Regulation of the apico-basolateral trafficking polarity of the homologous copper-ATPases ATP7A and ATP7B.

The homologous P-type copper-ATPases (Cu-ATPases) ATP7A and ATP7B are the key regulators of copper homeostasis in mammalian cells. In polarized epithelia, upon copper treatment, ATP7A and ATP7B traffic from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively. We characterized the sorting pathways of Cu-ATPases between TGN and the plasma membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN in limiting copper conditions, and in high copper, ATP7A traffics to basolateral membrane, whereas ATP7B traverses common recycling, apical sorting and apical recycling endosomes en route to apical membrane. Mass spectrometry identified regulatory partners of ATP7A and ATP7B that include the adaptor protein-1 complex. Upon knocking out pan-AP-1, sorting of both Cu-ATPases is disrupted. ATP7A loses its trafficking polarity and localizes on both apical and basolateral surfaces in high copper. By contrast, ATP7B loses TGN retention but retained its trafficking polarity to the apical domain, which became copper independent. Using isoform-specific knockouts, we found that the AP-1A complex provides directionality and TGN retention for both Cu-ATPases, whereas the AP-1B complex governs copper-independent trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B.

Zn-CD@Eu Ratiometric Fluorescent Probe for the Detection of Dipicolinic Acid, Uric Acid, and Ex Vivo Uric Acid Imaging.

Development of reliable methods for the detection of potential biomarkers is of the utmost importance for an early diagnosis of critical diseases and disorders. In this study, a novel lanthanide-functionalized carbon dot-based fluorescent probe Zn-CD@Eu is reported for the ratiometric detection of dipicolinic acid (DPA) and uric acid (UA). The Zn-CD@Eu nanoprobe was obtained from a simple room-temperature reaction of zinc-doped carbon dots (Zn-CD) and the EDTA-Eu lanthanide complex. Under optimal conditions, a good linear response was obtained for DPA in two concentration ranges of 0-55 and 55-100 µM with a limit of detection of 0.53 and 2.2 µM respectively, which is significantly below the infectious dosage of anthrax (∼55 µM). Furthermore, the Zn-CD@Eu/DPA system was employed for the detection of UA with a detection limit of 0.36 µM in the linear range of 0-100 µM. The fluorescent probe was successfully implemented for determining DPA and UA in human blood serum, sweat, and natural water bodies with considerable recovery rates. In addition, the potential of the nanoprobe for ex vivo visualization of UA was demonstrated in fruit fly (Drosophila melanogaster) as a model organism.

Coumarin-Based Noncytotoxicity Fluorescent Dye for Tracking Actin Protein in In-Vivo Imaging.

Drosophila shares maximum homology with the human disease-causing genes and thus has been employed to evaluate the toxicity of numerous compounds. Further, its distinguishable developmental stages, easy rearing, and short lifespan make it a perfect model organism to study toxicological properties of any new compound. The current study evaluates the toxic effect of a coumarin-based organic fluorescent dye, 7-hydroxy-4-methyl-8-((4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl)-2H-chromen-2-one (CTC), using Drosophila melanogaster as a model organism by studying different behavioral, screening, and staining techniques using Oregon-R flies. For toxicity assessment, one control fly group was compared with various flies that had been subjected to fed CTC dye orally of different concentrations (0.5, 1, 2.5, and 5 µg/mL). The 3rd instar larvae were checked for the larvae crawling assay. The crawling assay demonstrates that the speed and path of the treated larvae are almost equal to the control ones, which signifies the non-neurotoxic property of CTC. Trypan blue assay further suggested that the dye does not cause any major damage to the gut. Phalloidin staining revealed that the actin composition remains unaltered even after the CTC treatment, while the DAPI staining experiment indicates that CTC does not cause any nuclear damage to fly gut cells. However, at a concentration of 5 µg/mL, CTC causes developmental delay. The flies hatched after larval treatment of CTC do not show any structural defects, suggesting clearly that CTC is also nongenotoxic to Drosophila. The current studies propose CTC as a noncytotoxic and nongenotoxic dye to track actin protein in the model organism D. melanogaster.

Characterization and identification of inulin from Pachyrhizus erosus and evaluation of its antioxidant and in-vitro prebiotic efficacy.

Inulin is the polysaccharide obtained from different plant sources i.e. Wheat, Chicory, Jerusalem artichoke and Dahlia. In this study, Jicama (Pachyrhizus erosus) is used to isolate inulin using the microwave heating. The 1H NMR study reveals the presence of fructose and glucose unit which is the backbone of inulin. Further FT-IR and Raman confirmed the functional groups present in inulin. The UV-Vis spectroscopy analysis depicts the purity of the isolated inulin. The shape and size of the extracted inulin was determined from scanning electron microscopy and dynamic light scattering appeared as flat-flakes and 135 nm respectively. X-ray diffractogram showed semi-crystalline nature suggesting the stability of the extracted inulin. The isolated inulin has phenolic and flavonoid content of 8.1804 ± 6.26 mg gallic acid equivalent/g and 14.387 ± 4.192 mg rutin equivalent/g of dried polysaccharide respectively. The inhibition percentage of DPPH and FRAP of isolated inulin were found to be 75.74 ± 4.5% and 0.11 ± 0.007 respectively. The isolated inulin promotes the growth of probiotics like Enterococcus faecium (MZ540315) and Lactiplantibacillus plantarum (MZ540317). All the analysis suggest the isolated inulin has good prebiotic potential as the commercially available one. The current study proposes that isolated inulin can be used as a prebiotic in the future. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05619-6.

Synthesis of First Coumarin Fluorescent Dye for Actin Visualization.

Selective fluorescence imaging of actin protein hugely depends on the fluorescently labeled actin-binding domain (ABD). Thus, it is always a challenging task to image the actin protein (in vivo or in vitro) directly with an ABD-free system. To overcome the limitations of actin imaging without an ABD, we have designed a facile and cost-effective red fluorescent coumarin dye 7-hydroxy-4-methyl-8-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl-2H-chromen-2-one (CTC) for actin binding. The selective binding of the dye was investigated using the gut and eye of the model organism Drosophila melanogaster and C2C12 and SCC-9 cell lines. Our result suggests two major advantages of CTC over the dyes presently used for imaging actin proteins. First, the dye can bind to actin efficiently without any secondary intermediate. Second, it is much more stable at room temperature and exhibits excellent photostability. To the best of our knowledge, this is the first fluorescent dye that can bind to the actin protein without employing any secondary intermediate/actin-binding domain. These findings could pave the way for many biologists and physicists to successfully employ the CTC dye for imaging and tracking actin proteins by fluorescence microscopy in various in vivo and in vitro systems.

Aminoglycoside treatment alters hearing-related genes and depicts behavioral defects in Drosophila.

The hearing organ of Drosophila is present within the second segment of antennae. The hearing organ of Drosophila (Johnston's organ [JO]) shares much structural, developmental, and functional similarity with the vertebrate hearing organ (Organ of Corti). JO is evolving as a potential model system to examine the hearing-associated defects in vertebrates. In the vertebrates, aminoglycosides like gentamicin, kanamycin, and neomycin have been known to cause defects in the hearing organ. However, a complete mechanism of toxicity is not known. Taking the evolutionary conservation into account the current study aims to test various concentrations of aminoglycoside on the model organism, Drosophila melanogaster. The current study uses the oral route to check the toxicity of various aminoglycosides at different concentrations (50, 100, 150, 200, and 250 µg ml- 1 ). In Drosophila, many foreign particles enter the body through the gut via food. The aminoglycoside treated third instar larvae show defective crawling and sound avoidance behavior. The adult flies release lower amounts of acetylcholine esterase and higher amounts of reactive oxygen species than control untreated animals, accompanied by defective climbing and aggressive behavior. All these behavioral defects are further confirmed by the altered expression level of hearing genes such as nompC, inactive, nanchung, pyrexia. All the behavioral and genetic defects are reported as a readout of aminoglycoside toxicity.

Drosophila melanogaster as a model to understand the mechanisms of infection mediated neuroinflammation in neurodegenerative diseases.

The innate immune system primarily gets triggered by microbe infiltration, injury, stress, aging, and brain disorders. The hyperactivation of the innate immune system and neuroinflammatory reactions contributes to chronic age-related neurodegeneration. The mechanism for activation of the immune pathway is conserved between Drosophila melanogaster (D. melanogaster) and human being. Thus, D. melanogaster can serve as a model organism to decipher the cellular and molecular mechanism between infection and neurodegenerative diseases. In D. melanogaster, prolonged protective, excessive neuroinflammatory responses in the brain lead to neurodegeneration through antimicrobial peptides mediated neurotoxicity. The prolonged inflammation in the microglial cells helps in the progression of neurodegenerative disease. Therefore, the connection between inflammatory mechanisms in the brain and neurodegeneration pathogenesis in D. melanogaster is systematically reviewed.

Dietary infection of Enterobacter ludwigii causes fat accumulation and resulted in the diabetes-like condition in Drosophila melanogaster.

Bacteria as a foreign pathogen can alter the physiology and metabolism of the host. Many of the teeth infecting bacteria known to be associated with obesity and diabetes in various organisms. The current study aims to check the effect of teeth infecting bacteria Enterobacter ludwigii on model organism Drosophila melanogaster. To check the effect, various concentrations of E. ludwigii on fly physiology the bacteria were added to the fly food. Flies were allowed to grow and colonise in infected food. The offsprings were checked for the accumulation of lipid and fat. With the increase of bacteria within the gut the amount of lipid and fat increases. Alongwith the fat various biochemical parameters like glucose, trehalose, protein and triglyceride level found to be altered. Within the fly gut various metals, which have a role in the metabolism is altered. However, during colonisation within the gut, the morphology of the bacteria remains unaltered. In the adult fly, all the biochemical parameters like glucose, trehalose, protein and triglyceride level increased. The expression level of Dilp is upregulated. Altogether, the current study reports an infection of E. ludwigii causes the accumulation of fat and alters glucose metabolism in Drosophila melanogaster.

Simple techniques to study multifaceted diabesity in the fly model.

Diabetes and obesity are the two notorious metabolic disorders in today's world. Both diabetes and obesity are interlinked with each other and often referred to as 'Diabesity'. It is a complex and multi-organ failure disorder. Thus, many researches and tremendous efforts have been made toward prevention, treatment as well as early detection of diabesity. However, and still, there is a large gap in understanding the etiology as well as treatment of diabesity. Various animal models are also used to decipher the mechanism underlying diabesity. Among all the model organism, recently Drosophila melanogaster is gaining its importance to study diabetes, obesity, and other metabolic disorder. Various experimental methods like histological, biochemical, developmental, and behavioral assays are described in this study to detect diabetes as well as obesity in the fly model.

A Drosophila Model to Decipher the Toxicity of Nanoparticles Taken Through Oral Routes.

In recent era, nanoparticles (NPs) are widely used in food, medicine and body implants. Besides it's wide use being a foreign particle it may have some noxious effect on the body. To understand the mechanistic role of NPs toxicity, Drosophila appeared to be a superior model organism. Toxicity of several nanoparticles were accessed using Drosophila. The NPs, after oral route of exposure enter into the gut, crosses the barrier of peritrophic membrane and induces apoptosis. The toxicity of NPs within gut resulted in developmental delay, with decrease in pupa count, fly hatching along with weight loss. The adult fly hatched after nanoparticle treatment shows increasing phenotypic defect in various sensory organs as well as in different body parts. Besides phenotypic defect some of the nanoparticle results altered behavioural phenotypes like larva crawling or adult climbing. Alteration of both phenotypic as well as behavioural assay clearly hints that signalling pathway like Notch, Wnt, EGFR etc. get affected due to exposure of nanoparticle. Results from various labs prove that nanoparticle can mediate developmental defect by altering signalling pathways. Since many of the signalling pathways are conserved the effect seen in model organisms cannot be overlooked. All the nanoparticles used in food and medicine should be modified to nullify the toxic effect before used in food and medicine.

Can oral microbial infections be a risk factor for neurodegeneration? A review of the literature.

Neurodegenerative diseases, e.g., Alzheimer's and Parkinson's disease, etc., are serious life-threatening diseases, which involve degeneration of the neurons with time. Numerous studies have discussed the role of microbes in neurodegeneration. Oral cavity being the primary site of infection acts as a gateway for gigantic population of microbes to the human body. Oral infection is known to be associated with neurodegeneration. The current review summarizes various mechanisms due to which the oral microbiome can cause neurodegeneration.

Daphnia magna as a Model Organism to Predict the Teratogenic Effect of Different Compounds.

For aquatic ecosystem Daphnia magna is evolving as a model organism to check the teratogenicity of numerous compounds. D. magna can be easily cultured in the laboratory, and the teratogen effect of several compounds can be easily studied. The developmental stages are well studied in D. magna. All the developmental stages are transparent so the defect can be easily accessed. So, the postembryonic developmental changes can be easily studied after the exposure with teratogen. More importantly, D. magna also have a swimming behavioral phenotype. The behavioral defect can be easily accessed after teratogen exposure. The current chapter summarizes numerous protocols associated with embryo and adult staining and adult behavioral assays that can be used to access the teratogenicity of any unknown compound.

Exploration of Teratogenic and Genotoxic Effects on Model Organism Drosophila melanogaster.

Drosophila melanogaster is one of the crucial in vivo models in terms of analyzing the toxicity of various unknown chemicals. Every part of the fly serves as a model in metabolic and therapeutic approaches. Genotoxic and teratogenic compounds are exposed to Drosophila through the oral route. Further, the toxicity of genotoxic compounds is analyzed in Drosophila's gut, hemolymph, and phenotype. The toxicity of teratogen compounds is also analyzed using a Drosophila embryo. The current chapter summarizes several techniques that are used to detect the genotoxicity and teratogenicity of any unknown compound in this model.

Investigation of the effects of nanoplastic polyethylene terephthalate on environmental toxicology using model Drosophila melanogaster.

Plastic pollution has become a major environmental concern, and various plastic polymers are used daily. A study was conducted to examine the toxic effects of polyethylene terephthalate (PET) nanoplastics (NPLs) on Drosophila melanogaster. We have successfully synthesized PET NPLs and characterized using DLS, Zeta potential, TEM, HRTEM, SAED, XRD, FTIR, and Raman spectroscopy to gain crucial insights into the structure and properties. We fed PET NPLs to Drosophila to assess toxicity. ROS was quantified using DCFH-DA and NBT, and the nuclear degradation was checked by DAPI staining. Quantification of protein and activity of antioxidant enzymes like SOD, catalase depicted the adverse consequences of PET NPLs exposure. The dorsal side of the abdomens, eyes, and wings were also defective when phenotypically analyzed. These results substantiate the genotoxic and cytotoxic impact of nanoplastics. Notably, behavioral observations encompassing larval crawling and climbing of adults exhibit normal patterns, excluding the presence of neurotoxicity. Adult Drosophila showed decreased survivability, and fat accumulation enhanced body weight. These findings contribute to unraveling the intricate mechanisms underlying nanoplastic toxicity and emphasize its potential repercussions for organismal health and ecological equilibrium.

Ca@Cu-CD nanoprobe for dual detection of glycine and ex vivo glycine imaging.

Hydrothermally prepared copper-doped carbon dots (Cu-CDs) were modified with Ca2+, which serve as an excellent platform for the recognition of glycine. The feeble emission of Ca@Cu-CD increases substantially in the presence of glycine due to aggregation-induced emission. At the same time, there was a 5-fold increase in the current response of the Ca@Cu-CD modified electrode as compared to the control. The exceptional combination of fluorescence and conducting properties, along with Ca-glycine interaction, establishes our probe as a dual sensor for the detection of glycine in real serum samples. The limit of detection for this nonenzymatic fluorescence and electrochemical sensing are 17.2 and 4.1 nM, respectively. Furthermore, an extensive evaluation of the toxicity and bioimaging properties in fruit fly Drosophila melanogaster shows that the Ca@Cu-CD probe is not cytotoxic and can be applied for ex vivo imaging of glycine.

In Vivo Toxicological Analysis of the ZnFe2O4@poly(tBGE-alt-PA) Nanocomposite: A Study on Fruit Fly.

Recently, the use of hybrid nanomaterials (NMs)/nanocomposites has widely increased for the health, energy, and environment sectors due to their improved physicochemical properties and reduced aggregation behavior. However, prior to their use in such sectors, it is mandatory to study their toxicological behavior in detail. In the present study, a ZnFe2O4@poly(tBGE-alt-PA) nanocomposite is tested to study its toxicological effects on a fruit fly model. This nanocomposite was synthesized earlier by our group and physicochemically characterized using different techniques. In this study, various neurological, developmental, genotoxic, and morphological tests were carried out to investigate the toxic effects of nanocomposite on Drosophila melanogaster. As a result, an abnormal crawling speed of third instar larvae and a change in the climbing behavior of treated flies were observed, suggesting a neurological disorder in the fruit flies. DAPI and DCFH-DA dyes analyzed the abnormalities in the larva's gut of fruit flies. Furthermore, the deformities were also seen in the wings and eyes of the treated flies. These obtained results suggested that the ZnFe2O4@poly(tBGE-alt-PA) nanocomposite is toxic to fruit flies. Moreover, this is essential to analyze the toxicity of this hybrid NM again in a rodent model in the future.

Synergetic impact of MgO on PMMA-ZrO2 hybrid composites: Evaluation of structural, morphological and improved mechanical behavior for dental applications.

This work aims to demonstrate the effect of ZrO2 and MgO inclusion into the Poly(methyl methacrylate) (PMMA). To fabricate novel hybrid composites via heat cure method, various composites (PZM2, PZM4 and PZM6) were synthesized in the system [(95-x) PMMA + 5 ZrO2 + x MgO] (x = 2, 4, and 6) respectively. Density of the prepared composites were determined and varying between 1.035-1.152 g/cm3. X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) followed by EDAX and mechanical testing were performed to evaluate the fabricated composite properties. Moreover, to explore the structure of the fabricated composites the 13 C CP-MAS SSNMR and 1 H-13 C Phase-Modulated Lee Goldberg (PMLG) HETCOR Spectrum were recorded which clarify chemical shifting and motional dynamics of the composites. Mechanical tests were performed by UTM and the obtained parameters such as compressive strength, Young's modulus, fracture toughness, brittleness coefficient, flexural strength and flexural modulus are found to be in the range of 91-100 MPa, 0.48-0.51 GPa, 9.122-9.705 MPa.m1/2, 0.66-0.815, 51.03-42.78 MPa and 499-663 MPa respectively. Some more mechanical parameters such as proportional limit, elastic limit, failure strength, modulus of resilience and modulus of toughness were also calculated. Furthermore, tribological properties were also determined and the coefficient of friction (COF) was decreased by 17.4 % and 38 % for composite PZM6 at 20 N and 40 N as compared to the composite PZM2 and the lowest wear volume of 1.55 mm3 was observed for PZM2, whereas the maximum volume loss of 5.64 mm3 is observed for composite PZM6. To check out the biocompatibility, cytotoxicity and genotoxicity of the fabricated composites the Trypan-blue assay was also performed for PZM2 and PZM6 composites. Dissection on the gut of larvae was also performed on the both composites followed by DAPI and DCFH-DA staining. Therefore, these synthesized samples can be used for the fabrication of denture materials.

Antibacterial and cytotoxicity studies of pyrrolo-based organic scaffolds and their binding interaction with bovine serum albumin.

Two pyrrolo-based compounds, 1H-pyrrolo[3,2-b]pyridine-3-carboxylic acid (L1) and 1H-pyrrolo[3,2-c]pyridine-4-carboxylic acid (L2), were employed for the detection of bovine serum albumin (BSA) by UV-Vis and fluorescence spectroscopic methods in phosphate buffer solution (pH = 7). In the presence of L1 and L2, the fluorescence emission of BSA at 340 nm was quenched and concomitantly a red-shifted emission band appeared at 420 nm (L1)/450 nm (L2). The fluorescence spectral changes indicate the protein-ligand complex formation between BSA and L1/L2. An isothermal titration calorimetry (ITC) experiment was conducted to determine the binding ability between BSA and L1/L2. The binding constants are found to be 4.45 ± 0.22 × 104 M-1 for L1 and 2.29 ± 0.11 × 104 M-1 for L2, respectively. The thermodynamic parameters were calculated from ITC measurements (i.e. ∆rH = -40 ± 2 kcal/mol, ∆rG = -4.57 ± 0.22 kcal/mol and -T∆rS = 35.4 ± 1.77 kcal/mol), which indicated that the protein-ligand complex formation between L1/L2 with BSA is mainly due to the electrostatic interactions. The protein-ligand interactions were studied by performing molecular docking. Further, the antibacterial assay of L1 and L2 was conducted against gram-positive and gram-negative bacterial strains in an effort to address the difficulties caused by the co-occurrence of antimicrobial and multidrug-resistant bacteria. E. coli and S. aureus were significantly inhibited by L1 and L2. The L1 exhibits 13, 12 and 15 mm, whereas L2 exhibits a 2, 3 and 5 mm zone of inhibition against S. aureus, S. pyogenes and E. coli, respectively. In silico molecular docking of L1 and L2 was performed with bacterial DNA gyrase to establish the intermolecular interactions. Finally, the in vitro cytotoxicity activities of the ligands L1 and L2 have been carried out using drosophila.

Pph13 and orthodenticle define a dual regulatory pathway for photoreceptor cell morphogenesis and function.

The function and integrity of photoreceptor cells are dependent upon the creation and maintenance of specialized apical structures: membrane discs/outer segments in vertebrates and rhabdomeres in insects. We performed a molecular and morphological comparison of Drosophila Pph13 and orthodenticle (otd) mutants to investigate the transcriptional network controlling the late stages of rhabdomeric photoreceptor cell development and function. Although Otd and Pph13 have been implicated in rhabdomere morphogenesis, we demonstrate that it is necessary to remove both factors to completely eliminate rhabdomere formation. Rhabdomere absence is not the result of degeneration or a failure of initiation, but rather the inability of the apical membrane to transform and elaborate into a rhabdomere. Transcriptional profiling revealed that Pph13 plays an integral role in promoting rhabdomeric photoreceptor cell function. Pph13 regulates Rh2 and Rh6, and other phototransduction genes, demonstrating that Pph13 and Otd control a distinct subset of Rhodopsin-encoding genes in adult visual systems. Bioinformatic, DNA binding and transcriptional reporter assays showed that Pph13 can bind and activate transcription via a perfect Pax6 homeodomain palindromic binding site and the Rhodopsin core sequence I (RCSI) found upstream of Drosophila Rhodopsin genes. In vivo studies indicate that Pph13 is necessary and sufficient to mediate the expression of a multimerized RCSI reporter, a marker of photoreceptor cell specificity previously suggested to be regulated by Pax6. Our studies define a key transcriptional regulatory pathway that is necessary for late Drosophila photoreceptor development and will serve as a basis for better understanding rhabdomeric photoreceptor cell development and function.