic therapy and diagnostics of cancer. However, the formation of singlet oxygen is often unwanted for various light-sensitive compounds, e.g. it causes the photobleaching of fluorescent probes. In either case, during a development of new photosensitive chemicals and drugs there is a need to evaluate the amount of 1O2 formed during photoexcitation. The direct approach in measuring the amount of singlet oxygen is based on the detection of its luminescence at 1270 nm. However, this luminescence is usually weak, which implies the use of highly sensitive single-photon detectors. Thus the existing instruments are commonly complicated and expensive. Here we suggest an approach and report a device to measure the 1O2 luminescence using low-cost InGaAs avalanche photodiode and simple electronics. The measurements can be performed in stationary (not time-resolved) mode in organic solvents such as tetrachloromethane (CCl4), ethanol and DMSO. In particular, we performed spectral-resolved measurements of the singlet oxygen luminescence in CCl4 with the device and demonstrated high complementarity to literature data. The simple setup allows to evaluate the efficiency (or speed) of singlet oxygen generation and hence facilitates the development and characterization of new photosensitizers and other photosensitive chemicals.

Singlet oxygen stimulates mitochondrial bioenergetics in brain cells.

Oxygen, in form of reactive oxygen species (ROS), has been shown to participate in oxidative stress, one of the major triggers for pathology, but also is a main contributor to physiological processes. Recently, it was found that 1267 nm irradiation can produce singlet oxygen without photosensitizers. We used this phenomenon to study the effect of laser-generated singlet oxygen on one of the major oxygen-dependent processes, mitochondrial energy metabolism. We have found that laser-induced generation of 1O2 in neurons and astrocytes led to the increase of mitochondrial membrane potential, activation of NADH- and FADH-dependent respiration, and importantly, increased the rate of maximal respiration in isolated mitochondria. The activation of mitochondrial respiration stimulated production of ATP in these cells. Thus, we found that the singlet oxygen generated by 1267 nm laser pulse works as an activator of mitochondrial respiration and ATP production in the brain.

Wearable Laser Doppler Flowmetry Sensor: A Feasibility Study with Smoker and Non-Smoker Volunteers.

Novel, non-invasive wearable laser Doppler flowmetry (LDF) devices measure real-time blood circulation of the left middle fingertip and the topside of the wrist of the left hand. The LDF signals are simultaneously recorded for fingertip and wrist. The amplitude of blood flow signals and wavelet analysis of the signal are used for the analysis of blood perfusion parameters. The aim of this pilot study is to validate the accuracy of blood circulation measurements recorded by one such non-invasive wearable LDF device for healthy young non-smokers and smokers. This study reveals a higher level of blood perfusion in the non-smoker group compared to the smoker group and vice-versa for the variation of pulse frequency. This result can be useful to assess the sensitivity of the wearable LDF sensor in determining the effect of nicotine for smokers as compared to non-smokers and also the blood microcirculation in smokers with different pathologies.

Machine Learning Aided Photonic Diagnostic System for Minimally Invasive Optically Guided Surgery in the Hepatoduodenal Area.

Abdominal cancer is a widely prevalent group of tumours with a high level of mortality if diagnosed at a late stage. Although the cancer death rates have in general declined over the past few decades, the mortality from tumours in the hepatoduodenal area has significantly increased in recent years. The broader use of minimal access surgery (MAS) for diagnostics and treatment can significantly improve the survival rate and quality of life of patients after surgery. This work aims to develop and characterise an appropriate technical implementation for tissue endogenous fluorescence (TEF) and assess the efficiency of machine learning methods for the real-time diagnosis of tumours in the hepatoduodenal area. In this paper, we present the results of the machine learning approach applied to the optically guided MAS. We have elaborated tissue fluorescence approach with a fibre-optic probe to record the TEF and blood perfusion parameters during MAS in patients with cancers in the hepatoduodenal area. The measurements from the laser Doppler flowmetry (LDF) channel were used as a sensor of the tissue vitality to reduce variability in TEF data. Also, we evaluated how the blood perfusion oscillations are changed in the tumour tissue. The evaluated amplitudes of the cardiac (0.6-1.6 Hz) and respiratory (0.2-0.6 Hz) oscillations was significantly higher in intact tissues (p < 0.001) compared to the cancerous ones, while the myogenic (0.2-0.06 Hz) oscillation did not demonstrate any statistically significant difference. Our results demonstrate that a fibre-optic TEF probe accompanied with ML algorithms such as k-Nearest Neighbours or AdaBoost is highly promising for the real-time in situ differentiation between cancerous and healthy tissues by detecting the information about the tissue type that is encoded in the fluorescence spectrum. Also, we show that the detection can be supplemented and enhanced by parallel collection and classification of blood perfusion oscillations.

Author Correction: The light-oxygen effect in biological cells enhanced by highly localized surface plasmon-polaritons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The light-oxygen effect in biological cells enhanced by highly localized surface plasmon-polaritons.

Here at the first time we suggested that the surface plasmon-polariton phenomenon which it is well described in metallic nanostructures could also be used for explanation of the unexpectedly strong oxidative effects of the low-intensity laser irradiation in living matters (cells, tissues, organism). We demonstrated that the narrow-band laser emitting at 1265 nm could generate significant amount of the reactive oxygen species (ROS) in both HCT116 and CHO-K1 cell cultures. Such cellular ROS effects could be explained through the generation of highly localized plasmon-polaritons on the surface of mitochondrial crista. Our experimental conditions, the low-intensity irradiation, the narrow spectrum band (<4 nm) of the laser and comparably small size bio-structures (~10 µm) were shown to be sufficient for the plasmon-polariton generation and strong laser field confinement enabling the oxidative stress observed.

Anti-inflammatory and cell proliferative effect of the 1270 nm laser irradiation on the BALB/c nude mouse model involves activation of the cell antioxidant system.

Recently, many interdisciplinary community researchers have focused their efforts on study of the low-level light irradiation effects (photobiomodulation, PBM) as a promising therapeutic technology. Among the priorities, a search of new wavelength ranges of laser radiation to enhance the laser prospects in treatment of autoimmune and cancer diseases commonly accompanied by disorders in the antioxidant system of the body. The laser wavelengths within 1265-1270 nm corresponds to the maximum oxygen absorption band. Therefore, PBM effects on a model organism within this spectrum range are of particular interest for preclinical research. Here, we report comprehensive biomolecular studies of the changes in the BALB/c nude mice skin after an exposure to the continuous laser radiation at the 1270 nm wavelength and energy densities of 0.12 and 1.2 J/cm2. Such regime induces both local and systemic PBM effects, presumably due to the short-term increase in ROS levels, which in turn activate the cell antioxidative system.

Multimodal Optical Diagnostics of the Microhaemodynamics in Upper and Lower Limbs.

The introduction of optical non-invasive diagnostic methods into clinical practice can substantially advance in the detection of early microcirculatory disorders in patients with different diseases. This paper is devoted to the development and application of the optical non-invasive diagnostic approach for the detection and evaluation of the severity of microcirculatory and metabolic disorders in rheumatic diseases and diabetes mellitus. The proposed methods include the joint use of laser Doppler flowmetry, absorption spectroscopy and fluorescence spectroscopy in combination with functional tests. This technique showed the high diagnostic importance for the detection of disturbances in peripheral microhaemodynamics. These methods have been successfully tested as additional diagnostic techniques in the field of rheumatology and endocrinology. The sensitivity and specificity of the proposed diagnostic procedures have been evaluated.

Bifurcation in Blood Oscillatory Rhythms for Patients with Ischemic Stroke: A Small Scale Clinical Trial using Laser Doppler Flowmetry and Computational Modeling of Vasomotion.

We describe application of spectral analysis of laser Doppler flowmetry (LDF) signals to investigation of cerebrovascular haemodynamics in patients with post-acute ischemic stroke (AIS) and cerebrovascular insufficiency. LDF was performed from 3 to 7 days after the onset of AIS on forehead in the right and left supraorbital regions in patients. Analysis of LDF signals showed that perfusion in the microvasculature in AIS patients was lower than that in patients with cerebrovascular insufficiency. As a result of wavelet analysis of the LDF signals we obtained activation of the vasomotion in the frequency range of myogenic oscillation of 0.1 Hz and predominantly nutritive regime microcirculation after systemic thrombolytic therapy of the AIS patients. In case of significant stroke size, myogenic activity, and nutritive pattern microhaemodynamics were reduced, in some cases non-nutritive pattern and/or venular stasis was revealed. Wavelet analysis of the LDF signals also showed asymmetry in wavelet spectra of the LDF signals obtained in stroke-affected and unaffected hemispheres in the AIS patients. A mechanism underlying the observed asymmetry was analyzed by computational modeling of vasomotion developed in Arciero and Secomb (2012). We applied this model to describe relaxation oscillation of arteriole diameter which is forced by myogenic oscillation induced by synchronous calcium oscillation in vascular smooth muscle cells. Calculation showed that vasomotion frequency spectrum at the low-frequency range (0.01 Hz) is reciprocally modulated by myogenic oscillation (0.1 Hz) that correlates with experimental observation of inter-hemispheric variation in the LDF spectrum.

Photodynamic opening of blood-brain barrier.

Photodynamic treatment (PDT) causes a significant increase in the permeability of the blood-brain barrier (BBB) in healthy mice. Using different doses of laser radiation (635 nm, 10-40 J/cm2) and photosensitizer (5-aminolevulinic acid - 5-ALA, 20 and 80 mg/kg, i.v.), we found that the optimal PDT for the reversible opening of the BBB is 15 J/cm2 and 5-ALA, 20 mg/kg, exhibiting brain tissues recovery 3 days after PDT. Further increases in the laser radiation or 5-ALA doses have no amplifying effect on the BBB permeability, but are associated with severe damage of brain tissues. These results can be an informative platform for further studies of new strategies in brain drug delivery and for better understanding of mechanisms underlying cerebrovascular effects of PDT-related fluorescence guided resection of brain tumor.

Multimodal optical measurement for study of lower limb tissue viability in patients with diabetes mellitus.

According to the International Diabetes Federation, the challenge of early stage diagnosis and treatment effectiveness monitoring in diabetes is currently one of the highest priorities in modern healthcare. The potential of combined measurements of skin fluorescence and blood perfusion by the laser Doppler flowmetry method in diagnostics of low limb diabetes complications was evaluated. Using Monte Carlo probabilistic modeling, the diagnostic volume and depth of the diagnosis were evaluated. The experimental study involved 76 patients with type 2 diabetes mellitus. These patients were divided into two groups depending on the degree of complications. The control group consisted of 48 healthy volunteers. The local thermal stimulation was selected as a stimulus on the blood microcirculation system. The experimental studies have shown that diabetic patients have elevated values of normalized fluorescence amplitudes, as well as a lower perfusion response to local heating. In the group of people with diabetes with trophic ulcers, these parameters also significantly differ from the control and diabetes only groups. Thus, the intensity of skin fluorescence and level of tissue blood perfusion can act as markers for various degrees of complications from the beginning of diabetes to the formation of trophic ulcers.

Computational model of bladder tissue based on its measured optical properties.

Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a "generic" computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system "LAKK-M" was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.

Individual variability analysis of fluorescence parameters measured in skin with different levels of nutritive blood flow.

Fluorescence spectroscopy has recently become more common in clinical medicine. However, there are still many unresolved issues related to the methodology and implementation of instruments with this technology. In this study, we aimed to assess individual variability of fluorescence parameters of endogenous markers (NADH, FAD, etc.) measured by fluorescent spectroscopy (FS) in situ and to analyse the factors that lead to a significant scatter of results. Most studied fluorophores have an acceptable scatter of values (mostly up to 30%) for diagnostic purposes. Here we provide evidence that the level of blood volume in tissue impacts FS data with a significant inverse correlation. The distribution function of the fluorescence intensity and the fluorescent contrast coefficient values are a function of the normal distribution for most of the studied fluorophores and the redox ratio. The effects of various physiological (different content of skin melanin) and technical (characteristics of optical filters) factors on the measurement results were additionally studied. The data on the variability of the measurement results in FS should be considered when interpreting the diagnostic parameters, as well as when developing new algorithms for data processing and FS devices.

Calluna vulgaris root cells show increased capacity for amino acid uptake when colonized with the mycorrhizal fungus Hymenoscyphus ericae.

• Ericoid mycorrhizas are believed to improve N nutrition of many ericaceous plant species that typically occur in habitats with impoverished nutrient status, by releasing amino acids from organic N forms. Despite the ubiquity of mycorrhizal formation the mechanisms and regulation of nutrient transport in mycorrhizal associations are poorly understood. • We used an electrophysiological approach to study how amino acid transport characteristics of Calluna vulgaris were affected by colonization with the ericoid mycorrhiza fungus Hymenoscyphus ericae . • Both the V max and K m parameters of amino acid uptake were affected by fungal colonization in a manner consistent with an increased availability of amino acid to the plant. • The ecophysiological significance of altered amino acid transport in colonized root cells of C. vulgaris is discussed.

Technologic developments in the field of photonics for the detection of urinary bladder cancer.

Bladder cancer is a common cause of morbidity and mortality worldwide in an aging population. Each year, thousands of people, mostly men, are diagnosed with this disease, but many of them present too late to receive optimal treatment. As with all cancers, early diagnosis of bladder cancer significantly improves the efficacy of therapy and increases survival and recurrence-free survival rates. Ongoing research has identified many limitations about the sensitivity of standard diagnostic procedures in detecting early-stage tumors and precancerous changes. The consequences of this are often tumor progression and increased tumor burden, leading to a decrease in patient quality of life and a vast increase in treatment costs. The necessity for improved early detection of bladder cancer has spurred on research into novel methods that use a wide range of biological and photonic phenomena. This review will broadly discuss standard detection methodologies and their major limitations before covering novel photonic techniques for early tumor detection and staging, assessing their diagnostic accuracy for flat and precancerous changes. We will do so in the context of both cystoscopic examination and the screening of voided urine and will also touch on the concept of using photonic technology as a surgical tool for tumor ablation.

Substantiation of medical and technical requirements for noninvasive spectrophotometric diagnostic devices.

A scientific approach to the formulation of medical and technical requirements (MTRs) for noninvasive spectrophotometric diagnostic devices using optical technologies such as laser Doppler flowmetry and absorption spectroscopy is proposed. The theoretical modeling framework, metrological certification, and testing of these devices are still in the early stages of development. The theoretical estimation of the received signal levels for wavelengths between 514 and 940 nm is highly dependent on the blood volume level in the subject tissue. The proposed approach allows, in particular, the calculation of technical and metrological performance constraints of the instruments, such as the ranges of the sensitivity and power-related signal-to-noise ratios for different spectral channels and different biomedical (biochemical and physiological) parameters. Substantiation of specialized MTRs for the noninvasive spectrophotometric diagnostic devices can enable them to develop to the level of standardized measurement techniques.

Functional interaction of the SNARE protein NtSyp121 in Ca2+ channel gating, Ca2+ transients and ABA signalling of stomatal guard cells.

There is now growing evidence that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling in plants. Work from this laboratory first demonstrated that a soluble, inhibitory (dominant-negative) fragment of the SNARE NtSyp121 blocked K+ and Cl- channel responses to the stress-related hormone abscisic acid (ABA), but left open a question about functional impacts on signal intermediates, especially on Ca2+-mediated signalling events. Here, we report one mode of action for the SNARE mediated directly through alterations in Ca2+ channel gating and its consequent effects on cytosolic-free [Ca2+] ([Ca2+]i) elevation. We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor, SNAP, which promotes [Ca2+]i elevation independently of the plasma membrane Ca2+ channels. Consistent with these observations, Ca2+ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca2+]i, and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca2+]i transients. These observations offer primary evidence for the functional coupling of the SNARE with Ca2+ channels at the plant cell plasma membrane and, because [Ca2+]i plays a key role in the control of K+ and Cl- channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.

Selective targeting of plasma membrane and tonoplast traffic by inhibitory (dominant-negative) SNARE fragments.

Vesicle traffic underpins cell homeostasis, growth and development in plants, and is facilitated by a superfamily of proteins known as SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors] that interact to draw vesicle and target membrane surfaces together for fusion. Structural homologies, biochemical and genetic analyses have yielded information about the localization and possible roles of these proteins. However, remarkably little evidence is yet available that speaks directly to the functional specificities of these proteins in selected trafficking pathways in vivo. Previously, we found that expressing a cytosolic (so-called Sp2) fragment of one plasma membrane SNARE from tobacco and Arabidopsis had severe effects on growth, tissue development and secretory traffic to the plasma membrane. We have explored this dominant-negative approach further to examine the specificity and overlaps in Sp2 activity by generating a toolbox of truncated SNARE constructs and antibodies for transient expression and analysis. Using a quantitative ratiometric approach with secreted green fluorescent protein (secGFP), we report here that traffic to the plasma membrane is suppressed selectively by Sp2 fragments of plasma membrane SNAREs AtSYP121 and AtSYP122, but not of the closely related SNARE AtSYP111 nor of the SNARE AtSYP21 that resides at the pre-vacuolar compartment (PVC). By contrast, traffic of the YFP-tagged aquaporin fusion protein TIP1;1-YFP to the tonoplast was blocked (leading to its accumulation in the PVC) when co-expressed with the Sp2 fragment of AtSYP21, but not when co-expressed with that of AtSYP121. Export of secGFP was also sensitive to the Sp2 fragment of the novel, plant-specific SNARE AtSYP71 that was recently found to be present in detergent-resistant, plasma membrane fractions. Co-incubation analyses of the plasma membrane SNAREs with the regulatory subdomain included within the Sp2 fragments showed activity in destabilizing protein complexes, but only with the complementary SNAREs. We conclude that the Sp2 fragment action accurately reflects the known specificity and targeting of these SNAREs, implies functional overlaps that are of potential physiological interest, and underscores the use of a dominant-negative strategy in functional studies of a major subfamily of SNAREs in plants.