FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase.

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium-treated and polymer-embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX-conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosome-mitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.

Mitochondrial matrix RTN4IP1/OPA10 is an oxidoreductase for coenzyme Q synthesis.

Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O-methylation activity of COQ3. Rtn4ip1-knockout myoblasts had markedly decreased CoQ9 levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of dRtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.

GEN-Click: Genetically Encodable Click Reactions for Spatially Restricted Metabolite Labeling.

Chemical reactions for the in situ modification of biomolecules within living cells are under development. Among these reactions, bio-orthogonal reactions such as click chemistry using copper(I) and Staudinger ligation are widely used for specific biomolecule tracking in live systems. However, currently available live cell copper(I)-catalyzed azide/alkyne cycloaddition reactions are not designed in a spatially resolved manner. Therefore, we developed the "GEN-Click" system, which can target the copper(I)-catalyzed azide/alkyne cycloaddition reaction catalysts proximal to the protein of interest and can be genetically expressed in a live cell. The genetically controlled, spatially restricted, metal-catalyzed biorthogonal reaction can be used for proximity biotin labeling of various azido-bearing biomolecules (e.g., protein, phospholipid, oligosaccharides) in living cell systems. Using GEN-Click, we successfully detected local metabolite-transferring events at cell-cell contact sites.

A multifunctional peroxidase-based reaction for imaging, sensing and networking of spatial biology.

Peroxidase is a heme-containing enzyme that reduces hydrogen peroxide to water by extracting electron(s) from aromatic compounds via a sequential turnover reaction. This reaction can generate various aromatic radicals in the form of short-lived "spray" molecules. These can be either covalently attached to proximal proteins or polymerized via radical-radical coupling. Recent studies have shown that these peroxidase-generated radicals can be utilized as effective tools for spatial research in biological systems, including imaging studies aimed at the spatial localization of proteins using electron microscopy, spatial proteome mapping, and spatial sensing of metabolites (e.g., heme and hydrogen peroxide). This review may facilitate the wider utilization of these peroxidase-based methods for spatial discovery in cellular biology.

Low-volume plasma sampling for determination of dextromethorphan and dextrorphan in rat plasma: LC-MS/MS method and its application in pharmacokinetic study.

Dextromethorphan (DM) and its metabolite dextrorphan (DX) continue to draw the attention of researchers owing to their diverse pharmacodynamics. Thus, there are possibilities for repurposing DM. Most of the pharmacodynamics of DM needs further validation in different preclinical models. Also, it is necessary to correlate the pharmacodynamics with relevant pharmacokinetics data. Multiple bioanalytical techniques developed for this purpose primarily use a high sample processing volume. Since sample volume is a limiting factor for many preclinical models, an effort was taken to develop an alternative method suitable for handling low sample processing volumes. An efficient solid-phase extraction technique, robust liquid chromatographic (LC) separation and highly sensitive tandem mass spectrometric detection (MS/MS) showed suitability for use of a 30 µl sample processing volume. This led to the development of a highly specific, selective, accurate and precise-bio-analytical method for simultaneous quantification of DM and DX in rat plasma. The validated method was linear in the range of 0.196-403.356 ng/ml for DM and 0.102-209.017 ng/ml for DX. The application of the method was demonstrated through the estimation of pharmacokinetic parameters that showed good congruence with earlier studies.

COVID-19 detection on chest X-ray images using Homomorphic Transformation and VGG inspired deep convolutional neural network.

COVID-19 had caused the whole world to come to a standstill. The current detection methods are time consuming as well as costly. Using Chest X-rays (CXRs) is a solution to this problem, however, manual examination of CXRs is a cumbersome and difficult process needing specialization in the domain. Most of existing methods used for this application involve the usage of pretrained models such as VGG19, ResNet, DenseNet, Xception, and EfficeintNet which were trained on RGB image datasets. X-rays are fundamentally single channel images, hence using RGB trained model is not appropriate since it increases the operations by involving three channels instead of one. A way of using pretrained model for grayscale images is by replicating the one channel image data to three channel which introduces redundancy and another way is by altering the input layer of pretrained model to take in one channel image data, which comprises the weights in the forward layers that were trained on three channel images which weakens the use of pre-trained weights in a transfer learning approach. A novel approach for identification of COVID-19 using CXRs, Contrast Limited Adaptive Histogram Equalization (CLAHE) along with Homomorphic Transformation Filter which is used to process the pixel data in images and extract features from the CXRs is suggested in this paper. These processed images are then provided as input to a VGG inspired deep Convolutional Neural Network (CNN) model which takes one channel image data as input (grayscale images) to categorize CXRs into three class labels, namely, No-Findings, COVID-19, and Pneumonia. Evaluation of the suggested model is done with the help of two publicly available datasets; one to obtain COVID-19 and No-Finding images and the other to obtain Pneumonia CXRs. The dataset comprises 6750 images in total; 2250 images for each class. Results obtained show that the model has achieved 96.56% for multi-class classification and 98.06% accuracy for binary classification using 5-fold stratified cross validation (CV) method. This result is competitive and up to the mark when compared with the performance shown by existing approaches for COVID-19 classification.

Enzymatic Recording of Local Hydrogen Peroxide Generation Using Genetically Encodable Enzyme.

Reactive oxygen species (ROS) are endogenously generated in live cells and essential for cell signaling. However, excess ROS generation can cause oxidative damage to biomolecules, which are implicated in various human diseases, including aging. Here, we developed an in vivo hydrogen peroxide monitoring method using a genetically encodable peroxidase (APEX2)-based system. We confirmed that APEX2 is activated by endogenous H2O2 and generates phenoxyl radicals to produce biotinylated signals (i.e., biotin-phenol) and fluorescent signals (i.e., AmplexRed), which can be detected using a fluorescence microscope. We observed that all subcellular targeted APEX2s were activated by local H2O2 generation by menadione treatment. Among them, the endoplasmic reticulum lumen and lysosome-targeted APEX2 showed the highest response upon addition of menadione which implies that local H2O2 levels in those spaces are highly increased by menadione treatment. Using APEX2, we also found that a minimum amount of menadione (>10 µM) is required to generate detectable levels of H2O2 in all subcellular compartments. We also checked the local H2O2-quenching effect of N-acetylcysteine using our system. As APEX2 can be genetically expressed in diverse live organisms (e.g., cancer cell lines, mice, fly, worm, and yeast), our method can be effectively used to detect local generation of endogenously produced H2O2 in diverse live models.

Piecing Together a Puzzle of Exceptional Lesions: A Retrospective Study of a Potpourri of 160 Space-Occupying Lesions of the Central Nervous System.

Introduction Central nervous system (CNS) lesions are rare and histologically heterogenous, and carry serious potential for patient morbidity and mortality. A retrospective epidemiological review of CNS neoplasms is of great importance for future research because it can demonstrate the changes in the spectrum of CNS lesions of a population, unveil the possible associated risk factors, and indicate the potential therapeutic methods for various neoplastic and non-neoplastic lesions. Neurosurgeons have always shown an obsession with a good neuropathological diagnosis in intracranial and extracranial lesions. This obsession need not be overemphasized as it helps the clinician plan an adequate surgical/treatment strategy to optimize outcomes and minimize morbidity. Methods This study included a spectrum of 160 biopsies of patients with space-occupying lesions of the CNS during a period of two years (2019-2021). All the cases were studied and analyzed, and their histological typing/grading was done. The cases were graded and categorized according to the 2016 WHO Classification of CNS Tumors. Results Among 160 cases, the study showed a slight male preponderance of 100 (62.5%) cases. The maximum number of cases, 37 (23%) cases, was in the age group of 41-50 years. Clinically, the commonest complaints were headache and seizures. The most common location of tumor was supra-tentorial, comprising around 96 (60%) cases, of which 27 (28%) cases were located in the frontal lobe. There were four (2.5%) cases that had non-neoplastic lesions and the rest 156 (97.5%) cases had neoplastic lesions. Malignant lesions outnumbered the benign lesions, comprising of 82 (51.25%) cases. Among the neoplastic lesions, the highest cases were of astrocytoma, 48 (30.76%) cases, followed by meningioma, 42 (26.92%) cases. Also, 21 extremely rare and unusual cases were encountered. Conclusion The present study reflects the diversity of histopathological spectrum of CNS lesions in our center. In-depth studies from across various hospitals are required to have representative data on the incidence, epidemiological profile, and etiology of CNS lesions in India.

A chemical tool for blue light-inducible proximity photo-crosslinking in live cells.

We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.

Microplastics in polar regions: An early warning to the world's pristine ecosystem.

The menace of plastic which is polluting the ocean has emerged as a global problem. It is well-known to everyone that the ultimate end for most of the plastic debris is the ocean. The distribution of plastic rubbish in the oceans is strongly influenced by hydrodynamic properties of water. The continuous break down of plastic objects, as a consequence of thermal, chemical and biological processes along with various environmental factors, results into microplastics (MPs). The microplastics are those particles which are deriving pallets of plastic, having length of less than 5 mm or 0.2 in. Nowadays microplastics are everywhere in the waters all around the world. The high dispersion pattern of oceanic currents takes away microplastics in the entire ocean even to remote areas, like the Polar Regions. Microplastics are difficult to remove from the ocean and the ingestion of these particles by several consumers of different trophic levels like benthos, birds, and fishes is a threat to the diverse food webs and ecosystems. Different scientific investigations have ascertained that a significant concentration of MPs are present in various marine ecosystems globally including the Polar region (both Arctic and Antarctic), and in the upcoming future, the condition is expected to get worse. The objective of this review is to establish a baseline evidence for the availability of microplastics in the polar region. For this reason, the state of the art of knowledge on microplastics in Polar Regions was studied.

Photo-crosslinking: An Emerging Chemical Tool for Investigating Molecular Networks in Live Cells.

Studying protein-protein interactions (PPIs) is useful for understanding cellular functions and mechanisms. Evaluating these PPIs under conditions as similar as possible to native conditions can be achieved using photo-crosslinking methods because of their on-demand ability to generate reactive species in situ by irradiation with UV light. Various fusion tag, metabolic incorporation, and amber codon suppression approaches using various crosslinkers containing aryl azide, benzophenone, and diazirines have been applied in live cells. Mass spectrometry and immunological techniques are used to identify crosslinked proteins based on their capture transient and context-dependent interactions. Herein we discuss various incorporation methods and crosslinkers that have been used for interactome mapping in live cells.

BODIPY based colorimetric fluorescent probe for selective thiophenol detection: theoretical and experimental studies.

A BODIPY-based selective thiophenol probe capable of discriminating aliphatic thiols is reported. The fluorescence off-on effect upon reaction with thiol is elucidated with theoretical calculations. The sensing of thiophenol is associated with a color change from red to yellow and 63-fold enhancement in green fluorescence. Application of the probe for selective thiophenol detection is demonstrated by live cell imaging.