Patch-clamp technique to study mitochondrial membrane biophysics.

Mitochondria are double-membrane organelles crucial for oxidative phosphorylation, enabling efficient ATP synthesis by eukaryotic cells. Both of the membranes, the highly selective inner mitochondrial membrane (IMM) and a relatively porous outer membrane (OMM), harbor a number of integral membrane proteins that help in the transport of biological molecules. These transporters are especially enriched in the IMM, where they help maintain transmembrane gradients for H+, K+, Ca2+, PO43-, and metabolites like ADP/ATP, citrate, etc. Impaired activity of these transporters can affect the efficiency of energy-transducing processes and can alter cellular redox state, leading to activation of cell-death pathways or metabolic syndromes in vivo. Although several methodologies are available to study ion flux through membrane proteins, the patch-clamp technique remains the gold standard for quantitatively analyzing electrogenic ion exchange across membranes. Direct patch-clamp recordings of mitoplasts (mitochondria devoid of outer membrane) in different modes, such as whole-mitoplast or excised-patch mode, allow researchers the opportunity to study the biophysics of mitochondrial transporters in the native membrane, in real time, in isolation from other fluxes or confounding factors due to changes in ion gradients, pH, or mitochondrial potential (ΔΨ). Here, we summarize the use of patch clamp to investigate several membrane proteins of mitochondria. We demonstrate how this technique can be reliably applied to record whole-mitoplast Ca2+ currents mediated via mitochondrial calcium uniporter or H+ currents mediated by uncoupling protein 1 and discuss critical considerations while recording currents from these small vesicles of the IMM (mitoplast diameter = 2-5 µm).

Deficiency of mitochondrial calcium uniporter abrogates iron overload-induced cardiac dysfunction by reducing ferroptosis.

Iron overload associated cardiac dysfunction remains a significant clinical challenge whose underlying mechanism(s) have yet to be defined. We aim to evaluate the involvement of the mitochondrial Ca2+ uniporter (MCU) in cardiac dysfunction and determine its role in the occurrence of ferroptosis. Iron overload was established in control (MCUfl/fl) and conditional MCU knockout (MCUfl/fl-MCM) mice. LV function was reduced by chronic iron loading in MCUfl/fl mice, but not in MCUfl/fl-MCM mice. The level of mitochondrial iron and reactive oxygen species were increased and mitochondrial membrane potential and spare respiratory capacity (SRC) were reduced in MCUfl/fl cardiomyocytes, but not in MCUfl/fl-MCM cardiomyocytes. After iron loading, lipid oxidation levels were increased in MCUfl/fl, but not in MCUfl/fl-MCM hearts. Ferrostatin-1, a selective ferroptosis inhibitor, reduced lipid peroxidation and maintained LV function in vivo after chronic iron treatment in MCUfl/fl hearts. Isolated cardiomyocytes from MCUfl/fl mice demonstrated ferroptosis after acute iron treatment. Moreover, Ca2+ transient amplitude and cell contractility were both significantly reduced in isolated cardiomyocytes from chronically Fe treated MCUfl/fl hearts. However, ferroptosis was not induced in cardiomyocytes from MCUfl/fl-MCM hearts nor was there a reduction in Ca2+ transient amplitude or cardiomyocyte contractility. We conclude that mitochondrial iron uptake is dependent on MCU, which plays an essential role in causing mitochondrial dysfunction and ferroptosis under iron overload conditions in the heart. Cardiac-specific deficiency of MCU prevents the development of ferroptosis and iron overload-induced cardiac dysfunction.

Emerging Biomedical Applications of the Vesicular Stomatitis Virus Glycoprotein.

Nanoparticles (NPs) made of metals, polymers, micelles, and liposomes are increasingly being used in various biomedical applications. However, most of these NPs are hazardous for long- and short-term use and hence have restricted biomedical applications. Therefore, naturally derived, biocompatible, and biodegradable nanoconstructs are being explored for such applications. Inspired by the biology of viruses, researchers are exploring the viral proteins that hold considerable promise in biomedical applications. The viral proteins are highly stable and further amenable to suit specific biological applications. Among various viral proteins, vesicular stomatitis virus glycoprotein (VSV-G) has emerged as one of the most versatile platforms for biomedical applications. Starting with their first major use in lentivirus/retrovirus packaging systems, the VSV-G-based reagents have been tested for diverse biomedical use, many of which are at various stages of clinical trials. This manuscript discusses the recent advancements in the use of the VSV-G-based reagents in medical, biological research, and clinical applications particularly highlighting emerging applications in biomedical imaging.

Interferometric Imaging, and Beam-Formed Study of a Moving Type-IV Radio Burst with LOFAR.

Type-IV radio bursts have been studied for over 50 years. However, the specifics of the radio emission mechanisms is still an open question. In order to provide more information about the emission mechanisms, we studied a moving Type-IV radio burst with fine structures (spike group) by using the high-resolution capability of the Low-Frequency Array (LOFAR) on August 25, 2014. We present a comparison of Nançay Radioheliograph (NRH) and the first LOFAR imaging data of the Type-IV radio burst. The degree of circular polarization (DCP) is calculated at frequencies in the range 20 - 180 MHz using LOFAR data, and it was found that the value of DCP gradually increased during the event, with values of 20 - 30%. LOFAR interferometric data were combined with white-light observations in order to track the propagation of this Type-IV burst. The kinematics shows a westward motion of the radio sources, slower than the CME leading edge. The dynamic spectrum of LOFAR shows a large number of fine structures with durations of less than 1 s and high brightness temperatures ( T B ), i.e., 10 12 - 10 13 K. The gradual increase of DCP supports gyrosynchrotron emission as the most plausible mechanism for the Type IV. However, coherent emissions such as Electron Cyclotron Maser (ECM) instability may be responsible for small-scale fine structures. Countless fine structures altogether were responsible for such high T B . Supplementary Information: The online version contains supplementary material available at 10.1007/s11207-022-02042-0.

Identification, characterization and optimization of phosphate solubilizing rhizobacteria (PSRB) from rice rhizosphere.

Two billion people worldwide take rice (Oryza sativa L.) as a staple food. Phosphorus (P) and Nitrogen (N) are the major requirements of rice; although these are available in limited concentrations within rice growing regions. Among different types of Plant growth-promoting rhizobacteria (PGPR), Phosphate solubilizing rhizobacteria (PSRB) constitute an important class. These are known for plant growth promotion by enhancing P and N uptake. PSRB are nowadays used as biofertilizers to restore the soil health. Under the present investigation identification, characterization and optimization of phosphate solubilizing activity of these microbes at different pH, temperature and salt concentrations was carried out. Thirty-seven isolates were recovered from different regions of rice rhizosphere on Pikovskaya (PVK) agar among which 15 isolates were recovered from R.S. Pura, 12 isolates from Bishnah and 10 isolates were recovered from Akhnoor sector of Jammu, India. A prominent halo zone of clearance was developed around the colonies of 12 different isolates, indicating phosphate solubilization activity. Four distinct isolates were amplified, cloned and sequenced for taxonomic identification using 16S primers. The results indicated that PS 1, PS 2, PS 3, PS 4 were related to Pseudomonas aeruginosa, Bacillus subtilis strain 1, B. subtilis strain 2, B. subtilis strain 3, respectively. These strains when grown at a wide range of ecological factors showed maximum growth at pH between 6.8 and 8.8, temperature between 28 °C and 37 °C and salinity between 1% and 2%. Screening for phosphate solubilization activity revealed that the halo zone diameter formed by these isolates extended from 2.1 to 3.2 mm. The phosphate solubilizing efficiency (SE) ranged from 35.4 to 50.9 with highest value of 50.9 by PS4 and maximum P solubilization of 10.22 µg/ml was recorded by PS4 at 7th day. Phosphate solubilization activity of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus. MIC value for zinc sulphate heptahydrate in 12 isolates varied from 1 mg/ml to 6 mg/ml. Phosphate solubilization activity and MIC of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus.

Fusogenic Viral Protein-Based Near-Infrared Active Nanocarriers for Biomedical Imaging.

An effective drug delivery system (DDS) relies on an efficient cellular uptake and faster intracellular delivery of theranostic agents, bypassing the endosomal mediated degradation of the payload. The use of viral nanoparticles (VNPs) permits such advancement, as the viruses are naturally evolved to infiltrate the host cells to deliver their genetic material. As a proof of concept, we bioengineered the vesicular stomatitis virus glycoprotein (VSV-G)-based near-infrared (NIR) active viral nanoconstructs (NAVNs) encapsulating indocyanine green dye (ICG) for NIR bioimaging. NAVNs are spherical in size and have the intrinsic cellular-fusogenic properties of VSV-G. Further, the NIR imaging displaying higher fluorescence intensity in NAVNs treated cells suggests enhanced cellular uptake and delivery of ICG by NAVNs compared to the free form of ICG. The overall study highlights the effectiveness of VSV-G-based VNPs as an efficient delivery system for NIR fluorescence imaging.

Nematode Signaling Molecules Are Extensively Metabolized by Animals, Plants, and Microorganisms.

Many bacterivorous and parasitic nematodes secrete signaling molecules called ascarosides that play a central role regulating their behavior and development. Combining stable-isotope labeling and mass spectrometry-based comparative metabolomics, here we show that ascarosides are taken up from the environment and metabolized by a wide range of phyla, including plants, fungi, bacteria, and mammals, as well as nematodes. In most tested eukaryotes and some bacteria, ascarosides are metabolized into derivatives with shortened fatty acid side chains, analogous to ascaroside biosynthesis in nematodes. In plants and C. elegans, labeled ascarosides were additionally integrated into larger, modular metabolites, and use of different ascaroside stereoisomers revealed the stereospecificity of their biosynthesis. The finding that nematodes extensively metabolize ascarosides taken up from the environment suggests that pheromone editing may play a role in conspecific and interspecific interactions. Moreover, our results indicate that plants, animals, and microorganisms may interact with associated nematodes via manipulation of ascaroside signaling.

Post-operative analgesic effect of intraperitoneal ropivacaine with or without tramadol in laparoscopic cholecystectomy.

BACKGROUND AND AIMS: Intraperitoneal instillation of local anaesthetics has been shown to minimise post-operative pain after laparoscopic surgery. This study was aimed to evaluate the post-operative effect of intraperitoneal ropivacaine with and without tramadol in patients undergoing laparoscopic cholecystectomy. METHODS: Eighty patients undergoing laparoscopic cholecystectomy were randomised into two groups. Group R received 0.5% ropivacaine 18 mL with normal saline (NS) 2 mL and Group RT received 0.5% ropivacaine 18 mL with tramadol (100 mg, 2 mL) at the end of surgery intraperitoneally through the port. The pain score was monitored using a numerical rating scale (NRS) every 30 min till 4 h post-operatively and then at 6 h, 12 h and 24 h. The primary objective of the study was to compare the severity of pain between the groups. The secondary objectives were to compare the total dose of rescue analgesic and the time tofirst rescue analgesia between the groups Statistical analysis was performed using statistical package for the social sciences. Chi-square test and Mann Whitney U test were used for analysis. RESULTS: The pain score in Group RT was significantly lower than Group R at 2.5 h to 24 h (P = 0.005). Only 42.5% in Group RT demanded rescue analgesia as compared to 75% in Group R (P = 0.003). Total analgesic consumption of fentanyl was also reduced in the tramadol group (785 µg vs 1800 µg). No significant adverse effects were found. CONCLUSION: Intraperitoneal instillation of ropivacaine with tramadol reduces the post-operative pain and analgesic requirement in laparoscopic cholecystectomy as compared to ropivacaine alone.

Plant metabolism of nematode pheromones mediates plant-nematode interactions.

Microorganisms and nematodes in the rhizosphere profoundly impact plant health, and small-molecule signaling is presumed to play a central role in plant rhizosphere interactions. However, the nature of the signals and underlying mechanisms are poorly understood. Here we show that the ascaroside ascr#18, a pheromone secreted by plant-parasitic nematodes, is metabolized by plants to generate chemical signals that repel nematodes and reduce infection. Comparative metabolomics of plant tissues and excretions revealed that ascr#18 is converted into shorter side-chained ascarosides that confer repellency. An Arabidopsis mutant defective in two peroxisomal acyl-CoA oxidases does not metabolize ascr#18 and does not repel nematodes, indicating that plants, like nematodes, employ conserved peroxisomal ß-oxidation to edit ascarosides and change their message. Our results suggest that plant-editing of nematode pheromones serves as a defense mechanism that acts in parallel to conventional pattern-triggered immunity, demonstrating that plants may actively manipulate chemical signaling of soil organisms.

Protease Responsive Essential Amino-Acid Based Nanocarriers for Near-Infrared Imaging.

Delivery of the theranostic agents with effective concentration to the desired sites inside the body is a major challenge in disease management. Nanotechnology has gained attention for the delivery of theranostic agents to the targeted location. The use of essential amino-acid based homopolymers for the synthesis of biocompatible and biodegradable nanoparticles (NPs) could serve as a nanocarrier for delivery applications. In this study, poly-l-lysine (PLL) and salts were used to fabricate the NPs for the delivery of exogenous contrast agents. Here, indocyanine green (ICG) was encapsulated within these NPs, and a simple two-step green chemistry-based self-assembly process was used for the fabrication. The morphological and biochemical characterizations confirm the formation of ICG encapsulating spherical PLL NPs with an average diameter of ~225 nm. Further, a detailed study has been carried out to understand the role of constituents in the assembly mechanism of PLL NPs. Our results show a controlled release of the ICG from PLL NPs in the presence of the proteolytic enzyme. In-vitro cellular studies suggest that the PLL NPs were readily taken up by the cells showing their superior delivery efficiency of ICG in comparison to the free-form of the ICG.

Application of continuous-wave photoacoustic sensing to red blood cell morphology.

The feasibility of continuous wave laser-based photoacoustic (CWPA) response technique in detecting the morphological changes in cells during the biological studies, through the features extracted from CWPA signal (i.e., amplitude) is demonstrated here. Various hematological disorders (e.g., sickle cell anemia, thalesemia) produce distinct changes at the cellular level morphologically. In order to explore the photoacoustic response technique to detect these morphological changes, we have applied CWPA technique onto the blood samples. Results of our preliminary study show a distinct change in the signal amplitude of photoacoustic (PA) signal due to a change in the concentration of blood, which signifies the sensitivity of the technique towards red blood cell (RBC) count (related to hematological disease like anemia). Further hypotonic and hypertonic solutions were induced in blood to produce morphological changes in RBCs (i.e., swollen and shrink, respectively) as compared to the normal RBCs. Experiments were performed using continuous wave laser-based photoacoustic response technique to verify the morphological changes in these RBCs. A distinct change in the PA signal amplitude was found for the distinct nature of RBCs (swollen, shrink, and normal). Thus, this can serve as a diagnostic signature for different biological studies based on morphological changes at cellular level. The experiments were also performed using conventional pulsed laser photoacoustic response technique which uses nano-second pulsed laser and the results obtained from both PA techniques were validated to produce identical changes. This demonstrates the utility of continuous wave laser-based photoacoustic technique for different biological studies related to morphological cellular disorders.

Two-photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near-infrared dye: Application of anti-Kasha's rule for two-photon fluorescence imaging.

In recent years, two-photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near-infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two-photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two-photon excitation property of ICG to the second excited singlet (S2 ) state in aqueous solution. Furthermore, in this work, we demonstrate the two-photon cellular imaging application of ICG using direct fluorescence emission from S2 state for the first time. Our results show that two-photon excitation to S2 state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well-known "tissue-optical window." This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two-photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.

The effect of nanoencapsulation of ICG on two-photon bioimaging.

Multiphoton imaging, a highly effective diagnostic technique, has recently gained widespread attention for early-stage cancer detection. Tremendous efforts have been dedicated to explore various types of exogenous contrast agents for improved signal-to-noise ratio of multiphoton imaging. Indocyanine green (ICG), the only U. S. FDA approved near-infrared chromophore, has been recently used as an exogenous contrast agent for two-photon bioimaging. Despite its great potential applications in clinical settings, the conventional delivery method of ICG has limited applications due to its poor cellular uptake and optical stability in its free form. Herein, we report the effect of nanoencapsulation of ICG on two-photon bioimaging. For this study, ICG was encapsulated within poly-l-arginine (PLA) based nanoparticles for the first time. These nanoparticles were found to be biocompatible and biodegradable as the major constituents were salts and PLA. These nanoparticles were spherical with a mean diameter of ∼61 nm and exhibit higher photostability than free ICG. Additionally, nanoencapsulated ICG treated cells show enhanced contrast for two-photon bioimaging in comparison with its free form. In summary, nanoencapsulated ICG could serve as an exogenous chromophore for multiphoton imaging, which shows excellent delivery efficacy.

Quantitative Differentiation of Pneumonia from Normal Lungs: Diagnostic Assessment Using Photoacoustic Spectral Response.

Pneumonia is an acute lung infection that takes life of many young children in developing countries. Early stage (red hepatization) detection of pneumonia would be pragmatic to control mortality rate. Detection of this disease at early stages demands the knowledge of pathology, making it difficult to screen noninvasively. We propose photoacoustic spectral response (PASR), a noninvasive elasticity-dependent technique for early stage pneumonia detection. We report the quantitative red hepatization detection of pneumonia through median frequency, spectral energy, and variance. Significant contrast in spectral parameters due to change in sample elasticity is found. The tissue-mimicking phantom study illustrates a 39% increase in median frequency for 1.5 times the change in density. On applying to formalin-fixed pneumonia-affected goat lungs, it provides a distinct change in spectral parameters between pneumonia affected areas and normal lungs. The obtained PASR results were found to be highly correlating to standard histopathology. The proposed technique therefore has potential to be a regular diagnostic tool for early pneumonia detection.