Injectable biocompatible RAFT mediated nitroxide nanogels: A robust ROS-reduction antioxidant approach.

This work introduces novel nitroxide-based nanogels (NGs) crafted through controlled RAFT (Reversible Addition Fragmentation chain Transfer) polymerization, showcasing over 85% improved shelf-life compared to native superoxide dismutase (SOD) enzymes. These 30-40 nm NGs hold great promise for injectable delivery, effectively reducing foam cell formation and displaying potent antioxidant behavior against various reactive oxygen species (ROS), revolutionizing antioxidant therapy. Featuring a meticulously designed core-shell structure via precise RAFT polymerization, these NGs mimic SOD enzymatic activity with nitroxide-based antioxidants, providing unprecedented defense against ROS. Combining methacrylated 2,2,6,6-Tetramethyl-4-piperidyl methacrylate (PMA) and Glycidyl methacrylate (GMA) monomers with precisely synthesized nitroxyl radicals results in exceptional properties. Validated through comprehensive analytical methods, these NGs exhibit remarkable stability, halting foam cell formation even at high concentrations, and demonstrate notable biocompatibility. Their ability to protect low density lipoprotein (LDL) from oxidation for up to a month positions them at the forefront of combating cardiovascular diseases, especially atherosclerosis. This study pioneers injectable antioxidant therapy, offering an innovative approach to cardiovascular ailments. Targeting narrow plaques signifies a promising intervention, reshaping cardiovascular disease treatments. It highlights the potential of advanced drug delivery in biomedicine, promising more effective cardiovascular disease treatments.

Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

Target-Dependent Coordinated Biogenesis of Secondary MicroRNAs by miR-146a Balances Macrophage Activation Processes.

MicroRNAs (miRNAs) repress protein expression by binding to the target mRNAs. Exploring whether the expression of one miRNA can regulate the abundance and activity of other miRNAs, we noted the coordinated biogenesis of miRNAs in activated macrophages. miRNAs with higher numbers of binding sites (the "primary" miRNAs) induce expression of other miRNAs ("secondary" miRNAs) having binding sites on the 3' untranslated region (UTR) of common target mRNAs. miR-146a-5p, in activated macrophages, acts as a "primary" miRNA that coordinates biogenesis of "secondary" miR-125b, miR-21, or miR-142-3p to target new sets of mRNAs to balance the immune responses. During coordinated biogenesis, primary miRNA drives the biogenesis of secondary miRNA in a target mRNA- and Dicer1 activity-dependent manner. The coordinated biogenesis of miRNAs was observed across different cell types. The target-dependent coordinated miRNA biogenesis also ensures a cumulative mode of action of primary and secondary miRNAs on the secondary target mRNAs. Interestingly, using the "primary" miR-146a-5p-specific inhibitor, we could inhibit the target-dependent biogenesis of secondary miRNAs that can stop the miRNA-mediated buffering of cytokine expression and inflammatory response occurring in activated macrophages. Computational analysis suggests the prevalence of coordinated biogenesis of miRNAs also in other contexts in human and in mouse.

Co-evolutionary landscape at the interface and non-interface regions of protein-protein interaction complexes.

Proteins involved in interactions throughout the course of evolution tend to co-evolve and compensatory changes may occur in interacting proteins to maintain or refine such interactions. However, certain residue pair alterations may prove to be detrimental for functional interactions. Hence, determining co-evolutionary pairings that could be structurally or functionally relevant for maintaining the conservation of an inter-protein interaction is important. Inter-protein co-evolution analysis in several complexes utilizing multiple existing methodologies suggested that co-evolutionary pairings can occur in spatially proximal and distant regions in inter-protein interactions. Subsequently, the Co-Var (Correlated Variation) method based on mutual information and Bhattacharyya coefficient was developed, validated, and found to perform relatively better than CAPS and EV-complex. Interestingly, while applying the Co-Var measure and EV-complex program on a set of protein-protein interaction complexes, co-evolutionary pairings were obtained in interface and non-interface regions in protein complexes. The Co-Var approach involves determining high degree co-evolutionary pairings that include multiple co-evolutionary connections between particular co-evolved residue positions in one protein with multiple residue positions in the binding partner. Detailed analyses of high degree co-evolutionary pairings in protein-protein complexes involved in cancer metastasis suggested that most of the residue positions forming such co-evolutionary connections mainly occurred within functional domains of constituent proteins and substitution mutations were also common among these positions. The physiological relevance of these predictions suggested that Co-Var can predict residues that could be crucial for preserving functional protein-protein interactions. Finally, Co-Var web server (http://www.hpppi.iicb.res.in/ishi/covar/index.html) that implements this methodology identifies co-evolutionary pairings in intra and inter-protein interactions.

Rheb-mTOR activation rescues Aß-induced cognitive impairment and memory function by restoring miR-146 activity in glial cells.

Deposition of amyloid beta plaques in adult rat or human brain is associated with increased production of proinflammatory cytokines by associated glial cells that are responsible for degeneration of the diseased tissue. The expression of these cytokines is usually under check and is controlled at the post-transcriptional level via several microRNAs. Computational analysis of gene expression profiles of cortical regions of Alzheimer's disease patients' brain suggests ineffective target cytokine mRNA suppression by existing micro-ribonucleoproteins (miRNPs) in diseased brain. Exploring the mechanism of amyloid beta-induced cytokine expression, we have identified how the inactivation of the repressive miR-146 miRNPs causes increased production of cytokines in amyloid beta-exposed glial cells. In exploration of the cause of miRNP inactivation, we have noted amyloid beta oligomer-induced sequestration of the mTORC1 complex to early endosomes that results in decreased Ago2 phosphorylation, limited Ago2-miRNA uncoupling, and retarded Ago2-cytokine mRNA interaction in rat astrocytes. Interestingly, constitutive activation of mTORC1 by Rheb activator restricts proinflammatory cytokine production by reactivating miR-146 miRNPs in amyloid beta-exposed glial cells to rescue the disease phenotype in the in vivo rat model of Alzheimer's disease.

MicroRNA exporter HuR clears the internalized pathogens by promoting pro-inflammatory response in infected macrophages.

HuR is a miRNA derepressor protein that can act as miRNA sponge for specific miRNAs to negate their action on target mRNAs. Here we have identified how HuR, by inducing extracellular vesicles-mediated export of miRNAs, ensures robust derepression of miRNA-repressed cytokines essential for strong pro-inflammatory response in activated mammalian macrophages. Leishmania donovani, the causative agent of visceral leishmaniasis, on the contrary alters immune response of the host macrophage by a variety of complex mechanisms to promote anti-inflammatory response essential for the survival of the parasite. We have found that during Leishmania infection, the pathogen targets HuR to promote onset of anti-inflammatory response in mammalian macrophages. In infected macrophages, Leishmania also upregulate protein phosphatase 2A that acts on Ago2 protein to keep it in dephosphorylated and miRNA-associated form. This causes robust repression of the miRNA-targeted pro-inflammatory cytokines to establish an anti-inflammatory response in infected macrophages. HuR has an inhibitory effect on protein phosphatase 2A expression, and mathematical modelling of macrophage activation process supports antagonistic miRNA-modulatory roles of HuR and protein phosphatase 2A which mutually balances immune response in macrophage by targeting miRNA function. Supporting this model, ectopic expression of the protein HuR and simultaneous inhibition of protein phosphatase 2A induce strong pro-inflammatory response in the host macrophage to prevent the virulent antimonial drug-sensitive or drug-resistant form of L. donovani infection. Thus, HuR can act as a balancing factor of immune responses to curtail the macrophage infection process by the protozoan parasite.

Identification of Important Effector Proteins in the FOXJ1 Transcriptional Network Associated With Ciliogenesis and Ciliary Function.

Developmental defects in motile cilia, arising from genetic abnormalities in one or more ciliary genes, can lead to a common ciliopathy known as primary ciliary dyskinesia (PCD). Functional studies in model organisms undertaken to understand PCD or cilia biogenesis have identified 100s of genes regulated by Foxj1, the master regulator of motile ciliogenesis. However, limited systems based studies have been performed to elucidate proteins or network/s crucial to the motile ciliary interactome, although this approach holds promise for identification of multiple cilia-associated genes, which, in turn, could be utilized for screening and early diagnosis of the disease. Here, based on the assumption that FOXJ1-mediated regulatory and signaling networks are representative of the motile cilia interactome, we have constructed and analyzed the gene regulatory and protein-protein interaction network (PPIN) mediated by FOXJ1. The predicted FOXJ1 regulatory network comprises of 424 directly and 148 indirectly regulated genes. Additionally, based on gene ontology analysis, we have associated 17 directly and 6 indirectly regulated genes with possible ciliary roles. Topological and perturbation analyses of the PPIN (6927 proteins, 40,608 interactions) identified 121 proteins expressed in ciliated cells, which interact with multiple proteins encoded by FoxJ1 induced genes (FIG) as important interacting proteins (IIP). However, it is plausible that IIP transcriptionally regulated by FOXJ1 and/or differentially expressed in PCD are likely to have crucial roles in motile cilia. We have found 20 de-regulated topologically important effector proteins in the FOXJ1 regulatory network, among which some (PLSCR1, SSX2IP, ACTN2, CDC42, HSP90AA1, PIAS4) have previously reported ciliary roles. Furthermore, based on pathway enrichment of these proteins and their primary interactors, we have rationalized their possible roles in the ciliary interactome. For instance, 5 among these novel proteins that are involved in cilia associated signaling pathways (like Notch, Wnt, Hedgehog, Toll-like receptor etc.) could be 'topologically important signaling proteins.' Therefore, based on this FOXJ1 network study we have predicted important effectors in the motile cilia interactome, which are possibly associated with ciliary biology and/or function and are likely to further our understanding of the pathophysiology in ciliopathies like PCD.

Matrix-Assisted Regulation of Antimicrobial Properties: Mechanistic Elucidation with Ciprofloxacin-Based Polymeric Hydrogel Against Vibrio Species.

The design of a new drug material through modification of some well-known antibiotics to combat pathogenic bacteria must include a complete understanding of matrix regulation because the human body consists of primarily three types of matrices, that is, solid, semisolid, and liquid, all of which have a tendency to regulate antibacterial efficacy along with the bactericidal mechanism of several antimicrobial agents. Herein, matrix-dependent action of ciprofloxacin-based polymeric hydrogel scaffold was explored against a new species of Vibrio, namely, Vibrio chemaguriensis Iso1 ( V. chemaguriensis), which is resistant to most of the common antibiotics and possess genes that can be linked to pathogenicity. Ciprofloxacin was attached to the polymeric system consisting of hydrophilic polyethylene glycol methyl ether methacrylate (PEGMA) and zwitterionic sulphabetaine methacrylate (SBMA) with an antifouling nature via covalent linkage leading to effective polymer antibiotic conjugates (PACs) with linear and hyperbranched architectures. The hyperbranched PAC was transformed to a polymeric gel exhibiting greater antibacterial efficacy in solid matrix than that of the liquid one with a complete bactericidal effect and rod to spherical switching of bacterial cell followed by chain formation via "dual" contact activity and release mechanism through sustained removal of thiol-terminated ciprofloxacin fragment along with an equilibrium swelling and deswelling process. Lower killing efficacy was displayed in the liquid matrix with an intact cell morphology that is due to lack of forced contact between the cell wall and gel surface as well as entrapment of released bioactive fragment via an additional thick exopolysaccharide (EPS) layer, which represents a great challenge to modern medical sciences.

Leucine-Based Polymer Architecture-Induced Antimicrobial Properties and Bacterial Cell Morphology Switching.

To evaluate the comparative antibacterial activity of leucine-based cationic polymers having linear, hyperbranched, and star architectures containing both hydrophilic and hydrophobic segments against Gram-negative bacterium, Escherichia coli (E. coli), herein we performed zone of inhibition study, minimum inhibitory concentration (MIC) calculation, and bacterial growth experiment. The highest antibacterial activity in terms of the MIC value was found in hyperbranched and star architectures because of the greater extent of cationic and hydrophobic functionality, enhancing cell wall penetration ability compared to that of the linear polymer. The absence of the bacterial regrowth stage in the growth curve exhibited the highest bactericidal capacity of star polymers, when untreated cells (control) already reached to the stationary phase, whereas the bacterial regrowth stage with a delayed lag phase was critically observed for linear and hyperbranched architectures displaying lower bactericidal efficacy. Coagulation of E. coli cells, switching of cell morphology from rod to sphere, and lengthening due to stacking in an antimicrobial polymer-treated environment at the bacterial regrowth stage in liquid media were visualized critically by field emission scanning electron microscopy and confocal fluorescence microscopy instruments in the presence of 4',6-diamidino-2-phenylindole stain.

Recyclable Thermoresponsive Polymer-ß-Glucosidase Conjugate with Intact Hydrolysis Activity.

ß-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic ß-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of ß-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for ß-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

Monitoring aggregation of a pH-responsive polymer via proton exchange.

Understanding the changes in the macro-structure of amphiphilic pH-responsive polymers remains a relevant issue due to their potential use as drug delivery carriers. Since some of the amphiphilic polymers are known to exchange hydrogen ions with an aqueous solvent, we monitor the effective change of the surface to volume ratio of such polymer aggregates using solution-state nuclear magnetic resonance (NMR) spectroscopy. The surface to volume ratio with the help of UV-visible spectroscopy is shown to yield the average diameter of the polymer aggregates. We show that the proposed method not only satisfactorily corroborates the existing notions of how the aggregation of these polymers takes place as a function of pH, but also provides a quantitative estimate of the size of the aggregates.

Encapsulation induced aggregation: a self-assembly strategy for weakly pi-stacking chromophores.

Molecular assembly of weakly pi-stacking core-substituted naphthalene diimides (cNDIs) requires the participation of strong side-group interactions. Spatial confinement within a micellar core leads to locally elevated concentrations and reduced entropy that drives a rapid aggregation, often followed by a slower aggregate reorganization. Fast aggregation kinetics leads to self-sorting of aggregates.

Side-Chain Amino Acid-Based Cationic Antibacterial Polymers: Investigating the Morphological Switching of a Polymer-Treated Bacterial Cell.

Synthetic polymer-based antimicrobial materials destroy conventional antibiotic resistant microorganisms. Although these antibacterial polymers imitate the properties of antimicrobial peptides (AMPs), their effect on bacterial cell morphology has not been studied in detail. To investigate the morphology change of a bacterial cell in the presence of antimicrobial polymer, herein we have designed and synthesized side-chain amino acid-based cationic polymers, which showed efficient antibacterial activity against Gram-negative (Escherichia coli), as well as Gram-positive (Bacillus subtilis) bacteria. Morphological switching from a rod shape to a spherical shape of E. coli cells was observed by field emission-scanning electron microscopy analysis due to cell wall disruption, whereas the B. subtilis cell structure and size remained intact, but stacks of the cells formed after polymer treatment. The zone of inhibition experiment on an agar plate for E. coli cells exhibited drastic morphological changes at the vicinity of the polymer-treated portion and somewhat less of an effect at the periphery of the plate.

Identification of internalin-A-like virulent proteins in Leishmania donovani.

BACKGROUND: An active immune surveillance and a range of barriers to infection allow the host to effectively eliminate microbial pathogens. However, pathogens may use diverse strategies to subdue such host defences. For instance, one such mechanism is the use of leucine-rich repeat (LRR) proteins by pathogens (microbial) to cause infection. In this study, we aimed at identifying novel virulence factor(s) in Leishmania donovani, based on the possibility of lateral gene transfers of bacterial virulence factor(s) to L. donovani. METHODS: Rigorous homology searching protocols including Hidden Markov Model (HMM) and BLASTp based searches were employed to detect remote but significant similarities between L. donovani proteins and bacterial virulence factors. RESULTS: We found that some L. donovani proteins are similar to internalin-A (Inl-A) protein of Listeria monocytogenes, a surface LRR protein that helps mediate host cell invasion by interacting with E-cadherin on the cell membrane. However, to date, no such invasion mechanism has been reported in Leishmania donovani, the causative agent of visceral leishmaniasis. Moreover, a comparative LRR motif analysis of L. donovani Inl-A-like proteins against the Inl-A protein of L. monocytogenes revealed existence of characteristic consensus LRR regions, suggesting a reliable evolutionary relationship between them. Further, through rigorous three dimensional (3D) modeling of L. donovani Inl-A-like proteins and subsequent molecular docking studies we suggest the probability of human E-cadherin binding with the L. donovani Inl-A-like proteins. CONCLUSIONS: We have identified three potential candidates (UniProt ID: E9B7L9, E9BMT7 and E9BUL5) of Inl-A-like LRR containing proteins in L. donovani with the help of systematic whole genome sequence analysis. Thus, herein we propose the existence of a novel class of Inl-A-like virulence factor proteins in L. donovani and other Leishmania species based on sequence similarity, phylogenetic analysis and molecular modelling studies in L. donovani.

One-step integration of metal nanoparticle in photonic crystal nanobeam cavity.

A single step process of integrating a resonantly tuned silver nanoparticle into photonic crystal nanobeam cavities fabricated by focused ion beam milling is presented. Even though the quality factor of the cavities is reduced by a factor of 20, the emission peak at the cavity resonance is enhanced by 5-fold with respect to the cavities without the metal nanoparticle. The fluorescence is also compared before and after etching away the nanoparticle. Experimental quality factors and wavelength shifts are found to agree reasonably well with simulation values. These results are promising for future single photon emission studies involving the incorporation of quantum dot or NV center emitters into hybrid plasmonic/photonic crystal cavities for enhanced emission rates while retaining reasonably high quality factors.